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paclitaxel is an antineoplastic drug isolated from a bark extract of taxus brevifolia ( taxaceae ) the governments of the us and canada approved paclitaxel for sale in 1992 , and a parenteral solution of paclitaxel subsequently became commercially available in japan in 1997 .
paclitaxel is used clinically in the treatment of ovarian , breast , endometrial , stomach , and non - small cell lung cancers in japan .
the main adverse drug reactions to paclitaxel include gastrointestinal symptoms , peripheral neuropathy , arthralgia , muscular pain , nausea and vomiting , epilation , and pyrexia .
paclitaxel tends to be soluble in n , n - dimethylacetamide , acetonitrile , methanol , and ethanol but is relatively insoluble in water .
because 50% ethanol is used as the solvent for clinical paclitaxel injections , we hypothesized that impairment of specific central nervous system ( cns ) functions by ethanol or its cleavage product , acetaldehyde , as well as adverse reactions related to intoxication , may occur following treatment with this preparation .
thus , the possibility of adverse reactions following intake of ethanol accompanying paclitaxel administration should not be overlooked . since
many hospitals in japan are located in rural areas and are not conveniently accessible by public transportation , most patients drive to the hospital .
thus , it is important to consider the possible cns depressant actions of ethanol contained in injectable drug formulations , in order to reduce the risk of serious car accidents .
furthermore , in the road traffic act in japan , the breath ethanol concentration that constitutes drunk driving is 0.15 mg / l this threshold is lower than those in the uk , usa , and canada ( 0.40
mg / l ) , and those in australia , germany , and france ( 0.25 mg / l ) .
it is important to ensure that patients who receive paclitaxel injections containing ethanol do not have breath ethanol concentrations exceeding the legal threshold .
although research on plasma ethanol concentrations following paclitaxel administration has been published previously , only a few reports have evaluated the correlation between ethanol intake during chemotherapy and the ethanol concentration in exhaled breath . here , we investigated the concentration of ethanol in exhaled breath after chemotherapy with an intravenous paclitaxel infusion .
thirty japanese outpatients ( mean age 55 8.6 years [ range 3574 ] ; 2 male and 28 female ) who received treatment with paclitaxel ( 80330 mg / day ) for breast , ovarian , or gastric cancer were eligible subjects for this research .
this clinical study was approved by the institutional review board for clinical trials at gunma university hospital ( maebashi , japan ) .
written consent was obtained from all patients after they were informed of the study procedure .
the volume of ethanol administered and the infusion rate of ethanol were calculated from the volume of the paclitaxel infusion and the administration time .
immediately after administration of the intravenous infusion to a subject , a balloon - type gas detector tube ( kitagawa gas detector tube system ; komyo rikagaku kogyo kk , kanagawa , japan ) was used to measure the concentration of ethanol in exhaled breath .
the levels of aspartic acid aminotransferase ( ast ) and alanine aminotransferase ( alt ) were noted from the medical records , and the alcohol drinking history was taken from each patient .
correlations between the total amount of ethanol administered and the ethanol concentration in exhaled breath , and between the intravenous infusion speed and the ethanol concentration in exhaled breath , were calculated using pearson s correlation coefficient .
thirty japanese outpatients ( mean age 55 8.6 years [ range 3574 ] ; 2 male and 28 female ) who received treatment with paclitaxel ( 80330 mg / day ) for breast , ovarian , or gastric cancer were eligible subjects for this research .
this clinical study was approved by the institutional review board for clinical trials at gunma university hospital ( maebashi , japan ) .
written consent was obtained from all patients after they were informed of the study procedure .
the volume of ethanol administered and the infusion rate of ethanol were calculated from the volume of the paclitaxel infusion and the administration time . immediately after administration of the intravenous infusion to a subject ,
a balloon - type gas detector tube ( kitagawa gas detector tube system ; komyo rikagaku kogyo kk , kanagawa , japan ) was used to measure the concentration of ethanol in exhaled breath .
the levels of aspartic acid aminotransferase ( ast ) and alanine aminotransferase ( alt ) were noted from the medical records , and the alcohol drinking history was taken from each patient .
correlations between the total amount of ethanol administered and the ethanol concentration in exhaled breath , and between the intravenous infusion speed and the ethanol concentration in exhaled breath , were calculated using pearson s correlation coefficient .
the patient characteristics , the amount of paclitaxel administered , the speed of the intravenous infusion , and the concentration of ethanol in exhaled breath are summarized in table i. the average ethanol concentration in exhaled breath immediately after the intravenous infusion of paclitaxel was 0.028 0.015 mg / l ( range 0.000.06 ) .
ethanol concentrations in exhaled breath of individual patients hepatic function in all patients was assessed to be within the normal range , as indicated by ast and alt values of 1233 u / l and 1262 u / l , respectively .
the correlation coefficient between the total amount of ethanol administered via the intravenous infusion and the ethanol concentration in exhaled breath was weak ( r = 0.25 ; p = 0.055 ) [ figure 1a ] .
in contrast , the intravenous infusion speed had a relatively stronger positive correlation with the concentration of exhaled ethanol ( r = 0.49 ; p = 0.11 ) [ figure 1b ] .
relationship between the ethanol concentration in exhaled breath and ( a ) the total amount of ethanol administered via the intravenous paclitaxel infusion ; and ( b ) the speed of the paclitaxel infusion .
the patient characteristics , the amount of paclitaxel administered , the speed of the intravenous infusion , and the concentration of ethanol in exhaled breath are summarized in table i. the average ethanol concentration in exhaled breath immediately after the intravenous infusion of paclitaxel was 0.028 0.015 mg / l ( range 0.000.06 ) .
ethanol concentrations in exhaled breath of individual patients hepatic function in all patients was assessed to be within the normal range , as indicated by ast and alt values of 1233 u / l and 1262 u / l , respectively .
the correlation coefficient between the total amount of ethanol administered via the intravenous infusion and the ethanol concentration in exhaled breath was weak ( r = 0.25 ; p = 0.055 ) [ figure 1a ] .
in contrast , the intravenous infusion speed had a relatively stronger positive correlation with the concentration of exhaled ethanol ( r = 0.49 ; p = 0.11 ) [ figure 1b ] .
relationship between the ethanol concentration in exhaled breath and ( a ) the total amount of ethanol administered via the intravenous paclitaxel infusion ; and ( b ) the speed of the paclitaxel infusion .
more than 90% of ethanol is metabolized by alcohol dehydrogenase ( adh ) and aldehyde dehydrogenase 2 ( aldh2 ) in the liver it has been reported that people with low aldh2 activity show hereditary sensitivity to the effects of alcohol , and approximately 50% of japanese people are poor alcohol metabolizers thus , the percentage of japanese people who experience facial flush and heart palpitations in association with elevated blood aldehyde concentrations after drinking alcohol is larger than that of europeans and americans .
therefore , there is a greater risk of intoxication leading to a car accident in people who have poor ethanol metabolism , because the blood ethanol concentration remains high even after consumption of a relatively small amount of alcohol .
ethanol is eliminated primarily by a saturable ( michaelis - menten ) process hence , the half - life of ethanol changes according to the dose or the rate of administration .
paclitaxel injections contain 50% ( v / v ) ethanol ; thus , if 300 mg of paclitaxel is injected , 25 ml ethanol is also administered .
furthermore , because the first - pass effect does not apply to intravenous infusions , the effects of ethanol will be greater than with oral administration . in this study , an ethanol concentration in exhaled breath that exceeded the threshold for drunk driving , as specified in the road traffic act , was not detected in any patient , but there was one case that reached more than 40% of the threshold .
moreover , a previous report described several cases that exceeded the threshold defined by the law the relationship between the ethanol concentration in breath and that in blood has been investigated , and a method of deducing the blood concentration from the concentration in breath has been established .
moreover , when considering the cns effects , the ethanol concentration in breath ( which reflects the arterial blood ethanol concentration ) is considered to be a more suitable indicator than the venous blood ethanol concentration .
the ratio of venous blood ethanol concentrations to exhaled breath ethanol concentrations is approximately 2000 : 1 the average blood ethanol concentration estimated from our findings was 0.06 0.03 mg / ml .
webster et al . reported that the average plasma ethanol concentration after administration of paclitaxel in caucasian patients was 0.07 0.10 mg / ml when the average doses of paclitaxel in both studies ( 155 76 and 293 35 mg , respectively ) are taken into consideration , the estimated blood ethanol concentrations may have been a little higher in our study .
the difference in the body size between japanese and caucasian subjects may have affected this . because ethanol has a fast elimination rate , its concentrations steady state rapidly , and this is why the plasma ethanol concentration at the end of administration depends on the infusion speed .
thus , the ethanol concentration in exhaled breath after administration of paclitaxel is considered to be affected by the infusion speed but not by the total amount of ethanol administered .
there were several subjects who complained of facial flush or light - headedness after the end of the intravenous infusion , which may have been a response to the ethanol metabolite , acetaldehyde in these cases , markers other than the breath ethanol concentration should be considered , in order to assess the degree of intoxication . in general , patients with high sensitivity to ethanol tend to present with symptoms of alcohol impairment and also have impaired decision - making ability .
the gender bias of the patients should be mentioned as a limitation of this study . because most patients in the study were outpatients with breast cancer or ovarian cancer , the majority of the patients were female .
it has previously been shown that when the same dose of ethanol is administered to male and female subjects , higher blood concentrations are reached in females than in males , and this may have affected our results .
we have shown that the ethanol concentration in exhaled breath after administration of paclitaxel is affected by the infusion speed rather than by the total amount of ethanol administered . however ,
it is difficult to predict from this information which patients will show a high breath ethanol concentration .
hence , all outpatients receiving paclitaxel should avoid driving from hospital when possible and , if driving is unavoidable , they should drive only after taking a sufficient break .
the possible effects of the ethanol additive should be considered carefully when administering drugs , such as paclitaxel , with a high volume of ethanol additive .
|
backgroundethanol is included in certain injectable preparations of anticancer drugs to increase their solubility .
since the volume of ethanol in these preparations is approximately half of the total injection volume , the potential inhibitory effects of ethanol on the central nervous system can not be disregarded , especially considering that patients may drive immediately after administration of the medication .
therefore , the concentration of ethanol was examined in exhaled breath after administration of paclitaxel , an anticancer medication containing ethanol.methodsthe ethanol concentration in exhaled breath immediately after an intravenous infusion of paclitaxel was measured in 30 patients , using a balloon - type gas detector tube .
correlations between the concentration of ethanol in exhaled breath and the total amount of ethanol administered or the intravenous infusion speed were calculated.resultsthe mean ethanol concentration in exhaled breath was 0.028 0.015
mg / l .
the correlation between the ethanol concentration in exhaled breath and the total dose of ethanol was weak ( r2 = 0.25 ; p = 0.055 ) , while the intravenous infusion speed showed a stronger positive correlation with the concentration of ethanol in the breath ( r2 = 0.49 ; p = 0.11 ) .
the maximum concentration of ethanol measured in exhaled breath ( 0.06
mg / l ) was equivalent to 40% of the threshold for drunk driving , as specified in the road traffic act in japan.conclusionin this study , no patient had a breath ethanol concentration exceeding the legal threshold for drunk driving .
however , it is still advisable for patients to avoid driving after receiving paclitaxel injections .
when driving can not be avoided , patients should wait for a sufficient time after receiving the injection before driving .
|
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multiple sclerosis ( ms ) , received its name in 1955 , is an inflammatory disorder of brain and spinal cord where in body 's immune system incorrectly attacks its own central nervous system ( cns ) , causing variable and unpredictable symptoms ( some examples including slurred speech , blurred vision , loss of balance , poor coordination , tremors , numbness , extreme fatigue , problems with memory and concentration , paralysis , and blindness ) .
even though a lot of advances have been made in past few decades , exact causes of this disorder remains unknown(environmental factor combined with genetic predisposition ) .
there is a high cost associated with this disorder and it reduces life expectancy as well as quality of life .
unfortunately , there is no cure for ms , and most of the current drugs available in the market help treat the symptoms .
ms is not the same in any two people who have it , since the underlying cause is demyelination ( along with axon degeneration ) , which can target any brain area , resulting in a broad range of clinical symptoms ( compston and coles , 2002 ; alastair compston et al .
in addition , brain lesions can sometimes outnumber the clinical symptoms by as much as 10:1 , and lesions in noncritical areas may not result in obvious functional deficits , even if brain function is in fact altered .
there are four subtypes of the disease - relapsing - remitting ms , primary progressive ms , secondary progressive ms and progressive relapsing ms , which differ in relapse rate , clinical symptoms , trajectory , underlying causes and approaches to disease management .
combined all these aspects of the disease ( various locations for demyelination , clinically silent timeline , and a highly variable disease course ) make the approach to care and treatment complex ( compston and coles , 2008 ; trapp and nave , 2008 ) .
current behavioral and imaging methods used to assess disease severity are insufficient by themselves to allow confident prediction of disease progression .
the addition of functional modeling ( modeling that correlates the functional loss to disease progression ) and objective functional measurements ( imaging as well as other assessments combined with disease status ) may greatly improve such prediction .
currently , magnetic resonance imaging ( mri ) along with clinical manifestation of the disease are used as clinical tools for diagnosing ms ( filippi and grossman , 2002 ) . the 2010 revision to the mcdonald diagnostic criteria ( polman et al . ,
2011 ) uses a combination of lesion and clinical attacks for the most confident diagnosis of ms .
lesions must be disseminated in time or space or have positive identification from cerebral spinal fluid ( csf ) measure ( lublin et al . , 2014 ) .
unfortunately , discrepancy still exists between clinical symptoms and structural measurements , because of the poor correlation between the presence of lesions and symptoms .
the two common measures used for diagnosis and monitoring with mri are hyperintense lesions on dual - echo mr , which is a nonspecific measure of macroscopic tissue injury , and enhanced abnormalities on t1 lesion weighted images , which is a measure of lesions .
however , neither of these measures do provide extent and severity of inflammation , cellular component , or resultant tissue damage information ( filippi and grossman , 2002 ) .
thus , there is a need for supplemental information from alternative techniques to have a more holistic approach .
there are emerging techniques that hold promise to quantify the brain imaging data , and relate that with clinical outcomes .
some promising examples include magnetization transfer mri ( quantitative and continuous measure of loss of myelin and reduction in axonal density ) , diffusion weighted mri ( quantitative measure of size , shape , geometry , and orientation of tissues ) , proton magnetic resonance spectroscopy ( mrs , provides measure of two major pathologic aspects of ms - the active inflammatory / demyelinating process and axonal injury ) , functional mri ( provides functional information about brain activation during motor , sensitive , and cognitive tasks ) , high field strength imaging ( ex 7 t mri , leads to improved signal - to - noise ratio , speed , and resolution in both mri and mrs ) and transcranial magnetic stimulation ( tms , highly sensitive technique to evaluate cortico - spinal conduction abnormalities in ms ) .
since t1 scan is transient , it should be only used in patients that have frequent mri , and should not be used for comparison between subjects . instead ,
given the availability of multiple brain imaging modalities , the need still exists for creating a combined assessment tool .
quite a few studies have attempted to combine various measures ( petzold et al . , 2006 ; daumer et al . , 2007 , 2009 ; castro - borrero et al . , 2012 ; sormani , 2013 ; giffroy et al . ,
multiple sclerosis severity scale ( msss ) was developed to relate expanded disability status scale ( edss ) to the disease duration .
it has been shown that msss correlates with axonal biomarkers but not with glial biomarkers ( petzold et al . , 2006 ) .
this scale was tested on 195 ms patients to predict accrual of disability over time to see if current therapies have impact on disease severity over time .
pachner and steiner ( 2009 ) concluded that the current disease modifying drug therapies lack emphasis on disease severity .
an online analytical processing tool was developed that can be used for prognosis of near term future course of an individual patient ( daumer et al . , 2007 ) .
this tool is based on matching algorithm ( using statistical analysis ) and contains data of 1,059 patients .
outcome was to show the probable progression over time which can be useful for subjects , physicians , researchers and other professionals who counsel the patients . in an attempt to test the developing treatment methods , sormani ( 2013 ) explored the use of various biomarkers that can be used to predict the clinical response to interferon beta ( ifn- ) treatment .
he concluded with the need for precise , meaningful measures of disease progression , integrated with clinical measures to help with personalized treatment for ms .
a computational classifier was modeled that can be used for predicting short term course of ms ( bejarano et al . , 2011 ) .
it combined clinical data , with mri and motor evoked potential ( mep ) , and it was found that the model did good job on predicting short time scale disability .
a description of the use of various potential biomarkers with their pros and cons was created .
it ranges from markers for immunological activation , as well as markers for demyelination , axonal damage , oxidative stress , remyelination , glycoses , and details of specimen such as blood , urine , tears and csf ( bielecki et al . , 2010 ) .
( 2010 ) concluded with a need for unification and standardization of results of various measurements and techniques .
there have been many advances in modeling and designing tools and predictive capabilities for ms .
( 2015 ) studied 1,959 patients from 19952013 with clinically isolated syndrome and found that demographic and topographic characteristics had a low effect on prognostic factors for ms , while the presence of oligoclonal band ( oligoclonal bands are proteins called immunoglobulins and their presence indicates inflammation of the central nervous system ) had medium effect , and the number of lesions had the highest impact . in an another study ,
2014 ) recruited 652 patients and studied them for prediction factors that can be classified as disseminated in time ( dit ) , disseminated in space ( dis ) , or both , and concluded that there is a need of more predictive factor combinations to help risk for ms .
an analysis of 598 ms patients was performed in norway to determine factors responsible for life expectancy , and the conclusion was that high age at the onset was correlated with unfavorable prognosis ( riise et al . , 1988 ) . in another study on early prognostic features on the late course for ms in world war veterans
, it was found that pyramidal and cerebellar scores are the best predictors ( kurtzke et al . , 1972 ) .
wang et al . ( 2015 ) performed a study to combine in vivo biomarker using electrical vestibular stimulation and eye movement recording to measure evoked vestibular - ocular reflexed ( evor ) in 18 subjects with ms ( wang et al . , 2015 ) .
the goal was to measure the axonal conduction velocity to understand the myelin process and provide a way for assessing efficacy of novel reparative therapies in ms .
there still exists a need to for quantification , combination of various results from studies , and longer longitudinal studies .
lastly , a computational approach will be necessary for various biological , clinical , imaging findings to form an integrative modeling system that encompasses grey matter pathology , myelin sheath aqueous layers , energy metabolism , and perhaps most importantly , multi - scale or integrated modeling .
my recent paper ( chaubey and goodwin , 2016 ) is a computational modeling study which makes it possible to make quantitative predictions about the degree to which electrical signals will move more slowly through axonal pathways as a function of how much of their myelin sheath they have lost as a result of ms ( using data from diffusion tensor imaging ( dti ) , and other modalities ) .
the paper identifies a new biomarker for network failure in ms that should improve our ability to predict and track loss of sensory , motor and cognitive function in the disease and a better way to measure the efficacy of new treatments .
the potential also exists to relate this slow down to disease progression , and eventually , to disease prediction , in which we could use the existing model to predict the disease symptoms in subsequent years . a study in my research center ( brain sciences center , va medical center ) used magnetoencephalography ( meg ) resting state recordings as a functional biomarker to classify ms and various other patient populations to their respective groups , by assessing synchronous neural interactions at high temporal resolution as a measurement of the dynamic synchronous neural interactions , an essential aspect of brain function ( georgopoulos et al . , 2007 ) .
another study used similar analysis in 50 ms patients ( 31 rrms , 15 spms , 4 ppms ) and 214 healthy controls ( carpenter et al . , 2011 ) .
ancova was performed at each of the 30,628 sensor pairs ( pccs ) using group ( ms , control ) as a fixed factor and age , gender and handedness as covariates .
similar analyses were done using each ms phenotype ( rrms , spms , etc . ) as a factor and 300 ( out of 30,628 ) sensor pairs were found to have a significant group differences after correction for multiple comparisons .
there is a need to combine such objective measures of brain function with modeling techniques to improve assessment and prediction of the disease over what is possible using established methods . to list few direct advantages of these predictions : 1 )
these predictions can identify high - risk patients who require early and more aggressive therapies , 2 ) they can help patients with clinically isolated syndrome ( cis , one of the ms disease courses , refers to a first episode of neurologic symptoms , but it is not alone enough to have diagnosis of ms , as there needs to be two episodes disseminated in time or space ) to predict how likely and at what time frame the ms diagnosis is likely , 3 ) they can provide an individual plan for various subjects depending on their current status and symptoms ( bergamaschi and montomoli , 2016 ) .
there is a need for modeling to help direct research using testable predictions and fixing the current gap in knowledge .
there is enormous amount of data available , a lot of which is open access , and it is important to make sense of it all .
computational modeling can be an essential tool to help make use and sense of all this data and lead to better understanding , diagnosis , prediction of various diseases , especially ms . as there is no cure for ms , most treatments typically focus on slowing the progression of the disease and managing symptoms .
since tissue destruction and disease onset begins much earlier than the actual clinical symptoms appear , it will be important to test future therapeutics to detect early axonal loss , axonal dysfunction and efficiency of these treatment methods .
advances in imaging technology and use of new techniques need to be established and validated to determine the success of remyelination and cessation of old damage .
finally , a combination of modeling will allow for prediction and better quality of life for people with ms .
there is a huge need for a reliable and personalized disease prediction model of ms .
it would benefit patients with ms , as they can plan and select the best individualized treatment option .
it would be helpful to the clinician working with patients , especially early stage patients , to help with selection of the best therapeutic treatment options .
integration of modeling , biology , clinical and imaging data can help provide more personalized and reliable monitoring of disease progression and in turn lead to better disease prediction .
|
multiple sclerosis ( ms ) is a major cause of neurological disability in adults and has an annual cost of approximately $ 28 billion in the united states .
ms is a very complex disorder as demyelination can happen in a variety of locations throughout the brain ; therefore , this disease is never the same in two patients making it very hard to predict disease progression . a modeling approach which combines clinical , biological and imaging measures to help treat and fight this disorder
is needed . in this paper , i will outline ms as a very heterogeneous disorder , review some potential solutions from the literature , demonstrate the need for a biomarker and will discuss how computational modeling combined with biological , clinical and imaging data can help link disparate observations and decipher complex mechanisms whose solutions are not amenable to simple reductionism .
|
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the demand for blood and blood products in nigeria is high due to road traffic accidents , surgical and obstetrical blood loss , and anemia from other causes . despite several
the goal of clinical blood transfusion is to provide qualitative , safe , and adequate blood to recipient.1 the challenges in achieving these goals are enormous and serious efforts are been made globally , by government , donor agencies , and voluntary organizations towards its actualization .
the main sources of blood procurement are commercial ( remunerated ) blood donors , voluntary donors , and replacement donors .
a commercial blood donor offers a unit of blood for a fee paid by a contracted hospital vendor .
a replacement blood donor is a family member or relative of a patient , donating a unit of blood to be used for a specific patient , while a voluntary blood donor is a well - meaning member of the society who donates his or her blood without any inducement for use by a recipient not known to him or her .
there is a general policy on blood screening before utilization in which a donor blood is screened for transfusion transmissible viral pathogens to ensure that only safe blood is transfused .
it is a well - established fact that the safest source of blood is from a voluntary donor.24 in 1997 , world health organization ( who ) set a goal of achieving 100% voluntary donation by 202056 but as at 2010 only 57 of 126 countries surveyed had established this as a standard.7 blood transfusion is a known risk factor for transmission of infectious diseases including viral infections such as human immunodeficiency virus ( hiv ) , hepatitis b virus ( hbv ) , and hepatitis c virus ( hcv ) .
the risk of transmission is highest with blood procured from commercial donors.2489 there is still a high dependence on remunerated donors especially in developing nations such as nigeria .
several nations have made frantic efforts and are making significant improvement towards achieving 100% voluntary donor blood procurement but not so much has been achieved in nigeria .
only 5% of voluntary donation has been achieved in some major donor centers in nigeria.7 the objective of this investigation is to evaluate donor blood procurement and the associated risk of transfusion transmissible viral infections in a tertiary health facility in south - south nigeria .
records of the blood transfusion unit of the health facility from 1 january 2009 to 31 december 2009 were reviewed in order to identify the methods of donor blood procurement and screening results for hiv , hbv and hcv .
for every unit of blood , we studied the method of procurement ( commercial , replacement or volunteer blood donation ) and screening outcome . in this study , voluntary and
replacement donors were grouped together due to the lack of proper differentiation in some of the records , the number of voluntary donors were very few and their blood were actually directed to specific recipients .
blood from commercial donors is usually obtained from outside of the hospital through several vendors .
collection by the vendor is usually not supervised by hospital personnel but adheres to routine guidelines for phlebotomy .
the vendor brings the units of blood to the hospital in fulfillment of a contractual agreement .
blood from replacement donor or panel donor is collected at the blood transfusion unit of the hospital .
blood ( 450 ml ) is usually collected into a bag containing 63 ml citrate phosphate dextrose with adenine ( cpda ) and stored at 2 - 8c .
prior to phlebotomy , the donor 's blood group is determined and the packed cell volume ( pcv ) ascertained .
units of blood from commercial donors are inspected to determine good anticoagulation and exclude hemolysis .
the strip has two horizontal lines labeled control and patient bars . at room temperature , and using automatic micropipette , 50 l of plasma or serum is added to the sample pad . the test strip is read within 15 - 60 min . a single red line at position c ( control ) on the strip indicates a valid control . a similar line is found in patient bar if positive for either hiv-1 or hiv-2 .
the sample is negative for hiv if there is no colour change in the patient bar .
hepatitis b surface antigen screening is carried out using the clinotech 's one - step hepatitis b antigen test strip .
an already anticoagulated blood sample is collected into a clean tube and centrifuged at 3000 rpm for 10 min to obtain clear plasma at room temperature .
a test strip is then inserted into the test plasma to a maximum indicated by the manufacturers for 10 s and laid face up on a flat non - absorbent surface .
a test is positive if two transverse bands ( t = test and c = control ) are seen and negative when only the one band at control ( c ) is seen .
only units of donor blood found to have good collection and storage and free from these viral infections are offered for transfusion to patients .
all infected commercial donor blood was discarded , while infected replacement donors are not bled .
statistical analysis was conducted using the statistical package for social sciences ( spss ) software .
the unpaired student t - test was used to test the statistical significance between commercial and replacement donor status .
the test of association and risk factor for viral transfusion transmissible infection calculated using the chi - square test and odd ratios .
a total of 7,552 donors record were analyzed comprising 6,931(91.8% ) commercial and 621(8.2% ) replacement donors . 893 ( 11.8% ) commercial donor blood and 23(0.3% ) replacement donors test positive for viral transmissible infections [ table 1 ] .
the percentage of commercial donors tested positive was 12.9% comprising 7.2% for hiv only , 4.7% for hbv , 0.6% for hiv and hbv and 0.4% for hcv . while 3.8% of replacement donors tested positive for viral infection comprising of 2.6% , 1% and 0.2% for hiv , hbv and hcv respectively [ table 2 ] .
the chi square test was used to test the statistical significance of infection between commercial and replacement donors ( =45.07 , p<0.001 , od=3.845 ) .
the hospital blood demand and utilization has been on the increase . in this 12-month transfusion record study ,
a related work in the same hospital by enosolease et al.8 recorded 11,021 over a 3 years period ( january 2000 to december 2002 ) .
the hospital is still highly dependent on commercial donors as procurement from these donors accounted for 91.8% .
a majority of the replacement donors are medical students and doctors who donate to their unit patients ; some replacement donors are actually commercial donors recruited from commercial donor centers who disguise as patient relatives .
the risk of viral transfusion transmissible infection is highest with blood procured from commercial donors .
the findings are different from reports of earlier studies8 in the same centre but the prevalence of hiv in this study is comparable to that of the national hiv survey for 2010 which showed a rate of 7.5% in benin.10 this shows that the prevalence of hiv infection has increased over the years .
ejele et al.6 in a survey in niger delta nigeria reported lower prevalence rates , but the survey showed that commercial donors have the highest prevalence rates for viral transfusion transmissible infections .
the unacceptably high rate of infections among commercial donors may be attributed to the fact that most of the donors may belong to the high risked group .
it brings to fore the need to adhere to the strict practice of screening potential donors prior to donation .
furthermore , hospitals and donation centers should embrace and implement policies that will encourage voluntary blood donor scheme .
the donor blood samples that tested positive were not subjected to further testing to validate the results .
the donor blood samples that tested positive were not subjected to further testing to validate the results .
commercial blood donors are still the major source of blood to the hospital and they also have the highest prevalence of transfusion transmissible viral infections in this region thus constitute a major risk transmitting infections to potential recipients .
concerted efforts should be made towards encouraging voluntary blood donation , retention of donors through campaign and media enlightenment programmes so that reasonable progress can be made to achieve the who vision 2020 goal .
concerted efforts should be made towards encouraging voluntary blood donation , retention of donors through campaign and media enlightenment programmes so that reasonable progress can be made to achieve the who vision 2020 goal .
|
background : blood and blood products are scarce commodities .
the demand often outweighs the supply .
this study is directed at investigating the blood procurement sources and the risk of viral transfusion transmissible infection.materials and methods : the records of the blood transfusion unit of a tertiary health facility in south - south nigeria were studied .
the procurement and screening records from 1 january to 31 december 2009 were analyzed.results:7,552 donor records were analyzed , 6,931 were commercial donor and 621 replacement donors .
891 commercial donors were infected , 500 ( 7.2% ) were hiv positive , 323 ( 4.7% ) hbv positive , 42 ( 0.6% ) had hiv and hbv co - infection , while 28 ( 0.4% ) were hcv positive .
twenty - three replacement donors were infected , 16 ( 2.6% ) were hiv positive , 6 ( 1% ) were hbv positive , while 1 ( 0.2% ) were hcv positive .
none of the replacement donors had co - infection .
the risk of infection was significantly higher with commercial donor procurement ( x2=45.07 , p<0.001 , od=3.845).conclusion : commercial blood donors are still the major source of blood to the hospital and they also have the highest prevalence of transfusion transmissible viral infections in this region thus constitute a major risk transmitting infections to potential recipients .
|
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osteochondral lesions involve the articular cartilage and underlying subchondral bone , and most often affect the knee and the talus ( 1 ) .
osteochondral lesions of the femoral head are uncommon , and few studies have reported the imaging findings of such lesions . since this condition occurs in relatively young patients , and osteochondral damage puts joints at high risk of subsequent degeneration ( 2 ) , timely recognition of this disorder is important for proper treatment . herein , we review previous literature related to osteochondral lesions of the femoral head and 796report a case in which lesion involving the bilateral femoral heads was observed .
this lesion manifested as subchondral cysts according to the initial radiography images , and led to further evaluation by computed tomography ( ct ) arthrography and immediately obtained magnetic resonance imaging ( mri ) scan that revealed overlying articular cartilage defects .
a 20-year - old female patient was referred to our hospital from a local hospital .
her chief complaint was pain in both hips along with severe right side pain of approximately 2 months duration .
notably , however , she had been a taekwondo athlete in high school for several years . on clinical examination ,
plain radiography of the hip showed well - defined subchondral radiolucent lesions with a subtle sclerotic rim in the superior portion of the right femoral head ( fig .
the acetabuli in both sides of the hip displayed mild dysplasia with lower limit of normal ( center - edge angle , 24 - 25 degrees ) .
no signs of degeneration , such as joint space narrowing or juxta - articular sclerosis , were observed , except for subtle osteophyte around the head - neck junction of the right femur .
deformations of the femoral head , such as flattening , or the presence of crescent signs were also absent .
based on imaging findings , a provisional diagnosis of avascular necrosis with subchondral cysts could not be excluded . to further investigate the lesion ,
1b , c ) revealed several subchondral cysts that were hypointense on the t1-weighted images ( repetition time [ tr]/echo time [ te ] , 374/20 ) and hyperintense on the t2-weighted fat - suppressed images ( tr / te , 1500/29 ) .
the overlying portion in this region revealed a focal articular cartilage defect measuring about 10 15 mm in size , consistent with an osteochondral lesion .
no surrounding bone marrow edema was observed , and no cartilage damage was noted on the opposite site of the acetabulum .
the scintigraphy scan with 99 m technecium methylene diphosphonate showed no increased activity in the femoral head .
on arthroscopy , chondral damage was noted in the superior portion of the femoral head .
follow - up plain radiography of the hip performed about a year later revealed subchondral cysts in the superior portion of the left femoral head ( fig .
the patient reported no incidence of trauma in the meantime , other than mild hip discomfort .
1f ) scans of the left hip also showed several subchondral cysts , measuring about 14 13 mm in size , with associated overlying articular cartilage loss .
retrograde inspection of previous hip mri scans performed at the local referral hospital 2 months before the first visit to our hospital revealed lesions of the left femoral head with abnormal signal intensity , including hypointensity on the t1-weighted images and hyperintensity on the t2-weighted images ( fig .
this led us to hypothesize that the subchondral cyst lesion had progressed from a pre - existing lesion already present one year before .
the chondral defect was confirmed by arthroscopy and core decompression with a bone graft was performed , as had been done for the right side .
osteochondral lesions involve articular cartilage and the underlying subchondral bone , and mainly affect the knee and the talus ( 1 ) .
osteochondral lesions of the femoral head are rare and the prevalence is unclear ( 3 ) .
detailed reports of the imaging findings of such lesions are also quite limited . therefore , osteochondral lesions of the femoral head can be easily overlooked , unless radiologists are suspicious of osteochondral lesions that develop in the femoral region .
furthermore , osteochondral lesions of the femoral head are known to cause joint degeneration ( 2 ) .
we diagnosed the osteochondral lesion by identifying the accompanying overlying articular cartilage defects by ct arthrography and mri , which were performed for further evaluation .
we excluded a diagnosis of avascular necrosis due to the absence of a double line sign or a crescent sign of the femoral head on mri .
moreover , no signs indicative of primary osteoarthritis , such as joint space narrowing or juxta - articular sclerosis in both the acetabulum and femoral head , were observed .
the causes of osteochondral lesions have not yet been elucidated , and are likely to be multifactorial ( 4 ) .
one of the well - known causes of the osteochondral lesion of the femoral head is legg - calv - perthes disease . in this secondary lesion
, however , separated subchondral bone with articular cartilage from the joint surface is the main feature , which is different than our case .
other etiologies of osteochondral lesions in the hip include previous infection , genetics , ischemia or trauma ( 5 , 6 ) .
many studies have reported damages incurred by repetitive traumas ( 7 , 8 , 9 ) .
( 10 ) reported normal plain radiographic finding but mri detection of a subchondral lesion in an athlete with continuous hip pain , and suggested that although an athlete may not recall a specific incidence of past trauma , shearing injury of the cartilage by subluxation during joint rotation or impact damage caused by forces exerted by jumping could have occurred .
( 7 ) demonstrated that osteochondritis dissecans of the elbow can result from articular cartilage damage inflicted by repeated trauma .
nakamura et al . ( 6 ) reported an osteochondral lesion of the femoral head believed to have originated from repeated impact and high axial loads in a fencer .
our patient was previously an athlete , and so could have developed the osteochondral lesions by repetitive trauma , despite the lack of recollection of any particular past trauma .
( 10 ) also reported abnormal bone marrow signals from an osteochondral lesion on the femoral head .
this lesion exhibited hypointensity on the t1-weighted images and hyperintensity on the t2-weighted images , along with cartilage surface irregularities of the medial portion . in our case , although the cartilage surface irregularities were not immediately apparent from the previous mri scans taken at the local referral hospital , lesions displaying hypointensity on the t1-weighted images and hyperintensity on the t2-weighted images were present in the left femoral head .
an articular cartilage defect lesion accompanied by subchondral cysts was found about a year later , similar to the lesion previously observed on the right femoral head .
we concluded that an osteochondral lesion was already present at the time of the previous mri scan , and that this lesion progressed to the lesion with subchondral cysts .
it may often be necessary to distinguish osteochondral lesions from avascular necrosis in differential diagnoses .
the articular cartilage of the femoral head has been shown to be unaffected in the majority of cases of avascular necrosis , whereas this cartilage is often damaged in osteochondral lesions . moreover ,
avascular necrosis of the femoral head manifests itself as a somewhat broader lesion , often accompanied by serpiginous , low signal lines or crescent signs ( 11 ) . on the other hand ,
osteochondral lesions often reveal smaller - sized areas with wedge - shaped signal abnormalities , or a narrow inlet with deep nest - shaped lesions , as seen in this case ( 12 , 13 ) .
patient age , in particular being younger , is also helpful in distinguishing this condition from avascular necrosis ( 10 ) .
10 ) are thought to be indicative of lesions in the acute or subacute stage . in the chronic stage , however , a progressed degenerative bony lesion in the form of subchondral cysts , as seen in our case , or perhaps even a sclerotic change exhibiting hypointensity on the t1- and t2-weighted images , may be observed ( 6 ) . although some authors have reported that high uptake on the scintigraphy represents inflammation or lesion healing , no increased uptake was observed in our case , probably because the lesions are in the chronic stage .
in addition , young patients , especially those with a previous trauma or a history of being an athlete , are potential candidates for osteochondral lesions .
radiologists should look for bony lesions associated with articular cartilage defects in the femoral head , which could be indicative of an osteochondral lesion , similar to the lesions that are known to occur in the knee or the talus .
|
osteochondral lesions of the femoral head are uncommon and few studies have reported their imaging findings .
since joints are at risk of early degeneration after osteochondral damage , timely recognition is important .
osteochondral lesions of femoral head may often be necessary to differentiate from avascular necrosis . here , we report a case of osteochondral lesions on bilateral femoral heads .
this lesion manifested as subchondral cysts in initial radiographs , which led to further evaluation by computed tomography arthrography and magnetic resonance imaging , which revealed overlying cartilage defects .
|
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] |
diabetic retinopathy ( dr ) is a common complication of diabetes mellitus ( dm ) and is a leading cause of blindness worldwide .
diabetic macular ischemia ( dmi ) is an important category of diabetic retinopathy ( dr ) [ 2 , 3 ] . during the imaging study of the normal macula ,
an important hallmark is the capillary - free region called foveal avascular zone ( faz ) .
it is recognized that the faz can enlarge and can become irregular in dr and seems to get larger as the stage of retinopathy advances [ 2 , 3 ] .
dmi is characterized by the occlusion and loss of the macular capillary network or capillary dropout [ 4 , 5 ] .
this condition results in upregulation of growth factors , which contributes to the development of diabetic macular edema ( dme ) , the most frequent sight - threatening disorder in individuals with dr .
dmi is an irreversible category of diabetic maculopathy , and its presence limits the potential benefits of treatments for dr [ 3 , 5 , 6 ] .
the early treatment of diabetic retinopathy study ( etdrs ) established dmi standards that were determined using fluorescein angiography ( fa ) . according to the etdrs report 11 , clinically , there is a correlation between dmi and poor prognosis that varies according to the severity of the macular ischemia .
the anatomy of the retina appears to be altered in dmi , with thinning of retinal nerve fibre layer ( rnfl ) and outer retina and thickening of outer choroid . in retinal microcirculation
, blood supply is divided mainly into superficial capillary plexus ( scp ) and deep capillary plexus ( dcp ) .
choroidal circulation appears to be the most important blood supply to the central macula , including photoreceptor inner segment ( is ) band , which is most likely the most important consumer of oxygen .
it is likely that the dcp is responsible for up to 15% of the blood supply to the photoreceptors , especially during dark adaptation [ 8 , 10 , 11 ] .
fa has been the gold standard imaging modality since it was introduced in 1961 [ 7 , 12 , 13 ] however , it requires venipuncture , and reports of anaphylaxis and death related to contrast injections , despite being rare , have been documented .
in addition , the technique is costly and time - consuming , requiring up to 10 minutes for framing acquisition [ 4 , 8 , 10 , 11 ] .
spectral domain ( sd ) oct has emerged as a potential alternative for detecting macular nonperfusion in diabetic patients , but results are contradictory [ 1 , 2 , 13 ] .
macular ischemia may disrupt the normal flow of nutrients to the outer retina , but photoreceptor status on sd - oct remains controversial . in diabetic patients , the disruption of photoreceptors in sd - oct can indicate a manifestation of underlying dcp nonperfusion in patients with a relatively healthy macula [ 1416 ] .
oct angiography ( octa ) has been used for 3d mapping at microcirculation level [ 9 , 1316 ] .
it allows detection of retinal and choroidal structures via motion contrast imaging and high speed scanning , which detect blood flow by analysing signal decorrelations between scans [ 7 , 11 , 14 , 15 ] .
both inner and outer retinal capillary plexuses are imaged in contradistinction in conventional angiography , which does not effectively image the outer plexus .
this study aimed to compare the use of fluorescein angiography and octa in the diagnosis and quantification of dmi when applying a split - spectrum amplitude - decorrelation angiography algorithm ( ssada ) to improve the detection of flow signals in angiography with the purpose of offering a suitable sparing contrast alternative using 3 mm 3 mm octa for clinical investigations of diabetic patients [ 8 , 1520 ] .
this was a retrospective cross - sectional comparative study analysis conducted at federal university of goias and approved by ethics committee of the same institution . signed informed consent was obtained from each subject prior to enrolment .
imaging data were collected from patients who underwent fa and octa on the same day in a tertiary referral retina center in the period between january 1 , 2015 , and july 30 , 2015 .
exclusion criteria included significant cataract ( without surgical indications at the time of examination ) , previous retinal arterial or venous occlusion , inherited macular dystrophy , posterior segment inflammation , and macular degeneration or scarring of any cause .
subjects that presented motion artifacts during octa or poor signal strength were also not included in this study .
all subjects underwent a comprehensive ophthalmologic examination , including best - corrected visual acuity ( bcva ) on etdrs charts , + 78 d noncontact lens slit - lamp fundoscopy , color fundus photography , and fa and oct angiography , on a single day .
color fundus photographs were obtained and fa was performed using a digital retinal camera ( topcon trc 50dx ; topcon , paramus , nj ) .
the central macular nonperfusion area , faz boundaries , and the maximum height and area were manually delineated .
the faz area was measured using a single fa photograph , such as the octa angiogram of the scp and then divided into
( superior to 0.32 mm ) and small ( inferior to 0.32 mm ) groups .
group comprised both moderate and severe etdrs established grading groups [ 3 , 19 , 20 ] .
the quantification of macular nonperfusion in both fa and oct angiography captured images was completed using imagej software ( imagej , national institutes of health , bethesda , md ) .
octa instrument used was the rtvue xr avanti with angiovue software for octa ( optovue , inc . ,
fremont , ca ) , and imaging data were obtained using the split - spectrum amplitude - decorrelation angiography ( ssada ) software .
the algorithm was employed to improve the signal - to - noise ratio [ 8 , 1521 ] .
this instrument operates at ~840 nm wavelength , 70,000 a - scans per second , and a bandwidth of 50 nm .
the tissue resolution is 5 m axially and there is a 15 m beam width .
each b - scan consisted of 304 a - scans for a total of 2 304 304 a - scans per acquisition , with a total acquisition time of approximately 3 seconds .
the scanning area was captured in 3 mm 3 mm sections , and the acquired oct volumes were centered on the fovea [ 811 ] .
a 3 mm 3 mm octa image was used primarily because this size is the one that provides images with higher resolution in the used octa device [ 10 , 11 ] .
the ssada algorithm was used to generate a volumetric rendering of blood flow from the internal limiting membrane ( ilm ) to the choroid , and it allowed the direct visualization of normal and abnormal blood circulation [ 1821 ] . for the octa angiograms ,
it was used the automatic segmentation of the retinal layers at the level of the scp generated by the angiovue software in an orthogonal view . in order to correct for automated segmentation error and projection artifacts ,
the segmentation slab was manually adjusted , using corresponding structural oct b - scans as a guide for the placement of 2 segmentation lines : the inner located at 3 m beneath the internal limiting membrane and the outer boundary at 15 m beneath the inner plexiform layer .
this semiautomatic method permitted the readers to select images that pictured the largest extent of scp for subsequent quantitative analysis .
the dcp image was segmented with an inner boundary 15 m beneath the inner plexiform layer and an outer boundary at 70 m beneath the inner plexiform layer .
this section captured both layers of the outer capillary plexus , which sandwiched the inner nuclear layer .
all statistical analyses were performed using statistical package for social science 22.0 ( ibm spss ; ibm , armonk , ny ) .
fa and octa images were independently reviewed by two independent masked readers retina specialists ( jg and di ) who reached an agreement regarding the area ( mm ) of macular nonperfusion that was obtained using both methods according to etdrs report 11 .
intraclass correlation coefficient ( icc ) was used to estimate the agreement between individual measurements from both readers .
since the icc was consistently > 0.9 between the 2 readers , nonparametric wilcoxon signed - rank test was used to compare area measurements performed by 1 reader on fa and octa 3 mm 3 mm scans .
thirty - four eyes from 34 patients , including 20 ( 58.82% ) females and 14 ( 41.18% ) males , were enrolled and separated according macular status ( table 1 ) .
twenty - four eyes from 24 patients were placed in the group of patients with dmi , including 15 ( 62.5% ) females and 9 ( 37.5% ) males .
the mean ( sd ) age of the dmi population was 61.20 6.95 years .
the group without dmi comprised 10 patients , including 5 ( 50% ) females and 5 ( 50% ) males .
four eyes from 2 patients were excluded from the study due to a high quantity of motion artifacts .
patients with dmi presented a mean fa of 0.68 0.53 mm ( 95% ic , 0.460.90 ; p < 0.05 ) .
octa angiogram analysis demonstrated a mean of 0.58 0.35 mm ( 95% ic , 0.430.73 ; p < 0.05 ) .
the wilcoxon signed - rank test did not identify a significant difference between fa and octa in patients diagnosed with dmi ( p = 0.1374 ) .
patients without dmi presented a mean fa of 0.19 0.67 mm ( 95% ic , 0.140.24 ; p < 0.05 ) .
octa angiogram analysis demonstrated a mean of 0.20 0.79 mm ( 95% ic , 0.150.26 ; p < 0.05 ) .
the wilcoxon signed - rank test did not identify a significant difference between fa and octa in patients without dmi ( p = 0.9594 ) ( figure 1 ) .
the iccs for faz area measurements between 2 observers with respect to fa and octa were 0.96 ( ic : 0.360.71 ) and 0.92 ( ic : 0.350.79 ) , respectively , demonstrating the reproducibility and consistency of the methodology .
this result highlights the notion that the faz is most often clearly demarcated by a distinct foveal vascular ring and that its abnormalities , in addition to its capillary dropout , can be lucidly obtained from octa images .
etdrs research group connected the severity of macular nonperfusion to the potential for progression in dr .
in fact , in dr , the advanced deterioration of macular perfusion is the basis for macular ischemia , and developing a method to perceive perfusion maps may allow correlations between central ischemia and the different stages of dr .
however , it is an invasive method requiring venipuncture and contrast infusion ; it is a time - consuming test and provides only 2-dimensional images .
therefore , reports of anaphylaxis and death related to contrast injections have been documented , despite being rare .
octa is a noninvasive method , obtains highly detailed 3-dimensional images without requiring injection of a contrast dye , and allows faster acquisition of images ( figure 2 ) [ 9 , 10 , 1214 ] .
octa performed using a split - spectrum amplitude - decorrelation angiography ( ssada ) algorithm has already been shown to be useful for imaging microvascular changes in dr [ 811 , 14 , 20 ] .
cole et al . also observed macular nonperfusion in a diabetic patient in a 3 mm 3 mm octa that was centered on the fovea by applying a similar technology .
the 3 mm 3 mm octa central sections obtained using sd - oct allowed us to obtain a higher resolution over a small area ( figure 3 ) .
this area was sufficient for detecting central dmi , but it was not large enough to identify peripheral retinal nonperfusion .
high - resolution oct imaging allows measuring thickness of segmented retinal layers in angiographically apparent ischemic dr .
future octa devices improvements may provide clinicians the ability to obtain wider field images with better resolution . in the present study , statistical analysis
also did not indicate significant difference between area measurements obtained with fa and octa in patients diagnosed with dmi .
the same has occurred among patient without dmi regarding the measurement of normal faz area .
the icc for the zaf area between the 2 observers on fa and octa demonstrated the reproducibility and consistency of used methodology .
these results highlight the notion that faz is most frequently demarcated by a distinct foveal vascular ring and its abnormalities , in addition to its capillary dropout , can be lucidly obtained from octa imaging device .
first , all of the results were obtained during a single appointment , and there was no follow - up .
the present study demonstrates that fluorescein angiography and octa provide similar results when used to diagnose macular ischemia in diabetic patients . with further improvements
in summary , octa may provide images with higher details regarding macular status , becoming a novel imaging technique for the diagnosis of dmi , and may become an alternative to fa for this purpose .
the results also offer improved quantification of faz area in diabetic patients without dmi when compared to diabetic subjects with established macular ischemia .
|
purpose . to compare fluorescein angiography ( fa ) and optical coherence tomography angiography ( octa ) images of foveal avascular zone ( faz ) in patients with diabetic retinopathy ( dr ) with and without diabetic macular ischemia ( dmi )
. methods . the wilcoxon signed - rank test was used to compare area measurements and p values of < 0.05 were considered statistically significant .
fa and octa images were independently graded by 2 observers that reached agreement regarding quantitative dmi according established protocols .
the ischemic area was divided into
large macular ischemia ( superior to 0.32 mm2 ) and small ( inferior to 0.32 mm2 ) groups .
quantitative analyses of the faz were performed using custom software .
results .
thirty - four eyes from 34 diabetic patients were enrolled .
subjects with dmi presented a mean area on fa and octa of 0.68 0.53 mm2 and 0.58 0.35 mm2 , respectively ( p = 0.1374 ) .
patients without dmi presented a mean area on fa and octa of 0.19 0.67 mm2 and 0.20 0.79 mm2 , respectively ( p = 0.9594 ) .
the icc for the faz measurements between the 2 observers on fa and octa was 0.96 and 0.92 , respectively .
conclusion .
octa represents a novel technique for the diagnosis of dmi and it may become an alternative to fa for this purpose .
|
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in a recent breast
cancer study conducted by the clinical
proteomic tumor analysis consortium ( cptac ) , extensive global- and phospho- protein profiling were obtained for
a subset of breast cancer samples that have been extensively characterized
in the the cancer genome atlas ( tcga ) .
this is so far the first attempt to study protein
activities in breast cancer samples using sophisticated protein experiments
on a large scale .
then , by leveraging genomic analytical outputs from
tcga on these samples , we have the unique
opportunity to characterize and compare gene coexpression networks
and protein coexpression networks based on the same set of samples .
this is of great interest because knowledge about the common and different
coexpression patterns among rnas and proteins could improve our understanding
of complicated gene regulatory mechanisms in tumor samples and could
also facilitate the detection of important disease genes and therapeutical
targets . in the past decade , numerous statistical and machine
learning methods
have been proposed to construct gene gene regulatory networks
and protein protein regulatory networks , including coexpression
network methods , bayesian network methods , and gaussian graphical models .
review of these and other methods are available in
hecker et al . and lee et al .
; however , all of these proposed models are designed
to construct one network at a time .
presumably , we can use rna expression
data and protein expression data to construct two networks separately
and then compare , but such an approach is certainly less optimal ,
as gene expressions and protein expressions in one tumor sample are
closely related . on one hand ,
protein levels are regulated by their
rna levels , so we expect the two coexpression networks share common
structures . on the other hand ,
protein activities are subject to a
large amount of post - transcriptional modifications , and thus we also
expect to observe unique interaction patterns in each network .
this
motivates us to perform joint learning of both rna - seq and proteomic
networks , so we are able to borrow information across different data
sets and better capture the common correlation structure , which shall
then improve the overall accuracy of network estimation .
some of them have been specifically designed to estimate time - varying graphical
models in the context of time - series data .
proposed
likelihood - based methods for the joint estimation of multiple related
gaussian graphical models .
their approaches rely on two different
regularization schemes penalizing differences across partial correlation
structures of different classes . while both papers successfully demonstrate
the advantage of jointly modeling multiple networks over estimating
individual networks separately , the performance of the methods heavily
depends on the multivariate gaussian assumptions of gene expression
distributions , which may not hold in real biological systems .
recently , random - forest - based methods have been utilized for the
construction of grn .
random forest is a decision - tree - based
nonparametric method for building powerful prediction models .
its ensemble structure combined with its nonparametric
nature delivers excellent performance even when the sample size is
moderate .
the
key idea is to model the expression of each target gene as a function
of the expression of all other genes via random forest .
the superior
performance of genie3 was demonstrated in both dream 4 in - silico multifactorial
challenge and dream 5 network inference
challenge . in both challenges ,
, genie3 was further modified to estimate grn
based on time - series data .
proposed irafnet , another random - forest - based method that
combines different types of heterogeneous data , such as protein protein
interactions , transcription factor - dna binding , and gene knock - down
to estimate grn .
while these random - forest - based methods greatly advance
the field of grn construction , none of them is designed to estimate
simultaneously multiple related networks .
we propose jrf ( joint
random forest ) , a new algorithm for the simultaneous
estimation of protein coexpression and gene coexpression networks .
the key idea is to borrow information across different data by forcing
the class - specific tree ensembles to use the same genes for the splitting
rules . in this way
, regulatory relationships playing important roles
in multiple classes will be detected with better power .
after testing
the performance of our algorithm on several in silico experiments
and comparing jrf with different methods including genie3 the joint graphical lasso ( jgl ) , we applied jrf for the simultaneous construction
of gene coexpression networks and protein coexpression networks . for
our analysis , we considered gene expression from tcga and protein
expression from cptac data for 62 breast cancer patients .
we derived
the two networks and identified protein - specific hub genes and gene
modules , showing the unique contribution of proteomic data to breast
cancer research .
random forest
is a nonparametric algorithm that models a response variable via a
collection of decision trees .
specifically , each decision tree is
constructed based on a random subset of training samples , and the
splitting variable at each node of a decision tree is chosen from
a randomly sampled subset of predictors upon maximizing a certain
utility function ( e.g. , decrease in node impurity ) .
recently ,
huynh - thu et al . introduced genie3 , a
random - forest - based model for inferring gene regulatory networks ( grns ) .
in genie3 , first
, for each target gene k , its expression
is modeled as a function of the expression of all other genes via
random forest .
then , regulatory event ( j k ) is measured by ijk , the importance score of gene j in the random forest model of gene k.
specifically , the importance score ijk is defined as the summation
of node impurities across all nodes that utilize predictor j for the splitting rule divided by the total number of
trees in the random forest model of gene k. we start with some notations .
for each class g { 1 , ... , g } , denote the gene
expression data for p genes and ng individuals as xngpg , and
the ith observation of gene j under
class g as xijg .
an overview of jrf is shown in figure 1 , where , for simplicity , we consider only
two classes .
the key step of jrf is to construct g random forest models simultaneously using data from g classes when predicting the expression of a target gene k based on the expression of all other genes .
we propose
to use the same predictor variables for splitting rules in different
trees corresponding to different classes .
the goal is to borrow information
across different classes , so that regulatory relationships can be
better detected if there are coherent signals across different classes .
specifically , when we grow g decision trees in parallel
for g classes , at one node , we decide the
splitting variable based on the following procedure : ( 1 ) randomly
sample a set containing n genes from
the entire set of genes except gene k. ( 2 )
for each class , the best splitting rule based on each candidate
predictor in set is derived .
let be the subset of samples allocated to node
under class g. the splitting rule based on
the jth predictor is of the formwhere and are the subsets of samples in class g allocated to the left and right children of node
with a splitting rule based on the jth predictor .
for each predictor in set , the best threshold agj*( )
is derived as followswhereand with x representing the mean of set . in other
words , cgj is the decrease in node impurity
after splitting node in
the gth tree according to gene j. ( 3 ) finally , gene g * is chosen as the splitting
variable
of node for all classes based on the following criteria : because
jrf utilizes the same splitting variable
for corresponding nodes across all classes , predictors associated
with gene k in multiple classes are more likely to
be chosen for the splitting rules . for each step in the tree construction ,
( 13 )
only apply to classes for which is not a final
node . as in the original random forest model
, each tree grows until either the total number of observations allocated
to the final leaves falls below a certain prespecified threshold or
the maximum number of possible nodes is reached .
it is worth mentioning
that when the number of classes is one , jrf reduces to genie3 , the
original random forest model for network inference .
it is worth noting
that to implement jrf , data sets need to be standardized to mean zero
and unit variance .
importance scores depend on the scale of the data
and therefore variables ( genes ) need to be standardized before the
random forest models are fitted.figure 1jrf schematic . for simplicity ,
let us assume
that there are only
two classes and that each data contains the same number of samples .
for each target gene , gk
, we run random forest for each class and the two tree ensembles
are forced to share some structure as follows .
first , for each node
, the same set of genes s is randomly sampled from the entire set of genes ( a ) .
then , the
same variable in set s is chosen
for the splitting rule .
this is achieved by deriving the splitting
rule for any gene contained in set s ( b ) and choosing the best variable to maximize the sum of decrease
in node impurity over different classes ( c ) .
let us assume
that there are only
two classes and that each data contains the same number of samples .
for each target gene , gk , we run random forest for each class and the two tree ensembles
are forced to share some structure as follows .
first , for each node
, the same set of genes s is randomly sampled from the entire set of genes ( a ) .
then , the
same variable in set s is chosen
for the splitting rule .
this is achieved by deriving the splitting
rule for any gene contained in set s ( b ) and choosing the best variable to maximize the sum of decrease
in node impurity over different classes ( c ) . on the basis of the previously
described procedure , jrf constructs g random forest
models simultaneously for each target gene k and
returns a ranking of gene gene interactions based on importance
scores . to assess the performance of jrf in predicting the true interactions ,
receiver operating characteristic curves ( roc ) and precision - recall
curves can be computed by setting different thresholds on importance
scores . in this paper ,
jrf is compared with jgl , genie3-sep , and genie3-comb on several in silico experiments .
genie3-sep is genie3 used to estimate networks based on data from g classes separately , while genie3-comb is genie3 used to
estimate a unique network based on the union of data from all classes .
all random - forest - based algorithms ( jrf , genie3-comb , and genie3-sep )
provide a ranking of regulatory relationships based on importance
scores , and roc curves were computed by setting different thresholds
on these scores . instead , for jgl
, roc curves were constructed by
considering different values for the two parameters controlling the
level of sparsity ( as shown by figure s1 in the supporting information ) .
another approach to evaluate
the performance of jrf is choosing a proper cutoff value for importance
scores using permutation techniques .
let ijkg be the importance score
associated with the regulatory event ( j k ) in the gth class - specific tree ensemble .
specifically , this importance score is defined as where t is the number
of trees and is the set of nodes which utilize gene j for the splitting rule in the tree ensemble used to predict
gene k based on the gth class - specific
data . because in this paper we are interested in estimating undirected
networks , we derive importance score ij
kg for every edge ( j k ) as the average between importance
scores ikjg and ijkg , and
the final undirected networks can therefore be derived by applying
a cutoff on the edge importance scores . to derive a proper cutoff
value for importance scores , we utilize the following permutation - based
procedure : ( a ) for b { 1 , , b } , with b being the number of permutations : ( a.1 ) for any target gene k , we first permute
its sample order within each class and fit g random
forest models via jrf to predict the expression of gene k based on the expression of all other genes in g classes , respectively .
( a.2 ) repeat ( a.1 ) for all genes and
compute the final importance
scores for each edge in each class , which are denoted as { ippg(b)}g=1 g .
( b ) for each threshold , we
computewhere 1( ) is
the indicator
function , equal to one if event occurs and zero otherwise .
f( ) can serve as an approximation of the
false discovery rate ( fdr ) .
in the following
numerical studies , we use 0 = min{ : f( ) 0.001 } and declare an edge between j and k in class g if ij
the computational
complexity
of jrf is o(ptng=1glog(ng)ng ) , where p is the number of genes , t is the number
of trees in each forest , n is the number of variables
sampled at each node , g is the number of classes ,
and ng is the number
of samples in each class .
this complexity is in the same order as
the complexity of applying genie3 to estimate g networks
for g classes separately . in the contrast , the time
complexity of ggm - based approaches , such as jgl , often has the order
of p. so
when p is large , jrf and genie3 enjoy better computational
efficiency , which is supported by our numerical investigations ( see supporting information ) . in practice ,
the
number of trees ( t ) and the number of potential regulators
to be sampled at each node ( n ) are user - specified
parameters .
t is usually chosen sufficiently large
because the tree ensemble provides more accurate results as t increases . in our numerical studies , we reported results
for t = 1000 .
we also evaluated larger t values , such as 2000 and 5000 , but observed similar results ( data
not shown ) . for n ,
a common choice is where ( p 1 ) is
the number of predictors for each target gene . in general , large value
of n results in predictions with high bias ; while
low value results in predictions with high variance .
we implemented
an r package jrf using c routines wrapped with an
r interface .
similarly
to other tree - based algorithms for network construction , jrf can be
easily parallelized because the estimation process consists of p - independent subproblems .
a comparison between jrf , jgl ,
genie3-sep , and genie3-comb in terms of computational time is presented
in table s1 . for data generation
, we first considered
two networks containing 500 nodes connected by 498 and 249 edges ,
respectively .
in particular , network 1 is a network with two disjoint
components and network 2 consists of only the first component of network
1 . in this example
, we focused on two network topologies : power law and star topology ( figure s2 in supporting information ) . for each network topology , we simulated 20 replicates involving n = 50 samples from a gaussian graphical model .
network
structures and covariance matrices were simulated in the same way
as danaher et al .
( 2014 ) ( further details
can be found in the supporting information ) . besides this example
, we also consider a series of other different
simulation settings , including ( 1 ) two non - nested networks ; ( 2 ) five
non - nested networks ; ( 3 ) two nonrelated networks ; and ( 4 ) data from
non - gaussian distributions .
the expressions of 5864 proteins
for 62 breast cancer tumors ( proteome-ratio-norm-nona-normal.gct(04-jun-2014
version ) ) were downloaded from the cptac data coordinating center
( http://proteomics.cancer.gov/programs/cptacnetwork ) sponsored
by the national cancer institute . for three samples having technical
duplicates
we further standardized protein expression so that each
sample had median 0 and median absolute deviation ( mad ) 1 . finally ,
we excluded proteins with interquartile range < 70% quantile .
level - three rnaseq
data of breast tumor samples were obtained from the tcga web site
( http://tcga-data.nci.nih.gov/tcga/ ) .
first , we log - transformed
the data and then replaced all missing values with 0 . then , we standardized
each sample to have median 0 and mad 1 .
we focused on the 62 samples
contained in the proteomic data and excluded genes with more than
10% missing values .
then , we selected the top 10% genes with largest
interquartile range across samples .
as final subset , we considered
528 genes obtained by overlapping the set of 1464 selected genes and
the set of 1759 selected proteins . for each data
in this section ,
jrf was compared with
jgl , genie3-sep , and genie3-comb on several
in - silico experiments . in all implementations , we used t = 1000 and .
we evaluated
the performances of different
algorithms based on different network structures , sample sizes , and
number of genes .
figure 2 shows the average roc curves
of each method
over 20 replicates for network 1 and network 2 separately .
jrf outperforms
all other methods under all different settings , while genie3-sep is
the second best performer .
figure s3 shows
the boxplot of the area under the precision - recall curve ( aupr ) for
jrf , genie3-sep , and genie3-comb .
the advantage of jrf over genie3-sep is more
evident for network 2 , which contains only shared edges .
this is expected
because jrf should be able to detect common edges with better power .
mean of
roc curves for network 1 ( first column ) and network 2 ( second
column ) over 20 replicates for jrf ( red ) , genie3-comb ( green ) , genie3-sep
( blue ) , and jgl ( black ) .
for each replicate , we sampled 50 samples
from a gaussian graphical model based on the power law topology ( first
row ) and star topology ( second row ) . to further assess the performance of the proposed model ,
we included
more simulation scenarios in the supporting information .
in particular , in supporting information section 1.3 , we considered the case of non - nested networks sharing
some structure , while , in section 1.4 , we considered the case where
five different networks were estimated simultaneously .
as shown by figures s4 and s5 , jrf outperformed competitors
under all of these simulation scenarios .
in addition , it would be
interesting to evaluate the performance of jrf when class - specific
networks do not share any structure .
for this purpose , we simulated
two different networks with no common edge and applied different methods
on the corresponding data ( supporting information section 1.5 ) .
as shown in figure s6 ,
performance of jrf is very comparable to that of genie3-sep on this
simulation example .
this suggests that jrf is sufficiently robust
and works in the absence of common structure .
moreover , to assess
the performance of jrf in the presence of nonlinearities in the data ,
we further generated data examples using genenetweaver , an open - source software for the generation of
in - silico data from a set of ordinary and stochastic differential
equations ( supporting information section
1.6 ) .
as shown in the supplemental figure s7 and s8 , jrf again outperformed other algorithms in terms of both
area under the roc curve ( auc ) and area under precision - recall curve
( aupr ) .
finally , figure s9 shows results
for different algorithms in estimating networks with 1000 nodes ( further
details can be found in supporting information section 1.7 ) , in which we can see that jrf outperformed competitors
in terms of both auc and aupr .
it is worth mentioning that the increased
dimensionality did not negatively affect results , suggesting that
jrf can handle problem with high dimensionality . in this section ,
we assess the
performance of jrf and other algorithms on data sets involving different
number of samples .
figure 3 shows boxplots of auc over 20 replicates resulting from jrf ,
genie3-sep , and genie3-comb for different sample sizes , that is , n = 50 , 100 , 200 .
the aucs of jrf are significantly larger
than that of genie3-sep based on 20 replicates under all sample sizes .
as shown in the supplemental figure s10 , jrf outperforms competitors also in terms of aupr for different
samples sizes .
as shown , the smaller the sample size the bigger the
improvement of jrf over genie3-sep is .
this result is expected because
as the sample size increases genie3-sep should have sufficient information
to estimate the networks separately .
boxplot of auc over 20 replicates for
jrf ( red ) , genie3-comb ( green ) ,
and genie3-sep ( blue ) for different sample sizes , that is , n = 50 , 100 , and 200 .
data : samples simulated from a gaussian
graphical model on a power law topology . in this section ,
we compare jrf and
genie3-sep regarding their abilities to estimate class - specific edges .
we considered the 20 replicate data sets ( n = 100 ) ,
which was simulated based on the power law network topology in the
previous section . for both jrf and genie3-sep ,
edges were declared
using the permutation procedure illustrated above with b = 500 and an fdr threshold of 0.001 .
table 1a shows the performance of jrf and genie3-sep
in estimating network 1 and network 2 . for each network , we reported
the minimum and the maximum value of true positive rate ( tpr ) , false - positive rate ( fpr ) , and false discovery
rate ( fdr ) across 20 replicates . in particular , these
quantities are defined as tpr = tp/(tp + fn ) , fpr = fp/(fp + tn ) , and fdr = fp/(fp + tp ) , where tp is the number
of true positives , fp is the number of false positives , tn is the number of true negatives , and fn is the number of false negatives .
for both networks , jrf achieved
better power ( tpr ) than genie3-sep , while the resulting fprs were quite comparable .
table 1b shows a comparison of the two algorithms
regarding the detection of class - specific ( differential ) edges .
again ,
we computed the rate of true differential edges and false differential
edges and showed the minimum and the maximum value of these quantities
over 20 replicates . while the tprs for detecting
differential edges were similar for the two methods , the fprs of genie3-sep were significantly larger than that of jrf .
in addition ,
jrf leads to substantially lower fdrs for detecting
differential edges than genie3-sep .
for each quantity
( tpr , fpr , and fdr ) we show the minimum
value ( min ) and the maximum value ( max ) across 20 replicates .
note
that the true discovery rate ( tdr ) is defined as tdr = ( 1 fdr ) .
we applied jrf to construct coexpression
networks based on the cptac
global proteomics data set and tcga rnaseq data of 62 breast cancer
samples . for simplicity
, we will refer to the
coexpression networks constructed by jrf from global proteomics data
and rnaseq data as protein - network and rna - network . at fdr cutoff
of 0.001
, we detected 687 common edges shared by both networks , 502
edges unique to protein - network , and 382 edges unique to rna - network .
the topologies of the two networks are shown in figure 4 , while the full list of interactions can
be found in additional file 1 in the supporting information .
interestingly , a few network modules in protein - network
have a high percentage of edges unique to protein - network .
figure 5 highlights genes whose degrees ( i.e. , total
number of edges connecting to one gene ) in protein - network are much
higher than that in rna - network .
three out of the top five genes with
more protein - unique edges , c3 , cfb , and mpo , are related to immune
response functions .
it is well known that immune response plays a
major role in the patient response to chemotherapy treatments .
some of these genes have been shown to be predictive
of survival outcomes of breast cancer patients .
for example , c3 and
cp are predictive of chemoresistance in breast cancer patients , and high activity of mpo genotypes can enhance
efficacy of chemotherapy for early - stage breast cancer .
.
genes belonging to the mmp family have been linked to cancer progression
for their ability to degrade the extracellular matrix and may be implicated
in the formation of metastasis .
network based on proteomic
data ( a ) and rnaseq data ( b ) resulting
from jrf .
green edges ( 687 ) are shared between proteomic and rnaseq
data , red edges ( 502 ) are unique to proteomic data , while blue edges
are unique to rnaseq data ( 382 )
. only names of genes with at least
ten connecting edges are shown in the plot .
for both networks , the
full list of interactions can be found in additional file 1 .
number of connecting edges for top connected
genes in the network
based on proteomic data . for each gene , the green bar corresponds
to the number of connecting edges shared between proteomic and rnaseq
data ; the red bar indicates the number of protein - specific edges ;
while the blue bar indicates the number of rna - specific edges . for
each gene , we list its biological functions .
we then focused on network modules unique to protein - network .
we
derived network modules based on edge betweenness using function edge.betweenness.community
available in the r package igraph . in figure 6
, we show the protein - network
with gene modules enriched of at least one go category .
the full list
of genes contained in each module can be found in additional file
1 .
figure 6 shows ,
for each module , the most enriched go category and the corresponding
benjamini adjusted p - value .
two interesting protein - specific modules ( with more protein - specific
edges than shared edges ) are c1 and c2 .
fibroblast
growth factor receptor activity , as shown in figure 7 , genes are more tightly correlated
in the proteomic data compared with the rnaseq data .
various studies
have investigated the role of the fgfr pathway as a predictive marker
for breast cancer . as shown in figure 7 , fgfr2 and fgfr3
cerliani et al . reported a strong correlation between fgfr2 and fgfr3 based on protein
expression mentioning that there is no previous evidence of correlation
between these two proteins for breast cancer patients .
in addition ,
the interaction between fgfr2 and fgfr3 is contained in the string
database ( see table s2 ) .
similarly to
module c1 , genes in module c2 are higher correlated in the proteomic
data compared with the rnaseq data ( figure 7 ) .
this cluster contains genes such as apob ,
c3 , orm1 , and cp that have been recently shown to be predictive of
chemoresistance in breast cancer patients .
another
important gene in this module with a high number of connecting edges
is apoa1 ( the fifth highest connected node according to figure 5 ) .
this gene has been recently
identified as a potential target for disease progression using whole - genome
trancriptomic and whole proteomic .
as
shown in table s2 , 11 of the relationships
in figure 7 are contained
in the string database .
these results suggest that proteomic data
can provide complementary information to genomics data and help to
reveal important biological mechanisms .
, we show the most
enriched go category with corresponding benjamini adjusted p - value ( p ) .
in particular , only modules
reporting a significant enriched benjamini adjusted p - value ( p 0.01 ) are highlighted .
heatmap of the absolute value of correlation
between genes based
on proteomic and rnaseq data for module c1 ( a ) and c2 ( b ) in figure 6 . for each module
,
we include a pie plot showing the number of protein - specific interactions
( red ) and interactions shared across proteomic and rnaseq data ( green ) .
because we are interested
in assessing the utility of a joint learning , jrf was compared with
the standard random - forest algorithm that constructs the two networks
separately . for both methodologies , we derived undirected networks
using the same fdr cutoff ( 0.001 ) .
the protein - network resulting from
genie3-sep can be found in figure s11 in the supporting information .
as shown , 57% of the edges are shared across protein-
and rna - networks under jrf , while only 26% are shared under genie3-sep .
this finding can be explained by the fact that a joint learning can
facilitate the detection of common edges . in this section
,
we validate our findings using go categories . in particular , we consider
596 go terms containing fewer than 200 genes ( the list of go terms
can be found in tables s3s6 in the supporting information ) .
figure 8a shows the number of edges contained in at least one go term
for different fdr cutoff for both protein - networks .
first , we constructed
a network based on go terms where an edge ( a b ) was drawn between every pair of genes ( a , b ) sharing at least one go term .
then , a contingency
table was constructed considering variables network based
on go terms and network based on proteomic data
with categories
number of edges contained and number
of edges non - contained , and a fisher s exact test was
performed to derive enrichment p - values .
figure 8a , b shows that jrf
results in a network more overlapping with go terms than the standard
random - forest algorithm genie3-sep .
we validate protein - networks in figure 4 using go terms and
knockout signatures .
the first row contains network validation based on go terms . for our
analysis , we considered 596 go terms containing fewer than 200 genes .
( a ) number of edges contained in at least one go term for different
fdr cutoff .
the second row contains validation based on esr1 ( c ) and
gata3 ( d ) signatures . for a particular neighborhood size of either
esr1 or gata3
, we show the total number of knockout signatures contained
under jrf ( ) and genie3-sep ( ) . to further validate
our findings , we overlapped both networks with
esr1 and gata3 knock - down signatures .
we identified sirna knock - down
signatures of key transcription factors ( tfs ) of breast carcinoma
in mcf7 cells1 by accessing gene expression data available under gse31912
in gene expression omnibus ( further details
about these signatures can be found in section 2 of supporting information ) . for each network ,
a neighborhood
was defined by defining a threshold k for the number
of edges connecting a particular gene to the target gene of interest
( either esr1 or gata3 ) . for each network in figure 8
, we considered different values of k and counted the number of knockout signatures contained
in the neighborhood .
figure 8c , d shows the total number of signatures contained in k - size
neighborhoods for esr1 and gata3 , respectively . as shown , also in
this case , jrf results in a more enriched network .
figure s12 contains the same comparison for rna networks . in this paper , we developed jrf , a random - forest - based
algorithm for the simultaneous construction of gene coexpression and
protein coexpression networks .
jrf is designed to borrow information
across different expression data by selecting the same set of predictors
( genes ) as splitting variables in the random forest model corresponding
to each data . in this way , jrf is able to detect common relationships
( edges ) with better power and detect differential edges - specific to
individual data with fewer false positives .
because in this
study we were mainly interested in assessing the unique contribution
of proteomic data to breast cancer research , we focused our attention
on protein - specific hub genes and modules .
we identified two interesting
protein - specific modules containing potential targets for breast cancer
treatment .
in addition , we compared our algorithm to the original
random - forest algorithm , which constructs the two networks separately ,
and showed that our algorithm leads to networks more overlapping with
available knockout signatures and existing go terms .
the current
version of jrf requires the feature ( protein ) space
to be the same across different classes .
extension of jrf for handling
data involving different sets of variables remains as future work .
this problem arises in the protein domain because protein abundance
can be measured for different phosphorylation - sites that map to a
unique protein . as future work
, we will design a model able to jointly
estimate networks from phosphorylation - site abundance and rnaseq data ,
simultaneously . besides estimating gene - regulatory network and
protein - regulatory
network ,
as shown in the paper , jrf can be used in many other applications .
for example , we can apply jrf to estimate grns for the three breast
cancer subcategories ( luminal , basal , and her2 ) , grn for different
tissues , and grn for cancer and normal tissues .
another interesting
direction would be assessing the association between mirnas - genes
and mirnas - proteins simultaneously .
it is well known that the
downregulation of some mirnas may play an important role in the progression
of cancer .
therefore , a better understanding
of the interactions between mirna , mrna , and protein expression is
crucial to cast light on the potential disease mechanisms of cancer .
in this context , jrf can be easily utilized to jointly detect which
mirnas regulate genes and proteins .
jrf can be easily parallelized because the computation can be divided
into p - independent subproblems . on the contrary ,
for many existing methods such as bayesian networks and ggm - based
algorithms ,
the merit of jrf depends on the assumption that networks
of different
classes share some common structures . to assess the performance of
jrf in the absence of common structure , we conducted numerical studies
to investigate such cases and concluded that jrf is sufficiently flexible
to guarantee good performance even when nonrelated networks are considered . in jrf ,
we propose a permutation - based procedure
to derive proper cutoffs on importance scores for selecting confident
edges .
specifically , we introduce f( ) as an
approximation of the false discovery rate ( fdr ) of the selected edge
set based on cutoff ; however , in the numerical studies , we
observe that f( ) tends to underestimate fdr .
new methods to obtain more accurate estimate of fdr for
jrf warrant future research .
|
we focus on characterizing
common and different coexpression patterns
among rnas and proteins in breast cancer tumors . to address this problem ,
we introduce joint random forest ( jrf ) , a novel nonparametric algorithm
to simultaneously estimate multiple coexpression networks by effectively
borrowing information across protein and gene expression data .
the
performance of jrf was evaluated through extensive simulation studies
using different network topologies and data distribution functions .
advantages of jrf over other algorithms that estimate class - specific
networks separately were observed across all simulation settings .
jrf also outperformed a competing method based on gaussian graphic
models .
we then applied jrf to simultaneously construct gene and protein
coexpression networks based on protein and rnaseq data from cptac - tcga
breast cancer study .
we identified interesting common and differential
coexpression patterns among genes and proteins .
this information can
help to cast light on the potential disease mechanisms of breast cancer .
|
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in fixed orthodontic treatment , brackets were used for transferring orthodontic forces to the teeth . at first , to attach the brackets to the tooth , orthodontic bands were used and after welding brackets to bands , they were cemented to the tooth . in 1955 , buonocore introduced the acid - etch technique that was gradually used in different dental treatments . in 1965 ,
newman used direct bonding of orthodontic brackets that was considered as the first step in application of appliances with the improvement of esthetic presentation .
this technique was developed rapidly due to its simplicity , efficacy and providing more esthetic qualities . for achieving successful bonding ,
the bonding agent must penetrate to the enamel surface , have easy clinical use , dimensional stability and enough bond strength .
the bond strength of orthodontic brackets should be enough to not cause bonding failure and delay in treatment and it also should have adequate resistance against chewing forces and stresses from archwires .
on the other hand , easy debonding of the brackets without any damage to the teeth needs sufficient and safe bond strength . according to few stages for bonding of orthodontic brackets and related problems in the conventional system ,
other techniques such as application of self - etch primers or laser irradiation was suggested to simplify the bonding procedure . in the acid - etching technique , microporosity was produced on the enamel surface to provide micromechanical bonding .
enamel etching with phosphoric acid results in loss of the superficial layer of the enamel and dissolution of the enamel subsurface .
the amount of enamel loss depends on phosphoric acid concentration and the time of application .
laser etching was performed by the erbium family with two different wavelengths ( 2940 and 2780 nm ) .
this technique has some advantages such as having no vibration or heat and producing a surface which is acid resistant by altering the calcium to phosphor ratio and formation of less soluble compounds .
there are some studies which have evaluated the effect of laser etching on bond strength of orthodontic brackets with controversial results .
so , the purpose of this study was to compare shear bond strength ( sbs ) of orthodontic brackets bonded to enamel prepared by er : yag laser with two different powers and conventional acid - etching .
forty - five human premolars extracted for orthodontic purposes were selected for this study . in transillumination examination ,
the teeth showed healthy enamel on the buccal surface , without attrition , fracture , restoration , congenital anomalies and structural defects .
there was no history of chemical substance application such as hydrogen peroxide for these teeth .
after rinsing the teeth , they were placed in 0.5% chloramine t for inhibiting bacterial growth for 2 hours .
the teeth were divided into three groups according to conditioning method : group 1 : conventional etching with 37% phosphoric acid ; group 2 : laser irradiation by er : yag laser with output power of 1w ; and finally group 3 : laser irradiation with er : yag laser with output power of 1.5w . in group 1 ,
the samples were etched with 37% phosphoric acid gel ( 3 m , dental products , st.poul ) for 15 sec , then rinsed for 15 sec with water spray and dried with air spray for 10 sec in a 2 cm distance above the surface of the enamel .
laser irradiation in group 2 and 3 was carried out by er : yag laser ( us20d , deka , italy ) with a 2940 nm wavelength . the area was marked before irradiation . in group 2 ,
laser was used with an output power of 1w , energy of 100 mj and frequency of 10 hz .
these parameters were 1.5 w , 150 mj and 10 hz , respectively for group 3 .
the handpiece of laser was used 5 mm above the surface in non - contact mode and sweeping motion .
subsequently , the adhesive kit ( transbond xt , 3 m , unitek ) was used .
the adhesive paste was placed on the bracket base and the brackets were placed on the enamel with a 300 gr compressive force with gauge for 10 sec to produce uniform thickness .
the resin was polymerized by led ( mectron , starlight pro gac , italy ) with a 440480 nm wavelength and 400 mw / cm intensity for 40 sec . consequently
, the samples were thermocycled for 200 cycles between 5c and 55c water baths with 30 sec dwell time for each .
the specimens were mounted in auto - cure acrylic resin and the shear bond strength was measured by using a universal testing machine with a crosshead speed of 0.5 mm per second .
after debonding , the amount of resin remaining on the teeth was determined using the adhesive remnant index ( ari ) scored 1 to 5 ( table 1 ) by stereomicroscope ( nikon d - cs , japan ) with 10x magnification .
one - way analysis of variance was used to compare shear bond strengths and the kruskal - wallis test was performed to evaluate differences in the ari for different etching types .
the mean and standard deviation of the conventional acid - etch group , laser group ( 1w ) and laser group ( 1.5w ) was 3.82 1.16 , 6.97 3.64 and 6.93 4.87 , respectively .
there was no significant difference between laser group ( 1.5w ) and laser group ( 1w ) ( p=1.000 ) and conventional group ( p=0.085 ) , but there was a significant difference between laser group ( 1w ) and conventional group ( p=0.016 ) . according to graph 1 , the variances of values of the laser samples bond strengths was higher than the acid - etch group .
table 2 shows the frequency distribution of ari degrees in the three groups . according to the kruskal - wallis test
there are some studies which evaluate the enamel preparation by laser irradiation for orthodontic brackets .
the aim of this study was to assess the shear bond strength of orthodontic brackets bonded to enamel prepared by er : yag laser or acid - etch .
the bond strength of light curing composites may be influenced by thermal changes of the oral cavity and the quality of polymerization .
thermocycling is a common method for stimulating this condition ; therefore , we used this technique in this research .
the result of this study showed that both laser groups had higher bond strengths than the acid - etch group .
although this difference was significant between the laser group with an output power of 1w and the acid - etch group , the laser group with an output power of 1.5w showed no significant difference with the acid - etch group . according to usumez s study ,
laser irradiation with a power of 2w in comparison with the acid - etch technique showed similar shear bond strengths , but application of laser with a power of 1w showed a lower bond strength . on the other hand ,
gokcelik et al.s study which assessed the shear bond strength of samples prepared by er : yag laser and acid - etch found no significant difference between these two groups .
controversial results were obtained from different studies which evaluated the effect of laser irradiation compared to conventional methods due to different study designs and various parameters used in these studies .
morphological changes of enamel produced after laser irradiation depends on the energy density of the laser , the time of exposure , the distance of the laser handpiece from the surface and percentage of water irrigation .
samples irradiated with 1.5w power showed no significant difference compared to laser group with 1w power which is in agreement with the results obtained from basaran s study . in the present study ,
the laser groups showed higher bond strengths with higher standard deviations compared to the acid - etch group .
this finding reduced the credibility of laser application for enamel preparation , considered as an unfavorable characteristic . in similarity ,
usumez et al . reported higher distribution coefficient for shear bond strength of orthodontic brackets in laser prepared surfaces .
the reason may be related to the irregular etching pattern of surfaces irradiated by laser .
sasaki et al . found that preparation of enamel surfaces by er : yag laser can not be done homogeneously .
surfaces irradiated by laser showed some areas which were similar to unlased enamel surfaces but surface preparation by acid etch technique showed more homogeneous patterns which was like honey comb pattern that is favorable structure for adhesion process .
higher standard deviations in the laser groups may be associated with intrinsic nature differences of the teeth collected from different people , time of storage and environmental effects on the tooth after extraction . in order to control these problems ,
animal teeth can be used because numerous tooth samples can be provided from an animal . among different animals
maijer and smith stated that bond strength of 8 mpa is essential for orthodontic treatment . in this study ,
the mean of shear bond strength in the three groups was below the suggested value .
cerekja and cakirer showed that that thermocycling process reduced the shear bond strength of orthodontic brackets .
in addition , daub confirmed that this condition was due to differences in thermal expansion coefficients of the adhesive , brackets and enamel .
two thirds of the samples showed an ari degree of 4 or 5 which showed that the highest debonding happened in resin to teeth contact surface which needs less cleaning of debonded enamel leading to reduction of abrasion risk to the enamel , but it is better to have debonding in resin - bracket contact or inside the resin because the less adhesive remaining on the tooth , the more stress affecting the enamel surface in clinical condition , this kind of debonding is rare because providing favorable etching in enamel surface is difficult due to lack of controlling humidity , time and cooperation of patients in preparing the surfaces . in addition , the structural pattern of the bracket base makes debonding in the resin bracket contact surface uncommon .
in contrast to these results , lee in the evaluation of bonded brackets observed that samples prepared by acid - etch technique or er : yag laser irradiation showed more fracture pattern in resin - bracket contact surface .
these different results may be contributed to the debonding test procedure , which was tensile bond strength in lee s study .
valletta reported that debonding happened in bracket resin surface in tensile bond strength and in resin tooth contact surface in shear bond strength .
: yag laser with an output power of 2 w and frequency of 2 hz showed no significant increase in shear bond strength compared to the control group . in the present study , ari degree among the two laser groups and between laser groups and
in contrast , gokcelik showed higher ari degrees in er : yag laser compared to the acid - etch group . in laboratory conditions , loading forces to brackets were different from clinical conditions . in clinical conditions ,
besides , in the oral cavity , there are different kinds of stresses such as thermal changes , humidity and microbial plaque that make the simulation condition in laboratory difficult .
although bond strength tests are still far from ideal , attempts should be made to standardize these tests to make comparisons easier .
the shear bond strength of bracket to laser - prepared enamel with two different powers of 1 and 1.5 w was similar and laser groups showed higher bond strengths than the acid etch group .
however , high variances of values in bond strength of irradiated enamel should be considered to find the appropriate parameters for applying er : yag laser as a favorable alternative for surface conditioning .
|
introduction : the purpose of this study was to compare shear bond strength ( sbs ) of orthodontic brackets bonded to enamel prepared by er : yag laser with two different powers and conventional acid-etching.materials and methods : forty - five human premolars extracted for orthodontic purposes were randomly assigned to three groups based on conditioning method : group 1- conventional etching with 37% phosphoric acid ; group 2- irradiation with er : yag laser at 1 w ; and group 3- irradiation with er : yag laser at 1.5 w. metal brackets were bonded on prepared enamel using a light - cured composite .
all groups were subjected to thermocycling process .
then , the specimens mounted in auto - cure acryle and shear bond strength were measured using a universal testing machine with a crosshead speed of 0.5 mm per second .
after debonding , the amount of resin remaining on the teeth was determined using the adhesive remnant index ( ari ) scored 1 to 5 .
one - way analysis of variance was used to compare shear bond strengths and the kruskal - wallis test was performed to evaluate differences in the ari for different etching types.results:the mean and standard deviation of conventional acid - etch group , 1w laser group and 1.5w laser group was 3.82 1.16 , 6.97 3.64 and 6.93 4.87 , respectively.conclusion:the mean sbs obtained with an er : yag laser operated at 1w or 1.5w is approximately similar to that of conventional etching .
however , the high variability of values in bond strength of irradiated enamel should be considered to find the appropriate parameters for applying er : yag laser as a favorable alternative for surface conditioning .
|
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acute cytomegalovirus ( cmv ) infection is common in both immunocompromised and immunocompetent patients worldwide.12 the infection is often asymptomatic .
symptoms , when they occur , include fever , cervical lymphadenitis , and arthralgia.3 rarely , acute cmv infection may be complicated by pneumonia , colitis , myocarditis , pericarditis , or hemolytic anemia.4 vascular events caused by acute cmv infection , including thrombosis of the venous or arterial vascular system , are rarely reported in the english literature .
we report a case of acute cmv colitis in an immunocompetent adult that was complicated by vascular thrombosis and pulmonary embolism .
further , we present a review of the literature regarding the association between vascular events and cmv infection in immunocompetent patients .
a 78-year - old man was referred to our emergency department with a 3-day history of diarrhea and fever .
he had no recent travel history , did not use steroids , and had not experienced recent trauma .
he used medication for hypertension and diabetes mellitus , both of which were well controlled . on admission ,
his vital signs were : body temperature , 38.8 ; blood pressure , 145/85 mmhg ; respiratory rate , 18/min ; and pulse rate , 90/min . on physical examination , his pulse was regular and he had no cardiac murmurs , his chest was clear without signs of respiratory distress , and his abdomen was soft with hyperactive bowel sounds .
laboratory tests showed a white blood cell count of 8,270/mm ( normal : 3,99010,500/mm ) , a platelet count of 199,000/mm ( normal : 140,000450,000/mm ) , a prothrombin time of 14.10 seconds ( control : 11.16 seconds ) , an inr of 1.21 , an activated partial thromboplastin time of 26.0 seconds ( control : 27.4 seconds ) , and crp level of 16.7 mg / dl ( normal : < 0.8 mg / dl ) . a coagulation profile , including protein c ( 80% , normal : 70%140% ) , protein s ( 75% , normal : 60%130% ) and anti - thrombin iii ( 95% , normal : 75%125% ) , was normal .
. colonoscopy demonstrated multiple giant ulcers with skip lesions in the distal colon , sparing the rectum ( fig .
multiple forceps - biopsy tissue samples , taken from the coloniculcers , were sent for pathological examination .
however , 5 days post - colonoscopy , the patient experienced dyspnea and severe hypoxemia , necessitating emergent endotracheal intubation and ventilation .
a chest radiograph obtained post - intubation revealed an engorged main pulmonary trunk with an abrupt cutoff of pulmonary vascularity in the distal portions bilaterally , indicative of the " westermark sign " ( fig .
subsequently , a ct scan of the chest was performed that showed several filling defects within the pulmonary trunk and main pulmonary arteries ( fig .
histological evaluation of the colonic biopsy specimens revealed cytomegalic cells with densely staining nuclei and intranuclear inclusion bodies in the epithelium ( fig .
these epithelial cells were positive for monoclonal anti - cmv antibody on immunohistochemical analysis , indicative of cmv infection ( fig .
the patient had no obvious risk factors for thrombosis , and had no family history of coagulopathy .
follow - up ct scan of the patient 's chest showed complete resolution of the pulmonary emboli .
acute cmv infection in immunocompetent patients is common worldwide , with seroprevalence rates ranging from 40% to 100% , depending on country , socio - economic conditions , and age.1 colitis is the most common manifestation of severe cmv infection.2 patients with cmv colitis are generally asymptomatic ; if symptomatic , they may present with diarrhea , fever , bleeding , abdominal pain , and perforation .
4 vascular events are a very rare manifestation of severe cmv infection in immunocompetent individuals.2 these vascular events include thrombosis of the portal , superior mesenteric , or splenic vein ; cerebral venous thrombosis ; deep vein thrombosis ; and life - threatening pulmonary embolism .
567 vascular complications may be discovered only several weeks after the initial diagnosis of cmv infection . however , abgueguen et al . proposed that venous thrombosis in immunocompetent patients with cmv infection may not be as rare as previously reported.8 although the pathogenesis of cmv - induced vascular events is unknown ,
several mechanisms have been postulated , including direct damage to endothelial cells , increased levels of hemostatic indicators , induction of smooth muscle proliferation , enhanced platelet - derived growth factor production , and the presence of antiphospholipid antibodies.91011 vascular thrombosis in immunocompetent patients is usually associated with predisposing factors such as heterozygous factor v leiden mutation , presence of antiphospholipid antibodies , heterozygous prothrombin gene mutation , protein c and/or protein s deficiency , or oral contraceptive use.12 in contrast , this patient had no family history of coagulopathy , was not bed - ridden , and had no other risk - factors for thrombosis .
it is , therefore , likely that cmv infection was the precipitating factor for pulmonary embolism . in the diagnosis of vascular events caused by cmv infection ,
doppler ultrasound and ct scanning are the most useful diagnostic modalities.6 treatment of cmv - associated pulmonary embolism includes immediate administration of antiviral agents and anticoagulants .
advanced age , male gender , presence of immune - modulating comorbidities , and need for surgical intervention all negatively influence survival.13 although the cost - effectiveness of investigating for acute cmv infection in patients presenting with thrombosis has not yet been established , we believe that serum samples for cmv infection should be obtained and endoscopic examination should be considered in patients with idiopathic thrombosis . in conclusion , although pulmonary embolism caused by acute cmv colitis in immunocompetent patients is extremely rare , it should be considered in patients who present with severe respiratory symptoms . to the best of our knowledge ,
this is the first report of an immunocompetent patient with acute cmv colitis complicated by vascular thrombosis with pulmonary embolism in which no other obvious underlying predisposing factor for thrombosis was identified .
|
acute cytomegalovirus ( cmv ) infection occurs commonly in immunocompromised and immunocompetent patients , but is usually asymptomatic in the latter .
vascular events associated with acute cmv infection have been described , but are rare .
hence , such events are rarely reported in the literature .
we report a case of pulmonary embolism secondary to acute cmv colitis in an immunocompetent 78-year - old man .
the patient presented with fever and diarrhea .
colonic ulcers were diagnosed based on colonoscopy findings , and cmv was the proven etiology on pathological examination .
the patient subsequently experienced acute respiratory failure .
pulmonary embolism was diagnosed based on the chest radiography and computed tomography findings .
a diagnosis of acute cmv colitis complicated by pulmonary embolism was made .
the patient was successfully treated with intravenous administration of unfractionated heparin and intravenous ganciclovir .
|
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superparamagnetic iron oxide nanoparticles
( nps ) , with core diameters
of 315 nm , are used in a rapidly expanding number of applications
in the biomedical field ; the most common include cell labeling , hyperthermia , drug
delivery , and as contrast agents for magnetic
resonance imaging . for these applications ,
the iron oxide cores are coated with polymer , lipid , or other dispersants
to enable dispersion of nps in aqueous solutions containing biomolecules .
the most common method to stabilize iron oxide nps for biomedical
applications has been to enwrap them in a weakly adsorbed shell of
high molecular weight polymer ( often dextran ) or amphiphiles such as block copolymers or lipids .
however , weakly adsorbed shells lead to low polymer densities
on the particle surface that are further reduced with time under dilute
conditions , resulting in low colloidal stability .
chemically grafted
polymer shells are physically and chemically stable ; they can also
be made denser than physisorbed shells , and therefore they have received
increasing attention . with recent
improvements in the synthesis of nps there
has been a push toward more well - defined , core
spherical iron oxide nps can now be synthesized
with monodisperse size ( sd < 5% ) , but they are as - synthesized capped
with a strongly adsorbed , hydrophobic shell of oleate that is difficult
to replace ; these nps require stabilization
with a shell of linear , end - grafted polymer dispersants of sufficient
thickness and grafting density to ensure that the particles remain
monodisperse during application .
defined core shell architectures
enable the prediction of all colloidal properties and can be used
to define biological interactions through the attachment of organic
ligands .
the main advantage of grafting dispersants to the core is
that the hydrodynamic size of the nps , the stability of the shell ,
and the presentation of ligands can be precisely controlled , in contrast
to for nps with shells consisting of physisorbed high molecular weight
dispersants ; this critically determines np performance in a biological
fluid .
precise control over monodisperse
size , purity , and colloidal interactions is crucial for nps in biomedical
applications to control biodistribution and self - assembly of drug
delivery structures . achieving
high dispersant grafting density on monodisperse iron
oxide nanoparticles is challenging since the dispersant has to densely
replace the ( oleic acid ) organic shell carried by the as - synthesized
particles .
in addition , a major problem for the synthesis and characterization
of core shell nanoparticles is the efficient separation of
excess dispersants .
the peg - dispersant density on
nanoparticles is difficult to determine since continuous and complete
removal of excess dispersants from nanoparticle dispersions can lead
to particle aggregation and precipitation ; alternatively , not all
free ligands are removed , which leads to overestimation of the dispersant
density on the nanoparticles .
the actual stability and correct core shell
structure can not be determined if excess dispersants are not removed .
efficient separation methods can also contribute to nanoparticle degradation
and should be identified and avoided .
an irreversibly grafted
shell of > 2 chains / nm of peg(5
kda ) on polydisperse and irregularly shaped single nps was shown to
be required for colloidal stability under application conditions .
np curvature facilitates polymer grafting by
grafting - to of presynthesized polymer coils and chain grafting densities
up to 2.5 chains / nm have been claimed on iron oxide nps . until recently , only 0.5 chains / nm of peg(5 kda )
was reported grafted on spherical fe3o4 nps after ligand replacement by nitro - dihydrohyphenylalanine
( nitro - dopa)-peg(5 kda ) on monodisperse nanoparticles synthesized
using oleylamine as capping agent .
lak
and colleagues recently synthesized large , 25 nm in diameter , fe3o4 ferrimagnetic nanoparticles that were stabilized
through ligand replacement with nitrodopamine ( nda)-peg . due to their size
, these multidomain particles
are optimized for applications where brownian relaxation mechanisms
are desired , while particles with weaker magnetic interactions and
nel relaxation as the dominant relaxation mechanism require
iron oxide nps smaller than 15 nm . these large particles form
small aggregates in cell culture and pbs , which might be due to the
stronger long - range core
quantitatively addressed the problem of replacing oleic acid by ligand
exchange on superparamagnetic iron oxide nanoparticles .
full ligand replacement was not shown , although
the superiority of dopa and nitro - dopa anchor groups to displace oleic
acid from the surface was demonstrated .
these and other previous studies
did not address the effect of purification from excess ligands on
the results .
shell
nanoparticles under relevant conditions such as biological buffers
and elevated temperature was also not investigated .
we present
protocols to replace oleic acid for a dense spherical
brush peg - shell on monodisperse , monocrystalline , and spherical iron
oxide nanoparticles in the superparamagnetic size range of 310
nm diameter at physiological temperatures . the colloidal stability
in both phosphate buffered saline ( pbs ) and biofluids of these densely
pegylated core
shell particles significantly exceeds what has
previously been described for particles synthesized by ligand exchange .
importantly , we also present an evaluation of different purification
methods to separate pegylated particles from free peg , and we suggest
a protocol for iterated solvent precipitation and magnetic extraction
as most suitable to completely purify these nanoparticles at high
yield . only through this advancement
is the high grafting density
and colloidal stability conclusively demonstrated , with important
implications for correct comparison of previous and future results
on np ligand density .
all chemicals ( see supporting information for details ) were purchased from sigma - aldrich ,
all solvents were from roth , and peg was from jenkem and used as received
without further purification .
all nps and nitrocatechol ligands originated
from the same respective batch to ensure maximum reproducibility .
tem studies were performed on a fei tecnai
g2 20 transmission electron
microscope operating at 120 or 200 kv for high resolution imaging .
samples were prepared by dropping toluene dispersions ( as - synthesized
nps ) or aqueous dispersions ( pegylated nps ) onto a 300-mesh carbon - coated
copper grid and subsequently evaporating the solvent in air .
size
distributions were evaluated using the pebbles software package with a local intensity - model fitting algorithm .
thermograms were recorded on a mettler - toledo
tga / dsc 1 star system in the temperature range 25650 c
with a ramp of 10 k / min in synthetic air stream of 80 ml / s in order
to ensure complete combustion of ligands as nda was found to polymerize
by pyrolization under n2 .
70 l aluminum oxide crucibles
were filled with 0.51.5 mg of sample , and the total organic
content ( toc ) was evaluated as the mass loss fraction at 500 c
by horizontal setting .
h and c solution
spectra were collected on a bruker dpx operating at 300 mhz in cdcl3 and d6-dmso as solvents using
tms as an internal standard .
mid - ir powder spectra of the lyophilized
samples were collected on a single reflection bruker tensor 37 ftir
spectrometer with bruker platinum diamond single - reflection atr equipment
at a resolution of 4 cm by averaging 32 scans .
samples were measured
in type 0500 glass capillaries ( hilgenberg , germany ) with nominal
diameter of 1 mm and wall thickness of 10 m .
measurements were
carried out using a rigaku s - max 3000 saxs system equipped with a
copper - target microfocus x - ray tube micromax-002 + ( 45 kv , 0.88 ma )
with an energy of 8.05 kev , collimated through three pinholes ( 400 ,
200 , and 700 m ) to achieve a beam diameter at the sample position
of 210 m ( fwhm ) and a triton 200 2d multi wire gas - filled x - ray
detector ( 200 mm diameter of active area , spatial resolution 200 m ) .
data were acquired in the q - range from 0.01 to 0.26
with a measurement time of 28.800 s for
each scattering pattern at vacuum conditions better than 10 mbar .
subsequent data treatment included background correction based
on the measured transmission and radial integration with the saxsgui
2.8.03 software package .
superparamagnetic
oleic acid ( oa)-stabilized magnetite nanoparticles were synthesized
by thermal decomposition of an iron precursor according to a slightly
modified heat - up procedure described by hyeon et al .
briefly , for 9.6 nm nps a mixture of 50 ml of dioctyl ether
( oct2o ) and 7.04 ml of oa was heated to 100 c under
n2 .
1 ml of iron pentacarbonyl ( fe(co)5 ) was
injected rapidly , and the reaction mixture was heated to 290 c
with a temperature ramp of 3 c / min .
after aging for 1 h the
np dispersion was allowed to cool to room temperature and precipitated
thrice with ethanol ( etoh ) from toluene in order to remove excess
oa .
the size was controlled by the fe(co)5:oa ratio ; details
can be found in the supporting information ( table s1 ) .
6-nitrodopamine
hemisulfate ( nda - hso4 ) was synthesized according to literature
with slight modifications .
nda - peg(5
kda ) was synthesized by ( 1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylaminomorpholinocarbenium
hexafluorophosphate ( comu)-mediated peptide - coupling reactions ( see supporting information for experimental
details ) . typically , 1.3
g of wet ( from etoh
washing ) nps ( containing 0.11 g cores )
were dispersed in 30
ml of dmf ; the desired amount of nda - peg(5 kda ) , usually a 10-fold
excess ( with respect to the grafting density of 1 nda - peg / nm expected from preliminary measurements of the maximum achievable
grafting density by ligand replacement , e.g. , 1.9 g nda - peg(5 kda )
for 9.6 nm nps ) , was dissolved in dmf and mixed with the nps .
note
that only a low excess is needed for the ligand exchange , but to allow
for fast ligand exchange and easier determination of the efficiency
of the purification methods , a large excess was used .
the dispersion
was sonicated for 26 h at slightly elevated temperature ( 35 c ) .
preliminary tests showed that 26 h was sufficient to replace oa , and
longer sonication did not improve the exchange .
subsequently , the
mixture was extracted thrice with n - hexane ( 30 ml
each ) in order to remove released oleic acid .
afterward , the solvent
was evaporated ; the pegylated nps were lyophilized for 24 h and stored
as dry powder for further analysis and are referred to as
nps were obtained as a light brown powder
in 97% yield with respect to the amount of cores .
all purification
methods were applied after the extraction of the nps with n - hexane .
all nps were characterized and analyzed before
and after purification by tem , tga , ir , and dls .
additionally , nps
purified by precipitation and magnetic decantation were analyzed by
saxs .
0.3 g of the
nps after ligand replacement
( nps raw ) was dissolved in 2 ml of h2o and sonicated for
15 s. sephadex was swollen overnight in milli - q water at 40 c
and manually packed into a 25 2 cm column .
the length
of the column was sufficient to split the np sample in six to seven
identifiable , differently colored fractions ( figure s1 ) .
the eluates were collected and freeze - dried for further
analysis . for all np sizes fraction ii could be identified as the
main fraction . for two np sizes ( 9.6 and 7.1 nm )
this main fraction
was reapplied onto a fresh column , and the eluate was again characterized . the 1000 kda molecular weight
cutoff ( mwco ) membranes
were equilibrated for 10 min in 10% v / v etoh
and 30 min in h2o .
0.3 g of the raw nps were dissolved
in 5 ml of milli - q and dialyzed against 5 l of milli - q . to monitor
the dialysis progress ,
equivalents of 0.5 ml were taken after 1 , 2 ,
4 , 6.5 , and 24 h and freeze - dried .
the 9.6 and 7.1 nm nps were further
dialyzed for 48 h and the small nps ( 4.9 and 3.5 nm ) for 70 h. the
dispersions were lyophilized for further analysis by tem , tga , ir ,
and dls .
typically , 0.15 g of raw nps
were dispersed in 2 ml of milli - q and loaded into a 50 kda amicon - ultra-15
membrane centrifugation filter unit and centrifuged at 4500 g for 15 min .
the centrifuge effluent was collected , and
the nps were redispersed in 1 ml of milli - q .
this procedure was repeated
up to 15 times ; equivalents were taken after 5 , 7 , and 11 centrifugation
steps to track the removal of excess nda - peg .
all dispersions and
the collected effluents were freeze - dried for further analysis by
tem , tga , and dls .
typically 0.3
g of the raw nps ( containing 0.019 g cores for 9.6 nm nps ) were dissolved
in 10 ml of etoh by sonication and slight heating with a hot gun to
60 c .
the clear dispersion was poured into a small beaker and
mixed with 10 ml of ice - cold petrolether whereupon it got slightly
turbid .
the beaker was placed on a 5 5 cm 1 t magnet in the
fridge at 4 c , and the nps were soaked from the cloudy mixture
to the bottom of the beaker within a few minutes , dependent on the
np size .
the supernatant was decanted by holding back the nps with
the magnet , collected , combined , and freeze - dried for tga measurements .
this procedure was repeated 79 times ; after each step a small
sample of nps was kept and freeze - dried for tga analysis to monitor
the removal of excess ligands and impurities .
the final products after
the seventh step were freeze - dried and further analyzed .
the nps were
obtained as dark brown powder in 96% yield with respect to the cores .
freeze - dried samples
of all purification methods were stored at room temperature without
protection from light .
after 6 months dry storage , suspensions of
nps in milli - q water and pbs of the nps purified by dialysis ( 4.9
nm cores ) and magnetic decantation ( 7.1 nm cores ) were prepared ( 4
mg / ml ) and characterized by tem and dls .
the np
dispersions were stored for 4 months at 4 c to prevent microbe
growth and analyzed .
additionally a sample of 7.1 nm cores purified
by magnetic decantation was dissolved in pbs and water at 3 mg / ml ;
10% bovine calf serum was added to investigate if the nps aggregate
upon nonspecific protein adsorption .
dls was measured over a temperature
cycle from 207020 c in 5k steps with an equilibration
time of 5 min at each temperature .
spherical , monodisperse , and single - crystalline
superparamagnetic iron oxide nanoparticles were synthesized using
a modified heat - up method introduced by hyeon and co - workers . the np size could be precisely tuned in the
range of 310 nm without any further size selection process
by varying the fe(co)5 to oleic acid molar ratio ( see table
s1 in the supporting information for details ) .
the obtained nps were characterized by low- and high - resolution tem ,
tga , and atr - ftir .
figure 1 shows tem micrographs of monodisperse 3.5 0.3 , 4.9
0.4 , 7.1 0.4 , and 9.6 0.4 nm magnetite cores ,
calculated with pebbles by evaluation of 900 nps from multiple
hr and lr - tem micrographs of the respective batches .
the hr - tem ( inset
in figure 1c ) shows
the single - crystallinity of the nps with lattice spacing in the ( 311 )
direction of 0.26 nm .
the ring diffraction pattern ( inset figure 1d ) reveals the highly
crystalline structure of the nps .
the ratio of d - spacings
in the obtained pattern show good agreement with the jcpds database
numbers for maghemite or magnetite .
transmission
electron micrographs of as - synthesized monodisperse
oa - capped fe3o4 nps : ( a ) 3.5 nm .
( c ) 7.1 nm ; inset : high resolution tem image showing that the particles
are single - crystalline .
( d ) 9.6 nm ; inset : electron diffraction pattern
of 9.6 nm nps reveal the highly crystalline nature of the nps .
the grafting of nda - peg to the nanoparticle
cores was performed in large excess of peg in order to ensure fast
and full ligand replacement and to provide an easily distinguished
baseline for evaluation of different methods to remove unreacted dispersant .
the separation of all excess free peg - dispersant after grafting is
challenging , as indicated by previous work ; we performed a thorough comparison and analysis
of different methods that have been applied for core
shell
nanoparticle preparation : cross - linked dextran size - exclusion column
separation , dialysis , membrane centrifugation and precipitation
with magnetic decantation .
the different methods were compared on
their ability to remove excess peg to a stable value determined by
tga and their effect on nanoparticle stability .
the tga results with
corresponding nda - peg(5 kda ) grafting density are summarized for all
purification methods and core sizes in table 1 .
the total organic content ( toc ) was determined
as the mass loss fraction up to 650 c and converted to nda - peg(5
kda ) dispersant grafting density using the known molecular weight
of nda - peg , the average iron oxide core area determined by tem , and
a core density of 5.17 g / cm .
as described in the materials
and methods section , the nps were after column purification
collected in 67 dark brown , red , orange , and yellow fractions .
the same colors and amounts were observed for all np core sizes ( see figure 2b ) .
all samples passed
the column without visible sticking to the dextran matrix , which demonstrates
a high and homogeneous peg shell density on the particles .
fraction 2 could be identified as the main fraction for all sizes ;
it had the largest amount of nps with the smallest amount of nda - peg(5
kda ) .
figure 2 shows
the results of the column purification in terms of grafting density
based on measurement of organic content by tga . for two np sizes ( 9.6
and 7.1 nm )
the main fraction was dispersed in mq and reapplied onto
a fresh sephadex column .
for the 3.5 and 4.9 nm nps the amounts contained
in fraction 2 were too small for a second pass through the column .
the reapplied samples passed the column as a single band with a very
small fraction of particles that passed the column faster .
the organic
content of the reapplied fractions could be decreased from 66% to
49% ( 9.6 nm nps ) and 91% to 73% ( 7.1 nm nps ) by the second pass . for
the 9.6 nm nps
this translates into a grafting density of 1.0 nda - peg(5
kda)/nm , while for 7.1 nm nps a grafting density of 2
nda - peg(5 kda)/nm is calculated .
( a ) total organic content
of the column purification , measured
by tga and converted into nda - peg(5 kda)/nm using the
known molecular weight of nda - peg(5 kda ) and average core surface
area .
toc includes large excesses of free peg and sephadex and was
converted into dispersants / nm for comparison with the
other methods but does therefore not reflect the actual grafting density
of nda - peg .
( b ) fractions
of nps after column purification dispersed in water ( 9.6 nm , upper )
and freeze - dried ( 7.1 nm , lower ) .
dialysis has to be performed with membranes
having a pore size sufficiently large to allow diffusion of the dispersant
through the pores ; the mwco of membranes is however typically given
for equivalent protein or dextran sizes and can not be used directly
for comparison to highly solvated polymer molecular weights for which
the radius of gyration is much bigger than for e.g. protein of the
same molecular weight .
a rule of thumb of choosing a pore size > 5
that of the dialyzed molecule is often invoked .
this was confirmed
by attempting dialysis of dissolved peg(5 kda ) ( 0.2 g / ml against 5
l of milli - q ) in a membrane of 1214 kda mwco , i.e. , nominally
23 times the molecular weight of the nda - peg , which was used
in previous publications studying grafting - to and ligand replacement
of pegylated nps .
after 24 h only 50% of the free
peg was removed , and only 60% had been removed after 5 days despite
exchanging milli - q in the reservoir five times .
thus , choosing a too
low mwco results in remaining free dispersant and subsequent overestimation
of the dispersant grafting density by tga .
we therefore changed to cellulose - based
membranes with a mwco of 1000 kda , which nominally corresponds to
a cutoff hydrodynamic size of 37 nm ( assuming a scaling related
to equivalent sizes of dextran ) .
nps with a hydrodynamic radius rh between 15 and 23 nm ( for 7.1 nm cores ) were
still retained while free nda - peg(5 kda ) with an rh of 4 nm was presumably
removed from the dispersion .
figure 3a shows the removal of excess ligands by
dialysis for all sizes of nps .
large - core nps ( 7.1 and 9.6 nm ) dialyzed
for 24 h yielded an average grafting density of 1 nda - peg(5
kda)/nm determined by tga .
however , nps also started to
stick to the dialysis membrane after 24 h. tem of large nps ( 7.1 and
9.6 nm ) extracted from the dialyzed dispersion after 24 h showed aggregated
morphology ( figure 3b ) .
the 24 h dialyzed samples could be redispersed in water but showed
poor long - term stability .
visible aggregates were obtained after 1
week . continued dialysis of the large cores led to an even lower grafting
density ( 0.5 nda - peg(5 kda)/nm ) and increased
aggregation .
( a ) removal of excess ligands by dialysis with a 1000
kda mwco
membrane .
total organic content was measured by tga and converted
into nda - peg(5 kda)/nm using the known molecular weight
of nda - peg(5 kda ) and average core surface area determined by tem .
after 24 h the surface coverage was 1 nda - peg(5 kda)/nm independent of size , but core size - dependent aggregation
and precipitation were observed .
dialysis for 70 h lowered the grafting
density to 0.3 nda - peg(5 kda)/nm , resulting in colloidally
unstable dispersions .
( b ) typical tem of nanoparticles with 9.6 nm
core size that had visible aggregates ( inset ) grafted with nda - peg(5
kda ) after 24 h dialysis against water using a 1000 kda mwco cellulose
membrane .
nps with small core sizes ( 4.9
and 3.5 nm ) had grafting densities
of 0.8 and 1.7 nda - peg(5 kda)/nm , respectively , after
24 h dialysis and showed good colloidal stability .
the small nps were
dialyzed also for 70 h , which resulted in lowering of the average
grafting density to 0.3 nda - peg(5 kda)/nm . during
dialysis
the np suspension underwent visible color change
from light brown to almost black ( figure s2 in the supporting information ) , indicating that as dialysis proceeds ,
surface - bound nda - peg is ripped off in addition to the initial removal
of free nda - peg .
in contrast
to diffusion - driven
dialysis , a mwco of 50 kda was sufficient to remove excess nda - peg .
this can be ascribed to the convective flow and high forces induced
by the centrifugation that deform the free polymer to flow through
narrow pores .
figure 4 shows the results of the membrane centrifugation for the different
np sizes . ( a ) removal of excess ligands by repeated membrane centrifugation
with amicon 50 kda centrifugation filter units .
after 11 repetitions
all sizes show an average grafting density around 0.50.7 nda - peg(5
kda)/nm .
( b ) more centrifugation steps lead to a decrease
in grafting density and colloidal instability .
left to right : 4.6
nm nps after 5 , 4.6 nm nps after 15 , and 7.1 nm nps after 11 centrifugation
steps .
the average grafting density at this point was measured to
be 1 nda - peg(5 kda)/nm by tga , but the resuspended
nps were colloidally unstable , as can be seen by the visible aggregates
sticking to the vial in figure 4b . in order to study
if the grafting density stays constant ,
the centrifugation
steps were repeated up to 15 times .
as shown in figure 4a , the grafting density did not stay constant
but could be reduced to 0.4 nda - peg(5 kda)/nm .
these nps
could be redispersed in milli - q but showed visible aggregation and
precipitation after 2 days ( figure 4b ) .
analysis of the combined centrifugates showed a
99.8% organic composition , meaning that only ligands were removed
from the dispersion and all cores were retained by the membrane .
all
nps purified by this method showed strong aggregation and were therefore
not analyzed further . both column
separation and filtration ( dialysis and membrane centrifugation )
are
established methods , but they failed to produce a high yield of colloidally
stable nanoparticles for all sizes when the peg - shell was grafted
through ligand replacement .
we therefore attempted a variation on
precipitation that exploits the possibility to magnetize the nanoparticle
cores . while hydrophilic , polymer - stabilized superparamagnetic nanoparticles
can not be pulled out of aqueous solution using a fixed magnet , such
extraction is possible in solvent / nonsolvent mixtures in which the
poor solubility leads to formation of small aggregates . by dispersing
the particles in a solvent mixture of etoh with an addition of petrolether ,
a cloudy suspension of poorly dispersed core
the poorly suspended nps could be pulled to the
container wall by application of a fixed magnet .
the decantation
step has to be repeated several times due to the poor solubility also
of the free peg dispersant in the etoh / petrolether .
tga was therefore
performed after each decantation step to evaluate the stepwise reduction
in dispersant concentration ( figure 5a ) . after the third precipitation and magnetic decantation
step the grafting density stayed constant , interpreted as that no
further dispersant removal is possible and that all remaining dispersants
are strongly bound to the iron oxide core surface .
the grafting density
is within the measurement error independent of core size at 1
nda - peg(5 kda)/nm .
standard deviations were calculated
based on results measured on 67 different samples for each
core size .
the combined supernatants were analyzed with tga , and 1.5%
inorganic material was found , which means that this purification method
removes free nda - peg and no cores .
after reaching the constant grafting
density by precipitation and magnetic decantation the retained product
included 98% of the cores initially added for ligand replacement independent
of np size . ( a ) step - by - step removal of excess ligands by repeated precipitation / magnetic
decantation
total organic content was measured by tga and converted
into nda - peg(5 kda)/nm .
after three precipitations with
magnetic extraction the free dispersant is removed for all core sizes .
a stable grafting density of 1 nda - peg(5 kda)/nm independent of core size remains with a yield of 98% purified nps .
( b ) representative tem micrograph of well - dispersed , nda - peg - grafted
nps with 9.6 nm core diameter . in comparison to oa - capped nps
it is
possible to dry the nps well dispersed and without order on the grid
due to the steric repulsion of the added peg shell ( cf .
tem micrographs
of smaller cores purified by this method can be found in the supporting information ( figure s7 ) .
compared
to the oa - capped nps ( figure 1d ) , the interparticle distance is increased due to the bulky
nda - peg shell , and importantly an inspection of thousands of particles
did not reveal any aggregated cores .
this strongly suggests that the
peg shell was formed around individual cores and remained homogeneous .
the morphology of the np cores did not change after oa was replaced
by nda - peg . a color change from light brown to dark brown
the difficulty of achieving complete exchange of oa for
new dispersants previously reported by us and others demonstrates the need to
establish that the total organic content determined by tga is traced
exclusively to nda - peg .
significant amounts of other dispersants on
the core surface might alter the physicochemical properties of the
core shell particles .
atr - ftir analysis was performed to verify
the composition of the dispersant shell after purification ( figure 6a ; insets can be
found in figure s4 ) .
ir spectra were also
recorded on the complete supernatant collected from the precipitation
and magnetic decantation method .
the presence of nda - peg on the np
surface could be confirmed by several marker bands below 1000 cm as well as bands in the region from 1345 to 1240
cm and from 1144 to 840 cm that can be mainly attributed to ethylene glycol ch2 and
c o / c c stretching vibrations , respectively .
( a ) atr - ftir spectra of 7.1 nm iron oxide nanoparticles :
( a ) as - synthesized
cores with oa ligands , ( b ) nda - peg - nps after ligand exchange before
purification , ( c ) peg - nda - nps purified by precipitation and magnetic
decantation , ( d ) peg - nda - nps dialyzed 24 h , ( e ) peg - nda - nps purified
through sephadex g75 column ( fr ii ) , ( f ) sephadex g75 , ( g ) peg - nda ,
and ( h ) combined supernatants of all magnetic decantation steps .
labeling :
( ) 1960 cm oh - bending from peg - cooh , ( + )
1740 cm coor or cooh , ( ) 1702 cm ( c = o ) free oa , ( ) 1640 cm amide
conh or nh2 , ( ) 1074974 cm sugar band , ( ) 965 cm ( ( c = o)oh )
free oa ; 680400 cm np bands . ( b ) representative
tga ( solid lines ) and dsc ( dashed ) graphs of 4.9 nm nps measured in
synthetic air : oa - capped nps ( orange ) , nda - peg - nps raw
after ligand exchange before purification ( black ) , precipitation and
magnetic decantation ( green ) , dialyzed ( light blue ) , membrane filtration
with amicon ( dark cyan ) , column purified ( dark yellow ) , and pure ligand
nda - peg ( navy ) .
the amide stretching
vibrations at 1640 ( co ) and 1550 cm ( nh ) ( figure 6a ( ) ) that can be found in all spectra
except for the oa - np are a good indication of successful coupling
of nda to peg and its presence at the np surface ; this peak is slightly
larger for nps purified by magnetic decantation compared to dialyzed
nps .
a weak peak ( figure s4b ( ) )
at 1491 cm fits monodeprotonated nitrocatechol and is attributed to surface - bound nda - peg .
the band at 1728 cm ( figure s4a ( ) ) arises from residual carboxylic carbonyl groups
of free peg(5 kda ) that has not been coupled to nda and is found only
in the nonpurified sample and the combined supernatants .
a similar
trend for the band at 1960 cm ( figure 6a ( ) ) suggests a common
origin ; we therefore attribute this vibration to an overtone or combination
vibration of the out - of - plane oh bending mode of peg carboxylic acid
. the bands at 1730 and 1740 cm ( figure 6 ( + ) ) that were found in all
samples except in the precipitation purified nps and oa - nps might
originate from peg - cooh or coor groups from unreacted peg or cross - reacted
nda - peg ( o - ph ) . however , these byproducts were removed by precipitation / magnetic
decantation .
an important observation is that the nps purified
by column show
deviations from the ir absorption spectrum for iron oxide nanoparticles
purified by other means .
the column material sephadex dextran shows
strong similarities ( bands at 1074974 and 790740 cm , figure 5a ( ) , oh vibrations at 36003000 cm ) with these additional peaks in the spectrum , indicating that nps
purified by column are additionally covered with small amounts of
dextran .
a final important question is the presence of significant
amounts
of weakly or strongly bound oleic acid after ligand replacement and
purification .
in particular , free or weakly bound oleic acid can greatly
influence the colloidal stability .
the presence of free oleic acid
can be determined from absorptions at 1702 cm in
the ftir spectra ( figure 6 ( ) ) assigned to the c = o stretch of h - bonded
( dimeric ) alkylcarboxylic acids and from other bands characteristic
for free acid groups such as the oh in - plane and out - of - plane bending
at 1410 and 965 cm which is overlaid ( figure 6 ( ) ) .
the absence of the bands at 1702 cm in all samples
( also the raw sample ) demonstrates that free oa was successfully removed
by the dmf hexane extraction .
the presence of oleate can be evaluated at 1605 , 1520 , and 1410
cm corresponding to two asymmetric and one symmetric
stretching vibrations of the carboxylate headgroup . whereas the peaks at 1605 and 1520 cm are overlaid
by bands from the peg , a slight shoulder of the band at 1410 cm can be found in all samples .
this band , however ,
is more prominent in the raw and the dialyzed samples than in the
precipitated samples . for the column purified nps this peak
multistep
tga profiles observed below 400 c have been attributed
alternatively to vaporization of physisorbed oleic acid , to chemisorbed
oleate species with different binding strengths , or to partial cleavage of the capping agent .
our data ( figure 6b ) show that a significant second step in tga only
is observed when free or weakly bound impurities such as physisorbed
oleate or dextran are present .
a one - step profile is only observed
for nps purified by magnetic decantation .
we conclude from the
ir measurements that the ligand replacement
is not complete in any sample .
this is also suggested by that the
measured high surface coverage of 1 nda - peg(5 kda)/nm is still lower than the theoretical maximum surface coverage
of > 2 nda - dispersants / nm .
however ,
for samples purified by precipitation and magnetic extraction only
a minor amount of strongly complexed oleate remains on the particle
surface shielded by a dense peg brush .
if oleate with a negligible
desorption rate is distributed on the particle surface , its short
extension combined with the high peg - brush density still results in
excellent colloidal stability as shown in the next section .
the long - term stability of samples
that were purified by dialysis and magnetic decantation was investigated
with tem , dls , and saxs .
long - term stability of dialyzed samples was
only investigated for 4.9 nm core size as the dispersions of nps with
larger core diameters showed visible aggregation after a few days .
also , samples purified by membrane filtration were not subjected to
long - term stability studies as the dispersions showed visible aggregates
after 2 days . for long - term studies of core
shell nps
purified by precipitation and magnetic decantation , 7.1 nm nps were
dispersed at a high concentration of 4 mg / ml that speeds up aggregation
in both h2o and pbs .
pbs tests the stability at physiological
ionic strengths and is additionally known to cause aggregation and
precipitation of poorly stabilized iron oxide nps due to the strong
interaction of phosphates with iron ions .
tem , dls , and saxs were
measured after 1 week and 4 months .
for nps in water and pbs a rh of 16 and 23 nm ( intensity weighted ) , respectively ,
was measured by dls ( table 2 ) , which is in good agreement with previous findings for individually
stabilized core
shell np with nda - peg monolayer , considering the
length of hydrated nda - peg(5 kda ) to be 4 nm and the core 7.1 nm , resulting in a rh(theoretical ) of 15 nm .
these values are significantly
smaller than in other recent publications investigating direct ligand
replacement on oleic acid - capped nanoparticles with peg dispersants .
the narrow size distributions strongly support the interpretation
of individually stabilized and colloidally stable core
no precipitation and negligible
change in the hydrodynamic radius were found for nps in milli - q as
well as pbs after storage of the dispersions for 4 months at 4 c
( table 2 ) .
the rh of dialyzed 4.9 nm cores was
measured to be 16 nm and increased slightly after 4 months to 18 nm .
tem imaging of the nanoparticles stored for 4 months in milli - q and
pbs showed no trace of core aggregation or aging of the particles
( figure s5 ) .
the stability of the dialyzed
sample during storage , but not to further dialysis , indicates a degradation
mechanism related to the dialysis membranes .
scattering curves and fits for samples in milli - q and pbs
fresh and after 4 months are shown in figure 7b .
the high monodispersity of the cores is
evident from the scattering curves . a core size of 6.8 0.4
nm could be fitted to the data .
this
is in excellent agreement with the core size determined by image analysis
of the tem data of the same particles ( 7.1 0.4 nm ) .
after storage
for 4 months at 4 c there is no significant deviation in the
scattering attributable to aggregates , such as a structure factor
peak and/or a change in the low q range .
( a ) dls measurements
of 7.1 nm nps purified by magnetic decantation ;
3 mg / ml in pbs with 10% v / v fetal calf serum added .
temperature cycle
from 20 to 70 c ( circles ) and back ( triangles ) with 5 min equilibration
time at each temperature .
( b ) saxs curves of 7.1 nm nps in pbs and
h2o , freshly prepared and after storage for 4 months at
4 c .
inset : clear colloidal dispersion of nps from measurement
( a ) stored in pbs / fcs 10% v / v for 2 months at 4 c after temperature
cycling .
the colloidal stability of the
nps was further challenged by heating
the nps ( 3 mg / ml ) up to 70 c in pbs with 10% v / v fetal calf
serum ( fcs ) .
this test is applied to distinguish between particles
that show stability in serum but interact with denatured protein aggregates
and those for which interactions between core and denatured proteins
also do not occur.figure 7a shows the evolution of the hydrodynamic
radius with temperature .
no increase in rh or precipitation of nanoparticles during the temperature cycle was
observed .
the inset in figure 7b shows a picture of the same np dispersion subjected to temperature
cycling after being stored for 2 months at 4 c .
aggregation
or precipitation also can not be observed after storage ; the dispersion
remains clear and colloidally stable .
tem micrographs ( figure s6 ) show that the nps in pbs / fcs remain
well - dispersed after storage for 2 months in serum .
dialysis has to be performed with membranes
having a pore size sufficiently large to allow diffusion of the dispersant
through the pores ; the mwco of membranes is however typically given
for equivalent protein or dextran sizes and can not be used directly
for comparison to highly solvated polymer molecular weights for which
the radius of gyration is much bigger than for e.g. protein of the
same molecular weight .
a rule of thumb of choosing a pore size > 5
that of the dialyzed molecule is often invoked .
this was confirmed
by attempting dialysis of dissolved peg(5 kda ) ( 0.2 g / ml against 5
l of milli - q ) in a membrane of 1214 kda mwco , i.e. , nominally
23 times the molecular weight of the nda - peg , which was used
in previous publications studying grafting - to and ligand replacement
of pegylated nps .
after 24 h only 50% of the free
peg was removed , and only 60% had been removed after 5 days despite
exchanging milli - q in the reservoir five times .
thus , choosing a too
low mwco results in remaining free dispersant and subsequent overestimation
of the dispersant grafting density by tga .
we therefore changed to cellulose - based
membranes with a mwco of 1000 kda , which nominally corresponds to
a cutoff hydrodynamic size of 37 nm ( assuming a scaling related
to equivalent sizes of dextran ) .
nps with a hydrodynamic radius rh between 15 and 23 nm ( for 7.1 nm cores ) were
still retained while free nda - peg(5 kda ) with an rh of 4 nm was presumably
removed from the dispersion .
figure 3a shows the removal of excess ligands by
dialysis for all sizes of nps .
large - core nps ( 7.1 and 9.6 nm ) dialyzed
for 24 h yielded an average grafting density of 1 nda - peg(5
kda)/nm determined by tga .
however , nps also started to
stick to the dialysis membrane after 24 h. tem of large nps ( 7.1 and
9.6 nm ) extracted from the dialyzed dispersion after 24 h showed aggregated
morphology ( figure 3b ) .
the 24 h dialyzed samples could be redispersed in water but showed
poor long - term stability .
visible aggregates were obtained after 1
week . continued dialysis of the large cores led to an even lower grafting
density ( 0.5 nda - peg(5 kda)/nm ) and increased
aggregation .
( a ) removal of excess ligands by dialysis with a 1000
kda mwco
membrane .
total organic content was measured by tga and converted
into nda - peg(5 kda)/nm using the known molecular weight
of nda - peg(5 kda ) and average core surface area determined by tem .
after 24 h the surface coverage was 1 nda - peg(5 kda)/nm independent of size , but core size - dependent aggregation
and precipitation were observed .
dialysis for 70 h lowered the grafting
density to 0.3 nda - peg(5 kda)/nm , resulting in colloidally
unstable dispersions .
( b ) typical tem of nanoparticles with 9.6 nm
core size that had visible aggregates ( inset ) grafted with nda - peg(5
kda ) after 24 h dialysis against water using a 1000 kda mwco cellulose
membrane .
nps with small core sizes ( 4.9
and 3.5 nm ) had grafting densities
of 0.8 and 1.7 nda - peg(5 kda)/nm , respectively , after
24 h dialysis and showed good colloidal stability .
the small nps were
dialyzed also for 70 h , which resulted in lowering of the average
grafting density to 0.3 nda - peg(5 kda)/nm . during
dialysis
the np suspension underwent visible color change
from light brown to almost black ( figure s2 in the supporting information ) , indicating that as dialysis proceeds ,
surface - bound nda - peg is ripped off in addition to the initial removal
of free nda - peg .
first - generation , hydrophilic iron oxide nps were mainly stabilized
with dextran or carbohydrate derivatives physically adsorbed without using a separately
defined anchor such as nitrodopamine .
a second generation of polymer - coated
iron oxide nps could be said to have polydisperse cores but defined
polymer shells end - grafted with defined anchor groups .
shell architecture
allows for exact characterization and functionalization , which is
crucial to medical applications . a third generation with monodisperse
iron oxide cores to perfectly tailor uniform properties
several mutually contradicting conditions
during the ligand replacement reaction have to be fulfilled : ( 1 ) to
dissolve the capping agent ( oleic acid ) , ( 2 ) to solubilize the dispersant ,
( 3 ) to keep the dispersant at low coil size , quantitatively described
by e.g. rg , which determines the grafting
footprint , and ( 4 ) to provide the right conditions ( protonation ) of
the anchor group to irreversibly bind to the core .
we could
optimize the ligand exchange procedure within the boundaries
of the contradicting requirements and thus produce nps with grafting
densities consistently around 1 nda - peg(5 kda)/nm .
the
grafting densities of 1 nda - peg(5 kda)/nm for
monodisperse nps were shown to be sufficient to render the nps long - term
stable in biologically relevant media .
this is in contrast to what
was reported earlier for polydisperse systems . for polydisperse cores
a grafting density
of 2 nda - peg(5 kda)/nm was found as the threshold
for colloidal stability at temperatures above body temperature and
in biological fluids .
we hypothesize that the accurate exchange of
ligands into a homogeneously coated shell is what enables the improved
stability at a lower average grafting density for monodisperse , spherical
nps . on the other hand
, we were unable to achieve grafting densities
higher than 1 nda - peg(5 kda)/nm by direct ligand
replacement . that the grafting density was constant with respect to
core size indicates that surface curvature is not the determinant
of the ultimate grafting density by ligand replacement in the superparamagnetic
size range .
this is similar to previous observations for ligand replacement
of hydrophobic dispersants and nda - peg
grafting in the melt , for which within
the experimental error a difference in grafting density with respect
to curvature could not be demonstrated . in the present case
the constant
grafting density is lower and suggests that the size of the peg - coil
is the main determinant of the grafting density .
recent work
by e.g. mefford and co - workers reported very high ligand
densities from ligand replacement in their study of oleic acid displacement . in their study ,
one precipitation step followed by dialysis with
low mwco membranes was used to remove excess dispersant .
our results
show that this is insufficient to remove excess dispersants so their
unusually high dispersant density in comparison with literature can
likely be traced to this fact .
a higher polydispersity of the cores
can also have contributed to uncertainty in the grafting density .
, large monodisperse cores were grafted with nda - peg and purified
by repeated ultracentrifugation from aqueous solution , which is only
feasible with very large cores .
they report a grafting density of
1 nda - peg(5 kda)/nm , which is inconsistent with
the presented tga results that yield a grafting density within a large
interval of 528 nda - peg(5 kda)/nm .
this organic
content , which corresponds to an order of magnitude higher grafting
density than the theoretical maximum , demonstrates that also a single
centrifugation step and dialysis are insufficient to purify also large
core nps .
these samples even showed low colloidal stability , which
is typically associated with a low grafting density .
however , one
should keep in mind that stabilizing ferrimagnetic nanoparticles with
large cores using thin polymer brushes is more challenging than in
the superparamagnetic range . a different ligand replacement protocol
in this study
might also have left a higher fraction of destabilizing
or heterogeneously distributed oleate bound to the np surface . finally , it was recently shown that grafting of polymer shells
under solvent - free , polymer - melt conditions can lead to a near complete
coverage of end - grafted polymer dispersants , with unique shell structure
and colloidal properties as the result .
while this might seem superior we show here that optimized protocols
for grafting - to can yield the required colloidal properties for standard
medical applications .
our grafting - to by one - step ligand replacement
has the benefits of ( a ) providing near - total yields ( 98% ) under optimal
conditions , while melt - grafting requires considerable excess dispersant
and was reported with a maximum yield of 30% calculated on
nanoparticle cores ; ( b ) multifunctional particles can be constructed
by molar mixing of differently functionalized dispersants without
risking degradation of the functional group under the melt - grafting
conditions ; and ( c ) direct ligand replacement is easier to scale up
for synthesis of large batches .
purification of as - synthesized
core shell nps is crucial
for proper determination of np properties , and we emphasize the thorough
analysis that we have done of the efficiency and effect of different
purification methods as a central result of the presented work . to
our knowledge , there has been no previous study on the colloidal stability
of core
we
report a strong time - dependent degradation of the particles when dialyzed
through high mwco regenerated cellulose membranes .
cellulose ester
should be inert and not the cause of the observed particle degradation ,
which from tem clearly included the particle core .
a similar trend
was found for nps purified by membrane filtration : np degradation
in terms of removal of surface - bound nda - peg was observed , and a colloidally
stable product could not be obtained .
degradation of the core can
occur by that a dispersant with a strongly binding anchor group leaves
the core surface and removes the surface iron ion with it .
since the other purification methods do not
lead to this degradation , i.e. , the nda anchor is stably bound to
the core , we hypothesize that the degradation during dialysis and
membrane filtration involves an osmotic / mechanical strain related
to dispersant interaction with the pores of the membranes where the
particles are also found to precipitate .
stretching of the peg chain
leads to a spring - like increase in energy ; it could effectively weaken
the bond to the surface and lead to polymer detachment and the observed
surface dissolution .
size - exclusion column chromatography with
cross - linked dextran
as column material is often used for purification of biotechnological
samples in the size range of nanoparticles ; it was previously advocated
by us and others as a suitable way to purify core
shell nanoparticles
after grafting - to and simultaneously testing their grafting density
and colloidal stability .
dextran has high affinity to iron oxide and
has
been used in commercial contrast agent coatings of iron oxide nanoparticles .
our new results show that for highly but not extremely densely grafted
nps dextran can transfer from the column to the nps .
the cross - linked
dextran employed in a size - exclusion column should not transfer to
the nps , but the ir results unambiguously show its presence in the
eluted np fractions even after passing the same fraction through fresh
columns twice . although dextran was not found by ir for the same type
of np cores with grafting densities > 2 nda - peg(5 kda)/nm , a lower grafting density seems to
result in transfer of column material to the free core area .
the high
peg grafting density of 1 nda - peg(5 kda)/nm obviously
prevents further np aggregation or sticking to the column .
we interpret
this result as that transferred dextran also can stabilize nanoparticles
that are not extremely densely grafted .
this additional physisorbed
dextran coating can explain the high organic content in other particle
fractions that passed the column .
high - molecular - weight polymer physisorbed
to nps induce bridging interactions that cluster multiple cores ; this
leads to effective differences in size and column retention time ,
which spreads the nps over several fractions and pushes down the yield
of the main fraction . thus ,
despite the stability of the nps purified
by column chromatography , the application of cross - linked dextran
as column material can not be generally recommended for purification
that should yield particles with controlled shell properties .
the
choice of another column material with less strong interaction with
iron oxide could remedy this drawback .
however , the yield and throughput
would based on our study remain low compared to magnetic decantation . among
the many investigated purification methods , our study suggests
a combination of precipitation and magnetic decantation repeated in
multiple cycles as the preferred choice .
this method reproducibly
yields nps with exceptional long - term colloidal stability , at high
yield and without free dispersant .
this very high yield
is far superior to the inherent losses of nps when column purification
or dialysis is used and can be attributed to the efficient magnetic
extraction .
similar approaches have previously been applied in combination
with centrifugation , which is sensitive to the choice
of solvent ratio and polymer shell molecular weight for their success
and do not make use of the superparamagnetic core . the crucial step
for successful separation by magnetic decantation was to discard the
supernatant prior to sedimentation of precipitated excess peg , since
peg and densely peg - grafted nps have similar solubility .
aggregation
of the superparamagnetic cores enables rapid collection of the nps
within 25 min depending on their size ; the spontaneous precipitation
and sedimentation of the free peg takes 10 min .
the petrolether
used in the magnetic decantation also aided removal of remaining oa
from the nanoparticle sample that can not be removed by dialysis or
centrifugation , since oa was found in the removed supernatant .
a final major advantage of the magnetic decantation method is that
in addition to its high yield and purity it can be scaled up and automated .
large volumes and samples can be handled in a short time ( < 1 h
for > 3 cycles necessary for free dispersant removal ) , while dialysis
is inherently time - consuming ( relying on diffusion ) , membrane centrifugation
is limited by the volume of the filtration units , and columns are
difficult to scale .
we show magnetic decantation for synthesis of
gram quantities and further scaling up is possible .
we conclude that direct ligand replacement of oleic acid - coated
monodisperse iron oxide nps can be optimized to consistently yield
grafting densities of 1 nda - peg(5 kda)/nm regardless
of core size in the investigated superparamagnetic range ( diameter
310 nm ) .
shell nps colloidally stable over many
months in biologically relevant media as well as withstanding demanding
tests such as temperature cycling in serum .
the choice of purification
method is as important as the synthesis protocol for reproducible
results and greatly affects yield , purity , speed , and amount that
can be produced .
the best purification method that we found was magnetic
decantation , which compared to dialysis , centrifugation filtration ,
and sephadex size - exclusion chromatography provides the highest quality
product and a means to verify purity ; it also provides the highest
yield and sample volumes that can be extracted per time unit .
the
methods described in this work form the fundament for development
of a whole set of diversely functionalized nps that are suitable for
biomedical applications such as targeted mri , multifunctional superparamagnetic
nps , and drug delivery .
|
fundamental research
on nanoparticle ( np ) interactions and development
of next - generation biomedical np applications relies on synthesis
of monodisperse , functional , core shell nanoparticles free
of residual dispersants with truly homogeneous and controlled physical
properties .
still , synthesis and purification of e.g. such superparamagnetic
iron oxide nps remain a challenge . comparing the success of different
methods
is marred by the sensitivity of analysis methods to the purity
of the product .
we synthesize monodisperse , oleic acid ( oa)-capped ,
fe3o4 nps in the superparamagnetic size range
( 310 nm ) .
ligand exchange of oa for poly(ethylene glycol )
( peg ) was performed with the peg irreversibly grafted to the np surface
by a nitrodopamine ( nda ) anchor .
four different methods were investigated
to remove excess ligands and residual oa : membrane centrifugation ,
dialysis , size exclusion chromatography , and precipitation combined
with magnetic decantation .
infrared spectroscopy and thermogravimetric
analysis were used to determine the purity of samples after each purification
step .
importantly , only magnetic decantation yielded pure nps at high
yields with sufficient grafting density for biomedical applications
( 1 nda - peg(5 kda)/nm2 , irrespective of size ) .
the
purified nps withstand challenging tests such as temperature cycling
in serum and long - term storage in biological buffers .
dynamic light
scattering , transmission electron microscopy , and small - angle x - ray
scattering show stability over at least 4 months also in serum . the
successful synthesis and purification route
is compatible with any
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|
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] |
the incidence of autoimmune diseases ( aids ) such as systemic lupus erythematosus ( sle ) , rheumatoid arthritis , and primary biliary cirrhosis ( pbc ) has been increasing in china . until now ,
the antinuclear antibody ( ana ) test , wherein antibodies are detected by indirect immunofluorescence assay on hep-2 cells , is commonly used as an initial screening method .
the presence of ana is nonspecific and can be associated with many nonautoimmune factors , such as carcinoma , infection , pharmaceuticals , and environmental factors . as a result ,
an ana positive frequency in healthy individuals > 20% has been reported . on the other hand
, ana may exist several years before an aid can be diagnosed , with higher titer of ana being correlated closely with a higher risk of the onset of aids during adolescence and adulthood .
the epidemiologic characteristics of aid in different countries and districts varies . to our knowledge ,
information about the ana prevalence and the susceptibility or potential of aids in the general population of china is lacking .
we aimed to evaluate the ana prevalence in a wide range of the general chinese population ( aged 288 years ) .
to assess the ana positive rate among the general population of china , we conducted a cross - sectional study .
twenty thousand nine hundred seventy sera were taken from the physical examination center in baoding , hebei , china , from july 2011 to september 2013 ( figure 1 ) .
the sample size and male to female ratio conform to the standard of the sixth national census of china in 20102011 ( http://www.stats.gov.cn/tjsj/pcsj/rkpc/6rp/indexch.htm ) .
a total of 6800 children ( including 3300 girls and 3200 boys ) aged from 2 to 18 years were recruited from 3 public kindergartens , 5 middle schools , and 2 high schools .
the other participants consisted of 14,170 individuals ( 7220 women and 6950 men ) obtained by random sampling .
the study complied with the world medical association declaration of helsinki and was approved by the ethics committee of the first center hospital of bao ding .
to assess the ana positive rate among the general population of china , we conducted a cross - sectional study .
twenty thousand nine hundred seventy sera were taken from the physical examination center in baoding , hebei , china , from july 2011 to september 2013 ( figure 1 ) .
the sample size and male to female ratio conform to the standard of the sixth national census of china in 20102011 ( http://www.stats.gov.cn/tjsj/pcsj/rkpc/6rp/indexch.htm ) .
a total of 6800 children ( including 3300 girls and 3200 boys ) aged from 2 to 18 years were recruited from 3 public kindergartens , 5 middle schools , and 2 high schools .
the other participants consisted of 14,170 individuals ( 7220 women and 6950 men ) obtained by random sampling .
the study complied with the world medical association declaration of helsinki and was approved by the ethics committee of the first center hospital of bao ding .
sera were tested by indirect fluorescence on hep-2 cells according to the manufacturer s instructions ( euroimmun ag , lubeck , germany ) . titer 1:320 was considered to be positive . as a result , 243 positive samples were further tested by line immunoassay ( lia ) ( euroimmun ag ) for 15 specific autoantibodies ( ie , anti - nrnp , anti - sm , anti - ssa , anti - ro52 , anti - ssb , anti - jo-1 , anti - scl-70 , anti - cnepb , anti - dsdna , anti - his , anti - pcna , anti - nuk , anti - rib , anti - m2 , and anti - pmscl-70 ) .
euroblotmaster ( euroimmun ag ) and eurolinescan ( euroimmun ag ) were used to complete the operation and for test result interpretation , respectively .
spss for windows version 17.0 ( ibm - spss , inc , armonk , new york ) was used for statistical calculations .
spss for windows version 17.0 ( ibm - spss , inc , armonk , new york ) was used for statistical calculations .
the age and sex distribution of 20,970 participants are summarized in table i. the ages ranged from 2 to 88 years , with mean age 32 ( 19.7 ) years for both sexes .
the prevalence of ana in male and female participants by age groups are shown in table i. the overall prevalence of ana was 5.92% and correlated positively with age .
there were significant differences among each age group except age older than 80 years , as shown in table i ( p < 0.01 ) . in the female group ,
there are 2 sharp peaks in ana positivity at the 20-year and 40-year age groups . in 1243 ana - positive sera tested by lia ,
44.2% were positive for at least 1 of 15 specific ana antibodies . in terms of the effect of age and sex on ana positivity , there were significant differences among the 3 groups ( ie , 20 years , 2149 years , and 50 years ; = 275.04 ; p < 0.01 ) , and between male and female ( = 236.47 ; p < 0.01 ) , as shown in table i and table ii , respectively . the autoantibodies with the top-3 positive frequency were anti - ro-52 ( 19% ) , anti - m2 ( 17.8% ) , and anti - ssa ( 14.3% ) ; whereas anti - scl-70 , anti - jo-1 , and anti - sm were the less frequently detected antibodies among the autoantibodies detected ( table iii ) .
this is the first research into the prevalence of ana in the general chinese population .
satoh et al hold that the ana prevalence in the us population of individuals aged 12 years and older is 13.8% , and based on national health and nutrition examination survey 1999 - 2004 data , more than 32 million people in the united states are positive for ana .
other studies indicate that japanese show a 9.5% prevalence at a 1:100 cutoff level dilution , whereas for indians it is 12.3% .
anti - ro-52/ssa was the most detected antibody in both of those studies . in the present and another relevant study ,
based on that comparison among different nations , we hold the opinion that ana positive frequency in adults differs by geography .
sex and age had been assumed to be the factors that influenced ana positivity . in our study , ana prevalence correlated positively with age on the whole , with higher prevalence present in the 20- to 30-year and 40- to 50-year groups .
the latter peak is in line with the single peak at age 40 to 49 years described in a us population that was characterized by the physiologic stage of puberty and menopause , implying that estrogen may play an important role . as such
, some similarities of genetic deposition and hormone factors may have an influence on disease manifestations . between the sexes ,
our results indicate that the female to male ratio in ana positive cohorts is almost 4:1 , except in the 80 years group .
our study also showed that ana positivity in girls ( 8.2% ) was higher than in boys ( 2.5% ) .
this indicated that females have a higher ana titer than males even at a relatively immature stage . because ana - iif is a primary screening test for adolescent sle ,
hayashi et al reported that it is important to emphasize the ana titer as a prognostic marker in certain aids , and a 1:160 or 1:320 dilution titer is recommended .
although ana may be influenced by nonimmune factors , several articles show that the positive frequency is no more than 5% at a dilution titer of 1:160 in the general population . in our study , at the cutoff value of 1:320 titer , we found a similar result to previous studies in that older people were prone to higher frequency of ana positivity than younger people ( figure 2 ) .
consistent with the findings in a japanese population , we found anti - ro52/ssa was the most prevalent autoantibody in the general population .
ro-52/ssa is a useful serologic marker for sle and sjogren syndrome , myositis , systemic sclerosis , and pbc .
anti - ro / ssa and antichromatin had the highest predictive value for sle diagnosis .
the prevalence of patients with asymptomatic pbc in an asian population derived from previous studies seemed to be larger than that inferred from our study .
coincident to the data from japanese residents , our results showed that anti - scl-70 , anti - jo-1 , anti - nuk , and anti - sm were the least - detected antibodies .
it is a pilot study on a large - scale population without comprehensive consideration of other factors such as occupation , genetic deposition , and biochemical factors .
ana positive frequency is high in the general chinese population and differs in different sex and age groups .
the authors have indicated that they have no conflicts of interest regarding the content of this article .
|
backgroundthe incidence of autoimmune diseases such as systemic lupus erythematosus , rheumatoid arthritis , and primary biliary cirrhosis has increased significantly in china .
information about the susceptibility or potential of autoimmune diseases in the general population is lacking.objectiveto explore the prevalence of antinuclear antibody ( ana ) and its specificities in the general population in china.methodstwenty thousand nine hundred seventy sera samples were taken from the physical examination center in baoding , china .
indirect immunofluorescence and line immunoassays were used to detect ana and its specificities , respectively.resultssamples from females had a higher prevalence of ana than samples from males ( 2 = 278.55 ; p < 0.01 ) . for both sexes ,
the prevalence of ana positively correlated with age and there were significant differences among different age groups at 10-year intervals , except the 80 years group ( p < 0.05 ) .
one thousand two hundred forty - three ana - positive samples were further analyzed with line immunoassays .
there was a significant difference among age groups and between sex groups in terms of the specific autoantibodies ( p < 0.01 ) .
the autoantibodies with the top-3 positive frequencies were anti - ro-52 , anti - m2 , and anti-ssa.conclusionsthere was a high prevalence of ana positivity in the general chinese population that seemed to be influenced by sex and age and correlated with specific autoantibodies .
|
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] |
kaposi sarcoma ( ks ) is characterized by multicentric neoplastic proliferation of vascular endothelial cells .
aids - related ks has a rapidly progressive course with early mucosal and systemic involvement .
the incidence of aids - associated ks , especially the disseminated form , is very rare in india .
furthermore , the importance lies in early diagnosis and treatment which significantly affect the outcome .
we are reporting the case for its rarity in india ( probably the 18 case ) and to highlight the importance of the early institution of highly active antiretroviral therapy ( haart ) .
a 34-year - old male presented with multiple asymptomatic pigmented raised lesions over the trunk , bilateral upper and lower limbs for last two months .
he had history of fever and weight loss ( > 10% total body weight ) for the past six months .
history of swelling of bilateral lower limbs , scrotum , and penis was present since one month .
on examination , the patient had pallor , generalized lymphadenopathy , bilateral pitting pedal edema , and hepatosplenomegaly .
cutaneous examination revealed multiple discrete to grouped violaceous macules , papules , nodules and plaques measuring 0.2 cm 0.2 cm to 0.5 cm 3 cm over the trunk , bilateral upper and lower limbs [ figure 1 ] .
cutaneous lesions over the lower abdomen and left thigh were coalescing to give a cobblestone appearance [ figure 1 ] .
oral cavity showed two well - defined violaceous patches over the hard palate [ figure 2 ] . on investigation
, he had low hemoglobin ( 6.9 g / dl ) , deranged renal function parameters ( urea - 69 mg / dl and creatinine - 1.9 mg / dl ) and massive bilateral pleural effusion on chest x - ray .
serology for hiv-1 was positive by enzyme - linked immunosorbent assay and negative for hiv-2 , and his cd4 count was 573/mm .
histopathology of skin biopsy specimen revealed proliferation of endothelial cells with formation of slit - like vascular spaces in the upper and middle dermis .
spindle - shaped endothelial cells were arranged in solid cords and fascicles admixed with variable lymphoplasmacytic infiltrate , hemorrhage , and lymphatic - like dilated spaces [ figures 3 and 4 ] .
promontory sign , characterized by protruding of new vessels into the lumen of existing large vessel , was also demonstrated [ figure 5 ] . on immunocytochemistry ,
a final diagnosis of aids - associated disseminated ks was made but unfortunately the patient died before starting haart because of recurrent massive pleural effusion .
multiple violaceous macules , papules , nodules , and plaques over the lower abdomen and bilateral thigh , some of which are coalescing to give
cobblestone - like appearance two well - circumscribed violaceous patches present over the hard palate cord - like proliferation of spindle - shaped endothelial cells with areas of hemorrhage and variable lymphoplasmacytic infiltration in the dermis ( h and e , 100 ) spindle - shaped endothelial cells arranged in fascicles in the dermis ( h and e , 400 ) promontory sign - new blood vessels protruding into the lumen of existing blood vessel ( arrow ) ( h and e , 400 ) cd31 positive proliferating endothelial cells ( ihc , 100 )
human herpes virus-8 ( hhv-8 ) is considered as the putative causative agent of ks .
co - infection of hhv-8 with hiv promotes the oncogenic capabilities of hhv-8 , leading to the development of ks .
the low incidence of ks in india is thought to be because of the low prevalence of the hhv-8 infection , with a prevalence of 4.7% reported from south india .
cutaneous lesions of classic ks start over the extremities as violaceous patches which gradually progress to form plaques and nodules .
progression is slow in the classic form , and mucosal and systemic involvement is rarely seen .
the incidence of aids - associated ks is higher in homosexual males in comparison to females .
clinically , aids - related ks differs from the classical form because of its rapid clinical course and widespread dissemination that affect the mouth , lymph node , lungs , gastrointestinal tract and genitalia .
oral mucosal involvement can occur in all type of ks but more common in aids - associated ks .
common sites affected are hard palate and soft palate followed by gingiva , and dorsal tongue .
the presence of oral lesion not only points towards undiagnosed hiv infection but also its association with the disseminated form of the disease which have potential diagnostic and therapeutic implications . in 50% patients of ks , oral mucosa
is affected and in 20 - 25% , cases oral mucosa is the initial site of involvement . in indian patients , an involvement of both cutaneous and oral mucosa occurred in 61.53% cases while 38.46% had only cutaneous involvement .
our patient presented with two violaceous patches over hard palate which probably correlates with the rapidly progressive nature of the disease .
in contrast to most of the malignancies , lymph node involvement in ks is not associated with worse prognosis and is of no special clinical significance .
lung involvement in ks occurs in 20% of the patients and is the most life - threatening form of the disease .
patients commonly present with progressive dyspnea , nonproductive cough , fever , chest pain and pleural effusion .
our patient had recurrent massive pleural effusion probably due to involvement of the lungs leading to the demise of the patient . from a dermatologist 's point of view
, ks should be kept in mind while dealing with an hiv - seropositive case with violaceous raised lesions , as the prognosis depends on the timely initiation of haart therapy .
the closest mimics of ks are bacillary angiomatosis , pseudo - ks , lichen planus , malignant melanoma , pyogenic granuloma , and hemangioma .
immunohistochemical staining with hhv-8 clinches the diagnosis of ks , but in our case , we could not perform the test because of financial constraint .
aids - related ks responds well to haart therapy , and systemic chemotherapeutic drugs are indicated in nonresponsive patients or in patients with systemic involvement .
liposomal anthracyclines ( doxorubicin and daunorubicin ) are the united states food and drug administration approved first - line agent for treatment of ks . due to its side - effect profile ,
other systemic treatment modalities include interferon- and combination chemotherapies with adriamycin , bleomycin plus vincristine , or vinblastine . in conclusion ,
our case highlights the rapidly progressive nature of aids - associated disseminated ks and importance of the early institution of haart .
early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi 's sarcoma ( ks ) to achieve favorable prognosismucosal lesion can give a clue to the undiagnosed hiv .
early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi 's sarcoma ( ks ) to achieve favorable prognosis mucosal lesion can give a clue to the undiagnosed hiv .
early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi 's sarcoma ( ks ) to achieve favorable prognosismucosal lesion can give a clue to the undiagnosed hiv .
early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi 's sarcoma ( ks ) to achieve favorable prognosis mucosal lesion can give a clue to the undiagnosed hiv .
early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi 's sarcoma ( ks ) to achieve favorable prognosismucosal lesion can give a clue to the undiagnosed hiv .
early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi 's sarcoma ( ks ) to achieve favorable prognosis mucosal lesion can give a clue to the undiagnosed hiv .
|
aids - associated disseminated kaposi sarcoma ( ks ) is a rare entity , especially in india due to the low prevalence of human herpes virus-8 infections in indian population . due to its rapid and progressive nature
, early diagnosis and institution of highly active antiretroviral therapy is crucial in aids - associated ks , with a view to achieving favorable prognosis .
we report a case of disseminated ks in an hiv-1 positive patient , who presented with two months history of multiple violaceous patches and plaques over the trunk , bilateral upper limbs , lower limbs , and hard palate .
the patient died of recurrent massive pleural effusion before starting antiretroviral therapy .
this case is being reported due to the paucity of ks in the indian literature , especially the disseminated type and to highlight its rapidly progressive course which can be fatal .
|
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pelvic organ prolapse ( pop ) is a major health issue for women , affecting quality - of - life and requiring costly surgery.1,2 pop is an age - related disorder , and the prevalence increases as a population ages .
women who attain the age of 80 years have a greater than 10% risk of requiring surgery to treat pop.3 treatment of genital prolapse in elderly women is challenging and almost always depends on the medical condition and sexual activity level of the patient .
although pessaries are offered as first - line therapy to such patients,4,5 surgery may be indicated for patients who have failed or do not desire the lifelong use of a pessary .
various surgical techniques using laparotomy , laparoscopy , and vaginal surgery , including colporrhaphy ( with or without mesh placement ) , sacrospinous ligament fixation ( sslf ) , abdominal sacrocolpopexy ( asc ) , high sacrouterine ligament fixation , prolapse kit placement , colpocleisis , and constricting surgical procedures , have been utilized .
asc has an 80%85% long - term success rate , while sslf has a 70%75% long - term success rate.6,7 prolapse kits ( gynecare prolift ; ethicon , inc . ,
somerville , nj , usa ) have a success rate of 96.5%.8 however , all procedures may cause major complications , such as bladder injury , hemorrhage , wound dehiscence , rectal injury , and postoperative vault infection .
asc has disadvantages , including a long operative time , a long recovery time , possible injury to pelvic structures , hemorrhage , and ileus .
sslf also has disadvantages , including the possibilities of hemorrhage , infection , and nerve damage .
prolift kits can have adverse events , such as bladder injury and mesh erosion . on the other hand ,
obliterative and constrictive surgical procedures have lower levels of complications , a low risk of perioperative morbidity , and an extremely low risk of prolapse recurrence .
patients who are not interested in a functional vagina because they are not sexually active and have no desire to be so active in the future , are the best candidates for obliterative or constrictive vaginal surgery .
various techniques have been described , but lefort colpocleisis is the preferred procedure for obliterative surgery.911 although colpocleisis with uterine preservation is effective , drawbacks are apparent .
other drawbacks include late vaginal bleeding , ureteric occlusion or injury as a result of kinking in the distal ureter , a risk of gynecological cancer , vaginal discharge , and an uncomfortable feeling of fullness in the vagina.12,13 constricting surgical procedures are alternatives avoiding these drawbacks . the goal is to narrow the vaginal tube to close the defect .
we performed constricting anterior and posterior colporrhaphy , levator myorrhaphy , and high perineorrhaphy with concurrent hysterectomy , and investigated the feasibility , intraoperative complications , and short - term follow - up results of these constricting procedures in 54 patients aged 75 years or older .
we performed constricting anterior and posterior colporrhaphy , levator myorrhaphy , and high perineorrhaphy with concurrent hysterectomy , and investigated the feasibility , intraoperative complications , and short - term follow - up results of these constricting procedures in 54 patients aged 75 years or older .
this study was performed at the obstetrics and gynecology department at tepecik training and research hospital .
the study was approved by the institutional review board of tepecik education and research hospital and all participants provided informed consent . the hospital database was searched for cases treated between january 2011 and january 2014 ; we sought women over the age of 75 years who underwent surgery for pelvic floor disorders , with or without incontinence surgery . in total , 202 such women were identified . with the increase in life expectancy , an age of 75 years has been used as a cutoff for elderly in several studies.14,15 we used the same cutoff .
our study inclusion criteria were an age greater than 75 years and surgery for pop stage 2 or higher ( figure 1 ) , with vaginal hysterectomy , constricting anterior and posterior colporrhaphy , levator myorrhaphy , and a high perineorrhaphy , with or without a urinary incontinence procedure . a total of 123 patients who underwent other types of pop surgery were excluded .
demographic characteristics , including age , parity , mode of delivery , and the presence / absence of hypertension and diabetes , were recorded .
prolapse assessment was performed by a specialist , using the pop quantification ( pop - q ) system.16 objective recurrence was defined as stage 2 or higher pop .
subjective recurrence was defined as a perception of prolapse ( bulge symptoms ) by the patient .
regret and satisfaction were assessed using the decision regret scale pelvic floor disorders ( drs - pfd ) and satisfaction decision scale pelvic floor disorders ( sds - pfd ) forms.17 the drs - pfd is a five - item questionnaire with a five - point response scale .
the sds - pfd is a six - item questionnaire with a five - point response scale .
the pelvic floor distress inventory ( pfdi ) was used to assess quality of life outcomes.18 pfdi has three subscales : the urinary distress inventory , the colorectal - anal distress inventory , and the pelvic organ prolapse distress inventory .
patient answers are ranked on a likert scale , with 1= not at all , 2= somewhat , 3= moderately , and 4= quite a bit .
the mean value of all questions answered is then multiplied by 25 for the actual score ( range 0100 ) . the higher the score , the greater the perceived impact that pelvic floor dysfunction has on a patient s life .
results were presented as mean standard deviation ( sd ) or as median ( interquartile range ) .
preoperatively , all patients underwent a stress test in the standing position , at a bladder volume of 300 ml , with the pessary placed and the prolapse reduced .
the results of the cough stress test were recorded as positive if urine loss occurred with a cough , or as negative if no urine loss was noted .
they received an intravenous first - generation cephalosporin 1 hour before surgery and underwent a prophylactic antiseptic vaginal wash with iodine - containing soap before surgery .
after hysterectomy , the anterior midline of the vaginal cuff was used as the starting point for anterior vaginal wall incision .
a full - length and full - width dissection of the anterior vaginal wall from the underlying musculoconnective tissue was performed .
after dissection of the vaginal epithelium from the underlying pubocervical fascia , a vicryl # 2 suture was placed on the fascias , using a continuous circular clockwise motion .
the floor of the bladder was next pushed up as the purse - string suture was tied .
excess vaginal epithelium was excised , and the paravaginal connective tissue and vaginal epithelium were approximated in the midline , using a delayed absorbable suture .
after the anterior colporrhaphy , the vaginal cuff was closed with three interrupted 0-dexon sutures .
ovarian ligaments , the round ligaments , and the cardinal and uterosacral ligaments were attached to the vaginal vault to support the vaginal apex .
next , two allis clamps were placed at the 4- and 8-oclock positions of the hymenal ring .
the goal of vaginal incision was to allow separation of epithelium from the underlying connective tissue .
the plane of separation was continued as far laterally as necessary , in either direction , toward the pelvic sidewall to expose the puborectalis muscle , the bulbocavernosus muscle , and the perineal membrane .
excess vaginal epithelium was excised and the puborectalis muscle , the bulbocavernosus muscle , and the vaginal epithelium approximated in the midline , using a delayed absorbable suture .
finally , the vaginal introital epithelium was approximated with an absorbable running suture , rendering the resultant genital hiatus about 1 cm in length ( figure 3 ) .
a concomitant transobturator sling operation , using a safyre t ( promedon , cordoba , argentina ) polypropylene monofilament sling ( placed through a separate vaginal incision ) was performed in women for whom the stress test was positive .
after surgery , saline - soaked gauze was packed into the vagina , and a urethral catheter inserted .
preoperatively , all patients underwent a stress test in the standing position , at a bladder volume of 300 ml , with the pessary placed and the prolapse reduced .
the results of the cough stress test were recorded as positive if urine loss occurred with a cough , or as negative if no urine loss was noted .
they received an intravenous first - generation cephalosporin 1 hour before surgery and underwent a prophylactic antiseptic vaginal wash with iodine - containing soap before surgery .
after hysterectomy , the anterior midline of the vaginal cuff was used as the starting point for anterior vaginal wall incision .
a full - length and full - width dissection of the anterior vaginal wall from the underlying musculoconnective tissue was performed .
after dissection of the vaginal epithelium from the underlying pubocervical fascia , a vicryl # 2 suture was placed on the fascias , using a continuous circular clockwise motion .
the floor of the bladder was next pushed up as the purse - string suture was tied .
excess vaginal epithelium was excised , and the paravaginal connective tissue and vaginal epithelium were approximated in the midline , using a delayed absorbable suture .
after the anterior colporrhaphy , the vaginal cuff was closed with three interrupted 0-dexon sutures .
ovarian ligaments , the round ligaments , and the cardinal and uterosacral ligaments were attached to the vaginal vault to support the vaginal apex .
next , two allis clamps were placed at the 4- and 8-oclock positions of the hymenal ring .
the goal of vaginal incision was to allow separation of epithelium from the underlying connective tissue .
the plane of separation was continued as far laterally as necessary , in either direction , toward the pelvic sidewall to expose the puborectalis muscle , the bulbocavernosus muscle , and the perineal membrane .
excess vaginal epithelium was excised and the puborectalis muscle , the bulbocavernosus muscle , and the vaginal epithelium approximated in the midline , using a delayed absorbable suture .
finally , the vaginal introital epithelium was approximated with an absorbable running suture , rendering the resultant genital hiatus about 1 cm in length ( figure 3 ) .
a concomitant transobturator sling operation , using a safyre t ( promedon , cordoba , argentina ) polypropylene monofilament sling ( placed through a separate vaginal incision ) was performed in women for whom the stress test was positive .
after surgery , saline - soaked gauze was packed into the vagina , and a urethral catheter inserted .
of these 79 patients , 12 did not respond to phone calls , six patients moved or died , and seven patients did not agree to participate in the study .
there was no significant difference between the study group and the nonresponders / nonagreement cohorts in terms of age , body mass index , parity , comorbidities , and stage of prolapse ( data not shown ) .
the patients mean ( sd ) age was 79.7 ( 2.6 ) years , the mean ( sd ) body mass index was 29.14 ( 3.16 ) kg / m , and the mean ( sd ) parity was 3.51 ( 1.55 ) .
intraoperative complications occurred in only one patient : this was a bladder injury that was identified and repaired intraoperatively ( table 2 ) .
three patients fitted with midurethral slings had postoperative voiding difficulties , which spontaneously resolved within 3 days .
the mean follow - up time was 24.4 months after constricting surgery ( range : 8 to 44 months ) .
the mean sds - pfd score was 4.82 , suggesting a high satisfaction ( table 3 ) .
there were four cases ( 7% ) of de novo urge incontinence ( the symptoms resolved with anticholinergic medication ) .
two patients developed de novo stress urinary incontinence after the procedure and were given midurethral sling surgery .
no anatomical ( no prolapse beyond hymen ) or subjective recurrence of the prolapse was noted during follow up .
we found that treatment of advanced prolapse using a constricting technique in elderly women is a feasible , safe , and effective surgical option that may represent an alternative to colpocleisis and other techniques that install mesh or graft material .
the complication rates were low , but the operation time and hospital stay rather long in our study .
only one intra - operative complication occurred , and no patient developed prolapse recurrence during follow up .
the low complication and high success rates are similar to those of other studies exploring the colpocleisis procedure and new sling techniques .
lu et al12 reported on 63 elderly pop patients of stages 3 or 4 who underwent total or partial colpocleisis .
similarly , zebede et al10 reported a 98.1% anatomical success rate in elderly women after lefort colpocleisis .
cho et al20 showed that the anatomical success rate was 97.1% after total mesh repair using the prolift technique to treat advanced pop in elderly women .
although the complication and success rates were similar to those of obliterative techniques , the mean operating time was longer in our study.12,20 the ultimate decision on the approach and procedure when older women require pop should be based on the overall health , physical activity status , and future sexual activity plans of the patient , as well as on surgeon training , skills , and preference .
colpocleisis is less invasive , requires a shorter operative time , and has fewer surgical risks compared with constricting procedures .
when the pelvic anatomy limits or prevents colpocleisis , or if women chose hysterectomy after being provided with detailed information , a constricting anterior and posterior colporrhaphy , with levator myorrhaphy and high perineorrhaphy , is an option in elderly patients at low anesthesiological risk .
a decision to perform ( or not ) an incontinence procedure in such patients is difficult and should be individualized .
patients should be preoperatively evaluated for urinary incontinence and bladder function because the morbidity of postoperative stress incontinence must be balanced against the possibility of urinary retention .
hysterectomy is often suggested and performed at the time of pop repair because most of the common techniques for apical prolapse repair require hysterectomy . in sslf and asc ,
uterine - sparing procedures correct apical prolapse by attaching the lower uterus or cervix to a support structure .
these techniques are not widely used because most surgeons have not been trained to perform them .
also , the common wisdom has been that retaining the uterus increases the risk of recurrent prolapse , although there are no data to support this notion .
hysterectomy eliminates future cervical or intrauterine pathology but causes pelvic neuropathy and disruption of natural support structures .
uterine - sparing surgery has less impact on sexual function and may preserve fertility . in our study group ,
we performed constricting surgery in 79 ( 39% ) of 202 patients who had no desire to be sexually active .
we believe that this high rate of sexual inactivity is attributable to culturally induced religious beliefs in our country , to the effect that sexual activity is for the young . none of our patients reported having had intercourse in the previous 1 year .
it has been reported that recurrent anterior vaginal wall prolapse after conventional repair occurs in more than 30% of cases.21 to improve outcomes , synthetic graft materials have been introduced to reinforce native tissues .
it has been reported that anterior colporrhaphy with synthetic graft reinforcement significantly reduces anatomical recurrence , but no difference in symptomatic recurrence was noted , and the mesh erosion rate was high .
we preferred to not use mesh because of the high mesh erosion rate , and we performed constricting surgery because we did not expect a high recurrence rate .
the major limitations of our study are as follows : there was no control group that underwent either standard nonconstricting repair or colpocleisis ; preoperative questionnaires were not completed ; and all patients were of american society of anesthesiologists anesthesia risk class 1 or 2.22 it may be difficult to perform this procedure in patients of higher anesthesia risk classes . however , the performance of all surgeries by the same surgical team enhanced the validity of our study .
our results indicate that constricting anterior and posterior colporrhaphy , levator myorrhaphy , and high perineorrhaphy with concurrent hysterectomy , is a feasible , safe , and effective surgical option in elderly patients at low anesthesiological risk .
the decision to perform an incontinence procedure should be individualized based on preoperative findings after prolapse reduction .
constricting procedures should not be the sole forms of surgery offered for prolapse , being a part of the pelvic surgeon s armamentarium when treatment of severe pop is required .
|
objectiveswe performed constricting anterior and posterior colporrhaphy , levator myorrhaphy , and high perineorrhaphy with concurrent hysterectomy , and investigated the intraoperative complications , and short - term outcomes of these constricting procedures in patients aged 75 years or older.methodswe searched our hospital database for cases , between january 2011 and january 2014 , of women aged over 75 years who underwent surgery for pelvic organ prolapse of stage 2 or higher , via vaginal hysterectomy , constricting anterior and posterior colporrhaphy , levator myorrhaphy , and high perineorrhaphy , with or without treatment of urinary incontinence .
all volunteers were evaluated via pelvic examination using the pelvic organ prolapse quantification system , the modified decision regret scale
pelvic floor disorders form , the satisfaction decision scale pelvic floor disorders form , and the pelvic floor distress inventory form.resultsfifty-four patients were included in the study .
the mean follow - up time was 24.4 months after constricting surgery ( range : 844 months ) . there were four cases ( 7% ) of de novo urge incontinence ( the symptoms resolved upon prescription of anticholinergic medication ) .
two patients developed de novo stress urinary incontinence after the procedure and were treated via transobturator sling surgery using safyre t polypropylene monofilament slings .
no anatomical or subjective recurrence of prolapse was noted during the follow - up period .
no patient required additional surgery for recurrence of prolapse.conclusionconstricting anterior and posterior colporrhaphy , levator myorrhaphy , and high perineorrhaphy with concurrent hysterectomy is a feasible , safe , and effective surgical option in elderly patients at low anesthesiological risk . the decision to perform an incontinence procedure
should be individualized based on preoperative findings after prolapse reduction .
|
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the introduction of martensitic shape memory ( sm ) nickel - titanium ( niti ) alloy in the manufacturing of rotary endodontic instruments has led to relative increase in their flexibility , cutting efficiency and cyclic fatigue resistance .
these alloys were popularly termed as m - wire , cm - wire ( controlled memory ) and thermal memory wire by different manufacturers .
the unique nature of the martensitic sm alloy is the presence of stable martensite phase at body temperature . as the austenite is relatively more rigid ,
they remain in the deformed state even after the withdrawal of stress due to the absence of conventional stress - induced martensitic transformation as observed in superelastic ( se ) niti alloys .
although , deformed martensitic niti instruments recover their shape when heated during sterilization , few instruments undergo permanent plastic deformation making them destined for single use .
also , separation of rotary instruments during instrumentation without warning is a major hindrance for the success of root canal treatment .
therefore , a supplementary treatment that increases the martensite content of sm alloy would be ideal to overcome the drawbacks and improve the clinical performance of these instruments . cryogenic treatment ( ct )
is a supplementary procedure of exposing metals to very low temperatures , which affects the entire cross - section , unlike purely surface treatment . based on the soaking temperature ,
the process may be deep ct ( dct ) or shallow ct ( sct ) .
dct of se niti endodontic instruments has shown improved cutting efficiency and microhardness . the two main mechanisms responsible for the improvement in steel tool properties following ct are a complete transformation of retained austenite to the martensite phase and precipitation of micro - carbide particles . despite extensive reports on the enhancement of mechanical properties of steel tools following ct ,
their impact on the microstructure of sm niti alloys is not studied so far in the literature .
therefore , the aim of this preliminary study was to investigate the role of dry ct conditions on the microstructure of martensitic sm niti alloy .
experiments were conducted on ni-50.8 wt% ti sm alloy with austenite finish temperature ( af ) of 46c in the form of sheet ( 2.2 mm thickness ) and rod ( 9 mm diameter ) of same ingot which complies with the specifications of american society for testing materials ( astm ) f2063 - 05 .
five cylindrical specimens ( 10 mm height ; 9.0 mm diameter ) and five sheet specimens ( 15 mm 15 mm square ) were sectioned from the as - received niti sheet and rod with the help of wire electrical discharge machine ( aq300 l ; sodick , shaumburg , il ) .
the protocol for dry ct and the experimental setup has been followed earlier in the previous study .
the specimens were randomly divided into four experimental groups based on the soaking temperature and time with a control group as follows : dct 24 group : 185c ; 24 h , dct 6 group : 185c ; 6 h , sct 24 group : 80c , 24 h , sct 6 group : 80c , 6 h and control ( ctrl ) group : no treatment .
the constant cooling rate and warming rate used during dry ct was 1c / min ( 3 h ) and 0.6c / min ( 6 h ) respectively .
the cross - sections of five niti cylindrical specimens ( one specimens from each group ) were polished with a standard sequence of abrasives ( carbimet paper discs , buehler : 240 and 600 grit ) and alumina paste ( alpha micropolish , buehler : 6 m , 1 m and 0.5 m particle sizes ) .
the polished surfaces were etched using an etching solution of composition hf + hno3 + h2o with a volume ratio of 1:4:5 by swabbing technique .
the microstructures of the specimens were examined with an optical microscope ( leica dm4000 m led ; leica microsystems cms gmbh , germany ) and an scanning electron microscope ( sem ) ( cx-200 ; coxem ltd . , daejeon , south korea ) .
forty - five lines per mm were used for the calculation in both vertical and horizontal direction .
the elemental composition of different zones namely , the matrix , grain boundaries , martensitic variants and precipitates was determined for the sm alloy using scanning electron microscope ( cx-200 ; coxem ltd . ,
daejeon , south korea ) coupled to energy dispersive x - ray spectrometer ( sem - eds ) .
the quantitative analysis was performed in nonstandard analysis mode employing zaf correction methods using the software .
x - ray diffraction ( xrd ) study was performed on polished sheet specimen from each group at room temperature using x - ray diffractometer ( miniflex ii - c , rigaku , tokyo , japan ) .
the xrd system was operated under the following conditions : cu - k rotating anode ( = 1.5406 ) , 40 kv accelerating voltage , 40 ma beam current , 10-100 2-theta range at 0.02 steps and 2 s acquisition time per step . a silicon standard was used for calibration of the diffractometer ( 640b silicon powder xrd spacing , standard reference material ; nist , gaithersburg , md , usa ) . the ( hkl ) planes corresponding to the peaks of austenite , martensite and precipitates were identified using the icdd database ( pdf release 2004 ; international centre for diffraction data , newton square , pa , usa ) .
the protocol for dry ct and the experimental setup has been followed earlier in the previous study .
the specimens were randomly divided into four experimental groups based on the soaking temperature and time with a control group as follows : dct 24 group : 185c ; 24 h , dct 6 group : 185c ; 6 h , sct 24 group : 80c , 24 h , sct 6 group : 80c , 6 h and control ( ctrl ) group : no treatment .
the constant cooling rate and warming rate used during dry ct was 1c / min ( 3 h ) and 0.6c / min ( 6 h ) respectively .
the cross - sections of five niti cylindrical specimens ( one specimens from each group ) were polished with a standard sequence of abrasives ( carbimet paper discs , buehler : 240 and 600 grit ) and alumina paste ( alpha micropolish , buehler : 6 m , 1 m and 0.5 m particle sizes ) .
the polished surfaces were etched using an etching solution of composition hf + hno3 + h2o with a volume ratio of 1:4:5 by swabbing technique .
the microstructures of the specimens were examined with an optical microscope ( leica dm4000 m led ; leica microsystems cms gmbh , germany ) and an scanning electron microscope ( sem ) ( cx-200 ; coxem ltd . , daejeon , south korea ) .
forty - five lines per mm were used for the calculation in both vertical and horizontal direction . the elemental composition of different zones namely , the matrix , grain boundaries , martensitic variants and precipitates was determined for the sm alloy using scanning electron microscope ( cx-200 ; coxem ltd . ,
daejeon , south korea ) coupled to energy dispersive x - ray spectrometer ( sem - eds ) .
the quantitative analysis was performed in nonstandard analysis mode employing zaf correction methods using the software .
x - ray diffraction ( xrd ) study was performed on polished sheet specimen from each group at room temperature using x - ray diffractometer ( miniflex ii - c , rigaku , tokyo , japan ) .
the xrd system was operated under the following conditions : cu - k rotating anode ( = 1.5406 ) , 40 kv accelerating voltage , 40 ma beam current , 10-100 2-theta range at 0.02 steps and 2 s acquisition time per step . a silicon standard was used for calibration of the diffractometer ( 640b silicon powder xrd spacing , standard reference material ; nist , gaithersburg , md , usa ) .
the ( hkl ) planes corresponding to the peaks of austenite , martensite and precipitates were identified using the icdd database ( pdf release 2004 ; international centre for diffraction data , newton square , pa , usa ) .
optical microscopic images [ figure 1a ] of all etched specimens showed equiaxed grains with well - defined boundaries .
the average diameter of the grains was approximately 25 m with astm grain size number of 7.7 in all the groups .
table 1 shows the volume of martensite that is observed in the images of different groups . ( a ) optical micrograph of shape memory nickeltitanium ( niti ) alloy ( black circle denotes the martensite variant of the grain ) , ( b ) scanning electron microscope micrograph ( white circle denotes the martensite variant ) ; holes ( black arrow ) and precipitates ( white arrow ) in the niti matrix are present , ( c ) eds spectrum showing the elemental composition comparison of martensite and austenite phase in all the groups the martensite variants were well appreciated under higher magnification with the sem images [ figure 1b ] .
the eds spectrum reveals the elemental composition of the niti specimen , which is nickel - rich [ figure 1c ] .
the average elemental composition of the as - received sm alloy was ni 50.8 wt% and ti 48.4 wt% with trace elements [ table 2 ] .
the regional variations in the composition revealed an increase in ni content , especially in the precipitates and twinned regions .
elemental composition of sm niti alloy at different regions x - ray diffraction pattern [ figure 2 ] of the specimens at room temperature contained the following peaks : ( 110 ) , ( 200 ) and ( 211 ) atomic planes for austenite ; ( 11 ) , ( 002 ) and ( 012 ) atomic planes for martensite ; ( 203 ) , ( 205 ) and ( 220 ) atomic planes for tini3 precipitates ; ( 220 ) and ( 042 ) atomic planes for ti2 ni3 and ti3ni4 precipitates respectively .
average intensities and full width at half - maximum height of the peaks corresponding to austenite ( 211 ) and martensite ( 002 ) were used to describe the qualitative change in crystal size and content of the sm niti alloy [ table 1 ] .
x - ray diffraction patterns for the shape memory nickel - titanium alloy at room temperature . ( a ) all the groups .
optical microscopic images [ figure 1a ] of all etched specimens showed equiaxed grains with well - defined boundaries .
the average diameter of the grains was approximately 25 m with astm grain size number of 7.7 in all the groups .
table 1 shows the volume of martensite that is observed in the images of different groups . ( a ) optical micrograph of shape memory nickeltitanium ( niti ) alloy ( black circle denotes the martensite variant of the grain ) , ( b ) scanning electron microscope micrograph ( white circle denotes the martensite variant ) ;
holes ( black arrow ) and precipitates ( white arrow ) in the niti matrix are present , ( c ) eds spectrum showing the elemental composition comparison of martensite and austenite phase in all the groups
the martensite variants were well appreciated under higher magnification with the sem images [ figure 1b ] .
the eds spectrum reveals the elemental composition of the niti specimen , which is nickel - rich [ figure 1c ] .
the average elemental composition of the as - received sm alloy was ni 50.8 wt% and ti 48.4 wt% with trace elements [ table 2 ] .
the regional variations in the composition revealed an increase in ni content , especially in the precipitates and twinned regions .
x - ray diffraction pattern [ figure 2 ] of the specimens at room temperature contained the following peaks : ( 110 ) , ( 200 ) and ( 211 ) atomic planes for austenite ; ( 11 ) , ( 002 ) and ( 012 ) atomic planes for martensite ; ( 203 ) , ( 205 ) and ( 220 ) atomic planes for tini3 precipitates ; ( 220 ) and ( 042 ) atomic planes for ti2 ni3 and ti3ni4 precipitates respectively .
average intensities and full width at half - maximum height of the peaks corresponding to austenite ( 211 ) and martensite ( 002 ) were used to describe the qualitative change in crystal size and content of the sm niti alloy [ table 1 ] .
x - ray diffraction patterns for the shape memory nickel - titanium alloy at room temperature . ( a ) all the groups .
cryogenic treatment of sm niti alloys for endodontic use appears to be a promising field of research due to the expected transformation of retained austenite to martensite as observed in stainless steel alloys .
the sm alloy regains its shape on heating due to the martensite - austenite transformation , which make the file se before cooling .
deep cryogenic processing uses temperatures around 185c because this is a minimum temperature easily obtainable with liquid nitrogen .
the purposes of selecting raw niti specimens in this study are as follows :
the intention was to assess the effect of ct alone.the influence of thermomechanical processing and manufacturing methods ( machining / twisting ) were eliminated.the dimension of the specimens were customized according to the requirement.the complicated geometry of the endodontic files was avoided .
the influence of thermomechanical processing and manufacturing methods ( machining / twisting ) were eliminated .
the complicated geometry of the endodontic files was avoided . the structural analysis of the surface and near - surface ( 50 m )
the grain size of the niti specimens obtained from optical micrographs did not show major variations between the groups .
martensitic variants were observed in specimens of all the groups including the ctrl . this could be attributed to the af temperature of the sm niti alloy which is above the room temperature .
the volume of martensite obtained from micrographs in dct 24 group was relative higher ( 4.1% ) . in our previous study
, specimens treated with dct 24 conditions had showed a significant reduction in vickers hardness and reciprocatory sliding wear resistance of sm niti alloy ( unpublished observations ) .
the conversion of retained austenite into martensite with a substantial increase in the volume of martensite phase could be the reason for such peculiar behavior of the alloy .
although the difference in volume of martensite at a given site among the experimental groups was minimal , they do not actually reflect the exact volume of the entire cross - section of the alloy . in our previous experiments ,
dsc analysis on the as - received sm niti alloy used in this study showed two - stage ( austenite r - phase martensite ) phase transformation during cooling ( unpublished observations ) , which is similar to the transformation behavior of sm niti instruments .
ti3ni4 precipitates observed in the micrographs influence the martensitic transformation behavior of the alloy by favoring the formation of r - phase and altering the ni content of the matrix . the ni content in the matrix is reduced due to the presence of ni - rich intermetallic phases , which subsequently results in the increase of af temperature and overall martensite phase transformation temperature range .
the xrd spectra corresponding to 2 range of between 35 and 80 is of special interest as most of the austenite and martensite peaks were located .
the peak intensity of martensite ( 002 ) phase and the additional martensite peaks ( 11 and 012 ) was relatively high in the xrd pattern of dct 24 specimen [ figure 2 and table 1 ] .
this could be attributed to a prominent increase in the martensite volume of the specimen as observed from the micrograph .
crystallites and subsequently the grains in the as - received niti specimens preferentially grow along the martensite ( 002 ) lattice plane following ct . the ( 110 ) peak of the austenite phase was not observed in both control and experimental groups except dct 24 .
this could be due to overlapping of ( 110 ) peak with the prominent ( 11 ) martensite peak .
the presence of martensite phase at room temperature in the niti specimens could be attributed to the composition and heat treatment of the alloy . in dct 24
specimen , the crystallite size and peak intensity of austenite was reduced , whereas they are increased for martensite .
although minor variations in values were observed in other experimental groups , they can not be attributed to the variations in the ct parameters .
it is known that the fatigue resistance and flexibility of the sm rotary instruments is remarkably increased due to the presence of proportionately higher martensite phase .
therefore , the life of the rotary endodontic instrument made out of our new experimental alloy will be extended in a great way .
furthermore , soaking period plays an important role in altering the microstructure of sm niti alloy as far as dct is concerned .
longer soaking time of 24 h provides adequate time for the transformation of retained austenite to the martensite phase .
under the limitations of this study , deep dry ct with 24 h soaking period increases the martensite content of sm niti alloy without altering the grain size . by correlating our previous experiments and the current findings , deep dry ct with 24 h soaking period
could be recommended as a supplementary method in the manufacturing in order to extend the life of sm niti endodontic instruments .
further investigation is required to evaluate the effect of ct on the fatigue properties of the bulk specimens .
|
context : recent introduction of shape memory ( sm ) nickel - titanium ( niti ) alloy into endodontics is a major breakthrough .
although the flexibility of these instruments was enhanced , fracture of rotary endodontic instruments during instrumentation is an important challenge for the operator .
implementation of supplementary manufacturing methods that would improve the fatigue life of the instrument is desirable.aim:the purpose of this study was to investigate the role of dry cryogenic treatment ( ct ) conditions on the microstructure of martensitic sm niti alloy.materials and methods : experiments were conducted on ni-51 wt% ti-49 wt% sm alloy .
five cylindrical specimens and five sheet specimens were subjected to different ct conditions : deep ct ( dct ) 24 group : 185c ; 24 h , dct 6 group : 185c ; 6 h , shallow ct ( sct ) 24 group : 80c , 24 h , sct 6 group : 80c , 6 h and control group .
microstructure of surface was observed on cylindrical specimens with an optical microscope and scanning electron microscope at different magnifications .
subsurface structure was analyzed on sheet specimens using x - ray diffraction ( xrd).results : microstructures of all sm niti specimens had equiaxed grains ( approximately 25 m ) with well - defined boundaries and precipitates .
xrd patterns of cryogenically treated specimens revealed accentuation of austenite and martensite peaks .
the volume of martensite and its crystallite size was relatively more in dct 24 specimen.conclusions:dct with 24 h soaking period increases the martensite content of the sm niti alloy without altering the grain size .
|
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earlier studies have reported that either caloric restriction or the inactivation of nutrient - dependent pathways is able to increase life extension in different eukaryotes .
the extension of lifespan in various organisms is associated with increased resistance to oxidative stress ( 1 ) .
the findings of numerous studies using various models of organisms have provided indirect evidence for the hypothesis that reactive oxygen species ( ros ) production and subsequent induction of ros defense are essential contributors to longevity ( 2 ) . as in higher eukaryotes ,
the unicellular yeast , saccharomyces cerevisiae , has been the most used yeast model in aging studies ( 3 , 4 ) .
yeast lifespan can be measured through two methods : replicative aging refers to the number of divisions a single mother cell undergoes before death , whereas chronological lifespan measures the viability of cultures at the stationary phase of the growth curve ( 5 , 6 ) .
the fission yeast , schizosaccharomyces pombe , has been recently used as a model for chronological aging studies inasmuch as it is more similar to the last common ancestor of humans and fungi ( 7 ) .
nutrient restriction increases resistance to oxidative stress , reduces macromolecular damage , and promotes lifespan in s. pombe ( 8 - 10 ) .
roux et al . ( 11 ) revealed that the s. pombe gpa2 mutant exhibits not only a short lifespan but also impaired mitochondrial regulation and high production of ros .
( 12 ) reported that calorie restriction favors oxidative metabolism , ros production , and sty1 map kinase activation and this stress pathway favors lifespan extension . in s. pombe ,
glucose , which is a primary carbon source , is detected by g protein - coupled receptors and generates a signal via the camp - dependent protein kinase a ( pka ) ( 13 , 14 ) . the glucose sensing and signaling pathways have also been found to be involved in metabolic adaptation and cellular response to diverse stress agents ( 15 , 16 ) . in s. pombe , the multistep phosphorelay system and the mitogen - activated protein kinase ( mapk ) pathway govern the transcriptional regulation in response to oxidative stress , which is generated by the accumulation of ros ( 17 ) .
mapk sty1 plays an important role in the regulation of downstream targets through triggering two transcriptional activators , atf1 and pap1 , in oxidative stress response ( 18 ) .
these transcription factors activate or induce the expression of antioxidant genes such as ctt1 ( cytoplasmic catalase ) , gpx1 ( glutathione peroxidase ) , ttr1 ( thioredoxin reductase ) , trx2 ( thioredoxin ) , ntp1 ( neutral trehalase ) , pgr1 ( glutathione reductase ) , and sod1 ( superoxide dismutase , sod ) ( 19 ) .
trehalose ( , -1 , 1-diglucose ) is a storage disaccharide and is present in particularly high concentrations in resting and stressed yeast cells ( 20 ) .
trehalose 6-phosphate ( t6p ) is synthesized in s. pombe from glucose 6-phosphate and udp - glucose by t6p synthetase , encoded by the tps1 gene ( 21 ) , and converted to trehalose by t6p phosphatase , encoded by the tpp1gene ( 22 ) .
the breakdown of trehalose to glucose is catalyzed by the enzyme neutral trehalase , encoded by the ntp1 gene ( 23 ) . generally in yeast ,
the regulation of trehalose synthesis and breakdown is done by camp - dependent phosphorylation mechanisms ( 24 ) .
it has been reported that the ntp1 expression is regulated by the pathway of protein kinase cascade activated under osmotic or oxidative stress ( 23 , 25 ) or by the binding of the transcription factors to elements such as camp - response element under thermal stress ( 26 ) . in the present study , ird5 and ird11
were used to evaluate whether or not trehalose contributes to survival under moderate oxidative stress .
in a previous study , we identified two glucose - repression - resistant mutants , namely ird5 and ird11 ( 27 ) . in the ird11 mutant , the oxidative stress response
is affected by glucose signaling in a manner different from that caused by glucose deprivation ( 28 ) .
the ird5 mutant has a lower caloric intake owing to reduced glucose consumption efficiency ( 29 ) .
the inefficient glucose uptake in ird5 might be a cause of increased oxidative stress response .
accordingly , in the present study , through trehalose accumulation measurement and colony - forming unit ( cfu ) counting , we compared the ird mutants and the wild type to clarify the possible relationship between glucose signaling , oxidative stress response , and lifespan in s. pombe .
in this study , the wild type of s. pombe
lindner liquefaciens ( 972 h ) and its relative invertase mutants ( i.e. ird5 and ird11 ) , which are resistant to glucose suppression ( 27 ) , were used . the selective medium consisted of 0.5%
yeast extract and 3% sucrose and 400 g / ml 2-deoxy - d - glucose ( 2-dog ) was developed for the ird mutants .
the strains were cultured in the ye medium containing 3% glucose ( repressed condition ) and 0.5% glucose ( glucose starvation condition ) and the ye medium containing 0.1% glucose plus 3% glycerol ( de - repressed condition ) . all the chemicals were provided by sigma - aldrich , germany .
the exponentially growing s. pombe cells ( wild type and ird5 and ird11 ) under repressed conditions were split into two tubes . in the experimental group , 2 mm hydrogen peroxide ( h2o2 ) ( sigma - aldrich , germany ) was added to the medium , resulting in a mild level of oxidative stress in the s. pombe cells ( 30 ) .
after one hour , both experimental and control cells were removed by centrifugation and washed with sterile distilled water so that their trehalose content could be measured .
chronological lifespan analyses were done on the wild type and the ird mutant cells , grown under repressed and de - repressed conditions for 21 days .
usually one day ( the exponential phase ) after starting the cultures , and this time point was designated as day 0 , measurements were started and continued via sampling at 2-day intervals ( 3rd , 5th , ... , 21st days of the study ) .
this study was performed under three conditions : repressed ; de - repressed ; and stressed conditions . from each group cells ,
samples were collected on the 1st ( the exponential phase ) , 3rd , and 5th ( stationary phase ) days of the study , and trehalose accumulation was measured for each case .
trehalose was extracted and assayed as is described by parrou and francois ( 31 ) .
the amount of the trehalose contents of the samples was measured by treatment with trehalase ( sigma - aldrich , germany ) .
the amount of the generated glucose was determined enzymatically via the glucose oxidase - peroxidase system ( god - pod assay ) using a commercial kit ( fluitest-glu , biocon , germany ) .
statistical comparisons were made using the one - way analysis of variance ( anova ) module of graphpad prism 5 .
in this study , the wild type of s. pombe
lindner liquefaciens ( 972 h ) and its relative invertase mutants ( i.e. ird5 and ird11 ) , which are resistant to glucose suppression ( 27 ) , were used . the selective medium consisted of 0.5%
yeast extract and 3% sucrose and 400 g / ml 2-deoxy - d - glucose ( 2-dog ) was developed for the ird mutants .
the strains were cultured in the ye medium containing 3% glucose ( repressed condition ) and 0.5% glucose ( glucose starvation condition ) and the ye medium containing 0.1% glucose plus 3% glycerol ( de - repressed condition ) . all the chemicals were provided by sigma - aldrich , germany .
the exponentially growing s. pombe cells ( wild type and ird5 and ird11 ) under repressed conditions were split into two tubes . in the experimental group , 2 mm hydrogen peroxide ( h2o2 ) ( sigma - aldrich , germany ) was added to the medium , resulting in a mild level of oxidative stress in the s. pombe cells ( 30 ) .
after one hour , both experimental and control cells were removed by centrifugation and washed with sterile distilled water so that their trehalose content could be measured .
chronological lifespan analyses were done on the wild type and the ird mutant cells , grown under repressed and de - repressed conditions for 21 days . usually one day ( the exponential phase ) after starting the cultures , and this time point
was designated as day 0 , measurements were started and continued via sampling at 2-day intervals ( 3rd , 5th , ... , 21st days of the study ) .
this study was performed under three conditions : repressed ; de - repressed ; and stressed conditions . from each group cells ,
samples were collected on the 1st ( the exponential phase ) , 3rd , and 5th ( stationary phase ) days of the study , and trehalose accumulation was measured for each case .
trehalose was extracted and assayed as is described by parrou and francois ( 31 ) .
the amount of the trehalose contents of the samples was measured by treatment with trehalase ( sigma - aldrich , germany ) .
the amount of the generated glucose was determined enzymatically via the glucose oxidase - peroxidase system ( god - pod assay ) using a commercial kit ( fluitest-glu , biocon , germany ) .
statistical comparisons were made using the one - way analysis of variance ( anova ) module of graphpad prism 5 .
of the strains under study , ird5 showed the most longevity in both repressed and de - repressed conditions .
the ird11 mutant had a lower lifespan extension than the wild type under repressed condition , while it had a higher lifespan extension than the wild type under de - repressed condition . on the other hand ,
the lifespan extension of ird11 grown in de - repressed condition was similar to that of ird5 grown in repressed or de - repressed condition ( figure 1 ) .
a , grown in repressed condition ( ye-3% glucose ) ; b , grown in de - repressed condition ( ye-0.1% glucose plus 3% glycerol ) ; ( wt : wild type , r : repressed , dr : de - repressed condition ) . to comprehend the relationship between trehalose concentrations in the cells grown in repressed , de - repressed , and stressed conditions , the level of trehalose was estimated in the wild type and the mutant cells .
the intracellular trehalose concentrations during the period of growth under repressed , de - repressed , and stressed conditions relative to the wild type and mutant cells are shown in figure 2 a , figure 2 b , and figure 2 c , respectively .
the results obtained from trehalose content measurement in the three conditions were evaluated in two ways .
first , each group was evaluated individually and the trehalose contents of the groups on the 3rd and 5th days of the study were compared with those of the 1st day .
second , the trehalose content of each group was compared with that of the wild type . a , grown in repressed condition ( ye-3% glucose ) ; b , grown in repressed condition ( ye-0.1% glucose plus 3% glycerol ) ; c , grown in stressed condition ( 2 mm h2o2 , one hour ) .
the trehalose contents are expressed as mg glucose per 0.2 g of wet mass of the cells .
statistical significance was evaluated by one - way anova ( p < 0.05 - 0,001 ) .
( wt : wild type , r : repressed , dr : de - repressed , hp : oxidative stress conditions ) .
* , * * , * * * : each group was evaluated individually and the trehalose contents of the groups on the 3rd and 5th days of the study were compared with those of the 1st day . + , + + , + + + : trehalose content of each group is compared with that of the wild type .
when compared independently , as is depicted in figure 2 a , except for ird5 under repressed condition , the trehalose content was increased remarkably on the 3rd day of the study .
however , a comparison with the wild type showed a sharp decrease in the trehalose level in the ird mutant . as is evident from figure 2 b , under de - repressed condition
, the level of intracellular trehalose was notably increased in the stationary phase ( 3rd day ) , while no significant changes were detected in either ird5 or in ird11 . under stressed condition
( figure 2 c ) , during the stationary phase ( 3rd day ) , the trehalose level in ird11 was increased as a pattern similar to that observed in the wild type ; nevertheless , only trace amounts of alteration were seen in ird5 .
finally , concerning the wild type during the stationary growth phase , a dramatic decrease was observed in the trehalose content in ird5 ( 3rd and 5th days ) and ird11 ( 3rd day ) .
of the strains under study , ird5 showed the most longevity in both repressed and de - repressed conditions .
the ird11 mutant had a lower lifespan extension than the wild type under repressed condition , while it had a higher lifespan extension than the wild type under de - repressed condition . on the other hand ,
the lifespan extension of ird11 grown in de - repressed condition was similar to that of ird5 grown in repressed or de - repressed condition ( figure 1 ) .
a , grown in repressed condition ( ye-3% glucose ) ; b , grown in de - repressed condition ( ye-0.1% glucose plus 3% glycerol ) ; ( wt : wild type , r : repressed , dr : de - repressed condition ) .
to comprehend the relationship between trehalose concentrations in the cells grown in repressed , de - repressed , and stressed conditions , the level of trehalose was estimated in the wild type and the mutant cells .
the intracellular trehalose concentrations during the period of growth under repressed , de - repressed , and stressed conditions relative to the wild type and mutant cells are shown in figure 2 a , figure 2 b , and figure 2 c , respectively .
the results obtained from trehalose content measurement in the three conditions were evaluated in two ways .
first , each group was evaluated individually and the trehalose contents of the groups on the 3rd and 5th days of the study were compared with those of the 1st day .
second , the trehalose content of each group was compared with that of the wild type . a , grown in repressed condition ( ye-3% glucose ) ; b , grown in repressed condition ( ye-0.1% glucose plus 3% glycerol ) ; c , grown in stressed condition ( 2 mm h2o2 , one hour ) .
the trehalose contents are expressed as mg glucose per 0.2 g of wet mass of the cells .
statistical significance was evaluated by one - way anova ( p < 0.05 - 0,001 ) .
( wt : wild type , r : repressed , dr : de - repressed , hp : oxidative stress conditions ) .
* , * * , * * * : each group was evaluated individually and the trehalose contents of the groups on the 3rd and 5th days of the study were compared with those of the 1st day . + , + + , + + + : trehalose content of each group is compared with that of the wild type .
when compared independently , as is depicted in figure 2 a , except for ird5 under repressed condition , the trehalose content was increased remarkably on the 3rd day of the study . however , a comparison with the wild type showed a sharp decrease in the trehalose level in the ird mutant . as is evident from figure 2 b , under de - repressed condition , the level of intracellular trehalose was notably increased in the stationary phase ( 3rd day ) , while no significant changes were detected in either ird5 or in ird11 . under stressed condition
( figure 2 c ) , during the stationary phase ( 3rd day ) , the trehalose level in ird11 was increased as a pattern similar to that observed in the wild type ; nevertheless , only trace amounts of alteration were seen in ird5 .
finally , concerning the wild type during the stationary growth phase , a dramatic decrease was observed in the trehalose content in ird5 ( 3rd and 5th days ) and ird11 ( 3rd day ) .
the pka activated by glucose starvation and stress - activated mapk pathways regulate the transcription of downstream genes via protein - dna interactions at uas1 and uas2 of the fbp1 gene ( 32 ) .
the existence of cross - talking between sty1p and pka1p regulates not only glucose repression and oxidative stress response pathways but also trehalose accumulation .
( 25 ) reported that the expression of the ntp1 and tps1 genes in s. pombe is partially regulated by the sty1p kinase under salt - induced osmotic stress and conditions of slight oxidative stress and is fully dependent on this kinase under severe oxidative stress .
another study showed that cell viability may depend on capacity to rapidly degrade the trehalose that is accumulated during oxidative stress .
the authors indicated that glutathione reductase can be inhibited by trehalose in a dose - dependent manner . on the other hand , sanchez - fresneda et al .
( 33 ) reported that the stress - induced trehalose accumulation is hog1-independent in candida albicans .
the extension of lifespan in s. pombe is associated with both calorie restriction and increased resistance to oxidative stress ( 10 - 12 ) .
( 34 ) , our results suggest that lifespan extension in s. pombe may not be associated with oxidative stress resistance and trehalose accumulation .
the oxidative stress - resistant mutant ird11 , which is affected by glucose signaling in a manner different from that caused by glucose deprivation ( 28 ) , had a lower lifespan extension than the wild type under repressed condition and had a higher lifespan extension than the wild type under de - repressed condition .
indeed , the lifespan extension of ird11 grown in de - repressed condition was similar to that of ird5 grown in repressed or de - repressed condition .
we suggest that the longevity of the ird5 mutant grown in both repressed and de - repressed conditions might be , in part , in consequence of glucose depletion condition caused by a reduced glucose consumption rate in these cells compared to the ird11 mutant and the wild type .
because , contrary to ird11 , there is an adaptive response to oxidative stress in ird5 , caused by glucose sensing / signaling which emerges in glucose starvation ( 29 ) . in light of the results of the present study
, it seems that lifespan extension is mostly related to glucose sensing / signaling rather than oxidative stress response and trehalose accumulation .
|
background : glucose is the preferred carbon and energy source in most organisms and plays an active role in the regulation of many biological processes . however , an excess of glucose leads to such undesirable conditions as diabetes and age - related diseases . since schizosaccharomyces pombe homologous of many human genes , it offers several advantages for the investigation of the molecular mechanisms underlying human disease and aging studies .
we have identified two glucose - repression - resistant mutants ( ird5 and ird11 ) of s. pombe.objectives:we aimed to investigate the possible relationship between lifespan extension and oxidative stress response induced by exposure to hydrogen peroxide alongside the trehalose accumulation level by using the two s. pombe mutants ( i.e. ird5 and ird11 ) , which are repressed by glucose and are resistant to oxidative stress.materials and methods : we employed trehalose accumulation measurement and colony - forming unit ( cfu ) counting using the ird mutants in exponential and stationary phases and compared them to the wild type grown in repressed , de - repressed , and stressed conditions to clarify the possible relationship between glucose signaling , oxidative stress response , and lifespan in s. pombe.results:the lifespan of the ird5 mutant was significantly longer that of either the ird11 mutant or the wild type cells . under repressed condition
, the trehalose content was increased remarkably on the 3rd day of the study in the ird11 mutant and the wild type . under de - repressed condition
, the level of intracellular trehalose was notably increased on the 3rd day in ird11 . under stressed condition , the trehalose level in ird11
was increased on the 3rd day as a pattern similar to that observed in the wild type.conclusions:our results demonstrated no significant correlation between the ird5 lifespan and the trehalose concentration .
likewise , the correlation between lifespan extension , trehalose accumulation , and cellular resistance to hydrogen peroxide was not significant .
|
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acute pancreatitis is an inflammatory intra - abdominal process , which in approximately 1520% of patients presents in a severe form , with a gradual establishment of multiple organ dysfunction or local complications , including necrosis , pseudocyst , and abscess .
severe acute pancreatitis is a condition associated with high mortality , which is characterized by a complex and incompletely understood pathophysiological mechanism [ 2 , 3 ] .
the deficit in our understanding of the mechanism driving the inflammatory process in acute pancreatitis is a reason why our therapeutic strategy has failed to reduce mortality , despite ongoing research .
when acute pancreatitis leads to the establishment of acute kidney injury , there is a 5- to 10-fold rise in mortality , which can reach 70% [ 46 ] .
the prevention of acute kidney injury can be a useful strategy in the prevention of the morbidity and mortality associated with acute pancreatitis .
eugenol ( 1-allyl-4-hydroxy-3-methoxybenzene ) is a naturally occurring substance , found in the essential oil of commonly consumed spices such as clove oil as well as cinnamon , basil , and nutmeg oils .
it has many pharmacological properties which are mainly analgesic , anti - inflammatory , antioxidant , and vasodilatory action , while it has been shown to ameliorate kidney injury in a model of gentamycin - induced nephrotoxicity .
the aim of this study is to assess the possible reduction in the extent of acute kidney injury after administration of eugenol in an experimental model of acute pancreatitis .
106 male wistar rats , aged 3 - 4 months and weighing 220350 gr , were used in this study .
they were housed in cages under standard laboratory conditions ( 12 hr light - dark cycles , 2225c room temperature , and 5558% humidity ) , with free access to food and water . the animals were procured from the hellenic pasteur institute ( athens , greece ) .
the experiment took place at the elpen experimental research center ( pikermi , greece ) , while the histological analysis was carried out at the lab of histology , embryology , medical school , democritus university of thrace .
the experimental surgical procedures and the general handling of the animals conformed to the international guidelines of directive 86/609/eec on the protection of animals used for experimental and other scientific purposes .
the animals were randomly assigned in 3 groups : sham ( n = 20 ) , control ( n = 46 ) , and eugenol ( n = 40 ) .
the animals were anaesthetized initially by being placed in a glass box containing isoflurane and then through administration of 0.25 ml of butorphanol ( dolorex ; intervet / schering / plough animal health , boxmeer , holland ) by subcutaneous injection .
the animals were intubated with a 16 g venous catheter , which was then connected to a ventilator set at 70 breaths / min and a tidal volume of 3 ml .
after confirmation of the success of intubation , anaesthesia was maintained by a mixture of 93% o2 , 5% co2 , and 2% isoflurane .
briefly , after induction of anaesthesia and preparation of the surgical site , the abdomen was entered via a 3 cm midline incision under sterile conditions .
the biliopancreatic duct was identified and ligated near the duodenal wall with a 4 - 0 silk sutures ( in the control and eugenol groups , but not in the sham group )
. 1 ml of normal saline and 1 ml of 5% d5w were instilled in the abdominal cavity .
the abdomen was closed with vicryl 2 - 0 sutures . in the eugenol group ,
eugenol was administered by a nasogastric catheter in a dose of 15 mg / kg , while the sham and control groups received corn oil solution without eugenol .
postoperatively , analgesia was maintained through subcutaneous administration of 2 ml / kg butorphanol ( dolorex ; intervet / schering / plough animal health , boxmeer , holland ) .
euthanasia was performed at a predetermined time for each animal with the use of ketamine ( narcetan ; vetoquinol , buckingham , uk ) 0.30.6 ml and xylazine ( rompun ; bayer , uxbridge , uk ) 0.10.3 ml , followed by a midline laparotomy and exsanguination of the abdominal aorta .
time points for analysis were 6 , 12 , 24 , 48 , and 72 hours postoperatively .
serum samples for measurement of urea and creatinine as well as specimens from both kidneys for histopathological examination were acquired .
louis , mo , usa ) was purchased and prepared in an oily solution in the chemical laboratory of elpen pharmaceutical co. inc .
this was achieved with the admixture of pure eugenol in a corn oil solution in a concentration of 1.5 mg eugenol / ml .
samples were placed in 10% buffered formalin solution , and 4 m paraffin - embedded sections were stained with hematoxylin / eosin .
all specimens were evaluated by a pathologist blinded to the sequence of the biopsy specimens .
slides were evaluated with regard to 5 histopathological parameters and with the use of a semiquantitative scoring system as depicted on table 1 .
the scores of each individual parameter for each slide were added and a histopathological score was obtained for each specimen .
immunohistochemical staining was applied to detect the possible expression of inflammatory cytokines like il-6 , tnf- , and myeloperoxidase .
the following antibodies were used : myeloperoxidase ( rabbit polyclonal ) , dako ( a 0398 ) , diluted 1 : 400 tnf- ( rabbit polyclonal ) , abnova ( pab8016 ) , diluted 1 : 1000 , il-6 ( rabbit polyclonal ) , and abcam ( ab6672 ) , diluted 1 : 500 . the buffers , blocking solutions , secondary antibodies , avidin - biotin complex reagents , and chromogen were supplied in a detection kit ( envision hrp , mouse / rabbit detection system ( k 5007 ) , dako ) .
to inhibit endogenous peroxidase , the specimens were incubated with 3% h2o2 ( 200 ml h2o and 6 ml h2o2 ) for 15 min in a dark room .
before the primary antibody was applied , the sections were immersed in 10 mm citrate buffer ( ph 6.0 ) , rinsed in tris - buffered saline , and subsequently heated in a microwave oven ( 650800 w ) for three cycles of 5 min .
the slides were washed with tris - buffered saline before application of the primary antibody in order to reduce nonspecific binding of antisera .
sections were then briefly counterstained with mayer 's hematoxylin , mounted , and examined under a nikon eclipse 50i microscope ( nikon instruments inc , ny , usa ) .
the average labeling index was assessed according to the proportion of positive cells , after scanning the entire section of the specimen .
the results were graded as negative ( 0 ) for < 10% of stained cells , low ( 1 ) for > 10% and < 30% of cells stained , moderate ( 2 ) for > 30% and < 70% cells stained , and high expression ( 3 ) for > 70% cells stained ( table 1 ) .
the statistical analysis of the results was completed with the use of the 20th version of spss ( statistical package for the social sciences , spss inc . ,
we performed an analysis in which the data were treated as qualitative using fisher 's exact test ( this test was preferable to x
because of the small number of animals in each subcategory / time point ) .
evaluation of the different variables was performed to determine whether they were normally distributed ( kolmogorov - smirnov are shapiro - wilk ) .
the three different groups were then analyzed using the kruskal - wallis one - way analysis of variance test .
finally , the mann - whitney u test was further used to compare the groups in pairs .
these tests were applied to the overall sample and for each individual subgroup corresponding to individual time points ( 6 , 12 , 24 , 48 , and 72 hours postoperatively ) .
the difference between the eugenol and control groups is apparent at 48 and 72 hours after induction of pancreatitis ( figures 1 and 2 ) .
the histological score for these two groups is higher compared to the sham group at 48 and 72 hours and for the whole sample .
eugenol administration lowers hyperemia and dilation of renal parenchyma capillaries and the difference was statistically significant for the 48 and 72 hour time points and for the whole sample .
the eugenol group exhibited lower values than the control group and both exhibited higher values than the sham group .
the same was true for hyperemia and dilation of renal corpuscles capillaries for the 48- and 72-hour time points , but not for the whole sample .
there were no inflammatory infiltrations in any of the animals in our experimental model and measurement of this factor did not produce any results .
edema was reduced through the administration of eugenol and , again , the difference to the control group was significant for the 48- and 72-hour time points and the whole sample .
the control group had higher values than the sham group at 48 hours and also higher values than both the sham and eugenol groups at 72 hours .
when values of the whole sample were considered , the control group had higher values than the eugenol group , which in turn had higher values than the sham group .
analysis of the whole sample showed only higher values for the eugenol and control groups when compared to the sham group .
there was no clear difference regarding il-6 expression between the different groups ( figures 3 and 4 ) . on the contrary
there was a statistically significant difference between the eugenol and control groups 72 hours after induction of pancreatitis , while both groups exhibit higher tnf- expression than the sham group .
there was no statistically significant difference between the eugenol and control groups for mpo expression , although there was a trend toward higher expression for the control group after 72 hours .
eugenol administration resulted in lower serum levels of urea and creatinine especially at the 48- and 72-hour time points , compared to the control group .
urea and creatinine levels were higher for both the eugenol and control groups , when they were compared to the sham group ( figure 4 ) .
the results of this study suggest that eugenol attenuates the intensity of the histopathological changes and the expression of tnf- and mpo in the renal parenchyma , while lowering the values of serum urea and creatinine when administered in a rat acute pancreatitis experimental model .
to evaluate the extent of kidney injury , we decided to evaluate serum urea and creatinine levels and the histopathological changes in the kidney , as well as the expression of tnf- , il-6 , and mpo in the renal parenchyma .
the role of cytokines , such as tnf- and il-6 , in the pathophysiology of acute pancreatitis has been studied extensively and they have been found to contribute to the activation of the systematic inflammatory response process and multiorgan failure , which is a hallmark of severe acute pancreatitis and is , ultimately , correlated with the observed high mortality rates [ 10 , 11 ] .
the role of cytokines in acute kidney injury has been found to be equally important .
the cytokine - mediated inflammatory response has a central role in the pathophysiology of acute renal failure irrespective of its cause .
mpo has been used as a marker of neutrophil migration in acute pancreatitis studies and has been correlated to the severity of kidney injury [ 1214 ] .
the histopathological evaluation showed that the histologic score was lower for the eugenol group in comparison to the control group at 48 and 72 hours from the initiation of the inflammatory process ( means : 3.75/6.5 and 4.12/7.62 , resp . ) and this difference was statistically significant .
this difference between the two groups was also present for individual histological changes such as hyperemia and dilation of renal parenchyma and renal corpuscles capillaries and edema .
the difference observed in the degree of acute tubular necrosis and inflammatory infiltration was not statistically significant . regarding the expression of inflammatory mediators , tnf- levels were higher for the control group in comparison to the eugenol group with the difference reaching statistical significance at the 72-hour time point ,
while there was a trend for higher mpo expression in the control group at 72 hours , which was , however , not statistically significant .
in contrast , il-6 levels did not show the same correlation and there were no statistically significant differences between the eugenol and control groups .
we chose the bile - pancreatic duct ligation model as it is a well - characterized model of acute pancreatitis , which mimics acute pancreatitis caused by biliary obstruction , which is a frequent clinical scenario and results in multiorgan failure similar to that observed in humans [ 15 , 16 ] .
we have previously used this experimental model and we were able to show that it generates acute pancreatitis with histopathological changes in the pancreatic tissue including hemorrhage and necrosis .
out of a total of 106 animals , 6 died and the fact that they were all in the control group could be seen as further evidence supporting the protective role of eugenol .
it is possible that these animals would have exhibited signs of severe kidney injury , if they had survived until the predetermined time of euthanasia .
however , since the distal bile - pancreatic duct ligation model is not usually fatal , we can not directly attribute the death of these animals to the severity of acute pancreatitis .
eugenol has been shown to possess a multitude of pharmacological effects , some of which make it a likely candidate for use in the setting of acute pancreatitis and can explain the results observed in our study . the analgesic action of eugenol has been well documented and doses in the range of 40100 mg / kg have been shown to be effective in rat experimental models [ 1820 ] .
in addition , eugenol acts as an anti - inflammatory substance inhibiting cyclooxygenase and reducing the release of proinflammatory mediators such as il-1 , tnf- , and pge2 [ 2224 ] .
the antioxidative potential of eugenol has been studied in a number of , mainly in vitro , studies where it has been shown to bind to free oxygen radicals and attenuate the action of oxidative substances [ 2528 ] , while a recent study of gentamycin - induced nephrotoxicity offers insight into how eugenol can prevent kidney injury by reducing oxidative damage .
these combined properties of eugenol can be used to explain the observed reduction in tnf- expression , as well as the reduction of kidney inflammation .
eugenol administration causes a dose - dependent , reversible vasodilation through its effect on the endothelial cells [ 29 , 30 ] , which is comparable to nifedipine .
the potential of eugenol to inhibit the vasoconstriction that is associated with kidney injury points to another potential mechanism for its effect in the model of acute pancreatitis .
a number of authors have proposed strategies to reduce kidney injury caused by acute pancreatitis .
zhang et al . have tried dexamethasone administration in an experimental model of retrograde injection of sodium taurocholate in the pancreatic duct .
the dexamethasone group exhibited milder congestion of the glomerular capillary , swelling of the renal tubular epithelial cells , and less inflammatory cell infiltration than that of the control group , which was shown by the lower histological score at the 6- and 12-hour time points .
the same authors found a significant difference in the serum levels of tnf- in favor of the dexamethasone group , while expression of nf-b in the renal tissue was more pronounced in the dexamethasone group .
the same model has been used to study octreotide and baicalin ( 5,6,7-trihydroxyflavone-7-o - d - glucuronic acid ) .
the administration of these substances had a protective effect on the kidney and both the histological score and renal parenchyma nf-b expression were lower in comparison to the control group .
il-6 were reduced compared to the control group in another study with the same experimental protocol .
there have been a number of studies of plant derived substances , used in traditional chinese medicine .
ligustrazine proved to be protective for the kidney as was demonstrated by the lower creatinine levels and the milder histopathological changes in comparison to the control group . in another study ,
the administration of 3 traditional chinese medicine substances ( ligustrazine , kakonein , and panax notoginsenosides ) resulted in reduced mortality and milder histopathological changes in the rat kidney . finally , the model of induction of acute pancreatitis through sodium taurocholate administration was used for the study of poly(adp - ribose ) polymerase inhibition , through 3-aminobenzamide ( 3-ab ) administration .
the administration of 3-ab resulted in reduced mortality and a reduction in the increase of creatinine , tnf- , il-1b , and il-6 , milder histopathological changes , and reduced mpo expression in the kidney .
the half life of eugenol in the rat has been determined to be 18,3 hours ; therefore , at 72 hours , most of the initial dose would have been cleared from the circulation .
it is possible that a repeat administration of eugenol could further increase the therapeutic result .
moreover , the time frame of our protocol reached 72 hours , which was not adequate for the complete evaluation of the effect of eugenol .
indeed , a difference in the extent of kidney injury between the eugenol and control groups is first observed 48 hours after the onset of acute pancreatitis and it is greater at 72 hours .
in conclusion , the administration of eugenol in a rat model of acute pancreatitis was protective for the kidneys in our experimental model .
further research is necessary to determine the possible role of eugenol in the management of acute pancreatitis .
|
aim .
acute pancreatitis is an inflammatory intra - abdominal disease , which takes a severe form in 1520% of patients and can result in high mortality especially when complicated by acute renal failure .
the aim of this study is to assess the possible reduction in the extent of acute kidney injury after administration of eugenol in an experimental model of acute pancreatitis .
materials and methods .
106 male wistar rats weighing 220350 g were divided into 3 groups : ( 1 ) sham , with sham surgery ; ( 2 ) control , with induction of acute pancreatitis , through ligation of the biliopancreatic duct ; and ( 3 ) eugenol , with induction of acute pancreatitis and eugenol administration at a dose of 15 mg / kg . serum urea and creatinine , histopathological changes , tnf- , il-6 , and mpo activity in the kidneys were evaluated at predetermined time intervals .
results .
the group that was administered eugenol showed milder histopathological changes than the control group , tnf- activity was milder in the eugenol group , and there was no difference in activity for mpo and il-6 .
serum urea and creatinine levels were lower in the eugenol group than in the control group . conclusions .
eugenol administration was protective for the kidneys in an experimental model of acute pancreatitis in rats .
|
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] |
over the last few decades , a dramatic worldwide increase in infection rates by multidrug - resistant ( mdr ) pathogens has occurred , which is acknowledged as a public health crisis .
management of infections caused by these pathogens is often difficult due to the scarcity of available active drugs .
the last report of the european antimicrobial resistance surveillance system ( earss ) network , which includes 30 european countries , describes a general european - wide increase in antimicrobial resistance for the gram - negative pathogens under surveillance ( escherichia coli , klebsiella pneumoniae and pseudomonas aeruginosa ) .
high proportions of antimicrobial - resistant p. aeruginosa have been reported by many european countries . in a study performed in 2000 in spain , 41% of acinetobacter baumannii
indeed , the rate of carbapenem resistance has increased dramatically over the last decade , especially in the critical care setting .
an ominous emerging threat is the appearance of gram - negative microorganisms harboring new beta - lactamases that confer high - level resistance to all available classes of beta - lactam antibiotics . concerning gram - positive bacteria , methicillin - resistant staphylococcus aureus ( mrsa ) and enterococcus spp .
the incidence of mrsa infections seems to have remained stable over recent years , although this pathogen causes severe infections .
the issue of increasing incidence of mdr is clearly more complex in intensive care units ( icus ) , where selection pressure and emergence of resistance , as well as the risk of patient - to - patient transmission , are highest .
the spanish annual april - to - june icu national nosocomial infection surveillance study ( estudio nacional de vigilancia de infeccin nosocomial , [ envin ] ) confirms that multi - drug resistance is an unresolved problem in spanish intensive care , with worrisome rates of gram - negative mdr pathogens .
in addition , mdr microorganisms often do not cause true infection , but only colonization , constituting a hidden reservoir for the spread of these pathogens .
the prognosis of patients who develop nosocomial infection in the icu is poor , especially if an mdr pathogen is involved .
mortality rates and economic burden are significantly higher in infections caused by mdr pathogens , than in those caused by susceptible organisms . moreover ,
even the acquisition of an mdr pathogen , without concomitant infection , is associated with an increased risk of death , length of hospitalization , and cost .
the spanish society of intensive care medicine and coronary care units ( semicyuc ) and the spanish society of intensive care nursing ( seeiuc ) have recently completed their role as technical lead for two programs aimed at reducing icu - acquired infections , namely catheter - related bloodstream infections [ zero bacteremia ] and ventilator - associated pneumonia ( vap ) [ zero vap ] .
both projects were developed within a framework of zero tolerance . zero bacteremia and zero vap were both promoted by the spanish ministry of health , more than 200 icus participated , and the programs consisted of the implementation of evidence - based infection prevention bundles for catheter - related bloodstream infection and vap .
highly successful results for both initiatives confirmed that these practices could be systematically implemented across spain , could reduce the rates of these infections and could contribute to diminish antimicrobial use in the participating icus .
with the experience gained in the two previous projects , a new project named zero resistance was developed by the semicyuc with the support of the spanish ministry of health .
this project uses the same structure created for zero bacteremia and zero vap , which is based on coordination at national , regional and local levels .
a scientific expert committee ( sec ) for the development and implementation of this program was appointed as follows : semicyuc nominated nine intensivists chosen for their expertise in the field of prevention and management of infections in the critical care setting and seeiuc designated an intensive care nurse with experience in infection control .
a microbiologist , an epidemiologist , an infectious diseases specialist , and two technicians from the ministry of health with broad knowledge in the field were also incorporated .
the members of the sec reviewed the available evidence in pubmed indexed papers , including observational studies , clinical trials , guidelines , systematic reviews and meta - analyses .
the following databases were searched : medline , embase , the cochrane library , and centre for reviews and dissemination , including the national health service economic evaluation database and the health technology assessment database .
the implementation of bundles of effective measures , compared to individual interventions , has been proposed to reduce the incidence of catheter - related bloodstream infections or vap . with this concept in mind ,
the sec developed a bundle of 10 recommendations that was discussed and approved after review and analysis of the existing scientific literature .
admittedly , the evidence supporting some of the chosen recommendations is weak , but all were deemed to reach at least the level of
expert recommendation. no grading system was used to support the strength and quality of recommendations .
criteria for defining mdr pathogens vary from institution to institution and are also not uniform in the published literature , although the most highly resistant strains are readily recognizable . based on the pathogens considered most problematic in spanish icus ,
zero resistance collects information on episodes of infection and colonization of the pathogens listed in table 1 . finally ,
because acquiring an infection may be the result of errors in patient - care , all three programs were designed to reduce and prevent these by incorporating an integral patient safety program .table 1
definitions of multidrug - resistant bacteria monitored in the
zero resistance
program
microorganism
resistance marker
gram - positive
staphylococcus aureus
methicillin ( mrsa )
enterococcus spp.vancomycin ( vre)gram - negative
enterobacteriaceae
3rd generation cephalosporins ( particularly esbl - producing)carbapenems ( particularly carbapenemase - producing )
pseudomonas aeruginosa
3 antibiotic classes , including carbapenems , cephalosporins , piperacillin - tazobactam , flouroquinolones , aminoglycosides and colistin
acinetobacter baumannii
carbapenems
imipenem , meropenem or doripenem ; ceftazidime or cefepime ; ciprofloxacin or levofloxacin ; gentamicin , tobramycin or amikacin .
esbl : extended spectrum beta - lactamase ; mrsa : methicillin - resistant staphylococcus aureus ; vre : vancomycin - resistant enterococcus .
definitions of multidrug - resistant bacteria monitored in the
zero resistance
program
imipenem , meropenem or doripenem ; ceftazidime or cefepime ; ciprofloxacin or levofloxacin ; gentamicin , tobramycin or amikacin .
esbl : extended spectrum beta - lactamase ; mrsa : methicillin - resistant staphylococcus aureus ; vre : vancomycin - resistant enterococcus . the main objective of the zero resistance
project is reduction in the cumulative incidence of patients with icu - acquired mdr infections by 20% .
secondary objectives are to study the epidemiology of mdr infections in spanish icus , to be able to distinguish imported from icu - acquired cases , to promote and strengthen safety assurance in participating units , and to create a network of icus implementing safe , and evidence - based practices .
the primary aim of the bundle recommendations is reduction of the three most influential factors contributing to the development and transmission of mdr , namely : 1 ) adequate prescription of antibiotics ; 2 ) early detection and prevention of cross - colonization of mdr ; and 3 ) elimination of reservoirs .first recommendation : in each icu , at least one intensivist will be designated as responsible for the use of antimicrobials .
he / she should have extensive experience in infection control and in the treatment of severe infections .
analysis of antimicrobial use should include : review of the indication for antimicrobials , evaluation of the appropriateness of the antimicrobial and the correct administration ( dosing , intervals and duration),evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation.rationale : antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge .
moreover , many pathophysiological changes associated with severe acute illness or sepsis , like capillary leak , third spacing , increased volume of distribution , and impaired renal and/or liver function , affect antimicrobial pharmacokinetics / pharmacodynamics .
therefore , it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality .
this implies choosing optimal empirical antibiotics , appropriate mode of administration , and correct dosage .
administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations , which are associated with treatment failure and resistance development .prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock .
however , as soon as microbiological information is available , empiric therapy should be adapted , if appropriate , by either reduction in number and/or narrowing of antimicrobial spectrum .
in fact , de - escalation of empirical therapy is performed in less than 50% of patients .
recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression .second recommendation : empirically administer antimicrobials active against mdr pathogens only in cases of severe sepsis or septic shock and high risk of mdr pathogen(s ) based on patient risk factors and/or knowledge of local ecology . otherwise , narrow - spectrum or withholding of antimicrobials is recommended until microbiological results become available and targeted therapy with antibiotics active against mdr pathogens ( carbapenems , colistin , tigecycline , glycopeptides , daptomycin , linezolid ) should be started if needed . in all cases , samples for culture of the potential sources of infection
should be obtained before starting antibiotic therapy.rationale : early and adequate antimicrobial therapy is associated with increased survival in patients with severe sepsis and septic shock .
however , delaying antimicrobial therapy until microbiological confirmation is available has been shown to be associated with similar outcomes in febrile surgical icu patients compared to starting antimicrobials immediately after the clinical diagnosis of infection .
more recently , a quasi - experimental , before - after observational cohort study concluded that , after adjusting for confounders , aggressive antimicrobial therapy was an independent predictor of mortality . in the aggressive period , antimicrobial treatment was always started in patients suspected of having an infection after appropriate cultures were obtained . in the second period ( conservative strategy ) ,
antimicrobial treatment was started only after objective findings confirmed the infection .the main limitation of both studies is that they were carried out in surgical patients and data from medical units are lacking .
however , it is important to keep in mind that in febrile patients with severe sepsis or septic shock a delay in antimicrobial therapy may be fatal .
in addition , the choice of empirical antimicrobial therapy should be based on an updated knowledge of the local ecology .
therefore , it seems prudent to recommend starting empiric antimicrobials active against mdr pathogens immediately only in cases meeting criteria for severe sepsis or septic shock and risk factors for mdr pathogens .
obviously , efforts to reduce the delay of microbiological results ( use of rapid diagnostic techniques , direct contact with the microbiologist ) and close follow - up of the clinical course to rapidly detect signs of alarm are fully endorsed.third recommendation : in each unit , at least one nurse will be designated as leader of this project and responsible for infection control measures aimed at reducing transmission of mdr pathogens.rationale : success of quality control programs is particularly dependent on the involvement of all categories of healthcare professionals .
nurses play a critical role in preventing and controlling infectious diseases and measures to prevent patient - to - patient transmission are a significant component of care.a multidisciplinary team approach is necessary to develop and implement strategies to prevent infection in the critically ill patient .
the participation of nurses is of extraordinary importance for the success of infection control programs in intensive care .
in fact , most procedures performed to reduce the risk of nosocomial infection ( vascular catheter care , artificial airway care , mouth hygiene , etc . )
are part of the nurse s daily tasks.programs that have achieved significant reductions in nosocomial infection rates have designated at least one physician and one nurse in each icu as team leaders .
this model has also been implemented by successful programs designed to reduce nosocomial infection rates in the icu endorsed by semicyuc .
the zero resistance program clearly supports the nomination in every icu of a nurse leader responsible for infection control to reduce nosocomial infections and transmission of mdr pathogens.fourth recommendation : it is recommended to perform an active search for mdr pathogens in all patients on admission to the unit and at least once a week throughout their stay .
these samples will be processed to identify mdr pathogens according to the local epidemiology and in collaboration with the microbiology service and infection control team of each hospital.rationale : guidelines for mdr organisms include recommendations for routine screening cultures and contact precautions for patients after admission to high - risk units , e. g. , icus .
the implementation of contact precautions in patients colonized or infected with mdr is widely accepted .
in contrast , the use of routine surveillance cultures in mdr management is still a matter of debate and not widely performed .
initial screening is specially recommended for mrsa , although the same principles and practices apply to gram - negative mdr organisms , which actually now constitute the main threat.active surveillance programs are time and resource - consuming .
the type and number of samples are selected according to local resources and epidemiology and should include at least nasal , rectal and oropharyngeal swabs ( bronchial aspirates in intubated patients ) .
in addition , other samples may be necessary to control potential reservoirs ( infections , skin ulcers , etc.).concerning surveillance cultures , two approaches are acceptable : all patients are screened at icu admission or only those patients with at least one of the risk factors included in the checklist ( see fifth recommendation).fifth recommendation : at admission to the icu , a
checklist of risk factors ( table 2 ) must be completed to identify patients at high risk of mdr pathogen carriage .
patients meeting at least one of the risk factors must be cared for under application of contact precautions pending culture results.table 2
checklist of risk factors for carriage of multidrug - resistant ( mdr ) bacteria
risk factor
1 .
hospital admissionlasting > 5 days , during last 3 monthsyes no 2 .
institutionalized ( prison , healthcare and social centers , geriatric centers , etc.)yes no 3 . known colonization or infection with mdr pathogensyes no 4 .
antibiotic therapy 7 days in previous month ( particularly 3rd and 4th generation cephalosporins , flouroquinolones and carbapenems)yes no 5 .
comorbidities associated with high incidence of colonization or infection with mdr pathogens : cystic fibrosis , bronchiectasis , chronic skin ulcers , etc.yes no rationale : several risk factors associated with carriage of mdr at admission to the hospital or to the icu have been identified : prior antibiotic use , the presence of invasive devices and certain underlying diseases are the most frequently reported .
patients at risk of nosocomial pneumonia caused by mdr pathogens according to american thoracic society / infectious diseases society of america ( ats / idsa ) criteria are : current hospitalization of 5 days or more , prior antibiotic therapy , prior hospitalization , residence in a nursing home or extended - care facility , home infusion therapy within 30 days , chronic dialysis within 30 days , home wound care , family member with an mdr pathogen , and immunosuppression . however , in a prospective evaluation , although these criteria had an excellent negative predictive value ( 96% ) , they had a very low positive predictive value ( 18% ) for infection or colonization with an mdr pathogen at icu admission . in a case
control study , immunosuppression was not independently associated with mdr bacteria in the icu .in other studies , risk factors for specific pathogens , like mrsa or a. baumannii , have been identified in an attempt to establish control measures that limit spread .
this approach is particularly indicated in icus in which a particular microorganism causes the majority of episodes of colonization / infection.with this information , the sec generated a checklist ( table 2 ) for detection of patients at high risk of carrying mdr pathogens .
if one or more of these risk factors is present , screening cultures at icu admission is mandatory and the patient must be placed in contact isolation until culture results are negative for the target organisms .
the prospective validation of this checklist is one of the pending tasks of this program.sixth recommendation : compliance with preventive measures including those based on transmission mechanisms should be routinely measured.rationale : contact precaution and hand hygiene are the mainstay for reducing transmission of microorganisms .
briefly , contact precautions ( by staff and visitors ) consist of : hand hygiene and donning of gown and gloves immediately prior to room entry , and disposal of gown and gloves inside the patient s room , followed by hand hygiene immediately prior to leaving the room.adherence rates for contact precautions in icu settings with availability of all facilities were between 75 and 80% in one study .
correct practice includes : ( 1 ) use of a contact precautions sign for every patient colonized / infected by mdr pathogens ; ( 2 ) availability of contact precautions equipment at patient room entry ; ( 3 ) barrier disposal containers inside patient room ; and ( 4 ) monitoring of adherence to the contact precautions protocol by staff / visitors .
if there are no closed rooms , precautions must be tightened.to achieve the desired results , all staff members should watch compliance with preventive measures .
concerning this issue , the sec of zero resistance considers that nurses have a special responsibility in implementing effective prevention .
therefore , the rest of the hospital staff and visitors must follow their recommendations.seventh recommendation : all units should develop a cleaning protocol for rooms of patients with mdr pathogens.rationale : many published outbreaks of mdr pathogens detect a common source on environmental surfaces and in moist areas .
nevertheless , substantial improvements in cleaning and disinfection can be achieved by using standardized protocols in the icu [ 37 - 39 ] .
cleaning procedures must be adapted to the architectural characteristics of each unit and agreed upon with the cleaning staff and the nosocomial infection control committee .
this protocol should include fixed structures ( floors and walls ) as well as the bed ( including main structure , rails and mattress ) .
cleaning protocols for rooms occupied by patients with mdr pathogens must specify methodology , frequency of cleaning and disinfectant products . because different cleaning products are approved in each hospital ,
if deemed necessary , controls will be established to ensure mdr eradication .eighth recommendation : a file / document specifying the existing equipment in the icu and its respective cleaning protocols should be available and updated.rationale : any clinical or technological equipment could act as a microbiological reservoir for mdr pathogens . therefore , the first action is to remove all expendable materials , leaving work surfaces as free as possible .
equipment should be filed and information on the following aspects provided : staff responsible for cleaning , cleaning schedule and cleaning methodology ( disinfection , sterilization ) .
each healthcare worker is responsible for cleaning and disinfection of equipment for personal use ( stethoscopes , flashlights )
.ninth recommendation : to include products containing 4% chlorhexidine in daily patient hygiene if colonized or infected with mdr pathogens.rationale : several observational studies and single - center trials have concluded that daily chlorhexidine bathing of icu patients reduces the acquisition of mdr pathogens and the incidence of certain infections [ 40 - 43 ] .
a systematic review concluded that chlorhexidine body - washing may be effective in preventing carriage , and possibly bloodstream infections , with gram - positive mdr pathogens ( mrsa and vancomycin - resistant enterococci [ vre ] ) , whereas the evidence that this intervention eradicates carriage or prevents infection with gram - negative mdr pathogens is weak .in a recent randomized multicenter trial carried out in 13 icus , the effect of different infection control strategies on acquisition of mdr pathogens was assessed .
improved hand hygiene plus unit - wide chlorhexidine body - washing reduced acquisition , particularly of mrsa .
interestingly , in the context of sustained high level compliance of hand hygiene and chlorhexidine bathing , screening and isolation of carriers did not reduce acquisition rates of mdr pathogens .
more recently , a multicenter , open , crossover trial documented the clinical benefits of daily bathing with chlorhexidine - impregnated washcloths in reducing the risks of acquisition of mdr and the development of hospital - acquired bacteremia .chlorhexidine solutions must contain 0.16 grams of chlorhexidine ( digluconate ) per liter ( dissolve 20 ml of 4% chlorhexidine in 1 liter of warm water ) .
contraindications for chlorhexidine use and adverse reactions should be taken into account . because chlorhexidine is a cationic molecule , its activity can be reduced by natural soaps , various inorganic anions , non - ionic surfactants , and hand creams containing anionic emulsifying agents .
daily chlorhexidine bathing is simple to implement and relatively inexpensive and may be an important adjunctive intervention to barrier precautions to reduce acquisition and the subsequent development of infection.tenth recommendation : if an outbreak is suspected it is recommended to identify the causative organism with molecular typing methods.rationale : studies of outbreaks based on the phenotypic characteristics of microorganisms ( antigenic properties , metabolic or antibiotic resistance ) are limited and do not provide conclusive differences or similarities between them .
therefore , molecular typing methods , to be able to recognize epidemiologically - linked isolates derived from a common precursor microorganism , should be performed .
this will also provide understanding of the mechanism of transmission and dissemination and allow strategies to control and eradicate the epidemic to be designed .the zero resistance
program encourages hospitals without resources for molecular testing to send mdr isolates to a reference laboratory ( national center for microbiology , institute of health carlos iii ; ) , where the microbiological test will be performed free of charge .
first recommendation : in each icu , at least one intensivist will be designated as responsible for the use of antimicrobials .
he / she should have extensive experience in infection control and in the treatment of severe infections .
analysis of antimicrobial use should include : review of the indication for antimicrobials , evaluation of the appropriateness of the antimicrobial and the correct administration ( dosing , intervals and duration),evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation.rationale : antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge
. moreover , many pathophysiological changes associated with severe acute illness or sepsis , like capillary leak , third spacing , increased volume of distribution , and impaired renal and/or liver function , affect antimicrobial pharmacokinetics / pharmacodynamics .
therefore , it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality .
this implies choosing optimal empirical antibiotics , appropriate mode of administration , and correct dosage .
administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations , which are associated with treatment failure and resistance development .prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock .
however , as soon as microbiological information is available , empiric therapy should be adapted , if appropriate , by either reduction in number and/or narrowing of antimicrobial spectrum .
in fact , de - escalation of empirical therapy is performed in less than 50% of patients .
recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression .
review of the indication for antimicrobials , evaluation of the appropriateness of the antimicrobial and the correct administration ( dosing , intervals and duration ) , evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation .
rationale : antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge .
moreover , many pathophysiological changes associated with severe acute illness or sepsis , like capillary leak , third spacing , increased volume of distribution , and impaired renal and/or liver function , affect antimicrobial pharmacokinetics / pharmacodynamics .
therefore , it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality .
this implies choosing optimal empirical antibiotics , appropriate mode of administration , and correct dosage .
administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations , which are associated with treatment failure and resistance development .
prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock .
however , as soon as microbiological information is available , empiric therapy should be adapted , if appropriate , by either reduction in number and/or narrowing of antimicrobial spectrum .
in fact , de - escalation of empirical therapy is performed in less than 50% of patients .
recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression .
second recommendation : empirically administer antimicrobials active against mdr pathogens only in cases of severe sepsis or septic shock and high risk of mdr pathogen(s ) based on patient risk factors and/or knowledge of local ecology .
otherwise , narrow - spectrum or withholding of antimicrobials is recommended until microbiological results become available and targeted therapy with antibiotics active against mdr pathogens ( carbapenems , colistin , tigecycline , glycopeptides , daptomycin , linezolid ) should be started if needed . in all cases , samples for culture of the potential sources of infection
rationale : early and adequate antimicrobial therapy is associated with increased survival in patients with severe sepsis and septic shock . however , delaying antimicrobial therapy until microbiological confirmation is available has been shown to be associated with similar outcomes in febrile surgical icu patients compared to starting antimicrobials immediately after the clinical diagnosis of infection .
more recently , a quasi - experimental , before - after observational cohort study concluded that , after adjusting for confounders , aggressive antimicrobial therapy was an independent predictor of mortality . in the aggressive period , antimicrobial treatment was always started in patients suspected of having an infection after appropriate cultures were obtained . in the second period ( conservative strategy ) ,
the main limitation of both studies is that they were carried out in surgical patients and data from medical units are lacking .
however , it is important to keep in mind that in febrile patients with severe sepsis or septic shock a delay in antimicrobial therapy may be fatal .
in addition , the choice of empirical antimicrobial therapy should be based on an updated knowledge of the local ecology .
therefore , it seems prudent to recommend starting empiric antimicrobials active against mdr pathogens immediately only in cases meeting criteria for severe sepsis or septic shock and risk factors for mdr pathogens . obviously , efforts to reduce the delay of microbiological results ( use of rapid diagnostic techniques , direct contact with the microbiologist ) and close follow - up of the clinical course to rapidly detect signs of alarm are fully endorsed .
third recommendation : in each unit , at least one nurse will be designated as leader of this project and responsible for infection control measures aimed at reducing transmission of mdr pathogens . rationale : success of quality control programs is particularly dependent on the involvement of all categories of healthcare professionals .
nurses play a critical role in preventing and controlling infectious diseases and measures to prevent patient - to - patient transmission are a significant component of care .
a multidisciplinary team approach is necessary to develop and implement strategies to prevent infection in the critically ill patient .
the participation of nurses is of extraordinary importance for the success of infection control programs in intensive care .
in fact , most procedures performed to reduce the risk of nosocomial infection ( vascular catheter care , artificial airway care , mouth hygiene , etc . ) are part of the nurse s daily tasks .
programs that have achieved significant reductions in nosocomial infection rates have designated at least one physician and one nurse in each icu as team leaders .
this model has also been implemented by successful programs designed to reduce nosocomial infection rates in the icu endorsed by semicyuc .
the zero resistance program clearly supports the nomination in every icu of a nurse leader responsible for infection control to reduce nosocomial infections and transmission of mdr pathogens .
fourth recommendation : it is recommended to perform an active search for mdr pathogens in all patients on admission to the unit and at least once a week throughout their stay .
these samples will be processed to identify mdr pathogens according to the local epidemiology and in collaboration with the microbiology service and infection control team of each hospital .
rationale : guidelines for mdr organisms include recommendations for routine screening cultures and contact precautions for patients after admission to high - risk units , e. g. , icus .
the implementation of contact precautions in patients colonized or infected with mdr is widely accepted .
in contrast , the use of routine surveillance cultures in mdr management is still a matter of debate and not widely performed .
initial screening is specially recommended for mrsa , although the same principles and practices apply to gram - negative mdr organisms , which actually now constitute the main threat .
the type and number of samples are selected according to local resources and epidemiology and should include at least nasal , rectal and oropharyngeal swabs ( bronchial aspirates in intubated patients ) .
in addition , other samples may be necessary to control potential reservoirs ( infections , skin ulcers , etc . ) . concerning surveillance cultures ,
two approaches are acceptable : all patients are screened at icu admission or only those patients with at least one of the risk factors included in the checklist ( see fifth recommendation ) .
checklist of risk factors ( table 2 ) must be completed to identify patients at high risk of mdr pathogen carriage .
patients meeting at least one of the risk factors must be cared for under application of contact precautions pending culture results.table 2
checklist of risk factors for carriage of multidrug - resistant ( mdr ) bacteria
risk factor
1 .
hospital admissionlasting > 5 days , during last 3 monthsyes no 2 .
institutionalized ( prison , healthcare and social centers , geriatric centers , etc.)yes no 3 .
antibiotic therapy 7 days in previous month ( particularly 3rd and 4th generation cephalosporins , flouroquinolones and carbapenems)yes no 5 .
comorbidities associated with high incidence of colonization or infection with mdr pathogens : cystic fibrosis , bronchiectasis , chronic skin ulcers , etc.yes no
checklist of risk factors for carriage of multidrug - resistant ( mdr ) bacteria
rationale : several risk factors associated with carriage of mdr at admission to the hospital or to the icu have been identified : prior antibiotic use , the presence of invasive devices and certain underlying diseases are the most frequently reported .
patients at risk of nosocomial pneumonia caused by mdr pathogens according to american thoracic society / infectious diseases society of america ( ats / idsa ) criteria are : current hospitalization of 5 days or more , prior antibiotic therapy , prior hospitalization , residence in a nursing home or extended - care facility , home infusion therapy within 30 days , chronic dialysis within 30 days , home wound care , family member with an mdr pathogen , and immunosuppression . however , in a prospective evaluation , although these criteria had an excellent negative predictive value ( 96% ) , they had a very low positive predictive value ( 18% ) for infection or colonization with an mdr pathogen at icu admission . in a case
control study , immunosuppression was not independently associated with mdr bacteria in the icu . in other studies ,
risk factors for specific pathogens , like mrsa or a. baumannii , have been identified in an attempt to establish control measures that limit spread .
this approach is particularly indicated in icus in which a particular microorganism causes the majority of episodes of colonization / infection . with this information ,
the sec generated a checklist ( table 2 ) for detection of patients at high risk of carrying mdr pathogens .
if one or more of these risk factors is present , screening cultures at icu admission is mandatory and the patient must be placed in contact isolation until culture results are negative for the target organisms .
the prospective validation of this checklist is one of the pending tasks of this program .
sixth recommendation : compliance with preventive measures including those based on transmission mechanisms should be routinely measured .
rationale : contact precaution and hand hygiene are the mainstay for reducing transmission of microorganisms . adherence to these practices must be continuously reinforced and monitored .
briefly , contact precautions ( by staff and visitors ) consist of : hand hygiene and donning of gown and gloves immediately prior to room entry , and disposal of gown and gloves inside the patient s room , followed by hand hygiene immediately prior to leaving the room .
adherence rates for contact precautions in icu settings with availability of all facilities were between 75 and 80% in one study .
correct practice includes : ( 1 ) use of a contact precautions sign for every patient colonized / infected by mdr pathogens ; ( 2 ) availability of contact precautions equipment at patient room entry ; ( 3 ) barrier disposal containers inside patient room ; and ( 4 ) monitoring of adherence to the contact precautions protocol by staff / visitors .
if there are no closed rooms , precautions must be tightened . to achieve the desired results
, all staff members should watch compliance with preventive measures . concerning this issue , the sec of zero resistance
seventh recommendation : all units should develop a cleaning protocol for rooms of patients with mdr pathogens . rationale : many published outbreaks of mdr pathogens detect a common source on environmental surfaces and in moist areas .
nevertheless , substantial improvements in cleaning and disinfection can be achieved by using standardized protocols in the icu [ 37 - 39 ] .
cleaning procedures must be adapted to the architectural characteristics of each unit and agreed upon with the cleaning staff and the nosocomial infection control committee .
this protocol should include fixed structures ( floors and walls ) as well as the bed ( including main structure , rails and mattress ) .
cleaning protocols for rooms occupied by patients with mdr pathogens must specify methodology , frequency of cleaning and disinfectant products . because different cleaning products are approved in each hospital ,
eighth recommendation : a file / document specifying the existing equipment in the icu and its respective cleaning protocols should be available and updated .
rationale : any clinical or technological equipment could act as a microbiological reservoir for mdr pathogens . therefore , the first action is to remove all expendable materials , leaving work surfaces as free as possible .
equipment should be filed and information on the following aspects provided : staff responsible for cleaning , cleaning schedule and cleaning methodology ( disinfection , sterilization ) .
each healthcare worker is responsible for cleaning and disinfection of equipment for personal use ( stethoscopes , flashlights ) .
ninth recommendation : to include products containing 4% chlorhexidine in daily patient hygiene if colonized or infected with mdr pathogens .
rationale : several observational studies and single - center trials have concluded that daily chlorhexidine bathing of icu patients reduces the acquisition of mdr pathogens and the incidence of certain infections [ 40 - 43 ] .
a systematic review concluded that chlorhexidine body - washing may be effective in preventing carriage , and possibly bloodstream infections , with gram - positive mdr pathogens ( mrsa and vancomycin - resistant enterococci [ vre ] ) , whereas the evidence that this intervention eradicates carriage or prevents infection with gram - negative mdr pathogens is weak . in a recent randomized multicenter trial carried out in 13 icus ,
improved hand hygiene plus unit - wide chlorhexidine body - washing reduced acquisition , particularly of mrsa . interestingly , in the context of sustained high level compliance of hand hygiene and chlorhexidine bathing , screening and isolation of carriers did not reduce acquisition rates of mdr pathogens .
more recently , a multicenter , open , crossover trial documented the clinical benefits of daily bathing with chlorhexidine - impregnated washcloths in reducing the risks of acquisition of mdr and the development of hospital - acquired bacteremia .
chlorhexidine solutions must contain 0.16 grams of chlorhexidine ( digluconate ) per liter ( dissolve 20 ml of 4% chlorhexidine in 1 liter of warm water ) .
contraindications for chlorhexidine use and adverse reactions should be taken into account . because chlorhexidine is a cationic molecule , its activity can be reduced by natural soaps , various inorganic anions , non - ionic surfactants , and hand creams containing anionic emulsifying agents .
daily chlorhexidine bathing is simple to implement and relatively inexpensive and may be an important adjunctive intervention to barrier precautions to reduce acquisition and the subsequent development of infection .
tenth recommendation : if an outbreak is suspected it is recommended to identify the causative organism with molecular typing methods . rationale : studies of outbreaks based on the phenotypic characteristics of microorganisms ( antigenic properties , metabolic or antibiotic resistance ) are limited and do not provide conclusive differences or similarities between them . therefore , molecular typing methods , to be able to recognize epidemiologically - linked isolates derived from a common precursor microorganism , should be performed .
this will also provide understanding of the mechanism of transmission and dissemination and allow strategies to control and eradicate the epidemic to be designed .
program encourages hospitals without resources for molecular testing to send mdr isolates to a reference laboratory ( national center for microbiology , institute of health carlos iii ; ) , where the microbiological test will be performed free of charge .
the main objective of the zero resistance project is reduction in the cumulative incidence of patients with icu - acquired mdr infections by 20% .
secondary objectives are to study the epidemiology of mdr infections in spanish icus , to be able to distinguish imported from icu - acquired cases , to promote and strengthen safety assurance in participating units , and to create a network of icus implementing safe , and evidence - based practices .
the primary aim of the bundle recommendations is reduction of the three most influential factors contributing to the development and transmission of mdr , namely : 1 ) adequate prescription of antibiotics ; 2 ) early detection and prevention of cross - colonization of mdr ; and 3 ) elimination of reservoirs .first recommendation : in each icu , at least one intensivist will be designated as responsible for the use of antimicrobials .
he / she should have extensive experience in infection control and in the treatment of severe infections .
analysis of antimicrobial use should include : review of the indication for antimicrobials , evaluation of the appropriateness of the antimicrobial and the correct administration ( dosing , intervals and duration),evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation.rationale : antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge .
moreover , many pathophysiological changes associated with severe acute illness or sepsis , like capillary leak , third spacing , increased volume of distribution , and impaired renal and/or liver function , affect antimicrobial pharmacokinetics / pharmacodynamics .
therefore , it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality .
this implies choosing optimal empirical antibiotics , appropriate mode of administration , and correct dosage .
administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations , which are associated with treatment failure and resistance development .prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock .
however , as soon as microbiological information is available , empiric therapy should be adapted , if appropriate , by either reduction in number and/or narrowing of antimicrobial spectrum .
in fact , de - escalation of empirical therapy is performed in less than 50% of patients .
recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression .second recommendation : empirically administer antimicrobials active against mdr pathogens only in cases of severe sepsis or septic shock and high risk of mdr pathogen(s ) based on patient risk factors and/or knowledge of local ecology . otherwise , narrow - spectrum or withholding of antimicrobials is recommended until microbiological results become available and targeted therapy with antibiotics active against mdr pathogens ( carbapenems , colistin , tigecycline , glycopeptides , daptomycin , linezolid ) should be started if needed . in all cases , samples for culture of the potential sources of infection
should be obtained before starting antibiotic therapy.rationale : early and adequate antimicrobial therapy is associated with increased survival in patients with severe sepsis and septic shock . however , delaying antimicrobial therapy until microbiological confirmation is available has been shown to be associated with similar outcomes in febrile surgical icu patients compared to starting antimicrobials immediately after the clinical diagnosis of infection .
more recently , a quasi - experimental , before - after observational cohort study concluded that , after adjusting for confounders , aggressive antimicrobial therapy was an independent predictor of mortality . in the aggressive period , antimicrobial treatment was always started in patients suspected of having an infection after appropriate cultures were obtained . in the second period ( conservative strategy ) ,
antimicrobial treatment was started only after objective findings confirmed the infection .the main limitation of both studies is that they were carried out in surgical patients and data from medical units are lacking .
however , it is important to keep in mind that in febrile patients with severe sepsis or septic shock a delay in antimicrobial therapy may be fatal .
in addition , the choice of empirical antimicrobial therapy should be based on an updated knowledge of the local ecology .
therefore , it seems prudent to recommend starting empiric antimicrobials active against mdr pathogens immediately only in cases meeting criteria for severe sepsis or septic shock and risk factors for mdr pathogens .
obviously , efforts to reduce the delay of microbiological results ( use of rapid diagnostic techniques , direct contact with the microbiologist ) and close follow - up of the clinical course to rapidly detect signs of alarm are fully endorsed.third recommendation : in each unit , at least one nurse will be designated as leader of this project and responsible for infection control measures aimed at reducing transmission of mdr pathogens.rationale : success of quality control programs is particularly dependent on the involvement of all categories of healthcare professionals .
nurses play a critical role in preventing and controlling infectious diseases and measures to prevent patient - to - patient transmission are a significant component of care.a multidisciplinary team approach is necessary to develop and implement strategies to prevent infection in the critically ill patient .
the participation of nurses is of extraordinary importance for the success of infection control programs in intensive care .
in fact , most procedures performed to reduce the risk of nosocomial infection ( vascular catheter care , artificial airway care , mouth hygiene , etc . )
are part of the nurse s daily tasks.programs that have achieved significant reductions in nosocomial infection rates have designated at least one physician and one nurse in each icu as team leaders .
this model has also been implemented by successful programs designed to reduce nosocomial infection rates in the icu endorsed by semicyuc .
the zero resistance program clearly supports the nomination in every icu of a nurse leader responsible for infection control to reduce nosocomial infections and transmission of mdr pathogens.fourth recommendation : it is recommended to perform an active search for mdr pathogens in all patients on admission to the unit and at least once a week throughout their stay .
these samples will be processed to identify mdr pathogens according to the local epidemiology and in collaboration with the microbiology service and infection control team of each hospital.rationale : guidelines for mdr organisms include recommendations for routine screening cultures and contact precautions for patients after admission to high - risk units , e. g. , icus . the implementation of contact precautions in patients colonized or infected with mdr is widely accepted .
in contrast , the use of routine surveillance cultures in mdr management is still a matter of debate and not widely performed .
initial screening is specially recommended for mrsa , although the same principles and practices apply to gram - negative mdr organisms , which actually now constitute the main threat.active surveillance programs are time and resource - consuming .
the type and number of samples are selected according to local resources and epidemiology and should include at least nasal , rectal and oropharyngeal swabs ( bronchial aspirates in intubated patients ) .
in addition , other samples may be necessary to control potential reservoirs ( infections , skin ulcers , etc.).concerning surveillance cultures , two approaches are acceptable : all patients are screened at icu admission or only those patients with at least one of the risk factors included in the checklist ( see fifth recommendation).fifth recommendation : at admission to the icu , a
checklist of risk factors ( table 2 ) must be completed to identify patients at high risk of mdr pathogen carriage .
patients meeting at least one of the risk factors must be cared for under application of contact precautions pending culture results.table 2
checklist of risk factors for carriage of multidrug - resistant ( mdr ) bacteria
risk factor
1 .
hospital admissionlasting > 5 days , during last 3 monthsyes no 2 .
institutionalized ( prison , healthcare and social centers , geriatric centers , etc.)yes no 3 .
antibiotic therapy 7 days in previous month ( particularly 3rd and 4th generation cephalosporins , flouroquinolones and carbapenems)yes no 5 .
comorbidities associated with high incidence of colonization or infection with mdr pathogens : cystic fibrosis , bronchiectasis , chronic skin ulcers , etc.yes no rationale : several risk factors associated with carriage of mdr at admission to the hospital or to the icu have been identified : prior antibiotic use , the presence of invasive devices and certain underlying diseases are the most frequently reported .
patients at risk of nosocomial pneumonia caused by mdr pathogens according to american thoracic society / infectious diseases society of america ( ats / idsa ) criteria are : current hospitalization of 5 days or more , prior antibiotic therapy , prior hospitalization , residence in a nursing home or extended - care facility , home infusion therapy within 30 days , chronic dialysis within 30 days , home wound care , family member with an mdr pathogen , and immunosuppression .
however , in a prospective evaluation , although these criteria had an excellent negative predictive value ( 96% ) , they had a very low positive predictive value ( 18% ) for infection or colonization with an mdr pathogen at icu admission . in a case
control study , immunosuppression was not independently associated with mdr bacteria in the icu .in other studies , risk factors for specific pathogens , like mrsa or a. baumannii , have been identified in an attempt to establish control measures that limit spread .
this approach is particularly indicated in icus in which a particular microorganism causes the majority of episodes of colonization / infection.with this information , the sec generated a checklist ( table 2 ) for detection of patients at high risk of carrying mdr pathogens .
if one or more of these risk factors is present , screening cultures at icu admission is mandatory and the patient must be placed in contact isolation until culture results are negative for the target organisms .
the prospective validation of this checklist is one of the pending tasks of this program.sixth recommendation : compliance with preventive measures including those based on transmission mechanisms should be routinely measured.rationale : contact precaution and hand hygiene are the mainstay for reducing transmission of microorganisms .
briefly , contact precautions ( by staff and visitors ) consist of : hand hygiene and donning of gown and gloves immediately prior to room entry , and disposal of gown and gloves inside the patient s room , followed by hand hygiene immediately prior to leaving the room.adherence rates for contact precautions in icu settings with availability of all facilities were between 75 and 80% in one study .
correct practice includes : ( 1 ) use of a contact precautions sign for every patient colonized / infected by mdr pathogens ; ( 2 ) availability of contact precautions equipment at patient room entry ; ( 3 ) barrier disposal containers inside patient room ; and ( 4 ) monitoring of adherence to the contact precautions protocol by staff / visitors .
if there are no closed rooms , precautions must be tightened.to achieve the desired results , all staff members should watch compliance with preventive measures . concerning this issue , the sec of zero resistance
therefore , the rest of the hospital staff and visitors must follow their recommendations.seventh recommendation : all units should develop a cleaning protocol for rooms of patients with mdr pathogens.rationale : many published outbreaks of mdr pathogens detect a common source on environmental surfaces and in moist areas .
nevertheless , substantial improvements in cleaning and disinfection can be achieved by using standardized protocols in the icu [ 37 - 39 ] .
cleaning procedures must be adapted to the architectural characteristics of each unit and agreed upon with the cleaning staff and the nosocomial infection control committee .
this protocol should include fixed structures ( floors and walls ) as well as the bed ( including main structure , rails and mattress ) .
cleaning protocols for rooms occupied by patients with mdr pathogens must specify methodology , frequency of cleaning and disinfectant products . because different cleaning products are approved in each hospital ,
if deemed necessary , controls will be established to ensure mdr eradication .eighth recommendation : a file / document specifying the existing equipment in the icu and its respective cleaning protocols should be available and updated.rationale : any clinical or technological equipment could act as a microbiological reservoir for mdr pathogens .
therefore , the first action is to remove all expendable materials , leaving work surfaces as free as possible .
equipment should be filed and information on the following aspects provided : staff responsible for cleaning , cleaning schedule and cleaning methodology ( disinfection , sterilization ) .
each healthcare worker is responsible for cleaning and disinfection of equipment for personal use ( stethoscopes , flashlights )
.ninth recommendation : to include products containing 4% chlorhexidine in daily patient hygiene if colonized or infected with mdr pathogens.rationale : several observational studies and single - center trials have concluded that daily chlorhexidine bathing of icu patients reduces the acquisition of mdr pathogens and the incidence of certain infections [ 40 - 43 ] .
a systematic review concluded that chlorhexidine body - washing may be effective in preventing carriage , and possibly bloodstream infections , with gram - positive mdr pathogens ( mrsa and vancomycin - resistant enterococci [ vre ] ) , whereas the evidence that this intervention eradicates carriage or prevents infection with gram - negative mdr pathogens is weak .in a recent randomized multicenter trial carried out in 13 icus , the effect of different infection control strategies on acquisition of mdr pathogens was assessed .
improved hand hygiene plus unit - wide chlorhexidine body - washing reduced acquisition , particularly of mrsa . interestingly , in the context of sustained high level compliance of hand hygiene and chlorhexidine bathing , screening and isolation of carriers did not reduce acquisition rates of mdr pathogens .
more recently , a multicenter , open , crossover trial documented the clinical benefits of daily bathing with chlorhexidine - impregnated washcloths in reducing the risks of acquisition of mdr and the development of hospital - acquired bacteremia .chlorhexidine solutions must contain 0.16 grams of chlorhexidine ( digluconate ) per liter ( dissolve 20 ml of 4% chlorhexidine in 1 liter of warm water ) .
contraindications for chlorhexidine use and adverse reactions should be taken into account . because chlorhexidine is a cationic molecule , its activity can be reduced by natural soaps , various inorganic anions , non - ionic surfactants , and hand creams containing anionic emulsifying agents .
daily chlorhexidine bathing is simple to implement and relatively inexpensive and may be an important adjunctive intervention to barrier precautions to reduce acquisition and the subsequent development of infection.tenth recommendation : if an outbreak is suspected it is recommended to identify the causative organism with molecular typing methods.rationale : studies of outbreaks based on the phenotypic characteristics of microorganisms ( antigenic properties , metabolic or antibiotic resistance ) are limited and do not provide conclusive differences or similarities between them .
therefore , molecular typing methods , to be able to recognize epidemiologically - linked isolates derived from a common precursor microorganism , should be performed .
this will also provide understanding of the mechanism of transmission and dissemination and allow strategies to control and eradicate the epidemic to be designed .the zero resistance
program encourages hospitals without resources for molecular testing to send mdr isolates to a reference laboratory ( national center for microbiology , institute of health carlos iii ; ) , where the microbiological test will be performed free of charge .
first recommendation : in each icu , at least one intensivist will be designated as responsible for the use of antimicrobials . he / she should have extensive experience in infection control and in the treatment of severe infections .
analysis of antimicrobial use should include : review of the indication for antimicrobials , evaluation of the appropriateness of the antimicrobial and the correct administration ( dosing , intervals and duration),evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation.rationale : antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge .
moreover , many pathophysiological changes associated with severe acute illness or sepsis , like capillary leak , third spacing , increased volume of distribution , and impaired renal and/or liver function , affect antimicrobial pharmacokinetics / pharmacodynamics . therefore , it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality .
this implies choosing optimal empirical antibiotics , appropriate mode of administration , and correct dosage .
administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations , which are associated with treatment failure and resistance development .prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock .
however , as soon as microbiological information is available , empiric therapy should be adapted , if appropriate , by either reduction in number and/or narrowing of antimicrobial spectrum .
in fact , de - escalation of empirical therapy is performed in less than 50% of patients .
recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression .
review of the indication for antimicrobials , evaluation of the appropriateness of the antimicrobial and the correct administration ( dosing , intervals and duration ) , evaluation of de - escalation of antimicrobial therapy or even antimicrobial cessation .
rationale : antibiotic prescription in the critical care setting is a complex task that requires profound and extensive knowledge .
moreover , many pathophysiological changes associated with severe acute illness or sepsis , like capillary leak , third spacing , increased volume of distribution , and impaired renal and/or liver function , affect antimicrobial pharmacokinetics / pharmacodynamics . therefore , it is imperative to identify intensivists with a profound knowledge of infectious diseases in critically ill patients in order to improve prescription quality
. this implies choosing optimal empirical antibiotics , appropriate mode of administration , and correct dosage .
administration of antimicrobials to severely ill patients at dosages defined in studies conducted in healthy volunteers often achieves only suboptimal serum concentrations , which are associated with treatment failure and resistance development .
prompt and adequate antimicrobial therapy reduces morbidity and mortality in severe sepsis and septic shock . however , as soon as microbiological information is available , empiric therapy should be adapted , if appropriate , by either reduction in number and/or narrowing of antimicrobial spectrum .
in fact , de - escalation of empirical therapy is performed in less than 50% of patients .
recent studies have shown that de - escalation is safe even in critically ill patients with severe sepsis or immunosuppression .
second recommendation : empirically administer antimicrobials active against mdr pathogens only in cases of severe sepsis or septic shock and high risk of mdr pathogen(s ) based on patient risk factors and/or knowledge of local ecology .
otherwise , narrow - spectrum or withholding of antimicrobials is recommended until microbiological results become available and targeted therapy with antibiotics active against mdr pathogens ( carbapenems , colistin , tigecycline , glycopeptides , daptomycin , linezolid ) should be started if needed . in all cases , samples for culture of the potential sources of infection
rationale : early and adequate antimicrobial therapy is associated with increased survival in patients with severe sepsis and septic shock . however , delaying antimicrobial therapy until microbiological confirmation is available has been shown to be associated with similar outcomes in febrile surgical icu patients compared to starting antimicrobials immediately after the clinical diagnosis of infection .
more recently , a quasi - experimental , before - after observational cohort study concluded that , after adjusting for confounders , aggressive antimicrobial therapy was an independent predictor of mortality . in the aggressive period , antimicrobial treatment was always started in patients suspected of having an infection after appropriate cultures were obtained . in the second period ( conservative strategy ) ,
the main limitation of both studies is that they were carried out in surgical patients and data from medical units are lacking .
however , it is important to keep in mind that in febrile patients with severe sepsis or septic shock a delay in antimicrobial therapy may be fatal .
in addition , the choice of empirical antimicrobial therapy should be based on an updated knowledge of the local ecology .
therefore , it seems prudent to recommend starting empiric antimicrobials active against mdr pathogens immediately only in cases meeting criteria for severe sepsis or septic shock and risk factors for mdr pathogens . obviously , efforts to reduce the delay of microbiological results ( use of rapid diagnostic techniques , direct contact with the microbiologist ) and close follow - up of the clinical course to rapidly detect signs of alarm are fully endorsed .
third recommendation : in each unit , at least one nurse will be designated as leader of this project and responsible for infection control measures aimed at reducing transmission of mdr pathogens . rationale : success of quality control programs is particularly dependent on the involvement of all categories of healthcare professionals .
nurses play a critical role in preventing and controlling infectious diseases and measures to prevent patient - to - patient transmission are a significant component of care .
a multidisciplinary team approach is necessary to develop and implement strategies to prevent infection in the critically ill patient .
the participation of nurses is of extraordinary importance for the success of infection control programs in intensive care .
in fact , most procedures performed to reduce the risk of nosocomial infection ( vascular catheter care , artificial airway care , mouth hygiene , etc . ) are part of the nurse s daily tasks .
programs that have achieved significant reductions in nosocomial infection rates have designated at least one physician and one nurse in each icu as team leaders .
this model has also been implemented by successful programs designed to reduce nosocomial infection rates in the icu endorsed by semicyuc .
zero resistance program clearly supports the nomination in every icu of a nurse leader responsible for infection control to reduce nosocomial infections and transmission of mdr pathogens .
fourth recommendation : it is recommended to perform an active search for mdr pathogens in all patients on admission to the unit and at least once a week throughout their stay .
these samples will be processed to identify mdr pathogens according to the local epidemiology and in collaboration with the microbiology service and infection control team of each hospital .
rationale : guidelines for mdr organisms include recommendations for routine screening cultures and contact precautions for patients after admission to high - risk units , e. g. , icus .
the implementation of contact precautions in patients colonized or infected with mdr is widely accepted .
in contrast , the use of routine surveillance cultures in mdr management is still a matter of debate and not widely performed .
initial screening is specially recommended for mrsa , although the same principles and practices apply to gram - negative mdr organisms , which actually now constitute the main threat .
the type and number of samples are selected according to local resources and epidemiology and should include at least nasal , rectal and oropharyngeal swabs ( bronchial aspirates in intubated patients ) .
in addition , other samples may be necessary to control potential reservoirs ( infections , skin ulcers , etc . ) . concerning surveillance cultures ,
two approaches are acceptable : all patients are screened at icu admission or only those patients with at least one of the risk factors included in the checklist ( see fifth recommendation ) .
checklist of risk factors ( table 2 ) must be completed to identify patients at high risk of mdr pathogen carriage .
patients meeting at least one of the risk factors must be cared for under application of contact precautions pending culture results.table 2
checklist of risk factors for carriage of multidrug - resistant ( mdr ) bacteria
risk factor
1 .
hospital admissionlasting > 5 days , during last 3 monthsyes no 2 .
institutionalized ( prison , healthcare and social centers , geriatric centers , etc.)yes no 3 . known colonization or infection with mdr pathogensyes no 4 . antibiotic therapy 7 days in previous month ( particularly 3rd and 4th generation cephalosporins , flouroquinolones and carbapenems)yes no 5 .
comorbidities associated with high incidence of colonization or infection with mdr pathogens : cystic fibrosis , bronchiectasis , chronic skin ulcers , etc.yes no
checklist of risk factors for carriage of multidrug - resistant ( mdr ) bacteria
rationale : several risk factors associated with carriage of mdr at admission to the hospital or to the icu have been identified : prior antibiotic use , the presence of invasive devices and certain underlying diseases are the most frequently reported .
patients at risk of nosocomial pneumonia caused by mdr pathogens according to american thoracic society / infectious diseases society of america ( ats / idsa ) criteria are : current hospitalization of 5 days or more , prior antibiotic therapy , prior hospitalization , residence in a nursing home or extended - care facility , home infusion therapy within 30 days , chronic dialysis within 30 days , home wound care , family member with an mdr pathogen , and immunosuppression . however , in a prospective evaluation , although these criteria had an excellent negative predictive value ( 96% ) , they had a very low positive predictive value ( 18% ) for infection or colonization with an mdr pathogen at icu admission . in a case
control study , immunosuppression was not independently associated with mdr bacteria in the icu . in other studies ,
risk factors for specific pathogens , like mrsa or a. baumannii , have been identified in an attempt to establish control measures that limit spread .
this approach is particularly indicated in icus in which a particular microorganism causes the majority of episodes of colonization / infection . with this information ,
the sec generated a checklist ( table 2 ) for detection of patients at high risk of carrying mdr pathogens .
if one or more of these risk factors is present , screening cultures at icu admission is mandatory and the patient must be placed in contact isolation until culture results are negative for the target organisms .
the prospective validation of this checklist is one of the pending tasks of this program .
sixth recommendation : compliance with preventive measures including those based on transmission mechanisms should be routinely measured .
rationale : contact precaution and hand hygiene are the mainstay for reducing transmission of microorganisms .
briefly , contact precautions ( by staff and visitors ) consist of : hand hygiene and donning of gown and gloves immediately prior to room entry , and disposal of gown and gloves inside the patient s room , followed by hand hygiene immediately prior to leaving the room .
adherence rates for contact precautions in icu settings with availability of all facilities were between 75 and 80% in one study .
correct practice includes : ( 1 ) use of a contact precautions sign for every patient colonized / infected by mdr pathogens ; ( 2 ) availability of contact precautions equipment at patient room entry ; ( 3 ) barrier disposal containers inside patient room ; and ( 4 ) monitoring of adherence to the contact precautions protocol by staff / visitors .
if there are no closed rooms , precautions must be tightened . to achieve the desired results
, all staff members should watch compliance with preventive measures . concerning this issue , the sec of zero resistance
seventh recommendation : all units should develop a cleaning protocol for rooms of patients with mdr pathogens . rationale : many published outbreaks of mdr pathogens detect a common source on environmental surfaces and in moist areas .
nevertheless , substantial improvements in cleaning and disinfection can be achieved by using standardized protocols in the icu [ 37 - 39 ] .
cleaning procedures must be adapted to the architectural characteristics of each unit and agreed upon with the cleaning staff and the nosocomial infection control committee .
this protocol should include fixed structures ( floors and walls ) as well as the bed ( including main structure , rails and mattress ) .
cleaning protocols for rooms occupied by patients with mdr pathogens must specify methodology , frequency of cleaning and disinfectant products . because different cleaning products are approved in each hospital ,
eighth recommendation : a file / document specifying the existing equipment in the icu and its respective cleaning protocols should be available and updated .
rationale : any clinical or technological equipment could act as a microbiological reservoir for mdr pathogens .
therefore , the first action is to remove all expendable materials , leaving work surfaces as free as possible .
equipment should be filed and information on the following aspects provided : staff responsible for cleaning , cleaning schedule and cleaning methodology ( disinfection , sterilization ) .
each healthcare worker is responsible for cleaning and disinfection of equipment for personal use ( stethoscopes , flashlights ) .
ninth recommendation : to include products containing 4% chlorhexidine in daily patient hygiene if colonized or infected with mdr pathogens .
rationale : several observational studies and single - center trials have concluded that daily chlorhexidine bathing of icu patients reduces the acquisition of mdr pathogens and the incidence of certain infections [ 40 - 43 ] .
a systematic review concluded that chlorhexidine body - washing may be effective in preventing carriage , and possibly bloodstream infections , with gram - positive mdr pathogens ( mrsa and vancomycin - resistant enterococci [ vre ] ) , whereas the evidence that this intervention eradicates carriage or prevents infection with gram - negative mdr pathogens is weak . in a recent randomized multicenter trial carried out in 13 icus ,
improved hand hygiene plus unit - wide chlorhexidine body - washing reduced acquisition , particularly of mrsa . interestingly , in the context of sustained high level compliance of hand hygiene and chlorhexidine bathing , screening and isolation of carriers did not reduce acquisition rates of mdr pathogens .
more recently , a multicenter , open , crossover trial documented the clinical benefits of daily bathing with chlorhexidine - impregnated washcloths in reducing the risks of acquisition of mdr and the development of hospital - acquired bacteremia .
chlorhexidine solutions must contain 0.16 grams of chlorhexidine ( digluconate ) per liter ( dissolve 20 ml of 4% chlorhexidine in 1 liter of warm water ) .
contraindications for chlorhexidine use and adverse reactions should be taken into account . because chlorhexidine is a cationic molecule , its activity can be reduced by natural soaps , various inorganic anions , non - ionic surfactants , and hand creams containing anionic emulsifying agents .
daily chlorhexidine bathing is simple to implement and relatively inexpensive and may be an important adjunctive intervention to barrier precautions to reduce acquisition and the subsequent development of infection .
tenth recommendation : if an outbreak is suspected it is recommended to identify the causative organism with molecular typing methods . rationale : studies of outbreaks based on the phenotypic characteristics of microorganisms ( antigenic properties , metabolic or antibiotic resistance ) are limited and do not provide conclusive differences or similarities between them . therefore , molecular typing methods , to be able to recognize epidemiologically - linked isolates derived from a common precursor microorganism , should be performed .
this will also provide understanding of the mechanism of transmission and dissemination and allow strategies to control and eradicate the epidemic to be designed .
program encourages hospitals without resources for molecular testing to send mdr isolates to a reference laboratory ( national center for microbiology , institute of health carlos iii ; ) , where the microbiological test will be performed free of charge .
active implementation of this type of program is clearly necessary in order to achieve the desired results .
the agency for quality assurance of the spanish ministry of health will promote implementation in collaboration with the 17 regional healthcare authorities through dissemination , coordination and follow - up .
every autonomous region will create a coordinating team led by an intensivist , responsible for contacting hospital management .
the hospital management will notify their local infection and patient quality assurance committees and nominate a local coordinating team consisting of at least an intensivist and an intensive care nurse .
it is recommended that the local teams keep track of the number of healthcare workers , physicians , nurses and nurse aides that complete the web - based training modules and report their local educational indices to the regional coordinator .
the impact of zero resistance , as in all quality programs , must be measured using quality indicators that can be broken down into structure , procedure and outcome indicators .
obviously , outcome measures are of greater interest since they reflect all aspects of care and are the ultimate objectives of the intervention .
the proposed indicators are explained in detail in the program , but each local team should decide which indicators to monitor depending on the information systems and efforts necessary to obtain these measurements .
zero resistance program are committed to entering data required for calculation of the relevant indices in the web - based envin - helics registry .
data are recorded through a specific adaptation of the envin - helics web page .
summary descriptive statistics are available on - line for every individual unit , which can directly access its data on a daily basis .
bacterial resistance to antibiotics is growing day by day , particularly in hospitals , with a significant impact on mortality and morbidity .
the lack of new antibiotics , especially for gram - negative mdr pathogens , aggravates this serious problem as noted by numerous agencies and professional societies .
antibiotics are often incorrectly prescribed : inadequate antibiotics or incorrect dosage for a particular infection , administration of antibiotics for non - bacterial infections , and excessively long treatment courses are all frequent .
zero resistance is a project developed by the semicyuc with the technical support of the spanish ministry of health , with the main objective of reducing the cumulative incidence of patients with icu - acquired mdr by 20% .
this project contains a bundle of 10 recommendations aimed at improving prescription of antibiotics , detection and prevention of cross - colonization of mdr pathogens , and elimination of reservoirs .
this initiative includes an integral patient safety program and educational modules to facilitate its implementation .
adherence to the project and its results will be evaluated through a series of indicators .
|
this article is one of ten reviews selected from the annual update in intensive care and emergency medicine 2015 and co - published as a series in critical care .
other articles in the series can be found online at http://ccforum.com/series/annualupdate2015 .
further information about the annual update in intensive care and emergency medicine is available from http://www.springer.com/series/8901 .
|
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tissue culture methods and irradiation procedure have been described in . the three dimensional tissue culture model
( 3d epioral ) and media were purchased from mattek corporation ( ashland , ma ) .
the 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . in order to induce differentiation and stratification of the keratinocytes of the 3d cultures ,
then the tissues were irradiated ( dose of irradiation was 0 , 2 , or 12 gy ) at the city of hope ( duarte , ca ) facility .
subsequently the tissues were incubated for 6 h at 37 c with 5% co2 after irradiation .
then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna .
the rneasy plus mini kit ( qiagen , germantown , md ) was used to extract total rna of at least two identically treated tissues .
the integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) . only rna samples with absorbance ratio , a260/280 > 2.0 , and rin >
the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase ; the transcription reaction included aminoallyl utp ( aa - dutp ) and the subsequent product was conjugated to cy5 nhs ester ( ge healthcare life sciences , pittsburg , pa ) . the human whole genome onearray v4.3 ( phalanx biotech , san diego , ca ) was used to perform the dna microarray analysis .
for this purpose , a quantity of 0.025 mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42 c using the hybbag mixing system with 1 onearray hybridization buffer ( phalanx biotech ) and 0.01 mg / ml sheared salmon sperm dna ( promega , madison , wi ) . after the hybridization procedure
the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format .
the data from all the arrays of each experimental set were analyzed using rosetta resolver system ( rosetta biosoftware , usa ) .
testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver .
the error - weighted approach ( which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy ) was used to calculate the average expression values . in order to decide whether intensity data was significantly above background , p - values
were calculated to test the null hypothesis that expression is absent ( called p - value detected ) . a p - value detected < 0.05 indicated that the transcript specific to a given probe was truly present or expressed .
lastly , we also calculated p - values for determining whether genes were differentially expressed . rather than focusing solely on fold changes ,
rosetta resolver uses error - model - based hypothesis tests , which account for both fold change and expression level .
three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . using the r function boxplot , raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes .
this analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . for each sample
the scattered plots of raw and normalized log2 data were compared using the r function pairs .
pearson correlation tables were used to define the linear relationship of the two variables , raw and normalized data , in the scatter plots . for each technical repeat , we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of < 0.01 .
scatter plots showed that technical replicates had high repeatability and all the correlation values were > 0.953 .
our article , , emphasizes the differentially expressed genes of tissues that received different amounts of radiation ( i.e. 0 , 2 , or 12 gy ) , while herein we show the profiling of all the transcriptomic data resulting from these treatments .
we only used genes with | fold change | > 1.5 and p - value < 0.05 .
we input gene symbols into david bioinformatics , while throughout we used the default settings .
the threshold for significance was a benjamini - adjusted p - value < 0.05 . based on our hypothesis , irradiated samples ( compared to non - irradiated control samples ) would display patterns of gene expression consistent with the physiological effects of irradiation .
table 2 includes selected enriched categories from our qc enrichment analysis ( table s1 contains all enrichment analysis results ) .
that is , up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death , cell proliferation , oncogenesis , and ubiquitin conjugation .
also , down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division .
overall , these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in .
the following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics .
up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .
goterm cc = gene ontology cellular component , mf = molecular function , bp = biological process , and up_seq_feature = uniprot sequence feature .
other enriched categories are from the interpro , sp pir , smart , and kegg databases .
count = the number differentially expressed genes annotated with a given enriched term .
adj p - val = benjamini - adjusted p - values . more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file = function_annotation.html .
up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .
goterm cc = gene ontology cellular component , mf = molecular function , bp = biological process , and up_seq_feature = uniprot sequence feature .
other enriched categories are from the interpro , sp pir , smart , and kegg databases .
count = the number differentially expressed genes annotated with a given enriched term .
more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file = function_annotation.html .
tissue culture methods and irradiation procedure have been described in . the three dimensional tissue culture model
( 3d epioral ) and media were purchased from mattek corporation ( ashland , ma ) .
the 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . in order to induce differentiation and stratification of the keratinocytes of the 3d cultures ,
then the tissues were irradiated ( dose of irradiation was 0 , 2 , or 12 gy ) at the city of hope ( duarte , ca ) facility .
subsequently the tissues were incubated for 6 h at 37 c with 5% co2 after irradiation .
then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna .
the rneasy plus mini kit ( qiagen , germantown , md ) was used to extract total rna of at least two identically treated tissues .
the integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) . only rna samples with absorbance ratio , a260/280 > 2.0 , and
the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase ; the transcription reaction included aminoallyl utp ( aa - dutp ) and the subsequent product was conjugated to cy5 nhs ester ( ge healthcare life sciences , pittsburg , pa ) .
the human whole genome onearray v4.3 ( phalanx biotech , san diego , ca ) was used to perform the dna microarray analysis . for this purpose ,
a quantity of 0.025 mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42 c using the hybbag mixing system with 1 onearray hybridization buffer ( phalanx biotech ) and 0.01 mg / ml sheared salmon sperm dna ( promega , madison , wi ) . after the hybridization procedure ,
the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format .
the data from all the arrays of each experimental set were analyzed using rosetta resolver system ( rosetta biosoftware , usa ) .
testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver .
the error - weighted approach ( which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy ) was used to calculate the average expression values . in order to decide whether intensity data was significantly above background , p - values
were calculated to test the null hypothesis that expression is absent ( called p - value detected ) . a p - value detected < 0.05 indicated that the transcript specific to a given probe was truly present or expressed .
lastly , we also calculated p - values for determining whether genes were differentially expressed . rather than focusing solely on fold changes ,
rosetta resolver uses error - model - based hypothesis tests , which account for both fold change and expression level .
three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . using the r function boxplot , raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes .
this analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . for each sample
the scattered plots of raw and normalized log2 data were compared using the r function pairs .
pearson correlation tables were used to define the linear relationship of the two variables , raw and normalized data , in the scatter plots . for each technical repeat , we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of < 0.01 .
scatter plots showed that technical replicates had high repeatability and all the correlation values were > 0.953 .
our article , , emphasizes the differentially expressed genes of tissues that received different amounts of radiation ( i.e. 0 , 2 , or 12 gy ) , while herein we show the profiling of all the transcriptomic data resulting from these treatments .
we only used genes with | fold change | > 1.5 and p - value < 0.05 .
we input gene symbols into david bioinformatics , while throughout we used the default settings .
the threshold for significance was a benjamini - adjusted p - value < 0.05 . based on our hypothesis , irradiated samples ( compared to non - irradiated control samples ) would display patterns of gene expression consistent with the physiological effects of irradiation .
table 2 includes selected enriched categories from our qc enrichment analysis ( table s1 contains all enrichment analysis results ) .
that is , up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death , cell proliferation , oncogenesis , and ubiquitin conjugation .
also , down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division .
overall , these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in .
the following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics .
up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .
goterm cc = gene ontology cellular component , mf = molecular function , bp = biological process , and up_seq_feature = uniprot sequence feature .
other enriched categories are from the interpro , sp pir , smart , and kegg databases .
count = the number differentially expressed genes annotated with a given enriched term .
more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file = function_annotation.html .
up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .
goterm cc = gene ontology cellular component , mf = molecular function , bp = biological process , and up_seq_feature = uniprot sequence feature .
other enriched categories are from the interpro , sp pir , smart , and kegg databases .
count = the number differentially expressed genes annotated with a given enriched term .
more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file = function_annotation.html .
|
we evaluated a three - dimensional ( 3d ) human oral cell culture that consisted of two types of cells , oral keratinocytes and fibroblasts as a model of oral mucositis which is a debilitating adverse effect of chemotherapy and radiation treatment .
the 3d cell culture model was irradiated with 12 or 2 gy , and total rna was collected 6 h after irradiation to compare global gene expression profiles via microarray analysis . here
we provide detailed methods and analysis on these microarray data , which have been deposited in gene expression omnibus ( geo ) : gse62395 .
|
[
2,
2834,
1688,
110,
151,
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883,
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127,
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diabetes mellitus ( dm ) is a group of metabolic disorders , mainly characterized by impaired glucose metabolism and consequent hyperglycemia as the common feature . whereas cardiovascular complications are well described for dm , it is a relatively new consideration that diabetic patients are highly predisposed to cancer . in spite of some ethnic- , gender- , and age - specific differences , significantly increased risk of liver , pancreas , bladder and digestive tract cancer types
1general predisposition of diabetics to single cancer types : breast , endometrial , colorectal , pancreas , and liver cancers general predisposition of diabetics to single cancer types : breast , endometrial , colorectal , pancreas , and liver cancers many population studies indicate an increased risk for almost all kinds of cancer in dm with particular ethnic- as well as gender - dependent preferences ( fig . 2 ) .
2population studies in japan as an example of cancer predisposition of diabetics in years 19902003 population studies in japan as an example of cancer predisposition of diabetics in years 19902003 with respect to single dm - types , population studies have been carried out in different countries showing geographic particularities in cancer incidence . thus , long - term monitoring of a cancer - specific incidence in the subpopulation of type 1 diabetics was performed in sweden during 19651999 .
it is noteworthy that particular risk in childhood was demonstrated for endometrial , buccal - cavity , bladder as well as cervix cancer types ( fig .
, the highest risk has been registered for cancer of stomach , endometrium , cervix , and bladder .
3specific cancer incidence with respect to age in swedish diabetics type 1 registered in years 19651999 specific cancer incidence with respect to age in swedish diabetics type 1 registered in years 19651999 similar studies carried out for type 2 diabetes demonstrate liver , pancreas and bladder cancer to be the leading types ( fig . 4 ) .
4cancer risk for type 2 diabetics in north italy in years 19871996 cancer risk for type 2 diabetics in north italy in years 19871996
according to the current worldwide statistics , every 10 s one patient dies due to dm - related consequences : dm is currently the fourth leading cause of death .
furthermore , accumulating data demonstrate that , once appeared , cancer outcomes have worse prognosis for diabetics compared to non - diabetic oncologic patients ( fig . 5 ) .
5mortality by cancer in diabetic versus non - diabetic patients registered in netherlands in years 19952002 mortality by cancer in diabetic versus non - diabetic patients registered in netherlands in years 19952002 this general figures are well - supported by data collected in usa to treatment of single cancer types as represented in fig .
these data clearly indicate less success in treatment of cancer for dm - patients .
6increased mortality of diabetics versus non - diabetics for single cancer types as documented for patients treated in usa in years 19821998 increased mortality of diabetics versus non - diabetics for single cancer types as documented for patients treated in usa in years 19821998 the hindered treatment of oncologic diseases in diabetes care is , however , not the only reason of significantly increased cancer - related mortality of diabetics .
recent studies indicate that diabetics are generally predisposed to cancer development [ 29 , 1122 ] . here
we overview subcellular and molecular mechanisms that can contribute to cancer - provocation in diabetics .
increased oxidative stress has been implicated in molecular pathomechanisms of dm and majority of chronic diseases developed secondary to diabetes [ 2325 ] .
several studies demonstrated the excessive production of ros to be the common feature of both diabetes types [ 10 , 22 , 2628 ] .
disturbed glucose / insulin homeostasis initiates overall cellular stress leading to ir / reversible damage to subcellular structures [ 28 , 29 ] .
these findings indicate an imbalance between the increased production of ros and decreased dna - repair capacity . depending on a quality of cell - cycle controlling machinery ,
this imbalance can lead either to extensive apoptotic cell lost or proliferation of damaged cells . whereas the first process causes mainly tissue - degeneration , the latter predisposes to cancer development .
both degenerative alterations in damaged organs and predisposition to cancer have been reported for dm - patients .
extensive damage to dna as well as mutations has been demonstrated also for mitochondria in dm - patients [ 28 , 32 , 33 ] .
mitochondrial genetic background as well as mitochondrial stress - response is considered as an important contributor in predisposition to cancer in diabetics [ 32 , 34 , 35 ] . electron transfer chain ( etc ) is the main energy source essential for performance of all cellular functions . particularly , dna - repair is a highly energy - consuming machinery , the efficiency of which obligatory depends on the quality of mitochondria .
mitochondrial dna variations may affect a highly sensitive balance between etc - efficiency and production of ros in favor of the latter increasing , therefore , mutagenic effects of ros and decreasing energy production .
this is so called vicious circle resulting in mitochondrial dysfunction ( fig .
7 ) . as for mitochondrial protein repertoire , both quantitative and qualitative changes in nd1-nd6 ( nadh - dehydrogenases of the etc - complex i ) have been shown to contribute to the vicious circle resulting in premature aging and plenty of pathologies including neurodegeneration and cancer . in diabetics , the mitochondrial vicious circle has been implicated specifically in individual predisposition to breast cancer .
causes a dangerous imbalance between highly increased production of ros on one side and low energy production on the other side .
highly increased damage to chrdna and mtdna , remarkably decrease repair capacity as well as compromise cell - cycle control are direct consequences of stress - provoked mitochondrial dysfunction [ 22 , 26 , 28 , 33 , 36 ] mitochondrial vicious circle causes a dangerous imbalance between highly increased production of ros on one side and low energy production on the other side .
highly increased damage to chrdna and mtdna , remarkably decrease repair capacity as well as compromise cell - cycle control are direct consequences of stress - provoked mitochondrial dysfunction [ 22 , 26 , 28 , 33 , 36 ] patients with diabetic history are at increased risk of infection [ 3840 ] .
there is a growing body of evidence for compromised immune response in this patient cohort as a consequence of metabolic syndrome [ 4143 ] .
this imbalance can lead to highly increased incidence of viral infection potentiating cancer risk in diabetics [ 4346 ] .
currently it is already well - acknowledged that more than 15 % of viral infections are able to cause cancer in humans .
thus , human papillomavirus ( hpv ) infection is attributed to 80 % of all human cancers and was proposed to play a central role in molecular pathomechanisms of breast cancer [ 4850 ] .
integration of viral particles in human genome frequently results in activation of several proto - oncogenes , which in turn trigger tumorigenic mechanisms in affected cells [ 22 , 48 , 5153 ] .
thus , a viral activation of proto - oncogene c - myc is well described in literature .
thereby , a targeted integration of viral particles in human genome plays the crucial role for consequent cancer development [ 47 , 48 , 54 ] . in this context
, hpv demonstrates clear preference for its integration sites , mainly in regions situated closely to c - myc coding sequences .
once activated , c - myc protein suppresses the cell - cycle controlling activity of p53 , and allows , therefore , the development of new tumorigenic phenotype of transformed human cells [ 22 , 48 , 52 ] . in consensus , the activated synthesis of viral proteins e6 , e7 , e1 and e2 has been shown to be involved in cancer - related cell transformation [ 48 , 55 ] .
most relevant mechanisms for viral etiology of cancer predisposition , particularly , in dm - pathology are summarized in fig .
8clue to viral etiology in dm - provoked cancer : vicious circle is the particularity of metabolic syndrome with high risk of cancer development [ 22 , 26 , 28 , 33 , 36 , 3845 , 5156 ] clue to viral etiology in dm - provoked cancer : vicious circle is the particularity of metabolic syndrome with high risk of cancer development [ 22 , 26 , 28 , 33 , 36 , 3845 , 5156 ]
increased oxidative stress has been implicated in molecular pathomechanisms of dm and majority of chronic diseases developed secondary to diabetes [ 2325 ] .
several studies demonstrated the excessive production of ros to be the common feature of both diabetes types [ 10 , 22 , 2628 ] .
disturbed glucose / insulin homeostasis initiates overall cellular stress leading to ir / reversible damage to subcellular structures [ 28 , 29 ] .
these findings indicate an imbalance between the increased production of ros and decreased dna - repair capacity . depending on a quality of cell - cycle controlling machinery ,
this imbalance can lead either to extensive apoptotic cell lost or proliferation of damaged cells . whereas the first process causes mainly tissue - degeneration , the latter predisposes to cancer development .
both degenerative alterations in damaged organs and predisposition to cancer have been reported for dm - patients .
extensive damage to dna as well as mutations has been demonstrated also for mitochondria in dm - patients [ 28 , 32 , 33 ] .
mitochondrial genetic background as well as mitochondrial stress - response is considered as an important contributor in predisposition to cancer in diabetics [ 32 , 34 , 35 ] . electron transfer chain ( etc ) is the main energy source essential for performance of all cellular functions . particularly , dna - repair is a highly energy - consuming machinery , the efficiency of which obligatory depends on the quality of mitochondria .
mitochondrial dna variations may affect a highly sensitive balance between etc - efficiency and production of ros in favor of the latter increasing , therefore , mutagenic effects of ros and decreasing energy production .
this is so called vicious circle resulting in mitochondrial dysfunction ( fig .
7 ) . as for mitochondrial protein repertoire , both quantitative and qualitative changes in nd1-nd6 ( nadh - dehydrogenases of the etc - complex i ) have been shown to contribute to the vicious circle resulting in premature aging and plenty of pathologies including neurodegeneration and cancer . in diabetics , the mitochondrial vicious circle has been implicated specifically in individual predisposition to breast cancer .
causes a dangerous imbalance between highly increased production of ros on one side and low energy production on the other side .
highly increased damage to chrdna and mtdna , remarkably decrease repair capacity as well as compromise cell - cycle control are direct consequences of stress - provoked mitochondrial dysfunction [ 22 , 26 , 28 , 33 , 36 ] mitochondrial vicious circle causes a dangerous imbalance between highly increased production of ros on one side and low energy production on the other side .
highly increased damage to chrdna and mtdna , remarkably decrease repair capacity as well as compromise cell - cycle control are direct consequences of stress - provoked mitochondrial dysfunction [ 22 , 26 , 28 , 33 , 36 ]
there is a growing body of evidence for compromised immune response in this patient cohort as a consequence of metabolic syndrome [ 4143 ] .
this imbalance can lead to highly increased incidence of viral infection potentiating cancer risk in diabetics [ 4346 ] .
currently it is already well - acknowledged that more than 15 % of viral infections are able to cause cancer in humans .
thus , human papillomavirus ( hpv ) infection is attributed to 80 % of all human cancers and was proposed to play a central role in molecular pathomechanisms of breast cancer [ 4850 ] .
integration of viral particles in human genome frequently results in activation of several proto - oncogenes , which in turn trigger tumorigenic mechanisms in affected cells [ 22 , 48 , 5153 ] .
thus , a viral activation of proto - oncogene c - myc is well described in literature . thereby , a targeted integration of viral particles in human genome plays the crucial role for consequent cancer development [ 47 , 48 , 54 ] . in this context
, hpv demonstrates clear preference for its integration sites , mainly in regions situated closely to c - myc coding sequences .
once activated , c - myc protein suppresses the cell - cycle controlling activity of p53 , and allows , therefore , the development of new tumorigenic phenotype of transformed human cells [ 22 , 48 , 52 ] . in consensus , the activated synthesis of viral proteins e6 , e7 , e1 and e2 has been shown to be involved in cancer - related cell transformation [ 48 , 55 ] .
most relevant mechanisms for viral etiology of cancer predisposition , particularly , in dm - pathology are summarized in fig .
8clue to viral etiology in dm - provoked cancer : vicious circle is the particularity of metabolic syndrome with high risk of cancer development [ 22 , 26 , 28 , 33 , 36 , 3845 , 5156 ] clue to viral etiology in dm - provoked cancer : vicious circle is the particularity of metabolic syndrome with high risk of cancer development [ 22 , 26 , 28 , 33 , 36 , 3845 , 5156 ]
taking together the above given facts , we conclude that diabetics may be highly predisposed to cancer development specifically due to following contributors :
strong stress factors ( excessive metabolic alterations , disturbed glucose / insulin homeostasis , hormonal deregulation , insufficient detoxification ) with consequently excessive production of rosmitochondrial dysfunction with consequent low energy production , insufficient repair capacity and accumulating damage to both chromosomal and mitochondrial dnahigh risk for infectious disorders with consequently induced viral proto - oncogenic activity as well as activity of particular pathogenic bacterial forms such as helicobacter pylori . strong stress factors ( excessive metabolic alterations , disturbed glucose / insulin homeostasis , hormonal deregulation , insufficient detoxification ) with consequently excessive production of ros mitochondrial dysfunction with consequent low energy production , insufficient repair capacity and accumulating damage to both chromosomal and mitochondrial dna high risk for infectious disorders with consequently induced viral proto - oncogenic activity as well as activity of particular pathogenic bacterial forms such as helicobacter pylori .
adequacy of stress response , repair capacity as well as immune defence are highly individual for each patient and strongly depend on risk factors such as genetic background , age , environmental factors , nutrition , body culture , life style , etc .
thus , varying breast cancer risk in different ethnic and social groups is well documented [ 16 , 5860 ] . breast fat deposits and distribution increase risk for breast cancer in female and even in male patients [ 16 , 6165 ] .
in contrast , breast cancer development is significantly reduced in people with regular physical activity .
alcohol abuse and tobacco consumption are further contributors which remarkably increase risk of cancer development [ 17 , 20 , 6771 ] .
current biotechnology possesses sufficient power to estimate the severity of damage to subcellular structures , individual stress reactions and repair capacity . for example , by stress proteome profiling in peripheral leukocytes and blood plasma , individual stress reactions can be well estimated .
advanced predictive diagnostic approaches are currently close to clinical application and allow to select groups of risk and to estimate a predisposition to severe complications in diabetics [ 34 , 8284 ] .
much attention should be focused on preventive measures in diabetic healthcare , in order to restrict or even avoid severe secondary complications , such as cancer .
nowadays , it is increasingly clear that a well - balanced individually created diet is considered as an effective preventive measure and treatment of majority of chronic complications in diabetics [ 8688 ] .
|
diabetes mellitus ( dm ) is a lifelong progressive disease with high morbidity and mortality worldwide .
whereas cardiovascular complications are well - known for dm , increasing evidence indicates that diabetics are predisposed to cancer .
understanding of molecular pathomechanisms of cancer in dm is of great importance .
dysregulation of glucose / insulin homeostasis leads to increased production of reactive oxygen / nitrogen species ( ros / rns ) and consequent damage to chromosomal / mitochondrial dna , a frequent finding in dm .
long - term accumulation of modified / damaged dna is well - acknowledged as triggering cancer .
dna - repair is a highly energy consuming process provoking increased mitochondrial activity . particularly dangerous is a provoked activity of damaged mitochondria leading to a vicious circle lowering energy supply and potentiating ros / rns production .
mitochondrial dysfunction may be implicated in pathomechanisms of diabetes - related cancer .
high risk for infectious disorders and induced viral proto - oncogenic activity may further contribute to cancer provocation . much attention should be focused on preventive measures in diabetic healthcare , in order to restrict severe diabetes - related complications .
|
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pulmonary hypertension is an increasingly recognized complication of sickle - cell anaemia and a risk factor for early death [ 14 ] .
recurrent episodes of acute and subacute pulmonary crises reflecting in situ sickling within the lung has been postulated to be the primary event leading to pulmonary hypertension .
haemolysis may participate in its pathogenesis by limiting nitric oxide ( no ) bioavailability and producing vasculopathy [ 5 , 6 ] .
an initial study in howard university , usa , using echocardiographic assessment of tricuspid valve regurgitant jet velocity 2.5 m / sec as diagnostic criteria , demonstrated pulmonary hypertension in 32% of adult sickle - cell patients , and the prevalence appeared to increase with age of the patients . in patients
between 40 and 49 years old , the prevalence was 40% and increased to 5560% by age 50 and above .
other studies have documented prevalence rates between 20 and 40% [ 4 , 810 ] .
sickle - cell anaemia patients with pulmonary hypertension have a significantly increased mortality rate compared with patients without pulmonary hypertension .
sutton and colleagues reported 40% mortality rate in sickle - cell patients with pulmonary hypertension at 22 months after diagnosis ( odd ratio 7.86 ; 95% confidence interval = 2.6323.4 ) compared with sickle - cell patients without pulmonary hypertension .
castro et al . in a study of 34 adult sickle - cell patients who underwent right heart catheterization for evaluation of pulmonary hypertension found increased pulmonary artery pressure in 20 ( 58.8% ) on initial catheterization . during 2345 months of
follow up , 11 of these 20 ( 55.0% ) died compared with 3 of the 14 without pulmonary hypertension ( 21.0% ) .
every 10 mmhg increase in mean pulmonary artery pressure was associated with 1.7 increase in mortality ( 95% confidence interval = 1.12.7 ; cox proportional hazard model , p = 0.028 ) .
the study was aimed at comparing the clinical and electrocardiographic findings in sickle - cell anaemia patients with raised pulmonary artery pressure with those of patients without pulmonary hypertension .
the study was carried out in the adult outpatient sickle - cell clinic and the cardiac centre of the university of nigeria teaching hospital ( unth ) , enugu , nigeria .
the study subjects were drawn from adult patients ( age 18 years ) , attending the adult sickle - cell clinic of the hospital , who had haemoglobin genotype ss on haemoglobin electrophoresis , were in steady state , and consented to participate in the study .
steady state is defined as absence of any crisis in the preceding four weeks , absence of any symptoms , or signs attributable to acute illness .
a total of sixty two sickle - cell anaemia patients , and sixty two age- and sex - matched normal controls were studied .
resting 12-lead electrocardiography was performed on all subjects using cardioline ar-600 model electrocardiography machine at a paper speed of 25 mm / s and standardized at 0.1 mv / mm . a single observer analyzed the electrocardiogram .
measurements of the heart rate , cardiac axis , pr- interval , qrs duration , and qtc interval were done in the standard fashion .
heart rate correction of the qt interval was performed using bazett 's formula ( qtc = qt/ rr ) .
the dispersion of p - wave , qrs , and qtc intervals was measured manually under magnifying glass by the same observer and was taken as the difference between the maximum and minimum values of each parameter on standard 12-lead electrocardiogram . left ventricular hypertrophy on electrocardiogram was based on sokolow and lyon voltage criteria , while right ventricular hypertrophy was based on the criteria described by allenstein and mori ( dominant or tall r - waves or rs pattern in a vr , v1 , and v2 with deep s - wave in i , avl , v5 , and v6 ) .
echocardiography was done using hewlett packard sonos 2500 echocardiography machine with 3.7 mhz transducer .
pulmonary artery flow acceleration time was obtained from a doppler signal of the pulmonary flow in the parasternal short - axis view at the aortic valve level and was described as the time from onset to peak flow velocity . the mean pulmonary artery pressure ( mean pap )
was calculated from the formula : mean pap ( m
m / h
g ) = 90 ( 0.62 a
t ) , where at is the acceleration time of the pulmonary artery flow .
pulmonary hypertension was defined as calculated mean pulmonary artery pressure 30 mm / hg [ 4 , 7 ] .
ethical clearance for the study was obtained from the ethical committee of the university of nigeria teaching hospital , enugu , nigeria .
the declaration of helsinki 's recommendations for guiding physicians in biomedical research involving human subjects were followed .
data were presented as means standard deviation for continuous variables and as proportions for categorical variables
. comparisons of continuous variables between groups were made with independent student 's t - test .
for discrete variables , distribution between groups was compared with chi - square test and fishers exact test as appropriate ( where an expected cell is less than 5 ) .
multivariate pearson 's correlation coefficient was used to determine the relations between clinical , electrocardiographic data and mean pulmonary artery pressure .
all statistical analyses were carried out using the statistical packages for social sciences ( spss inc . ,
chicago , illinois ) software version 11.0 and epi - info version 3.4 . statistical tests with 2-tailed probability values less than 0.05 were considered statistically significant .
the age , gender , and anthropometric parameters of the patients and controls are shown in tables 1 and 2 .
values for pulmonary artery pressure in the patients and controls are shown in table 3 .
elevated pulmonary artery pressures ( pap ) as defined by pap 30 mmhg was demonstrated in ( 26 ) 41.9% of patients with sickle - cell anaemia and in ( 2 ) 3.2% of the controls ;
= 26.571 , df = 1 , p < 0.001 ( figure 1 ) .
p - wave duration , qtc interval , and qtc dispersion were increased in patients with pulmonary hypertension ( table 5 ) .
significant correlation was found between mean pap and ( 1 ) frequency of crisis ( spearman correlation = 0.320 ; p = 0.011 ) , ( 2 ) body mass index ( pearson 's correlation = 0.297 ; p = 0.019 ) , and ( 3 ) qtc interval ( pearson 's correlation 0.261 ; p = 0.040 ) , ( table 6 ) .
the prevalence of pulmonary hypertension in adult nigerian sickle - cell anaemia patients in this study was 41.9% .
this value is slightly higher than previous report of 25% by aliyu et al . in northern nigeria .
however , in that study , neither the mean pap nor the mean age of the subjects was stated .
previous studies have relied on tricuspid regurgitant jet velocity as an indirect estimate of pap .
this has resulted in an underestimation of the prevalence of pulmonary hypertension in sickle - cell anaemia patients .
results of right heart catheterization for evaluation of pulmonary artery pressures in sickle - cell patients have shown prevalence rate of 58.8% .
autopsy studies suggest that up to 75% of sickle - cell anaemia patients have histological evidence of pulmonary arterial hypertension at the time of death .
mean pap derived from the echocardiographic estimation of pulmonary artery flow acceleration time in steady - state patients as was used in this study has been shown to correlate significantly with values from cardiac catheterization .
several studies have corroborated the role of vaso - occlusive crisis and acute chest syndrome in initiating nitric oxide depletion resulting in the pathological changes in pulmonary hypertension [ 6 , 19 ] .
this study identified a positive correlation between pulmonary artery pressure and frequency of vaso - occlusive crisis .
patients with pulmonary hypertension were found to have reduced body mass index when compared with patients without pulmonary hypertension .
this difference could be due to increased disease severity in patients with pulmonary hypertension .
recurrent insitu vaso - occlusive crisis in the pulmonary vascular bed with resultant pulmonary hypertension and right ventricular hypertrophy has been demonstrated in sickle - cell disease patients .
this is in keeping with the finding by this study of a significantly increased prevalence of right ventricular hypertrophy in patients with pulmonary hypertension .
right ventricular hypertrophy in these patients explains the significant positive correlation between pulmonary artery pressure and qtc interval .
the significance of increased p - wave duration and the spatial dispersion of qtc - interval noted in this study in patients with pulmonary hypertension are unclear .
qtc dispersion is a measure of the disparity among qtc intervals in various electrocardiographic leads and reflects the variability of myocardial repolarization .
this corroborates the finding in a study by akgul et al . in turkey which recorded higher qtc dispersion in sickle - cell patients with pulmonary hypertension .
pulmonary hypertension in adult sickle - cell anaemia patients is significantly associated with electrocardiographic evidence of right ventricular hypertrophy , increased p - wave duration , qtc interval , and qtc dispersion and correlates significantly with frequency of vaso - occlusive crisis and qtc interval .
the observations by this study tend to suggest that these parameters could be useful for early detection and prevention of pulmonary hypertension in patients with sickle - cell anaemia .
|
pulmonary hypertension is an emerging complication of sickle cell anaemia with associated increased risk of mortality . in order to evaluate the clinical and electrocardiographic findings in adult sickle - cell patients with pulmonary hypertension ,
a cross sectional study was conducted on sixty two sickle cell anaemia patients and sixty two age and sex matched normal controls .
elevated pulmonary artery pressures ( pap ) , defined by pap 30 mm hg on echocardiography , was demonstrated in 41.9% of patients with sickle cell anaemia and in 3.2% of the controls ;
2 = 26.571 , p < 0.001 .
right ventricular hypertrophy , increased p - wave duration , qtc interval , and qtc dispersion were significantly associated with pulmonary hypertension .
significant correlation was found between mean pap and ( 1 ) frequency of crisis ( spearman correlation = 0.320 ; p = 0.011 ) , ( 2 ) body mass index ( pearson 's correlation = 0.297 ; p = 0.019 ) , and ( 3 ) qtc interval ( pearson 's correlation 0.261 ; p = 0.040 ) . pulmonary hypertension in adult sickle anaemia patients is associated with electrocardiographic evidence of right ventricular hypertrophy , and correlates significantly with frequency of vaso - occlusive crisis , and qtc interval .
the observations by this study tend to suggest that these parameters could be useful for early detection and prevention of pulmonary hypertension in patients with sickle cell anaemia .
|
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in the last decade we have witnessed a dramatic increase both in the proportion and absolute number of bacterial pathogens presenting multidrug resistance to antibacterial agents .
organizations such as the us centers for disease control and prevention ( cdc ) , the european centre for disease prevention and control ( ecdc ) and the world health organization ( who ) are considering infections caused by multidrug - resistant ( mdr ) bacteria as an emergent global disease and a major public health problem .
the emergence of resistant microorganisms , either by mutations or the acquisition of mobile genetic elements carrying resistance genes , may take place irrespective of the presence of antibacterial agents .
it is the exposure to these drugs what provides the necessary selective pressure for the rise and spread of resistant pathogens .
therefore , the driving force behind the increasing rates of resistance can ultimately be found in the abuse and misuse of antibacterial agents , whether used in patients and livestock or released into the environment .
antimicrobial resistance has become a global health threat that will require the coordinated action of many different stakeholders to tackle antibiotic resistance at its very root .
the aim of the meeting organized jointly by b - debate ( bio - cat ) and the barcelona institute for global health ( isglobal ) in partnership with the european society of clinical microbiology and infectious diseases ( escmid ) and the spanish network for research in infectious diseases ( reipi ) was to generate debate among the main stakeholders ( i.e. policy makers , public health authorities , regulatory agencies , pharmaceutical companies and the scientific community at large ) and come up with a coordinated set of strategies to fight antimicrobial resistance in a multifaceted approach .
the meeting focused on three major areas : antimicrobial resistance in animals and the food chain ; in the environment and the community ; and within the healthcare setting .
the widespread use of antimicrobial agents in animals and the food chain constitutes an important source of antimicrobial resistance , although the impact of such use on human health remains controversial .
massive amounts of antibiotics have been used as growth promoters as well as for prophylaxis and the treatment of infections among farm animals and in aquaculture , increasing the selective pressure on both commensal and pathogenic microorganisms that can spread to humans through direct contact and via the food chain or indirectly from the environmental pollution of farm effluents .
interventions to limit the emergence and spread of resistant bacteria in the animal setting may include the following : ( a ) banning antibiotic use as growth promoters and limiting its use for other nontherapeutic applications , ( b ) reducing the dissemination of mdr bacteria through the food chain by improving farm biosecurity and developing alternative treatment strategies and increasing hygienic conditions and practices along the food chain , ( c ) developing education programs , mainly directed at veterinarians , farmers , and food handlers and ( d ) linking surveillance systems on antibiotic resistance established for humans and animals .
the european food safety authority ( efsa ) is playing an essential role in detecting emerging risks in the area of mdr bacteria within the food industry .
several proposals have been made by efsa for the harmonization of monitoring and reporting of resistant bacteria , such as : ( a ) agreeing a comprehensive set of antibacterial agents to be included in the monitoring plans , ( b ) reinforcing antimicrobial resistance monitoring in sentinel bacteria , ( c ) conducting active monitoring programs in healthy animals based on randomized sampling plans and ( d ) harmonizing of epidemiological values .
antibiotics that have become critical for human health should be clearly identified and their use restricted to humans only in order to avoid cross - resistance . in this respect , the who has established a list of essential antimicrobial agents for human use to be avoided in nonhuman interventions .
compliance with the who recommendations , however , is neither mandatory nor regulated . who has also initiated different collaborative programs to tackle foodborne antimicrobial resistance through the promotion of national coordination and integrated surveillance , prevention and control of antibiotic resistance in the food chain and the improvement of awareness on antibiotic use and risk of resistance among veterinarians and the food industry .
antimicrobial resistance in the community has steadily been increasing during the last decades , especially regarding resistance to quinolones , carbapenems and third - generation cephalosporins .
the latter relates to the increased prevalence of extended - spectrum -lactamases among enterobacteriaceae and with the spread of high - risk clones such as escherichia coli st131 .
surveillance studies of antimicrobial resistance and antibiotic consumption have drawn attention to this phenomenon and should be used to drive political campaigns to contain resistance .
the task of the cdc / ecdc is crucial in identifying , assessing and communicating current and emerging human health threats on antimicrobial resistance .
the latest ecdc report on antimicrobial resistance surveillance in europe ( http://www.ecdc.europa.eu/en/publications/publications/antimicrobial-resistance-surveillance-europe-2012.pdf ) showed that methicillin - resistant staphylococcus aureus prevalence is stabilizing or even decreasing in some countries , while resistance to third - generation cephalosporins in particular and multidrug resistance ( three or four antibacterial agents ) in general continues to show a sharp and widespread increase in e. coli and klebsiella pneumoniae .
currently , antimicrobial consumption data from the european union and countries belonging to the european economic area / european free trade association are expressed as a number of defined daily doses ( ddd ) per 1000 inhabitants and per day .
complementary to ddd , the number of packages per 1000 inhabitants and per day are also reported , depending on the availability of data on packages from the national surveillance networks .
information on packages is deemed to improve the understanding and interpretation of differences in the levels and trends of antimicrobial consumption observed between and within countries , as the ddd system can not take into account changes in package content .
in addition , a drug resistance index that aggregates information about antibiotic resistance and antibiotic used into a single composite measure has also been proposed .
such drug resistance index , similar to the way the dow jones is used in economics , would allow the continuous quantitation of antibiotic effectiveness overtime in particular geographic areas .
as stated above , antibiotic abuse has greatly contributed to speed up the development of antibiotic resistance , and in this regard human medicine has played a key role .
inappropriate prescribing ( whether caused by obsolete guidelines or pharmaceutical pressures ) , over - the - counter antibiotic availability and self - medication reflect a general lack of awareness on the global threat that antibiotic resistance poses to our society . educational programs on the rational use of antibiotics addressed to primary care physicians , drug vendors and the community in general must be enforced to ease the pressure on prescribers and reduce antibiotic consumption .
similarly , the prescription of delayed receipts conditioned to the remission or worsening of clinical symptoms might also contribute to such reduction .
additional measures should include up - to - date local antibiotic prescribing guidelines , active reporting on antibiotic prescribing and consumption and the enforcement of local surveillance programs on antibiotic resistance .
the implementation of such measures , however , needs substantial legislation amendments and increased funding , which depend on a strong commitment by policy makers at both national and international scales .
of particular note is the use of antibiotics in low - income countries , where there are additional factors contributing to the emergence of resistance , including ( a ) less potent activity of some antibacterial agents ( including counterfeit drugs ) , ( b ) over - the - counter availability , with insufficient dosages , ( c ) lack of diagnostic laboratories and ( d ) poor level of sanitation , facilitating the familiar and community spread of resistant organisms .
the excessive use of antimicrobial agents to treat both humans and animals has also caused the accumulation of these compounds in the environment , and the impact of such accumulation on the emergence of antibiotic resistance should not be underrated .
antibacterial agents have several routes of entry into the environment , such as sewage from the community or hospitals through manure and water bodies .
the accumulation of antibacterial agents further selects resistant microorganisms , turning the environment into a gigantic reservoir for antibiotic resistance genes that feeds on the constant and increasing environmental pollution .
wastewater treatment plants have become a hot spot for horizontal gene transfer and the coselection of genetic determinants providing resistance to antibiotics , pollutants , heavy metals , biocides , disinfectants , or detergents .
the current legislation on water quality mainly focuses on the presence of indicator microorganisms but does not address the antibiotic concentrations of sewages and treatment plants .
strategies to mitigate the risks of environmental exposure should be aimed at improving industrial systems for sanitation and decontamination of hospital sewage water .
the concurrence of high antibiotic consumption , critically ill patients and a permanent influx of pathogenic species within the healthcare setting nurtures the development of resistance and provides an ideal scenario for the dissemination of resistant microorganisms and horizontal transfer of resistance genes .
the degree of antimicrobial resistance in these settings depends on intrinsic factors related to the particular idiosyncrasies of each centre as well as on external factors such as the influx of resistant pathogens that originate in the community .
intrinsic differences in resistance rates between hospitals can be attributed to use of individual rooms vs. two or three bedrooms or open units , staffing , antibiotic stewardship , environmental cleaning , adherence to hand hygiene precautions and infection control programs .
antimicrobial consumption also shows huge interhospital differences that might be partially explained by case mix as well as by differences in the flow of patients carrying resistance bacteria that are transferred from other healthcare facilities . in order to minimize the unwanted consequences of antimicrobial use , implementation of antimicrobial stewardship programs
ideally , antimicrobial stewardship should be designed to include the following : ( a ) passive educational measures ( antibiotic guidelines , educational sessions ) , ( b ) active interventions ( clinical rounds , prospective audits , reassessment of antibiotic perspectives ) , ( c ) restrictive measures ( limiting antibiotics on the hospital formulary , reporting of susceptibility by the microbiology laboratory , antibiotic order form , preauthorization ) , and ( d ) supplemental measures ( computer - assisted management programs , multidisciplinary stewardship teams , consultancy services ) .
in addition , infection control measures should be planned according to the microorganisms of interest , case mix of patients and whether endemic or epidemic .
risk assessment in target pathogens needs to consider ( a ) the pathogen itself , as there are different dosing strategies for different species , ( b ) time of exposure the duration of treatment should be kept for as short as possible , ( c ) drug exposure related to risk in an inverse u relationship , ( d ) patterns of drug exposure , ( e ) inoculum size , with different dosing for high - load ( pneumonia ) and low - load infections ( surgically treated complicated skin and soft tissue infection ) and ( f ) combination therapy to suppress resistance .
we use breakpoints to categorize microorganisms as susceptible ( treatable with the agent in question ) and resistant ( not treatable with the agent ) to guide therapy . at the international level , breakpoints are officially determined by two main institutions : the clinical and laboratory standards institute ( clsi ) in the united states , which is a not - for - profit membership organization sponsored by the industry and the medical community ; and the european committee on antimicrobial susceptibility testing ( eucast ) , a joint effort among escmid , ecdc , and the european medicines agency ( ema ) .
breakpoints are reviewed at regular intervals because changes in doses , administration modes and indications for therapy occur and new resistance mechanisms are discovered .
differences among breakpoints suggested by clsi or eucast are subtle but significant for certain antimicrobial classes , and such differences reflect the use of divergent criteria as well as different conflicts of interest .
in addition , there is also a plethora of likewise national organizations providing breakpoints and guidelines ( i.e. bsac , ca - sfm , crg , din , nwga , srga , among others ) that all together contribute to a very poor standardization of procedures .
an international harmonization of breakpoints is clearly needed , and in order to achieve it , either all countries must adopt the standards of one of the two main committees ( clsi or eucast ) , or a novel international breakpoint committee must be created that embraces all of the above .
the rapid detection of resistance mechanisms plays an important epidemiological role in surveillance studies to evaluate the potential dissemination of resistance genes . from the therapeutic point of view
, however , this information does not exclude the presence of other mechanisms of resistance affecting the same antibacterial agent .
hence , its presence has a high predictive value for resistance but not for susceptibility .
the prompt identification of the antimicrobial susceptibility of a microorganism , on the other hand , ensures the administration of the correct treatment and reduces the need for broad - spectrum drugs , limiting the emergence of antimicrobial resistance .
molecular methods have shortened the time to detect specific resistance mechanisms and the development of next generation sequencing technologies has increased the number of sequenced bacterial genomes at an exponential rate
. a better understanding of the molecular basis of antimicrobial resistance has facilitated the development of bioinformatic tools to identify antibiotic resistance genes in bacterial genomes , such as arg - annot , resfinder , and the card database .
in addition , mass spectrometry techniques have proven extremely useful in the rapid identification of bacterial species and their use in the detection of resistance profiles to certain classes of antibiotics has provided excellent results .
similarly , advanced applications of nanoparticles and bacterial microencapsulation to clinical are very promising and might be fully developed in the years to come .
the development of novel therapeutic strategies , however , seems to have reached a dead end . despite the urgent need to find new antibacterial products , many pharmaceutical companies have abandoned antibiotic drug discovery programs .
several factors have contributed to this situation : ( a ) difficulty predicting the development of resistance adding risk to research and development ( r&d ) investment , ( b ) significant scientific bottlenecks , ( c ) complex and divergent regulatory requirement and ( d ) the challenge of the commercial model there is high investment but low returns compared to alternative r&d investment . relaunching antimicrobial drug discovery and development should be a global priority , and some initiatives have been suggested to encourage investment by the pharmaceutical industry , such as extending the period of exclusivity for certain antibiotics ( i.e. the gain ( generating antibiotics incentives now ) act ) and amending regulatory requirements to accelerate access to antibiotics for serious infections for which there are few , if any , alternatives . promoting research and development of novel antimicrobial drugs needs to address the issue of the challenging commercial model and come up with strategies to reconcile public health needs with an attractive economic model for the pharmaceutical industry .
initiatives such as the 10 20 initiative , promoted by the infectious diseases society of america ( idsa ) , that attempts to produce ten new systemic antibiotics by the year 2020 , or the european innovative medicine initiative ( http://www.imi.europa.eu ) , supporting collaborative research projects between the pharmaceutical industry and the academia , combine public and private funding to invigorate antimicrobial drug research .
one of these research projects to tackle antimicrobial resistance is combacte ( combatting bacterial resistance in europe ) , which aims to give antibiotic drug development a much - needed boost by pioneering new ways of designing and implementing efficient clinical trials for novel antibiotics . fortunately , there are also a handful of small- and medium - size companies as well as multiple research groups that are investing in antimicrobial drug discovery , although such initiatives will eventually have to rely on larger corporations to proceed through the expensive phase 2/3 of clinical development . in this regard ,
efforts are being made by ema to relax the regulations for clinical trials , a measure thought to cheapen and accelerate the release of new drugs to the market .
table 1 summarizes the different strategies that are being used to discover and develop drugs to fight bacteria .
the current global threat of antimicrobial resistance and the urgent need to control it and to find new antibacterial products has prompted the different stakeholders to take action in integrating research and public health , and in maintaining and promoting the national and international antimicrobial resistance research community .
the joint programming initiative on antimicrobial resistance is a collaborative research initiative supported by 18 european countries plus canada as a response to this threat .
it has defined a strategic research agenda under the assumption that only a collaborative effort will provide the necessary critical mass and scientific expertise to answer the most important and urgent research questions related to antimicrobial resistance .
other actions promoted by nonprofit entities such as the world alliance against antibiotic resistance ( waaar , france ) , antibiotic action ( uk ) , react ( sweden ) and the antibiotic resistance initiative ( isglobal , spain ) , among others , are also playing an important role in this process .
to summarize , the following measures can be taken to prevent the emergence and spread of antibiotic resistance worldwide : ( a ) rational use of antibiotics in all settings , ( b ) implementation of infection control measures in healthcare settings , ( c ) development of strategies to mitigate the risks of environmental exposure , ( d ) development of rapid diagnostic tests , ( e ) promotion of research on antibacterial resistance prevention and surveillance , ( f ) promotion of research and development of novel antimicrobial strategies and antibacterial agents and ( g ) improved general awareness of antibiotic use and risk of increasing resistance .
the flow of resistance and the main interventions that are needed to prevent the threat of antibiotic resistance at specific key points are summarized in fig . 1 .
the factors that have led us to the current situation and the measures proposed to circumvent it are no novelty to the scientific community , and much has been written about it .
implementing those measures will demand a concerted action of all stakeholders involved ( policy makers , public health authorities , regulatory agencies , pharmaceutical companies and the scientific community at large ) , but above all , it will require strong regulatory modifications and a great deal of investment .
the treatment of patients infected with drug - resistant pathogens is much more expensive as a result of longer hospitalization times and the use of more expensive last - resort drugs .
the annual economic burden associated with the treatment of antibiotic - resistant infections has been estimated to be between $ 21,000 and $ 34,000 million in the united states alone , and around 1500 million in europe , which includes the economic impact associated with the number of days of lost productivity , estimated to be approximately 450 million each year in europe . a global and coordinated initiative to tackle antibiotic resistance
will be needed to persuade the general population and policy makers of the advantages , both medical and economic , of combating the threat of antimicrobial resistance .
|
in the last decade we have witnessed a dramatic increase in the proportion and absolute number of bacterial pathogens resistant to multiple antibacterial agents .
multidrug - resistant bacteria are currently considered as an emergent global disease and a major public health problem . the b - debate meeting brought together renowned experts representing the main stakeholders ( i.e. policy makers , public health authorities , regulatory agencies , pharmaceutical companies and the scientific community at large ) to review the global threat of antibiotic resistance and come up with a coordinated set of strategies to fight antimicrobial resistance in a multifaceted approach .
we summarize the views of the b - debate participants regarding the current situation of antimicrobial resistance in animals and the food chain , within the community and the healthcare setting as well as the role of the environment and the development of novel diagnostic and therapeutic strategies , providing expert recommendations to tackle the global threat of antimicrobial resistance .
|
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the study was conducted in 100 households of 9 affected villages of engil district on the edge of herat city .
households were included if they were located on 3 randomly selected transect lines and if members of the households owned either cattle or sheep . because there are no reliable estimates on seroprevalence of cchf in afghanistan , we estimated prevalence of immunoglobulin ( ig ) g to be 7% ( 2,3 ) .
a sample size of 160 persons was required to detect this prevalence at the 95% confidence level . allowing for cluster sampling at the household level ,
320 persons was the target sample size ( 90 households , assuming an average of 3.5 persons > 15 years of age per household ) .
from each household , all members > 15 years were surveyed , if they gave consent .
sheep and cattle were selected as livestock types , and 1 or 2 animals per household were randomly selected .
inclusion criteria for humans were residing in a livestock - owning household , giving informed consent , being > 15 years of age , willing to answer the risk factor questionnaire , and willing to give 5 ml of blood .
for collection of blood samples and vector specimens from sheep and cattle , permission was given by the head of household .
a standardized , structured , and pretested questionnaire that covered individual data for each participant , including personal details , exposure variables , and self - reported disease history , was used .
animal data were collected from each household and included each animal s origin , tick exposure , age , and sex .
blood samples from human participants were collected by trained health workers according to standard procedures .
blood samples were centrifuged at room temperature at the local laboratory on the day of collection .
serum was separated , frozen , and transported to kabul for storage and onward shipment . a sandwich / indirect elisa detected specific igg at a 1:100 dilution for all human samples by using the vector - best diagnostic kit , ( vector - best , novosibirsk , russia ) .
we have previously compared this kit with an in - house elisa ( us army medical research institute for infectious diseases , fort detrick , md , usa ) , and the results were comparable in all samples tested ( l. mustafa et al . , unpub .
we used an in - house elisa using the ibar 10200 strain of cchf as antigen ( us army medical research institute for infectious diseases ) and anti - species igg horseradish peroxidase conjugated ( kpl inc . ,
gaithersburg , md , usa ) to test for igg in all animal serum specimens .
data were analyzed by using stata version 8 ( statacorp , college station , tx , usa ) .
the primary outcome was seropositivity among household members , with secondary outcomes being seropositivity in animals and the presence of virus in ticks .
exposure factors for seroprevalence were identified on an a priori basis and appropriate measures of statistical significance were applied to detect differences at the 95% confidence level .
the study was approved by the ethics boards of namru-3 and the afghan public health institute , afghanistan . in total
igg seroprevalence was 37/330 ( 11.2% , 95% confidence interval [ ci ] 8.015.1 ) .
of all the potential explanatory variables , only 2 factors were associated with an elevated risk of igg positivity : daily contact with cattle ( 33/264 [ 12.5% ] vs. 1/52 [ 1.8% ] ; = 5.1 , p = 0.02 ) and exposure to raw animal skins ( 24/144 [ 16.7% ] vs. 12/176 [ 6.8% ] ; = 7.7 , p = 0.006 ) .
self reported clinical illness ( fever ) occurred in 55% of participants over a 5-month reporting period . among the participants
, 20.8% reported that they had had an illness involving bleeding from teeth , gums , and or other parts of the body , but this event was not associated with igg positivity or with age group .
these results suggest that the risk for cchf exposure is uniformly high among the population .
the oldest age group shows an approximate lifetime risk for exposure and seroconversion of 17% ( 95% ci 10.2%25.8% ) .
ninety - two cattle and 40 sheep were included , and serologic analysis of their blood samples was conducted .
seroprevalence was 79.1% ( 95% ci 69.0%87.1% ) among cattle and 75.0% ( 95% ci 57.0%88.5% ) among sheep .
prevalence was uniformly high regardless of age , sex , or origin of the animals , suggesting that the disease is highly endemic in the livestock population . among our sample ,
ticks ( n = 259 ) from domestic animals were predominantly adult hyalomma marginatum ( 94.6% ) .
engorged females were found more on cattle than on sheep ( 43% and 27% , respectively ) .
seroprevalence in this population of animal owners is higher than in other reported studies from the region ( 3,4 ) , and the risk for exposure appears approximately uniform .
the route of transmission to humans is either through the bite of ticks or through contact with infected animals or animal products .
this second route of transmission is probably more important in iran and afghanistan than tick - borne transmission .
control of cchf requires control of the disease vector , and surveillance is necessary to ensure optimum timing of interventions such as livestock dipping or sponging because tick abundance is highly seasonal . further research on rates of antibody acquisition among humans and animals , virus transmission dynamics , and effectiveness of disease control measures is required .
cchf is a regional public health concern of larger than previously acknowledged significance and requires control mechanisms from both the health and agriculture sectors .
|
in response to an outbreak of crimean - congo hemorrhagic fever in western afghanistan , we measured immunoglobulin g seroprevalence among household members and their animals . seroprevalence was 11.2% and 75.0% in humans ( n = 330 ) and livestock ( n = 132 ) , respectively .
persons with frequent exposure to cattle had an elevated risk of being immunoglobulin g positive .
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cancer , which is commonly a harbinger of imminent patient death , is a group of diseases characterized by uncontrolled growth and spread of abnormal cells and aberrant cell behavior , which leads to expansive masses that destroy surrounding normal tissue and is able to attack vital organs resulting in disseminated disease .
it is an inevitable matter of concern in the medicinal chemistry era and an increasing burden to the population . according to estimates from the international agency for research on cancer ( iarc ) , the global burden is expected to grow to 21.4 million new cancer cases and 13.2 million cancer deaths by 2030 .
there were 12.7 million new cancer cases in 2008 worldwide , of which 5.6 million were in the economically developed countries and 7.1 million were in the economically developing countries .
the corresponding estimates for total cancer deaths in 2008 were 7.6 million ( about 21,000 cancer deaths a day ) , 2.8 million in the economically developed countries and 4.8 million in the economically developing countries [ 1 , 2 ] .
pathophysiology of all cancers involves the malfunction of genes that control cell growth , division , and death .
cancers evolve through multiple changes resulting from a combination of hereditary and environmental factors , which mutate genes encoding critical cell - regulatory proteins .
the new generations of anticancer drugs affect the signals that promote or regulate the cell cycle , growth factors and their receptors , signal transduction pathways , and pathways affecting dna repair and apoptosis rather than targeting the direct synthesis of dna .
chalcone derivatives of diverse chemical architectures are quite significant in anticancer drug discovery and hence are in the center of attention of drug hunters .
anticancer activity of chalcone might be due to molecular alteration such as induction of apoptosis , dna and mitochondrial damage , inhibition of angiogenesis , tubulin inhibition , kinases inhibition , and also drug efflux protein activities .
chalcone is chemically 1,3-diaryl-2-propen-1-one ( figure 1 ) in which the two aromatic rings are joined by a three - carbon ,-unsaturated carbonyl system , representing a class of flavonoids that occur naturally in fruits and vegetables .
chalcones are also metabolic precursors of some flavonoids and isoflavonoids . the plants containing chalcone are traditionally employed for therapeutical purposes [ 37 ] .
chalcones were exploited well for their wide application in pharmacological area . it is reported that chalcones have several advantages such as poor interaction with dna and low risk of mutagenicity .
hence , some clinically useful anticancer drugs have reported genotoxicity due to their interaction with amino groups in nucleic acids ; chalcones may be devoid of these side effects due to their structural flexibility .
literature reveals that natural and synthetic chalcones are desirable to elicit cytotoxic and apoptotic activity ( figure 2 ) .
they have been reviewed not only in the articles mentioned in the text but also in more recent articles , which have eluded the authors [ 911 ] .
this review ( surveyed database , 20132015 ) is complementary to the researchers for optimizing the lead chalcone as potential anticancer scaffold with increased potency .
cell death in tumors is a passive degradative reaction known as necrosis , most likely due to inadequate angiogenesis within the tumor cell . on the other hand ,
apoptotic cell death includes a number of gene families , and altering proapoptotic pathways specifically in tumors is something of a holy grail for oncology and medicinal chemistry .
when cell production rate exceeds the cell loss rate through mitosis , it results in net tumor .
apoptosis is often referred to as an altruistic cell suicide process , where damaged dna in the cell produces signals , resulting in both repair and apoptotic pathways , and if repair can not be done then the cell undergoes apoptosis in a manner like better dead than wrong .
the cells which harbour mutant p53 will have a survival advantage over normal cells . in response to dna damage ,
normal cells upregulate p53 which acts as a transcription factor for cell cycle arrest and apoptosis ; thereafter , p53-mutant cells become unable to carry out this protective arrest or apoptosis and might survive with what otherwise would turn to lethal genetic damage , perhaps explaining why p53 mutations are so common in human cancers . the decision of apoptosis
is largely played out on the mitochondrial surface between three major families : the so - called three horsemen of apoptosis .
the final executioner proteases called caspases play pivotal role by cleaving critical substrates such as dna repair enzymes and cytoskeletal proteins , but they are stored as zymogens bound to an apoptotic adenosine triphosphate , apoptosis - activating factor-1 ( apaf-1 ) , which is the mammalian homologue of the nematode caenorhabditis elegans cell death protein , ced-4 .
once cytochrome c is released , it binds to the cytosolic protein apaf-1 to facilitate the formation of apoptosome , which in turn activates apoptotic caspases .
however , proapoptotic bcl-2 family proteins such as bax which is upregulated by p53 can activate apoptosis by releasing cytochrome c ( cyt - c ) from mitochondria , where apaf-1 activation takes place [ 2 , 6 , 12 , 13 ] .
literature on anticancer chalcones highlights the employment of three pronged strategies , namely , structural manipulation of both aryl rings , replacement of aryl rings with heteroaryl scaffolds , and molecular hybridization through conjugation with other pharmacologically interesting scaffolds for enhancement of anticancer properties .
various substitutions on both aryl rings ( a and b ) of the chalcones , depending upon their positions in the aryl rings , appear to influence anticancer activity by interfering with various biological targets . similarly , heterocyclic rings , either as ring a or as ring b in chalcones , also influence the anticancer activity shown by this class of compounds .
hybrid chalcones formulated by chemically linking chalcones to other prominent anticancer scaffolds such as benzodiazepines , benzothiazoles , and imidazolones have demonstrated synergistic or additive pharmacological activities too .
anticancer activity of three naphthyl chalcones , 1 , 2 , and 3 , shown in figure 3 was investigated by winter et al .
, these chalcones were found to possess concentration- and time - dependent cytotoxicity on murine lymphoblastic leukemia cell line ( l1210 ) .
chalcones 1 , 2 , and 3 induced apoptosis via an activated caspases - dependent pathway .
the activities of caspase-8 , caspase-9 , and caspase-12 were found to increase after the treatment of l1210 cells with the cc50 of 30 m of chalcone 1 and 40 m of chalcones 2 and 3 .
it was found that chalcones 1 and 2 induced an increase of proapoptotic proteins bax , bid , and bak ( only chalcone 2 ) , as well as a decrease in antiapoptotic bcl-2 expression .
these chalcones also induced an increase in death receptor fas and a decrease in p21 and p53 expression .
chalcones 1 and 2 exert a statistically significant increase in cytochrome c release from mitochondria to cytoplasm .
cytochrome c release mediates caspase-9 activation , while the apoptosis inhibitors from bcl-2 family prevent the efflux of cytochrome c from mitochondria .
chalcone 3 seems to act by a different mechanism to promote cell death , as it did not change the mitochondrion - related proteins nor did it induce the cytochrome c release .
chalcones 1 , 2 , and 3 induced an increase in cell calcium concentration and an increase in chop ( c / ebp homologous protein ) expression which is the transcription factor involved in endoplasmic reticulum stress , together with an increase in caspase-12 activity , suggesting that chalcones could induce an endoplasmic reticulum ( er ) stress .
they did not result in the arrest of g0/g1 , s , or g2/m phases of the cell cycle in contrast to paclitaxel , which was used as a positive control in this study and induced a statistically significant enrichment of g2/m fraction .
they presented a selectivity index ( si ) higher than one , indicating that chalcones show higher selectivity to leukemic cells when compared to nontumoral cells ( vero = monkey kidney cells and nih/3t3 = murine fibroblast ) .
the only exception was chalcone 3 which presented the same toxicity in l1210 and vero cells .
the selectivity index ( si ) corresponds to cc50 ( concentration of the compounds , which results in 50% of cell viability ) of chalcones on nontumoral cell lines ( vero and nih/3t3 ) divided by cc50 determined for cancer cells ( l1210 ) .
2,4,5-trimethoxychalcones 4 , 5 ( figure 3 ) were found to reduce the viability of human k562 acute myeloid leukemia cell and human jurkat acute lymphoid leukemia cell significantly and caused a cytotoxic effect in both cell lines in a concentration- and time - dependent manner .
they were found to have low ic50 values ( 4 to 8 m ) against both cell lines and did not have a cytotoxic effect on normal human lymphocytes .
the mechanism of action involves the reduction of cell proliferation which has been shown by a reduction in the expression of cell proliferation marker ki67 .
cell death induced by 2,4,5-trimethoxychalcones was due to induction of apoptosis through the reduction of the mitochondrial membrane potential , reduction in bcl-2 expression , increase in bax expression , increase in active caspase-3 , and activating the intrinsic pathway and execution phase of apoptosis .
histone deacetylases ( hdacs ) are enzymes which catalyze removal of acetyl groups from lysine residues .
hdacs play a critical role in epigenetic gene regulation and hence control multiple cellular events .
hence , hdacs expressions and/or activity are deregulated in various cancer subtypes , leading to a target for anticancer therapy .
coumarin - containing compounds 6a , 6b , and 6c ( figure 3 ) were found to inhibit total hdac activity by 2050% at 100 m in chronic myeloid leukemia k562 and histiocytic lymphoma u937 cell lines .
the activity of the compounds was compared to peripheral blood mononuclear cells ( pbmcs ) of healthy donors , which showed no effect on pbmc viability . in comparison to 6a ,
compound 6b , with a methoxy group at r3 , presented increased levels of inhibition .
compound 6b was tested against seven hdac isoenzymes ( 1 , 2 , 3 , 6 , 8 , 10 , and 11 ) and acted as a pan - hdaci with ic50s between 12 and 85 m .
6b inhibited hdac3 with an ic50 at 12 m and may serve as a lead for targeting this nuclear isoenzyme .
chalcone 7 ( figure 3 ) with monomethoxy group at the ortho position showed a significant effect on downregulation of cancer cell proliferation and viability in three different leukemia cell lines ( k562 , jurkat , and u937 ) .
the executioner caspase cleavage analyses indicated that the cytotoxic effect mediated by chalcone 6 is due to induction of apoptotic cell death by caspase-3 and caspase-7 activation .
the cytotoxic effect was cell type - specific and targeted preferentially cancer cells as peripheral blood mononuclear cells ( pbmcs ) from healthy donors were less affected by the treatment compared to k562 , jurkat , and u937 leukemia cells .
it was observed that compound 6 obeyed lipinski 's rule of five which indicates drug - likeness property of that molecule .
2-hydroxy-2,4,6-trimethoxy-5,6-naphthochalcone , 8 ( figure 4 ) , had most effectively inhibited the clonogenicity of sw620 colon cancer cells .
the ic50 value for compound 8 was 14.5 1.1 m against sw620 cells .
mechanistically , compound 8 triggered cell cycle arrest at g2/m phase , followed by an increase in apoptotic cell death , which involves the disruption of microtubular networks .
the mitotic spindle network is essential for appropriate segregation of chromosomes between the two daughter cells at cell division , where disruption leads to apoptosis .
compound 8 was found to destabilize microtubule assembly during mitosis , associated with failure of mitotic spindle formation , in turn blocking mitosis at the prometaphase or metaphase - anaphase transition .
however , the study did not distinguish between the dna damage checkpoint and mitotic checkpoint pathways of apoptosis .
trail ( tumor necrosis factor- ( tnf- ) related apoptosis - inducing ligand ) is a member of the tnf superfamily of cytokines .
tnf family members contain highly conserved carboxyl - terminal domains and induce receptor trimerization for the transduction of intracellular signal .
trail binds to four different receptors , two of which are death receptors 4 and 5 ( dr4 and dr5 , resp . ) , induces apoptosis , and decoys receptors , dcr1 and dcr2 , which lack the cytoplasmic death domain for transducing apoptotic death signals and protect cells from trail induced cell death by interfering with signaling through dr4 and dr5 .
it is generally believed that transformed tumor cells are more susceptible to trail - mediated cell death owing to the selective loss of decoy receptors .
chalcone 9 ( figure 4 ) ( ic50 = 4.39 m ) containing an amino ( -nh2 ) group was the most potent and selective against trail - resistant human colon cancer ( ht-29 ) and nontoxic against normal human embryonic kidney ( hek-293 ) cells .
it was observed that a large portion of ht-29 cells had undergone late apoptosis and/or necrosis after treatment with 5-fluorouracil ( 5 m ) .
these findings were consistent to show that chalcone 33 induces apoptosis rather than necrosis in ht-29 cells .
it increases the death receptors expression ( trail - r1 and trail - r2 ) and proapoptotic markers ( p21 , bad , bim , bid , bax , smac , caspase-3 , and caspase-8 ) as well as reducing the antiapoptotic markers ( livin , xiap , and hsp27 ) .
anthraquinone based chalcone compounds were synthesized and evaluated for their anticancer potential , where compounds 10a , 10b , and 10c ( figure 5 ) showed promising activity in inhibition of cervix adenocarcinoma ( hela ) cells with ic50 values ranging from 2.36 to 2.73 m and low cytotoxicity against healthy human fetal lung fibroblast cell lines ( mrc-5 ) .
all the compounds cause the accumulation of cells in s and g2/m phases in a dose - dependent manner and induce caspase-3-dependent apoptosis and noncovalent interaction with the minor groove of the double - helical ct - dna and the damage of dna double helix structure .
significant increase of p18 bax in treated hela cell line yielded consistency with the higher cytotoxic efficacy of compounds which implies the involvement of mitochondria in apoptotic process . synthesis and antiproliferative activity of a series of novel coumarin - chalcone hybrids were done by singh et al .
. the most potent molecule of the series was compound 11 ( figure 5 ) .
regression of tumors induced by hela cell xenografts in nod scid mice was found after oral administration of this molecule .
it inhibited proliferation of cervical cancer cells ( hela and c33a ) by inducing apoptosis and arresting cell cycle at g2/m phase .
apoptosis was due to induction of caspase - dependent intrinsic pathway and alterations in the cellular levels of bcl-2 family proteins .
the mitochondrial transmembrane potential also got highly depleted in compound 11 treated cells due to an increase in bax / bcl-2 ratio and intracellular ros .
compound 11 induced release of cytochrome c into the cytosol and activation of initiator caspase-9 and executioner caspase-3 and caspase-7 .
tumor suppressor protein p53 and its transcriptional target puma ( p53 upregulated modulator of apoptosis ) were upregulated , suggesting their role in mediating the cell death . based on dose response curves ,
the calculated ic50 ( after 48 h treatment ) was 4.7 1.0 m for c33a cells and 7.6 2.0 m for hela cells .
similar results were found in colony formation assay , where significant decrease in cellular colonies was observed in compound treated groups .
chalcone 11 was apparently nontoxic to the animals as they did not show any loss of weight during experiment .
cell population with fragmented nuclei increased in a time - dependent manner with the addition of chalcone 11 .
significant increase in apoptotic cells with fragmented nuclei , which is consistent with the changes in nuclear morphology and flow cytometry analysis , indicated induction of apoptosis by chalcone 11 .
novel isoxazolyl chalcones were synthesized and evaluated for their activities in vitro against human non - small lung cancer cells ( h1792 , h157 , a549 , and calu-1 ) .
compounds 12a and 12b ( figure 6 ) were identified as the most potent anticancer agents with ic50 values ranging from 1.35 to 2.07 m and from 7.27 to 11.07 m against h175 , a549 , and calu-1 cell lines , respectively .
the structure - activity relationship study showed that compounds bearing electron withdrawing groups ( ewg ) at the 2-position of the phenyl ring in aryl group were more effective .
compounds 12a and 12b increased the percentage of cell in sub - g0 phase and decreased the percentage of cell in g0/g1 phase .
these results suggested that 12a and 12b induced apoptosis in a549 cells in a dose - dependent manner .
they have upregulated the dr5 in a dose - dependent manner which suggests the extrinsic pathways of apoptosis .
cleaved bands of caspase-8 , caspase-9 , caspase-3 , and poly ( adp - ribose ) polymerase ( parp ) also indicated the induction of apoptosis .
imine derivatives of hybrid chalcone analogues 13a , 13b , 13c , and 13d ( figure 6 ) containing anthraquinone scaffold were synthesized and evaluated for their in vitro cytotoxic activity against hela , ls174 , and a549 cancer cells .
compound 13c with furan ring linked to imino group showed potent activity against all target cells with ic50 values ranging from 1.76 to 6.11 m , which are comparable to cisplatin .
generally , introduction of electron withdrawing substituents such as -cl and -cf3 in metaposition of the phenyl ring was found to exert good cytotoxicity .
compounds did not have tendency to significantly accumulate the cells in sub - g1 phase , except compound 13a , which led to an increase in sub - g1 ( observed only in cells undergoing apoptosis ) phase in a549 cells as well as changes in g2/m arrest of a549 and ls174 cells .
compounds induced the activities of caspase-3 and caspase-8 in treated hela cells and exhibited strong antiangiogenic activity by showing potent inhibitory potential against matrix metalloproteinases ( mmp-2 ) secretion .
strong implication of mmp-2 is found in angiogenesis and promotes tumor metastatic potential playing crucial role not only in invasion but also in determination of cancer cell transformation , growth , apoptosis , and signal transduction pathways .
3-phenylquinolinylchalcone derivatives , 14 and 15 ( figure 6 ) , were synthesized and evaluated for their antiproliferative activities .
compound 14 was active against the growth of non - small cell lung cancer cells h1299 and skbr-3 breast cancer cells with ic50 values of 1.41 and 0.70 m , respectively , which was more active than the positive topotecan ( ic50 values of 6.02 and 8.91 m , resp . ) .
compound 15 exhibited an ic50 value of less than 0.10 m against the growth of mda - mb231 in a dose- and time - dependent manner and was noncytotoxic to the normal mammary epithelial cell ( h184b5f5/m10 ) .
the proportion of cells was decreased in g1 and accumulated in g2/m phase after 24 h treatment of compound 15 , while the hypodiploid ( sub - g0/g1 phase ) cells increased .
compound 15 caused activation of caspase-3 , cleavage of parp , and consequently the cell death .
caspase-3 is an executioner caspase whose activation leads to the cleavage of key cellular proteins including dna repair enzyme poly ( adp - ribose ) polymerase which is involved in dna repair predominantly in response to environmental stress and is important for the maintenance of cell viability .
-cyano bis(indolyl)chalcone 16 ( figure 6 ) was found to be the most potent and selective against a549 lung cancer cell line ( ic50 = 0.8 m ) in vitro which is time - dependent .
the activity is due to the presence of methoxy and fluoro substituents on indole rings .
it was found to enhance tubulin polymerization suggesting the activity as microtubule stabilizing agents , though it was a preliminary study .
nf-b pathway has been shown to be critical to survival of lung cancer cells , and many natural products obtained from plants were found to inhibit the activation of this pathway .
two cardamonin analogs , 4,4-dihydroxylchalcone , 17 , and 4,4-dihydroxy-2-methoxychalcone , 18 ( figure 6 ) , were found to suppress the activation of nf-b pathway in lung cancer cells .
compound 17 inhibited the survival of primary lung cancer cells with ic50 values of 0.44 0.05 and 0.16 0.03 m , respectively , against two lung cancer cells tested .
compound 18 inhibited their survival with similar ic50 values ( 0.56 0.12 m and 0.65 1.36 m ) .
immunoblot analysis displayed that caspase-3 and parp were both cleaved in the two lung cancer cell lines treated with compounds 17 and 18 , indicating the activation of apoptotic pathway .
subsequent estimation of the phosphorylation level of ikk , nf-b pathway stimulating kinase , by immunoblotting assay demonstrated that both decreased ikk phosphorylation in lung cancer cell lines including a549 , nci - h460 , and nci - h1668 and lead to the accumulation of ib and ib and the export of nuclear p65 transcription factor . from the immunofluorescent staining
, it was shown that transcription factor p65 , which is a downstream effect of nf-b pathway , was sequestered in the cytoplasm of a549 cells treated by compound 17 or compound 18 .
incorporation of luciferase reporter system also results in transcription activity of nf-b in lung cancer cells treated with compound 17 or compound 18 and demonstrated that relative luciferase expression was significantly reduced in compound 17 or compound 18 treated lung cancer cells .
in addition , the transcription of target genes of nf-b pathway , bcl-2 , and survivin was found to be suppressed in lung cancer cells treated with the two compounds .
. higher concentration of the two compounds greatly inhibited the growth of tumors by 71.0 and 79.1% , respectively , although inhibitory effect was not significant at lower concentration .
compared with compound 17 , compound 18 appeared to be slightly more potent to retard the growth of established tumors .
-carboline based chalcones were synthesized and evaluated for their cytotoxic activity against a panel of human cancer cell lines .
compounds 19a and 19b ( figure 6 ) showed very good antiproliferative activity against breast cancer cell line ( mcf-7 ) , where ic50 values were recorded at 2.25 m and 3.29 m , respectively .
furthermore , compound 19a markedly induced dna fragmentation and apoptosis in breast cancer cells , which is an essential step in the cells undergoing apoptosis .
the molecular mechanism to induce apoptosis in human hepatoma smmc-7721 cells was investigated by using 2,4-dihydroxy-6-methoxy-3,5-dimethylchalcone , 20 ( figure 7 ) , isolated from the buds of cleistocalyx operculatus .
compound 20 induced apoptosis via a mitochondria dependent pathway involving inhibition of bcl-2 expression leading to disintegration of the outer mitochondrial membrane .
further downstream of the apoptosis cascade , compound 20 , increases caspase-3 and caspase-9 activities .
angiogenesis and invasion are two highly interconnected events in cancerous cells where involvement of multiple molecules and pathways takes place .
hypoxia inducible factor-1 ( hif-1 ) has been identified as an important protein involved in those events .
activated hif-1 controls the expression of genes involved in tumor cell immortalization , stem cell maintenance , metabolic reprogramming , autocrine growth , and treatment failure , thereby making it an important target for the development of new antitumor therapy .
a novel series of chalcone derivatives were synthesized and their biological activities against hif-1 were evaluated by wang et al .
it has been found that compound 21 ( figure 7 ) exhibited inhibitory effects on hif-1 at 10 m concentration by downregulating the expression of hif-1 and hif-1 under hypoxic conditions .
the hif inhibition rate has been found to be 83.0 16.9% , while topotecan , hif-1 inhibitor , was used as control and for comparison it has the inhibition rate of 82.8 2.9% . in addition ,
compound 21 displayed inhibitive effect against the migration and the invasion of hep3b ( human liver cancer cell line ) and human umbilical vein endothelial cells ( huvec ) and tube formation of huvecs and significant antiangiogenic and anti - invasive activities . from the in vivo study
it has been found that 21 could retard tumor growth of hep3b xenograft models and reduced cd31 and mmp-2 expression in tumor tissues .
cd31 ( cluster of differentiation 31 ) is platelet endothelial cell adhesion molecule ( pecam-1 ) , which is a protein found to be expressed in certain tumors .
compound 27 was well tolerated and was found to be nontoxic up to 200 mg / kg in swiss mice in acute intravenous toxicity .
the molecular mechanisms of growth inhibition in human bladder cancer cell lines t24 and ht-1376 by chalcone 22 ( figure 8) are due to the induction of apoptosis , cell cycle progression inhibition by regulating cell cycle related factor like significant reduction in the expression of cyclin a and cyclin b1 , decreases in the expression of cdc2 , and also increases in the expression of p21 and p27 .
so , chalcone may cause cell cycle arrest by reducing the complex of cdc2/cyclin b due to downregulation of multiple g2/m regulating proteins .
it triggers the mitochondrial apoptotic pathway by releasing cytochrome c from the cytoplasm with an increase in caspase-9 and activating caspase-3 .
it is also increases the expression of proapoptotic proteins bax and bak , which are responsible for the initiation of mitochondrial apoptotic pathway , and decreases the expression of antiapoptotic proteins bcl-2 and bcl - xl , while overexpression of bcl-2 and bcl - xl is associated with enhanced oncogenic potential .
chalcone inhibits the nf-b activation by increasing the expression of ib in cytoplasm , leading to apoptosis .
the copper chelate complex 23 ( figure 8) showed promising activity with ic50 value of 5.95 m against pc3 cancer cell line in vitro .
the estimated ic50 value for doxorubicin was 8.7 m , which was used as positive control .
it was found that cancer cells have high affinity to copper ions ; thus , upon chelation of chalcones with copper they are thought to be more liable to target cancer cells in microdendron architecture .
it was assumed that copper ions might act as penetration enhancers of chalcones into cancer cells .
even if copper ions are noncytotoxic to cancer cells , they might act as inhibitors of drug efflux proteins characteristic to cancer cells .
drug efflux proteins , such as p - glycoproteins ( pgp ) , breast cancer resistance protein ( bcrp ) , and multidrug resistance - associated protein 1 ( mrp1 ) , are responsible for drug resistance of cancer cells .
the high cytotoxic activity is attributed to the presence of the p - methylphenyl moiety of ring b and the copper ion .
hybrids of n-4-piperazinyl - ciprofloxacin - chalcone were prepared by abdel - aziz et al .
national cancer institute ( nci ) selected 5 compounds , namely , 24a , 24b , 24c , 24d , and 24e ( figure 9 ) , to carry out in vitro one - dose anticancer assay against full nci 60 cell lines derived from leukemia , lungs , colon , renal , melanoma , cns , prostate , ovarian , and breast cancer cell lines .
of the selected compounds , 24a and 24d exhibited the highest ability to inhibit the proliferation of different cancer cell lines while undergoing one - dose anticancer test . among the nine tumor subpanels tested for
, compound 24a was found to have broad - spectrum antitumor activity with selectivity ratios ranging between 0.42 and 1.87 at the gi50 level without producing selectivity , while compound 24d showed high selectivity towards the leukemia subpanel with selectivity ratio of 6.71 at gi50 level .
moreover compound 24d showed remarkable anticancer activity against most of the tested cell lines with gi50 ranging from 0.21 to 57.6 m .
furthermore , compounds 24c and 24e have shown remarkable topo - ii inhibitory activity compared to etoposide at 100 m and 20 m concentrations . compounds 24c and 24e were found to be potent topo - i inhibitors at 20 m concentration when compared to camptothecin .
the results that were obtained indicated that introduction of n-4-piperazinyl moiety of ciprofloxacin into the chalcones derivatives dramatically increases the anticancer activity of the tested compounds severalfold higher than that of the activity shown by weak anticancer ciprofloxacin .
increase in antiproliferative activity of the tested compounds might be the result of either synergistic effect of the chalcone derivatives and/or alteration of the physicochemical properties of ciprofloxacin . to determine the dna unwinding properties of 24c and 24e , amsacrine ( m - amsa ) , a well - known dna intercalator , was chosen as a standard where a supercoiled phot1 dna was used as a substrate since unwinding of the double strand of dna helix is a practical characteristic of intercalating drugs .
amsacrine blocked unwinding of phot1 dna in the presence of excess topo - i in dose - dependent manner , while compounds 24c and 24e did not block dna unwinding at high concentration up to 1000 m treatment .
the assay indicated that 24c and 24e do not interact with dna but interact with topoisomerases for their inhibitory activity .
compound 25 ( figure 9 ) with a propionyloxy group at the 4-position of the left phenyl ring showed the most potent inhibition activity which inhibited the growth of hepg2 , a549 , a375 , smmc-7221 , and k562 cancer cell lines with ic50 values of 0.15 , 0.36 , 0.62 , 0.61 , and 0.52 m , respectively .
compound 25 was also shown to arrest cells in the g2/m phase of the cell cycle and inhibit the polymerization of tubulin .
molecular docking study suggested that compound 25 binds into the colchicine binding site of tubulin . in summary , these findings suggest that compound 23 is a promising new antimitotic compound for the potential treatment of cancer .
a new series of pyrano chalcone derivatives containing indole moiety synthesized by wang et al . ( 2014 ) were evaluated for antiproliferative activities . among those ,
compound 26 ( figure 9 ) , with a propionyloxy group at the 4-position of the phenyl ring a and n - methyl-5-indolyl on the b ring displayed the most potent cytotoxic activity against all tested cancer cell lines including multidrug resistant phenotype , which inhibits cancer cell growth with ic50 values ranging from 0.22 to 1.80 m .
the tested cell lines were smmc-7221 , hepg2 ( liver ) , pc-3 ( prostate ) , a549 ( lung ) , k562 ( leukemia ) , hct116 ( colon ) , skov3 ( ovarian ) , mcf-7 ( breast ) , vincristine resistant hct-8 ( hct-8/v ) , and taxol resistant hct-8 ( hct-8/t ) .
compound 26 was the most potent towards hepg2 cells with ic50 value of 0.22 m .
colchicine ( 2.5 m ) and paclitaxel ( 2.5 m ) were used as a reference .
compound 26 produced cytotoxic activity in normal human liver cell line ( lo2 ) above the concentration of 10 m .
it significantly induced cell cycle arrest in g2/m phase and inhibited the polymerization of tubulin .
it was also shown from molecular docking analysis that compound 26 binds at the colchicine binding site of tubulin .
chalcone 26 also exerted potent anticancer activity in hepg2 xenografts in balb / c nude mice , where the growth of tumors in mice treated with chalcone 26 ( 30 mg / kg ) was reduced by 56.58% compared with that in control mice treated with vehicle only at day 35 . positive control reduced the growth of tumors by 25.93% .
cell division cycle 25 ( cdc25 ) , which includes cdc25a , b , and c homologues , a subfamily of dual - specificity protein tyrosine phosphatases , plays a role in the regulation of the cell cycle .
the cdc25a and cdc25b isoforms are overexpressed in primary tissue samples from various human cancers , which is strongly associated with tumor aggressiveness and poor prognosis .
cdc25b controls cell cycle progression by catalyzing removal of covalently attached contiguous phosphates and the subsequent activation of cyclin - dependent kinase 1 ( cdk1 ) .
cdc25b overexpression has been documented in a variety of human cancers , including head and neck and colon , and non - small cell lung cancer validates its oncogenic potential .
a series of 2-hydroxy-4-isoprenyloxychalcone derivatives were evaluated for the inhibition of cdc25b activity by zhang et al .
( 2014 ) . among those , two compounds , 27 and 28 ( figure 9 ) , have been found to have the potent inhibition activity and significantly inhibited cdc25b with inhibition rates of 99.95% and 99.75% , respectively , at a dose of 20 g / ml , which is similar to the known tyrosine phosphatase inhibitor sodium orthovanadate . in vitro cytotoxic activity assays showed that compounds 27 and 28 were potent against hct116 , hela , and a549 cells .
they were particularly cytotoxic against colon hct116 cancer cell line with ic50 values of 0.89 and 1.76
irinotecan ( ic50 = 2.14 mol / l ) , a clinically relevant camptothecin , was chosen as a reference drug . in colo205
xenograft balb / c nude male mice , compound 27 produced a tumor volume inhibition of about 50% .
a new series of pyrazole chalcones were screened for the in vitro anticancer activities against mcf-7 ( human breast adenocarcinoma ) and hela ( human cervical tumor cells ) cell lines .
compound 29 ( figure 9 ) showed the highest inhibition in human mcf-7 and hela cell lines with an ic50 value of 0.018 g / ml and 0.047 g / ml , respectively .
the ic50 for standard doxorubicin in hela and mcf-7 cells was 1.15 g / ml and 1.84 g / ml , respectively .
new prenylated chalcone , 30 ( figure 9 ) , named as renifolin c , was isolated from whole desmodium renifolium plants and cytotoxicity was evaluated against five human tumor cell lines ( nb4 , a549 , shsy5y , pc3 , and mcf7 ) using the mtt method , using paclitaxel as a positive control .
the ic50 values were 6.4 m and 8.5 m against nb4 and pc3 cell lines and above 10 m for the other cell line tested .
various benzylidene pregnenolones were synthesized and in vitro cytotoxicity studies were performed by banday et al .
compounds 31a and 31b ( figure 9 ) were found to be active against the hct-15 ( colon ) and mcf-7 ( breast ) cell lines .
chalcone 31a has ic50 value of 1.02 m and 0.79 m , respectively , against hct-15 and mcf-7 , while 31b has 0.81 m and 1 m , respectively .
chalcone - coumarin hybrid 33 ( figure 10 ) ( ic50 = 0.53 m ) displayed cytotoxicity against human lymphoblastic leukemia cell line ( molt-3 ) , where etoposide ( ic50 = 0.05 m ) was taken as a standard .
compounds 32 and 33 shown in figure 10 displayed higher cytotoxic potency against hepg2 cells than the control drug , etoposide .
the hybrid 33 ( ic50 = 4.26 m ) was potent but was found to be toxic toward noncancerous african green monkey kidney cell line ( vero ) .
though analog 32 displayed cytotoxic activity against hepg2 with ic50 value of 8.18 m , it was found to be nontoxic against vero cells .
it has been shown from molecular docking study that compounds could snugly occupy the colchicine binding site of -tubulin .
( 2013 ) . among them , potent chalcone 34 ( figure 10 ) induced apoptotic cell death of a wide variety of tumor cell lines including multidrug resistant human tumor cell lines in the nanomolar range by attacking microtubules .
flow cytometric study showed that chalcone 34 significantly induced cell cycle arrest in g2/m phase at 0.2 m concentration . in vitro immunofluorescence
staining and tubulin polymerization assay displayed that chalcone 34 promoted tubulin polymerization into microtubules and caused microtubule stabilization similar to paclitaxel , which is suggesting that chalcone 34 is a microtubule stabilizer via enhancing the rate of tubulin polymerization .
then , in in vivo study , 34 exhibited potent inhibitory activities in human hepg2 xenograft tumor models .
chalcone derivatives , named parasiticins c , 35 and 36 ( figure 10 ) , were isolated from the leaves of cyclosorus parasiticus .
the in vitro cytotoxic activities were evaluated against lung cancer a549 , hepatocellular carcinoma hepg2 , breast cancer mcf-7 and mda - mb-231 , leukemia all - sil , and pancreatic cancer sw1990 .
compounds 35 and 36 exhibited substantial cytotoxicity against all six cell lines , especially toward hepg2 with the ic50 values of 1.60 and 2.82 m , respectively , which is comparable to the doxorubicin positive control ( 1.19 m ) .
they induced apoptosis in the hepg2 cell line . in the clonogenicity assay on hepg2 cell line , capacity of single cancer cells to generate colonies
compound 35 showed more potent cytotoxicity than 36 , suggesting that the presence of an additional lactone ring fused enhances the cytotoxic activity .
a fission yeast , schizosaccharomyces pombe , was utilized in bioassay guided screening on several plant extracts to search for anticancer agents by snchez - pic et al .
it was found that an extract from leaves of corema album belonging to family ericaceae had antiproliferative activity .
2,4-dihydroxychalcone , 37 ( figure 11 ) , the promising cytotoxic compound , was found in this extract and cytotoxic activity is likely due to its ability to induce dna damage which blocks cell cycle progression at the g2/m transition in a concentration of 8 m to 20 m .
compound 37 could induce dna damage or interfere with dna replication , which would eventually result in the activation of the cell cycle checkpoints , thus blocking cell cycle in g2 . in subsequent study ,
deletions of rad3 , chk1 , and cds1 , three key kinases required for dna checkpoints operating at g2/m , were done .
rad3 is required in order to signal downstream of either dna replication problems or dna damage , whereas chk1 transmits dna damage - induced signals and cds1 signals in response to defective dna replication .
it was found that both rad3 and chk1 deletions suppressed the cell cycle block produced by compound 37 , while cds1 deletion showed partial suppression , suggesting that the g2/m block observed in compound 37 is mostly caused by the activation of dna damage checkpoint response .
methyl methanesulfonate ( mms ) , an alkylating compound that induces dna damage , was used as control .
thus , compound 37 ( 820 m ) directly or indirectly causes damage to the dna which results in a rad3-dependent and chk1-dependent cell cycle block at the g2/m transition .
cathepsins have emerged as a potential target for chemoprevention and anticancer therapy . increased levels of cathepsin b and cathepsin h in a variety of tumors have shown their contribution towards invasion and metastasis .
molecule 38 ( figure 12 ) has been found to be potent among the synthesized inhibitors , which inhibits cathepsin b and h. the ki ( inhibition constant ) value against cathepsin b was 6.18 10 m and 2.8 10 m for cathepsin h. the inhibition was of competitive type .
2-hydroxychalcones have been evaluated as potential inhibitors to these cysteine proteases probably due to presence of ,-unsaturated carbonyl moiety in the molecule .
-ch2sh moiety present at the active site of enzymes can interact with the electron deficient center present in the chalcones inhibiting the enzymes activity .
nitrosubstitution is useful for an effective interaction with cathepsins b and h. its inhibition to cathepsin b as compared with cathepsin h indicates that cathepsin b 's active site is more susceptible to these compounds as compared to cathepsin h. the results were also found to be consistent when compared with in silico docking studies .
cotreatment with a chemosensitizer such as compounds 39a and 39b ( figure 13 ) and trail was found to be more efficient to treat gbm ( glioblastoma multiforme ) than treatment with trail alone .
gbm is one of the most aggressive forms of human malignant brain tumors , characterized by rapid growth , extensive invasiveness , and robust neoangiogenesis . against human crt - mg astroglioma cells by using the lactate dehydrogenase ( ldh ) release assay , it was found that compounds 39a and 39b with trail ( 25 ng / ml ) showed good cytotoxicity .
2-ethylamino and substituted triazole groups are promising templates in the backbone of chalcone to develop trail sensitizers as anticancer agents .
mdr ( multidrug resistance ) can be defined as the ability of cancer cells to survive exposure to different chemotherapeutic agents and is a major cause of treatment failure in human cancers .
4-hydroxy-2,6-dimethoxychalcone , 40 ( figure 14 ) , was isolated from polygonum limbatum with ic50 values of 2.54 m against multidrug resistant cem / adr5000 leukemia cells .
p - glycoprotein - expressing and multidrug resistant cem / adr5000 cells were much more sensitive toward compound 40 than doxorubicin ( ic50 = 195.12 m ) , which is a reference drug .
compound 40 ( figure 14 ) has been found to arrest the cell cycle between g0/g1 phases , which is via the strong induction of apoptosis by disrupting mitochondrial membrane potential ( mmp ) and an increased production of reactive oxygen species ( ros ) in the studied leukemia cell line .
it was found that caspases were not involved in the apoptotic pathway induced by compound 40 .
the presence of hydroxyl ( -oh ) and methoxy ( -och3 ) substituents influences the activity of compound 40 .
the membrane transporter p - glycoprotein ( p - gp ) , encoded by mdr1 gene , is the main mechanism responsible for resulting in decreased intracellular drug accumulation in human mdr cancers .
mitogen activated protein ( map ) kinase pathway is involved in p - gp - mediated mdr .
dimer of chalcone named as tomoroside b , 41 ( figure 14 ) , was isolated from the aerial parts of helichrysum zivojinii .
the combined effects of chalcone dimer 41 and tipifarnib ( the inhibitor of map kinase signaling cascade , a farnesyltransferase inhibitor ) in mdr cancer cell line , nci - h460/r , were found .
these results point to the ability of compound 41 to enhance tipifarnib efficacy in mdr cancer cells as it reduced the ic50 for tipifarnib from 9.0 m ( applied alone ) to 3.8 m .
activity was compared to tipifarnib employing human sensitive non - small cell carcinoma cell line nci - h460 ( ic50 = 198 m for compound 41 alone ) , its multidrug resistant ( mdr ) counterpart nci - h460/r ( ic50 = 172 m for compound 41 alone ) , and spontaneously immortalized normal human keratinocyte cell line hacat .
the concentration around 100 m of compound 41 significantly inhibited the growth of normal hacat cells , considering that hacat are normal , but transformed cells with immortalized phenotype ; these results point to the potential of compound 41 to exert the cancer preventive effects .
compound 41 increased the expression of hif-1 , probably by exerting antioxidant and redox status modulator effect .
compound 41 increased the expression of hif-1 and acted synergistically with tipifarnib probably by inhibiting the map kinase prosurvival signaling . to identify potent anticancer agents against the cisplatin - resistant human ovarian cancer cell line
the two most active compounds were 42 ( gi50 = 3.87 m ) and 43 ( gi50 = 3.95 m ) ( figure 14 ) .
it was found that compounds 42 and 43 trigger cell cycle arrest at the g2/m phase and apoptotic cell death in a2780/cis cancer cells .
aurora a kinase is known to be involved in cell cycle arrest at the g2/m phase . in vitro aurora a kinase assay of compound 42 showed the half - maximal inhibitory concentration ( ic50 ) of 66.4 m . because aurora a kinase may not be a real molecular target of compound 42 , gi50 value of compound 42 is higher than the ic50 value of 42 .
chalcone architecture linked with various substitutions , such as methoxy , amino , and hydroxyl , and alteration of both rings has been found to be effective in making it a potential candidate in the anticancer armamentarium .
the related studies showed excellent cytotoxic activities of chalcones in the different cell death pathways , not just acting in blocking the process of cell division .
inhibitory concentrations in nanomolar range were impressive to show its ability to arrest cell division .
furthermore , besides preclinical study , clinical study of chalcone may yield the development of research in this field .
it is expected that this review may give the medicinal chemists a basis to get in touch with the recent updates and will be helpful for enrichment in this field .
|
cancer is an inevitable matter of concern in the medicinal chemistry era .
chalcone is the well exploited scaffold in the anticancer domain .
the molecular mechanism of chalcone at cellular level was explored in past decades .
this mini review provides the most recent updates on anticancer potential of chalcones .
|
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cytomegalovirus ( cmv ) colitis is common among immunocompromised patients who are more prone to opportunistic infections .
after the infection of cmv is detected by histology or serology , antiviral treatment is usually chosen initially . since cmv disease
can not be diagnosed on clinical symptoms alone , diagnosis and treatment are sometimes delayed . here
, we report a case of a hemorrhagic cmv colitis that occurred in an immunocompromised patient after a colectomy due to colon cancer and was successfully treated with ganciclovir .
an 85-year - old woman underwent esophagogastroduodenoscopy and colonoscopy for investigation of anemia . a laterally spreading tumor of the granular type , 85 mm in diameter ,
although magnifying image - enhanced colonoscopy suggested a tubulovillous adenoma , surgical resection , not endoscopic mucosal resection , was chosen because of the size of the tumor . however , as she had a medical history including breast cancer , myocardial infarction , general edema due to chronic renal failure ( oral administration of prednisolone 5 mg / day ) , diverticulosis of sigmoid colon , and bilateral ureteral stent placement due to retroperitoneal fibrosis , she did not want to take medical treatment anymore but to monitor her own condition for a while .
after 3 months , she finally agreed to have surgery and was admitted to our hospital .
the patient underwent ileocecal resection followed by stapled functional end - to - end anastomosis ( fig .
the postoperative course was favorable ; dietary intake and oral drug administration were started on postoperative day 4 and both flatus and stool passed .
however , she had sudden abdominal pain and heavy bloody discharge on postoperative day 11 .
although conservative treatment , including blood transfusion therapy for anemia , was performed initially , there was no improvement after a week .
in addition to the endoscopic findings , the patent 's immunocompromised state suggested possible cmv colitis . because cmv antigen was confirmed in peripheral blood samples , intravenous antiviral treatment was started from the postoperative day 23 ( ganciclovir , 150 mg / day ) .
after 4 days of the treatment , bloody discharge stopped , and at 8 days , improvement of abdominal pain was observed .
the antiviral treatment was continued for 2 weeks until the serological confirmation of negative cmv antigen .
after improvement of the colitis - related symptoms , the patient was discharged from our hospital .
cmv infection often develops a latent infection with no signs or symptoms after acute infection .
although most people are exposed to cmv in their lifetime , typically only the patients with weakened immune systems , such as aids , malignancy , organ transplant or bone marrow transplant , and steroid or immunosuppressive treatments , become ill by reactivating cmv and present with cmv pneumonia , gastroenteritis , retinitis , and encephalitis .
cmv disease is often diagnosed by pathologic and serologic confirmation because clinical symptoms are not specific .
furthermore , colonoscopic findings of cmv colitis mimic many conditions , including pseudomembranous colitis , ischemic colitis , ulcerative colitis and crohn 's disease . in our case , as we initially considered the bleeding from the diverticulum or anastomosis , conservative treatment was chosen .
after one week of conservative therapy , there was no sign of improvement in bloody discharge ; therefore , we decided to perform colonoscopy .
we were hesitant to perform colonoscopy due to our patient having had a colectomy . according to the meta - analysis of cmv colitis in immunocompetent hosts , the highest mortality rates were associated with immune modulating conditions that include 16 patients . of those analyzed ,
5 patients ( 31.3% ) received colectomy as initial treatment and 8 patients ( 50% ) and 6 patients ( 37.5% ) had coexisting renal failure and diabetes mellitus , respectively . in our case
, as the patient was given steroids due to chronic renal failure and the colonoscopy revealed the longitudinal ulcer at the anastomosis , we were able to suspect cmv colitis and confirmed the diagnosis serologically .
because endoscopic biopsy was not performed , cmv infection was not evaluated histologically . of note
, we performed immunohistochemistry of cmv protein using resected colon cancer specimen , though cmv - positive stain was not detected .
this result seems to relate with the fact that cmv is not associated with carcinogenesis and reactivation of latent cmv infection causes cmv colitis [ 5 , 6 ] .
the first choice of treatment of cmv infection is antiviral therapy [ 7 , 8 ] .
systemic antiviral treatment has provided significant advances and has resulted in dramatically improved outcomes [ 7 , 8 ] .
treatment time usually ranges from 1 to 4 weeks , depending on the treatment effect such as improvement of the symptoms and/or confirmation of the negative cmv antigen . in our case , despite the necessity of reducing the dose of ganciclovir due to renal failure , the patient showed good response to systemic therapy and did not require further treatment . fortunately , as she did not show uncontrollable heavy bleeding or symptoms of bowel perforation , surgical resection was not needed .
the case we mentioned above can potentially occur in immunocompromised patients . with recent progress in medicine
, the number of high - risk patients who undergo surgery is increasing ; therefore , the reactivation of latent cmv infection in immunocompromised patients should be actively considered for the differential diagnosis , leading to timely diagnosis and appropriate treatment .
|
we report a case of hemorrhagic cytomegalovirus ( cmv ) colitis , occurring in a postoperative patient due to a weakened immune system .
an 85-year - old woman with a medical history , including chronic renal failure treated with oral administration of prednisolone , underwent colectomy due to an ascending colon cancer . while the postoperative course was favorable , she exhibited acute severe abdominal pain and massive bloody discharge after 11 days of surgery .
her colonoscopic examination showed multiple longitudinal ulcers on the anastomosis .
in addition to these endoscopic findings , her past medical history helped suggest cmv colitis . because serological testing revealed positive cmv antigen , she was finally given a diagnosis of cmv colitis and received intravenous ganciclovir for the initial treatment .
hemorrhagic cmv colitis after colectomy is an important postoperative complication ; we therefore present our case with diagnosis and treatment experience .
|
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healthcare is provided by public and private hospitals , and there are 5 major burns units . in january 2005 , pustular lesions were observed on the foot of a patient in 1 of the burns units , and similar lesions subsequently appeared on other patients .
local health authorities were informed of the outbreak , and an investigatory team confirmed reports of similar infections in the city s other burns units , with retrospective identification of at least 19 probable cases occurring over a 5-month period .
most patients had a fever ( 39.0c40.5c ) for 23 days , followed by the appearance of an eruption(s ) .
lesions were typically small , rounded , umbilicated , and nodular in appearance with an erythematous base ( figure 1a ) .
they contained cheesy pustular material and increased in size ( from 12 mm to > 1 cm in diameter ) and severity over 78 days .
the lesions involved burn wounds and intact skin surrounding them , with the unhealed margins of the wounds being covered by a layer of thick yellow secretion .
some patients had a sparse rash ; others had closely spaced lesions that produced a cobblestone appearance .
a single lesion developed on a paramedical staff member s finger , and lesions developed around 1 patient s insertion site for an intravenous line ( figure 1b ) .
in all instances , the disease was self limiting , and patients recovered in 34 weeks .
c ) orthopoxvirus particles detected by electron microscopy ( em ) examination of negatively stained grids prepared from pustular material ( magnification 73,000 ) .
d ) transmission em examination of ultrathin sections of infected vero cell cultures showing classic intracytoplasmic orthopoxvirus factories and maturing virus particles ( magnification 21,000 ) .
results of bacteriologic and mycologic examination of biopsy samples , impression smears , and swab samples from lesions were negative .
histopathologic examination showed extensive ulceration and granulation , with epidermal necrosis and subepidermal edema plus acute and chronic inflammatory cell infiltration .
impression smears and biopsy tissues were sent to the special pathogens unit , national institute for communicable diseases ( nicd ) , sandringham , south africa , and to the health protection agency ( hpa ) , centre for emergency preparedness and response , porton down , salisbury , united kingdom .
electron microscopy of negative - stained grids prepared at hpa and ncid laboratories from pustular material showed orthopoxvirus particles , and examination of ultrathin sections prepared from infected vero cell cultures at hpa found classic orthopox intracytoplasmic virus factories and particle maturation sites ( figure 1c , d ) .
pcr was performed on nucleic acid extracted from the samples , using primers specific for regions of the orthopoxvirus hemagglutinin gene ( at nicd ) and b5r membrane protein gene ( at hpa ) .
after nucleotide sequences were determined for the pcr products , phylogenetic analyses were conducted in relation to corresponding orthopoxvirus sequences obtained from genbank , using methods described elsewhere ( 14,15 ) .
the causative agent was found to cluster with buffalopoxvirus isolates within the vaccinia subgroup of orthopoxviruses ( figure 2 ) , and 3 patients from 2 separate burns units were shown to be infected with the identical virus , which was distinct from other known buffalopoxvirus isolates . to investigate the possibility of a shared source of infection , 17 samples of saline , antimicrobial drug ointments , petroleum jelly , cotton dressings , and swabs in common use were obtained from the 5 burns units and tested by pcr at nicd ; no results were positive .
inquiries led to the suggestion that the outbreak was probably propagated by transfer of infected patients between burns units .
this hypothesis was confirmed when a policy to isolate all new admissions , including referrals from other burn centers , for their first 2 weeks in a unit successfully controlled transmission of new endogenous cases .
maximum likelihood phylogenetic tree based on a 955-nt alignment of the karachi isolate and 33 orthopoxvirus sequences of the b5r gene from genbank constructed with clustalw ( www.ebi.ac.uk/clustalw/index.html ) and tree - puzzle ( http://bioweb.pasteur.fr/seqanal/interfaces/puzzle.html ) ; figures at nodes represent puzzle support values . the orthopoxvirus types are indicated to the right .
control measures included education of staff , single - room or cohort isolation of patients infected or suspected of being infected with buffalopoxvirus , and reinforcement of infection - control practices , such as hand disinfection after contact with any patient . to reduce virus load in the environment ,
the measures proved effective in reducing transmission within burns units , but they did not prevent the sporadic arrival of newly infected patients .
buffalopoxvirus outbreaks reported to date have been geographically restricted , and human cases have been limited to persons with direct exposure to infected animals , usually in rural communities ( 111 ) .
this reported outbreak uniquely involved nosocomial infections in 5 widely separated burns units in karachi , pakistan .
however , buffaloes are the most common dairy animal in pakistan , even within the city limits of karachi , and buffalo fat , particularly in the form of butter or ghee , sometimes is used at home as a dressing for burns . thus , burn patients newly infected with buffalopoxvirus
due to disparity in the sophistication and cost of the care provided at the burns units in karachi , patients are often transferred or move themselves between units , thus facilitating the possible spread of infection . in this outbreak , 6 of the 19 patients with putative cases of buffalopoxvirus infection are known to have transferred between burns units during treatment .
the infection was of low virulence for humans . delay in recognizing and investigating the outbreak is cause for concern and can be ascribed to poor awareness and lack of resources .
|
during 5 months in 20042005 , buffalopoxvirus infection , confirmed by virus isolation and limited nucleic acid sequencing , spread between 5 burns units in karachi , pakistan .
the outbreak was related to movement of patients between units .
control measures reduced transmission , but sporadic cases continued due to the admission of new patients with community - acquired infections .
|
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we have observed a case of renal involvement complicated by granular corneal dystrophy type ii ( gcd2 ) .
gcd2 , also known as avellino corneal dystrophy ( cd ) , is an autosomal dominant disorder caused by a mutation in the transforming growth factor--induced ( tgfbi ) gene .
this mutation can be found in several distinct autosomal dominant genetically determined cases of cd ; however , it is not known whether this mutation produces other clinical manifestations other than cd .
tgfbi proteins ( tgfbip ) interact with several extracellular matrix ( ecm ) components [ 3 , 4 ] .
we believe that our study was a type of oculorenal syndrome associated with a tgfbi mutation , which remains to be acknowledged .
the following clinical laboratory values were noted : serum urea nitrogen ( bun ) , 14.9 mg / dl ; creatinine ( cre ) , 0.79 mg / dl ; total cholesterol , 189 mg / d ; total protein , 6.4 g / dl ; and albumin , 3.9 g / dl .
the levels of c - reactive protein , immunoglobulins ( ig ) , and total complement , c3 , c4 , and c1q were all normal .
tests for antinuclear antibody , hepatitis b virus surface antigen , hepatitis c virus antibody , and cryoglobulins were all negative .
renal ultrasound and computed tomography revealed normal kidneys . a kidney biopsy , performed using light microscopy , revealed 11 glomeruli , 1 of which was obsolete or sclerosed ( fig 1a ) .
light microscopy did not demonstrate any remarkable changes in the glomeruli ( fig 1b ) .
focal tubular atrophy with dilation of peritubular capillaries and focal infiltration of small round cells were observed .
after 7 years , the patient developed mild hypertension and began taking 4 mg / day of losartan potassium .
the patient 's mild proteinuria ( 11.5 g / g cre ) continued , and her renal function was mildly decreased .
after 10 years , the patient was re - admitted for additional evaluation of proteinuria .
laboratory testing revealed the following : urinary protein level of 1.5 g / day , bun level of 15.0 mg / dl , and cre level of 0.94 mg / dl .
approximately 2 years before her second admission , the patient complained of mild blurred vision and was diagnosed with cd .
slit - lamp examination revealed a large number of gray - white central granular and linear opacities in both eyes ( fig 2 ) ; therefore , we diagnosed her condition as gcd2 .
a second kidney biopsy was performed under light microscopy , revealing 18 glomeruli , 6 of which were obsolete or sclerosed ( fig 3a ) .
segmental double contours of the glomerular capillary walls were also observed ( fig 3c ) .
focal tubular atrophy with mild interstitial inflammation , dilation of peritubular capillaries , and segmental thickening of tubular basement membranes ( tbm ) were observed .
the subendothelial space was widened , and irregularity of the glomerular basement membrane ( gbm ) was segmentally observed .
segmental irregular thinning , basket - waving , duplication , lamellation , and reticulation of gbm and tbm were observed partially and slightly ( fig 3e
immunostaining of the -5 chains of type iv collagen was normal . upon her renal pathological findings
, we assumed the existence of a genetic cause . after obtaining informed consent , we collected dna from the patient .
the genome dna was extracted from the whole blood , and targeted next - generation sequencing of candidate genes for inherited renal diseases was negative ( online suppl .
. real - time polymerase chain reaction using a simple buccal swab ( avellino labs universal test ; alut ) revealed tgfbi heteromutation ( r124h ) .
we speculated that this condition was a novel case of oculorenal syndrome associated with tgfbi mutation .
her 74-year - old father had the same mutation of tgfbi ( r124h ) and was diagnosed with relatively mild gcd2 .
her 77-year - old mother and 26-year - old daughter , however , did not have mutated tgfbi ( r124h ) or gcd2 .
her father had no proteinuria but had a slightly elevated level of urinary n - acetyl--d - glucosaminidase ( 3.0 u / g cre ) ; however , his renal function was normal ( serum cre , 0.79 mg / dl ) .
subsequently , the patient was again treated in our outpatient clinic with 4 mg / day of losartan potassium but with no immunosuppressive agents .
these two conditions may coincidentally coexist ; however , findings demonstrating an association between renal involvement and gcd2 have been presented .
the tgfbip ( also known as ig - h3 , keratoepithelin ) is a 68-kda ecm protein with four evolutionary conserved fasciclin-1 domains and a carboxy - terminal arg - gly - asp sequence .
this protein participates in many physiological processes , including morphogenesis , adhesion / migration , tumorigenesis , angiogenesis , wound healing , and inflammation .
tgfbip is found in ecm of several human tissues and is abundant in the cornea .
mutations of the human tgfbi gene have been linked to several autosomal dominant multiple types of cd , including gcd2 .
almost all cases of gcd2 are caused by tgfbi gene mutations ( 5q31 ) , particularly p.arg124his ( r124h ) . in a previous study ,
tgfbi gene mutation was estimated to have a prevalence of at least 11.5 affected people per 10,000 individuals in korea . according to embryonic expression studies using a mouse knock - out model of tgfbi , schorderet et al .
mutations in adhesion and ecm molecules , such as integrins and laminin-2 , play an important role in the pathogenesis of focal segmental glomerulosclerosis ; however , thus far , the relationship between tgfbi mutation and kidney disease has not been established . in an autopsy patient with tgfbi - related cd , pathologic deposits caused by tgfbip accumulation
were only observed in the cornea and in no other tissue or organ , including the kidney ; however , an electromicroscopic examination was not performed in that report .
tgfbip was present in the capsule and tbm of the developing kidney and was predominantly localized in the epithelial cells of the collecting ducts as well as the distal proximal tubules .
tgfbip is secreted into the extracellular space and may bind to fibronectin , laminin , and type i , ii , and iv collagens as well as integrins [ 12 , 13 ] .
proteomic analysis revealed that tgfbip is a component of glomerular ecm [ 3 , 4 ] , and it exhibits protein - protein interactions between the following ecm proteins : -2 macroglobulin ; -1 , -2 chain type i collagen ; -1 chain type ii collagen ; -1 , -2 , -3 , and -4 chain type iv collagen ; fibronectin ; and fibrillin-1 .
proteoglycans directly bind to tgfbip and affect collagen vi aggregation and possibly the interaction between integrin and collagen vi .
binding allows tgfbip , including ecm proteins , to play an important role in cell - collagen signaling interactions that comprise bm , bone formation , and development as well as cell migration and growth . in our patient ,
various pathological findings of gbm and tbm and widening of the subendothelial space of gbm were observed by electron microscopy .
a negative genetic analysis of well - known monogenic kidney diseases prompted us to consider that tgfbi mutation could affect the bm of the developing kidneys and produce such bm findings .
the pathophysiological mechanisms and the incidence of this condition or genotype - phenotype correlation for tgfbi mutations are not obvious .
if detailed examination of the corneas is not performed , gcd2 may not be observed until middle - age and older . it should be noted that detailed ocular examinations , including cornea assessments , are valuable when diagnosing nephropathy associated with tgfbi mutations .
presently , alut testing is beneficial for laser - assisted in situ keratomileusis to protect patients from accelerated vision loss . as demonstrated with our patient , this test is easy and safe to perform when diagnosing patients with nephropathy associated with tgfbi mutations . in conclusion ,
we have reported the first case of a unique nephropathy complicated by tgfbi - related cd .
tgfbi - related nephropathy remains unknown and is difficult to diagnose without electron microscopic examination .
further reports should be accumulated to determine whether the incidence of renal diseases associated with this mutation may presently be more frequent .
patients with tgfbi - related cds , including gcd2 , should be examined for renal abnormalities .
the authors have no ethical conflicts to disclose , and the patient provided informed consent .
|
many types of inherited renal diseases have ocular features that occasionally support a diagnosis .
the following study describes an unusual example of a 40-year - old woman with granular corneal dystrophy type ii complicated by renal involvement .
these two conditions may coincidentally coexist ; however , there are some reports that demonstrate an association between renal involvement and granular corneal dystrophy type ii .
granular corneal dystrophy type ii is caused by a mutation in the transforming growth factor--induced ( tgfbi ) gene .
the patient was referred to us because of the presence of mild proteinuria without hematuria that was subsequently suggested to be granular corneal dystrophy type ii .
a kidney biopsy revealed various glomerular and tubular basement membrane changes and widening of the subendothelial space of the glomerular basement membrane by electron microscopy .
however , next - generation sequencing revealed that she had no mutation in a gene that is known to be associated with monogenic kidney diseases .
conversely , real - time polymerase chain reaction , using a simple buccal swab , revealed tgfbi heteromutation ( r124h ) .
the tgfbi protein plays an important role in cell - collagen signaling interactions , including extracellular matrix proteins which compose the renal basement membrane .
this mutation can present not only as corneal dystrophy but also as renal disease .
tgfbi - related oculorenal syndrome may have been unrecognized .
it is difficult to diagnose this condition without renal electron microscopic studies . to the best of our knowledge ,
this is the first detailed report of nephropathy associated with a tgfbi mutation .
|
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we now appreciate that the immune system recognizes cancer and that patients with strong natural immune reactions have better survival compared with those with weak responses .
this concept was first demonstrated in patients with colorectal cancer ( crc ) by pages et al .
here they demonstrated that the immune contexture , defined by number , type and location of tumour immune infiltrates in primary tumours , are key prognostic factors for disease - free and overall survival ( os ) in crc patients .
importantly , the immunoscore , an immune - based classification strategy derived from three aspects of the immune contexture , proved superior over the standard tumour node metastases classification in predicting outcomes in survival and relapse of crc patients and their response to therapy . collectively , a good immunoscore , defined as high densities of tumour - infiltrating lymphocytes particularly with a t helper type 1 ( th1 ) and cytotoxic orientation , was associated with longer disease - free survival and/or improved os .
this correlation was first documented in crc but has now been extended to a number of other cancers , including melanoma , head and neck , breast , ovarian and lung .
importantly , recognition that cancer development and progression is influenced by the host immune system has led to the development of a conceptual framework called cancer immunoediting , ' which explains the dual host - protective and tumour - promoting roles of the immune system .
furthermore , the demonstration that cancers actively evade immune destruction has led to its inclusion as an emerging hallmark of cancer in 2011 .
tumours can escape immune detection and destruction by a number of mechanisms that have been previously discussed .
these can include : ( i ) downregulation of antigen and major histocompatibility complex class i ( mhc i ) expression , ( ii ) production of immunosuppressive mediators ( for example , interleukin-10 ( il-10 ) , transforming growth factor- , adenosine , vascular endothelial growth factor - a ( vegf - a ) , indoleamine 2,3-dioxygenase ( ido ) ) to dampen effector cell activation and ( iii ) recruitment of immune cell subsets ( t regulatory cells ( tregs ) and myeloid - derived suppressor cell ( mdsc ) ) to suppress effector immune cell function and facilitate its growth and metastases .
in addition to these pathways , we now understand that physiological negative feedback loops that curtail t - cell activation , to prevent autoimmunity , can be exploited by tumours to limit anti - tumour responses .
for example , after activation , t cells upregulate checkpoint receptors such as cytotoxic t - lymphocyte antigen 4 ( ctla-4 ) , which bind to b7 ligands with higher affinity than the co - stimulatory receptor cd28 , thus attenuating effector t - cell function .
other t - cell checkpoint receptors that limit activated t - cell effector function include programmed cell death-1 ( pd-1 ) , b and t lymphocyte attenuator ( btla ) , t - cell immunoglobulin mucin 3 ( tim3 ) and lymphocyte activation gene-3 ( lag-3 ) .
recently , it has been demonstrated in melanoma patients that tumours can also counter attack from activated t cells though another mechanism termed adaptive immune resistance . in this study ,
tumour - infiltrating lymphocytes producing inflammatory cytokines such as interferon- in the tumour microenvironment upregulated pd - l1/l2 expression on tumours , which delivers an inhibitory signal to pd-1-expressing t cells . over the past 5 years
, the use of antibodies to target checkpoint receptors ( also termed checkpoint blockade ) has emerged as one of the most effective ways to overcome tumour - induced immunosuppression and re - activate an endogenous anti - tumour response . in 2011 , anti - ctla-4 ( ipilimumab ) was the first immunomodulatory monoclonal antibody approved by the food and drug administration for the treatment of patients with advanced melanoma on the basis of improved survival benefits .
recently , second - generation checkpoint inhibitors , anti - pd-1 and anti - pd - l1 , have induced better anti - tumour efficacy ( 31% objective response rate ) compared with ipilimumab ( 6% ) . with the recent observation of further increase in anti - tumour efficacy ( 53% ) through the combination of anti - ctla-4 and anti - pd-1
clinical trials are currently in different stages of accrual and development to assess checkpoint receptor blockade in combination with ( 1 ) chemotherapies , ( 2 ) radiotherapy , ( 3 ) small - molecule inhibitors , ( 4 ) vaccines and ( 5 ) co - stimulatory receptors ( for example , cd40 , ox40 ( also known as cd134 ) , cd137 , gitr ( glucocorticoid - induced tumour necrosis factor ( tnf ) receptor - related protein ) and cd27 .
the induction of iraes is an important factor to consider when attenuating co - stimulatory and co - inhibitory immune receptors for cancer immunotherapy .
thought also has to be given as to how targeting of these receptors in combination or with other therapeutics may induce combination - specific iraes that may not be predicted or observed by monotherapies .
iraes are thought to be related to therapy - associated cytokine release and t - cell - mediated organ infiltration and different from bona fide autoimmunity as they normally resolve following therapy or with corticosteroids or anti - tnf therapy .
iraes can generally involve the gastrointestinal , liver , skin , nervous and endocrine systems ( figure 1 ) . in a pooled analysis of 325 patients
treated with 10 mg kg of anti - ctla-4 , iraes of any grade were observed in 72.3% .
similar proportions were also observed in patients treated with anti - pd-1 alone , although with less severity . in most cases , iraes are mostly mild and generally manageable with appropriate treatment algorithms now having been developed .
this includes dose interruption / discontinuation and the use of systemic high - dose corticosteroids .
specifically , grade 3 or 4 iraes , which developed in each of these patients ( 14% for anti - pd-1 and 25% for anti - ctla-4 ) warranted attention , as in extreme cases these can be severe and life threatening .
patients who resolve their grade 2 iraes can recommence treatment , although any grade 3 or 4 iraes ( with the exception of grade 3 skin toxicity ) is a contraindication to further therapy with ipilimumab .
importantly , almost all patients ( 93% ) developed iraes following concurrent anti - pd-1 and anti - ctla-4 monoclonal antibody therapy with an increase in grade 3 or 4 iraes ( 50% ) being observed compared with those treated with monotherapy alone . moving forward , the better safety and clinical efficacy of targeting pd-1
( most likely due to its role in regulating t - cell activation in peripheral tissues ) suggest that it will be the checkpoint receptor of choice to combine with other therapies for cancer treatment .
nevertheless , one may predict that the act of combining immunotherapies increases the proportion of patients who may develop severe iraes .
this may limit the number of patients who can fully benefit from combination cancer therapies due to discontinuation , as seen in 21% of patients treated with anti - ctla-4 and anti - pd-1 .
iraes generally manifest during the induction phase of treatment ( initial 912 weeks of therapy ) and organ - specific iraes appears to follow kinetics of appearance . in the case of ipilimumab where iraes of skin and mucosa develop after about 36 weeks , this is followed by diarrhoea / colitis around week 5 and liver toxicity and endocrinopathies around week 6 .
more recently , an update on patients treated with anti - pd-1 reported on 2 years of safety monitoring and found that similar to what has been reported for ipilimumab , most adverse events tended to occur within the first 6 months of therapy .
hence , the goal of therapies targeting checkpoint receptors in combination with other approaches should aim to generate a strong therapeutic index over the induction period of therapy .
questions that have to be answered include whether it is possible to release / activate anti - tumour immunity without development of iraes if the right combination , dosing and scheduling is utilized .
alternatively , is the development of iraes a predictor of anti - tumour responses ? in one study , it was reported that ipilimumab - treated patients who developed grade 3 or 4 iraes were more likely to achieve a clinical response compared with those who had no iraes .
interestingly , in a retrospective analysis of 498 patients pooled from four phase ii clinical trials treated with different doses of ipilimumab , feng et al . reported that higher exposure ( dose ) was associated with better survival , albeit at a greater risk of developing iraes in the grade 3 or higher category .
a caveat is that this correlation is observed simply because the longer the patients responding are treated , the greater the possibility of developing iraes .
however , the study by feng et al . also suggested that patients who did not experience iraes during the induction period were not likely to experience it during maintenance therapy with ipilimumab .
it would be interesting to perform retrospective analysis to determine whether these group of patients also did not benefit as much clinically .
potentially these analyses may help answer the question of whether tumour reactive and autoreactive t cells are the same or distinct .
it should be noted that , in some studies , there were patients , albeit at a low proportions , who developed durable responses with no associated iraes .
analysis of these patient cohorts may provide clues as to the requirements necessary to release anti - tumour immunity without inducing iraes . for patients who discontinued combination therapy due to iraes ,
do they still benefit clinically and what is the minimum period of therapy required to allow immune activation and/or release of immune suppression ?
a recent report suggests this is likely , where patients with advanced melanoma treated with ipilimumab were found to survive for up to 10 years , confirming the durability of the overall survival ( os ) trend observed .
these results , the largest analysis of os for 1861 patients , included 2 phase iii trials , 8 phase ii trials and 2 retrospective trials .
their findings demonstrated that in 1015% of the total patients who responded , 22% of patients can achieve 310 years os and this os reaches a plateau at 3 years .
this was observed regardless of dose ( 3 or 10 mg kg ) , previous treatment history and whether they had been kept on a maintenance therapy with ipilimumab .
analysis of safety data , which were not included in these results , may shed light on the association of iraes ' severity with os as well as clinical benefits obtained following therapy cessation .
in addition to checkpoint receptors , agonistic antibodies targeting co - stimulatory receptors belonging to the tnf receptor family ( cd40 , ox40 , cd137 , gitr and cd27 ) are also being assessed .
these receptors , in general , act through activation of effector cell function and are currently being evaluated for their anti - tumour efficacy in various phase i / ii clinical trials . in particular , the data for agonistic antibodies targeting cd40 and cd137 ( 4 - 1bb ) are most mature where some objective responses , as well as corresponding iraes , have been reported . in a number of phase i clinical trials ,
agonistic anti - cd40 monoclonal antibodies have been evaluated against solid tumours or b - cell non - hodgkin 's lymphoma .
objective responses were reported in a proportion of these patients , with the most common iraes being primarily grade 2 cytokine release syndrome , which manifests as transient chills , fever and rigors and mild elevations in liver enzymes and decreases in circulating platelet numbers .
interestingly , iraes such as colitis or dermatitis , which are normally associated with checkpoint receptor blockade and can result in dose interruption / discontinuation , were not observed in targeting cd40 , suggesting the comparative safety of anti - cd40 .
targeting of another co - stimulatory receptor cd134 ( ox40 ) in one phase 1 clinical trial was also safe as no acute toxicities were reported at the dose tested .
by contrast , grade 3/4 iraes ( severe liver toxicity ) were observed in patients treated with anti - cd137 ( urelumab ) particularly at high doses , resulting in the termination of a number of phase i trials .
currently , lower doses of anti - cd137 are being evaluated for their safety in a phasei / ii clinical trial with reports suggesting anti - tumour activity can still be elicited without significant liver toxicity . the experience with ipilimumab and immunotherapies in general is their kinetics of response is much slower but more durable compared with those seen with cytotoxics .
recently , a long - term follow - up study of 177 patients treated with ipilimumab reported a median time to achieve complete response of 30 months , consistent with slow kinetics of tumour regression .
similarly , the kinetics of anti - pd-1 responses is atypical with some patients responding rapidly as early as 8 weeks into therapy , whereas other patients displayed new tumours or growth of existing tumours before responding .
in addition , some patients continued to respond despite cessation of anti - pd-1 therapy .
strikingly , a different kinetics of response was observed in patients treated with the maximum tolerated doses of anti - pd-1 and anti - ctla-4 , given in combination .
impressively , these patients all had tumour reduction of 80% ( classified as deep response ) or more at their first scheduled assessment at 12 weeks .
this is in contrast to patients who were given anti - ctla-4 and anti - pd-1 in a sequenced regime .
this suggests anti - tumour responses can be rapid if the right combination is given at optimal dosage and timing .
the question now is whether ( 1 ) maintenance dosage is required for patients treated with combination therapies who responded rapidly so as to minimize their risk of further developing more iraes or ( 2 ) can patients who developed grade 3 or 4 iraes and ceased therapy still benefit and can one correlate the number of doses they received before cessation , with clinical benefit to determine what is the minimum period of therapy required ?
( 3 ) if iraes are predictors of response , are there biomarkers that can be used to determine which patients will develop them and thus enable monitoring and prophylactic treatment to prevent their escalation into grade 3 or 4 toxicities and cessation of treatment ?
given that many patients with iraes treated with systemic corticosteroids and/or anti - tnf resolve their symptoms rapidly if treated when symptoms first appear , it would suggest serum tnf and other inflammatory cytokines like interleukin-6 could be measured as biomarkers and targeted .
there seems to be a consensus that corticosteroids or anti - tnf administration do not impede anti - tumour immune responses following checkpoint blockade .
given that most patients will develop some form of iraes following immunomodulatory therapies , particularly in combination , it would suggest concurrent administration of anti - tnf and/or corticosteroids with combination therapies is an option that can be investigated .
the demonstration that targeting checkpoint receptors activates endogenous anti - tumour immunity and leads to significant clinical benefit in different cancer types now spurs the question of how their efficacy can be further improved through rationale combination approaches . in advanced melanoma ,
concurrent anti - ctla-4 and anti - pd-1 therapy resulted in clinical benefits in 50% of patients , which to date is the best result obtained with cancer immunotherapies .
nevertheless , 50% of patients did not respond , and in some cancer types such as pancreas and prostate , checkpoint blockade does not appear to provide any significant clinical benefits .
thus , early - phase clinical trials are exploring how checkpoint inhibitors combine with other agents , including ( 1 ) conventional therapies ( radiotherapy , chemotherapy ) , ( 2 ) alternative immunotherapies ( vaccines , cytokine , adoptive cellular therapy ) , ( 3 ) targeted therapies ( braf or vascular endothelial growth factor inhibitors ) or ( 4 ) immunomodulatory antibodies . with combination immunotherapies increasingly being tested in clinical trials , iraes will be an ongoing issue that has to be dealt with ( as discussed above ) as it may limit their usage .
furthermore , even when both agents have regulatory approval and have distinct mechanisms of action and toxicity profile , unexpected specific iraes induced by the combination can occur .
this was illustrated by a recent clinical trial that reported on the unexpected hepatotoxicity observed with melanoma patients after concurrent treatment with a braf inhibitor ( vemurafenib ) and ipilimumab , resulting in cessation of the trial . given the beauracracy , cost and time associated with conducting clinical trials , utilizing preclinical mouse models that can more accurately model tumour immunity and iraes may allow more informed assessment of
which therapies can be combined to induce optimal therapeutic index ( figures 2 and 3 ) .
tumour - associated autoimmune syndrome is very rare in patients with cancer , such as melanoma - associated retinopathy and vitiligo .
however , new data suggest that it is not merely a side effect of robust anti - tumour immunity .
the development of vitiligo is considered to be a benefit to the prognosis of the patient with malignant melanoma . in a preclinical study , mice with
vitiligo generated more cd8 memory t cells , which protected them from melanoma expressing the same melanocyte antigens . in another study , inflammatory monocytes or dendritic cells accumulated in the skin
were shown to be capable of killing melanoma cells as well as normal melanocytes . however , whether the relationship of tumour - associated autoimmune syndrome and tumour immunity is dependent on the innate immune system or adaptive immune system or both requires further investigation .
in contrast , iraes are not true autoimmune diseases , as they typically resolve with cessation of therapy and appropriate immunosuppressive regimes .
in addition , the experience with ipilimumab and immunotherapies in general is their kinetics of response is much slower but more durable compared with those seen with cytotoxics . thus utilizing spontaneous mouse tumour models
may better mimic the response kinetics that is observed in the clinic and may potentially allow for the development of any therapy - induced toxicity .
these models can be divided into those that are carcinogen - induced such as methylcholanthrene ( mca)-induced fibrosarcomas and 7,12-dimethylbenz[]anthracene ( dmba)/12-o - tetradecanoylphorbol-13-acetate ( tpa)-induced skin papillomas or genetically engineered mouse tumour models , which have enforced expression of oncogenes and/or the loss of function of tumour suppressors , often in a tissue - specific and/or temporally controlled manner ( figure 2 ) .
examples include the her2/neu or pymt transgenic mice to mimic breast cancer , the mt / ret model of spontaneous metastatic melanoma and braftyr - creerpten mice in which 4-hydroxytamoxifen ( 4-ht ) induces de novo melanoma as well as the use of adenoviral vectors encoding cre recombinase to selectively introduce mutations in the oncogene kras and the tumour - suppressor gene tp53 in the pulmonary epithelia to induce autochthonous lung tumours .
interestingly , it has been noted that carcinogen - induced models are very immunogenic , whereas germline mutation models are often not , most likely due to the former carrying more passenger mutations , thereby generating more neoantigens that can potentially be recognized by the immune system .
this was elegantly illustrated in two studies investigating the role of t cells in selecting for non immunogenic sarcomas using either a mouse model of sarcomagenesis driven by cre recombinase - triggered activation of the kras oncogene and inactivation of the trp53 tumour - suppressor gene or a mca - induced mouse model of sarcoma . in the first model , sarcomas that developed
did not appear to be recognized by adaptive immunity as these tumours grew equally well when transplanted into wild - type or rag-2-deficient mice .
on the contrary , mca - induced sarcomas , which had many passenger mutations in addition to kras and trp53 , were demonstrated to generate tumour - specific t cells , which could then selectively sculpt tumour immunogenicity .
interestingly , mouse mca - induced sarcomas were also found to have qualitative and quantitative genomic mutation profiles to carcinogen - induced human cancers , such as smoker 's lung cancer .
thus carcinogen - induced mouse models of cancer may better mimic cancers that are immunogenic .
in contrast , transplantable tumours , which grow more rapidly and are employed as an initial model to assess the therapeutic potential of combination therapies , may be less useful . as most transplantable tumours are grown subcutaneously on the flanks of mice ,
an alternative approach is to inoculate them orthotopically to better mimic the tumour environment from which they originated .
indeed , immunotherapies that demonstrate efficacy against a renal tumour grown subcutaneously were not as effective when the same tumour was transplanted into the kidney .
although checkpoint blockade in patients commonly induces iraes , such events have rarely been commented upon or observed in preclinical mouse models , even when 3 different pathways are targeted .
it is possible that while treated mice appeared outwardly healthy , closer examination of animals may have revealed the presence of biochemical autoimmunity , which is frequently observed in patients even if they do not display clinical symptoms .
in addition , observable iraes may not have manifested owing to the generally short nature of preclinical mouse models experiments or , perhaps , the strain of mice used may be more resistant to developing iraes compared with humans .
this was elegantly shown in a recent study where repeated dosing of anti - ctla-4 in sjl / j mice over 5 weeks was able to induce hypophysitis , an irae that appears to be associated with ipilimumab treatment .
testing checkpoint blockade in combination or with other therapies in mouse strains more prone to development of autoimmune symptoms ( for example , nod , sjl / j ) may be one strategy to assess the development of tumour immunity and autoimmunity .
furthermore , it has also been shown that repeated dosing of agonistic antibodies to co - stimulatory receptors such as gitr induced anaphylaxis in tumour - bearing mice that was caused by serum antibodies , and dependent on cd4 t cells , b cells , basophils , platelet - activating factor and gitr . alternatively , to observe iraes in mice , a more dramatic alteration of the immune system such as the complete and systemic depletion of tregs may be required .
intratumour treg depletion by antibodies such as anti - ctla-4 ( now a recognized mechanism ) does not seem sufficient to induce iraes in mice , as are observed in humans .
in addition , heterogeneity in the kinetics of response is normally seen in patients after immunotherapy , such as those that were treated with anti - pd-1 , and thus a tumour model that mimics what is observed in the clinic will be useful . using the de novo model of mca - induced fibrosarcoma , we observed that complete treg depletion in dereg mice bearing established mca - induced tumours displayed a similar heterogeneous range of tumour responses to that observed in the clinic . in our study , no overt autoimmunity with treg depletion was observed , but this may be explained by the potential compensatory effect of other nontransgenic regulatory cells in the dereg mice .
comparative experiments in foxp3-dtr knock - in mice , where autoimmunity is more easily generated upon treg depletion , are currently underway in our laboratories . in clinical trials ,
concurrent blockade of ctla-4 and pd-1 checkpoint receptors induced deep and rapid anti - tumour responses in contrast to the slower response kinetics seen in patients given anti - ctla-4 and anti - pd-1 in a sequenced - regime .
this suggests that anti - tumour responses can be rapid if the right combination is given at optimal dosage and timing .
it was not made clear if patients who had the rapid deep objective response had high / low grade iraes .
indeed , we recently reported the use of the poorly immunogenic b16f10 melanoma model to characterize a very heterogeneous anti - tumour effect of the immune response induced by complete treg depletion in dereg mice .
strikingly , the duration of the tumour immune system interaction induced in individual treg - depleted mice positively correlated with their propensity to develop vitiligo .
a rapid complete tumour rejection was not associated with the development of autoimmunity ; however , a proportion of mice that suppressed but did not effectively clear b16f10 melanoma did develop vitiligo .
we would postulate that approaches that combine with treg depletion to rapidly reject tumours may also diminish iraes .
currently , cancer patients who have chronic autoimmunity are generally precluded from clinical trials with checkpoint blockade .
however , patients with certain autoimmune diseases ( pernicious anaemia , crohn 's disease , ulcerative colitis , systemic lupus erythematosis and psoriasis ) are generally thought to be at higher risk of cancer due to the underlying dysregulation of their immune system , as well as a consequence of their treatment regime .
thus , it is imperative we understand how and if toxicities induced by combination therapies in this cohort of patients differ to cancer patients who do not have underlying autoimmunity , that is , whether the therapies will be more toxic . given that many tumour - associated antigens are self - antigens , and that self - reactive t / b cells are increased in patients with autoimmunity , should a case be made that cancer patients with stable underlying autoimmunity be allowed treatment with immunotherapies as they may respond better ?
of course , these patients have to be monitored stringently owing to their increased risk of developing iraes or have their autoimmune symptoms exacerbated .
nevertheless , studies in this cohort of patients may potentially help in answering the question of how much overlap they are between tumour reactive and autoreactive t cells as well as their relative importance in different cancers .
the foxp3-dtr mouse represents one model where we can evaluate how combination immunotherapies attenuate tumour immunity / iraes . in this
setting , tumour - bearing foxp3-dtr mice can be conditionally depleted of their tregs to mimic the maximum release of suppression on all immune cells and can then be treated with different immunotherapies / agents .
potentially , this may allow us to assess how different co - inhibitory / co - stimulatory receptors combine with each other or with other therapies to attenuate anti - tumour immunity / iraes .
although clinical observations suggest that autoimmune effectors are intricately involved in tumour cell killing , evidence using antigen - specific preclinical mouse models indicates that , in some cases , the immune system can selectively target tumour tissue while sparing self . indeed
how self antigen - specific t cells target tumour versus healthy tissues , and how they are regulated in different microenvironments , is still unclear . to address this issue , miska et al .
evaluated the ability of specific t effector cells to kill tumour versus healthy cells expressing the same self antigen in the same animal .
their studies concluded that tumours suppressed autoimmune t effector cells locally without distal impairment , suggesting that higher levels of suppression exist in tumours compared with healthy tissues expressing the same self antigen .
their studies also highlighted an increased potency of regulatory mechanisms such as treg and mdscs in tumours , although the molecular mechanisms responsible for this increased suppression remain to be elucidated .
thus antigen - specific cd4 or cd8 t cells that are unable to eliminate tumours can still mediate destruction of healthy tissues , suggesting that the threshold of inducing autoimmunity is lower than tumour immunity .
this implies that systemic administration of immunotherapies that result in t - cell activation will almost always induce autoimmunity unless a substantial antigenic difference is identified between tumour target and healthy tissue .
thus strategies that can alleviate immune suppression specifically at the tumour site will be the way forward for next - generation combination therapy approaches .
another issue for consideration is that many of the cancer patients may have underlying metabolic syndrome such as obesity and diabetes , which have repeatedly been associated with increased incidence for some common cancers .
how would combination therapies impact on this cohort of patients in terms of tumour immunity / iraes ?
this question may be assessed by testing immunotherapies with the obese diabetic mouse ( ob / ob ) model .
the durable clinical benefits obtained with immune checkpoint blockade have reinvigorated the field of cancer immunotherapy . moving forward ,
checkpoint blockade looks set to be explored in combination with other therapies to further improve their therapeutic efficacy and their impact on more cancer types .
however , the proportion of patients developing severe iraes following combination immunotherapies most likely will increase . utilizing relevant preclinical mouse models of cancer that better model tumour
|
the current excitement surrounding cancer immunotherapy stems particularly from clinical data involving agents mediating immune checkpoint receptor blockade , which have induced unprecedented efficacy against a range of tumours compared with previous immunotherapeutic approaches .
however , an important consideration in targeting checkpoint receptors has been the emergence of associated toxicities termed immune - related adverse events ( iraes ) . in light of the clinical benefits observed after co - blockade of checkpoint receptors and data from preclinical mouse models , there is now a strong rationale to combine different checkpoint receptors together , with other immunotherapies or more conventional therapies to assess if clinical benefits to cancer patients can be further improved .
however , one may predict the frequency and severity of iraes will increase with combinations , which may result in premature therapy cessation , thus limiting the realization of such an approach .
in addition , there is a limit to how many different combination therapies that can be tested in a timely manner given the legal , regulatory and budgetary issues associated with conducting clinical trials .
thus , there is a need to develop preclinical mouse models that more accurately inform us as to which immunotherapies might combine best to provide the optimal therapeutic index ( maximal anti - tumour efficacy and low level iraes ) in different cancer settings . in this review
we will discuss the iraes observed in patients after checkpoint blockade and discuss which mouse models of cancer can be appropriate to assess the development of tumour immunity and iraes following combination cancer immunotherapies .
|
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hemophagocytic lymphohistiocytosis ( hlh ) secondary to infectious disease is an important entity especially in tropics where infectious diseases are rampant and still pose a major threat .
a timely diagnosis and prompt treatment can improve the clinical outcome of this otherwise potentially fatal condition ! this article aims to alert the clinicians that in persistent unresolved fever especially in tropics , a diagnosis of secondary hlh should be given due consideration and we present 6 cases in this paper .
all patients presented between march 2012 and march 2013 ( details shown in table 1 ) and fulfilled the revised criteria of hlh as listed in table 2 . of them ,
the mean age at diagnosis was 33.83 years ( range : 20 to 64 years ) , with a male : female ratio of 2:1 .
three patients presented with evidence of hepatomegaly and/or splenomegaly . all of the patients had at least a bi- or trilineage cytopenia , elevated liver enzymes and hyperferritinemia .
two of the cases were secondary to dengue fever and one secondary to disseminated tuberculosis , one secondary to pulmonary tuberculosis , one secondary to malaria ( falciparum ) and one secondary to leptospirosis .
all patients had good recovery and none of them relapsed at a median follow up of 4 months .
characteristics of secondary hlh patients m male ; f female , y yes ; n no ; csa cyclosporine ; na not available proposed hlh diagnostic criteria , 2009 ravenous macrophage - hemophagocytosis of rbcs by macrophage seen
hlh is a hyper - inflammatory condition which may be familial or occur secondary to autoimmune diseases or infection , malignancy or other triggers . despite advances in the diagnostic work up of febrile patients ,
hlh remains elusive from the diagnostic capabilities of many clinicians and continues to be a potentially fatal disease entity .
the underlying pathophysiology of the disease constitutes an unrestrained immune activation with defective macrophage function regulation .
an excessive activation of macrophages leads to a cytokine storm in the host and leads to host tissue damage and organ dysfunction associated with the syndrome .
excessive pro - inflammatory or defective anti - inflammatory responses leading to this cytokine storm can be triggered by host factors or environmental agents .
accordingly , a hlh arising in the setting of an underlying genetic mutation is termed familial hlh , in the setting of an underlying rheumatologic disease like rheumatoid arthritis is termed as macrophage activation syndrome ( mas ) and in the setting of an underlying infection it is termed as reactive or secondary hemophagocytic syndrome ( hps ) or secondary hlh . case reports describing mas post bone marrow transplant in patients with juvenile rheumatoid arthritis , or secondary to sle or dermatomyositis or other autoimmune diseases have been reported .
macrophage and neutrophil activation is a hallmark in conditions like stills disease which can lead to hyperinflammatory condition with hlh .
overproduction of proinflammatory cytokines , uncontrolled activation of t cells , and macrophages associated with decreased natural killer cell and cytotoxic cell functions seem to be the hallmarks of the immunologic abnormalities in mas .
recent human and murine investigations suggest that all hpss should be differentiated based on etiology and pathogenesis as treatment strategies for each may vary .
however , all etiologies lead to a state of hyper ferritin levels . the precise mechanism of ferritin as a trigger or a bystander in pathogenesis needs to be explored .
the reactive or infection - associated hlh remains a relatively important and yet unfortunately an underdiagnosed entity especially in the tropical world .
various combinations of high grade fever sometimes a second spike of fever after a brief period of recovery which coincides with fresh cytopenias , unresponsiveness to broad - spectrum antibiotics , new onset organomegaly or sudden increase in size of organomegaly in the setting of an infectious disease are some of the diagnostic clues for this disease . in case of infections ,
a simple blood investigation that shows elevated levels of serum ferritin should raise the suspicion of a coexisting hlh .
a tentative diagnosis of hlh for initiation of immunosuppressive therapy can be done when clinical and lab abnormalities exist as defined in revised 2009 hlh protocol . also , in the resource poor settings , a single value of ferritin more than 10,000 in the absence of iron overload conditions like hemochromatosis and thalassemia syndromes can act as a surrogate marker for hlh with a sensitivity of 90% and specificity of 96% .
hps secondary to infections has been classified as a separate entity under international classification of diseases by world health organization .
other viruses like dengue , herpes , cmv , hiv have been reported to have hps secondary to them .
secondary hps has frequently been associated with intracellular pathogens that stimulate th1 immune response . of the bacterial infections ,
other reports with organisms like salmonella , leptospirosis , malaria , toxoplasmosis , leishmenia , rickettsia and other organisms have been postulated in secondary hlh .
although case reports and case series have frequently reported the reactive hlh secondary to infectious causes especially in the tropical countries , it continues to remain as an under diagnosed and under - reported entity .
the treatment of secondary hlh includes aggressive treatment of underlying condition along with immunosuppressive therapy .
however , recent data suggest to a less intense immunosuppressive therapy . as opposed to the familial hlh ,
where allogenic stem cell transplant is the only curative treatment , most of the infection associated hlh cases respond to a course of corticosteroids .
some patients may need additional treatment with drugs like etoposide and cyclosporine ; however , a full course of hlh 2004 protocol is rarely required in them . in our case
series , all patients were initiated with dexamethasone at a dose of 10 mg / m / day .
three patients with hlh secondary to underlying tuberculosis , malaria and leptospirosis [ case 2 , 5 , 6 in table 1 ] each responded to steroid monotherapy . within 2 days of starting steroids their fever reduced with improvement in cytopenias .
one patient with dengue fever had a ferritin values more than 100,000 along with severe pancytopenia .
she was given two doses of etoposide iv at a dose of 100 mg / m ( reduced dose ) at weekly intervals .
other two patients were given one single dose of etoposide following which they became symptomatically better .
one patient with dengue and one with tuberculosis however had delayed recovery of platelet count and they were started on oral cyclosporine .
csa was given for duration of three months following which it was tapered and stopped .
this is in tune with the other case reports and case series of secondary hlh .
one possible explanation for this is removal of inciting agent by means of effective antibacterial therapy . because of its powerful proapoptotic activity
describe their unique experience with stem cell transplant in treating secondary hlh in an adolescent .
srinivas et al . in their systemic review of hemophagocytosis syndrome ( hps ) in tropics found infectious trigger as the cause in 51% of the adult patients .
leishmenia was seen in 40.6% , rickettsia in 18.8% malaria in 15.6% and enteric fever in 9.4% .
56% patients were secondary to viruses , 26% secondary to dengue virus , 17.3% were secondary to ebv and 8.7% each to cmv and parvovirus b 19 .
most of the literature on hlh in tropics is centered on few hospitals and includes case reports and case series .
larger studies and trials are required to throw more light on this potentially fatal condition and unfold the mysteries of ravenous macrophages . in india ,
hlh associated with dengue fever and malaria with a high parasite index has been documented . in one study from india
, the dengue virus has been found to be the most common agent causing hlh in children .
many of the previous case reports of hlh are reported in complicated dengue fever like dengue hemorrhagic syndrome .
crohn 's disease and immunosuppression are associated with an increased risk for developing secondary hlh , although none of our patients had underlying disease or immunosuppressed status .. however , cases with classical dengue syndrome have also been reported from indian subcontinent . in our series also , we report two cases of classical dengue fever with secondary hlh .
our cases add to the existing literature of handful of cases of hlh in dengue .
although more common in tropics a case of elderly female has been described by cdc , usa in which hlh secondary to possible dengue infection proved fatal and physicians in west need to be alerted about possible travel acquired dengue which can have fatal complications like secondary hlh .
describe the time - lines of six cases of confirmed dengue with varying severities of hemophagocytosis .
both our patients presented with fever , pancytopenia , organomegaly , high ferritin and fulfilled the criteria of hlh and responded to corticosteroids and etoposide .
hlh secondary to malaria was reported in a young man ( case number 5 ) who had persistent fever , falling counts despite treatment with antimalarials .
had described one of the first cases of hemophagocytosis secondary to malaria ( falciparum ) which resolved with antimalarials .
park et al . discusses four case reports of hlh secondary to vivax malaria all of which resolved with antimalarials .
however , studies in the pediatric population show degree of parasitemia is associated with severity of disease .
similarly our case was secondary to p falciparum malaria with severe parasitemia seen on peripheral smear .
the patient responded to corticosteroids .. hlh secondary to disseminated tuberculosis has been described in past .
pristilla et al . in their review analyzed 36 cases of tuberculosis with secondary hlh .
they found fever to be the most common presenting symptom and evidence of extra - pulmonary tuberculosis was found in 83% of cases . in our series ,
case number 2 a young boy with fever , weight loss , military tuberculosis and choroid tubercles was diagnosed as a case of disseminated tuberculosis .
no significant improvement with anti tubercular treatment prompted us to look for other causes and was diagnosed as secondary hlh based on clinical features , high ferritin and bone marrow evidence of hemophagocytosis .
steroids were added to his treatment course and he showed significant improvement within 2 weeks . similarly an elderly female was diagnosed to have hlh secondary to pulmonary tuberculosis and was started on steroids .
however , she did not improve and was further treated with iv etoposide and oral cyclosporine and showed complete remission of symptoms and evidence of hemophagocytosis on follow up after 4 week .
this is consistent with earlier studies which showed negative tuberculin test should never preclude the possibility of overwhelming tubercular infection in hlh .
leptospirosis is a spirochete which is commonly prevalent in coastal belt of south india . to the best of our knowledge ,
no case of hlh secondary to this disease in adult has been reported . the diagnostic challenge in making appropriate diagnosis has been discussed in one of the previous case reports from taiwan in a young male who presented with shock and had evidence of reactive hemophagocytosis .
our patient was a fisherman from the endemic area who presented with fever and oliguria .
in due course he developed hepatosplenomegaly , severe cytopenia , esr of 5 , high ferritin and bone marrow evidence of hemophagocytosis .
in conclusion with limited experience and lack of guidelines for treatment of tropical hlh , a high index of suspicion is something clinicians should bear in mind .
|
hemophagocytic lymphohistiocytosis ( hlh ) is a potentially fatal hyper inflammatory condition , if not recognized and treated in time .
a high index of suspicion can help identify the condition early .
this condition can occur in the primary or secondary form .
secondary hlh or hemophagocytic syndrome ( hps ) secondary to infections is an important clinical entity especially in tropical world . in this article
, we share our experience with this entity and make an attempt to explore literature about ravenous macrophages which occurs secondary to infections .
it is a series of six cases of hlh secondary to infectious disease in our center in a coastal city in south india over last one year with follow up .
|
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] |
acetone is the simplest ketone compound . in general , acetone is not considered harmful , and the world health organization has not classified acetone as carcinogenic . however , its prolonged inhalation can not only cause irritation of the mucous membranes , headaches , confusion , and narcotic effects , but lead to coma as well [ 15 ] . for etiological reasons ,
acetonaemia is classified to be of endogenous and exogenous origin [ 6 , 7 ] .
multiple toxicities and physiopathological conditions result in ketosis ( acetonaemia particularly ) , including diabetes mellitus ( dm ) , starvation coupled with physiologic stress , prolonged exercise , during pregnancy , and ethanol toxicity , other alcohol ingestions , drug toxicities , inborn errors of ketone metabolism , alcoholic ketoacidosis , delirium tremens , and hypothermia [ 69 ] .
the main symptom of dm is a high blood glucose concentration depending on insulin deficiency . in this case
the body can not fully use glucose but could use fatty metabolism instead of glucose for energy .
dm , especially diabetes and autoimmune associated diseases , thyroid disease , and diseases that can accompany diabetes ( hypertension , cardiovascular disease , cerebrovascular disease , and renal insufficiency ) can cause pathological changes in most of the tissues , organs , and biological fluids depending on lipotoxicity and glucotoxicity .
a lot of medical , chemical , and medicolegal investigations have been carried out with the determination of acetone in blood and other biological fluids [ 2 , 6 , 7 , 10 ] . over the decades
, several methods have been used for its determination in biological samples . in the beginning
, colorimetric methods were developed and used for the determination of acetone in plasma [ 1113 ] .
these methods have common disadvantages such as the lack of specificity and detection limit . in recent decades , gas chromatographs equipped with flame ionization detectors or mass spectrometric detectors
have been developed for determination of acetone concentrations in body fluids and in expired air [ 1418 ] .
enzymatic methods are more specific but more complex and have long assay times and gas chromatographic methods , although widely used , are applied with difficulty as routine tests .
the determination of acetone in the blood is most important in clinical diagnostic laboratory studies .
there are three ketone bodies , while the two main ketone bodies are acetoacetate ( acac ) and 3-b - hydroxybutyrate ( 3hb ) , the third ketone body ; acetone ( ac ) is found minimum level .
ketone bodies are produced by the liver and used peripherally as an energy source when glucose is not readily available [ 9 , 19 ] .
ketone bodies are three water - soluble compounds that are produced as by - products when fatty acids are broken down for energy in the liver and kidney .
ketone bodies are produced from acetyl - coa mainly in the mitochondrial matrix of hepatocytes when carbohydrates are so scarce that energy must be obtained from breaking down fatty acids [ 9 , 20 ] .
acetone can not be converted back to acetyl - coa , so it is excreted in the urine or exhaled .
recently , blood or urine testing kits have been used to test for the presence of acetone in clinical biochemistry laboratories .
acetone can also be quantified by sampling the human blood and testing by gas chromatography .
described a rapid and simple hplc procedure that can be used for the routine measurement of acetone in biological fluids , such as plasma and urine . according to fujii et al .
, it is proposed that liquid chromatography with fluorescence ( lc - fl ) seems to be useful for the determination of acetone in the saliva . in this paper , we present a rapid and simple hplc technique using 2,4-dnph as a derivatizing reagent for quantitative determination and metabolomic research of acetone in biological fluid such as human blood .
2,4-dnph ( sigma - aldrich , 97% ) , acetone ( merck , 99.8% ) , acetonitrile ( sigma - aldrich , 99.8% ) , and methanol ( merck , 99.8% ) were used in this study .
for the chromatographic analysis , thermo scientific dionex ultimate 3000 hplc with a thermoacclaim - c18 ( 15 cm 4.6 mm 3 m ) column and uv - vis dad were used .
the deionized water was 18.2 mcm ( millipore direct - q3 uv ) and was used throughout the experiments .
for the biochemical analyses , cobas 6000 ( roche , germany ) autoanalyzer was used for blood glucose levels .
qualitative analysis of total ketones in urine was evaluated by iris iricel 2000 analyzer ( icem velocity ) . in the first stage of our study , following a 12-hour fasting venous blood samples were taken from patients admitted to hospital of the faculty of medicine , canakkale onsekiz mart university .
the human blood and urine samples were directly collected into a tube ( without a collection device ) between 08:30 and 11:00 am .
clinical biochemistry laboratory blood and urine glucose tests were studied for routine biochemistry using a urine autoanalyzer .
the patients were divided into high blood glucose and urine ketone positive subjects ( group 1 ) and high blood glucose and urine ketone negative example subjects ( group 2 ) .
the patients with hyperglycemia were 8 females and 7 males ( age : 2187 ; n = 15 ) , while 5 female and 2 male patients had positive urine ketones ( age : 2168 , n = 7 ) and 5 male and 3 female patients had negative urine ketones ( age : 5587 , n = 8) .
the blood glucose levels varied between 110 and 320 mg / dl in our patients ( table 1 ) . in the second stage of the study , to determine the probable positive acetone , its quantitative analysis was carried out in biological fluids using the hplc technique .
the blood samples were immediately prepared for hplc analysis carried out within 8 hours after sample collection .
plasma specimens were deproteinized with acetonitrile ( 1 : 1 , v / v ) ; 2,4-dnph is added to the supernatant ( filtered blood samples ) and treated with acetonitrile ( 2 : 1 , v / v ) to prevent crystallization of the synthesized phenylhydrazone .
an aliquot ( 20 microliters ) of the reaction mixture was subjected to hplc at ambient temperature using thermoacclaim - c18 ( 15 cm 4.6 mm 3 m ) column and uv - vis dad with ( methanol / acetonitrile ) water as eluent at a flowrate of 1 ml min and detection at 365 nm [ 2 , 3 ] .
the experimental procedures were conducted in accordance with the ethical standards of the helsinki declaration and approved by the canakkale onsekiz mart university human research ethics committee . written informed consent was obtained from all the subjects .
the 2,4-dinitrophenylhydrazone standards were prepared by mixing a and b solutions : ( a ) : 0.40 g of 2,4-dnph dissolved in 2.00 ml of h2so4 + 3.00 ml of h2o + 10.0 ml ethanol ; ( b ) : 0.50 g or 1.00 ml of the acetone standard dissolved in 20.0 ml ethanol .
after this mixing , a precipitate was formed in each case , isolated through filtration , and dried in vacuum [ 3 , 24 , 25 ] .
acetone was added into its 2,4-dnph derivatives by mixing 1.00 ml of a solution containing 200 mg/100 ml of 2,4-dnph with 1.0 ml of h3po4 , and 4.00 ml of the human serum .
after 2 h , a 40.0 l aliquot was withdrawn and analyzed by the hplc technique [ 3 , 2427 ] .
chromatographic separation was achieved in a thermoacclaim - c18 ( 15 cm 4.6 mm 3 m ) column at uv - vis dad detector ( max 365 nm ) .
the injection volume was 20.0 l and the detection was performed at 365 nm .
the following gradient was used : ( methanol / acetonitrile ) ( 8 : 2 ) water 60 : 40 ( v / v ) .
elution was achieved at retention time ( tr ) 12.10 and flow - rate of 1 ml min .
standard calibration curve was prepared with acetone ( 0.5 , 2.5 , 5.0 , 10 , and 20 mmol l ) and 40 l 2,4-dnph in acetonitrile .
human samples were prepared by adding 40 l 2,4-dnph and 500 l acetonitrile to 200 l human serum .
the calibration curve was constructed by plotting the peak area of the 2,4-dnph derivative of acetone ( y ) versus the concentration of acetone ( x , mmol l ) by linear regression ( n = 4 ) .
the equation was found as y = 0.7361x + 0.0877 with a 0.9967 , correlation coefficient ( r ) .
the method proposed was validated as described in ich guidelines in parameters of linearity , limit of detection and quantification , accuracy , and precision .
the linearity of the method was determined at four concentration levels ranging from 0.5 to 20 mmol l. the calibration curves were constructed by plotting the peak area of the 2,4-dnph derivative of acetone ( y ) versus concentration of acetone ( x ) . the slope , y - intercept , and correlation coefficient were calculated . to check the linearity ,
the lod was estimated using signal - to - noise ratio of 3 : 1 or ( 3 s / m ) and loq as 10 : 1 or ( 10 s / m ) , at which accuracy and standard deviation were within 20% as per ich [ 28 , 32 , 34 , 35 ] .
intraday accuracy and precision were performed for acetone at 5.0 mmol l in replicate ( n = 3 ) .
accuracy ( expressed as recovery ) and precision ( expressed as relative standard deviation ) should not deviate by 15% of the nominal concentration .
as can be seen from figure 1 , the retention time ( tr ) was obtained as 3.80 min .
the most efficient separation of acetone as its 2,4-dnph was obtained with a thermoacclaim c18 column ( 15 cm 4.6 mm 3 m ) at retention time ( tr ) 12.10 min and flowrate of 1 ml min using a ( methanol / acetonitrile ) water elution gradient . no elution problem for acetone as its 2,4-dinitrophenylhydrazone derivative was observed .
typical hplc chromatogram of 0.681 mmol l acetone as its 2,4-dnph derivative is given in figure 2 .
the hplc chromatogram of 15 mmol l acetone in human blood in the first patient of group 1 is given in figure 3 . in our study , the patients were determined as high blood glucose and urine ketone positive subjects ( group 1 ) and high blood glucose and urine ketone negative example subjects ( group 2 ) .
the blood glucose levels , given in table 1 , vary between 110 and 320 mg / dl in our patients , except for sample 7 .
many patients in our study have dm disease , except samples numbered 4 , 5 , 11 , 13 , and 15 ( table 1 ) .
the calibration plot of peak area against concentration was obtained linear in the range 0.5 to 20 mmol l. the regression equation and correlation coefficient were obtained as y = 0.7361x + 0.0877 with a 0.9967 , correlation coefficient ( r ) .
so this value is smaller than 2.2 , and calibration curve is linear . for the human plasma samples , fcritical value for calibration curve at 3 degrees of freedom ( p = 0.05 ) is 3.18 .
so , this value is smaller than fcritical value , and obtained values are appropriate .
the lod and loq were found as 0.041 and 0.136 mmol l , respectively .
the accuracy for acetone in human plasma samples expressed as recovery was found as 98% . for interday assay ,
the accuracy for acetone in human plasma samples expressed as recovery was found as 96% .
2,4-dnph can be used to qualitatively detect the carbonyl functionality of a ketone such as acetone or aldehyde functional group .
a positive test is signaled by a yellow , orange , or red precipitate known as a dinitrophenylhydrazone .
if the carbonyl compound is aromatic , then the precipitate will be red ; if aliphatic , then the precipitate will have a more yellow color .
the reaction between 2,4-dnph and a ketone such as acetone is shown below:(1)rrco+c6h3no22nhnh2c6h3no22nhncrr+h2othis reaction can be described as a condensation reaction , with two molecules joining together with loss of water .
it is also considered an addition - elimination reaction : nucleophilic addition of the -nh2 group to the c = o carbonyl group , followed by the removal of an h2o molecule .
2,4-dnph does not react with other carbonyl - containing functional groups such as carboxylic acids , amides , and esters . for carboxylic acids , amides , and esters
, there is resonance associated stability as a lone pair of electrons interacts with the p - orbital of the carbonyl carbon resulting in increased delocalization in the molecule .
also with carboxylic acids there is the effect of the compound acting as a base , leaving the resulting carboxylate negatively charged and hence unable to be attacked by this nucleophile [ 36 , 37 ] .
in this study , an analytical method was applied for the quantitative determination of acetone in human blood .
the determination was carried out using a uv - vis dad detector with hplc . in most of our patients ' blood samples ,
higher levels of acetone have been measured in the patients who have high level of blood glucose and positive urine ketone ( group 1 ) .
the hplc method based on the labeling of acetone with 2,4-dnph seems to offer a rapid , low cost , sensitive , selective , and reproducible methodology for quantification of the acetone level in clinical samples such as human blood .
the hplc method described here overcomes many of the problems in the determination of acetone in biological fluids and the preanalytical errors .
the volatile ketone is promptly stabilized by conversion into its dnph derivative and rapidly determined without recourse to a solvent extraction step .
the method uses very inexpensive reagents . as can be stated by brega et al .
, the proposed hplc method can therefore be used to great advantage over current gas chromatographic methods ; it can be used in experiments requiring multiple samples and specific activity determination for the routine measurement of acetone in diabetic patients and in biological monitoring of exposed workers .
we also presented acetone as a useful tool for the hplc - based metabolomics investigation of endogenous metabolism and quantitative clinical diagnostic analysis . in the medical and medicolegal practice
however , the detection and early identification of acetone could be used as an initial indicator of detection of all these physiopathological conditions and the biological monitoring test and to determine further diagnostic management and timely treatment .
determination of acetone levels in blood may be a valid clinical approach in the symptomatic or / and nonsymptomatic cases in the literature for determining treatment strategy and controlling glycemic levels of patients .
consequently , the advanced studies which evaluate blood and urine ketone bodies level together should be performed in different physiopathological conditions including clinical and forensic toxicological studies .
|
using high - performance liquid chromatography ( hplc ) and 2,4-dinitrophenylhydrazine ( 2,4-dnph ) as a derivatizing reagent , an analytical method was developed for the quantitative determination of acetone in human blood .
the determination was carried out at 365 nm using an ultraviolet - visible ( uv - vis ) diode array detector ( dad ) . for acetone as its 2,4-dinitrophenylhydrazone derivative , a good separation was achieved with a thermoacclaim c18 column ( 15 cm 4.6 mm 3 m ) at retention time ( tr ) 12.10 min and flowrate of 1 ml min1 using a ( methanol / acetonitrile ) water elution gradient .
the methodology is simple , rapid , sensitive , and of low cost , exhibits good reproducibility , and allows the analysis of acetone in biological fluids .
a calibration curve was obtained for acetone using its standard solutions in acetonitrile .
quantitative analysis of acetone in human blood was successfully carried out using this calibration graph .
the applied method was validated in parameters of linearity , limit of detection and quantification , accuracy , and precision .
we also present acetone as a useful tool for the hplc - based metabolomic investigation of endogenous metabolism and quantitative clinical diagnostic analysis .
|
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cytauxzoon felis is a tick - transmitted protozoan parasite that can cause cytauxzoonosis in wild and domestic felids .
cytauxzoonosis in domestic cats has been reported throughout central , south - eastern and south - central usa .
oklahoma is considered enzootic for c felis and iowa ( ia ) is a non - enzootic state but borders other enzootic states .
domestic cats infected with c felis often show severe , acute clinical signs characterized by fever , inappetence , anorexia , dyspnea and icterus .
onset of disease typically occurs 1014 days after c felis - infected ticks feed on naive cats and progresses quickly , with fatalities reported 17 days after onset of clinical signs .
a recent study demonstrated 60% survival in c felis - infected cats that received a combination therapy of azithromycin and atovaquone with supportive care . a free - roaming cat is a domestic cat that has been born and raised without or limited contact to humans and is unsocialized .
as free - roaming cats live outdoors and are exposed to ticks , they are favorable populations in which to examine and monitor the distribution and range of cytauxzoonosis . our objective was to determine the prevalence of c felis infections in free - roaming cats in enzootic north - central oklahoma ( ok ) and non - enzootic central ia .
blood samples were collected from free - roaming cats in stillwater , ok , and ames , ia , as part of community trap
cats were trapped mainly in north - central ok and in central ia . in ok , all cats were sedated with a mixture of tiletamine hydrochloride and zolazepam hydrochloride ( telazol ; zoetis ) , ketamine hydrochloride ( ketamine ; putney ) and xylazine ( anased ; akorn ) . in ia
, a mixture of ketamine hydrochloride ( ketamine ; putney ) , dexmedetomidine hydrochloride ( precedex ; orion pharma ) , buprenorphine ( simbadol ; zoetis ) and butorphanol tartrate ( torbugesic - sa ; zoetis ) was used for sedation in 20122013 . in 2014 ,
butorphanol tartrate ( torbugesic - sa ; zoetis ) was added to the mixture but only for fractious cats .
the approximate ages of cats were determined based on dentition , and cats that were 46 months or older were selected for blood collection to increase the chance of finding c felis - infected cats .
cats were placed in dorsal recumbency , fur around the neck was clipped and 70% isopropyl was sprayed in the area . approximately 1 ml blood was collected from the jugular vein and was immediately placed in an edta collection tube .
genomic dna was extracted from peripheral whole blood samples using genejet whole blood dna purification mini kit ( thermo scientific ) .
briefly , 200 l whole blood was mixed with 20 l proteinase k solution and 400 l lysis solution . after incubating the sample at 56c for 10 mins ,
the prepared mixture was transferred to the spin column and centrifuged for 1 min at 6000 g ( ~8000 rpm ) .
the column was then washed with 500 l wash buffer i and centrifuged again for 1 min at 8000 g ( ~10,000 rpm ) .
the column was then washed with wash buffer ii and centrifuged for 3 mins at 20,000 g ( 14,000 rpm ) .
dna was extracted with 200 l preheated ( approximately 56c ) pcr - quality water added to the center of the column membrane to elute genomic dna .
the sample was incubated for 2 mins at room temperature and centrifuged for 1 min at 8000 g ( ~10,000 rpm ) .
a conventional pcr was performed to amplify the c felis small subunit rrna ( ssu - rrna ) .
cycling conditions of nested pcr were as follows : denaturation at 95c for 5 mins , annealing at 54c for 1 min and extension at 72c for 1 min .
pcr products were separated on 1.75% agarose gel and viewed with ultraviolet light . positive control templates consisted of dna extracted from whole blood of a cat that died from an infection of c felis , whereas negative control reactions used dna isolated from purified water .
pcr products were purified using a qiaquick pcr purification kit ( qiagen ) and amplicons were sequenced by eurofin genomics ( huntsville , al ) .
ninety - five percent confidence intervals ( cis ) were calculated according to sterne s exact method , using quantitative parasitology 3.0 .
a total of 380 blood samples were collected from january to may 2014 in ok .
three of 380 ( 0.8% ; 95% ci 0.222.3% ) samples showed approximately 250 bp bands , which were targeted product size for c felis .
blast comparison of these three sequences showed 100% identity to c felis ( eg , l19080 , ay531524 , ay679105 , af399930 , gu903911 ) .
all c felis - infected cats were male and older than 12 months of age .
two infected cats were from stillwater , ok , and caught separately in march and april respectively .
one c felis - infected cat originated from drumright , ok , in march ( figure 1 ) .
a total of 292 blood samples were collected from august 2012 to april 2014 in ia .
neuter return ( tnr ) programs were conducted in stillwater , ok , and ames , ia .
the prevalence of c felis ( 0.8% ) in north - central ok found in the current study was lower than the previously reported 3.4% in domestic cats in ok .
rizzi et al reported a difference in prevalence of c felis infection depending on geographic locations within ok ; 13/77 ( 16.9% ) cats were infected with c felis in eastern ok , while 10/602 ( 1.7% ) cats were infected with c felis in north - central ok .
rizzi et al postulated differences in the prevalence of c felis within ok could include strain variation in virulence of c felis , differences in immunologic responses of cats to infection with c felis and differences in c felis inoculation from ticks .
three different genotypes of c felis , ribosomal internal transcribed spacer regions ( its ) a , b and c , have been demonstrated , and itsb and itsc seem to be more pathogenic with a higher mortality rate than itsa . although it has not been established which genotype is more prevalent or pathogenic than others in ok , genotypic variations could influence prevalence because cats infected with the more pathogenic genotype are likely to succumb to infection . as cats become infected with c felis through tick bites , prevalence of c felis infection in domestic cats
is likely affected by geographic variation of the abundance and activity of ticks and reservoir hosts .
our study supports the report of rizzi et al , which demonstrated that the prevalence of c felis in a given enzootic area can vary from location to location and from the population of cats sampled .
dna of c felis was not detected in 291 blood samples collected from free - roaming cats in central ia , a non - enzootic state that borders enzootic states .
one case of c felis infection in a domestic cat has been reported in south - western ia along the missouri river .
unfortunately , this report did not provide details regarding travel history , age , or sex of the infected cat . in our study ,
blood samples were collected from relatively young cats , and that might have influenced the results as those cats had experienced less time being exposed to tick vectors .
as the geographic distribution and range of a americanum expands northwards , it is important to keep monitoring free - roaming cat populations where cytauxzoonosis has not been considered enzootic .
historically , only bobcats were thought to be reservoirs for c felis ; however , reports have indicated domestic cats that survive acute cytauxzoonosis become chronically infected with c felis and can also serve as a source of c felis infection .
as free - roaming outdoor cats encounter ticks , the source of c felis infections , they are an excellent population to monitor the expansion of c felis distribution in the usa .
|
case summary cytauxzoonosis is a tick - borne disease of cats , and oklahoma ( ok ) , usa , is considered an enzootic state . to determine the prevalence of cytauxzoon felis ,
blood was collected from free - roaming cats , as they are frequently exposed to tick vectors .
our objective was to determine the prevalence of c felis infection in free - roaming cats in north - central oklahoma and central iowa ( ia ) .
infection with c felis was determined using dna extracted from blood and pcr amplification .
blood was collected from 380 free - roaming cats between january and april in 2014 in ok .
dna from c felis was detected in 3/380 ( 0.8% ; 95% confidential interval [ ci ] 0.222.3% ) . in ia
, 292 blood samples were collected between 2012 and 2014 .
no c felis - infected cats were detected ( 0 ; 95% ci 00%).relevance and novel information the prevalence of c felis ( 0.8% ) in north - central ok reported herein was lower than the previously reported 3.4% in domestic cats in ok .
our study supports that the prevalence in a given enzootic area can vary by location and from the pool of cats sampled .
none of 291 ( 0% ) cats were infected with c felis in central ia . to date , only one case of cytauxzoonosis in a domestic cat has been reported in ia .
it is important to monitor cats for c felis infections in northern us states , as geographic distribution of amblyomma americanum expands northward . as free - roaming cats have more contact with the tick vectors of c felis , this population allows us to monitor the expansion of c felis distribution .
|
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in order to collect a set of images useful to test the algorithm , we run 10 plasma samples using 2d - ge . 2d - ge was performed according to maresca et al .
, with each sample run in duplicate . for the first - dimension of electrophoresis ,
200 g of plasma protein samples ( approximately 3 l ) were applied to 18-cm linear ipg strips 47 ( ge healthcare , uppsala , sweden ) and focused until 72,000 v / h were reached .
prior to sds page , the ipg strips were equilibrated twice for 15min in equilibration buffer ( 50 mm tris
hcl ph 8.8 , 6 m urea , 30% glycerol ( v / v ) , 2% sds ( w / v ) and traces of bromophenol blue ) containing 1% dtt ( w / v)for the first equilibration step and 2.5% iodoacetamide ( w / v ) for the second step , respectively .
page was performed on 12.5% polyacrylamide gels at 60 ma / gel at 16 c and terminated when the dye front reached the lower end of the gel . after sds page
, the gels were stained overnight with rubps in fixing solution ( 30% ethanol and 10% acetic acid ) according to the manufacturer s instructions .
the gel images were acquired by gel - doc - it 310 imaging system ( uvp , upland , ca ) , with the following setting
exposure 7 s , gain 3 and three different apertures : 6 , 7 and 8 .
aperture is expressed as f - stop ( e.g. , f2.8 or f/2.8 ) : the smaller is the f - stop number ( or f / value ) , the larger is the aperture . using a larger exposure yields a larger number of spots and , at the same time , more saturated areas . in our case , only the image acquired at the lower exposure ( figure 1c ) could be properly analysed by commercial and academic software currently available , while the saturation in some areas in the other two images ( figure 1a and b ) prevented accurate evaluation of protein expression . using the algorithm described in this work , we have been able to approximate the intensity values of the non - saturated spots within the saturated area . after image scanning ,
the 2d - ge images are noisy and usually affected by several artefacts such as scratches , air bubbles and spikes .
scratches and air bubbles are unintentionally introduced by the operator , while spikes are caused by the presence of precipitated staining particles or imperfect polyacrylamide gel matrix . in particular , spikes are a sort of impulsive noise because of the small area of high intensity .
the aim of reducing speckles in a 2d - ge image ( despeckling ) is to remove the noise without introducing any significant distortion in quantitative spot volume data .
noise suppression methods employed in commercial software packages for 2d - ge image analysis are based on spatial filtering , particularly the most widely used techniques are based on local median filter .
although several other methods have been developed , the advantage of using the median filter is the easy application , since it normally requires the setting of only one main parameter , the filter kernel , which is generally related to the noise size . at the same time
, the size of the filter matrix also accounts for the major impact exerted on the resulting image .
we evaluated the performance of filter size on a set of test images from 3 3 to 7 7 and observed remarkable deterioration for larger filter size , due to the decreased the contrast of the image .
therefore , a 3 3 filter was chosen , which was capable of removing most of the spikes , while preserving the shape of the spots .
the result of a 3 3 median filter on a sample image is shown in figure 5 .
a portion of gel with spikes in 2d and 3d view was shown in figure 5a and c , respectively .
the results of a 3 3 median filter are shown in figure 5b and d accordingly .
saturation effects in 2d gel images could occur for several reasons : ( i ) intense staining , causing complete absorption of light passing through the spot , ( ii ) long exposure time during the acquisition step , ( iii ) too high protein concentration , or ( iv ) accumulation of pigment on top of high intensity spots , leading to the loss of dynamic linear range .
saturation effects might also result from manipulation of image data with image processing software that is not designed for quantitative image analysis ( e.g. , photoshop ) .
for instance , visually - enhanced images often show plateau areas in the high and low intensity regions .
the commercial software is able to detect a saturated area when the intensity reaches the maximum value of grey - scale .
however , in most cases saturated areas are very similar to plateau zones , i.e. , where the pixels have similar intensity without reaching the maximum value of grey - scale . in these cases
, the software might also fail to detect the plateau regions , causing the operator to underestimate the problem . in order to identify the plateau regions
, we implemented a morphological filter , inspired by the rolling - ball algorithm described by sternberg , which allows segmentation of the plateau zones .
this method is based on a structural element ( se ) defined by a circle of given radius ( rd ) and a grey - scale value tolerance ( gvt ) . in particular , for each pixel ( x , y ) of the image i , the se is defined as a circular neighbourhood of rd:(1a)se=(s , t)ini/(s - x)2+(t - y)2<rdwhere(1b)i=(0,image width)[0,image height]denotes the spatial domain of the image .
the gvt represents the rate of grey values of the pixel in the centre of the ses ( figure 6b ) .
for instance , setting the parameter to 10 , the rd is 10 pixels and the gvt in the 10% of grey values of the pixel at position ( x , y ) .
the centre of se is moved along each pixel of the image and the maximum and minimum grey values of the pixels for each point ( x , y ) within the given rd are calculated .
when the difference between maximum and minimum is less than gvt , the area defined by the local operator is considered as a plateau area .
it is worth noting that , when the value of the parameter is low , the rd of the ball is small and only few pixels are included in se .
we examined the performance with rd values from 10 to 25 and for this analysis we selected a value of 15 .
after localizing the plateau regions , we segmented the image to identify the isolated protein spots containing plateau areas on each gel image .
the segmentation procedure yields a set of image segments , consisting of connected neighbouring pixels enclosed by a spot boundary .
the image is considered as a landscape , and the segmentation is to identify all the local minima in the landscape and then find the catchment basins associated with each local minima . the boundary between several catchment basins is called a watershed . for 2d - ge each separated catchment basin is considered as an isolated protein spot .
unlike the usual watershed segmentation , only the plateau areas in our method have been assigned to a catchment basin .
finally , the grey values of the pixels inside the spot , excluding the region identified as a plateau area , were used in the gaussian extrapolation step to recover the distribution of the unsaturated spot .
the final step consists of reconstructing the saturated spots resulting from high exposure images and approximating the unknown grey values in the plateau region .
this has been done considering the unsaturated spot to be described by an analytical function , depending on a restricted set of parameters . in particular
, we assumed each cross section of the spot intensity along both vertical and horizontal axes to be approximated by a generalized gaussian distribution .
namely , for each value of the y - coordinates we considered a function of the form:(2)f(x , m(y),(y),x0,b)=m(y)(y)exp-|x - x0(y)|bb(y)b for b = 2 , eq .
( 1b ) defines the kernel of a standard gaussian distribution centred in x0(y ) , where (y ) and m(y ) is the standard deviation and the maximum of intensity values , respectively .
note that , unlike the other parameters , b does not depend on y , assuming that the approximating gaussian can have different maximum , center and variance in different sections .
,nyand the corresponding intensities { iij } , we determine the set of parameters ( m , , x0 , b ) for which the function defined in ( 2 ) fits at best the values of the intensity in the unsaturated region . in practice , we have to minimize an error function that defines how good a particular parameter set is . for example , a standard least - squared criterion can be used(4)em(yj),(yj),x0(yj),b=i=1nxfxi , m(yj),(yj),x0(yj),b - iij2 however , the error measure can be defined in different ways , e.g. , according to different norms or including different weighs for the different parameters and/or the different pixels . in particular ,
we modified ( 4 ) in order to control the variation of the parameters for different sections . in our case the error function ( 5 ) can be formulated as follows(5)em(yj),(yj),x0(yj),b = em(yj),(yj),x0(yj),b+m(yj)-m(yj-1)2+(yj)-(yj-1)2+x0x0(yi)-x0(yj-1)2 for positive values of the parameters ( m , , x0 ) , the problem is then reduced to finding the parameters yielding the minimum of the selected error function .
one possibility is to perform an exhaustive search on all the values of a pre - defined parameters space .
however , if the size of the parameter space is large , a more effective newton
after image scanning , the 2d - ge images are noisy and usually affected by several artefacts such as scratches , air bubbles and spikes .
scratches and air bubbles are unintentionally introduced by the operator , while spikes are caused by the presence of precipitated staining particles or imperfect polyacrylamide gel matrix . in particular , spikes are a sort of impulsive noise because of the small area of high intensity .
the aim of reducing speckles in a 2d - ge image ( despeckling ) is to remove the noise without introducing any significant distortion in quantitative spot volume data .
noise suppression methods employed in commercial software packages for 2d - ge image analysis are based on spatial filtering , particularly the most widely used techniques are based on local median filter .
although several other methods have been developed , the advantage of using the median filter is the easy application , since it normally requires the setting of only one main parameter , the filter kernel , which is generally related to the noise size . at the same time
, the size of the filter matrix also accounts for the major impact exerted on the resulting image .
we evaluated the performance of filter size on a set of test images from 3 3 to 7 7 and observed remarkable deterioration for larger filter size , due to the decreased the contrast of the image .
therefore , a 3 3 filter was chosen , which was capable of removing most of the spikes , while preserving the shape of the spots .
the result of a 3 3 median filter on a sample image is shown in figure 5 . a portion of gel with spikes in 2d and 3d view was shown in figure 5a and c , respectively .
the results of a 3 3 median filter are shown in figure 5b and d accordingly .
saturation effects in 2d gel images could occur for several reasons : ( i ) intense staining , causing complete absorption of light passing through the spot , ( ii ) long exposure time during the acquisition step , ( iii ) too high protein concentration , or ( iv ) accumulation of pigment on top of high intensity spots , leading to the loss of dynamic linear range .
saturation effects might also result from manipulation of image data with image processing software that is not designed for quantitative image analysis ( e.g. , photoshop ) .
for instance , visually - enhanced images often show plateau areas in the high and low intensity regions .
the commercial software is able to detect a saturated area when the intensity reaches the maximum value of grey - scale .
however , in most cases saturated areas are very similar to plateau zones , i.e. , where the pixels have similar intensity without reaching the maximum value of grey - scale . in these cases ,
the software might also fail to detect the plateau regions , causing the operator to underestimate the problem . in order to identify the plateau regions
, we implemented a morphological filter , inspired by the rolling - ball algorithm described by sternberg , which allows segmentation of the plateau zones .
this method is based on a structural element ( se ) defined by a circle of given radius ( rd ) and a grey - scale value tolerance ( gvt ) . in particular , for each pixel ( x , y ) of the image i , the se is defined as a circular neighbourhood of rd:(1a)se=(s , t)ini/(s - x)2+(t - y)2<rdwhere(1b)i=(0,image width)[0,image height]denotes the spatial domain of the image . the se is depicted in figure 6a .
the gvt represents the rate of grey values of the pixel in the centre of the ses ( figure 6b ) . for instance , setting the parameter to 10 , the rd is 10 pixels and the gvt in the 10% of grey values of the pixel at position ( x , y ) .
the centre of se is moved along each pixel of the image and the maximum and minimum grey values of the pixels for each point ( x , y ) within the given rd are calculated .
when the difference between maximum and minimum is less than gvt , the area defined by the local operator is considered as a plateau area .
it is worth noting that , when the value of the parameter is low , the rd of the ball is small and only few pixels are included in se .
we examined the performance with rd values from 10 to 25 and for this analysis we selected a value of 15 .
after localizing the plateau regions , we segmented the image to identify the isolated protein spots containing plateau areas on each gel image .
the segmentation procedure yields a set of image segments , consisting of connected neighbouring pixels enclosed by a spot boundary .
the image is considered as a landscape , and the segmentation is to identify all the local minima in the landscape and then find the catchment basins associated with each local minima .
the boundary between several catchment basins is called a watershed . for 2d - ge each separated catchment basin is considered as an isolated protein spot . unlike the usual watershed segmentation , only the plateau areas in our method have been assigned to a catchment basin .
finally , the grey values of the pixels inside the spot , excluding the region identified as a plateau area , were used in the gaussian extrapolation step to recover the distribution of the unsaturated spot .
the final step consists of reconstructing the saturated spots resulting from high exposure images and approximating the unknown grey values in the plateau region .
this has been done considering the unsaturated spot to be described by an analytical function , depending on a restricted set of parameters . in particular
, we assumed each cross section of the spot intensity along both vertical and horizontal axes to be approximated by a generalized gaussian distribution .
namely , for each value of the y - coordinates we considered a function of the form:(2)f(x , m(y),(y),x0,b)=m(y)(y)exp-|x - x0(y)|bb(y)b for b = 2 , eq .
( 1b ) defines the kernel of a standard gaussian distribution centred in x0(y ) , where (y ) and m(y ) is the standard deviation and the maximum of intensity values , respectively .
note that , unlike the other parameters , b does not depend on y , assuming that the approximating gaussian can have different maximum , center and variance in different sections . the reconstruction problem can be formulated as follows .
,nyand the corresponding intensities { iij } , we determine the set of parameters ( m , , x0 , b ) for which the function defined in ( 2 ) fits at best the values of the intensity in the unsaturated region . in practice , we have to minimize an error function that defines how good a particular parameter set is .
for example , a standard least - squared criterion can be used(4)em(yj),(yj),x0(yj),b=i=1nxfxi , m(yj),(yj),x0(yj),b - iij2 however , the error measure can be defined in different ways , e.g. , according to different norms or including different weighs for the different parameters and/or the different pixels . in particular , we modified ( 4 ) in order to control the variation of the parameters for different sections . in our case the error function ( 5 ) can be formulated as follows(5)em(yj),(yj),x0(yj),b = em(yj),(yj),x0(yj),b+m(yj)-m(yj-1)2+(yj)-(yj-1)2+x0x0(yi)-x0(yj-1)2 for positive values of the parameters ( m , , x0 ) , the problem is then reduced to finding the parameters yielding the minimum of the selected error function .
one possibility is to perform an exhaustive search on all the values of a pre - defined parameters space .
however , if the size of the parameter space is large , a more effective newton
mn conceived the study , developed the algorithm for the detection of overexposed areas and plateau regions .
mn and ac drafted the manuscript with the help of ef . all authors read and approved the final manuscript .
|
analysis of images obtained from two - dimensional gel electrophoresis ( 2d - ge ) is a topic of utmost importance in bioinformatics research , since commercial and academic software available currently has proven to be neither completely effective nor fully automatic , often requiring manual revision and refinement of computer generated matches . in this work , we present an effective technique for the detection and the reconstruction of over - saturated protein spots .
firstly , the algorithm reveals overexposed areas , where spots may be truncated , and plateau regions caused by smeared and overlapping spots .
next , it reconstructs the correct distribution of pixel values in these overexposed areas and plateau regions , using a two - dimensional least - squares fitting based on a generalized gaussian distribution .
pixel correction in saturated and smeared spots allows more accurate quantification , providing more reliable image analysis results .
the method is validated for processing highly exposed 2d - ge images , comparing reconstructed spots with the corresponding non - saturated image , demonstrating that the algorithm enables correct spot quantification .
|
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] |
although the human genome sequence was released a decade ago , the role of functional noncoding rnas ( ncrnas ) is much less understood compared with their coding counterparts .
several previous studies have demonstrated that the human genome is pervasively transcribed ( 14 ) , but thoroughly cataloging all the rna species ( especially ncrna ) is challenging .
undiscovered ncrnas might be rare , transient or beyond the detection limits of conventional approaches .
furthermore , ncrnas also tend to be idiosyncratic to species and tissues ( 5,6 ) .
nevertheless , advances in rna - seq have provided a new method of surveying the whole transcriptome to an unprecedented degree .
recent genome - wide studies revealed tens of thousands of novel transcripts , the majority of which were long noncoding rnas ( lncrnas , > 200 nt ) ( 49 ) .
although a few dozen lncrnas have been characterized to some extent and are reported to have critical roles in diverse cellular and disease development processes ( 6,1014 ) , the biogenesis and function of most lncrnas remain unclear .
accurate and quantitative assessment of coding potential is the first step toward comprehensive annotation of newly discovered transcripts .
until now , prediction of coding potential heavily relied on sequence alignment , either pairwise homology search for protein evidence such as that used in the coding - potential calculator ( cpc ) and portrait methods ( 15,16 ) or multiple alignments to calculate the phylogenetic conservation score such as that used in the phylogenetic codon substitution frequencies ( phylocsf ) and rnacode methods ( 17,18 ) .
alignment - based approaches are particularly useful for highly conserved protein - coding genes and , to a lesser extent , short genes encoding housekeeping or regulatory rnas ( e.g. snrnas , snorna , transfer rna ) .
however , these approaches can not immediately apply to all the novel transcripts because of several intrinsic limitations .
first , most newly discovered transcripts are lncrnas , which tend to be lineage specific and less conserved ( 5,6 ) .
for example , only 29 of 550 lncrnas identified from zebrafish had detectable sequence similarity with putative mammalian orthologs ( 6 ) , and only 993 of 8195 human lncrnas have orthologous transcripts in other species ( 5 ) .
second , considerable fractions of lncrnas are overlapped with either the sense or antisense strand of protein - coding genes .
these lncrnas can not be correctly classified by homology searching because they would have significant matches to protein - coding genes ( 3,8,19 ) .
third , the reliability of alignment - based approaches largely depends on the quality of alignments ( 20 ) .
this is problematic because most widely used multiple - sequence alignment tools use heuristics and do not guarantee optimal alignments .
for instance , cpc and phylocsf took 2 days to evaluate the coding potential of 14 353 lncrnas identified by cabili et al .
this problem is getting more attention as massive - scale rna sequencing is increasingly being performed .
consequently , a more accurate , robust and faster method that does not rely on sequence alignment is needed to distinguish ncrnas , especially lncrnas , from protein - coding genes . here , we present coding - potential assessment tool ( cpat ) , an alignment - free program , which uses logistic regression to distinguish between coding and noncoding transcripts on the basis of four sequence features .
cpat is highly accurate ( 0.967 ) and extremely efficient ( 10 000 times faster than cpc and phylocsf , and 50 times faster than portrait ) .
cpat needs only the sequence or coordinate file as input , and it is straightforward to use .
we expanded the availability of cpat to a larger scientific audience via a web interface , which allows users to submit sequences and receive the prediction results back almost instantaneously ( http://lilab.research.bcm.edu/cpat/index.php ) .
coding - potential prediction is essentially a binary decision problem , which makes logistic regression a suitable approach . as an alignment - free method , all selected features ( predictor variables )
the first feature was the maximum length of the open reading frame ( orf ) .
orf length is one of the most fundamental features used to distinguish ncrna from messenger rna because a long putative orf is unlikely to be observed by random chance in noncoding sequences . despite the simplicity , orf length has high concordance with more sophisticated discrimination methods and remains the primary criterion in almost all coding - potential prediction methods ( 21 ) .
the second feature was orf coverage defined as the ratio of orf to transcript lengths .
this feature also has good classification power , and it is highly complementary to , and independent of , the orf length ( supplementary figures s1 and s3 ) . some large bona fide ncrnas may contain putative long orfs by random chance ( 5 ) , and thus can not be classified correctly by orf length alone .
fortunately , those large ncrnas usually have much lower orf coverage than protein - coding rnas ( figure 1b ) .
figure 1.score distribution between coding ( red ) and noncoding ( blue ) transcripts for the four linguistic features selected to build the logistic regression model ; training data set containing 10 000 coding and 10 000 noncoding transcripts were used .
score distribution between coding ( red ) and noncoding ( blue ) transcripts for the four linguistic features selected to build the logistic regression model ; training data set containing 10 000 coding and 10 000 noncoding transcripts were used .
the third feature we used was the fickett testcode score ( termed fickett score hereafter ) , which is a simple linguistic feature that distinguishes protein - coding rna and ncrna according to the combinational effect of nucleotide composition and codon usage bias ( 22 ) .
briefly , the fickett score is obtained by computing four position values and four composition values ( nucleotide content ) from the dna sequence .
the position value reflects the degree to which each base is favored in one codon position versus another .
for example , position value of a ( apos ) is calculated as follows :
cpos , gpos and tpos are determined in the same way .
these eight values are then converted into probabilities ( p ) of coding using a lookup table provided in the original article .
each probability is multiplied by a weight ( w ) for the respective base , where the value of w reflects the percentage of time each parameter alone successfully predicts coding or noncoding function for the sequences of known function .
finally , the fickett score is calculated as follows :
the fickett score is independent of the orf , and when the test region is 200 nt in length ( which includes most lncrna ) , this feature alone can achieve 94% sensitivity and 97% specificity , with
the fourth feature we used was hexamer usage bias ( termed hexamer score hereafter ) .
this may be the most discriminating feature because of the dependence between adjacent amino acids in proteins ( 23 ) .
the hexamer score can be computed in numerous ways ; here , we used a log - likelihood ratio to measure differential hexamer usage between coding and noncoding sequences . for a given dna sequence , we calculated the probability of the sequence under the model of coding dna and under the model of noncoding dna , and then we took the logarithm of the ratio of these probabilities as the score of coding potential .
we used f ( hi ) ( i = 0 , 1 , , 4095 ) and f ( hi ) ( i = 0 , 1 , , 4095 ) to represent in - frame hexamer frequency , calculated from coding and noncoding training data sets ( described below ) , respectively .
for a given hexamer sequence s = h1 , h2 , , hm ,
hexamer score determines the relative degree of hexamer usage bias in a particular sequence .
positive values indicate a coding sequence , whereas negative values indicate a noncoding sequence . we build a logistic regression model using these four linguistic features as predictor variables .
a test was used to evaluate whether our logit model with predictors fits the training data significantly better than the null model , which had only an intercept .
we built a high - confidence training data set to measure the prediction performance of our logit model .
this data set contained 10 000 protein - coding transcripts selected from the refseq database ; all transcripts had high - quality protein sequences annotated by the consensus coding sequence project .
we evaluate the model with a 10-fold cross - validation and measured its sensitivity , specificity , accuracy , precision and area under the curve ( auc ) characteristics .
the receiver operating characteristic ( roc ) curve and precision recall ( pr ) curve were generated using rocr package ( 24 ) .
we also built a nonparametric two - graph roc curve for selecting the optimal cpat score threshold that maximizes the sensitivity and specificity of cpat while minimizing misclassifications .
we built an independent test data set to compare the performance of cpat with that of cpc , phylocsf and portrait .
this test set composed of 4000 high - quality protein - coding genes ( refseq annotated ) and 4000 lncrnas from a human lncrna catalog ( 5 ) .
assuming that all 4000 lncrnas are truly noncoding sequences , we could compute the sensitivity , specificity , accuracy and precision of the algorithms to measure their performance .
those 528 genes were equally assigned to the true - negative and false - positive categories .
the abbreviations in the equations below are as follows : fn , false negative ; fp , false positive ; tn , true negative ; tp , true positive
all four selected features were concordantly higher in coding transcripts and lower in noncoding transcripts ( figure 1 ) .
we plotted three major features ( orf size , fickett score and hexamer score ) in a three - dimensional space to evaluate their combinatorial effect ( figure 2 ) .
coding and noncoding transcripts in our training data set were grouped into two distinct clusters , indicating good concordance between features .
the test p value was < .001 ( = 23 548.44 ; degrees of freedom = 4 ) , indicating that the logit model as a whole fits significantly better than the null model .
ten - fold cross - validation showed that cpat could achieve very high accuracy , with an auc of 0.9927 ( figure 3a ) .
we also provide the pr curve because the roc curve can be misleading when the test data are largely skewed ( figure 3b ) .
we use nonparametric two - graph roc curves to determine an optimal cpat score threshold that maximizes the discriminatory power ( figure 3c and d ) . according to figure 3d ,
a score threshold of 0.364 gave the highest sensitivity and specificity ( 0.966 for both ) for human data .
figure 2.three-dimensional plot shows combinatorial effects of fickett score , hexamer score and orf size on 10 000 coding genes ( red dots ) and 10 000 noncoding genes ( blue dots ) .
dashed curves represent the 10-fold cross - validation ; solid curves represent the averaged curve from 10 validation runs .
( d ) two - graph roc curve is used to determine the optimum cutoff value .
three - dimensional plot shows combinatorial effects of fickett score , hexamer score and orf size on 10 000 coding genes ( red dots ) and 10 000 noncoding genes ( blue dots ) .
dashed curves represent the 10-fold cross - validation ; solid curves represent the averaged curve from 10 validation runs .
( d ) two - graph roc curve is used to determine the optimum cutoff value .
we compared the performance of cpat with that of cpc , phylocsf and portrait ( protein - independent support vector machine model ) using an independent test data set composed of 4000 coding genes and 4000 noncoding genes .
a multiple alignment of 45 vertebrate genomes , including that of human , was downloaded from the ucsc ( university of california , santa cruz ) genome browser and was used as the input alignment for phylocsf . in general , cpat ( sensitivity : 0.96 , specificity : 0.97 ) had greater classification power compared with all other programs ( figure 4 ; supplementary tables s1 and s2 ) .
although cpc had the highest sensitivity ( 0.99 ) , it suffered from poor specificity ( 0.74 ) .
one possible explanation is that a significant proportion of ncrnas has a certain degree of sequence similarity to protein - coding genes .
phylocsf had the least sensitivity ( 0.90 ) and the lowest specificity ( 0.63 ) .
part of the reason for these outcomes is that nonconserved transcripts can not be processed by phylocsf .
if we consider those 528 nonconserved transcripts as noncoding , the specificity increased from 0.63 to 0.69 , and the sensitivity remained unchanged .
cpat achieved highest overall accuracy ( 0.97 ) when compared with cpc ( 0.87 ) , phylocsf ( 0.76 ) and portrait ( 0.92 ) .
cpat s excellent discriminatory power was further demonstrated by the greatest separation between the score distributions of coding and noncoding sequences ( figure 5 ) . unlike cpc , phylocsf and portrait , choosing a smaller cpat score threshold to increase the sensitivity will not sacrifice too much specificity .
figure 4.performance comparison between cpat , cpc , phylocsf and portrait using roc curves .
figure 5.cumulative curves of coding - potential assessment score for ( a ) cpat , ( b ) portrait , ( c ) cpc and ( d ) phylocsf .
cumulative curves of coding - potential assessment score for ( a ) cpat , ( b ) portrait , ( c ) cpc and ( d ) phylocsf .
one could argue that phylocsf underperformed in this study because we used whole transcripts for testing rather than consecutive protein - coding exons and intergenic regions as used in its original article ( 17 ) . to address this issue , we compiled another single - exon test data set consisting of 184 protein - coding and 278 noncoding transcripts . the test results with this data set indicated that cpat ( sensitivity : 0.962 , specificity : 0.842 ) still outperformed phylocsf ( sensitivity : 0.832 , specificity : 0.588 , supplementary figure s2 ) .
( 25 ) , phylocsf ( sensitivity 0.91 , specificity 0.99 ) has better performance than cpat ( sensitivity 0.50 , specificity 0.98 ) .
this is reasonable because lncrnas in our test data set are poorly conserved , whereas lncrnas in lin et al .
test data set are highly conserved because they are taken from multiple - sequence alignments of three closely related drosophila species .
hence , we argue that phylocsf works better if the transcripts are highly conserved , which are rare to find in lncrnas ( 5,6 ) .
this also highlights the achilles heel of the alignment - based methods for detecting lncrnas .
in contrast , the dramatic decrease in cpat s sensitivity is due to the lack of orf information in lin et al .
test data set , which is largely composed of individual exons , and not exon - length complete transcripts .
this , however , will not limit the application scope of cpat because most full - length transcripts can be constructed at the current sequencing depth ( 8) .
we measured the computational speed of cpat , cpc and phylocsf on a sample of 200 sequences randomly selected from the test data set .
cpat took 0.67 s to process the data , and it was four orders of magnitudes faster than both cpc [ 11 945 s ( 3.3 h ) ] and phylocsf [ 11 737 s ( 3.3 h ) ] . furthermore ,
computational time for the phylocsf did not include the time spent preparing multiple - alignment files for analysis .
portrait was significantly faster than cpc and phylocsf , and therefore all 8000 test genes were used to evaluate its speed : cpat took 23.83 s to process the test set , and it was 48 times faster than portrait [ 1146.30 s ( 19 min ) ] .
a number of linguistic features characterizing coding rna sequences have been developed over the past 30 years .
these include maximum orf size , dinucleotide usage , codon usage bias , hexamer usage bias , nucleotide composition bias between codon positions and imperfect periodicity in base occurrences ( 23,26 ) . among these features , we selected orf features ( size and coverage ) because of their discriminatory power and ease of calculation ( 21 ) . in - frame hexamer score
was selected because it has the highest prediction accuracy ( average of sensitivity and specificity ) as evaluated by fickett and tung in 1992 ( 23 ) .
fickett score was selected because it simultaneously captures the compositional bias and position asymmetry , which are orthogonal to the orf features .
supplementary figure s3 shows the performance of these individual features as well as the combined feature set .
the combined feature set has very high sensitivity and specificity ( > 0.966 ) , leaving very little room for further improvement .
annotation of genomes has always been a challenging task for biologists , and these efforts have been accelerated by deep transcriptome sequencing .
distinguishing between protein - coding and noncoding sequences is the first and arguably the most crucial step in genome annotation .
detecting the coding - potential of these transcripts via alignment - based software is intractable .
we developed cpat , a highly accurate alignment - free method , which uses a logistic regression model to discriminate between coding and noncoding transcripts using pure linguistic features . compared with other tools ,
cpat is more robust , markedly faster and more convenient to use . taken together , cpat is able to accurately assess the coding potential of tens of thousands of transcripts in real - time , and will be a valuable tool for the rapidly growing rna - seq community .
source code was implemented in c and python and is freely available at : http://code.google.com / p / cpat/. the web server was implemented in php , mysql and apache , with support for all major browsers : http://lilab.research.bcm.edu/cpat/index.php .
supplementary data are available at nar online : supplementary tables 1 and 2 and supplementary figures 13 .
department of defense prostate cancer program [ pc094421 to w.l . ] ; the cancer prevention and research institute of texas [ rp110471-c3 to w.l . ] ; the center for individualized medicine ( cim ) at mayo clinic ( to j.p.k . ) . funding for open access charge : cancer prevention and research institute of texas [ rp110471-c3 to w.l . ] .
|
thousands of novel transcripts have been identified using deep transcriptome sequencing .
this discovery of large and
hidden transcriptome rejuvenates the demand for methods that can rapidly distinguish between coding and noncoding rna . here
, we present a novel alignment - free method , coding potential assessment tool ( cpat ) , which rapidly recognizes coding and noncoding transcripts from a large pool of candidates . to this end
, cpat uses a logistic regression model built with four sequence features : open reading frame size , open reading frame coverage , fickett testcode statistic and hexamer usage bias .
cpat software outperformed ( sensitivity : 0.96 , specificity : 0.97 ) other state - of - the - art alignment - based software such as coding - potential calculator ( sensitivity : 0.99 , specificity : 0.74 ) and phylo codon substitution frequencies ( sensitivity : 0.90 , specificity : 0.63 ) .
in addition to high accuracy , cpat is approximately four orders of magnitude faster than coding - potential calculator and phylo codon substitution frequencies , enabling its users to process thousands of transcripts within seconds .
the software accepts input sequences in either fasta- or bed - formatted data files .
we also developed a web interface for cpat that allows users to submit sequences and receive the prediction results almost instantly .
|
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] |
, it must develop tolerance to paternal antigens to avoid a lethal immunologic attack against the fetus . on the other hand
, it must preserve the ability to fight infections from a multitude of commensal and environmental pathogens .
disruption of this balance can have devastating consequences , including preterm labor and death of the fetus and/or mother .
the normal maternal immune response to pregnancy is increasingly recognized as a dynamic process , with changes in the maternal pro / anti - inflammatory profile occurring at different stages of gestation [ 13 ] . despite this recognition , a complete , longitudinal immunologic profile of normal
such analyses are critical for understanding the response to specific infectious and immunologic diseases that show disproportionately negative outcomes during pregnancy , such as influenza and ulcerative colitis .
one approach to such immune profiling is through the use of multiplex cytokine arrays , allowing for simultaneous quantification of many proteins with a very small amount of plasma or serum .
others have used multiplex arrays to study the maternal cytokine milieu , but the studies have been predominantly limited time point cross - sectional [ 68 ] or case - control [ 911 ] in design .
the results of some longitudinal studies have been published in recent years , although results have been based on relatively few time points ( 5 or fewer ) [ 13 ] . in this study
, we applied the multiplex array approach to evaluate the changes in 42 cytokines in rich detail during pregnancy .
the samples are from a cohort of 16 pregnant women , with each subject sampled a median of 18 times . because this represents a significant increase in the number of tests per subject compared to previous studies
, we also describe a strategy addressing within - subject correlation with repeated measurements using unconditional growth modeling . with this approach
, we have been able to detail the typical longitudinal variation of serum cytokines throughout pregnancy in this cohort of women .
each participant had a serum sample collected and cryopreserved biweekly from study enrollment during the first trimester ( average of 9.7 weeks ' gestation for first samples ) until 34 weeks ' gestation , then weekly from 34 weeks until delivery .
as a result of this repeated sampling , a median of 18 samples per participant were obtained longitudinally throughout pregnancy .
samples were collected under a protocol approved by the mayo clinic institutional review board ( irb ) that accrued participants from 1987 to 1988 .
information regarding date of the blood draw , date of delivery , outcome of the delivery , and any major complications that the woman experienced during pregnancy ( hypertensive disorders , preterm labor , and infections ) was available and helped identify appropriate participants to include .
only women who carried their pregnancies to term and experienced no pregnancy complications were included in this study .
we only used samples from subjects who had specimens obtained throughout the entire course of pregnancy and whose complete , longitudinally collected specimens appeared intact and nondesiccated .
most cytokines are stable in storage at 80 degrees c for up to 2 years , but stability beyond that time is not well known . therefore , before proceeding with studies on the samples of interest , we first tested protein levels of a small number of individually stored serum aliquots from other subjects archived on this protocol . since we found detectable levels of cytokines in the pregnant serum samples that were within range of control samples , we proceeded with the entire study involving a cohort of 16 healthy , primigravid women who completed a term pregnancy without any significant complications .
samples were assayed when freshly thawed to avoid freeze - thaw cycles , which are known to degrade cytokines .
contemporary plasma samples from 11 nonpregnant control subjects were collected under a separate irb - approved protocol for collection of biospecimens from healthy individuals within the same health system in 2010 and similarly assessed on the same 96-well plates for comparison and as a quality control measure . for contemporary nonpregnant control samples ,
peripheral venous blood was drawn into heparinized vacutainer tubes that were processed and separated into plasma and peripheral blood mononuclear cells ( pbmcs ) following gradient centrifugation using ficoll - paque ( ge healthcare , uppsala , sweden ) .
plasma was collected and immediately frozen at 80c in 1 ml aliquots until use .
protein levels for 42 cytokines , chemokines , and growth factors ( table 1 ) were measured using the milliplex map human cytokine / chemokine kit ( millipore , billerica , ma , usa ) per manufacturer 's instructions . to minimize the potential for interassay variability , each subject had all of their longitudinal specimens analyzed on the same plate .
protein concentrations were determined using a linear regression standard curve from each plate generated using the high pmt concentrations with sensitivity from 3.2 to 2,000 pg / ml . all samples were tested in duplicate with the mean value of the measurements used for statistical analyses .
comparison of baseline ( first trimester ) and end of pregnancy levels of cytokines / growth factors was carried out between the 16 primigravid subjects and the 11 nonpregnant control subjects using the kruskal - wallis tests .
there were extremes of values that fell outside of the limits of the multiplex array .
for cytokines below the limit of detection ( ld ) , we assigned a value that was half the lowest detectable value for the assay as previously described .
analytes above the detectable range were assigned a threshold concentration . to obtain a visual representation of overall patterns in the highly dimensional raw data
as an additional multivariate technique to identify patterns within the data , principle components analysis ( pca ) was also completed on the raw values obtained from the multiplex array . to assess the within - subject correlation , we determined change of cytokines and growth factors from a baseline time point the values obtained from the participants ' first blood draw in the first trimester by taking the log of 1 plus fold change from the baseline value : log(1 + x ) , where x = ( cytokine concentration of the sample of interest / cytokine concentration of the baseline sample ) to analyze trends over time .
first , we graphically assessed the growth trajectory for the 42 cytokines using smoothing splines .
we then fit unconditional mean models ( umm ) for the 42 cytokines in order to estimate the variance components : the within - subject variance ( ) and the between - subject variance ( 0 ) . estimating the two variance components helps to determine whether there is sufficient variation to warrant further analysis and enabled computations of the intraclass correlation coefficient , , which describes the proportion of the total outcome variation that lies between subjects .
next , we fit unconditional growth models ( ugm ) , which allowed random slope but not random intercept because the starting value of all patients was the same ( baseline value = 0.3 ) due to normalization to each individual 's baseline value . as both gestational age in weeks and trimester
can be used as the time covariate for growth curve models , and as the trimester can be treated as either a continuous or a categorical variable ( depending on whether we assume linear relationship between trimester and outcome variables ) , six different models were fitted for each cytokines as follows : ( 1 ) model with only the gestational age in weeks as the time covariate , ( 2 ) model with only the trimester ( continuous ) as the time covariate , ( 3 ) model with only the trimester ( categorical ) as the time covariate , where the variance - covariance pattern was assumed to be autoregressive , ( 4 ) model with both gestational age in weeks and continuous trimester as covariates , allowing random slope for gestational time in weeks , ( 5 ) model with both gestational age in weeks and continuous trimester as a covariate , allowing random slope for trimester , and ( 6 ) model with both gestational age in weeks and continuous trimester as a covariate , allowing random slopes for both gestational age in weeks and trimester .
we then compared the six models and the unconditional mean model for each of the cytokines based on logic and statistical fitness , using akaike information criteria ( aic ) .
because of the exploratory nature of this study , there was no correction for multiple comparisons .
the multiplex was designed to assess 42 factors , but the limits of detection were exceeded with three factors , and there were 5 cytokines that fell below the limit of detection in > 50% of the samples .
pdgf - aa , pdgf - ab / bb , and rantes had cytokine levels above detection limits of the assay in 9.9% , 11.2% , and 34.9% of the samples , respectively ( table s1 ; see supplementary material available online at http://dx.doi.org/10.1155/2015/952571 ) .
il-2 , il-3 , il-4 , il-13 , and tnf had > 50% of the samples below the detection limit ( table s1 ) .
differences in cytokine profiles between baseline ( first trimester ) pregnancy samples and nonpregnant control samples could be identified by pca ( figure 1 ) .
specifically , we identified significantly higher levels of gro , tgf , egf , pdgf - aa , and pdgf - ab / bb and significantly lower levels of scd40l , ip-10 , il-6 , il-17 , il-13 , and mcp-1 in the baseline pregnancy samples ( table 1 ) . when all longitudinal samples were included in a pca , no difference
next , we evaluated whether those differences apparent in early gestation were also present at the end of pregnancy . when comparing the final independent samples obtained at the end of pregnancy to the healthy control samples , the following remained significantly elevated in pregnant women : egf ( 330.6 versus 24.8 pg / ml , p = 0.0013 ) , gro ( 1,153.6 versus 341.6 pg / ml , p < 0.001 ) , sil-2ra ( 17.9 pg / ml versus lld , p = 0.02 ) , and tgf ( 15.8 versus 1.8 pg / ml , p = 0.0006 )
meanwhile , only eotaxin was lower in women at the end of pregnancy compared to normal controls ( 33.4 versus 330.0 pg / ml , p < 0.001 ) . to begin to assess within - subject variation , we clustered the raw data ( figure 2 ) , where the issue of correlation within subjects becomes visually apparent .
empirical growth plots demonstrated that the within - subject variation differs among different women ( figures 3 and 4 show a representative example using il-15 , and the remainder of the plots can be viewed in supplementary figures ) .
for example , patient o displays very large variation in the majority of the cytokines tested , whereas patients b , c , f , and g have comparatively steady levels for most cytokines .
also , high variability in one cytokine does not imply high variability in other cytokines for the individual women .
high interclass correlation ( 0.7 ) was observed for scd40l , rantes , il-10 , tnf , il-7 , and gm - csf ( table s2 ) .
this means that the majority of the variability in these cytokines is attributed to variance between subjects .
the interclass correlations were comparatively low ( < 0.3 ) for il-3 , il-4 , mcp-3 , and ip-10 , suggesting more variation within subjects for these cytokines .
the final model for mdc ( table 2 ) was ugm with gw , with a decreasing trajectory .
the estimated variance for the random slope is 0.000001632 , suggesting that the variability of slope among patients is quite small .
on the other hand , the average scatter of an individual 's outcome around her own trajectory is larger ( the estimated variance is 0.000937 , p < 0.0001 ) , suggesting that there might be some other important covariates that are not included in the model .
however , including gestational age in weeks as a covariate does substantially improve the fit of the model .
when comparing the estimated within - subject variance of the ugm with that from the umm , we found that the linear gestational age in weeks helps to explain 34% of the within - subject variation in mdc .
the estimated fixed effect suggests that mdc decreases over time ( the estimated slope is 0.00163 , with p 0.0001 , suggesting that the slope is significantly lesser than 0 , figure s2 ) .
eight other cytokines demonstrated statistical significance , although with increasing trajectories , with ugm and gw as a covariate : il-1 , il-6 , il-8 , il-12p70 , il-13 , il-15 , ip-10 , and flt3-ligand ( table 2 ) .
six cytokines showed association with trimester as a linear variable : il-1ra , il-3 , il-9 , il-12p40 , ifn2 , and scd40l ( table 3 ) .
il-1ra showed the greatest percent explanation by inclusion of linear trimester in the model . when comparing the estimated within - subject variance of the umm with that from the ugm
, we found that the linear trimester helps to explain 27% of the within - subject variation in il-1ra .
seven other cytokines showed significant associations with trimester as a categorical variable : il-4 , ifn , g - csf , tgf- ,
tnf- , sil-2ra , and mip-1 ( table 4 ) . the final model for both egf and vegf was umm with intercept only , leading to the conclusion that both egf and vegf do not change over time .
time - dependent ugm models were identified but did not meet statistical significance for the following cytokines : il-2 , il-5 , pdgf - aa , pdgf - ab , mip-1 , gro , mcp-1 , mcp-3 , rantes , il-17 , il-7 , eotaxin , fgf-2 , il-10 , tnf , il-1ra2 , fracktalkine , and gm - csf . therefore
, approximately 50% of cytokines measured by multiplex array did not show any association with gestational week or trimester .
our results suggest that the third trimester of pregnancy is characterized by an increasingly inflammatory ( e.g. , il-1 , il-6 , il-12 , il-15 , ip-10 , and scd40 ligand ) as well as counterregulatory ( e.g. , il-1ra and flt3-ligand ) milieu compared to earlier stages of pregnancy .
our superimposed growth curves of il-15 ( figure 4 ) and il-1 ( figure s3 ) , the cytokines with the strongest association with gestational week as a linear covariate , suggest that this change begins at approximately week 20 and peaks just after week 30 .
this is consistent with other reports of systemic immune activation as well as counterregulation in the latter part of pregnancy [ 3 , 1719 ] .
increasing il-1 and il-15 may predominantly be related to production from placental tissues [ 20 , 21 ] or mononuclear phagocytes [ 22 , 23 ] in late gestation .
the precise immunologic mechanisms responsible for this shift can not be determined by our study , but if the source of these cytokines is predominantly the innate immune system , one potential stimulus to these cytokines ' secretion is cell - free fetal dna . a recently proposed model links rising circulating levels of fetal dna to maternal innate immune activation via toll - like receptor- ( tlr- ) 9 on neutrophils and macrophages , leading to increasing inflammatory cytokine release , that may result in an immune cascade ultimately leading to parturition .
a novel finding of this study is the decline in mdc levels throughout pregnancy ( figure 5 ) .
mdc is chemoattractant for immature dendritic cells and type 2-biased t cells , and it is possible that decreasing mdc levels may reflect a gradual shift away from a self - amplifying type 2 immune response as pregnancy progresses .
il-9 is cytokine that has been recognized for decades but only recently had its cell of origin identified : the innate lymphoid cell ( ilc ) .
the role of il-9 , which has historically been categorized as a th2 cytokine prior to the recognition of ilc , is currently unknown in normal pregnancy .
because only serum was available for analysis , we can not confirm the cellular source of the changes observed with this cohort .
nonetheless , increasing transcription levels of several innate immune components , including cd14 , multiple tlr genes , and il-1b , have been observed in peripheral blood leukocytes of women in the third trimester , suggesting that the changes in concentrations of the cytokines identified in our study could plausibly be due to changes in peripheral blood cellular composition .
further studies will be necessary to determine whether placental tissues , circulating leukocytes , stromal cells , or other sources also contribute to the changes reflected in our results .
although these results show statistical significance , it is also noteworthy that the slopes of the variance were all fairly small for each of the analytes and that gestational week did not explain any more than 40% of the variation observed in any of the cytokines . pregnancy clearly alters the maternal immune milieu , but the changes from baseline in the setting of normal pregnancy are subtle and more variable in some women than in others .
what clinical factors contribute to this variation observed between women in the setting of a normal first pregnancy is not entirely known .
blood draws and resulting data were relatively sparse in the first trimester due to women not returning as frequently for study blood draws , even though they were scheduled per protocol , relative to the second and third trimesters .
it is also important to note that many analytes were not detectable in samples ; therefore , it is possible that a different method besides multiplex array on serum or plasma , for example , elisa for individual cytokines / growth factors , may be more sensitive to detect change over time .
previous longitudinal studies of maternal cytokines during pregnancy have shown some concordance with our results .
investigated the change of maternal plasma cytokines from early to midgestation in a large cohort ( approximately 1,000 patients ) and found that il-12 and ifn levels increased , while il-2 and gm - csf levels decreased as pregnancy progressed .
our results confirmed increasing il-12 levels , as well as an increase of ifn in the third trimester compared to the first .
however , kraus et al . published maternal multiplex elisa results from 50 women tested at each trimester as well as postpartum and showed that ifn levels decreased throughout gestation .
denney et al . also found that basal levels of ifn decreased throughout gestation , as did levels of tnf , il-1 , and il-6 in serum samples of 45 healthy pregnant women collected during each trimester .
another recent study demonstrated decreasing il-1 levels throughout gestation . while our study did not confirm any time - dependent change of tnf , we instead observed increases in il-1 and il-6 throughout pregnancy , in contrast to these studies .
clearly , individual heterogeneity exists , and our smaller sample size may account for some of these differences with prior studies .
although not the original focus of this study , we also identified several differences in cytokine / growth factor milieu when comparing pregnant women to healthy controls .
notably , the first trimester of pregnancy was characterized by an increase in several growth factors ( gro , egf , tgf , and pdgf ) and a relative decrease in inflammatory markers ( scd40l , il-6 , il-17 , ip-10 , eotaxin , and mcp-1 ) .
eotaxin , a potent chemoattractant for eosinophils during allergic reactions , has been similarly described to be suppressed during pregnancy by kraus et al . .
overall , the tolerance induction required for successful pregnancy at the level of the fetomaternal interface may also be observed in a relatively tolerogenic , growth factor - rich maternal systemic environment . the difference in sample source when comparing
the archived samples from healthy pregnant women ( serum ) and normal controls ( plasma ) may contribute to these findings .
for example , eotaxin , pdgf , egf , vegf , and soluble cd40 ligand have previously been shown to be significantly different in serum postclotting as compared to plasma samples .
because of this potential source of variation in comparing our historical to contemporary samples , our results will require validation with similarly processed samples of the same source .
however , egf levels have been reported to be approximately 65 pg / ml in serum of healthy individuals , well below our median 489.4 pg / ml in our samples obtained from pregnant subjects , suggesting that our results may hold true despite differences in sample source .
similarly , serum levels of cd40l have been shown to be 830-fold higher in serum as compared to plasma , suggesting again that the very low levels identified in the serum of our cohort of pregnant women may indeed be reflective of the pregnancy state .
our study is unique in that the longitudinal samples were obtained frequently throughout the course of pregnancy .
because of this , our study has the potential to more richly describe individual maternal immune variation over time . however , our study is limited by the relatively small sample size , the lack of pre- or postpartum samples , and potentially the age of the specimens . to address these limitations ,
we have recently conducted a larger longitudinal study of maternal immune changes during pregnancy , with peripheral blood samples obtained monthly throughout gestation as well as 6 weeks postpartum .
tolerance at the fetomaternal interface is complex and technically challenging to study in humans longitudinally . as a result , many recent studies have sought to understand how pregnancy changes maternal systemic immunity .
the possibility exists of maternal peripheral blood not completely or accurately reflecting the local changes within decidual tissues .
an example of such a discrepancy in our study is that of ip-10 , which was low in first - trimester pregnancy compared to healthy controls in this cohort
. ip-10 is secreted by decidual natural killer cells and is key to trophoblast migration and thus has previously been shown to be elevated during pregnancy [ 31 , 32 ] .
on the other hand , the decidual t cell compartment at parturition has recently been shown to closely reflect that of peripheral blood , suggesting that for some components of the immune system the maternal peripheral blood is reasonable to study .
furthermore , sampling maternal peripheral blood is much more feasible to perform longitudinally as compared to more invasive means and may still lend insight into the maternal adaptation to pregnancy .
successful pregnancy has previously been described as predominantly a type 2 immune response - biased phenomenon [ 3436 ] . in light of the collective evidence
, it seems that the description of pregnancy in terms of type 2 immune responses may be oversimplified , both in the overall description of key cytokines / growth factors as well as in the temporal dynamics , and it is in need of further refinement .
|
several recent studies have shown differences in the maternal immune milieu at different phases of pregnancy , but most studies have been cross - sectional or of relatively few time points .
levels of 42 cytokines were determined using a multiplex bead - based assay on archived serum from a cohort of pregnant women ( n = 16 ) at median of 18 time points tested , from the first trimester through to parturition , per woman .
unconditional growth modeling was then used to determine time - dependent changes in levels of these cytokines .
macrophage - derived chemokine ( mdc , aka ccl22 ) decreases as pregnancy progresses .
il-1 , il-6 , il-8 , il-12p70 , il-13 , il-15 , ip-10 , and flt3-ligand increase as a function of gestational weeks , and ifn2 , il-1ra , il-3 , il-9 , il-12p40 , and soluble cd40 ligand increase as a function of trimester . as
pregnancy normally progresses , a maternal shift away from a type 2-biased immune response and toward an inflammatory / counterregulatory response is observed .
|
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] |
all bacterial strains recovered from blood were identified from available microbiology department records of all 13 hospitals in san francisco county from january 1 , 1996 , to march 31 , 1999 . for three hospitals ( 4,5,9 ) ,
data from 1996 had been purged and were no longer available . for each isolate ,
information was also obtained on the ward , age , and gender of the patient .
only the first positive blood culture of a given species was included for a single patient throughout the study period , regardless of susceptibility pattern .
cultures positive for s. epidermidis were considered representative of clinical bacteremia if at least two isolates with identical susceptibility patterns were obtained from a minimum of two separate sets of blood cultures .
all other cultures positive for s. epidermidis were excluded , as were other common skin contaminants ( e.g. , propionibacterium acnes , peptostreptococcus , corynebacterium ) .
all but one hospital used automated systems ( vitek [ biomerieux vitek , hazelwood , mo ] or microscan [ baxter laboratories , west sacramento , ca ] ) for the susceptibility testing of gram - negative bacteria .
all hospitals used kirby - bauer disk - diffusion techniques for the evaluation of susceptibility profiles for streptococcus pneumoniae and other streptococcal species according to national committee for clinical laboratory standards ( nccls ) guidelines .
one hospital ( 12 ) performed all susceptibility testing using kirby - bauer disk - diffusion techniques .
all microbiology laboratories used mic breakpoints established by the nccls . the annual number of blood culture sets processed by each microbiology laboratory was also obtained .
data from five hospitals were obtained as text files or microsoft excel ( redmond , wa ) files and subsequently imported into a microsoft access ( redmond , wa ) database .
data from four hospitals were obtained in printed form , scanned as image files , converted into text files using textbridge pro 98 ( scansoft inc .
data from the remaining four hospitals were obtained from stored index cards , entered manually into the database , and verified for accurate entry .
we also obtained bed size and census data for all 13 hospitals in san francisco county .
total hospital admissions were tabulated from quarterly administrative records from 1996 through 1998 . for each hospital ,
the number of isolates for each species was tabulated for each year and for the entire study period . only species for which the total number of countywide isolates exceeded 100 during the study period
was calculated as the yearly number of organisms with intermediate or full resistance divided by the total number of organisms isolated in san francisco county that year .
because of laboratory variability in susceptibility testing , not all isolates are included in descriptions of proportional resistance .
means were calculated from the annual countywide percentages in each of the 4 years studied .
percent annual resistance was determined for any antibiotic tested in 50 isolates and analyzed for increasing or decreasing annual trend from 1996 through 1999 .
strains with full or intermediate resistance to an antibiotic were counted as resistant in all statistical analyses .
organisms demonstrating increasing or decreasing annual resistance to a given antibiotic ( p<0.05 ) were further described by calculating mean proportional resistance over the study period according to categories of hospital , ward , patient age ( in 10-year intervals ) , and patient gender .
spearman rank tests were used to determine any correlation between hospital indices ( beds , annual admissions , average length of stay ) and proportional antibiotic resistance .
p values remained unchanged with respect to alpha level ( 0.05 ) after removal of the three hospitals missing data from 1996 .
the number of isolates for each species was tabulated for each year and for the entire study period . only species for which the total number of countywide isolates exceeded 100 during the study period
was calculated as the yearly number of organisms with intermediate or full resistance divided by the total number of organisms isolated in san francisco county that year .
because of laboratory variability in susceptibility testing , not all isolates are included in descriptions of proportional resistance .
means were calculated from the annual countywide percentages in each of the 4 years studied .
percent annual resistance was determined for any antibiotic tested in 50 isolates and analyzed for increasing or decreasing annual trend from 1996 through 1999 .
strains with full or intermediate resistance to an antibiotic were counted as resistant in all statistical analyses .
organisms demonstrating increasing or decreasing annual resistance to a given antibiotic ( p<0.05 ) were further described by calculating mean proportional resistance over the study period according to categories of hospital , ward , patient age ( in 10-year intervals ) , and patient gender .
spearman rank tests were used to determine any correlation between hospital indices ( beds , annual admissions , average length of stay ) and proportional antibiotic resistance .
p values remained unchanged with respect to alpha level ( 0.05 ) after removal of the three hospitals missing data from 1996 .
a total of 11,573 bacterial strains were recovered from blood cultures by the 13 hospitals . after excluding duplicate cultures , we had 8,072 remaining clinical isolates .
information on hospital size , census , and blood culture volume is provided in table 1 . despite being distinct and nonadjoining ,
74,600 sets of blood cultures were processed each year in san francisco county ; 9.9% of these were positive for bacterial species .
staphylococcus aureus ( 1,858 ) , escherichia coli ( 1,634 ) , and s. pneumoniae ( 725 ) were the most common organisms .
the numbers of s. aureus , enterococcus faecalis , bacteroides fragilis , e. coli , and serratia marcescens increased annually during the 4-year period .
fourteen species had > 100 isolates and were considered for further analysis ( figure 1 ) .
all bacterial species that were isolated from blood in > 100 persons , january 1996 through march 1999 , all hospitals in san francisco county , california .
the proportion of mrsa and e. faecium resistant to vancomycin ( vre faecium ) increased annually ( figure 2 ) .
countywide , the proportion of mrsa isolates rose from 18.1% ( 1996 ) to 26.1% ( 1999 ) ( p<0.001 ) . in total , mrsa constituted 22.4% of all s. aureus isolates , including 19.6% of emergency department isolates and 15.9% of isolates in the outpatient setting ( table 2 ) .
methicillin resistance in s. aureus isolates was < 15% in patients < 20 years of age and > 20% in all other age groups , with the exception of 30- to 39-year - olds ( 17.6% ) ( table 3 ) .
increase in percentage of staphylococcus aureus isolates resistant to methicillin and increase in percent of enterococcus faecium resistant to vancomycin on a yearly basis from 1996 through the first quarter of 1999 .
proportional resistance refers to the proportion of isolates of that species that is resistant to the indicated antibiotic .
proportional resistance refers to the proportion of isolates of that species that is resistant to the indicated antibiotic .
percentages for small numbers of total isolates should be cautiously interpreted . during the study period , 124
vre isolates and 157 vancomycin - sensitive enterococcus isolates were unspeciated . among the speciated e. faecium isolates , the percentage resistant to vancomycin rose from 0% to 66.7% in the 4-year period ( p<0.001 ) .
vre faecium was most frequently isolated from inpatient adult wards , but six isolates were cultured from emergency department and outpatient settings .
over 66% of e. faecium isolates from skilled nursing facilities were resistant to vancomycin ( table 2 ) .
vre isolates exceeded 40% in all age groups with the exception of 10-year - olds ( 22.2% ) ( table 3 ) . of note ,
vre faecium isolates showed increasing annual resistance to doxycycline ( from 30% to 68% ; p = 0.02 ) . even after the skilled nursing facility ( hospital 13 ) was excluded , the percentage of mrsa and vre faecium isolates varied substantially among individual hospitals ( mrsa 12.5% to 37.5% , vre faecium 12.5% to 80.0% ) .
there was no correlation between proportional resistance and number of icu or total hospital beds , number of annual admissions , or average length of icu or total hospital stay .
however , for both organisms , there was increasing proportional resistance among adult wards in the following order : outpatient wards , emergency department , medical and surgical floors , medical and surgical icus , and skilled nursing facility wards .
notably , a substantial number of vre and mrsa isolates were cultured within the first 24 to 48 hours of hospital admission ( figure 3 ) .
plot of the number of methicillin - resistant staphylococcus aureus ( mrsa ) and vancomycin - resistant enterococcus ( vre ) isolates by hospital day of admission .
an early peak is noted , corresponding to patients entering the hospital with mrsa or vre bacteremia .
proportional resistance increased from 10.8% ( 21 isolates ) in 1996 to 14.6% ( 27 isolates ) in 1998 , but this increase was not statistically significant .
proportional resistance was highest at the extremes of age ( 16.4% in patients > 70 years of age and 19.3% in patients < 10 years of age ( table 3 ) .
countywide , e. coli ( 1,634 isolates ) , klebsiella pneumoniae ( 428 isolates ) , and pseudomonas aeruginosa ( 260 isolates ) were the most frequently isolated gram - negative bacilli .
this was true in all inpatient and outpatient wards with the exception of skilled nursing facilities , where proteus mirabilis was the most common gram - negative isolate after e. coli .
proportional resistance by ward for selective gram - negative organisms is shown in table 4 .
icu = intensive care unit among e. coli isolates , resistance to trimethoprim - sulfamethoxazole averaged 28% and resistance to ciprofloxacin averaged 3% .
increasing annual resistance to ticarcillin - clavulanate was seen in both e. coli ( 6% to 16% , p = 0.03 ) and k. pneumoniae ( 0% to 18% , p = 0.007 ) isolates .
p. aeruginosa isolates showed increasing annual countywide resistance to ciprofloxacin ( 7% to 21% , p = 0.005 ) , ceftazidime ( 6% to 16% , p = 0.02 ) , and imipenem ( 2% to 18% , p = 0.004 ) ( figure 4 ) .
resistance to each of these three antibiotics exceeded 10% in adult icu and adult medical and surgical wards .
resistance to gentamicin ( 15% ) and piperacillin - tazobactam ( 12% ) also increased but was not statistically significant .
yearly percent resistance to ciprofloxacin , ceftazidime , imipenem , and piperacillin in pseudomonas aeruginosa isolates from blood .
increasing proportional resistance occurred in three of the four antibiotics commonly used to treat this organism .
annual number of isolates tested to each antibiotic is given at the top of each graph .
there were 182 e. cloacae and 116 s. marcescens isolates from january 1996 through march 1999 .
s. marcescens isolates also showed increasing annual proportional resistance to gentamicin ( 0% to 14% , p = 0.02 ) and piperacillin ( 4% to 29% , p = 0.01 ) . resistance to ceftazidime , which can be predictive of inducible and extended - spectrum - beta - lactamases , was found in the following overall mean proportions in the study period : e. coli ( 1% ) , p. mirabilis ( 1% ) , k. pneumoniae ( 1% ) , s. marcescens ( 8% ) , p. aeruginosa ( 13% ) , and e. cloacae ( 39% ) .
only p. aeruginosa isolates demonstrated an increasing linear annual trend ( p = 0.02 ) .
the proportion of mrsa and e. faecium resistant to vancomycin ( vre faecium ) increased annually ( figure 2 ) .
countywide , the proportion of mrsa isolates rose from 18.1% ( 1996 ) to 26.1% ( 1999 ) ( p<0.001 ) . in total , mrsa constituted 22.4% of all s. aureus isolates , including 19.6% of emergency department isolates and 15.9% of isolates in the outpatient setting ( table 2 ) .
methicillin resistance in s. aureus isolates was < 15% in patients < 20 years of age and > 20% in all other age groups , with the exception of 30- to 39-year - olds ( 17.6% ) ( table 3 ) .
increase in percentage of staphylococcus aureus isolates resistant to methicillin and increase in percent of enterococcus faecium resistant to vancomycin on a yearly basis from 1996 through the first quarter of 1999 .
proportional resistance refers to the proportion of isolates of that species that is resistant to the indicated antibiotic .
proportional resistance refers to the proportion of isolates of that species that is resistant to the indicated antibiotic .
percentages for small numbers of total isolates should be cautiously interpreted . during the study period , 124
vre isolates and 157 vancomycin - sensitive enterococcus isolates were unspeciated . among the speciated e. faecium isolates , the percentage resistant to vancomycin rose from 0% to 66.7% in the 4-year period ( p<0.001 ) .
vre faecium was most frequently isolated from inpatient adult wards , but six isolates were cultured from emergency department and outpatient settings .
over 66% of e. faecium isolates from skilled nursing facilities were resistant to vancomycin ( table 2 ) .
vre isolates exceeded 40% in all age groups with the exception of 10-year - olds ( 22.2% ) ( table 3 ) . of note
, vre faecium isolates showed increasing annual resistance to doxycycline ( from 30% to 68% ; p = 0.02 ) . even after the skilled nursing facility ( hospital 13 )
was excluded , the percentage of mrsa and vre faecium isolates varied substantially among individual hospitals ( mrsa 12.5% to 37.5% , vre faecium 12.5% to 80.0% ) .
there was no correlation between proportional resistance and number of icu or total hospital beds , number of annual admissions , or average length of icu or total hospital stay .
however , for both organisms , there was increasing proportional resistance among adult wards in the following order : outpatient wards , emergency department , medical and surgical floors , medical and surgical icus , and skilled nursing facility wards .
notably , a substantial number of vre and mrsa isolates were cultured within the first 24 to 48 hours of hospital admission ( figure 3 ) .
plot of the number of methicillin - resistant staphylococcus aureus ( mrsa ) and vancomycin - resistant enterococcus ( vre ) isolates by hospital day of admission .
an early peak is noted , corresponding to patients entering the hospital with mrsa or vre bacteremia .
proportional resistance increased from 10.8% ( 21 isolates ) in 1996 to 14.6% ( 27 isolates ) in 1998 , but this increase was not statistically significant .
proportional resistance was highest at the extremes of age ( 16.4% in patients > 70 years of age and 19.3% in patients < 10 years of age ( table 3 ) .
countywide , e. coli ( 1,634 isolates ) , klebsiella pneumoniae ( 428 isolates ) , and pseudomonas aeruginosa ( 260 isolates ) were the most frequently isolated gram - negative bacilli .
this was true in all inpatient and outpatient wards with the exception of skilled nursing facilities , where proteus mirabilis was the most common gram - negative isolate after e. coli .
proportional resistance by ward for selective gram - negative organisms is shown in table 4 .
icu = intensive care unit among e. coli isolates , resistance to trimethoprim - sulfamethoxazole averaged 28% and resistance to ciprofloxacin averaged 3% .
increasing annual resistance to ticarcillin - clavulanate was seen in both e. coli ( 6% to 16% , p = 0.03 ) and k. pneumoniae ( 0% to 18% , p = 0.007 ) isolates .
aeruginosa isolates showed increasing annual countywide resistance to ciprofloxacin ( 7% to 21% , p = 0.005 ) , ceftazidime ( 6% to 16% , p = 0.02 ) , and imipenem ( 2% to 18% , p = 0.004 ) ( figure 4 ) .
resistance to each of these three antibiotics exceeded 10% in adult icu and adult medical and surgical wards .
no isolates resistant to ciprofloxacin were cultured from pediatric wards ( table 4 ) . resistance to gentamicin ( 15% ) and piperacillin - tazobactam ( 12% ) also increased but was not statistically significant . yearly percent resistance to ciprofloxacin , ceftazidime , imipenem , and piperacillin in pseudomonas aeruginosa
annual number of isolates tested to each antibiotic is given at the top of each graph .
there were 182 e. cloacae and 116 s. marcescens isolates from january 1996 through march 1999 .
s. marcescens isolates also showed increasing annual proportional resistance to gentamicin ( 0% to 14% , p = 0.02 ) and piperacillin ( 4% to 29% , p = 0.01 ) .
resistance to ceftazidime , which can be predictive of inducible and extended - spectrum - beta - lactamases , was found in the following overall mean proportions in the study period : e. coli ( 1% ) , p. mirabilis ( 1% ) , k. pneumoniae ( 1% ) , s. marcescens ( 8% ) , p. aeruginosa ( 13% ) , and e. cloacae ( 39% ) .
only p. aeruginosa isolates demonstrated an increasing linear annual trend ( p = 0.02 ) .
san francisco county has a population of approximately 735,000 and covers 46.7 square miles ( 26 ) .
it comprises multiple racial and ethnic groups ( black 10.9% , hispanic 13.9% , asian 29.1% , and native american 0.5% ) and is served by 13 hospitals .
we have shown that county surveillance of bacterial resistance is a useful addition to local hospital surveillance , particularly as antibiotic resistant bacteria increasingly spread from hospital to hospital and into the community at large . across the county , annual proportions of mrsa and vre isolates significantly increased over the 4-year period .
these data allow us to distinguish countywide outbreaks and trends from single - hospital changes in resistance patterns , and enable infection control efforts to expand or narrow to the appropriate scale . with awareness programs , county surveillance can broaden physicians knowledge of their hospital s effects on the community , as well as the effects of neighboring hospitals on resistance patterns in their particular hospital .
additionally , county surveillance that includes subcategorization of isolates by ward is invaluable in identifying patients at high risk and locations for transmission of resistant bacteria .
not surprisingly , we report our highest proportion of mrsa and vre isolates from icu and nursing home units .
large interhospital differences can lead to further study of ward practices that foster or abate transmission .
awareness can prompt hospital infection control personnel to ensure well - described preventive measures such as swabbing and isolation precautions for vre and mrsa in icu settings ( 2730 ) and nasopharyngeal swabbing and eradication of mrsa in hemodialysis wards ( 1,31 ) .
whether or not these represent true community - acquired strains or strains from patients recently released from hospital settings , they suggest that highly resistant bacterial outpatient infections and infectivity are increasing , a result consistent with recent studies ( 19,21,22 ) .
we also evaluated whether the wide variability in the proportions of resistant bacteria among san francisco hospitals was linked to hospital indices .
in contrast to previous nationwide sampling studies , none of this variability was correlated with the number of hospital icu beds ( 3 ) , total beds ( 1,2,31 ) , annual admissions ( 32 ) , or annual mean length of stay .
this may be due to our small number of hospitals , leading to limited power to detect such correlations .
alternatively , local community and hospital factors ( e.g. , increasing care of moderately ill patients at home , increasing home intravenous antibiotics , active transfer of patients between hospitals , and community - acquired resistant organisms ) may now be diminishing the effect of hospital size , census and length of stay on proportional resistance .
the 28% trimethoprim - sulfamethoxazole resistance in e. coli raises questions about the optimal empiric treatment of urinary tract infections in patients at high risk for bacteremia or urosepsis .
likewise , distinguishing resistance in the outpatient versus inpatient setting can guide empiric therapy in the appropriate setting .
second , in studying a larger populace , we can obtain sufficient numbers to study uncommon organisms .
similarly , countywide surveillance provides a means to identify and confirm novel resistant pathogens . in our study ,
one mrsa isolate with intermediate resistance to vancomycin and three vancomycin - resistant s. epidermidis isolates were noted in microbiology laboratory reports . as with the organisms recently reported in the united states ( 3537 ) ,
these were reported from nontertiary hospitals . nevertheless , our reports are unconfirmed and likely represent laboratory error .
however , if surveillance could be expedited to real - time use , such reports could be investigated and confirmed rather than dismissed . at its worst ,
third , countywide surveillance engenders further hypotheses and research regarding interhospital and community transmission of resistant organisms .
for example , our finding that 20% of emergency department s. aureus isolates are methicillin - resistant provides a flag to further study which county areas have the highest percentages of resistant s. aureus and which risk factors are involved ( e.g. , recent hospital admission , associated hemodialysis centers or nursing homes , or intravenous drug use ) .
the finding that p. mirabilis bacteremia is more common in nursing home wards raises questions about preventing urinary tract infections in that setting .
the relative lack of fluoroquinolone resistance in pediatric gram - negative isolates is likely due to the avoidance of fluoroquinolones in children because of potential detrimental cartilage effects .
the study of fluoroquinolone - resistant organisms during the transition years from pediatric to adult medicine may provide insight into the quantity and duration of antibiotic needed to produce selection pressure , as well as the speed and durability of emerging resistance .
chart review would have been an invaluable addition in distinguishing between community - acquired and recent hospital or nursing home acquisition of resistant organisms .
second , we did not collect or confirm isolates ; thus , although our results reflect microbiologic data actually presented to ordering physicians , they are subject to laboratory differences in speciation and susceptibility determination .
notably , not all organisms were fully speciated or tested against the antibiotics of interest .
third , we do not provide information on antibiotic use , which is known to be a major determinant of bacterial antibiotic resistance .
fourth , countywide trends can be driven by trends seen in the largest hospitals , particularly since smaller hospitals often lack sufficient numbers of isolates to make statistical analyses meaningful .
this was notable for our data on proportional increases in vre , which was largely driven by three hospitals . on the contrary , mrsa trends
were not limited to a few hospitals , nor were they limited to the largest hospitals in the county .
this would expedite surveillance and allow real - time collection and identification of unusually resistant organisms , as well as provide sentinel data regarding countywide outbreaks .
in addition , linking of patient information to microbiologic data would have expedited acquisition of sex , gender , and ward information .
furthermore , despite nccls guidelines , a fair amount of variability exists in laboratory practices and susceptibility panels .
further standardization of these practices would help ensure the reliability of merging data among hospitals . without a doubt , the greatest utility of countywide surveillance lies in its ability to ask screening questions that prompt a more thorough investigation of specific hospitals , wards , or age groups at particular risk for acquiring or transmitting highly resistant organisms .
we have shown how several such questions were raised by our surveillance of bacteremias in san francisco county and described its many advantages .
we have further defined our limitations and difficulties in performing such surveillance , in the hope that this will be helpful to further similar surveillance efforts .
|
countywide antibiotic resistance patterns may provide additional information from that obtained from national sampling or individual hospitals .
we reviewed susceptibility patterns of selected bacterial strains isolated from blood in san francisco county from january 1996 to march 1999 .
we found substantial hospital - to - hospital variability in proportional resistance to antibiotics in multiple organisms .
this variability was not correlated with hospital indices such as number of intensive care unit or total beds , annual admissions , or average length of stay .
we also found a significant increase in methicillin - resistant staphylococcus aureus , vancomycin - resistant enterococcus , and proportional resistance to multiple antipseudomonal antibiotics .
we describe the utility , difficulties , and limitations of countywide surveillance .
|
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aniridia is a rare bilateral congenital malformation of iris due to neuro - ectodermal developmental defect secondary to a mutation in pax6 gene located on chromosome 11p13 .
the incidence of aniridia is estimated to be 1 in 60,000 - 100,000 live births.1 it exists in both sporadic and familial form .
it may occur in isolation or associated with number of syndromes such as wagr ( wilm 's tumor , bilateral aniridia , genitourinary abnormalities and mental retardation ) .
the pax6 mutations cause a spectrum of ocular malformations , of which aniridia is the major sign .
other ocular defect that may be associated includes microcornea , corneal opacity , glaucoma , cataract , subluxated lens , foveal and optic nerve hypoplasia .
we report two cases of familial total aniridia associated with microcornea , high myopia and aphakia .
a 40-year - old male patient presented with marked dimness of vision for last 1 year .
his elder brother ( only sibling ) had visual acuity of 6/6 with -1.5 dioptre sphere ( dsph ) without any other ocular abnormalities .
ocular examination revealed best corrected visual acuity in right eye ( re ) counting finger at 3 feet and 3/60 in left eye ( le ) with + 7.0 dsph .
slit lamp examination showed bilateral total aniridia with superiorly subluxated cataractous lens in the re [ figure 1 ] and aphakia in the le .
the radius of corneal curvature was 8.8 mm in the re and 8.6 mm in the le .
the refractive power of cornea of the re in dioptre ( d ) was 39.0 in both vertical and horizontal meridian .
the anterior chamber depth in the re was 1.54 mm and in the le was 1.48 .
the axial length was 25.57 mm and 25.34 mm in the re and le respectively .
gonioscopy of be revealed rudimentary frill of iris tissue in all quadrants and the angle was open .
aniridia with superiorly subluxated lens dislocated lens in vitreous a 7-year - old girl , the only child of case i , had poor vision since early childhood .
her best corrected visual acuity was 6/36 be with -7.0 dsph and -1.0 dioptre cylinder ( dcyl ) at 90. she had bilateral total aniridia , microcornea , microspherophekic lens [ figure 3 ] and horizontal nystagmus .
average corneal diameter of be was 9.5 mm . radius of corneal curvature was 8.6 mm in the re and 8.3 mm in the le .
the refractive power of cornea of the re was 38.5d in vertical and 37.5d in horizontal meridian .
the diameter of lens was 7.9 mm in be and iop in be with goldmann applanation tonometry was 14 mm hg .
gonioscopy of be revealed rudimentary frill of iris tissue in all quadrants and the angle was open .
fundus examination of be revealed normal optic disc with foveal hypoplasia [ figure 4 ] .
a 40-year - old male patient presented with marked dimness of vision for last 1 year .
his elder brother ( only sibling ) had visual acuity of 6/6 with -1.5 dioptre sphere ( dsph ) without any other ocular abnormalities .
ocular examination revealed best corrected visual acuity in right eye ( re ) counting finger at 3 feet and 3/60 in left eye ( le ) with + 7.0 dsph .
slit lamp examination showed bilateral total aniridia with superiorly subluxated cataractous lens in the re [ figure 1 ] and aphakia in the le .
the radius of corneal curvature was 8.8 mm in the re and 8.6 mm in the le .
the refractive power of cornea of the re in dioptre ( d ) was 39.0 in both vertical and horizontal meridian .
the anterior chamber depth in the re was 1.54 mm and in the le was 1.48 .
the axial length was 25.57 mm and 25.34 mm in the re and le respectively .
gonioscopy of be revealed rudimentary frill of iris tissue in all quadrants and the angle was open .
a 7-year - old girl , the only child of case i , had poor vision since early childhood .
her best corrected visual acuity was 6/36 be with -7.0 dsph and -1.0 dioptre cylinder ( dcyl ) at 90. she had bilateral total aniridia , microcornea , microspherophekic lens [ figure 3 ] and horizontal nystagmus .
average corneal diameter of be was 9.5 mm . radius of corneal curvature was 8.6 mm in the re and 8.3 mm in the le .
the refractive power of cornea of the re was 38.5d in vertical and 37.5d in horizontal meridian .
the diameter of lens was 7.9 mm in be and iop in be with goldmann applanation tonometry was 14 mm hg .
gonioscopy of be revealed rudimentary frill of iris tissue in all quadrants and the angle was open .
fundus examination of be revealed normal optic disc with foveal hypoplasia [ figure 4 ] .
aniridia is a rare congenital abnormality transmitted as an autosomal dominant trait with 85 - 90% penetrance.2 microcornea may rarely be associated with aniridia.34 high myopia is again a very rare association with aniridia and microcornea.5 both these abnormalities were present in our case .
aniridia and lenticular abnormalities like cataract and subluxated lens may co - exist.134 ] case i had subluxated cataractous lens in the re and aphakia in the le due to spontaneously dislocated lens in vitreous cavity in the le . to the best of our knowledge , in the literature of aniridia association of aphakia
aniridia may be associated with defects in angle of anterior chamber and glaucoma , but the findings were not present in our cases .
foveal hypoplasia that may be associated with aniridia was present in our case.2 none of the patients was mentally retarded and thorough systemic examination was normal .
our cases highlight the rare association of congenital aniridia with microcornea , aphakia and high myopia .
|
aniridia is a rare congenital malformation that may be associated with various ocular and systemic manifestations .
we describe two cases of familial total aniridia associated with microcornea , high myopia and dislocated lens .
no systemic abnormality was noted in any of the cases .
|
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the global burden of disease 2004 data estimated that 186 million ( 2.9% ) of the world s
population were severely disabled and another 797 million ( 12.4% ) had moderate long - term
disability .
the average global
prevalence of moderate and severe disability in children is 93 million ( 5.1% ) .
however , the situation with regard
to children with disabilities has often been neglected .
the proportion of children among all age groups suffering
both moderate and severe disability is higher in low- and middle - income countries than in
high - income countries .
previous research on childhood disability in low- and middle - income countries focused on
intellectual and hearing disabilities , and little is known about other types of
disabilities .
presence of disability is considered as a key component of quality - of - life
evaluation .
children with
disabilities continue to face discrimination and restricted access to social services up to
adulthood .
for example , vision problems
correctable by glasses accounted for 40 percent of disabled children not attending school in
brazil .
development of principles and standard scales for disabilities that are sensitive to
cultural and resource differences has long been desired .
after the united nations
international seminar on measurement of disability in 2001 , the washington group on
disability statistics ( washington group ) was formed under united nations sponsorship and
developed a set of general disability measures that have been used by several countries in
censuses and surveys .
the questions use the world health organization s international
classification of functioning , disability , and health as a conceptual framework and focus on
functioning in basic actions .
the disability status of the population has been a concern in vietnam due to prolonged
periods of war . in 2006 ,
the
washington group disability measures were translated into vietnamese and included in the
questionnaire of the vietnam household living standard survey .
economic burden had been expressed as a percentage
of health - care expenditure relative to household income , with the rationale that a high
percentage means that it is likely to force household members to cut their consumption of
other minimum needs , trigger productive asset sales or high levels of debt , and lead to
impoverishment .
a study
on data from the world health survey in 20022004 in 14 developing countries showed that
households with disabilities experienced higher ratios of health - care to total household
expenditure than households without disabilities in two - thirds of countries . although the vietnamese
government is developing a universal health insurance system , households with members
suffering disabilities have to pay extra expenses for examination , treatment ,
rehabilitation , and other costs related to health - care use .
further , the disability statuses of children differ according to the socioeconomic statuses
of their families .
data from the world health surveys in 14 countries showed that persons
with disabilities are significantly worse off in two or more dimensions of economic
well - being ( education , employment , assets / living conditions , household expenditures , and
household expenditures on health care ) . on the other hand , study of the multiple indicator cluster
survey data in 20 countries did not indicate a consistent relationship .
pattern of associations differ by
countries and type of disabilities ; there are still controversial issues that need to be
resolved based on evidence .
the objectives of this study were to assess the economic burden of disability in different
functional domains among school - aged children in vietnam and to evaluate the association
between presence of disability and demographic and household characteristics of children , as
well as between economic burden of disability and demographic and household characteristics
of children , using a large national representative sample .
the vietnam
household living standard survey has been conducted nationwide by the general statistics
office , ministry of planning and investment , vietnam in 1993 , 1998 and every 2 years since
2002 .
survey households were chosen using a multistage stratified cluster sampling process
in which representative clusters were selected from all 64 provinces in vietnam , and a
random sample of households was selected within each cluster .
information collected
regularly in every survey included basic demographic characteristics , educational
attainment , health care use , and employment for all household members ; household income
and expenditure ; and housing condition .
information was collected through face - to - face
interviews with household heads , and key commune officials .
the survey employed intensive
interviewer training , standardized measurement tools and techniques , and instrument
pretesting .
the reason for selecting the survey conducted in 2006 was that it was the latest survey
that had collected information about disability .
the survey was conducted among a total of
9,189 households in may and september 2006 .
use of the vietnam household living standard survey data for this study was approved by
the general statistics office , ministry of planning and investment , vietnam .
the data set
used in this study was provided by the general statistics office and did not contain
personally identifiable information .
two types of main outcome variable were presence of difficulties that were result of some
physical or mental health problems , and health - care expenditure in the past 12 months .
presence of difficulties was assessed according to the standard set of questions
recommended by the washington group .
the validity of this scale in the surveys in asian
and pacific countries confirmed previously .
the questions aimed to identify difficulties in six
functional domains : vision ( vision difficulties or problems ) , hearing ( hearing limitation
or problems ) , remembering or concentrating ( problems with remembering or thinking that
contribute to difficulty in doing daily activities ) , mobility ( limitation or problems
getting around on foot ) , self - care ( problems with taking care of yourself independently ) ,
and communication ( problems with talking , listening , or understanding speech such that it
contributes to difficulty in doing daily activities ) .
furthermore , the response categories
captured the degree or severity of the difficulty ; the response options were no , at least
some difficulty , at least a lot of difficulty , and unable to do it at all . in this study ,
we dichotomized the response into no and yes ( at least some difficulty or more ) . information regarding expenses related to use of medical services was collected for each
household member who had used health - care services in the past 12 months .
the expenses
included out - of - pocket payment for medical service , treatment , and other related costs ,
such as bonuses for medical staff , and transportation fees .
the unit of measurement was
1000 vietnamese dong ( vnd ) ( equivalent to 0.063 - 0.066 united states dollars during the
survey periods ) .
we did not include payment for non - prescribed medicine , medical tools ,
health insurance , and aid , because information about them was not collected at the
individual level .
independent variables included sex , age , area ( urban , and rural ) , household income in the
past 12 months , educational attainment , and occupation of household head .
age was
categorized into three groups : 610 , 1114 , and 1517 years , each corresponding to modal
age for a primary school , a lower secondary school , and an upper secondary school ,
respectively .
household income was calculated as total household revenue minus total
expenditure for the revenue - generating activities and divided into quintiles .
we analyzed all
children ( n=9,882 ) between 6 and 17 years of age in survey households .
the analysis did
not account for the intra - household correlation because a small proportion of households
( 7.6% ) had more than one child with disabilities .
the data set included the household
sampling weight for each household , which had been calculated as the inverse of its
household selection probability .
we used the weight to take into account the multistage
sampling design for all analyses .
the prevalence of difficulty in six functional domains was calculated by sex , age , area ,
household income , educational attainment , and occupation of household head .
the difference
in prevalence by these characteristics was assessed with a logistic regression
analysis .
the multivariable logistic regression analysis required ten cases per independent
indicator variable in the model . in the subsequent analysis , a combined variable for
difficulty in hearing , remembering or concentrating , mobility , self - care , or communication
was used instead of using separate variables for difficulty in each domain .
the frequency
of having difficulty in vision was high enough for a multivariable analysis , and this
domain was not combined .
the per capita household income was calculated by dividing household income by the number
of household members .
then the ratio of the mean health - care expenditure to the mean per
capita household income in the past 12 months was computed for each difficulty status .
to
compare the ratio by difficulty status , a dichotomous variable to indicate whether the
ratio was greater than 0.05 or not was created and used as a dependent variable in the
logistic regression models .
the choice of 0.05 was based on the lower threshold value for
catastrophic impact of health - care expenditure share used by van doorslaer .
the ratio of the mean health - care expenditure to the mean per capita household income was
also compared by characteristics of subjects among all children , among children with
difficulty in vision , and among children with difficulty in hearing , remembering or
concentrating , mobility , self - care , or communication .
the odds ratio was adjusted for sex ,
age , area , household income in the past 12 months , educational attainment , and occupation
of household head .
the vietnam
household living standard survey has been conducted nationwide by the general statistics
office , ministry of planning and investment , vietnam in 1993 , 1998 and every 2 years since
2002 .
survey households were chosen using a multistage stratified cluster sampling process
in which representative clusters were selected from all 64 provinces in vietnam , and a
random sample of households was selected within each cluster .
information collected
regularly in every survey included basic demographic characteristics , educational
attainment , health care use , and employment for all household members ; household income
and expenditure ; and housing condition .
information was collected through face - to - face
interviews with household heads , and key commune officials .
the survey employed intensive
interviewer training , standardized measurement tools and techniques , and instrument
pretesting .
the reason for selecting the survey conducted in 2006 was that it was the latest survey
that had collected information about disability .
the survey was conducted among a total of
9,189 households in may and september 2006 .
use of the vietnam household living standard survey data for this study was approved by
the general statistics office , ministry of planning and investment , vietnam .
the data set
used in this study was provided by the general statistics office and did not contain
personally identifiable information .
two types of main outcome variable were presence of difficulties that were result of some
physical or mental health problems , and health - care expenditure in the past 12 months .
presence of difficulties was assessed according to the standard set of questions
recommended by the washington group .
the validity of this scale in the surveys in asian
and pacific countries confirmed previously .
the questions aimed to identify difficulties in six
functional domains : vision ( vision difficulties or problems ) , hearing ( hearing limitation
or problems ) , remembering or concentrating ( problems with remembering or thinking that
contribute to difficulty in doing daily activities ) , mobility ( limitation or problems
getting around on foot ) , self - care ( problems with taking care of yourself independently ) ,
and communication ( problems with talking , listening , or understanding speech such that it
contributes to difficulty in doing daily activities ) .
furthermore , the response categories
captured the degree or severity of the difficulty ; the response options were no , at least
some difficulty , at least a lot of difficulty , and unable to do it at all . in this study ,
we dichotomized the response into no and yes ( at least some difficulty or more ) . information regarding expenses related to use of medical services was collected for each
household member who had used health - care services in the past 12 months .
the expenses
included out - of - pocket payment for medical service , treatment , and other related costs ,
such as bonuses for medical staff , and transportation fees .
the unit of measurement was
1000 vietnamese dong ( vnd ) ( equivalent to 0.063 - 0.066 united states dollars during the
survey periods ) .
we did not include payment for non - prescribed medicine , medical tools ,
health insurance , and aid , because information about them was not collected at the
individual level .
independent variables included sex , age , area ( urban , and rural ) , household income in the
past 12 months , educational attainment , and occupation of household head .
age was
categorized into three groups : 610 , 1114 , and 1517 years , each corresponding to modal
age for a primary school , a lower secondary school , and an upper secondary school ,
respectively .
household income was calculated as total household revenue minus total
expenditure for the revenue - generating activities and divided into quintiles .
we analyzed all
children ( n=9,882 ) between 6 and 17 years of age in survey households .
the analysis did
not account for the intra - household correlation because a small proportion of households
( 7.6% ) had more than one child with disabilities .
the data set included the household
sampling weight for each household , which had been calculated as the inverse of its
household selection probability .
we used the weight to take into account the multistage
sampling design for all analyses .
the prevalence of difficulty in six functional domains was calculated by sex , age , area ,
household income , educational attainment , and occupation of household head .
the difference
in prevalence by these characteristics was assessed with a logistic regression
analysis .
the multivariable logistic regression analysis required ten cases per independent
indicator variable in the model . in the subsequent analysis , a combined variable for
difficulty in hearing , remembering or concentrating , mobility , self - care , or communication
the frequency
of having difficulty in vision was high enough for a multivariable analysis , and this
domain was not combined .
the per capita household income was calculated by dividing household income by the number
of household members .
then the ratio of the mean health - care expenditure to the mean per
capita household income in the past 12 months was computed for each difficulty status . to
compare the ratio by difficulty status ,
a dichotomous variable to indicate whether the
ratio was greater than 0.05 or not was created and used as a dependent variable in the
logistic regression models .
the choice of 0.05 was based on the lower threshold value for
catastrophic impact of health - care expenditure share used by van doorslaer .
the ratio of the mean health - care expenditure to the mean per capita household income was
also compared by characteristics of subjects among all children , among children with
difficulty in vision , and among children with difficulty in hearing , remembering or
concentrating , mobility , self - care , or communication .
the odds ratio was adjusted for sex ,
age , area , household income in the past 12 months , educational attainment , and occupation
of household head .
among 9,882 children , 397 ( 4.0% ) children had at least one type of disability .
table 1table 1prevalence of difficulty in six functional domains by demographic and household
characteristics of childrennfunctional domainsvisionhearing , remembering or concentrating ,
mobility , self - care , or communicationtotalhearingrememberingor concentratingmobilityself - carecommunicationoverall9882186(1.9%)223(2.3%)49(0.5%)98(1.0%)58(0.6%)82(0.8%)93(0.9%)sexboy503271(1.4%)119(2.4%)27(0.5%)53(1.1%)29(0.6%)51(1.0%)52(1.0%)girl4850114(2.4%)104(2.1%)22(0.5%)45(0.9%)29(0.6%)31(0.6%)41(0.8%)p=0.002p=0.459p=0.614p=0.539p=0.849p=0.047p=0.328age6 - 10313233(1.1%)89(2.8%)16(0.5%)33(1.1%)12(0.4%)44(1.4%)31(1.0%)11 - 14364181(2.2%)63(1.7%)17(0.5%)32(0.9%)19(0.5%)19(0.5%)30(0.8%)15 - 17310972(2.3%)71(2.3%)16(0.5%)33(1.1%)27(0.9%)20(0.6%)31(1.0%)trendp<0.001trendp=0.197trendp=0.909trendp=0.979trendp=0.017trendp=0.003trendp=0.971areaurban2198104(4.7%)39(1.8%)8(0.4%)14(0.6%)12(0.6%)21(1.0%)13(0.6%)rural768482(1.1%)184(2.4%)41(0.5%)84(1.1%)46(0.6%)61(0.8%)80(1.0%)p<0.001p=0.090p=0.271p=0.066p=0.814p=0.484p=0.044household income1st quintile ( lowest)254218(0.7%)82(3.2%)20(0.8%)35(1.4%)18(0.7%)26(1.0%)37(1.5%)2nd quintile234128(1.2%)60(2.6%)15(0.6%)30(1.3%)19(0.8%)19(0.8%)26(1.1%)3rd quintile194832(1.7%)35(1.8%)8(0.4%)17(0.9%)5(0.2%)12(0.6%)15(0.8%)4th quintile157542(2.7%)21(1.4%)4(0.3%)11(0.7%)5(0.3%)11(0.7%)7(0.4%)5th quintile ( highest)147465(4.4%)24(1.6%)2(0.1%)5(0.3%)12(0.8%)13(0.9%)9(0.6%)trendp<0.001trendp<0.001trendvp<0.001trendp<0.001trendp=0.545trendp=0.503trendp=0.001educational attainment of household
headprimary school graduate or none277938(1.4%)49(1.8%)16(0.6%)20(0.7%)10(0.4%)19(0.7%)16(0.6%)lower secondary school graduate312265(2.1%)79(2.5%)22(0.7%)43(1.4%)22(0.7%)24(0.8%)39(1.3%)upper secondary school graduate121944(3.6%)19(1.5%)2(0.2%)6(0.5%)7(0.6%)11(0.9%)2(0.2%)junior college degree , bachelor s degree , or
higher276139(1.4%)76(2.8%)9(0.3%)29(1.0%)20(0.7%)28(1.0%)36(1.3%)trendp=0.523trendp=0.064trendp=0.054trendp=0.789trendp=0.151trendp=0.116trendp=0.079occupation of household headleaders , professionals , or staffs in any
fields72535(4.8%)11(1.6%)2(0.3%)3(0.4%)7(1.0%)8(1.1%)3(0.4%)skilled workers in personal services and sales24510(3.9%)5(2.0%)2(0.8%)3(1.2%)002(0.8%)skilled workers in agriculture and fisheries4065(1.2%)8(2.0%)2(0.5%)5(1.2%)0(0.1%)2(0.4%)4(1.0%)skilled handicraftsman and other skilled manual
workers109238(3.5%)18(1.6%)3(0.3%)10(0.9%)5(0.5%)6(0.5%)5(0.5%)assemblers and machine operators2695(1.9%)3(1.0%)2(0.7%)2(0.7%)1(0.4%)1(0.4%)1(0.4%)unskilled workers636076(1.2%)157(2.5%)35(0.6%)64(1.0%)43(0.7%)57(0.9%)66(1.0%)armed forces1801(4.4%)01(4.4%)1(4.4%)1(4.4%)1(4.4%)not working76718(2.3%)20(2.6%)3(0.4%)10(1.3%)1(0.2%)7(1.0%)10(1.3%)p<0.001p=0.402p=0.719p=0.582p=0.104p=0.442p=0.226data for per capita household income and educational attainment of the household head
were missing in 2 cases and 1 case , respectively .
shows the prevalence of difficulty in six functional domains marginally and by
demographic and household characteristics of the 9,882 children .
the overall prevalence of
difficulty were 1.9% for vision , 0.5% for hearing , 1.0% for remembering or concentrating ,
0.6% for mobility , 0.8% for self - care , and 0.9% for communication .
data for per capita household income and educational attainment of the household head
were missing in 2 cases and 1 case , respectively .
table 2table 2relation between having difficulties and demographic and household
characteristics of childrendifficulties in visiondifficulties in hearing , remembering or
concentrating , mobility , self - care or communicationor(95%ci)or(95%ci)sexboyreferencereferencegirl1.70(1.23 , 2.37)p=0.0020.91(0.69 , 1.20)p=0.513age610referencereference11142.10(1.35 , 3.27)p=0.0010.61(0.43 , 0.86)p=0.00415172.09(1.34 , 3.28)p=0.0010.84(0.60 , 1.18)p=0.319trend p=0.001trend p=0.300areaurbanreferencereferencerural0.34(0.23 , 0.50)p<0.0011.04(0.69 , 1.56)p=0.842household income1st quintile ( lowest)referencereference2nd quintile1.35(0.73 , 2.49)p=0.3330.83(0.58 , 1.19)p=0.3013rd quintile1.50(0.82 , 2.73)p=0.1840.60(0.39 , 0.92)p=0.0204th quintile1.93(1.05 , 3.55)p=0.0340.46(0.27 , 0.77)p=0.0045th quintile ( highest)2.54(1.39 , 4.66)p=0.0030.55(0.32 , 0.94)p=0.029trend
p=0.001trend p=0.002educational attainment of household
headprimary school graduate or nonereferencereferencelower secondary school graduate1.25(0.81 , 1.93)p=0.3191.57(1.08 , 2.27)p=0.017upper secondary school graduate1.30(0.77 , 2.17)p=0.3271.08(0.59 , 1.98)p=0.804junior college degree , bachelor s degree , or
higher0.82(0.49 , 1.35)p=0.4261.47(1.02 , 2.11)p=0.040trend p=0.380trend p=0.124occupation of household headleaders , professionals , or staffs in any
fieldsreferencereferenceskilled workers in personal services and sales0.87(0.38 , 1.96)p=0.7311.17(0.39 , 3.53)p=0.782skilled workers in agriculture and fisheries0.43(0.16 , 1.21)p=0.1111.09(0.43 , 2.76)p=0.858skilled handicraftsman and other skilled manual
workers1.00(0.59 , 1.70)p=0.9990.88(0.39 , 1.97)p=0.757assemblers and machine operators0.36(0.13 , 0.98)p=0.0450.63(0.17 , 2.35)p=0.490unskilled workers0.48(0.29 , 0.80)p=0.0041.19(0.62 , 2.30)p=0.606armed forces3.27(0.38 , 27.94)p=0.279not working0.67(0.33 , 1.36)p=0.2631.32(0.59 , 2.95)p=0.492the odds ratios were adjusted for all other characteristics .
shows the adjusted odds ratios of difficulties in vision as well as those in
hearing , remembering or concentrating , mobility , self - care , or communication for each
characteristics of subjects .
difficulties in vision were more prevalent among girls than
boys , among children who were 1117 years of age than those who were 610 years of age , and
in urban areas than rural areas .
with regard to the association between difficulty and
household income , the direction of association differed between difficulty in vision and
difficulty in the other domains : household income of a child with difficulty in vision was
higher than that of a child without difficulty in vision , whereas household income of a
child with difficulty in domains other than vision was lower than that of a child without
difficulty in domains other than vision .
the odds ratios were adjusted for all other characteristics . with regard to all subjects ,
the average per capita household income was 7441.2 thousand
vietnamese dong , or 469491 united states dollars ; per capita total household expenditure
was 5350.6 thousand dong , or 337353 dollars ; and health - care expenditure was 95.0 thousand
dong , or 6 dollars .
table 3table 3per capita household income and health - care expenditure in the past 12
monthsper capitahousehold income(1000 vnd )
[ a]health - careexpenditure(1000 vnd )
[ b][b]/[a][b]/[a]>0.05mean(sd)mean(sd)share of inpatientcare expenditure%or(95%ci)overall7441.2(9043.1)95.0(887.9)54.9%0.01284.6%children with difficulties in specific
domainsvisionno7382.2(9052.6)81.3(709.5)53.4%0.01104.4%referenceyes10521.5(7969.7)807.4(3901.1)62.9%0.076714.6%4.78(2.95 , 7.73)p<0.001p<0.001p<0.001hearing , remembering or concentrating ,
mobility , self - care , or communicationno7467.9(9059.6)83.3(792.7)50.2%0.01124.4%referenceyes6284.0(8234.2)601.0(2735.9)83.1%0.095613.3%3.13(2.04 , 4.80)p<0.001p<0.001p<0.001hearingno7454.2(9061.6)94.8(888.4)54.9%0.01274.6%referenceyes4827.1(2777.5)133.6(783.7)54.2%0.02776.8%1.33(0.49 , 3.62)p=0.578p<0.001p<0.001remembering or concentratingno7461.8(9073.7)91.6(845.4)53.6%0.01234.5%referenceyes5391.7(4743.5)430.1(2845.4)82.3%0.079814.3%3.22(1.78 , 5.81)p<0.001p<0.001p<0.001mobilityno7441.8(9055.9)85.7(810)51.2%0.01154.5%referenceyes7341.7(6615.2)1656.5(4567.1)87.0%0.225624.8%7.49(3.90 , 14.38)p<0.001p<0.001p<0.001self - careno7438.1(9014.6)86.7(807.7)51.3%0.01174.5%referenceyes7809.8(12029.1)1079.7(4023.4)89.7%0.138216.2%3.99(2.06 , 7.74)p<0.001p<0.001p<0.001communicationno7460.0(9069.7)89.7(825.3)52.8%0.01204.5%referenceyes5456.8(5231.6)649.4(3447.4)84.8%0.119014.9%3.46(1.91 , 6.28)p<0.001p<0.001p<0.001p - values compairing per capita household income or healthcare expenditure by
difficulty status were obtained with mann - whitney u tests .
the odds ratios compared
the odds of [ b]/[a]>0.05 between children with a specific disability and those
without .
the odds ratios were adjusted for sex , age , area , household income , and
educational attainment and occupation of household head .
compares the ratio of health - care expenditure to per capita household income
between children with a specific disability and those without .
the proportion of children with a ratio greater than 0.05 was 4.6% and was higher among
children with difficulty in vision , remembering or concentrating , mobility , self - care , and
communication than those without these respective difficulties . among children with
difficulty in vision ,
both household income and health - care expenditure were higher among
children without difficulty in vision , respectively .
on the other hand , among children with
difficulty in hearing , remembering or concentrating , mobility , self - care , or communication ,
household income was lower and health - care expenditure was higher than among children
without difficulty , respectively .
p - values compairing per capita household income or healthcare expenditure by
difficulty status were obtained with mann - whitney u tests .
the odds ratios compared
the odds of [ b]/[a]>0.05 between children with a specific disability and those
without .
the odds ratios were adjusted for sex , age , area , household income , and
educational attainment and occupation of household head .
table 4table 4ratio of health - care expenditure to per capita household income among all
children who were 617 years of ageper capita householdincome [ a](1000
vnd)health - careexpenditure [ b](1000
vnd)[b]/[a][b]/[a]>0.05mean(sd)mean(sd)%or(95%ci)overall7441.2(9043.1)95.0(887.8)0.01284.6%areaurban12349.6(13779.1)126.6(945.6)0.01034.0%referencerural6036.5(6480.7)86.0(870.4)0.01424.8%0.91(0.69 , 1.22)p=0.535household income1st quintile ( lowest)2607.1(642.9)44.8(448.6)0.01725.3%reference2nd quintile4324.0(465.6)91.3(746.9)0.02116.0%1.13(0.87 , 1.45)p=0.3643rd quintile6215.7(642.9)130.5(1500.5)0.02104.6%0.82(0.61 , 1.10)p=0.1914th quintile9090.6(1068.7)96.1(685)0.01063.0%0.52(0.36 , 0.75)p=0.0015th quintile ( highest)20582.2(17665.2)139.3(754)0.00683.0%0.53(0.36 , 0.80)p=0.002trend p<0.001educational attainment of household
headprimary school graduate or none6528.2(6541.5)84.6(681.4)0.01304.9%referencelower secondary school graduate7223.7(6977.3)107.5(1138.1)0.01494.8%1.03(0.80 , 1.33)p=0.793upper secondary school graduate11528.2(14240.1)136.2(1135.5)0.01184.5%1.13(0.79 , 1.63)p=0.497junior college degree , bachelor s degree , or
higher6800.9(9819.9)73.2(564.8)0.01084.2%0.86(0.66 , 1.13)p=0.279trend p=0.319occupation of household headleaders , professionals , or staffs in any
fields14587.6(19354.9)120.7(693.7)0.00832.9%referenceskilled workers in personal services and sales12555.6(18497.1)142.9(929.3)0.01144.5%1.35(0.61 , 2.99)p=0.459skilled workers in agriculture and fisheries8658.4(11815.4)99.3(548.3)0.01155.8%1.76(0.91 , 3.41)p=0.092skilled handicraftsman and other skilled manual
workers7300.0(5111.7)114.2(801.1)0.01565.2%1.45(0.82 , 2.56)p=0.205assemblers and machine operators11464.5(9493.2)84.7(662.9)0.00743.9%1.25(0.53 , 2.97)p=0.612unskilled workers6082.9(5630.6)92.5(992.7)0.01524.7%1.25(0.75 , 2.10)p=0.389armed forces14426.3(5345.6)40.6(82.1)0.00280.0%not working8288.8(10242.1)51.9(157.5)0.00634.7%1.40(0.76 , 2.58)p=0.280the odds ratios were adjusted for sex , age , and all other characteristics .
compares the ratio of health - care expenditure to per capita household income
among all children who were 617 years of age by their characteristics . the proportion of
children with a ratio being greater than 0.05 was higher among children from the poorer
households .
table 5table 5ratio of health - care expenditure to per capita household income among children
with difficulty in visionper capita householdincome [ a](1000
vnd)health - careexpenditure [ b](1000
vnd)[b]/[a][b]/[a]>0.05mean(sd)mean(sd)%or(95%ci)overall10521.5(7969.7)807.4(3901.1)0.076714.6%-areaurban13450.2(9074.0)308.2(1432.3)0.02298.0%referencerural6811.5(3937.7)1439.8(5603.3)0.211423.1%5.19(1.53 , 17.60)p=0.008household income1st quintile ( lowest)2941.3(498.8)296.4(1044.2)0.100816.6%reference2nd quintile4319.3(477.3)191.0(365.9)0.044223.3%2.29(0.41 , 12.59)p=0.3423rd quintile6293.8(694.8)1665.8(7964.4)0.264710.7%0.83(0.11 , 6.03)p=0.8534th quintile8979.4(900.8)705.1(2693.2)0.07859.4%0.87(0.15 , 5.21)p=0.8805th quintile ( highest)18369.0(8705.7)853.5(2665.9)0.046515.6%3.24(0.61 , 17.27)p=0.168trend p=0.383educational attainment of household
headprimary school graduate9437.2(9528.8)586.8(2222.6)0.062217.4%referencelower secondary school graduate8397.7(4406.0)1601.3(6128.6)0.190720.0%1.31(0.47 , 3.64)p=0.611upper secondary school graduate14054.0(10112.1)197.9(343.0)0.01419.9%0.81(0.16 , 4.07)p=0.796junior college degree , bachelor s degree , or
higher11153.5(7011.5)392.1(2088.0)0.03528.4%0.51(0.11 , 2.37)p=0.386trend p=0.303occupation of household headleaders , professionals , or staffs in any
fields13808.2(5597.7)524.8(1932.6)0.03808.6%referenceskilled workers in personal services and sales12739.0(7626.8)1212.3(4290.6)0.09527.9%0.68(0.04 , 12.59)p=0.798skilled workers in agriculture and fisheries7388.3(1640.2)1292.3(2707.2)0.174920.2%2.80(0.14 , 54.00)p=0.496skilled handicraftsman and other skilled manual
workers7424.0(4390.7)1068.1(3417.2)0.143917.1%1.08(0.17 , 6.79)p=0.935assemblers and machine operators10534.1(3370.4)630.5(860.6)0.059941.9%9.18(0.95 , 88.46)p=0.055unskilled workers9511.7(8433.8)876.3(5236.1)0.092116.8%1.15(0.21 , 6.20)p=0.871not working14710.4(13221.9)205.4(290.6)0.01406.2%0.52(0.04 , 7.47)p=0.628the odds ratios were adjusted for sex , age , and all other characteristics .
compares the ratio of health - care expenditure to per capita household income
among children with difficulties in vision by their characteristics .
in rural areas ,
household income was lower and health - care expenditure was higher compared with urban areas ,
among children with a difficulty in vision .
the proportion of children with a ratio greater
than 0.05 was higher in rural areas than urban areas .
table 6table 6ratio of health - care expenditure to per capita household income among children
with difficulty in hearing , remembering or concentrating , mobility , self - care , or
communicationper capita householdincome [ a](1000
vnd)health - careexpenditure [ b](1000
vnd)[b]/[a][b]/[a]>0.05mean(sd)mean(sd)%or(95%ci)overall6284.0(8234.2)601.0(2735.9)0.09613.3%areaurban12824.1(16921.9)282.8(675.7)0.02215.4%referencerural4907.6(3477.6)668.0(2991.4)0.13612.9%1.34 ( 0.29 , 6.26)p=0.710household income1st quintile ( lowest)2618.1(632.8)349.0(2097.2)0.13311.9%reference2nd quintile4322.5(506.4)749.1(2560.5)0.17317.0%1.05 ( 0.32 , 3.43)p=0.9293rd quintile6222.9(687.4)937.6(4695.9)0.15111.1%0.15 ( 0.03 , 0.86)p=0.0334th quintile9108.4(1140.7)61.4(85.8)0.0070.0%-5th quintile ( highest)21188.9(18713.3)1074.0(2424)0.05124.0%0.59 ( 0.15 , 2.23)p=0.432trend p=0.033educational attainment of household
headprimary school graduate4801.7(3129.5)319.8(1365.7)0.06710.1%referencelower secondary school graduate5816.4(4466.5)861.3(3858.1)0.14814.2%1.40 ( 0.33 , 5.85)p=0.648upper secondary school graduate11232.7(22455)1471.4(3432.9)0.13138.4%12.30 ( 2.29 , 66.07)p=0.003junior college degree , bachelor s degree , or
higher6515.3(6914.2)298.1(1508.6)0.0468.4%0.70 ( 0.13 , 3.68)p=0.673trend p=0.972occupation of household headleaders , professionals , or staffs in all
fields24946.8(26821.6)1751.4(3303.5)0.07028.6%referenceskilled workers in personal services and sales5541.4(1866.3)92.3(143.1)0.01725.7%1.40 ( 0.04 , 43.55)p=0.848skilled workers in agriculture and fisheries6949.9(4327.8)1062.1(2487.6)0.15335.9%0.92 ( 0.10 , 8.60)p=0.944skilled handicraftsman and other skilled manual
workers6118.2(3232.9)264.8(378.6)0.04329.3%0.42 ( 0.05 , 3.73)p=0.439assemblers and machine operators8885.6(5298.1)969.6(932.7)0.10972.2%-unskilled workers4917.6(4013.0)611.4(3070.9)0.1247.6%0.09 ( 0.01 , 0.65)p=0.017armed forces10092.00.0 - 0.0%-not working5985.4(4888.2)79.9(121.6)0.01315.5%0.35 ( 0.02 , 5.65)p=0.462the odds ratios were adjusted for sex , age , and all other characteristics .
compares the ratio of health - care expenditure to per capita household income
among children with difficulties in hearing , remembering or concentrating , mobility ,
self - care , or communication by their characteristics .
the proportion of children with a
ratio greater than 0.05 was higher among children from the poorer households .
the goodness - of - fit of the multivariable logistic regression model was higher for analysis
in children with difficulties than in all children : the pseudo r of the
multivariable logistic regression model was 0.013 among all children , 0.163 among children
with difficulty in vision , and 0.231 among children with difficulty in hearing , remembering
or concentrating , mobility , self - care , or communication .
we used a large national representative sample to present the economic burden of
disabilities by different functional domains among school - aged children and its association
with demographic and household characteristics of children in vietnam .
the ratio of health - care
expenditure to per capita household income was higher among children who had a difficulty in
vision , remembering or concentrating , mobility , self - care , or communication compared with
those who did not have the respective difficulty .
there was a relation between household
income and the presence of difficulty in vision , as well as between household income and the
presence of difficulty in hearing , remembering or concentrating , mobility , self - care , or
communication : difficulty in vision was more prevalent in richer households ; on the other
hand , difficulty in hearing , remembering or concentrating , mobility , self - care , or
communication was more prevalent in poorer households . in the vietnam household living standard survey 2006 ,
the estimated prevalence of difficulty in
vietnamese children ranged from 0.5% for hearing to 1.9% for vision .
the prevalence of
disability among children in low- and middle - income countries has varied across studies . for
example
, the prevalence of visual impairment varied between 0.1% and 12.5% ; the prevalence
of hearing impairment between 0.4% and 19.7% ; and the prevalence of intellectual disability
between 0.09% and 18.3% .
the
prevalence of difficulty estimated in this study was within the range of previous reports in
low- and middle - income countries .
households with children with a disability spent more on health care than households
without such children . regarding difficulty in vision ,
the increase in the economic burden
of health care among children with disability was caused by substantially higher health - care
expenditure .
the higher household income among children with difficulty in vision than
children without this difficulty diluted the influence of the increase in the health - care
expenditure for difficulty in vision .
the share of inpatient care expenditure relative to
total health - care expenditure among children with difficulty in vision was not as high as
that among children with disabilities in other domains .
this low share of inpatient care is
consistent with the fact that among the pediatric population in the uk , the proportion of
children who received hospital eye services and required hospital admission was low ( less
than 10% ) and with the fact that the total cost of ophthalmic surgery and inpatient eye
services was less than that of ophthalmology outpatient eye services
. regarding difficulties in other domains , the difference in the ratio was caused by both
relatively lower income and substantially higher health - care expenditure among children with
a disability than those without the respective disability .
households with children with
difficulty in hearing , remembering or concentrating , mobility , self - care , or communication
tended to be poorer than those without children with difficulties . with the exception of
difficulty in hearing ,
there were systematic differences in the mechanisms of high economic burden of
disability between difficulty in vision and difficulty in other domains . in our analysis among children 617 years of age , a higher prevalence of disability was
observed in the poorer households with regard to disability in hearing , remembering or
concentrating , mobility , self - care , and communication . with regard to difficulty in vision
,
on the other hand , the prevalence was higher among those better off . in studies among adult
populations ,
it has been
reported that a larger percentage of children among those who are less wealthy were screened
positive with the disability questions , but the association was not consistent .
the possible reason for the reverse
trend with regard to difficulty in vision observed in this study is longer study hours among
children from better off households than those from worse off households . in vietnam ,
besides engaging in the standard school curriculum , children are enrolled in many kinds of
academic tutorials to improve their knowledge and skills .
these extra classes have
proliferated , since teachers often organize home - based classes to supplement their income
.
extra classes mean an
increased workload for the participating students compared with those not participating
. the
location can be at school , a teacher s house , or a student s house .
lengthy exposure to such
factors as inadequate indoor illumination in the reading environment or a chair or table
unsuitably large for a child s body size are reported to adversely impact a student s visual
acuity .
in addition to known risk factors
of visual difficulties , such as retinopathy of prematurity and family history of high
refractive error , these
environmental conditions and lifestyles are now suggested as modifiable causes .
the increase
in the working hours of children under uncontrolled conditions is considered as a potential
concern for child visual development .
the authors hypothesized that the different impact of health - care expenditure by
demographic and household characteristics of subjects was captured by the fitness of the
regression model to the observed data .
the model , which included socioeconomic factors ,
namely household income , educational attainment , and occupation of the household head ,
explained the variation in the ratio of health - care expenditure to the per capital household
income better in the analysis including only children with a disability than in the analysis
including all children .
this finding was interpreted as indicating that the association
between socioeconomic status and household burden was stronger ; in other words , the impacts
of differences in socioeconomic status on the household burden of health - care expenditure
were higher for children who had disabilities than for those who did not .
socioeconomically
disadvantaged children not only suffered from higher risk of disability but also experienced
heavier disadvantage from their socioeconomic status when they had a disability compared
with those who were well off . more attention
should be paid to reducing the disadvantage
caused by poor socioeconomic conditions when households have children with disabilities .
the
results of the present study will be useful for the design of equitable health systems in
vietnam .
this study used a nationwide household sample that was representative for the whole
country , that is , the 8 regions , urban / rural areas and 64 provinces in vietnam .
the sample
size was large , allowing us to reliably estimate the prevalence of difficulties and to
conduct multivariable analyses .
the information was self - reported , and there is a potential
for information bias ; however , the magnitude of the bias is considered to be low because of
several reasons : the survey employed high - quality interviewer training and standardized data
collection procedures across geographic regions ; the response rates were high , and there
were only a small amount of missing data ; and validated measurement scale for disability was
used , and the calculated prevalence of disability was within the range from studies in other
countries .
income was measured as a sum of all reported revenue and costs for all types of
activities .
health - care expenditure was collected for each member of family and asked
separately for inpatient and outpatients care .
the survey was cross - sectional and suitable for estimating prevalence and national average
income and expenditure .
however , no temporal relation was definitively confirmed because of
the possibility of reverse causation . to ascertain if poor socioeconomic status causes
difficulty or difficulty leads to poor socioeconomic status or both , further studies are
necessary .
if
the poor families had difficulty in accessing health care and did not receive all the needed
care , their health - care expenditure would tended to be lower than needed , leading to
underestimation of the potentially higher burden of disability among the poor households .
data included siblings living in the same household , and underestimation of standard errors
was possible , because the analyses did not take the intraclass correlation into account .
the
magnitude of the bias , however , is considered to be small because there was only one child
with a disability in most of the households .
health - care needs of children with a disability pose a higher burden on households with
lower financial resources .
visual disability was more prevalent among children from richer
households , whereas other disabilities were more prevalent among children from poorer
households .
|
objective : the aim of this study was to assess the economic burden of
disability of school - aged children and to evaluate the association between disabilities
and household socioeconomic status , as well as the economic burden of disability and
household socioeconomic status in vietnam.materials and methods : nationally representative data for 9,882 children
aged 6 to 17 years from the vietnam household living standard survey 2006 were used .
disabilities were measured in six basic functional domains , including vision , hearing ,
remembering or concentrating , mobility , self - care , and communication .
we evaluated the
association between area , household income , educational attainment , or occupation of
household head , and each difficulty .
the ratio of health - care expenditure to per capita
household income was compared by presence of a disability as well as household
socioeconomic status.results : the prevalence of difficulty was 1.9% for vision and 2.3% for at
least one of the other five domains .
difficulty in vision was more prevalent in the richer
households ( p=0.001 ) , whereas difficulty in the other five domains was more prevalent in
the poorer households ( p=0.002 ) .
the ratio of health - care expenditure to per capita
household income was greater than 0.05 in 4.6% of children . the adjusted odds ratio of
children with difficulty in vision having a health - care expenditure share greater than
0.05 compared with children without difficulty was 4.78 ( 95% ci : 2.95 , 7.73 ; p<0.001 ) ,
and that for difficulty in the other five domains was 3.13 ( 95% ci : 2.04 , 4.80 ;
p<0.001 ) . among children with difficulty in at least one of the five domains other than
vision , the proportion of children with a health - care expenditure share greater than 0.05
was higher among children from the poorer households ( p=0.033).conclusions : children with a disability spent more on health care relative
to their income than those without .
visual disability was more prevalent among children
from the richer households , whereas other disabilities were more prevalent among children
from the poorer households .
|
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acute obstructive suppurative pancreatic ductitis ( aospd ) is a rare complication of chronic pancreatitis that has been described in only seven previous case reports since 1995 .
aospd is defined as suppuration from the pancreatic duct ; however , in contrast to the pancreatic infections that typically complicate chronic pancreatitis , it is not associated with pancreatic pseudocyst , abscess or necrosis .
a 33-year - old female presented in july 2015 , with complaints of pain in epigastric region and multiple episodes of vomiting .
pain was dull aching and radiating to back associated with multiple episodes of bilious vomiting . on per abdomen examination , there was tenderness in epigastrium and right hypochondrium .
laboratory investigations revealed an elevated total leukocytes count ( 10 000/cumm ) , with normal serum amylase ( 41.9 units / l ) and serum lipase ( 29.4 units / l ) .
, multiple radio - opaque shadows were seen corresponding with distal part of pancreas ( fig .
ultrasonography was suggestive of hyper - echoic and atrophic pancreas with main pancreatic duct dilated upto 1 cm and 6 mm calculus in the main pancreatic duct at the level of pancreatic head . on computed tomography ( ct ) , multiple , conglomerated calcifications
were noted in the head and tail of pancreas and just proximal to ampulla of vater resulting in dilatation of the common bile duct , common hepatic duct and main pancreatic duct ( fig .
2 ) .
figure 1:radiograph showing multiple radio - opaque shadows corresponding to the distal part of pancreas .
intra - operative photograph showing frank pus on opening of pancreatic duct . on august 2015 , patient underwent endoscopic retrograde cholangio - pancreatography ( ercp)-guided stone removal with common bile duct ( cbd ) stent placement at other center .
two months later , patient came back with fever and pain in epigastric region with multiple episodes of bilious vomiting . on examination , there was tenderness all over the abdomen .
patient was taken for exploratory laparotomy . on exploration of pancreas main pancreatic duct aspiration revealed frank pus ( fig .
the presence of a stone was noted at the tail of pancreas , which was crushed and extracted .
longitudinal pancreaticojejunostomy with roux - en - y jejunojejunostomy was done . on microbiological examination of pus
she was started on pancreatic lipase 25 000 iu twice daily for additional 3 months .
chronic pancreatitis is an inflammatory and fibrosing disease of the exocrine pancreas characterized by irreversible morphological changes and permanent loss of function . according to the marseille rome classification of 1988
, chronic pancreatitis is used to refer to recurrent or persistent abdominal pain that is associated with irreversible and ongoing inflammatory destruction of exocrine parenchyma and , eventually , islets . a new clinical entity termed as aospd
was described by weinman et al . , defining it as a rare complication of chronic pancreatitis with suppuration of the pancreatic duct not associated with pancreatic pseudocyst , abscess or necrosis .
aospd has been described in only seven previous case reports since 1995 . while the pathogenesis of aospd is not completely understood , chronic pancreatitis , prior sphincterotomy , pancreatic stasis secondary to pancreatic duct obstruction and diabetes mellitus
patients undergoing biliary sphincterotomy have a common or shared biliary and pancreatic sphincter ; duodenal contents might also reflux into the pancreatic duct , in effect seeding the pancreatic duct .
if pancreatic duct stones were obstructing the outflow of the duct , a ductal infection could result .
this would be analogous to calculus obstruction of the biliary tree resulting in acute bacterial cholangitis .
the normal pancreas is usually resistant to infection , presumably because of the presence of bacteriostatic and bacteriocidal agents elaborated in pancreatic secretions . a chronically diseased pancreas may be much more susceptible to infection because these same antibacterial agents may be significantly impaired or diminished .
a pancreatic duct contaminated by duodenal reflux and obstructed by intraductal stones , in the setting of chronic pancreatitis , would seem a sufficient setting for the development of acute suppuration of the pancreatic duct .
diabetes mellitus , while not present in our patient , predisposes patients to a variety of uncommon infections and may play an additive role in infection with klebsiella .
this bacterium is usually contracted by oral route and one can only speculate that during previous instrumentation of the ampulla of vater , duodenal contents contaminated with this unusual organism may have refluxed into the pancreatic duct resulting effectively in seeding of the pancreatic juice . however , the presence of bacteria in the pancreatic duct is not by itself sufficient to cause suppuration .
fujimori et al . reported a case of aospd in the setting of peripheral blood stem cell transplantation for acute myeloid leukemia and subsequent chronic leucopoenia .
tajima et al . described aospd in the setting of pancreatic cancer ; as it can predispose to infection from biliary or pancreatic obstruction .
in all the reported cases the diagnosis of aospd was made on identification of pus in the pancreatic duct on ercp . in our case ,
the diagnosis was made on exploring the main pancreatic duct before performing a pancreaticojejunostomy . in all previous cases , patients complaints resolved post - ercp stent . here , the definite treatment was a longitudinal pancreaticojejunostomy with roux - en - y jejunojejunostomy ( modified puestow procedure ) .
surgery results in opening of the pancreatic capsule , which alleviates interstitial pressure , whereas longitudinal anastomosis ensures full drainage of the whole pancreatic duct length . as aospd is a rare complication of chronic pancreatitis it should be kept in mind while coming across cases with dilated main pancreatic duct and chronic pancreatitis .
once diagnosed , aospd should be treated with prompt intravenous antibiotic treatment with pancreatic duct decompression , which can be achieved either through ercp stenting or surgical decompression specifically longitudinal pancreaticojejunostomy with roux - en - y anastomosis .
surgical management should be preferred as ercp stenting can further lead to biliary reflux into the pancreatic duct after ercp sphincterotomy , which lead to further infection of the pancreatic duct .
however , as only seven cases have been reported since 1995 , there appears to be some other factors contributing to its pathogenesis .
thus , further study into this rare entity shall help provide more information in the near future .
|
abstractacute obstructive suppurative pancreatic ductitis ( aospd ) is a rare complication of chronic pancreatitis that has been described in only seven previous case reports since 1995 .
we report a case of a 33-year - old female a known case of chronic pancreatitis with computed tomography suggestive of dilated main pancreatic duct with multiple calcifications . on exploration ,
pancreatic duct aspiration revealed frank pus .
pus was drained after opening the pancreatic duct and longitudinal pancreaticojejunostomy was done .
patient was relieved of her symptoms after surgery . in conclusion ,
aospd should be considered in long standing cases of chronic pancreatitis .
aospd appears to respond quickly after drainage procedure like longitudinal pancreaticojejunostomy and should be considered the treatment of choice .
|
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what is new?the applicability of clinical practice guidelines to primary care has been questioned for individual conditions such as hypertension and depression , and concerns have been raised about guidelines promoting overtreatment of low - risk populations.until now , evidence from a systematic appraisal of the relevance to primary care of published guidelines has been lacking.nearly two - third of the research cited in support of national institute for health and care excellence guideline recommendations for primary care was of uncertain relevance to primary care patients , with little or no acknowledgment of this uncertainty.guideline development groups should more clearly identify which recommendations are intended for primary care and uncertainties about the relevance of the supporting evidence to primary care patients , to avoid potential overtreatment and adverse effects .
the applicability of clinical practice guidelines to primary care has been questioned for individual conditions such as hypertension and depression , and concerns have been raised about guidelines promoting overtreatment of low - risk populations.until now , evidence from a systematic appraisal of the relevance to primary care of published guidelines has been lacking.nearly two - third of the research cited in support of national institute for health and care excellence guideline recommendations for primary care was of uncertain relevance to primary care patients , with little or no acknowledgment of this uncertainty.guideline development groups should more clearly identify which recommendations are intended for primary care and uncertainties about the relevance of the supporting evidence to primary care patients , to avoid potential overtreatment and adverse effects .
the applicability of clinical practice guidelines to primary care has been questioned for individual conditions such as hypertension and depression , and concerns have been raised about guidelines promoting overtreatment of low - risk populations . until now ,
evidence from a systematic appraisal of the relevance to primary care of published guidelines has been lacking .
nearly two - third of the research cited in support of national institute for health and care excellence guideline recommendations for primary care was of uncertain relevance to primary care patients , with little or no acknowledgment of this uncertainty .
guideline development groups should more clearly identify which recommendations are intended for primary care and uncertainties about the relevance of the supporting evidence to primary care patients , to avoid potential overtreatment and adverse effects .
clinical practice guidelines are an increasingly important driver of decisions about patient care . they have been defined as recommendations intended to optimize patient care that are informed by a systematic review of evidence and an assessment of the benefits and harms of alternative care options .
guidelines have traditionally been developed to simply provide guidance for clinical decision making , but they are becoming embedded in the structure of uk primary care through their translation into indicators of quality of care in a national pay for performance financial incentive scheme ( the quality and outcomes framework ) and through the development of quality standards to inform decisions on health care planning and commissioning .
this increasing use of guidelines to develop incentives and standards for primary care may lead to more patients at lower risk of adverse outcomes receiving treatment and exposure to potential adverse effects .
groups developing guidelines about the care of primary care patients will use the current best evidence from primary care or lower risk populations where it exists . if high - quality primary care evidence is not found , the best evidence available may be from a secondary care or higher risk population .
this entirely appropriate approach leads to problems when a guideline development group ( gdg ) assumes that the evidence from research conducted on a higher risk population can automatically be applied to a lower risk primary care population .
if uncertainty about the evidence is not explicitly acknowledged , the integrity of the guideline is compromised and patient harm may result .
the benefits of treatment are usually lower in populations at lower risk of adverse outcomes , whereas the risk of harm from adverse treatment effects remains constant .
patients seen in primary care typically have less severe illness than those in hospital , and so evidence from trials conducted in secondary care may have limited relevance and result in harms outweighing benefits .
an example of taking evidence from a higher risk population and applying it to a lower risk population is the quality and outcomes framework indicator and national institute for health and care excellence ( nice ) heart failure guideline recommendation that all primary care patients with chronic heart failure ( including low grade ) should be offered -blockers and ace inhibitors .
this indicator is supported by evidence generalized from higher risk populations ( new york heart association grades iii
iv ) , in which there is clear evidence of benefit , to lower risk populations , in which the evidence of benefit is more equivocal .
the potential harm is the adverse effects of -blockers experienced by some patients , and the substantial risk of acute kidney injury from ace inhibitors , which may account for a tenth of the increase in hospital admissions because of an acute kidney injury .
it is therefore uncertain what the balance of harms and benefits might be in a typical primary care patient , and a general practitioner needs to know about this uncertainty when , for example , considering prescribing a -blocker to a patient with a relative contraindication to a -blocker therapy from a comorbid condition .
this vital information about uncertainty and the balance of benefits and harms is hard to find in the quality and outcomes framework guidance or nice guideline , which presents a single approach rather than acknowledging that there are several acceptable alternatives for low - risk patients .
another example where it is hard for the user of a clinical guideline to know about the balance of benefits and harms for a typical primary care patient is the quality and outcomes framework incentive to prescribe aspirin or an alternative antiplatelet to all patients with peripheral arterial disease , most of whom do not have symptoms and are managed in primary care .
the evidence that antiplatelet therapy can reduce serious vascular events comes primarily from a large subgroup analysis of the antithrombotic trialists ' collaboration meta - analysis in high - risk patients and a similar review conducted by nice .
however , the authors caution that their results may not be applicable to low - risk patients , and others have calculated that the number of potential reductions in coronary heart disease events exceeded the number of potential precipitated adverse bleeding events only for patients with a 1% or greater annual risk of coronary heart disease events .
a third example is chronic kidney disease ( ckd ) , where there is evidence of benefit to high - risk populations but no evidence of benefit in people with early - stage ckd at a low risk of future disease . both primary care physicians and
specialists have expressed concerns about potential harms from overtreatment resulting from expanding definitions of ckd in guidelines .
a small pilot study suggested that the evidence base for primary care guidelines might not be relevant to most primary care patients , with important implications for patient safety , and we wanted to systematically examine the evidence base for clinical guidelines used in primary care .
we used guidelines from the nice as it has been a leading provider of evidence - based clinical guidelines in the united kingdom since 2002 .
institute of medicine 's standards for trustworthy guidelines [ 1,1618 ] and with the international consensus that guidelines should be developed using an explicit and transparent process that minimizes distortions , biases , and conflicts of interest ; should base recommendations on a systematic review of the existing evidence ; should include experts and patient representatives on a multidisciplinary gdg ; and should consider important patient subgroups and patient preferences .
the development of nice clinical guidelines follows a well - established process . when a topic has been chosen , a national collaborating centre ( ncc ) is commissioned to develop the guideline .
the ncc prepares the scope which sets out what the guideline will and will not cover and recruits the gdg .
review questions are then developed to guide the evidence review and synthesis by the technical team , which the gdg uses to formulate recommendations .
we aimed to measure the percentage of primary care relevant publications in the cited evidence base for nice primary care recommendations , for all nice guideline recommendations for primary care published over 2 years .
we reviewed the scope of all 45 clinical guidelines published by nice in 2010 and 2011 and excluded any guideline explicitly aimed at non
five academic family physicians on the research team classified recommendations in the remaining guidelines as specific , relevant , or not relevant to primary care , using our previously piloted methods .
two reviewers independently rated each recommendation and then discussed any discrepancies by telephone and/or e - mail .
if they could not reach consensus on a recommendation , it was sent to a third reviewer and then classified according to the majority view .
reviewers used the following definitions , along with examples : primary care specific recommendations inform decisions that are almost always made by primary care providers such as general practitioners and primary care relevant recommendations inform decisions that could be made by health professionals in either primary care or another setting .
we included guidelines if more than 50% of the recommendations were judged relevant to primary care or if at least one recommendation was specific to primary care .
we reviewed all publications cited as evidence for included recommendations and classified each publication according to whether the study population had been selected from primary care or community settings ( ie , not from hospital outpatient or inpatient populations ) .
we classified publications as unclear if we could not obtain this information from these or related publications .
we considered systematic reviews as a single publication and included them if at least one study in the review had been conducted on a primary care or community population .
publications were excluded from the study and not considered further if they referred to other guidelines , nonsystematic reviews , or references that only appeared in the guideline appendices with no apparent link to the recommendations .
the primary outcome was the percentage of included cited publications that were relevant to primary care .
secondary outcomes were guideline development center , primary care membership of gdg , recommendations for further primary care research , whether grading of recommendations assessment , development and evaluation ( grade ) was used to assess the quality of evidence , and the clarity of links from the recommendation back to the supporting evidence base .
twenty - two of the 45 guidelines were assessed as relevant to primary care ( fig . 1 ) .
these 22 guidelines contained 1,185 recommendations , and the reviewers considered 777 of them to be relevant to primary care .
the two independent reviewers initially rated 981 ( 82.8% ) recommendations in the same category of specific , relevant , or not relevant to primary care , with discrepancies predominantly between the categories of primary care
specific or relevant . after discussion between the two reviewers , full agreement was reached on 1,166 ( 98.4% ) of recommendations .
of the 777 recommendations , 282 were excluded as they were based on gdg consensus in the absence of evidence or were standard clinical practice ( such as a recommendation to take a history from the patient ) , leaving 495 ( 42% , range by guideline 9100% ) recommendations that were both evidence based and relevant to primary care ( table 1 ) .
the links in the guideline from recommendation back to supporting research sometimes appeared to have become blurred in the process of developing the guideline .
the research was considered relevant to primary care in 590 ( 38% , range by guideline 674% ) of these publications ( table 1 ) .
the relevance to primary care was unclear in 67 publications , in which the research population was not clearly described in the original articles .
there was a wide variation across guidelines in the percentage of both recommendations and supporting publications that were relevant to primary care . in some guidelines ( eg , pregnancy and complex social factors ) both the recommendations ( 100% ) and the evidence base ( 74% ) were judged to be relevant to primary care .
other guidelines ( eg , chronic heart failure and constipation in children and young people ) were relevant to primary care ( 68% and 72% , respectively ) and yet were based on relatively little evidence from primary care ( 10% and 17% ) .
guidelines in mental health and women 's and children 's health had the highest percentages of supporting publications that were relevant to primary care ( 51% and 52% ) . the number of primary care providers on gdgs ranged from 0 to 4 ( 12% of all gdg membership ; table 2 ) .
the percentage of primary care providers on gdgs was not associated with the percentage of primary care relevant studies ( correlation coefficient , 0.15 ) .
further research in primary care was called for in 6 of the 22 full guidelines .
nine of the 22 included guidelines used elements of grade to assess the quality of included evidence [ 2123 ] .
nearly two - third of the research cited in support of nice guideline recommendations for primary care was of uncertain relevance to primary care patients , with little or no acknowledgment of this uncertainty in the published guideline .
only 38% of cited publications were based on patients typical of primary care . in many guidelines ,
the link from evidence to recommendation was not explicit . where evidence was available , many studies did not report the setting or population used for the research .
the low percentage ( 38% ) of studies relevant to primary care is likely to be due to the lack of suitable primary care research .
this correctly requires gdgs to extrapolate from research that has been conducted on other , often higher risk , populations .
the difficulty for the guideline user is not so much that the evidence is inevitably incomplete , but rather that it is not clear which recommendations are supported by primary care based relevant evidence and which by evidence from other clinical settings or by consensus of the gdg .
judgments about the relevance of the evidence to different populations made using grade 's indirectness
domain were useful but presented as a minor part of grade which did not come through clearly in final guidelines .
family physicians are experienced in managing clinical uncertainty and making decisions with incomplete information , and when deciding about treatment for a patient , they need to know the extent to which guideline evidence can be applied to that individual . information about uncertainty in the research , and potential applicability to a patient in primary care , is not currently easily accessible in guidelines in the way it needs to be to improve clinical decision making in primary care and avoid accusations of guideline bias .
other authors have noted the difficulties in applying guidelines where there are important differences between trial participants and typical primary care patients in hypertension .
only one group has looked specifically at whether guidelines are supported by evidence from primary care : a group in the netherlands looked at 13 guidelines for depression and screened 804 publications , to find only two studies based in primary care .
our research is the first systematic appraisal of national guidelines , and our results show that uncertain applicability of cited research to patients in primary care is a problem shared to a greater or lesser degree by all the guidelines reviewed .
strengths of the research include that it was based on a large sample of all recent nice guidelines and used piloted methods .
the estimate of 38% of evidence being based on patients typical of primary care is likely to be an overestimate rather than an underestimate , as we only included guidelines with most recommendations relevant to primary care ( in which the proportion of primary care evidence is likely to be greater than in other guidelines ) .
the biggest challenge for the research team was to classify the guidelines , recommendations , and research studies in turn as either relevant to primary care or not .
we used transparent and piloted methods , but the choice between relevant and not relevant was not always straightforward , and other groups might make different decisions in the gray areas .
we responded to this uncertainty using a generous definition for classifying study populations as relevant to primary care , which again would tend to make the 38% an overestimate rather than an underestimate .
future research is needed into the dynamics and approaches to reaching informal consensus used by gdgs and the influence of variable factors such as professional background and conflicts of interest .
internationally , primary and community care is usually of lower technology than hospital care , and so contextualizing research within the primary care setting is relevant to both developed and developing countries
. the extent to which research in high - technology settings can be considered relevant for guideline users in low - technology settings in low- and middle - income countries is under - researched .
frameworks such as the appraisal of guidelines for research & evaluation instrument ( agree ii ) to assess the quality of practice guidelines and the grade tool , which includes an indirectness
domain to assess the applicability to different populations , have been developed to improve the integrity of clinical guidelines .
nice and other national guideline developers contribute to and follow this good practice , have primary care membership of gdgs , try to ensure that secondary care content experts do not dominate discussions regarding recommendations for primary care , and involve the public in the process , and these frameworks and methods should continue to be developed and applied .
however , despite all the good practice , it is still hard for a primary care clinician to find the information they need to guide a decision about the potential benefits and harms of treatment .
guideline developers intend to present their recommendations with enough information for the user to track back to the strength of the underlying evidence , should they need to do so , but we found that tracking back to the evidence was usually very difficult .
the relevance of published clinical practice guidelines to primary care patients is still too opaque .
an enhanced approach is needed to avoid the assumption of generalizability of research evidence from high - risk research populations to low - risk primary care patients and reduce potential overtreatment and avoidable harm .
more explicit consideration of the primary care context is needed at all stages of development , to reduce dependence on the influencing skills of a primary care provider on the gdg .
the relevance to primary care should be carefully considered at initial scoping . for relevant guidelines , the initial review questions for the evidence search
should then be considered specifically for relevance to primary care , perhaps as a subgroup in the search , and negative findings reported if evidence is not found .
primary care relevance could be considered in terms of setting , severity of illness , or risk group depending on the guideline 's intended audience .
the guideline should be specific about where primary care research has or has not been used , including limitations or lack of evidence , and research recommendations where relevant primary care evidence is lacking should be clearly badged .
uncertainties in the evidence should always be clearly presented and not lost in the understandable desire to produce straightforward recommendations . a shift in focus during guideline development to outcomes ( such as health gain ) as opposed to simple processes ( such as pharmaceutical prescription ) has been proposed and should be encouraged .
an enhanced primary care perspective could come from either greater involvement from the outset of primary care professionals with relevant content expertise where feasible or from more explicit guidance to development groups combined with systematic checks at all stages of guideline development .
checks for compliance with development manuals and frameworks such as grade and agree ii would help ensure , for example , that grade profiles properly address issues of indirectness . at least one nice committee has already started to consider research data that have been reanalyzed to show the results separately for low- and high - risk populations , with a consequent change in the decision made .
success will be when a clinical guideline user can quickly and accurately determine the broad relevance of each recommendation to their patient , including uncertainties , and can easily track back from the recommendation to the underlying evidence base , should they wish to do so .
|
objectivesprimary care patients typically have less severe illness than those in hospital and may be overtreated if clinical guideline evidence is inappropriately generalized .
we aimed to assess whether guideline recommendations for primary care were based on relevant research.study design and settingliterature review of all publications cited in support of national institute for health and care excellence ( nice ) recommendations for primary care .
the relevance to primary care of all 45 nice clinical guidelines published in 2010 and 2011 , and their recommendations , was assessed by an expert panel.resultstwenty-two of 45 nice clinical guidelines published in 2010 and 2011 were relevant to primary care .
these 22 guidelines contained 1,185 recommendations , of which 495 were relevant to primary care , and cited evidence from 1,573 research publications .
of these cited publications , 590 ( 38% , range by guideline 674% ) were based on patients typical of primary care.conclusionnearly two - third ( 62% ) of publications cited to support primary care recommendations were of uncertain relevance to patients in primary care .
guideline development groups should more clearly identify which recommendations are intended for primary care and uncertainties about the relevance of the supporting evidence to primary care patients , to avoid potential overtreatment .
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as a country develops , the types of diseases that affect its population shift from primarily infectious , such as diarrhea and pneumonia , to primarily noncommunicable diseases , such as cardiovascular disease ( cvd ) , obesity , and cancer.1 the global burden of cvd has increased , mostly in wealthy countries .
however , many low and middleincome countries now face a growing burden from the modern health risks ( physical inactivity , tobacco ) , while still fighting an unfinished battle with traditional health risks ( undernutrition , sanitation).1 , 2 , 3 cvds are now the leading cause of mortality in the world , causing 17.5 million deaths in 2012 , which represented 31% of total global mortality.4 in france , cvds are the second major cause of mortality the first among women and represent nearly one third ( 28% ) of total mortality.5
optimistically , simple and costeffective measures such as dietary changes could reduce the obesity epidemic and the resultant cvd burden , as it has been estimated that lifestyle choices can account for up to 40% of premature cvd deaths.5 in the past few decades , associations between individual nutritional factors and chronic diseases , especially cvd , have been widely investigated .
more recently , alternative approaches that consider the whole diet ( for example , dietary indices reflecting diet quality ) have been used , making it feasible to capture nutrient interactions in the food matrix.6 , 7 in particular , the adherence to a traditional mediterranean dietary pattern , including a high consumption of plant foods and olive oil , low intakes of saturated fat and sugar , and a low / moderate consumption of wine , has been associated with a lower risk of cvd and mortality in many epidemiological studies,8 , 9 as well as with lower levels of inflammation.10 , 11 , 12 indeed , the association between inflammatory mechanisms and the development of cvd has been largely brought to light.13 , 14 , 15 inflammatory pathways promote thrombosis , a late complication of atherosclerosis responsible for myocardial infarctions ( mi ) and most strokes.14
to assess the adherence to a dietary pattern , several dietary scores or indices have been developed , such as the dietary inflammatory index ( dii ) .
this index was developed in 200916 and then updated in 2014.17 the dii aims to measure the inflammatory potential of the diet based on the pro and antiinflammatory properties of its various components , including macronutrients , vitamins , minerals , flavonoids , specific food items , and energy intake , according to the existing literature based on tissue culture , and animal and human studies . to our knowledge
, only 3 published studies have investigated the association between the dii and the risk of cardiovascular disease .
all 3 studies reported a protective role of a more antiinflammatory diet.18 , 19 , 20 these recent studies have begun to fill an important gap in understanding the relationship between systemic inflammation a phenomenon with multiple underlying causes and cardiovascular disease , by focusing specifically on diet , which appears to be a key modifiable factor involved in inflammatory pathways.10 , 11 , 12 additionally , to the best of our knowledge , no study has yet investigated the association of the dii with the incidence of specific cvd subtypes . in order to contribute to filling this research gap , we assessed the prospective association between the inflammatory potential of the diet , as measured by the dii , and the incidence of overall cvd and subtypes of cvd namely , mi ; strokes ; angina pectoris and revascularization interventions in a large cohort of french adults with a longterm followup .
subjects were selected from the supplmentation en vitamines et minraux antioxydants ( su.vi.max ) study .
the su.vi.max study was a randomized , doubleblind , placebocontrolled primary prevention trial conducted between 1994 and 2002 .
it included 12 741 french adults ( women aged 3560 years and men aged 4560 years ) .
its primary aim was to evaluate the potential efficacy of daily supplementation with antioxidant vitamins and minerals ( ascorbic acid , vitamin e , carotene , selenium , and zinc ) delivered at nutritional ( ie , nonpharmacologic ) doses on the prevention of cancer , ischemic heart disease , and overall mortality.21 the trial phase ended in 2002 and health events monitoring was pursued until september 2007 .
the su.vi.max study was registered at clinicaltrials.gov ( number nct00272428 ) and was conducted according to the guidelines laid down in the helsinki declaration of 1975 as revised in 1983 , and was approved by the ethics committee for studies with human participants of pariscochin hospital ( ccpprb no .
all subjects provided written informed consent . in this analysis , we selected men and women who provided at least 3 valid 24hour dietary records during the first 2 years of followup ( n=8108 ) and who had no missing data for covariables ( 7743 ) to be included in the models ( figure ) . selection of participants for our analyses , su.vi.max study , france
. cvd indicates cardiovascular diseases ; dii , dietary inflammatory index ; su.vi.max , supplmentation en vitamines et minraux antioxydants . throughout the entire su.vi.max study trial phase ( 19942002 ) , participants were asked to provide a 24hour dietary record every 2 months , for a total of 6 records per year covering all days of the week and all seasons of the year . for these analyses ,
mean dietary intakes were calculated across all available , valid 24hour dietary records obtained during the first 2 years of followup , in order to obtain a proxy for habitual dietary intakes .
dietary records were considered invalid if energy intake was < 100 or > 6000 kcal / day .
additionally , men reporting < 800 kcal / day and women reporting < 500 kcal / day across one third or more of records were excluded to account for energy underreporting .
an instruction manual including validated photographs of generic foods and portion sizes was provided to facilitate coding food portions.22 a food composition table was used to estimate nutrient intake.23 we used the dii to determine the inflammatory potential of the diet .
this index relies on information contained in 1943 published studies reporting association between nutritional factors and inflammatory markers.17 briefly , points were assigned to every food parameter on the basis of relevant articles found in the literature , according to whether each food parameter increased ( + 1 ) , decreased ( 1 ) , or had no ( 0 ) effect on 6 inflammatory biomarkers : interleukin1b , interleukin4 , interleukin6 , interleukin10 , tumor necrosis factor , and creactive protein .
the dii computed from the 24hour records in this study includes data on 36 of the 45 food parameters comprising the dii : carbohydrate , protein , total fat , energy , alcohol , fiber , cholesterol , saturated fatty acids , monounsaturated fatty acids , polyunsaturated fatty acids , omega 3 , omega 6 , niacin , thiamin , riboflavin , vitamin b6 , vitamin b12 , iron , magnesium , vitamin a , vitamin c , vitamin d , vitamin e , folic acid , carotene , anthocyanidins , flavan3ol , flavonols , flavonones , flavones , isoflavones , garlic , ginger , pepper , onion , and tea .
dietary intakes for these parameters were first standardized using a world mean and sd , derived from nutritional databases from 11 populations around the world , then transformed to percentiles and multiplied by literaturederived inflammatory points for each food parameter , then summed up .
dii scores were computed on the basis of participantspecific mean nutrient and food intakes , which were obtained by averaging intakes across all available dietary records , as explained in the first paragraph of the dietary data section ( ie , dii scores were not first calculated per day and then averaged ) .
more details on the score computation can be found elsewhere.17
information about health was selfreported via a monthly questionnaire during the followup .
other sources of information regarding outcome included yearly clinical examinations . in case of no contact with a participant for a long period , or nonattendance at the yearly visit ,
an investigation was launched to determine the reasons . in case of a suspected event ,
investigations were conducted to obtain relevant medical data ( clinical , biochemical , histological , and radiological reports ) from participants , physicians , and/or hospitals .
all cardiovascular events were reviewed and validated by an independent expert committee , unaware of the supplementation group assignment , and then classified using the international classification of diseases , 10th revision , clinical modification ( codes i20i24 , i64).24 first , we considered as outcome the occurrence of all cvds .
sociodemographic data , including sex , date of birth , education ( primary education only , up to secondary education , higher education ) , as well as information on marital status ( couple or single ) , smoking status ( neversmoked , former or current smoker ) , and physical activity level were obtained by a ( nonvalidated ) baseline questionnaire .
the items concerning physical activity inquired whether a participant had a regular physical activity , and if yes , whether they estimated this activity to be equivalent to 1 hour of walking per day .
body mass index was calculated using baseline weight and height measurements performed by trained personnel according to standardized procedures , using an electronic scale ( seca , hamburg , germany ) and a wallmounted stadiometer .
subjects contributed persontime up to the date of the cardiovascular event ( n=292 ) , death ( n=193 ) , the last completed questionnaire or last contact ( n=1704 ) or september 1 , 2007 ( n=5554 ) , whichever occurred first . for analyses concerning subclasses of cvd , participants who reported another cvd event than the one studied during followup were included as noncases for the studied cvd and censored at the date of that diagnosis .
nutritional intakes were energyadjusted using the residual method,25 and thus presented as adjusted ( least squares ) means with 95% ci .
values for all other quantitative variables were presented as means ( sd ) , and values for qualitative variables as percentages .
we estimated hazard ratio ( hr ) of cvd risk and 95% cis both for the continuous dii score and for quartiles of dii scores , using cox proportional hazard models.26 graphic methods ( log log [ survival ] versus log time plots ) were used to check for proportional hazards assumptions for this study . the lowest quartile of dii score ( q1 , reflecting the most antiinflammatory diet ) was used as the reference category .
the first model was crude ( model 0 ) , with only age as the timescale in the cox model .
a second model was adjusted for age ( timescale in the cox model ) , sex , and daily energy intake without alcohol ( continuous , in kcal / day ) ( model 1 ) .
a multivariate model was further adjusted for number of available 24hour dietary records ( continuous ) , smoking status , physical activity , education level , marital status , and treatment allocation group ( placebo or active ) ( model 2our main model ) .
two additional models ( 3a and 3b ) were created in order to verify whether our results would remain stable even when adjusting for baseline body mass index ( continuous , in kg / m ) and for alcohol intake ( continuous , in g / day ) , respectively .
the reason why model 3b was , in addition to body mass index , further adjusted for alcohol intake is that alcohol consumption is considered in a linear manner for the computation of the dii score while only moderate alcohol consumption is associated with a reduced cvd risk.27 the same models were used for both overall cvds and analyses focused on cvd subtypes . in order to verify the robustness of our findings
first , we reanalyzed our data after removing participants who developed a cardiovascular event during the first 2 years of followup .
second , we selected only participants with at least six 24hour records during the first 2 years of followup in order to improve the accuracy of nutritional estimates .
finally , we repeated our analyses with a dii score in which dietary intakes for all parameters were first adjusted for overall energy intake , using the energy density method ( [ crude intakes / total energy intake]1000 ) , named dii 2 .
all tests were 2sided and p<0.05 was considered statistically significant , except for interaction tests for which statistical power is often weak in observational studies.28 thus , p<0.10 was applied for these models .
interactions between the dii score ( modeled both as a continuous variable and as quartiles ) and sex and intervention group were investigated .
all analyses were performed using sas software ( version 9.3 ; sas institute inc , cary , nc ) .
subjects were selected from the supplmentation en vitamines et minraux antioxydants ( su.vi.max ) study .
the su.vi.max study was a randomized , doubleblind , placebocontrolled primary prevention trial conducted between 1994 and 2002 .
it included 12 741 french adults ( women aged 3560 years and men aged 4560 years ) .
its primary aim was to evaluate the potential efficacy of daily supplementation with antioxidant vitamins and minerals ( ascorbic acid , vitamin e , carotene , selenium , and zinc ) delivered at nutritional ( ie , nonpharmacologic ) doses on the prevention of cancer , ischemic heart disease , and overall mortality.21 the trial phase ended in 2002 and health events monitoring was pursued until september 2007 .
the su.vi.max study was registered at clinicaltrials.gov ( number nct00272428 ) and was conducted according to the guidelines laid down in the helsinki declaration of 1975 as revised in 1983 , and was approved by the ethics committee for studies with human participants of pariscochin hospital ( ccpprb no .
in this analysis , we selected men and women who provided at least 3 valid 24hour dietary records during the first 2 years of followup ( n=8108 ) and who had no missing data for covariables ( 7743 ) to be included in the models ( figure ) .
cvd indicates cardiovascular diseases ; dii , dietary inflammatory index ; su.vi.max , supplmentation en vitamines et minraux antioxydants .
throughout the entire su.vi.max study trial phase ( 19942002 ) , participants were asked to provide a 24hour dietary record every 2 months , for a total of 6 records per year covering all days of the week and all seasons of the year . for these analyses ,
mean dietary intakes were calculated across all available , valid 24hour dietary records obtained during the first 2 years of followup , in order to obtain a proxy for habitual dietary intakes .
dietary records were considered invalid if energy intake was < 100 or > 6000 kcal / day .
additionally , men reporting < 800 kcal / day and women reporting < 500 kcal / day across one third or more of records were excluded to account for energy underreporting .
an instruction manual including validated photographs of generic foods and portion sizes was provided to facilitate coding food portions.22 a food composition table was used to estimate nutrient intake.23 we used the dii to determine the inflammatory potential of the diet .
this index relies on information contained in 1943 published studies reporting association between nutritional factors and inflammatory markers.17 briefly , points were assigned to every food parameter on the basis of relevant articles found in the literature , according to whether each food parameter increased ( + 1 ) , decreased ( 1 ) , or had no ( 0 ) effect on 6 inflammatory biomarkers : interleukin1b , interleukin4 , interleukin6 , interleukin10 , tumor necrosis factor , and creactive protein .
the dii computed from the 24hour records in this study includes data on 36 of the 45 food parameters comprising the dii : carbohydrate , protein , total fat , energy , alcohol , fiber , cholesterol , saturated fatty acids , monounsaturated fatty acids , polyunsaturated fatty acids , omega 3 , omega 6 , niacin , thiamin , riboflavin , vitamin b6 , vitamin b12 , iron , magnesium , vitamin a , vitamin c , vitamin d , vitamin e , folic acid , carotene , anthocyanidins , flavan3ol , flavonols , flavonones , flavones , isoflavones , garlic , ginger , pepper , onion , and tea .
mean and sd , derived from nutritional databases from 11 populations around the world , then transformed to percentiles and multiplied by literaturederived inflammatory points for each food parameter , then summed up .
dii scores were computed on the basis of participantspecific mean nutrient and food intakes , which were obtained by averaging intakes across all available dietary records , as explained in the first paragraph of the dietary data section ( ie , dii scores were not first calculated per day and then averaged ) .
other sources of information regarding outcome included yearly clinical examinations . in case of no contact with a participant for a long period , or nonattendance at the yearly visit ,
an investigation was launched to determine the reasons . in case of a suspected event ,
investigations were conducted to obtain relevant medical data ( clinical , biochemical , histological , and radiological reports ) from participants , physicians , and/or hospitals .
all cardiovascular events were reviewed and validated by an independent expert committee , unaware of the supplementation group assignment , and then classified using the international classification of diseases , 10th revision , clinical modification ( codes i20i24 , i64).24 first , we considered as outcome the occurrence of all cvds .
sociodemographic data , including sex , date of birth , education ( primary education only , up to secondary education , higher education ) , as well as information on marital status ( couple or single ) , smoking status ( neversmoked , former or current smoker ) , and physical activity level were obtained by a ( nonvalidated ) baseline questionnaire .
the items concerning physical activity inquired whether a participant had a regular physical activity , and if yes , whether they estimated this activity to be equivalent to 1 hour of walking per day .
body mass index was calculated using baseline weight and height measurements performed by trained personnel according to standardized procedures , using an electronic scale ( seca , hamburg , germany ) and a wallmounted stadiometer .
subjects contributed persontime up to the date of the cardiovascular event ( n=292 ) , death ( n=193 ) , the last completed questionnaire or last contact ( n=1704 ) or september 1 , 2007 ( n=5554 ) , whichever occurred first . for analyses concerning subclasses of cvd
, participants who reported another cvd event than the one studied during followup were included as noncases for the studied cvd and censored at the date of that diagnosis .
nutritional intakes were energyadjusted using the residual method,25 and thus presented as adjusted ( least squares ) means with 95% ci .
values for all other quantitative variables were presented as means ( sd ) , and values for qualitative variables as percentages .
we estimated hazard ratio ( hr ) of cvd risk and 95% cis both for the continuous dii score and for quartiles of dii scores , using cox proportional hazard models.26 graphic methods ( log log [ survival ] versus log time plots ) were used to check for proportional hazards assumptions for this study .
the lowest quartile of dii score ( q1 , reflecting the most antiinflammatory diet ) was used as the reference category .
the first model was crude ( model 0 ) , with only age as the timescale in the cox model .
a second model was adjusted for age ( timescale in the cox model ) , sex , and daily energy intake without alcohol ( continuous , in kcal / day ) ( model 1 ) .
a multivariate model was further adjusted for number of available 24hour dietary records ( continuous ) , smoking status , physical activity , education level , marital status , and treatment allocation group ( placebo or active ) ( model 2our main model ) .
two additional models ( 3a and 3b ) were created in order to verify whether our results would remain stable even when adjusting for baseline body mass index ( continuous , in kg / m ) and for alcohol intake ( continuous , in g / day ) , respectively .
the reason why model 3b was , in addition to body mass index , further adjusted for alcohol intake is that alcohol consumption is considered in a linear manner for the computation of the dii score while only moderate alcohol consumption is associated with a reduced cvd risk.27 the same models were used for both overall cvds and analyses focused on cvd subtypes . in order to verify the robustness of our findings
first , we reanalyzed our data after removing participants who developed a cardiovascular event during the first 2 years of followup .
second , we selected only participants with at least six 24hour records during the first 2 years of followup in order to improve the accuracy of nutritional estimates . finally , we repeated our analyses with a dii score in which dietary intakes for all parameters were first adjusted for overall energy intake , using the energy density method ( [ crude intakes / total energy intake]1000 ) , named dii 2 .
all tests were 2sided and p<0.05 was considered statistically significant , except for interaction tests for which statistical power is often weak in observational studies.28 thus , p<0.10 was applied for these models .
interactions between the dii score ( modeled both as a continuous variable and as quartiles ) and sex and intervention group were investigated .
all analyses were performed using sas software ( version 9.3 ; sas institute inc , cary , nc ) .
these analyses included a total of 7743 participants ( 3197 men and 4546 women ) .
mean age at baseline ( sd ) was 51.94.7 and 47.16.6 years for men and women , respectively .
baseline characteristics of the study population are presented by sexspecific quartiles of the dii in table 1 . given the relatively large size of our sample , the following comparisons of characteristics across quartiles should be interpreted with caution , as not every statistically significant result is biologically and clinically meaningful .
compared to those with a lower dii score ( q1 ) , reflecting an antiinflammatory diet , participants with higher dii scores ( q4 ) , ( ie , more proinflammatory ) were slightly younger , less educated , less physically active , smokers , and less often postmenopausal women .
a higher dii score also was associated with slightly higher glycemia , slightly higher total blood cholesterol concentration , and slightly higher blood pressure , both systolic and diastolic .
in addition , a higher dii score was associated with lower total daily energy intake , as well as a lower fruit and vegetable consumption , a lower intake of mono and polyunsaturated fatty acids including n3 and n6vitamins ( c , e , b6 , b9 , and b12 ) , carotene , calcium , magnesium , sodium , iron , iodine , potassium , phosphorus , and fibers but a higher intake of saturated fatty acids and a higher alcohol consumption ( table 2 ) .
baseline characteristics across sexspecific quartiles of the dii score , su.vi.max study , france ( n=7743 ) values for quantitative variables are means ( sd ) , except for the dii where mean ( interquartile range ) is presented .
dii indicates dietary inflammatory index ; hdl , highdensity lipoprotein ; htm , hormonal treatment for menopause ; ldl , lowdensity lipoprotein ; su.vi.max , supplmentation en vitamines et minraux antioxydants .
oral contraceptive use : 3621 ; postmenopausal women : 4197 ; htm medication : 1092 ; glycemia : 7514 ; cholesterol : 7669 ; hdl and ldl cholesterol : 7618 ; systolic and diastolic blood pressure : 6422 .
nutritional data across sexspecific quartiles of the dii score , su.vi.max study , france ( n=7743 ) values are means ( sds ) or least squares means ( 95% ci ) , as specified .
dii indicates dietary inflammatory index ; mufa , monounsaturated fatty acids ; pufa , polyunsaturated fatty acids ; sfa , saturated fatty acids ; su.vi.max , supplmentation en vitamines et minraux antioxydants .
carbohydrate , fat , and protein are presented as percentage of total daily energy intake .
data were energyadjusted using the residual method , and thus presented as least squares means ( 95% cis ) . during the 13year followup ( mean duration was 11.4 years , corresponding to 87 932 personyears of observation ) ,
the distribution was as follows : 93 mi , 58 strokes , 128 angina pectoris and revascularization interventions , and 13 sudden deaths . in the crude model 0 ,
we observed a positive association between the dii score , reflecting a proinflammatory diet , and risk of cvd ( hrq4 versus q1=1.73 , 95% ci=1.252.39 ) ( table 3 ) .
however , this association was no longer observed after adjustment for confounding factors in any of the models ( 1 , 2 , 3a , or 3b ) .
interactions tested between the dii score and sex or intervention group were not significant , whether the dii was modeled as a continuous variable or as quartiles .
association between the dii and incidence of overall cardiovascular disease , su.vi.max study , france ( n=7743 ) the values listed next to
model 0model 3b are hazard ratio ( 95% cis ) , estimated through cox proportional hazard models , corresponding to sexspecific quartiles of the dii .
model 2 : model 1+supplementation group , number of 24h records , education level , marital status , smoking status , and physical activity .
dii indicates dietary inflammatory index ; iqr , interquartile range ; q , sexspecific quartile ; su.vi.max , supplmentation en vitamines et minraux antioxydants .
p for trend ( obtained by modeling dii quartiles as an ordinal variable ) . when separating cvds into subclasses ( table 4 ) , being in the highest dii score quartile was associated with a higher incidence of mi , as compared to being in the lowest quartile ( hrquartile 4 versus quartile 1=2.24 , 95% ci : 1.084.67 ) in the fully adjusted model ( model 2 ) , although the hr for a onepointincrease in dii was not statistically significant ( hr continuous dii=1.11 , 95% ci=0.971.27 ) .
no significant relationship was found regarding incidence of cerebrovascular accident or angina pectoris and revascularization intervention .
association between the dii and incidence of subclasses of cardiovascular disease , su.vi.max study , france ( n=7743 ) values are hazard ratio ( 95% cis ) , estimated through cox proportional hazard models , corresponding to sexspecific quartiles of the dii .
persontime per quartile ( q ) in days : q1 , 22 432 ; q2 , 22 117 ; q3 , 21 912 ; q4 , 21 471 .
model 2 : model 1+supplementation group , number of 24h records , education level , marital status , smoking status , and physical activity .
ap indicates angina pectoris ; dii , dietary inflammatory index ; mi , myocardial infarction ; q , sexspecific quartile ; ri , revascularization intervention ; su.vi.max , supplmentation en vitamines et minraux antioxydants .
three sets of supplemental analyses were conducted in order to investigate the stability of our results concerning mi .
first , when removing participants with cardiovascular events declared during the first 2 years of followup ( n=7602 ) , findings were very similar ( table 5 ) .
next , when selecting participants with at least six 24hour records ( n=6362 ) , findings were strengthened ( hrq4 versus q1 for mi risk=3.23 , 95% ci=1.377.62 ) , and the hr for a onepointincrease in dii was significantly different from 1.00 ( hr continuous=1.18 , 95% ci=1.011.38 ) ( table 6 ) .
finally , in analyses using a dii score based on energyadjusted nutritional parameters , the results were broadly consistent with those of our main analysis , although the observed association was weaker ( table 7 ) .
association between the dii and incidence of myocardial infarction among participants without early cardiovascular events , su.vi.max study , france ( n=7602 ) values are hazard ratio ( 95% cis ) , estimated through cox proportional hazard models , corresponding to sexspecific quartiles of the dii .
model 2 : model 1+supplementation group , number of 24h records , education level , marital status , smoking status , and physical activity .
dii indicates dietary inflammatory index ; iqr , interquartile range ; q , sexspecific quartile ; su.vi.max , supplmentation en vitamines et minraux antioxydants .
association between the dii and incidence of myocardial infarction among participants with at least six 24h dietary records , su.vi.max study , france ( n=6362 ) values are hazard ratio ( 95% cis ) , estimated through cox proportional hazard models , corresponding to sexspecific quartiles of the dii .
model 2 : model 1+supplementation group , number of 24h records , education level , marital status , smoking status , and physical activity .
dii indicates dietary inflammatory index ; iqr , interquartile range ; q , sexspecific quartile ; su.vi.max , supplmentation en vitamines et minraux antioxydants .
association between a dii calculated on the basis of energyadjusted nutritional parameters ( dii 2 ) and incidence of myocardial infarction , su.vi.max study , france ( n=7743 ) values are hazard ratio ( 95% cis ) , estimated through cox proportional hazard models , corresponding to sexspecific quartiles of the dii .
model 2 : model 1+supplementation group , number of 24h records , education level , marital status , smoking status , and physical activity .
dii indicates dietary inflammatory index ; iqr , interquartile range ; q , sexspecific quartile ; su.vi.max , supplmentation en vitamines et minraux antioxydants .
in this large cohort study of middleaged french adults , a prospective association was found between a proinflammatory diet , as reflected by higher dii scores , and the incidence of myocardial infarction over a 13year period , even after adjusting for a wide range of potential confounders including sociodemographic data and lifestyle habits .
no statistically significant relationship was observed for overall cvd risk , stroke , or angina pectoris and revascularization intervention incidence . to the best of our knowledge
, no previous study had investigated the association between the dii score and different subtypes of cvd . as indicated by the distribution of nutrient intakes by dii quartiles in our study ( table 2 )
, lower dii scores do not simply reflect diets with a higher antiinflammatory potential , but also diets that are generally healthier .
thus , our findings also should be interpreted in the light of studies that have found a preventive role of diets with an overall high quality with respect to mi incidence .
for example , lower risks of mi were found among individuals exhibiting a healthy diet , as defined by a score above 27 points on a slightly modified version of the smartdiet index in the troms study,29 and defined by being in the top quintile of recommended food score in a prospective cohort of swedish men.30 the mediterranean diet also has been related to a lower mi incidence.31
the dii was associated with increased mi risk but not with other cvd outcomes in our study .
a possible explanation could be that inflammation is more strongly linked to mi than other subtypes of cvd .
indeed , the infiltration and retention of lowdensity lipoprotein cholesterol in the arterial intima initiates an inflammatory response in the artery wall , involving the binding of blood cells , especially monocytes , which then differentiate into macrophages and internalize a broad range of molecules including oxidized lowdensity lipoprotein particles .
ultimately , the macrophages are transformed into foam cells , the prototypical cells in atherosclerosis .
several endogenous and microbial molecules can ligate patternrecognition receptors ( tolllike receptors ) on these cells , leading to the release of proinflammatory cytokines and ultimately to tissue damage .
as activated macrophages produce inflammatory cytokines that can destabilize lesions and initiate thrombus formation , all of these inflammatory reactions can finally lead to the activation and rupture of plaques , and mi.13 , 14 , 15
angina pectoris and revascularization interventions which are generally conducted among highrisk people before any cardiovascular event might reflect the early stages of inflammatory mechanism,14 compared to mi , which reflects the cumulative effect of advancedstage inflammation in the artery . on the other hand , ischemic or hemorrhagic
strokes may or may not be linked to mechanisms of inflammation32 ; nevertheless , it was hard to separate the 2 types in our study . to the best of our knowledge , only 3 other studies have investigated the association between the dii score and overall cvd risk using a prospective design .
two of these studies that were based on the spanish cohorts predimed ( prevencin con dieta mediterrnea)18 and sun ( seguimiento universidad de navarra)19 have investigated quartiles of the dii , and have found a positive association , showing a linear dose response trend , with overall cvd risk .
the respective hr , comparing the fourth with the first quartile , were 1.73 ( 95% ci=1.152.60),18 and 2.03 ( 1.063.88),19 respectively .
another study , conducted in a cohort of australian men,20 has investigated the dii as a binary variable ( negative versus positive scores ) ; it also found positive dii scores to be associated with an elevated overall cvd risk : odds ratio=2.07 ( 1.20 , 3.55 ) . other studies have focused on other dietary indexes . for instance , a higher level of adherence to the french dietary guidelines , as reflected in programme national nutrition santguideline score ( pnnsgs ) , was associated with a lower risk of cvd.33 this result is consistent with those obtained examining other diet quality indexes.34 , 35 nevertheless , these dietary indices do not focus on the inflammatory potential of the diet , and thus the measured association may reflect different mechanisms of action . some limitations of this study should be acknowledged .
first , the subjects in our analysis , who were volunteers participating in a nutritional intervention study,21 generally had a higher educational level than the general population , and probably a higher general interest in nutrition .
furthermore , we included only participants who had completed at least 3 dietary records during the first 2 years of the study ; thus , they may have been particularly compliant and health conscious .
moreover , participants of the su.vi.max cohort were aged between 35 ( women ) or 45 ( men ) and 60 years old at baseline .
thus , any generalization of these results to younger or older subjects should be done cautiously ; further studies may be needed in other populations .
additionally , the limited number of cases may have impaired our ability to detect some of the hypothesized associations , especially for specific cvd subtypes . finally ,
these findings are based on observational data ; thus , residual confounding can not be ruled out .
strengths of the present study include its large sample size and its prospective design , allowing us to assess the association between baseline inflammatory potential of the diet and cvd risk through a long followup period of 13 years .
in addition , our clinical and dietary data exhibited a high level of accuracy , with a mean of nine 24hour dietary records per participant .
finally , we used an index that was specifically designed to measure the inflammatory potential of any diet : the dii was based on peerreviewed literature focusing specifically on inflammation ; furthermore , this index can be adapted to virtually any dietary assessment method that provides estimates of nutrient intake ; and it is standardized to dietary intakes from representative populations around the world , thus facilitating easy quantitative comparisons across studies .
the results of this study provide new arguments for dietary prevention of mi , suggesting that favoring antiinflammatory nutritional compounds and limiting proinflammatory foods and nutrients may contribute to lower the risk of mi .
this is in line with the major role of inflammation in the development of atherosclerosis , the most important contributor to the growing burden of cvd .
the authors responsibilities were as follows : galan , hercberg , and kesseguyot were responsible for developing the concept , design , and protocol of the study and for coordinating data collection ; neufcourt performed the statistical analysis and wrote the article ; kesseguyot provided methodological guidance and had primary responsibility for the final content ; neufcourt , assmann , fezeu , touvier , graffouillre , shivappa , hbert , wirth , hercberg , galan , julia , and kesseguyot were involved in interpreting results and editing the manuscript for important intellectual content .
this work was supported by grants from the fondation coeur et artres , the french national research agency ( agence nationale de recherche , anr ) ( grant no .
anr05pnra010 ) and the french ministry of health ( direction gnrale de sant , dgs ) .
assmann was supported by a doctoral fellowship from the ecole doctorale galile , university of paris 13 , sorbonne paris cit .
drs shivappa , hbert , and wirth were supported by the united states national institute for diabetes , digestive and kidney diseases ( grant no .
dr james r. hbert owns controlling interest in connecting health innovations llc ( chi ) , a company planning to license the right to his invention of the dietary inflammatory index ( dii ) from the university of south carolina in order to develop computer and smartphone applications for patient counseling and dietary intervention in clinical settings .
|
backgroundcardiovascular diseases ( cvd ) are the leading cause of death in the world , and diet plays a major role in cvd incidence , especially through lipid oxidation mechanisms .
this , in turn , leads to tissue inflammation and formation of atheromatous plaques.methods and resultsour objective was to evaluate the association between the inflammatory potential of the diet and the incidence of overall cvd or its subclasses .
we included 7743 participants from the supplmentation en vitamines et minraux antioxydants ( su.vi.max ) cohort .
all cardiovascular events were recorded using selfreported information or clinical visits , and were validated . the dietary inflammatory index ( dii )
was computed using repeated 24hour dietary records ( mean=9.53.4 records / subject ) .
hazard ratio and 95% ci for outcomes ( cvd and subclasses ) were estimated across sexspecific quartiles of the dii using cox proportional hazard models .
a total of 292 cardiovascular events were recorded and validated during an average of 11.4 years of followup : 93 myocardial infarctions , 58 strokes , 128 angina pectoris and revascularization interventions , and 13 sudden deaths . when considering cvd subclasses , a diet with proinflammatory properties , as expressed by higher dii scores , was significantly associated with a higher risk of myocardial infarction ( hazard ratioquartile 4 versus quartile 1=2.24 , 95% ci : 1.084.67 ) .
no significant association was observed between the dii score and stroke or both angina pectoris and revascularization intervention.conclusionsa proinflammatory diet , as measured by a higher dii score , was prospectively associated with a higher risk of myocardial infarction .
promotion of a diet exhibiting antiinflammatory properties may help prevent myocardial infarctions .
|
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diabetic nephropathy ( dn ) is the leading cause of chronic kidney disease and the primary diagnosis in more than 40% of new patients on dialysis in several parts of the world including the united states .
identifying the factors that contribute to the pathogenesis of dn is a critical step towards halting its progression .
connective tissue growth factor ( ctgf ; ccn2 ) is a matricellular protein involved in modulation of the extracellular environment and plays a role in the development and progression of diabetic complications [ 211 ] . in dn , increased renal ctgf expression has been described both in glomeruli and in the tubulointerstitium .
urinary ctgf ( uctgf ) is elevated in dn and correlates with markers of disease severity such as urinary albumin excretion and glomerular filtration rate ( gfr ) [ 1214 ] .
furthermore , uctgf has been shown to correlate with progression of microalbuminuria in diabetic patients .
however , to interpret elevated uctgf in diabetes , it is crucial to understand the mechanisms behind this elevation . both increased intrarenal production and elevated plasma ctgf
have been suggested to account for elevated uctgf in dn [ 13 , 14 ] .
we have shown in healthy volunteers and normoglycemic mice that blockade of proximal tubular reabsorption results in a major increase in uctgf .
the aim of this study is to evaluate which factors contribute to elevated uctgf in diabetes . in diabetic mice , we measured the fractional excretion ( fe ) of both endogenous ctgf and recombinant ctgf ( i.e. , not intrarenally derived ctgf ) and examined the renal expression pattern of ctgf .
moreover , we examined the association between uctgf and urinary markers in type 1 diabetic patients .
diabetes was induced in nineteen 1012-week - old male c57bl/6 mice ( harlan , horst , netherlands ) by a single intraperitoneal injection of 200 mg / kg streptozotocin ( stz , sigma , st .
louis , mo , usa ) in sodium citrate buffer ( 100 mmol / l , ph 4.5 ) .
blood glucose was determined one week after injection of stz ( medisense precision xtra , abbott , abbott park , il , usa ) .
slow - release insulin pellets were implanted subcutaneously to stabilize the condition of the diabetic animals ( linbit , linshin , scarborough , on , canada ) .
ten weeks after stz , renal function parameters and fractional excretion of ctgf were determined .
two miniosmotic pumps ( model 1003d , alzet , cupertino , ca , usa , 100 l reservoir volume , release rate of 1 l / h ) were implanted intraperitoneally under isoflurane anaesthesia .
the other pump was used for simultaneous infusion of recombinant human ctgf ( rctgf , 38 pmol / h ) in fourteen of the diabetic mice and four of the control mice .
we used the proteolytic aminoterminal fragment of ctgf since this is the predominant form of ctgf detected in plasma and urine [ 13 , 1719 ] . to exclude
that rctgf had an effect on endogenous ctgf mrna expression in the kidney , we infused four control mice and five diabetic mice with vehicle .
the mice injected with rctgf were used to determine fe of rctgf and intrarenally derived uctgf ( see below ) .
forty - eight hours after pump implantation , mice were put in metabolic cages overnight for timed urine collection .
plasma was collected before and after the urine collection , ctgf levels were determined , and time - weighted averages of plasma ctgf were calculated . all samples and metabolic cages
all experiments were approved by the animal ethical committee of the university of utrecht and performed in accordance with national guidelines for the care and handling of animals .
three hundred and forty - nine well - characterized adult type 1 diabetic patients were selected from the outpatient clinic at steno diabetes center ( copenhagen , denmark ) .
forty - three of the patients with diabetic nephropathy had been previously analysed in a longitudinal study examining the impact of losartan on uctgf .
the study was approved by the ethical committee of copenhagen county and performed in accordance with the declaration of helsinki .
demographic and clinical data were recorded , including age , sex , duration of diabetes , and body mass index .
creatinine was determined in venous blood samples using the cobas mira plus ( roche , basel , switzerland ) .
hba1c was determined using variant high - performance liquid chromatograph ( bio - rad laboratories , hercules , ca , usa ) .
the estimated gfr ( egfr ) was calculated using the modification of diet in renal disease study method .
albumin excretion rate ( aer ) was determined in 24 h urine collections using turbidimetry .
macroalbuminuria was defined as albuminuria > 300 mg/24 h , microalbuminuria as albuminuria 30300 mg/24 h , and normoalbuminuria as albuminuria < 30 mg/24 h. sixty patients were excluded from analysis because of incomplete data due to insufficient sample availability or incomplete patient characteristics .
ctgf levels in plasma and urine were determined by sandwich elisa , using specific antibodies ( fibrogen ) directed against distinct epitopes in the aminoterminal fragment of ctgf , detecting both full length ctgf and the n - fragment ( n - ctgf ) , as described previously .
two elisa assays were used : an assay for detection of human or rctgf in either human or mouse samples and an assay for detection of rodent ctgf in mouse samples . the antibody used for detection in the human ctgf assay does not cross -
maxisorp microtiter plates ( nunc , rochester , ny , usa ) were coated with 0.4 g antigen ( fitzgerald industries international , acton , ma , usa ) . after washing , 20-time diluted samples were incubated with biotinylated detection antibody ( chicken anti - human 1-microglobulin ; icl , newberg , or , usa ) .
after washing , wells were incubated with hrp - conjugated streptavidin and binding was detected by measuring hrp activity using o - phenylenediamine as chromogenic substrate .
urinary 2-microglobulin ( 2 m ) ( anogen , mississauga , on , canada ) , heart - type fatty acid - binding protein ( h - fabp ) ( hytest , turku , finland ) , immunoglobulin g subclass 4 ( igg4 ) , kidney injury molecule-1 ( kim-1 ) , and neutrophil gelatinase - associated lipocalin ( ngal ) ( r&d systems , minneapolis , mn , usa ) were measured by elisa , as described previously .
urinary n - acetyl--glucosaminidase ( nag ) was measured using a modified enzyme assay according to lockwood and bosmann and corrected for nonspecific conversion ( haemoscan , groningen , netherlands ) .
prior to the animal experiments , 5% fitc - inulin ( sigma - aldrich , zwijndrecht , netherlands ) was dissolved in 0.9% nacl by heating in boiling water and dialyzed to remove residual free fitc .
the dialyzed fitc - inulin solution was sterilized by filtration through a 0.20 m syringe filter ( corning , new york , ny , usa ) .
fluorescence of plasma and urine was measured within hours after collection in black 96-well plates ( fluotrac , greiner bio - one , kremsmnster , austria ) in a fluostar optima ( bmg labtech , offenburg , germany ) at 485 nm excitation and 538 nm emission .
plasma and urine samples were buffered to ph 7.4 by dilution with hepes 50 mmol / l ph 7.4 ( 5- and 10-fold , resp . ) before fluorescence measurement .
matrix correction was applied for the standard curves . for routine histological examination and scoring of tubular atrophy ( ta ) ,
formalin - fixed , paraffin - embedded ( ffpe ) tissue sections were stained with periodic acid - schiff ( pas ) .
ta was defined as the presence of tubules with thickened tubular basement membranes and/or atrophic cells lining the tubules , with loss of brush border .
ta was scored by two skilled observers in ten randomly selected cortical areas on 100 magnification .
the following semiquantitative scale was used : 0 : no ta ; 1 : > 010% ta ( > 010% of tubuli in the field shows atrophy ) ; 2 : 1025% ta ; 3 : 2550% ta ; 4 : 5075% ta ; 5 : 75100% ta .
photographs were taken on a nikon eclipse e800 microscope with a nikon dxm1200 digital camera using the nikon act-1 software version 2.70 ( nikon netherlands , lijnden , netherlands ) .
briefly , 6 m thick ffpe tissue sections were dewaxed and rehydrated , incubated with proteinase k , postfixed , prehybridized , and hybridized with a 542-nt antisense ctgf dig - labeled riboprobe for 16 h at 70c .
upon washing , bound dig was detected using alkaline phosphatase - conjugated sheep anti - dig and nbt / bcip ( roche , almere , netherlands ) .
for isolation of cortical mrna , about 30 mg of the renal pole was cut using a scalpel . for medullary mrna , fifteen 10 m cryosections per sample were put on a glass slide and medulla was identified based on location and morphology and microdissected using a scalpel .
the remaining tissue was stained with hematoxylin and eosin to check the accuracy of medullary microdissection .
total rna was isolated using the rneasy rna isolation kit ( qiagen benelux , venlo , netherlands ) .
rna was reverse - transcribed with superscript ii reverse transcriptase ( invitrogen , paisley , uk ) . quantitative reverse transcription pcr ( qrt - pcr )
was performed on an applied biosystems 7900ht fast real - time pcr system ( applied biosystems , nieuwerkerk aan den ijssel , netherlands ) .
expression levels of ctgf and the internal references , tbp and gapdh , were determined using applied biosystems inventoried taqman gene expression assays , containing primers and probe .
urinary ctgf , fractional ctgf excretion , ctgf mrna ( fold change ) , urinary markers , and human plasma ctgf data were logarithmically transformed to allow parametric analysis .
urinary igg4 was dichotomized as greater than or less than the detection limit because of a high proportion of subjects with undetectable levels .
differences were calculated using student 's t - test or mann - whitney u test where appropriate .
correlations between variables were evaluated by pearson 's and spearman 's correlation coefficients ( r and , resp . ) where appropriate .
multiple linear regression analysis was performed to identify parameters independently associated with uctgf . to explore the association between clusters of biomarkers and uctgf
, we used mean standard deviation scores ( z - scores ) , a method previously described by schram et al . .
for each individual , the values of each marker were expressed as a z - score , that is , ( value in the individual minus the mean value in the study population ) divided by the standard deviation .
the proximal tubular reabsorption ( ptr ) z - score was then calculated as ( z - score of 1 m + z - score of 2m)/2 , the proximal tubular injury ( pti ) z - score as ( z - score of kim-1 + z - score of nag + z - score of ngal)/3 , and the combined proximal tubule ( pt ) z - score as ( ptr z - score + pti z - score)/2 .
this approach was used in order to avoid underestimating the associations between the different markers and uctgf . for all comparisons ,
the statistical analysis was performed using pasw statistics software version 18.03 for macintosh ( spss inc . , chicago , il , usa ) and graphpad prism software version 4.03 for windows ( graphpad software , san diego , ca , usa ) . in the diabetic mice study ,
urinary ctgf expressed per g creatinine provided similar results ( see supplementary figures in the supplementary material available online at http://dx.doi.org/10.1155/2015/539787 ) .
fractional excretion ( fe ) of ctgf was calculated as follows:(1)fectgf = ctgfurineinulinplasmactgfplasmainulinurine100%.assuming that rctgf and endogenous ctgf ( ectgf ) are handled similarly by the kidney and taking into account that rctgf can only appear in urine by filtration from plasma whereby ferctgf is only determined by ctgf filtered from the plasma ( and not by intrarenal ctgf production ) , the relative contribution of plasma - derived ectgf to urinary ectgf could be estimated as follows:(2)fraction of uectgf derived from plasma = ferctgffeectgf.the relative contribution of intrarenally derived ctgf to urinary ectgf could be estimated as follows:(3)fraction of uectgf derived from a local intrarenal source=1ferctgffeectgf.one diabetic mouse was excluded from this calculation because of unreliable measurements ( below lower limit of quantification ) .
the absolute amount of urinary ectgf derived from an intrarenal source was estimated as follows:(4)excretion of intrarenally - derived ectgf = excretion of ectgf1ferctgffeectgf.renal clearance of fitc - inulin was calculated by the standard formula .
diabetes was induced in c57bl/6 mice with stz which caused pronounced hyperglycemia within one week . at the end of the study period the diabetic mice had developed dn , for example , increased albuminuria and decreased gfr ( table 1 ) .
urinary excretion of ctgf was markedly elevated in all diabetic mice ( 999 fmol/24 h ( 1902946 ) , p < 0.0001 ) while in control mice uctgf was measurable in only a minority , with a maximum of 57 fmol/24 h. plasma ctgf was mildly increased in diabetic mice ( 340 pmol / l ( 290370 ) versus 230 pmol / l ( 185295 ) in control mice , p = 0.003 ) . to establish the influence of diabetic kidney disease on the renal handling of ctgf
, we infused recombinant human ctgf ( rctgf ) and fitc - inulin simultaneously by miniosmotic pumps . for detection of rctgf levels we used an elisa that does not cross - react with endogenous ctgf ( ectgf ) .
this allowed us to study urinary excretion of plasma derived ctgf independent from intrarenally produced ctgf , since urinary recombinant ctgf is exclusively derived from filtered plasma rctgf . on the contrary , urinary endogenous ctgf ( uectgf )
since ctgf is almost completely filtered from the plasma ( sieving coefficient 0.74 ) and rctgf clearance and fitc - inulin clearance were not differentially affected between control and diabetic mice ( p = 0.53 , data not shown ) , fractional excretion of rctgf ( ferctgf ) could be regarded as a measure of tubular ctgf passage with increased ferctgf reflecting reduced tubular reabsorption of ctgf .
we observed that in diabetic mice ferctgf was strongly increased ( 72-fold , p = 0.004 , figure 1(a ) ) , while in control mice tubular reabsorption of ( filtered ) rctgf was virtually complete .
ferctgf showed a tight linear correlation with urinary excretion of endogenous ctgf ( uectgf ) in diabetic mice ( r = 0.95 , p < 0.0001 , slope 1.1 on a logarithmic scale ( figure 1(b ) ) ) and emerged as an independent determinant of uectgf ( = 1.3 , p < 0.001 ) in a multivariate model that included the following parameters : ferctgf , plasma ctgf , cortical and medullary ctgf gene expression ( derived from qrt - pcr data of microdissected kidney , see below ) , and gfr .
we observed no obvious glomerular damage in diabetic animals , but significant tubular atrophy was observed ( figures 1(c ) and 1(d ) ) , which correlated with uectgf ( r = 0.62 , p = 0.005 , figure 1(e ) ) .
this is consistent with tubular function as a major determinant of elevated uectgf . to investigate the relative contribution of plasma - derived ectgf to uectgf
, we compared the fractional excretion ( fe ) of recombinant and endogenous ctgf . assuming that rctgf and ectgf are handled similarly by the kidney , comparison of ferctgf and feectgf allowed us to estimate the contribution of plasma - derived ectgf to uectgf , the ratio ferctgf / feectgf representing the fraction of urinary ectgf derived from plasma .
this revealed that only 37 ( 2955)% of urinary ectgf could be accounted for by plasma - derived ectgf , implying that most urinary ctgfs in diabetes must be derived from an intrarenal source and excreted into the tubular lumen ( figure 2 ) . due to the extremely low uectgf in healthy animals ,
the relative contribution of plasma - derived ectgf to uectgf in the healthy situation could not be established . to investigate the localization of intrarenal ctgf production
this showed that ctgf expression was hardly detectable in control kidneys but abundant in diabetic kidneys , where it was mainly present in glomeruli and medullary tubules ( figure 3(a ) ) . in agreement , qrt - pcr of microdissected kidney revealed that both cortical and medullary ctgf mrna were significantly increased in diabetic animals compared with controls ( 2.5-fold and 2.7-fold , resp . ,
we did not observe differences in ctgf mrna expression in cortex or medulla between animals infused with rctgf or with vehicle , both in diabetic mice and in nondiabetic controls ( data not shown ) .
intrarenally derived uctgf showed a tight linear correlation with ferctgf ( r = 0.92 , p < 0.0001 , slope 1.0 on a logarithmic scale , figure 4(a ) ) .
cortical ctgf expression also correlated with intrarenally derived uctgf ( r = 0.78 , p = 0.002 , figure 4(b ) ) , while no such correlation was observed for medullary ctgf expression ( p = 0.12 , figure 4(c ) ) . in a multivariate model including cortical and medullary gene expression ferctgf remained the only independent determinant of intrarenally derived uctgf ( = 1.1 , p < 0.001 ) .
these data suggest that tubular function is also an important determinant of intrarenally derived ctgf in the urine . to investigate the determinants of uctgf in human diabetes
, we studied the associations of uctgf with tubular markers and a glomerular marker in a cohort of patients with diabetes mellitus type 1 .
the low - molecular - weight proteins 2 m and 1 m were used as markers for reduced tubular reabsorption [ 27 , 28 ] .
kim-1 , ngal , and nag were used as markers for proximal tubular damage [ 29 , 30 ] and h - fabp as a marker for distal tubular damage [ 31 , 32 ] . in univariate analysis , uctgf
correlated with each of the damage markers ( table 3 ) . in a multivariate linear regression model containing age , sex , egfr , plasma ctgf , duration of diabetes , bmi , and hba1c ,
each of the tubular markers remained independently associated with uctgf while the glomerular damage marker igg4 lost its significance ( table 3 ) .
when igg4 , the proximal tubular reabsorption , and proximal tubular injury z - scores ( or the combined proximal tubular z - score ) and h - fabp were included simultaneously into the model , both proximal and distal tubular markers , but not igg4 , were independently related to uctgf ( table 3 ) .
these findings suggest that also in human diabetes uctgf is dependent on tubular status , with elevated uctgf reflecting both proximal tubular dysfunction and pathology in the distal tubule .
understanding the different determinants of elevated uctgf in diabetes is essential for its proper interpretation as biomarker and pathogenic factor .
previously , we have shown that in the healthy kidney filtered ctgf is almost completely reabsorbed in the proximal tubules by megalin - mediated endocytosis and that impairment of tubular reabsorption results in increased urinary excretion of ctgf ( in close correlation with that of 2 m ) . here
we show that also in diabetes tubular damage is a major determinant of elevated uctgf .
our findings indicate that in addition to reduced proximal reabsorption of uctgf increased intrarenal ctgf production plays a role .
both in human and in experimental diabetes , uctgf was independently associated with decreased tubular reabsorption . while in experimental diabetes
uctgf correlated very tightly with tubular dysfunction , the association in human diabetes was somewhat less prominent .
there was a clear independent association between uctgf and distal tubular damage marker h - fabp , suggesting increased ctgf secretion by the distal nephron .
this conforms to the increased distal tubular ctgf mrna expression that we observed in diabetic mice , although the correlation between uctgf and medullary ctgf mrna did not reach significance .
tubular damage might thus contribute to uctgf in two ways , by ( 1 ) decreased proximal reabsorption of ctgf and ( 2 ) increased expression and luminal secretion of ctgf in the distal nephron .
possibly , solute overload to the distal nephron due to proximal tubular dysfunction may cause distal tubular injury and induce distal tubular ctgf expression .
although we are not aware of any clinical , experimental , or in vitro studies addressing ctgf expression specifically in medullary tubules in diabetic kidney disease , increased tubular ctgf protein expression has been reported before , both in human and in experimental diabetes , and was postulated to contribute to tubulointerstitial fibrosis via paracrine effects at the basolateral membrane [ 4 , 3335 ] .
however , secretion of ctgf at the apical membrane of the distal tubular cell into the tubular lumen has not been described .
it would be useful to have matched kidney biopsies from diabetic patients with known plasma and urine ctgf levels to explore the associations between increased uctgf and pctgf , tubular dysfunction , and local production of ctgf .
although dn is traditionally viewed as a primarily glomerular disease , tubulointerstitial injury is also a major feature of dn with important prognostic significance [ 36 , 37 ] .
tubular injury was shown to be an early event in the pathogenesis of dn and tubular proteinuria could identify patients susceptible to dn even earlier than albuminuria alone [ 3741 ] .
renal ctgf protein overexpression at 5-year duration of diabetes predicted 10-year albuminuria values , while at 5-year albuminuria values did not differ from nondiabetic controls .
elevated uctgf reflecting tubular damage might thus be a valuable prognostic marker in dn and useful for early detection of patients at risk . as compared to established markers of tubular damage ,
ctgf is of particular interest because it might also enhance tubulointerstitial fibrosis [ 4 , 34 , 35 ] .
however , the true clinical value of uctgf in diabetes still needs to be established . in this study , we used a single high dose of stz to induce diabetic kidney disease in mice .
since stz has been associated with acute tubular injury , we can not exclude that part of the tubular damage observed in our dn model might be directly related to stz toxicity .
however , at 10 weeks after infusion of stz , we expect that the animals have recovered from the acute toxic effect of stz . instead , they have developed kidney disease in which both chronic glomerular and tubular damage are present , features that are also present in human diabetic kidney disease . to deduce the relative contributions of local production and tubular dysfunction in uctgf , a dn model with both glomerular and tubular features was required . since both features are not always present in non - stz pure dn models , which typically manifest a primarily , if not exclusively , glomerular and sometimes vascular phenotype , we selected the stz model for our studies . in our mouse model of diabetic kidney disease , we observed that most uctgf had a local renal source .
in addition to increased medullary ctgf mrna expression , ctgf production was increased in glomeruli , which is in agreement with previous reports [ 34 , 35 , 4446 ] .
however , it remains unclear how much intrarenally derived uctgf has a glomerular source and how much is secreted at the apical membrane of the distal tubular cell .
the tight independent linear association of intrarenally derived uctgf with ferctgf suggests that reduced tubular reabsorption of ctgf originating from a source upstream the proximal nephron , that is , the glomerulus , plays an important role . in human diabetes , but not in experimental diabetes , plasma ctgf emerged as an independent determinant of uctgf .
this suggests that in human diabetes the amount of ctgf filtered from the plasma in the glomeruli also contributes to uctgf , in addition to tubular damage .
plasma ctgf was shown to predict end - stage renal disease and mortality in macroalbuminuric patients . for uctgf
this has not been investigated yet , but the independent association with plasma ctgf and tubular damage suggests promising biomarker value . in conclusion ,
urinary ctgf excretion in diabetes is elevated as a result of both increased local production and reduced reabsorption due to tubular dysfunction .
we submit that urinary ctgf may be a useful biomarker reflecting both glomerular and tubulointerstitial hallmarks of diabetic kidney disease .
|
connective tissue growth factor ( ctgf ; ccn2 ) plays a role in the development of diabetic nephropathy ( dn ) .
urinary ctgf ( uctgf ) is elevated in dn patients and has been proposed as a biomarker for disease progression , but it is unknown which pathophysiological factors contribute to elevated uctgf .
we studied renal handling of ctgf by infusion of recombinant ctgf in diabetic mice .
in addition , uctgf was measured in type 1 dn patients and compared with glomerular and tubular dysfunction and damage markers . in diabetic mice ,
uctgf was increased and fractional excretion ( fe ) of recombinant ctgf was substantially elevated indicating reduced tubular reabsorption .
fe of recombinant ctgf correlated with excretion of endogenous ctgf .
ctgf mrna was mainly localized in glomeruli and medullary tubules .
comparison of fe of endogenous and recombinant ctgf indicated that 60% of uctgf had a direct renal source , while 40% originated from plasma ctgf . in dn patients ,
uctgf was independently associated with markers of proximal and distal tubular dysfunction and damage . in conclusion ,
uctgf in dn is elevated as a result of both increased local production and reduced reabsorption due to tubular dysfunction .
we submit that uctgf is a biomarker reflecting both glomerular and tubulointerstitial hallmarks of diabetic kidney disease .
|
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human cells show a different ddr during mitosis than in other cell cycle phases . during most of the cell cycle ,
after cells reach the point of no return , however , dsbs on mitotic chromosomes do not trigger cell - cycle delay or arrest ; and the cells rather proceed through mitosis even if they contain unrepaired dsbs or fragmented chromosomes.(32 ) indeed , the sensitivity to ionizing radiation is higher in mitotic cells than in interphase cells.(33,34 ) moreover , dsb introduction into mitotic cells by etoposide treatment induces massive chromosome aberrations in the next cell cycle in a cancer cell line ( fig .
thus , dsbs during mitosis are very toxic to cells because of the induction of severe genomic instability .
effect of etoposide - induced double - strand breaks ( dsbs ) during mitosis on genomic instability of mitotic chromosomes .
representative images of chromosome spreads from etoposide - treated ( + etoposide ) and non - treated ( etoposide ) in hct116 human colon cancer cells arrested in mitosis .
during mitosis , dsbs activate pikks to induce phosphorylation of h2ax , and mdc1 as well as the mrn complex are recruited to the dsb sites , as seen during interphases ( fig .
however , the recruitment of rnf8 , rnf168 , 53bp1 , and brca1 to dsb is largely suppressed during mitosis ( fig .
1b , d , right).(30,33 ) the mechanism of the suppression and its purpose were not well understood .
two groups recently showed that localization of rnf8 and 53bp1 to dsbs is inhibited during mitosis.(35,36 ) the first work done by orthwein et al .
( 2014 ) showed that , in human cells , rnf8 and 53bp1 are phosphorylated during mitosis by cdk1 and that inhibition of the phosphorylation restores their localization to dsb sites ( fig .
orthwein et al . also identified t198 on rnf8 as a cdk1 phosphorylation site and showed that rnf8-t198a , a phosphorylation - defective protein , can localize to mitotic chromatin after dsb introduction .
rnf8 prepared from mitotic extracts can not bind to mdc1 in vitro , but rnf8 prepared from cdk1 activity - inhibited cells interacts with mdc1 in vitro .
they also found that t1609 and s1618 on 53bp1 are phosphorylated during mitosis ( fig .
3a ) and that 53bp1-t1609a / s1618a double mutant protein can localize to mitotic chromatin and restores dsb repair in cells expressing rnf8-t198a .
their study confirmed that t1609 is phosphorylated by cdk1 , whereas s1618 is a target of plk1 in vitro .
these show that there are at least two distinct mechanisms to regulate the recruitment of these ddr effector proteins.(35 ) domain structure of 53bp1 and comparison of cdk1 and plk1 sites among xrcc4 orthologs .
( b ) domain structure of xrcc4 and conservation of phosphorylation sites among various species .
s326 in the c - terminus of human xrcc4 ( hs ) is a potential cdk1 phosphorylation site .
the gray and black boxes show xlf and dna ligase iv binding sites , respectively .
phosphorylation of cdk1 or plk1 sites are shown in mouse ( ms ) , chicken ( gg ) , zebrafish ( de ) , and budding yeast ( sc ) .
dna ligase iv and xlf binding sites in mouse , chicken , and zebrafish were predicted by aligning with the dna ligase iv and xlf binding sites of human xrcc4 using t - coffee software .
( 2014 ) indicated that t1609 and s1618 of 53bp1 are hyperphosphorylated in the absence of pp4c phosphatase in human cell lines.(36,37 ) they analyzed the function of pp4c phosphatase during ddr and found that these sites are phosphorylated specifically during mitosis.(36 ) these mitosis - specific phosphorylation sites are located in the ubiquitin - dependent recruitment motif of 53bp1 , a recognition site of ubiquitinated h2a , which is required for the localization of the protein to dsb sites.(1517 ) both of the reports(35,36 ) showed that dna damage during mitosis in cells expressing phosphorylation - defective 53bp1-t1609a / s1618a mutant , which restores its localization to dsb sites , leads to increased micronuclei formation .
this suggests that restoration of dsb repair during mitosis causes defects in proper chromosome segregation and that inhibition of dsb repair during mitosis serves to maintain genomic stability .
in addition to micronuclei formation , orthwein et al . showed that restoration of accumulation of rnf8 and 53bp1 to damaged mitotic chromosomes increases telomere fusions , which seems to be mediated by dsb repair such as c - nhej.(35 ) therefore , suppression of dsb repair by phosphorylation of rnf8 or 53bp1 by cdk1 or plk1 may protect the genome during mitosis .
in particular , suppression of rnf8- and 53bp1-dependent dsb repair during mitosis may prevent telomere fusions .
two groups showed that inhibition of the dna - pkcs suppresses micronuclei formation induced by mitotic dsbs in 53bp1-t1609a / s1618a - expressing or 53bp1-t1609a / s1618a rnf8-t198a - expressing cells , suggesting and that the core c - nhej complex plays a role in the segregation defects.(35,36 ) recently , we showed another dsb repair suppression mechanism by modulating the c - nhej core complex during mitosis.(38 ) dsb repair is largely suppressed during mitosis , but a substantial level of repair still occurs.(38 ) c - nhej causes formation of anaphase bridges , which are bridge - like dna structures that span daughter chromosomes and frequently induce genomic instability through inappropriate chromosome segregation.(39,40 ) in our study , severe chromosome aberrations such as dicentric and fragmented chromosomes are induced by mitosis - specific dsb introduction by transient treatment with etoposide ( fig .
2).(38 ) this observation raises a possibility that anaphase bridges are caused by dicentric chromosome formation by interchromosomal connections between telomeres or other chromosomal loci .
these results suggest that there is a process to connect sister chromatids or individual chromosomes in ddr during mitosis .
dna ligase iv - dependent c - nhej contributes to dicentric chromosome formation through telomere fusion in cells with dysfunctional telomeres,(41 ) which act as dsb ends.(42 ) therefore , we hypothesized that c - nhej is involved in the formation of anaphase bridges .
both xrcc4 and xlf are regulatory subunits of the dna ligase iv complex and are essential for its activity.(4345 ) anaphase bridge formation is reduced after xrcc4 knockdown , suggesting that c - nhej contributes to the formation of some of these bridges during mitosis.(38 ) knockdown of the hr - specific factor xrcc3 has almost no effect on anaphase bridge formation , indicating that c - nhej is more critical than hr for the formation of anaphase bridges in mitotic cells .
our studies strongly suggest that inappropriate use of the c - nhej pathway causes chromosome bridges , which lead to chromosome aberrations from mitotic dsbs .
a phosphoproteomics study revealed that many sites are phosphorylated during mitosis , including those on xrcc4,(46 ) so we evaluated cell - cycle regulation of xrcc4 phosphorylation.(38 ) this mitosis - specific phosphorylation of xrcc4 contributes to the suppression of anaphase bridge formation after induction of dsbs during mitosis.(38 ) the mitosis - specific phosphorylation of xrcc4 depends on both cdk1 and plk1 activities.(38 ) s326 is a putative cdk phosphorylation site , and s326a substitution substantially reduces mitosis - specific phosphorylation of xrcc4 ( fig .
3b).(38 ) interestingly , the s326 phosphorylation residue overlaps with the polo box recognition motif.(47 ) both s256 and s326 are phosphorylated during mitosis.(46 ) because s256 resides in a putative plk1 phosphorylation site ( fig .
3b ) , cdk - dependent phosphorylation of s326 might prime the mitosis - specific phosphorylation of xrcc4 by plk1 .
although the ortholog of xrcc4 has not yet been identified in nematode or fission yeast , most of the dna ligase iv subunits have been identified in various species ( table 1 ) .
we previously analyzed cell - cycle regulation of lif1p , which is the s. cerevisiae ortholog of xrcc4.(4850 ) lif1p is phosphorylated by cdks from s phase through mitosis and this phosphorylation plays a role in suppressing c - nhej in s to m phases through the pathway dependent on sae2 , the s. cerevisiae ortholog of ctip.(49 ) these findings prompted us to assess the conservation of the phosphorylation sites among xrcc4 orthologs .
although the locations of the phosphorylation sites are different , both s. cerevisiae lif1p and human xrcc4 have one putative target site for cdk1 and multiple sites for plk1 ( fig .
both the cdk1 and plk1 sites near the c - terminus were conserved between human and mouse ( fig .
3b ) . in chicken , there are two cdk1 sites near the c - terminus and one plk1 site . however , in zebrafish , although the cdk1 site near the c - terminus was not conserved , there are four cdk1 sites in other locations .
three of them are located downstream of the dna ligase iv binding site and overlap with plk1 sites ( fig .
all of the xrcc4 orthologs have cdk1 or plk1 phosphorylation sites downstream region of the dna ligase iv interaction domain .
thus , the function of mitosis - specific phosphorylation of xrcc4 is likely to be conserved among many species .
we showed that xrcc4 phosphorylation - defective mutant ( xrcc4-s326a ) restores rapid repair of mitotic dsbs associated with more anaphase bridge formation.(38 ) this suggests that mitotic xrcc4 phosphorylation is involved in suppressing c - nhej to prevent chromosome instability in human cells .
ctip promotes dsb end resection in hr and a - nhej.(51 ) because anaphase bridge formation is increased in ctip - depleted cells,(38 ) ctip may have a function to suppress anaphase bridges , possibly through the a - nhej pathway .
brca1 , which is important for recruitment of ctip to dsb sites , does not localize to mitotic dsb sites.(33 ) however , only the function of ctip in hr , but not in a - nhej , is dependent on interaction with brca1.(52 ) moreover , in xenopus m - phase extract , ctip can bind mitotic chromatin.(53 ) thus , ctip may function as an a - nhej factor in suppression of genomic instability during mitosis .
alternatively , ctip - dependent end resection may have functions to suppress anaphase bridge formation .
conservation of dna ligase iv complex subunits among species although dna ligase iv ( catalytic subunit ) localizes to mitotic chromosomes , xrcc4 does not,(54 ) suggesting that the dna ligase iv complex function during mitosis is different from those in other cell cycles . because complex formation of the dna ligase iv with xrcc4 is believed to be essential for its activity , an activity of the dna ligase iv complex may be modified during mitosis .
we confirmed the difference in the localization of dna ligase iv and xrcc4 during mitosis.(38 ) like wild - type protein , xrcc4 phosphorylation - defective mutant protein also fails to localize to mitotic chromosomes , showing that the phosphorylation of xrcc4 is not responsible for inhibiting the localization to chromosomes during mitosis .
on the basis of these findings , we propose that there may be two mechanisms to suppress c - nhej : the reduced recruitment of c - nhej - specific factors to mitotic chromosomes , and mitosis - specific phosphorylation of xrcc4 by modulating the function of the dna ligase iv complex . taken together ,
these results suggest that xrcc4 , a key regulatory subunit of the dna ligase iv complex , is required not only for c - nhej in interphase but also for suppression of c - nhej during mitosis to prevent genomic instability in human cells .
cells prevent carryover of dna lesions at the g2/m checkpoint because dna damage as well as the dna repair process are toxic during mitosis .
as described above , there are several types of dsb repair suppression systems that occur during mitosis .
however , mitotic cells do not undergo the second level of ddr : neither 53bp1 nor brca1 localizes to dsb sites .
mitosis - specific phosphorylation of 53bp1 and rnf8 prevents their localization to dsb sites.(35,36 ) because 53bp1 and brca1 localization to dsb sites is important for nhej and hr , respectively , dsb repair pathways should be largely suppressed on mitotic chromosomes ( fig .
we found that the third level of ddr is also suppressed or modified by mitosis - specific phosphorylation of xrcc4 , a component of the core c - nhej complex.(38 ) even if dsb repair pathways are largely suppressed by mitosis - specific phosphorylation of 53bp1 and rnf8 , considerable levels of dsb repair still occur and cause genomic instability during mitosis .
phosphorylation of xrcc4 during mitosis slows dsb repair.(38 ) thus , xrcc4 phosphorylation has some functions to modulate dna ligase iv complex activity ( fig .
in addition , xrcc4 fails to localize to mitotic chromatin.(54 ) the failure of xrcc4 to localize to mitotic chromosomes also may modify the composition of the dna ligase iv complex .
ctip , but not rad51 , is recruited to mitotic chromatin.(53 ) this also suggests that there is the third level of suppression mechanisms in hr and that ctip has some functions to prevent genomic instability without the requirement of brca1 localization .
( a ) double - strand break ( dsb ) induces histone h2ax phosphorylation ( h2ax ) by atm .
( b ) cdk1 and plk1 phosphorylate rnf8 and 53bp1 to inhibit 53bp1 and brca1 localization of dsb sites .
( c ) cdk1 and plk1 phosphorylate xrcc4 , a regulatory subunit of the dna ligase iv complex , to suppress canonical non - homologous end joining ( c - nhej ) activity .
ctip - dependent alternative non - homologous end joining ( a - nhej ) may prevent anaphase bridge formation .
cells use multiple safeguards to prevent genomic instability by suppression or modification of dsb repair activities during mitosis , through the activation of mitotic kinases , cdk1 and plk1
. the molecular mechanisms of mitosis - specific phosphorylation and the change in localization of the dna ligase iv component xrcc4 are still unknown .
analyzing how dna ligase iv activity in mitotic cells differs at the molecular level from during other cell - cycle phases is critical to understand the mechanisms of these controls .
another c - nhej component , dna - pkcs also phosphorylated by plk1 , is dephosphorylated by pp6 and is required for accurate chromosome segregation.(55 ) it is also interesting to analyze how those c - nhej factors are involved in accurate mitosis.(56 ) active dsb repair during mitosis affects chromosome segregation , which often results in apoptosis , aneuploidy , or other chromosome aberrations .
thus , studies of dsb repair control during mitosis are important to understand the origin of genomic instability , which causes cell tumorigenesis . in addition ,
activation of c - nhej during mitosis causes severe damage to growing cells like cancer cells , but the activation of nhej by itself would not be detrimental to interphase cells
. this property would be useful for the development of anticancer drugs in the future .
|
double - strand breaks ( dsbs ) are one of the severest types of dna damage .
unrepaired dsbs easily induce cell death and chromosome aberrations . to maintain genomic stability , cells have checkpoint and dsb repair systems to respond to dna damage throughout most of the cell cycle .
the failure of this process often results in apoptosis or genomic instability , such as aneuploidy , deletion , or translocation .
therefore , dsb repair is essential for maintenance of genomic stability . during mitosis
, however , cells seem to suppress the dna damage response and proceed to the next g1 phase , even if there are unrepaired dsbs . the biological significance of this suppression is not known . in this review , we summarize recent studies of mitotic dsb repair and discuss the mechanisms of suppression of dsb repair during mitosis .
dsb repair , which maintains genomic integrity in other phases of the cell cycle , is rather toxic to cells during mitosis , often resulting in chromosome missegregation and aberration .
cells have multiple safeguards to prevent genomic instability during mitosis : inhibition of 53bp1 or brca1 localization to dsb sites , which is important to promote non - homologous end joining or homologous recombination , respectively , and also modulation of the non - homologous end joining core complex to inhibit dsb repair .
we discuss how dsbs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability .
|
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epilepsy is the second most prevalent neurological disorder ( \documentclass[12pt]{minimal }
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} { } \(\sim 1\ )
\end{document}% prevalence ) affecting approximately 67 million people worldwide with up to 75% from developing countries .
eeg is important in developing countries for various clinical uses , including evaluation of suitability for epilepsy surgery and treatment of status epilepticus .
epilepsy diagnosis is complicated by the fact that electrical potentials recorded from scalp electroencephalogram ( eeg ) have poor sensitivity to localized epileptiform activity , especially high frequency oscillations ( hfos ) which now attract a lot of attention as a promising marker of epileptogenesis and ictogenesis . misdiagnosis of epilepsy is very common in patients of all ages and occurs in up to 50% of the patients , .
the hfos occupy the gamma ( 30100 hz ) , ripple ( 100200 hz ) and fast ripple ( 200500 hz ) bands along with delta , theta , alpha and beta bands making up the remainder of the spectral content of eeg .
appearance of fast eeg rhythms at the onset of seizures in humans and animal models of epilepsy has been described in many studies and first reported in humans by fisher et al . and allen et al . .
fast rhythms are especially evident in intracranial recordings in patients considered for surgical treatment of their drug - resistant epilepsy . in recent years , there has been a surge in the number of publications on hfos and their role in epilepsy ( for reviews , see , . numerous studies on animal models and epilepsy patients have revealed that : 1 ) hfos are one of the most common early manifestations of seizures recorded within minutes before seizure onset ; 2 ) hfos appear to be the most likely eeg correlate of a seizure onset zone ( soz ) ; and 3 ) the removal of hfo - generating areas correlates with good surgical outcome , , , . thus , hfos appear to be an excellent marker for the epileptogenic zone but their recording from scalp with conventional disc electrodes is difficult .
eeg has been a very useful research and clinical instrument for almost a century since its first human use by hans berger in 1920s .
the distance to the brain and the relatively large electrical impedance of the skull significantly reduce small electrical signals generated by individual neurons . to be detectable at the scalp ,
the signal should be produced by a relatively large population of neurons acting more or less synchronously at scales of at least several centimeters . only in this case
does the sum of individual neuronal signals become large enough to be detected at the scalp .
synchronous activity within neuronal assemblies is now considered an important mechanism of information processing , and this synchrony is expressed through neuronal oscillations occurring at different frequencies spanning several orders of magnitude from infraslow ( \documentclass[12pt]{minimal }
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} { } \(\sim 0.01\ )
\end{document } hz ) up to ultrafast ( \documentclass[12pt]{minimal }
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} { } \(\sim 1000\ )
\end{document } hz ) ( for a comprehensive review of brain oscillations , see .
there is an intricate relationship between temporal and spatial scales of brain oscillations in that higher frequency oscillations ( e.g. , high gamma band > 60 hz ) usually occur at smaller distances between sources and involve smaller neuronal populations .
this is because more precise synchronization dictated by shorter oscillatory periods can be achieved only through shorter neuronal connections with shorter propagation times or non - synaptic communication such as ephaptic interactions and gap junctions .
long distance connections have longer propagation times and therefore synchronization at larger distances is less precise and preferentially occurs at lower frequencies ( e.g. , within delta , theta , alpha and beta bands ) . as a result , high frequency oscillations > 60 hz usually reflect activity within well localized neuronal assemblies at spatial scales of a few mm , which are not typically visible from the scalp .
thus , conventional eeg has lower sensitivity to high frequency components of brain activity , .
conventional scalp eeg is also contaminated by noise represented by non - brain electrical activity such as ocular artifacts , scalp muscle potentials , and the electrocardiogram .
additional noise is occasionally produced by abnormally large electrode impedance , electrode movement , amplifier drifts , etc .
the noise can be as large as , or even larger than , the brain potential of interest and represents a significant problem for electroencephalographers and neurologists .
this noise shows high spatial coherence due to smearing effects of the head volume conductor . conventional disc eeg recordings also have reference electrode problems .
a common average reference and concentric electrodes have been proposed to resolve the reference electrode problems . however , in the common average reference recordings , it is possible that components present in most of the electrodes but absent or minimal in the electrode of interest may appear as ghost potentials .
thus , there is a strong need to develop new types of electrodes beyond conventional disc electrodes . to overcome the poor signal - to - noise ratio and reference problems of the disc electrodes besio et al .
have developed the tripolar concentric ring electrode ( tcre ) , a transformative electrode configuration .
the tcre consists of three electrode elements - outer ring , middle ring , and the central disc ( fig . 1 , b ) .
it is distinctively different from the disc electrode which has a single element ( fig .
the novelty of the tcre lies in a new principle of recording electrical activity of the brain from the scalp through its three closely spaced elements .
it can provide three separate signals from the three electrode elements used to record two bipolar differential signals for the tripolar laplacian derivation first described in as a weighted sum { 16*(m - d)-(o - d ) } where o , m , and d are the potentials on the outer ring , middle ring , and central disc , respectively .
the tripolar signal is a ' hardware realization ' of the laplacian i.e. , the second spatial derivative of the scalp signal . unlike other software methods ( ' laplacian montages ' in conventional eeg systems ) which approximate the laplacian by taking differences between disc electrodes placed , at best , > 1 cm apart
, the tcre performs the laplacian automatically and takes bipolar differences of the surface potentials from closely spaced ( \documentclass[12pt]{minimal }
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} { } \(\sim 1\ )
\end{document } mm ) concentric electrode elements with our custom preamplifier . with the electrode elements so close to each other , globally originating sources such as eye blinks , muscle or motion - related artifacts
contribute nearly equally and are attenuated sharply when bipolar differences are performed by the preamplifier . compared with disc signals , eeg recorded with the tcre ( teeg as described by ) has about a 4-fold ( 374% ) increase in signal - to - noise ratio ( snr ) and less than one - tenth ( 8.27% ) the mutual information between signals recorded from two adjacent tcres ( which suggests a significant attenuation of volume conductance effects and , as a result , higher spatial resolution ) , .
the tcre also has strong attenuation of various artifacts , 100 db at one radius from the electrode .
figure 1.conventional disc electrode ( a ) and tripolar concentric ring electrode ( b ) . conventional disc electrode ( a ) and tripolar concentric ring electrode ( b ) .
the current study presents preliminary data from patients with epilepsy in whom teeg was recorded in parallel with clinical conventional scalp eeg .
the goal is to demonstrate that tripolar electrodes may provide a unique opportunity to record hfos from scalp and thus improve diagnosis of epilepsy and localization of the seizure onset and the irritative zones .
their implementation into clinical practice may eventually help determine where to place and reduce the number of intracranial grids and depth electrodes to be implanted during presurgical evaluation of patients .
patients for this study were recruited from the national institute of neurology and neurosurgery ( ninn ; ten patients ) and rhode island hospital ( rih ; two patients ) .
patients were referred by the epilepsy clinic in each institution with the diagnosis of drug resistant epilepsy using the international league against epilepsy criteria .
diagnosis of epilepsy and epileptic seizures was based on the international classification of seizures 1981 and epileptic syndromes 1989 .
ictal and non - ictal recordings were obtained ; for non - ictal recordings lateralization and localization of the irritative zone ( iz ) was determined whereas in ictal recordings the soz or iz was identified with the appearance of behavioral changes associated with focal low voltage , fast activity , flattening or slow wave interruption . to verify that tcres can be used to record physiological signals we recorded from three healthy subjects , one at stanford school of medicine and two at the university of rhode island ( uri ) placing conventional disc electrodes over the occipital lobe ( at the o1 and o2 locations of the 1020 international electrode system with reference at a1 and ground electrode placed on the right collar bone ) .
two tcres were placed right next to the conventional electrodes at the o1 and o2 locations ( o1 ' and o2 ' positions , respectively ) . the tcre eeg signals ( teeg )
were first preconditioned with our custom made preamplifiers ( gain \documentclass[12pt]{minimal }
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} { } \(=20\ )
\end{document } ) and then passed to the bipolar inputs of the ( stanford - eeg-1200 , nihon kohden corporation , tokyo , japan ; uri - grass technologies aura ltm-64 , grass technologies , west warwick , ri ) clinical eeg recording system .
all signals were recorded with 1 to 120 hz band - width at 500 s / s and stored to the hard drive for post processing .
the subjects were asked to close and open their eyes repeatedly to record the presence or absence of alpha rhythms , respectively .
the study 's recording protocol was designed in such a way as to avoid any interference with clinical eeg recording and evaluation . at both the ninn and rih during the attachment of clinical conventional disc electrodes ( referred to as ' eeg electrodes ' or ' eeg signals ' in the subsequent text ) ,
the patient 's scalp was cleaned with nuprep and then eeg electrodes were placed at the 1020 international electrode system locations using ten-20 paste .
collodion was also used to help hold all electrodes in place at the ninn . to obtain teeg recordings in parallel to the clinical eeg
, the tcres were placed just behind the disc electrodes in locations close to the 1010 sites and attached to the scalp with ten-20 paste ( fig .
the ground was placed on the forehead ( rih , ninn ) and the reference electrode was placed on the forehead at rih and on the oz location at ninn .
clinical eeg was recorded with the comet as40 system ( grass technologies , west warwick , ri ) and stored separately for further clinical evaluation .
the eeg sampling rate was 200 samples per second ( s / s ) and the low - pass filter was 70 hz ( ninn and rih ) .
the teeg data were pre - amplified with the gain equal to either 6 ( for eight patients ) or 100 ( for four patients ) and amplified and digitized with an aura ltm-64 system ( grass technologies , west warwick , ri ) at different sampling frequencies for different patients . for four patients
the data were filtered 1100 hz and digitized at 200 s / s , another six were filtered 1200 hz and digitized at 400 s / s and for the remaining two patients the data were filtered 1250 hz and 1500 hz respectively and digitized at 1600 s / s . the 60 hz notch filter was used for all patients .
the recording sessions at the ninn usually lasted for six hours , from around 7 am to 1 pm .
for the two patients at rih , the recording was stopped shortly after the patient had a seizure ( 130 minutes total ) and for the other the recording lasted 66 minutes .
the ninn recording protocol included requests to patients to be sleep deprived the night before coming for a video - eeg monitoring and all patients signed an additional consent form as antiepileptic drugs dosage was reduced by half the previous day of the recording .
recorded data were reviewed by board certified neurologists and seizure onset time and duration were determined for each seizure .
seizure onset time was defined as the beginning of the first observable seizure pattern in either eeg or teeg .
note : t7/p7 and t8/p8 are the same as t3/t5 and t4/t6 in 1020 nomenclature .
the blue rings are for standard 1020 electrode locations . the 105 montage with tcres ( red ) placed near the 1020 locations .
note : t7/p7 and t8/p8 are the same as t3/t5 and t4/t6 in 1020 nomenclature .
the data were stored on the hard drive and exported from twin ( grass technologies , west warwick , ri ) into ascii text files that were then imported to matlab ( mathworks , natick , ma ) and converted to .mat files . for the teeg ,
next we used a modified version of the algorithm reported by gardner et al . for detection of hfos combined with the visual inspection to rule out high frequency artifacts .
the algorithm performed a continuous short - time fourier transform to calculate the power within a particular frequency band over consecutive and half - overlapping one - second epochs using a hamming tapering window . as a result
simple thresholding was also used to find events which had power significantly higher than the interictal background eeg .
then the time periods when the hfo power was exceeding its average plus two standard deviations were found .
frequency bands starting at high gamma band ( \documentclass[12pt]{minimal }
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} { } \(\sim 60\ )
\end{document } hz ) and up to the upper bound of the band - pass filter that varied for individual patients were searched for hfos .
we selected segments up to an hour prior to the onset of the seizures and generated spectrograms to follow the temporal dynamics of pre - seizure hfos .
for the patients who had epileptiform activity only but no seizures we calculated spectrograms on 10 minute long segments centered on the periods of epileptiform activity .
for the patients who did not have either epileptiform activity or seizures we performed spectral analysis on multiple 10 minute long segments at various times during the recording . to assess the relationship between teeg channels containing hfos preceding seizures and the soz the ratio between the number of teeg channels with hfos in the soz and the total number of teeg channels with hfos was calculated for each patient whose seizure was recorded .
we also calculated the ratio between the total number of teeg channels with hfos and the total number of teeg channels to assess how widespread the hfos were for each patient .
both ratios were averaged for the five patients with seizures included in this study . only the channels containing hfos preceding the seizures were assessed .
soz or iz was determined for each patient independently by three epileptologists ( iemj , jng , and rsf ) based on eeg data and videos only .
the epileptologists did not have access to teeg data and were not aware of the hfo detection results .
in one patient the third soz was determined by the location of resection by the surgeon . a disjunctive ( logical or )
that is all channels determined as part of the soz ( iz ) by either one of the epileptologists were considered to be a part of the soz ( iz ) .
first , we demonstrate the suitability of tcres to provide a sound eeg signal sensitive to physiological rhythms such as alpha activity as well as their insensitivity to myogenic activity which often corrupts scalp eeg recordings especially in the high frequency range > 30 hz .
if the tcre attenuates volume conductance effects and has a greater sensitivity to local sources , how good is it for recording global brain rhythms such as alpha activity ?
figure 3 , ( a / c ) shows the signals that were recorded with eyes closed / open and their corresponding power spectral densities are presented in fig . 3 , ( b / d ) .
the alpha waves were present in teeg ( top trace , blue ) coinciding with the corresponding alpha rhythm recorded by conventional disc electrodes ( middle and bottom traces , red and green respectively ) ( fig .
the alpha waves were blocked in both teeg and eeg when the eyes were open ( fig .
3 , c , d ) . thus , this result demonstrates the ability of tcres to record physiological rhythms .
figure 3.global physiological rhythms such as alpha activity observable with eyes closed can be recorded by tcres .
panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 . the middle trace ( red )
both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) .
the bottom trace ( green ) is from the o1 electrode with respect to the a1 reference electrode .
panel b : the power spectral densities for the signals of panel a. panels c and d are the signals and power spectral densities when the eyes are open .
it is evident that the alpha waves are present in both teeg and eeg records , specifically between 4 and 5 seconds ( panel a ) , which is confirmed by the spectra displaying a peak just below 10 hz ( panel b ) but are not seen in panels c and d. global physiological rhythms such as alpha activity observable with eyes closed can be recorded by tcres .
panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 .
both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) .
the bottom trace ( green ) is from the o1 electrode with respect to the a1 reference electrode .
panel b : the power spectral densities for the signals of panel a. panels c and d are the signals and power spectral densities when the eyes are open .
it is evident that the alpha waves are present in both teeg and eeg records , specifically between 4 and 5 seconds ( panel a ) , which is confirmed by the spectra displaying a peak just below 10 hz ( panel b ) but are not seen in panels c and d. a relative insensitivity of tcres to myogenic activity is demonstrated by teeg and eeg records obtained from the same subject during head movements ( fig .
the bottom trace ( green ) it is evident that from about 3 to 6 seconds there is a high - frequency contamination in the bipolar disc recordings that is significantly attenuated in the teeg ( top trace , blue ) .
this high - frequency interference coincided with head movements and is most likely caused by muscle artifacts .
4 shows the power spectral densities for the traces in panel a. the teeg power ( blue ) has the lowest power in the higher frequencies range where the muscle artifact is prominent in the records from the conventional bipolar disc signals . from our experience , such attenuation of myogenic artifacts
panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 .
both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) .
the bottom trace ( green ) is from the o1 disc electrode with respect to the a1 reference electrode .
conventional eeg is heavily corrupted by muscle artifacts which are evident from approximately 3 to 6 seconds of the record ( middle [ red ] and bottom [ green ] traces ) coinciding with head movements by the subject .
these artifacts are nearly unnoticeable in the teeg ( top trace [ blue ] ) .
panel b : the power spectral densities for the signals shown in panel a. the power spectrum for the teeg ( blue ) has much less high frequency power ( > 30 hz ) due to myogenic activity .
panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 .
both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) .
the bottom trace ( green ) is from the o1 disc electrode with respect to the a1 reference electrode .
conventional eeg is heavily corrupted by muscle artifacts which are evident from approximately 3 to 6 seconds of the record ( middle [ red ] and bottom [ green ] traces ) coinciding with head movements by the subject .
these artifacts are nearly unnoticeable in the teeg ( top trace [ blue ] ) .
panel b : the power spectral densities for the signals shown in panel a. the power spectrum for the teeg ( blue ) has much less high frequency power ( > 30 hz ) due to myogenic activity .
we separated the patients into 3 groups : ( 1 ) patients who had clinical seizures ( n\documentclass[12pt]{minimal }
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simultaneously recorded eeg and teeg and the corresponding spectrograms from representative patients are shown in fig . 5 and 6 .
5 the patient was a 42-year - old woman with seizure onset at 9 years .
she had been diagnosed with right temporal lobe epilepsy and had undergone a right temporal lobectomy but seizures persisted .
she was on clonazepam ( cnz ) , lamotrigine ( ltg ) and phenytoin ( pht ) at the time of the study . in figure 5
conventional eeg data are on panels a , b , e , f , and i and teeg data are on panels c , d , g , h , and j. the conventional eeg data were obtained from the bipolar montage ( channel f8-f4 ) .
this patient had a generalized seizure ( onset at approximately 610 seconds in the time scale of panels a - d in fig .
the seizure activity is evident by large increases in signal amplitude and power at all frequencies .
panels e , f , g , and h are eleven - second eeg and teeg segments ( marked by the black line in panel c ) shown at higher sequential resolution .
note the series of high gamma - band hfos between approximately 60 to 80 hz ( highlighted by ellipse in panel c ) occurring about every two seconds which are clearly evident in the teeg but not eeg , starting approximately 10 min prior to the generalized seizure activity ( compare panels a and c as well as e and g ) . with further zoom in panels i and j showing two - second segments
, one can see a high frequency burst in the teeg ( black horizontal line in the panel j ) which is not present in the eeg ( panel i ) .
conventional eeg ( bipolar montage , channel f8-f4 , panels b , f and i , 170 hz , 200 s / s ) , teeg ( from a tripolar electrode placed right behind the f8 position ( f8 ' ) , panels d , h and j , 1100 hz , 200 s / s , ) and their spectrograms ( panels a , e and panels c , g for eeg and teeg , respectively ) starting \documentclass[12pt]{minimal }
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} { } \(\sim 10\ )
\end{document } min before and continuing during a generalized seizure with onset marked with the dashed vertical black line .
panels e - h : eleven - second eeg and teeg segments ( marked by the black line in panel c ) are shown at higher temporal resolution .
note a series of high gamma - band bursts hfos at 6080 hz ( highlighted by ellipse in c ) occurring about every 2 s , which are clearly seen in the teeg spectrogram only .
panels i , j : further zoom - in of two - second segment shows one hfo in teeg ( black horizontal line in the panel j ) while this hfo is absent in eeg ( panel i ) .
figure 6.panel b shows 12 minutes of bipolar eeg from fp2-f4 ( 170 hz , 200 s / s ) .
panel e shows 30 seconds of eeg from panel b at the onset of the generalized seizure ( dashed line ) .
note the high gamma - band burst hfos just prior to the partial seizure ( highlighted by ellipse in panel c ) .
conventional eeg ( bipolar montage , channel f8-f4 , panels b , f and i , 170 hz , 200 s / s ) , teeg ( from a tripolar electrode placed right behind the f8 position ( f8 ' ) , panels d , h and j , 1100 hz , 200 s / s , ) and their spectrograms ( panels a , e and panels c , g for eeg and teeg , respectively ) starting \documentclass[12pt]{minimal }
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} { } \(\sim 10\ )
\end{document } min before and continuing during a generalized seizure with onset marked with the dashed vertical black line .
panels e - h : eleven - second eeg and teeg segments ( marked by the black line in panel c ) are shown at higher temporal resolution .
note a series of high gamma - band bursts hfos at 6080 hz ( highlighted by ellipse in c ) occurring about every 2 s , which are clearly seen in the teeg spectrogram only .
panels i , j : further zoom - in of two - second segment shows one hfo in teeg ( black horizontal line in the panel j ) while this hfo is absent in eeg ( panel i ) .
panel b shows 12 minutes of bipolar eeg from fp2-f4 ( 170 hz , 200 s / s ) .
panel e shows 30 seconds of eeg from panel b at the onset of the generalized seizure ( dashed line ) .
note the high gamma - band burst hfos just prior to the partial seizure ( highlighted by ellipse in panel c ) .
this patient was a 45 year old male , with onset of epilepsy at 22 years .
his seizures consisted of simple partial seizures followed by complex partial with or without secondarily generalization .
he was on carbamazepine ( cbz ) and ltg at the time of the recording .
his magnetic resonance imaging showed left mesial temporal sclerosis and a right frontal venous angioma .
panel d shows teeg recorded from location fp2 ' directly behind the fp2 disc electrode .
, we also found gamma band bursts ( \documentclass[12pt]{minimal }
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} { } \(\sim 70\ )
\end{document } hz ) in the teeg , approximately three minutes before the seizures .
6(c ) highlights the high gamma - band burst hfos in the teeg at location fp2 ' approximately 4 min prior to the partial seizure .
the hfos are present throughout the pre - seizure spectrogram but became more consolidated around 70 hz about 3 min prior to the seizure .
it is also important that the teeg during the tonic seizure was less contaminated with muscle and movement artifacts than the eeg ( compare panels e and f ) .
also note the higher power in eeg ( compare panels a and c ) from approx .
400 to 650 s when the patient was still anxious / disoriented and moving while recovering from the seizure . during that same period
overall , hfos preceding seizures were present in teeg data from all five patients whose seizures were recorded .
for these five patients , out of all the teeg channels that contained hfos an average of 78.2% were also in the soz ( iz ) ( patient minimum : 42.9% ; maximum : 100% ) .
the average percentage of the total number of teeg channels recorded that contained hfos is equal to 35.5% ( patient minimum : 5.3% ; maximum : 73.7% ) .
figure 7 portrays the relationship between the clinical soz ( iz ) and hfo - containing channels for two patients that we present the spectrograms for in fig .
5 ( panel a ) and fig . 6 ( panel b ) respectively .
figure 7.panel a shows the relationship between the clinical seizure onset zone ( soz ) and hfo - containing channels for the patient from fig .
panel b shows the relationship between the clinical irritative zone ( iz ) and hfo - containing channels for the patient from fig .
soz or iz was determined for each patient independently by three epileptologists ( iemj , jng , and rsf ) based on eeg data and videos only .
panel a shows the relationship between the clinical seizure onset zone ( soz ) and hfo - containing channels for the patient from fig .
panel b shows the relationship between the clinical irritative zone ( iz ) and hfo - containing channels for the patient from fig .
soz or iz was determined for each patient independently by three epileptologists ( iemj , jng , and rsf ) based on eeg data and videos only .
it has been shown that eeg is frequently contaminated by artifacts originating from various sources such as scalp muscles , eye blinks , eye movements , or patient movement .
state what is intuitively obvious ; artifacts obscuring the eeg at the time of seizure onset can greatly hinder the interpretation of the recorded seizures . during a tonic or tonic - clonic seizure ,
muscle activity is very prominent and is reflected in the eeg by random , high frequency signals .
. showed that when eegs were moderately contaminated with muscle artifacts blind source separation techniques based on canonical correlation provides signals that are more reliable for source estimation than raw eeg .
they also found that in cases of severe muscle contamination of the eeg or when the muscle artifact had different spatial information than the spatial information of the epileptiform event , the blind source separation techniques based on canonical correlation did not work well .
in general , a major reason why eeg has not reached its full potential in epilepsy diagnosis is due to artifact contamination .
we have found that the tcre and the teeg automatically attenuate myogenic activity and movement artifacts .
in contrast to the typical examples of artifact contaminated conventional bipolar eeg , the teeg is much less contaminated by the muscle activity . it should be noted that conventional approaches to remove muscle artifacts by digital signal processing ( such as filtering ) lead to significant loss of information since the gamma band activity and hfos in general are within the bandwidth of the electromyogram ( emg ) . in other words , removing the emg will inevitably remove high frequency components of the brain activity , which is not a desirable result .
the teeg , although not as obvious , may also be contaminated with emg ( fig .
4 , a , top blue trace ) but from a much more local source .
the emg from more distant sources have nearly equal contributions on the elements of the tcre and are cancelled out when the potentials on the elements are subtracted from each other with bipolar differences . in this study , we show , in a limited number of patients , that high - power hfo activity is detected at specific locations on the scalp surface in the teeg records of patients with epilepsy .
this hfo activity was apparent prior to seizures . although there may have been hfo activity at various times in the teeg during the recordings the bandwidth of the hfo activity narrowed sharply and the power increased just prior to the seizure .
we did not see the hfo activity in the conventional eeg , even when using bipolar montages which somewhat attenuates global artifacts .
in some instances this may partially be due to the low - pass filter used to record the eeg .
the eeg low - pass filter was set to 70 hz and this cutoff frequency may have partially filtered out gamma band activity . however , the gamma band activity detected in the teeg was often well below 70 hz , in one case as low as 63 hz , and in these cases the gamma band activity was still undetected in the conventional eeg . when a low - pass digital filter was applied to the teeg at 70
hz the hfos were still present in the teeg for patients whose hfos were below 70 hz .
we do not know what the upper limit is for detecting hfos on the scalp .
in rats we have found an increase in power up to 300 hz during ptz - induced seizure electrographic activity recorded with teeg . in two patients having seizures
for whom the data were filtered 1250 hz and 1500 hz respectively and digitized at 1600 s / s we have detected hfo preceding seizures in gamma , ripple , and fast ripple bands at frequencies up to 425 hz .
a potential disadvantage of recording with tcre is deemphasis of non - artifactual signals with wide spatial distribution , for example , spike - waves .
we intend to develop the circuitry to record both teeg and an eeg approximation from the same sensor in the near future . despite a limited sample size of patients having seizures in this study ( 5 patients ) we were able to investigate a possible correlation between the clinically determined soz ( iz ) and the location of hfos detected by tcres .
hfos preceding seizures were present in teeg data from all five patients whose seizures were recorded in an average of 35.5% of the patient 's teeg channels . out of those channels containing hfos
an average of 78.2% were within soz ( iz ) determined independently by three epileptologists based on eeg data and videos . in all five patients who had recorded seizures , at least one channel in the soz exhibited hfos ( in one out of five patients hfos were present in a single teeg channel ) . in two patients no specific soz was determined but rather an iz .
for those two patients hfos were present in 7 and 4 teeg channels respectively out of 20 ( 35% ) and 19 ( 21% ) channels total respectively which is on par with the population average of 35.5% .
examples of the relationship between the soz ( iz ) and hfo - containing channels are presented in fig .
the third epileptologist ( jng ) mentioned 4 hz activity over central leads but no clear electrographic onset .
soz definitions from all three epileptologists ( iemj , jng , and rsf ) overlapped for two channels ( fp2 and f4 ) over the right frontal lobe .
these preliminary results support that teeg is capable of detecting hfos from the scalp , which are not usually seen in conventional eeg records .
further studies with more patients are needed to more accurately correlate locations of ( presumably abnormal ) hfo activity with clinically determined soz as well as the resected tissue in surgical cases .
due to the improved signal quality of teeg we hope that in the future we will be able to use teeg to detect seizures and trigger stimulation to attenuate seizures .
we have shown that transcranial focal electrical stimulation ( tfs ) via tcres in rat models was effective in reducing penicillin - induced myoclonic jerks , , pilocarpine - induced status epilepticus ( an extreme form of continuing seizures ) and in a third model , we applied tfs in rats treated by pentylenetetrazole ( ptz ) .
tfs significantly reduced ptz - induced hypersynchrony at the beta and gamma frequencies , most recently we developed an automatic non - invasive seizure control system based on tfs and tested it successfully on rats .
|
epilepsy is the second most prevalent neurological disorder ( \documentclass[12pt]{minimal }
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} { } \(\sim 1\ )
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} { } \(\sim 67\ )
\end{document } million people worldwide with up to 75% from developing countries .
the conventional electroencephalogram is plagued with artifacts from movements , muscles , and other sources .
tripolar concentric ring electrodes automatically attenuate muscle artifacts and provide improved signal quality .
we performed basic experiments in healthy humans to show that tripolar concentric ring electrodes can indeed record the physiological alpha waves while eyes are closed .
we then conducted concurrent recordings with conventional disc electrodes and tripolar concentric ring electrodes from patients with epilepsy .
we found that we could detect high frequency oscillations , a marker for early seizure development and epileptogenic zone , on the scalp surface that appeared to become more narrow - band just prior to seizures .
high frequency oscillations preceding seizures were present in an average of 35.5% of tripolar concentric ring electrode data channels for all the patients with epilepsy whose seizures were recorded and absent in the corresponding conventional disc electrode data .
an average of 78.2% of channels that contained high frequency oscillations were within the seizure onset or irritative zones determined independently by three epileptologists based on conventional disc electrode data and videos .
|
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in advanced stages , solid tumors can metastasize to various organs , including the central nervous system . in particular , breast , lung and prostate cancer metastases
current operative concepts are focused on lesions to the vertebra , while metastases to non - osseous spinal structures are rare and usually associated with advanced disease .
the ongoing advancements in oncologic therapies have led to improvements in the life expectancy of cancer patients , even in palliative situations .
therefore , an increasing incidence of the formerly rare symptomatic leptomeningeal metastases ( lm ) should be expected .
lm cause obstruction of cerebrospinal fluid ( csf ) circulation and compression of neurologic tissues .
the differential diagnosis of lm includes ruling out a wide range of malignant and benign conditions , such as congenital and degenerative lesions , infectious and autoimmune diseases and neurinoma . the radiologic discrimination between metastases and neurinomas is primarily based on distinct neuroimaging features , particularly number , size and growth pattern . whereas lm are often encountered as multiple small nodules at ower spinal structures ( e.g. the cauda equina ) , presumably due to gravity
, neurinomas appear as single lesions in the neuroforamen and might present at any height .
the clinical presentation of lm depends on the location and growth - pattern , often resulting in general symptoms such as nausea and head - aches due to interruption of the csf flow and , later signs of myelopathy due to compression of the spinal cord . on the other hand
, neurinomas usually affect single nerve roots with typical clinical symptoms in the associated dermatomes and peripheral nerves .
the present study reports the first case of a solitary leptomeningeal metastasis , mimicking a benign dumbbell - shaped neurinoma of a cervical nerve - root that was successfully treated with surgery and adjuvant radiochemotherapy ( rct ) .
a 47-year - old woman was referred to our institution because of progressive unilateral arm elevation weakness with an onset 8 weeks prior .
she presented with a clearly visible atrophy of the biceps and triceps muscles and substantial reduction of both muscular strength in arm elevation and elbow flexion and extension .
neurological examination revealed hyperactive biceps and triceps tendon reflexes and hypesthesia confined to c57 dermatomes , indicating cervical myelopathy .
the patient was diagnosed with breast cancer ( bc ) 9 years earlier and underwent unilateral mastectomy ( pt2 m , pn3a , m0 , g2 , r0 , estrogen receptor ( er ) immunoreactive score ( irs ) 12 , progesterone receptor ( pr ) irs 4 , and human epidermal growth factor receptor 2 ( her2/neu ) negative ) .
five years later , supraclavicular lymph node - metastases on her right side were found ; again , the patient declined chemotherapy .
six months later , a brain metastasis was diagnosed and rct was initiated ( corticosteroids , bevacizumab and capecitabine ) .
the patient rejected being treated with zoledronate for suspected bone metastasis , and she aborted rct 14 months later . until the occurrence of the current neurologic symptoms , regular follow - up examinations showed that the patient had stable disease . after admission , total spine and cranial magnetic resonance imaging ( mri )
scans were performed and revealed the previously diagnosed right parietal intracerebral metastasis and a solitary lesion in the cervical spinal canal .
the spinal tumor was located in the right neural foramen between c3/4 with an extraforaminal extension , thereby compressing the cervical plexus at levels c35 .
thus , the neuroimaging findings were consistent with a dumbbell - shaped neurinoma ( fig .
an abdominal staging computed tomography ( ct ) scan and a three - phase bone scintigraphy showed no further lesions .
the case was reviewed at an interdisciplinary tumor board , and according to the histological findings , resection followed by adjuvant therapy was recommended .
surgery was performed under general anesthesia using a ventral approach to the cervical spine , with the patient in the supine position .
after microsurgical exposure , the tumor presented as completely intradural with the characteristic appearance of a neurinoma ( fig .
2 ) . following resection of the intraspinal tumor and corpectomy , the vertebral bodies c3 and c4 were replaced ( pyramesh - cage , medtronic gmbh , meerbusch , germany ) and ventral plate - stabilization ( cslp , synthes gmbh , umkirch , germany ) from c25 was performed ( fig .
the intraoperative rapid frozen section showed disorganized nerve root tissue with cell nests of unknown entity .
the definitive histological examination of the tissue revealed epitheloid cells with irregular and hyperchromatic nuclei and scattered mitosis arranged in nests embedded in stroma rich in collagen fibers ( fig .
immunohistochemical staining revealed that the tumor cells were positive for cytokeratin ( ck ) 8 ( fig .
the final histopathology report specified the tumor as a metastatic lesion originating from the previously diagnosed bc .
postoperatively , the patient presented with minor dysphagia , which was treated effectively by logopedic therapy .
while the muscular weakness of the arm showed no significant changes during the inpatient stay , there was a clear improvement in the hypesthesia at the c57 dermatomes .
the patient was transferred to a medical oncology service and received combined rct . at the last follow - up ,
3 months after the operation , the neurologic symptoms were unaltered , and a spinal mri showed no signs of local recurrence ( fig . 5 ) .
a 47-year - old woman was referred to our institution because of progressive unilateral arm elevation weakness with an onset 8 weeks prior .
she presented with a clearly visible atrophy of the biceps and triceps muscles and substantial reduction of both muscular strength in arm elevation and elbow flexion and extension .
neurological examination revealed hyperactive biceps and triceps tendon reflexes and hypesthesia confined to c57 dermatomes , indicating cervical myelopathy .
the patient was diagnosed with breast cancer ( bc ) 9 years earlier and underwent unilateral mastectomy ( pt2 m , pn3a , m0 , g2 , r0 , estrogen receptor ( er ) immunoreactive score ( irs ) 12 , progesterone receptor ( pr ) irs 4 , and human epidermal growth factor receptor 2 ( her2/neu ) negative ) .
five years later , supraclavicular lymph node - metastases on her right side were found ; again , the patient declined chemotherapy .
six months later , a brain metastasis was diagnosed and rct was initiated ( corticosteroids , bevacizumab and capecitabine ) .
the patient rejected being treated with zoledronate for suspected bone metastasis , and she aborted rct 14 months later . until the occurrence of the current neurologic symptoms , regular follow - up examinations showed that the patient had stable disease .
after admission , total spine and cranial magnetic resonance imaging ( mri ) scans were performed and revealed the previously diagnosed right parietal intracerebral metastasis and a solitary lesion in the cervical spinal canal .
the spinal tumor was located in the right neural foramen between c3/4 with an extraforaminal extension , thereby compressing the cervical plexus at levels c35 .
thus , the neuroimaging findings were consistent with a dumbbell - shaped neurinoma ( fig . 1a
the case was reviewed at an interdisciplinary tumor board , and according to the histological findings , resection followed by adjuvant therapy was recommended .
surgery was performed under general anesthesia using a ventral approach to the cervical spine , with the patient in the supine position .
after microsurgical exposure , the tumor presented as completely intradural with the characteristic appearance of a neurinoma ( fig .
2 ) . following resection of the intraspinal tumor and corpectomy , the vertebral bodies c3 and c4 were replaced ( pyramesh - cage , medtronic gmbh , meerbusch , germany ) and ventral plate - stabilization ( cslp , synthes gmbh , umkirch , germany ) from c25 was performed ( fig .
the intraoperative rapid frozen section showed disorganized nerve root tissue with cell nests of unknown entity .
the definitive histological examination of the tissue revealed epitheloid cells with irregular and hyperchromatic nuclei and scattered mitosis arranged in nests embedded in stroma rich in collagen fibers ( fig .
immunohistochemical staining revealed that the tumor cells were positive for cytokeratin ( ck ) 8 ( fig .
the final histopathology report specified the tumor as a metastatic lesion originating from the previously diagnosed bc .
postoperatively , the patient presented with minor dysphagia , which was treated effectively by logopedic therapy .
while the muscular weakness of the arm showed no significant changes during the inpatient stay , there was a clear improvement in the hypesthesia at the c57 dermatomes .
the patient was transferred to a medical oncology service and received combined rct . at the last follow - up ,
3 months after the operation , the neurologic symptoms were unaltered , and a spinal mri showed no signs of local recurrence ( fig .
these intraforaminal lesions are usually amenable to microsurgical resection with excellent local disease control and good clinical outcome .
differential diagnosis comprises eliminating a multitude of conditions , including primary malignant neoplasms and metastatic disease to the leptomeninges .
a range of primary solid malignancies is known to spread into the spine and mimic the growth pattern of intraforaminal tumors . while the incidence of neurinoma reaches 0.30.5/100,000 persons per year , lm of solid tumors are rare and
still , lm represents an important neurologic complication of systemic bc with an estimated frequency of 343% , based on clinical research and autopsies .
interestingly , the incidence of lm is on the rise , supposedly because of more effective therapies and therefore , longer survival of bc patients . in neurinomas , a dumbbell - shaped growth pattern accounts for approximately 6% of the cases and these most often affect the cervical followed by the lumbar spine .
in contrast , in neuroimaging and autopsy , lm usually occurs as multiple small nodules . yet , with mri they present with a signal similar to nerve sheath tumors , which tend to be greater in size and occur as solitary masses .
while neurinomas are usually located close to cervical and lumbar nerve roots , lm are most frequently found in the lumbar and sacral regions among the conus and cauda equina .
this might be explained by the possible mechanisms of metastasis : besides direct transdural invasion , hematogenous or lymphatic dissemination , the most likely route is tertiary spreading from brain tumors via the csf and the effect of gravity might explain the caudal location of the lm
. therefore , localization and growth pattern are the most important indicators for the differential diagnosis .
still , in the present case , the localization , growth pattern and mri signal were concordant with a cervical neurinoma .
typical clinical signs of spinal neurinomas are segmental pain , root pain and motor deficits with varying onset and course , usually progressing slowly .
lm on the other hand are typically associated with headaches and nausea due to disruption of the csf circulation and neurologic deficits of sensory and motor function in the case of compression of the spinal cord or nerve roots .
the majority of motor sensory dysfunction caused by lm is found to be of a myelopathic pattern rather than the effect of a single nerve root , and most often affects the lower extremities . while the initial manifestation of lm can occur between several weeks and 17 years following the primary diagnosis of bc , rapid deterioration after the clinical diagnosis is common and the prognosis is poor .
mean survival after diagnosis of lm is about 7.5 months and one - year survival is 1022% . the described case presented with an atypical clinical presentation , consistent with a large neurinoma affecting the cervical plexus .
lm therapy is palliative and aims to prevent further neurologic deterioration and improve the quality of life . for lm , radiotherapy
is widely accepted , yet has not been proven to affect overall survival , and systemic as well as local chemotherapy are the focus of recent studies . until now
the present case report emphasizes the importance of the differentiation between benign neurinomas and solitary lm in cancer patients . surgical treatment combined with adjuvant or neoadjuvant therapy
however , a literature search shows that no conclusive optimal treatment has yet been put forth . considering the growing number of medium- and long - term cancer survivors , future studies should focus on the improvement of diagnostic and therapeutic measures for patients with lm .
written informed consent was obtained from the patient for publication of this case report and accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request .
we have no personal or financial conflicts of interest related to the preparation and publication of this manuscript .
following the statutes of the local ethics committee of the university hospital of cologne , no judgment is necessary for case reports with retrospective data - collection of clinical data when the informed consent of the patient is available .
c.k.b . was involved in conception and design of the study , interpretation of the data , and writing of the manuscript .
were involved in the literature analysis and critical review of the manuscript ; p.e . and
all authors approved the final version of the manuscript.key learning pointsrecognizing the importance of the differentiation between benign neurinomas and solitary leptomeningeal metastasis in cancer patients.recognizing , that surgical treatment combined with adjuvant or neoadjuvant therapy can improve neurologic function and lessen pain.a literature search shows that no conclusive optimal treatment has yet been put forth.considering the growing number of medium- and long - term cancer survivors , future studies should focus on the improvement of diagnostic and therapeutic measures for patients with leptomeningeal metastasis . recognizing the importance of the differentiation between benign neurinomas and solitary leptomeningeal metastasis in cancer patients.recognizing , that surgical treatment combined with adjuvant or neoadjuvant therapy can improve neurologic function and lessen pain.a literature search shows that no conclusive optimal treatment has yet been put forth.considering the growing number of medium- and long - term cancer survivors , future studies should focus on the improvement of diagnostic and therapeutic measures for patients with leptomeningeal metastasis . recognizing the importance of the differentiation between benign neurinomas and solitary leptomeningeal metastasis in cancer patients . recognizing , that surgical treatment combined with adjuvant or neoadjuvant therapy can improve neurologic function and lessen pain .
considering the growing number of medium- and long - term cancer survivors , future studies should focus on the improvement of diagnostic and therapeutic measures for patients with leptomeningeal metastasis .
recognizing the importance of the differentiation between benign neurinomas and solitary leptomeningeal metastasis in cancer patients.recognizing , that surgical treatment combined with adjuvant or neoadjuvant therapy can improve neurologic function and lessen pain.a literature search shows that no conclusive optimal treatment has yet been put forth.considering the growing number of medium- and long - term cancer survivors , future studies should focus on the improvement of diagnostic and therapeutic measures for patients with leptomeningeal metastasis . recognizing the importance of the differentiation between benign neurinomas and solitary leptomeningeal metastasis in cancer patients .
recognizing , that surgical treatment combined with adjuvant or neoadjuvant therapy can improve neurologic function and lessen pain .
considering the growing number of medium- and long - term cancer survivors , future studies should focus on the improvement of diagnostic and therapeutic measures for patients with leptomeningeal metastasis .
|
introductionspinal metastases are frequently encountered in patients with breast cancer . because of
recent improvements in oncologic therapies a growing incidence of symptomatic leptomeningeal metastases ( lm ) should be expected .
the differential diagnosis of lm comprises a wide range of conditions , including neurinoma . the radiologic discrimination between metastases and neurinomas is primarily based on distinct neuroimaging features , particularly number , size and growth pattern.presentation of casewe report the first case of a solitary leptomeningeal metastasis of a cervical nerve - root , which mimicked a benign dumbbell - shaped neurinoma , using neuroimaging and visualized intraoperatively .
the tumor was successfully treated with surgery followed by adjuvant radiochemotherapy ( rct).discussionwhile the patient history directs towards a metastasis , the localization , growth pattern and mri signal were concordant with a cervical neurinoma .
the current literature is not conclusive concerning the optimal choice of treatment ; the therapy is strictly palliative and indications for surgery remain individual decisions .
however , due to recent improvements in survival of patients with lm require reconsideration of established strategies.conclusionthe present case report and the reviewed literature point towards a growing clinical relevance of symptomatic lm in cancer patients and their possible atypical presentations and locations .
|
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intentional replantation ( intr ) is described as the intervention of purposely removing a tooth and following some extra - oral procedures , replacing it into its socket . despite increasing tendency to be a favored treatment alternative in cases of periodontally hopeless teeth ( pht ) , enhancement of periodontal tissue support and thus , increasing the lifetime and functional quality of replanted teeth is still one of the main concerns .
supportive treatments have been suggested to improve results obtained with intr . despite the contribution of these to periodontal health , only periodontal pockets and inflammation
were evaluated , and no detailed information was given about periodontal and mucogingival state . because of thin tissue phenotype , pht in mandibular anterior area may frequently subject to gingival recession in case of periodontal destruction .
therefore , mucogingival problem should be potentially considered in performing intr . although reports involving intr point out outcomes about survival and reduction of inflammation around treated teeth , there is limited data indicating the importance of mucogingival state .
in addition , no report represented treatment of pht with a two - step procedure involving intr and free gingival graft ( fgg ) .
therefore , objective of this report is to present 15-month results of treatment including intr and mucogingival surgery .
the report also aims to underline the contribution of supporting approaches to intr procedure in terms of periodontal tissue support and amount of keratinized gingiva .
a 20-year - old female was referred to kirikkale university periodontology department in june 2011 with mobility and discomfort in mandibular left lateral incisor .
clinically , tooth had malposition , severe periodontitis with 3 mobility , inflammation , and recession [ figure 1 ] .
baseline measurements included plaque index , gingival index , probing depth ( from the gingival margin [ gm ] to base of pocket ) , attachment level ( from cemento - enamel junction [ cej ] to base of pocket ) , gingival recession ( from cej to gm ) , and keratinized gingiva ( from gm to mucogingival junction ) [ table 1 ] .
radiographically , the root had huge radiolucent area demonstrating crater - shaped bony defect starting from the coronal third reaching to apical with completely lost buccal + lingual walls [ figure 2 ] .
pretreatment view of the mandibular left lateral incisor demonstrating significant coronal and lateral displacement with hyperemic and edematous gingiva baseline and follow - up clinical parameters pretreatment radiographic view . supporting alveolar bone
was completely lost after treatment planning , informed consent was signed , and remaining treatments were completed .
one week after endodontic therapy , intr was performed using the technique applied by demiralp et al . in brief , the tooth was extracted and put on a sterilized moisturized sponge [ figure 3 ] . after scaling and root planning , 100 mg /
ml tetracycline hcl ( boehringer , ingelheim am rhein , germany ) was applied to the root surface for 5 min .
the surface was rinsed with sterile distilled water for 1 min , granulation tissue inside the socket was gently debrided and tooth was replaced into the socket .
tooth was repositioned in most possible ideal position to eliminate traumatic occlusion resulted due to pathologic migration .
tooth stabilization was achieved from the incisal third with composite restorative material ( 3 m filtektm z250 , 3 m espe , st .
after intr , doxycycline was prescribed for 7 days and oral hygiene activities were continued .
professional cleaning was made at 3 week , and maintenance visits were scheduled . the tooth was gently extracted and the root surface was scaled and root planed three months after , radiographic view was uneventful , and pocket depths were shallow [ table 1 ] .
however , inflammation and mucogingival stress ( no keratinized tissue ) was detected [ figure 4 ] .
as the second step , fgg was utilized in accordance with the technique described by miller [ figure 5 ] .
sutures were removed 10 days following surgery and toothbrushing was discontinued during this time . recall visits including professional prophylaxis were arranged . at 15 months , splint was removed , and mobility was reevaluated .
three months following replantation procedure . note the lack of keratinized tissue ( a ) recipient site prepared for the graft .
( c ) view following graft suturation wilcoxon test was performed to evaluate the time - dependent change of the clinical parameters and significance level was set at p = 0.05 .
compared with baseline , significant improvement was detected at 6 , 9 , and 15 months periodontal measurements [ table 1 ] .
although gingival recession did not change at 6 months postsurgical visit , a slight decrease was observed in the subsequent follow - up .
gingiva was healthy with its firm view and no bleeding on probing was detected [ figure 6 ] .
radiographically , the amount of radiolucent area was diminished , and hard tissue formation was detectable around the root apex with no root resorption [ figure 7 ] . although slight discoloration occurred in attached gingiva and tooth toward the end of follow - up , patient was satisfied with outcomes of treatment .
note that the splint was removed and the gingiva was firm and healthy with a slight color change fifteen months posttreatment periapical radiographic view .
in modern periodontology , growing amount of evidence indicates that several therapeutic modalities can successfully result in keeping pht in place .
cortellini et al . compared outcomes of periodontal treatment with extraction + prosthetic replacement for pht and suggested that last step therapeutic procedures should be considered before extraction of pht .
intr , another last resort therapy , has demonstrated successful results in the treatment of pht . in the present report
, significant hard tissue formation was obtained , and replanted tooth was successfully retained during 15 months . even though fgg was proposed as supportive in mucogingival problems and prosthodontic and orthodontic treatments ,
a mucogingival problem was detected after intr and it was decided to utilize fgg around the replanted tooth . following surgery , in addition to the resolution of inflammation , 8 mm keratinized gingiva and minimal root coverage was obtained . consequently , it can be recommended that mucogingival problems and supportive treatment alternatives should be considered following intr procedure in which time the inflammation dissolves and clear decision can be made to perform mucogingival surgery .
enhancement of periodontal tissue support and thus , increasing the lifetime and functional quality of the replanted teeth is still one of the main concerns . in 2006 , tzm et al . described use of platelet rich plasma ( prp ) in intr of pht .
eighteen - month follow - up results of their case demonstrated new bone formation around the root apex .
two study groups combined different regenerative options , including enamel matrix derivatives ( emd ) , bone grafts , membranes and prp with intr to improve tissue support and suggested use of these materials with intr in order to have more successful and predictable results .
evaluated results of intr of pht with emd + demineralized freeze - dried bone allograft ( dfdba ) and showed that emd + dfdba might develop the outcomes of intr . despite comments of these literatures , only periodontal pocket depth and gingival inflammation parameters were evaluated , and no detailed information was given about mucogingival state . to the best of our knowledge ,
the present case is the first one demonstrating use of fgg to increase periodontal tissue support of a replanted tooth .
following utilization , inflammation symptoms were reduced , and minimal root coverage was obtained with keratinized gingiva enhancement .
in this case - report , 15-month results of mucogingival surgery after intr were presented . consequently , treated pht survived with healthy gingiva , reduced pocket depth , acceptable mucogingival relationship and new hard tissue formation . from the results of this single case lack of baseline periapical radiograph and standardized radiographic measurements , intr supported with fgg may be speculated as an alternative to keep pht for a period of time .
|
the aim of the report is to document 15-month results of mucogingival surgery applied following intentional replantation ( intr ) .
a mandibular left lateral incisor with severe periodontitis and malposition was replanted and splinted following root canal treatment .
three months after intr , a free gingival graft ( fgg ) was placed .
fifteen months follow - up of the case demonstrated significant improvement in periodontal healing parameters and significant bone fill was detected around the root apex .
as a conclusion , intr may be speculated as a convenient alternative to keep the periodontally hopeless teeth and supportive approaches such as fggs may improve the outcomes following intr .
|
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slipped capital femoral epiphysis ( scfe ) is an adolescent hip disorder with displacement of the capital femoral epiphysis from the metaphysis through the physis .
most scfes are varus ( medial and posterior epiphyseal displacement relative to the metaphysic ) but can occasionally be valgus ( lateral and superior epiphyseal displacement ) [ 14 ] .
the vast majority are idiopathic ; atypical scfes are those that occur due to an endocrine disorder [ 57 ] , renal failure osteodystrophy , or radiation therapy [ 7 , 9 , 10 ] .
the literature regarding the epidemiology and demographics of scfe first requires a general knowledge of the disorder .
the traditional clinical classification was acute , chronic , and acute on chronic [ 1216 ] , based on the patient 's history , physical examination , and roentgenograms .
an acute scfe is defined as those with symptoms for <3 weeks with an abrupt displacement through the proximal physis .
acute - on - chronic scfes are those with chronic symptoms initially and the subsequent development of acute symptoms .
newer classifications are more clinically useful , depend upon physeal stability , and predict the prognosis regarding subsequent avascular necrosis .
a stable scfe is defined as one where the child is able to ambulate , with or without crutches .
an unstable scfe is defined as one where the child can not ambulate , with or without crutches .
the radiographic classification depends upon the presence or absence of a hip effusion on ultrasonography [ 19 , 20 ] .
the absence of metaphyseal remodeling and the presence of an effusion define an unstable scfe ; metaphyseal remodeling and the absence of an effusion define a stable scfe .
unstable scfes have a much higher incidence of avascular necrosis ( up to 50% in some series ) compared to stable scfes ( nearly 0% ) .
radiographs demonstrate an inferior and posterior slip of the proximal femoral epiphysis relative to the metaphysis .
the gradual slip demonstrates radiographic signs of remodeling on the superior and anterior femoral metaphysis , and periosteal new - bone formation at the epiphyseal - metaphyseal junction posteriorly and inferiorly . in the early slip
as such it is usually seen early only on the lateral view , and both anteroposterior and lateral radiographs must be obtained .
other radiographic signs of an early scfe are the metaphyseal blanch sign of steel and klein 's line .
the metaphyseal blanch sign of steel is a radiographic double density seen on the anteroposterior view at the level of the metaphysis ; this double density reflects the posterior cortical lip of the epiphysis as it is beginning to slip posteriorly and is radiographically superimposed upon the metaphyseal density .
klein 's line is drawn along the anterior or superior aspect of the femoral neck ; the epiphysis should normally intersect this line .
in an early scfe the epiphysis will be flush with or even below this line ( figure 1 ) .
the first is the amount of displacement of the epiphysis on the metaphysis ( figure 2 ) .
a mild scfe is defined as epiphyseal - metaphyseal displacement < 1/3 the width of the metaphysis ; a moderate scfe 1/31/2 ; a severe scfe > 1/2 ( figure 2(a ) ) .
this method is less accurate than angular measurement since distinct landmarks are difficult to determine due to metaphyseal remodeling in the gradual stable scfe .
angular measurement uses the epiphyseal - shaft angle on the frog - lateral radiograph ( figure 2(b ) ) and is categorized into 3 groups : mild < 30 , moderate 3050 , and severe >
this classification is important for long - term prognosis as mild and moderate slips have a much better long - term prognosis than severe slips which demonstrate a more rapid development of degenerative hip disease [ 26 , 27 ] .
scfe has been known by many different names since its first description in the late 19th and early 20th centuries . prior to the last 40 years or so ,
even now there is no actual mesh ( medical subject heading ) term for slipped capital femoral epiphysis ( scfe ) .
slipped capital femoral epiphysis or scfe , must be searched as a keyword phrase . to ensure capture of all the published literature ,
older terms were also searched as keywords or keyword phrases . therefore the terms used to search for scfe
the last strategy was to combine these two sets of terms : ( slipped or slipping or sliding or displaced or displacement or detached or detachment or separated or separation ) combined with ( femur or head or capitis or epiphysis or epiphyseal or epiphysiolysis or epiphysiolyses ) .
the databases searched were pubmed ( 19472010 ) ( http://www.ncbi.nlm.nih.gov/pubmed/ ) , ovid medline ( 19472010 ) , embase ( 19472010 ) , worldcat ( 18802010 ) ( books and theses ) ( http://firstsearch.oclc.org/ ) , google scholar ( 18802010 ) ( http://scholar.google.com/ ) , web of knowledge ( 19872010 ) , and indexcat ( 18801961 ) ( index catalogue of the library of the surgeon - general 's office ) ( http://www.indexcat.nlm.nih.gov/ ) .
exclusion criteria were those manuscripts discussing surgery , therapy , rehabilitation , and any foreign language articles without an english abstract .
individual orthopaedic journals were also searched for articles published prior to 1966 that predate electronic medline indexing , including journal of bone and joint surgery ( american and british ) , clinical orthopaedics and related research , and acta orthopaedica scandinavica .
age groups were limited to those < 18 years old ; duplicate citations were removed .
these 1407 manuscripts were reviewed to find those that discussed any of the topics regarding scfe and epidemiology , demographics , incidence , prevalence , race , gender , family history , inheritance , genetics , age , bone age , weight ( either birth weight or normal weight ) , height , growth , maturation , any other anthropometric characteristics , seasonal variation , hormone , endocrine , congenital anomalies , collagen , immunoglobulin , and opposite hip .
of these 1407 manuscripts , 114 provided ample information and are the contents of this paper .
conventional quotation for scfe incidence in the literature is the number of cases per 100,000 ( usually for the age range 8 to 15 years old , although some use < 25 years of age ) and is used throughout this paper .
the incidence ( table 1 ) ranges from 0.2 per 100,000 in eastern japan to 17.15 in the northeastern united states .
recent studies indicate that the overall incidence in the united states is 10.8/100,000 , similar to 10.08 in boys in the kelsey et al .
's study . in japan it has increased to 2.22/100,000 for boys and 0.76/100,000 for girls . in olmsted county , minnesota ,
the incidence is increasing in some areas compared to previous reports ; in new mexico it has nearly tripled ( 6.05 versus 2.13 ) compared to an earlier study . in scotland
, the incidence has increased from 3.78 in 1981 to 9.66 in 2000 , correlated with rising obesity .
the incidence of scfe in southern sweden increased in rural areas for boys ( 7.5 versus 4.8 ) but decreased for girls ( 2.2 versus 3.8 ) .
no differences were noted in connecticut in white children between urban and nonurban settings ( 3.19 for both ) although there was an increased incidence in africans living in urban areas compared to nonurban areas ( 7.95 versus 1.35 ) .
there are differences by geographic region . in a recent study the highest incidence was in the northeast at 17.15 and the lowest in the midwest at 7.69 .
this is similar to an earlier study where the incidence in the northeast was 10.08 and 2.13 in new mexico .
race is classified using the definitions of eveleth and tanner : caucasians , africans in africa and of african ancestry , asiatics ( amerindians , hispanics , indonesian - malays ) , indo - mediterraneans ( inhabitants of the near east , north africa , and indian subcontinent ) , and australian aborigines and pacific island peoples .
the relative racial frequency ( rrf ) of scfe , with whites at 1.0 , is 4.5 for pacific islanders , 2.2 for africans , 1.05 for amerindian ( native americans and hispanics ) , 0.5 for indonesian - malay ( chinese , japanese , thai , vietnamese , etc . ) , and 0.1 for indo - mediterranean peoples ( near east , north african , or indian subcontinent ancestry ) .
more recent data indicates that these numbers , relative to caucasians , is 5.6 for polynesians , 3.9 for blacks , and 2.5 for hispanics ( figure 3(a ) ) [ 29 , 31 ] .
in new zealand the rrf was 5.6 for the maori peoples and 4.2 for other pacific islanders . in singapore the rrfs , relative to chinese , are 9.6 for indo - mediterraneans ( indian ) , 2.8 for malay , and 3.0 for mixed ( eurasians ) ( figure 3(b ) ) .
thus indo - mediterraneans ( indian ) had a 9-fold increased incidence compared to indo - malay ( chinese ) .
this is in contrast to another study where indo - mediterraneans had a 5 times lower frequency of scfe compared to indo - malays ( all groups
these racial differences most likely reflect the average body weights for each racial group and further support the significant role that obesity and mechanical stress plays in the etiology of scfe .
a less likely explanation is racial variability in acetabular depth and femoral head coverage ; the acetabulae in african children were deeper than white children in one study , but not in another study .
early in this century 90% occurred in males but has now decreased to ~60% . in a review of 4343 children ,
there are differences in gender by race , with indo - mediterraneans having the highest proportion of boys ( 90% ) and polynesians an equal male / female ratio .
early in this century the average age was much higher with a gradual decrease over time ( figure 4(a ) ) .
fifteen years ago the average age was 13.5 years for boys and 12.0 years for girls and is now 12.0 years for boys and 11.2 years for girls ( table 3 ) .
in scotland , the age has dropped from 13.4 to 12.6 years over a 20-year period .
one study demonstrated different ages at presentation by racial group ( figure 4(c ) ) .
the physiologic age range during which scfe occurs is less variable than the chronologic age range ( figure 5 ) , termed the narrow window [ 56 , 63 ] . the average symptom duration for stable scfes is 4 to 5 months ( with minimal difference by gender ) . in 2482 children ,
the average symptom duration was 4.3 months ; 4.5 months for boys and 3.6 months for girls ( table 4 ) .
two recent studies have noted a decrease in symptom duration to 2 - 3 months [ 46 , 88 ] .
although there is a general correlation between symptom duration and scfe severity there is considerable variability ( figure 6(a ) ) . for any given individual child , slip severity and
symptom duration is unique ; in a large population , there is a weak positive correlation with slip severity and symptom duration .
mild scfes have a shorter duration of symptoms than severe scfes ( figure 6(b ) ) which suggests that all scfes start at a similar age ; those children with severe scfes just had a longer symptom duration and time to presentation than those with milder scfes .
in a review of 328 stable scfes this was documented ; a child with a stable scfe was 2.0 times more likely to have a moderate or severe scfe if > than 12.5 years at diagnosis than if < 12.5 years at diagnosis , and a child with a stable scfe is 4.1 times more likely to have a moderate or severe scfe if the duration of symptoms is > than 2 months than if 2 months .
other positive predictors of increasing time to diagnosis are knee / distal thigh pain , medicaid coverage , lower family income , and a stable scfe .
slip severity and symptom duration is greater in children presenting with knee pain compared to other symptoms [ 8891 ] . in a study of 45 children ,
15 presented solely with knee pain having an average symptom duration of 8.3 months ; 74% of the scfes were severe .
the remaining 30 presented with other symptoms with an average symptom duration of 6.5 months ; 24% of the scfes were severe .
reviewed 87 children with stable scfes , 20 had a delayed diagnosis , with 8 ( 40% ) being severe . in leeds ,
england , those children with scfe diagnosed in the emergency department compared to general practitioners had a shorter symptom duration ( 95 versus 119 days ) and fewer severe scfes ( 8 versus 21% ) .
one study , however , did not see a difference in symptom duration in scfe children where the diagnosis had been initially missed due to the absence of hip pain ( average symptom duration 127 days and 146 days in those whose diagnosis was and was not missed ) .
it would seem reasonable that as symptom duration increases radiographic changes should concomitantly progress ( more metaphyseal remodeling ) . the presence or absence of a positive klein line and metaphyseal remodeling ( superior and anterior smoothing , inferior and posterior buttressing ) were directly correlated to symptom duration but with significant variability .
those with more metaphyseal changes had higher bmis , interpreted as the larger body mass per unit height resulting in more bony reaction / adaptation according to wolf 's law .
the age at diagnosis of the 1st scfe correlates with scfe severity and is also related to the narrow physiologic window [ 56 , 63 ] . subtracting the average symptom duration for the mild , moderate , and severe categories from the average age at presentation results in a similar average age at onset ( figure 6(c ) ) , confirming the narrow bone age window .
50% of children with scfe are > 95th percentile weight for age [ 30 , 9496 ] .
age at diagnosis decreases with increasing obesity ; 12.4 years for those over the 95th percentile weight for age and 14.3 years for those under the 10th percentile weight for age .
recently body mass index ( bmi ) [ 97 , 98 ] has been used to evaluate body habitus ; the average bmi in scfe children is 2530 kg / m or > 85th percentile [ 39 , 46 , 99102 ] .
the average bmi of children with bilateral scfe is higher than with unilateral scfe ( 26.8 versus 31.1 kg / m ) .
obese children have decreased femoral anteversion , and scfe children have femoral retroversion even in the noninvolved hip [ 103 , 104 ] .
this , along with biomechanical studies demonstrating an increased physeal stress in retroverted hips and the many demographic studies associating obesity with scfe , supports the theory that obesity is intimately involved in the development of most idiopathic scfes . in latitudes north of 40 scfe presents more frequently in the late summer and autumn months ( figure 7(a ) ) [ 34 , 58 , 59 , 61 , 62 , 106 , 107 ] .
subtracting symptom duration from the time of presentation demonstrates that the scfe onset is in the early summer / late spring months ( figure 7(b ) ) .
the reported proportion of bilaterality depends upon the study , method of radiographic measurement , race , and treatment ( table 5 ) .
most series report 18 to 50% bilaterality with recent follow - up studies into adulthood quoting 63% [ 34 , 109 , 110 ] and 66% .
bilaterality is more frequent in africans ( 34% ) than hispanics ( 17% ) , whites ( 17% ) , or asians ( 18% ) .
treatment may affect risk of bilaterality ; bilaterality is 36% with in situ fixation and 7% spica cast treatment .
close attention is mandatory to the opposite normal hip in those children with a unilateral scfe treated by in situ fixation .
the age at presentation is younger in children who present with a unilateral scfe that later develops bilateral scfes compared to those who do not develop bilateral scfes [ 17 , 50 , 59 , 113116 ] .
this age difference is seen in both chronologic age ( 12 versus 13 years of age ) [ 17 , 30 , 115 , 116 ] and bone age .
in one study the overall difference in chronologic age in those with a unilateral scfe that became bilateral compared to those that stayed unilateral was 1 year ( 12 versus 13 years ) ; in another study 0.9 years for girls ( 11.0 versus 11.9 years ) and 2.2 years ( 12.1 versus 14.2 years ) for boys ; and in a 3rd study 11.5 versus 12.7 years , with those children aging < 12 years having a unilateral scfe demonstrated a 3.8 times increased risk of developing a contralateral scfe . in a study of 50 children with unilateral scfe using a modified oxford hip bone age , a contralateral scfe occurred in 85% of hips having a score of 16 , 11% of hips having a score of 21 , and never when the score was 22 . once the triradiate cartilage has closed , the risk of a contralateral scfe is only 4% , although another study did not find any association between the status of the triradiate cartilage , other skeletal maturity markers , and subsequent bilaterality .
the risk of bilaterality is increased when the posterior slope angle of the capital femoral epiphysis is higher [ 119 , 120 ] . in children with bilateral scfes , 5060% present with simultaneous bilateral involvement . in those children with sequential bilateral scfes ,
8090% of the second scfes occur within the first 18 months after the first scfe ( figure 8) [ 17 , 29 , 30 , 34 , 50 , 67 , 102 , 121 , 122 ] .
tests for a disease are evaluated in terms of sensitivity , specificity , positive predictive value , and negative predictive value .
sensitivity is the proportion of individuals in a tested population who actually have the disease and are identified as having it with the test .
specificity is the proportion of individuals in a tested population who do not have a given disease and are identified as not having it with the test .
the probability that a person with a positive result truly has the disease is the positive predictive value ; the probability that a person with a negative test truly does not have the disease is the negative predictive value .
when evaluating a child with a newly diagnosed scfe , the underlying etiology immediately comes into consideration .
this is for both diagnostic concerns with potential medical issues as well as orthopaedic treatment .
the underlying medical issues involve significant anesthetic concerns ; treatment involves the question of prophylactic fixation of the opposite hip when the child presents with a unilateral scfe . any
test that can give an accurate negative predictive value for a child with a scfe not having an atypical scfe is important .
if the test has a high negative predictive value , the clinician can be confident that the scfe is idiopathic .
the history of prior radiation therapy is usually discovered in the initial evaluation . however , many times it is the orthopaedic surgeon who picks up on an underlying medical problem as the etiology of the scfe ( e.g. endocrine dysfunction , renal osteodystrophy ) . in 2001
the age - weight test was described to assist in the differentiation between an idiopathic and atypical scfe .
the age - weight test is defined as negative when age < 16 years and weight 50th percentile and positive when beyond these boundaries ( table 6 ) .
is the child having an atypical scfe , absence of disease is the child having an idiopathic scfe .
the demographics of 433 children ( 285 idiopathic , 148 atypical ) with 612 scfes were studied to define predictors of atypical scfes .
there was significant variability in both age and bilaterality between the atypical and idiopathic groups ( figure 9 ) .
multiple logistic regression analysis demonstrated that age and weight were predictors of an atypical scfe . for two patients of equal weight , those < 10 , or > 16 years of age are 4.2 times more likely to have an atypical scfe ; for two patients of equal age , those < 50th percentile weight are 8.4 times more likely .
the probability of a child with a negative test having an idiopathic scfe is 93% , and a child with positive test having an atypical scfe is 52% . in the same year
. showed that the height at diagnosis was an important predictor of an endocrinopathy . the sensitivity and negative predictive value of detecting an underlying endocrinopathy in a patient with a scfe who was < 10th percentile in height for age were 90.2% and 98% .
the age - weight test was further confirmed in 2006 , along with the definition of the height and age - height tests .
the age - height test was defined using the same cells as the age - weight test except that the percentiles applied to height rather than weight .
the height test is defined as positive if the child 's height is 10th percentile and negative if > 10th percentile for age .
the height test has a positive and negative predictive value of 75% and 97% . of these three tests ,
the height test is likely to be the most useful in the differentiation between a typical and atypical scfe if the height of a child can be obtained .
when the height can not be obtained , the age weight test will result in a similar negative predictive value but with a lower positive predictive value .
thus the weight , and where possible , the height of any child newly diagnosed with a scfe should be obtained to assist in the differentiation between an atypical and idiopathic scfe .
familial scfe was first noted in the english language literature in 1940 with many more subsequent descriptions [ 6574 , 7680 , 8387 ] .
proposed patterns of genetic transmission include x - linked dominant , autosomal dominant with variable penetrance , and autosomal recessive [ 68 , 71 , 73 , 77 ] .
hla phenotype studies in children demonstrate no common findings ( table 7 ) [ 74 , 75 , 7982 , 86 ] .
other skeletal abnormalitiesin most children with scfe , femoral anteversion is decreased . in a 3-dimensional ct scan study of 30 scfes
there was a reduction in femoral anteversion from 12.7 to 7.0 as well as a reduction in the femoral neck shaft angle from 141 to 134. in another ct scan study of 25 scfes a reduction of femoral version 1 was noted in the involved hip and 7 in the uninvolved hip in unilateral cases . in a 3rd ct scan study of 25 scfes femoral
anteversion was 9.8 and 25 in a control group of children between the ages of 8 and 16 years .
patients with acute scfes have less reduction in anteversion compared to those with chronic scfes ( 9.3 versus 0.7 ) in the involved hip and uninvolved hip ( 15.7 versus 11.8 ) , with an average difference in version in acute scfes of 6.7 and in chronic scfes of 13.9 .
there are also no abnormalities in tibial torsion in children with stable scfes .genu recurvatum has been fleetingly described in scfe [ 132 , 133 ] .
it is believed that this represents a gradual anterior slip of the proximal tibial epiphysis , with the physes perhaps universally weak or susceptible to large stresses from obesity .
surprisingly , tibia vara ( blount 's disease ) is very rarely associated with scfe [ 134136 ] even though both conditions typically occur in obese children .
peroneal spastic flatfoot has been associated in certain cases with scfe .increase width of the symphysis pubis and unaffected hip joint cartilage space has been observed , suggesting an abnormality of cartilage formation or maturation on a macroscopic level rather than a microscopic level .
skewness of the symphyseal joint has also been described on standing radiographs of the pelvis .
in most children with scfe
, femoral anteversion is decreased . in a 3-dimensional ct scan study of 30 scfes
there was a reduction in femoral anteversion from 12.7 to 7.0 as well as a reduction in the femoral neck shaft angle from 141 to 134. in another ct scan study of 25 scfes a reduction of femoral version 1 was noted in the involved hip and 7 in the uninvolved hip in unilateral cases . in a 3rd ct scan study of 25 scfes femoral
anteversion was 9.8 and 25 in a control group of children between the ages of 8 and 16 years .
patients with acute scfes have less reduction in anteversion compared to those with chronic scfes ( 9.3 versus 0.7 ) in the involved hip and uninvolved hip ( 15.7 versus 11.8 ) , with an average difference in version in acute scfes of 6.7 and in chronic scfes of 13.9 .
it is believed that this represents a gradual anterior slip of the proximal tibial epiphysis , with the physes perhaps universally weak or susceptible to large stresses from obesity .
surprisingly , tibia vara ( blount 's disease ) is very rarely associated with scfe [ 134136 ] even though both conditions typically occur in obese children .
increase width of the symphysis pubis and unaffected hip joint cartilage space has been observed , suggesting an abnormality of cartilage formation or maturation on a macroscopic level rather than a microscopic level .
skewness of the symphyseal joint has also been described on standing radiographs of the pelvis .
other nonskeletal associationsimmune complexes in the synovial fluid and synovium of the hip , but not in the serum , have been found in a high percentage of scfe cases [ 140 , 141 ] .
this is different from the synovitis of the knee or hip caused by other disorders where immune complexes are rarely found .
fluorides in the water , used to reduce dental caries , also strengthen the physes and metaphyseal bone ; however , no difference in the incidence of scfe was seen in children with or without fluoride exposure .
immune complexes in the synovial fluid and synovium of the hip , but not in the serum , have been found in a high percentage of scfe cases [ 140 , 141 ] .
this is different from the synovitis of the knee or hip caused by other disorders where immune complexes are rarely found .
fluorides in the water , used to reduce dental caries , also strengthen the physes and metaphyseal bone ; however , no difference in the incidence of scfe was seen in children with or without fluoride exposure .
the current incidence of scfe ranges from 0.33/100,000 to 24.58/100,000 children 8 to 15 years of age , depending upon gender and ethnicity .
there is significant variability within racial groups and the relative frequency , relative to caucasians at 1.0 , is 5.6 for polynesians , 3.9 for blacks , and 2.5 for hispanics .
the average age is 12.0 years for boys and 11.2 years for girls ; obese children present earlier than light weighted children .
the physiologic age range during which scfe occurs is less variable than the chronologic age range .
although there is a general correlation between symptom duration and scfe severity , there is considerable variability .
the majority of children are obese with > 50% of children > 95th percentile weight for age ; the average bmi in scfe children is 2530 kg / m or > 85th percentile .
bilaterality ranges from 18 to 50% and is more frequent in africans compared to hispanic , white , and indo - malay peoples . in children with bilateral involvement , 5060% present with simultaneous scfes ; 8090% of those who present with a unilateral scfe and
the age at presentation is younger for those who present with a unilateral scfe and develop contralateral involvement compared to those who do not develop a contralateral scfe .
the age - weight , age - height , and height test are useful to differentiate between an idiopathic and atypical scfe .
|
the etiology of slipped capital femoral epiphysis ( scfe ) is unknown with many insights coming from epidemiologic / demographic information .
a systematic medical literature review regarding scfe was performed .
the incidence is 0.33/100,000 to 24.58/100,000 children 8 to 15 years of age .
the relative racial frequency , relative to caucasians at 1.0 , is 5.6 for polynesians , 3.9 for blacks , and 2.5 for hispanics .
the average age is 12.0 years for boys and 11.2 years for girls .
the physiologic age when scfe occurs is less variable than the chronologic age .
the average symptom duration is 4 to 5 months .
most children are obese : > 50% are > 95th percentile weight for age with average bmi is 2530 kg / m2 .
the onset of scfe is in the summer when north of 40n .
bilaterality ranges from 18 to 50% . in children with bilateral involvement , 5060% present with simultaneous scfes and those who present with a unilateral scfe and
subsequently develop a contralateral scfe do so within 18 months .
the age at presentation is younger for those who present with a unilateral scfe and later develop a contralateral scfe .
the age - weight , age - height , and height test are useful to differentiate between an idiopathic and atypical scfe .
|
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] |
proteomics has emerged as a valuable tool
for the identification
of biomarkers associated with human disease including those resulting from very subtle changes in normal cell
functions and signaling pathways .
proteomic technology has advanced
to a state where thousands of proteins can be identified within complex
samples , yet disease - based studies using fresh or frozen tissues are limited
by a lack of available specimens for longitudinal clinical investigations .
in contrast , there are millions of archival formalin - fixed , paraffin - embedded
( ffpe ) tissues for which the clinical course of disease and response
to therapy has been established .
ffpe tissue studies are affected
greatly by sample collection , tissue processing , and archival time .
though these factors are difficult to control
in archival samples , improvements in techniques such as mtraq have
made relative quantitation of disease biomarkers in ffpe tissue possible .
improving protein extraction and detection of less abundant protein
biomarkers is also of critical importance in proteomic analysis .
protein
modifications by formaldehyde treatment and histological processing significantly limit the use ffpe tissues for proteomic analyses .
this has prevented proteomic studies of the clinical course of diseases ,
such as prostate and breast cancer , that evolve slowly or where the
time between treatment and recurrence is long .
coupling proteomic
investigations with the retrospective pathology information available
from archival ffpe tissues would produce a wealth of practical information
on human diseases .
some are practical for slide - mounted ffpe tissue , such as
quantitative fluorescence imaging analysis ( qfia ) , which is reproducible
and sensitive for specific standardized proteins , or maldi - imaging
mass spectrometry ( ms ) .
other encouraging mass spectrometry
( ms)-based proteomic studies of ffpe tissues have appeared in the
recent literature ; however , these investigations have typically been
restricted to minute tissue specimens , such as those obtained by laser
capture microdissection .
further , some studies report high rates of
false - positive protein identification and are limited to the analysis
of tryptic digests of ffpe tissues by liquid chromatography ms
( lc ms ) .
our laboratory has been studying the reactions
of formaldehyde
with proteins and ways to reverse these reactions .
using proteins in aqueous solution , we demonstrated that
the majority of protein formaldehyde adducts and cross - links were
consistently reversed with mild heating following the removal of excess
formaldehyde by dialysis .
we then developed
a tissue surrogate , which consists of one or more proteins that form
a gel - like plug when treated with formaldehyde at protein concentrations
exceeding 75 mg / ml . these tissue surrogates have sufficient physical
integrity to be processed using normal histological methods .
a variety of extraction buffers and heating
protocols were examined for their ability to recover proteins from
tissue surrogates .
protein recovery was generally modest , and studies
with multiprotein tissue surrogates revealed extraction bias , meaning
that the composition of the solubilized proteins did not match that
of the corresponding tissue surrogate .
subsequent studies showed that
the ethanol dehydration step of histology caused most formaldehyde - treated
proteins to adopt conformations enriched in -sheets , leading
to the formation of protein aggregates where the -sheets form
a dense network of intermolecular formaldehyde cross - links .
we proposed that the difficulty of rehydrating
these stabilized protein aggregates was the primary impediment to
recovering proteins from ffpe tissue .
pressure promotes water
penetration into the inner core of proteins ,
causing denaturation , whereas heat alone causes protein unfolding
followed by aggregation .
consequently ,
we hypothesized that the combined effects of heat and elevated pressure
would facilitate the rehydration of the highly cross - linked protein
aggregates in ffpe tissues .
the increased exposure to water should
greatly improve protein solubilization while simultaneously promoting
the reversal of protein formaldehyde adducts and cross - links .
initial physical studies on tissue surrogates supported this hypothesis and suggested that the effect of pressure was
to reduce the size of protein aggregates through increased water penetration ,
rather than to increase the rate of reversal of protein formaldehyde
adducts and cross - links directly .
encouraged by these results , we extracted ffpe mouse liver tissues
with heat augmented by elevated hydrostatic pressure , with the goal
of reducing extraction bias , improving the recovery of intact proteins ,
and obtaining tryptic digests that more closely resemble those from
matched fresh - frozen tissue .
tissue tek oct compound was purchased from
sakura , usa , amicon ultra 3k centrifugal filters were purchased from
millipore , and 37% formaldehyde and pierce detergent removal columns
were obtained from thermo fisher . precast nupage bis - tris 412%
gels , 2-(n - morpholino)ethanesulfonic acid
sds
running buffer , and the silverquest staining kit were purchased from
life technologies .
tissue extracts were
heated under a pressure of 40,000 psi ( 276 mpa ) using both home - built
and commercial instruments .
the home - built instrument consisted of
a 2-ml capacity ms-1 stainless steel reaction vessel coupled to a
manually operated high pressure piston screw pump available from high
pressure equipment company ( erie , pa ) .
the temperature of the pressure
vessel was regulated by a eurotherm 2132 temperature controller ( leesburg ,
va ) connected to an aluminum heating collar surrounding the reaction
vessel .
the tissue extract ( 2 ml ) was added directly to the reaction
vessel using a syringe .
the construction and operation of this pressure
system has been described in detail previously .
the commercial instrument was a model nep 2320 barocycler ( pressure
biosciences ) modified by the manufacturer to hold isobaric pressure
and to provide temperature control up to 95 c .
the tissue extract
( 2 ml ) was added to a ft500 sample tube , which was capped and placed
in the pressure vessel of the barocycler .
the liver from a female balb / c
mouse was obtained under the secondary use provision from the department
of laboratory animal medicine of the armed forces institute of pathology
( usa ) .
half of the liver was immediately frozen in tissue - tek oct
compound , divided into several equal - sized pieces , and stored at 80
c .
the other half was fixed for 48 h at 4 c in 10% formalin .
the fixed liver tissue was washed for 30 min with distilled water
and dehydrated through a graded series of alcohols ( 70 , 85 , and 100%
by volume ) and two changes of xylene , 30 min each .
recovery experiments , 10 m sections of ffpe mouse liver were
cleared of paraffin by incubating the sections through two changes
of xylene for 10 min each .
the sections were rehydrated through a
series of graded alcohols for 10 min each2 changes each of
100% ethanol , 85% ethanol , and 70% ethanol and then incubated
in distilled water for a minimum of 30 min , as described previously .
matched fresh - frozen and ffpe liver tissue sections
were analyzed after 30 days , and again after 1 year , of storage .
the ability
of elevated hydrostatic pressure combined with heat to improve the
recovery of proteins from ffpe mouse liver tissue was evaluated using
two heat - based ffpe proteomic protocols recently reported in the literature .
for each protocol , the experimental procedure was followed exactly
as published , but with the heating step divided into two arms , or
variations . in the first arm
, the ffpe tissue extract was heated at
the temperature and for the length of time reported in the original
method . in the second experimental arm ,
the temperature and length
of time was identical , but the experiment was performed under a pressure
of 40,000 psi .
the two experimental arms were then analyzed using
identical gel electrophoresis and lc / ms conditions .
the protein content
of the tissue extracts was determined using a bca protein assay kit
( pierce ) .
following deparaffinization and
rehydration , the tissue was cut into small pieces and suspended in
36 ml of 50 mm tris - hcl , ph 7 , 2% sds ( extraction buffer 1 ,
eb1 ) as described by shi et al .
the tissue
suspension was homogenized with three 5-s cycles of sonication on
ice using a probe - tip sonicator , and the resulting homogenate was
split into two equal fractions .
one fraction ( protocol-1a ) was incubated
at 100 c for 30 min followed by 80 c for 2 h at atmospheric
pressure ( 14.7 psi ) in a sand bath .
the second fraction ( protocol-1p )
was incubated at 100 c for 30 min followed by 80 c for
2 h at 40,000 psi using our hand - built pressure instrument .
a 750
m section ( approximate thickness ) of matched fresh - frozen liver
was cut with a razor blade and homogenized in 22.5 ml of eb1 by sonication
as described above .
proteins were extracted using two methods : incubation
of the homogenate in an ice bath for 2.5 h ( protocol-1fi ) or incubation
at 100 c for 30 min followed by 80 c for 2 h at atmospheric
pressure using a sand bath ( protocol-1fh ) .
following deparaffinization and rehydration , the
tissue was cut into small pieces and suspended in 36 ml of
100 mm tris - hcl , ph 8 , 100 mm dtt , 4% sds ( extraction buffer 2 , eb2 )
as described by ostasiewicz et al . and
homogenized with three 5-s cycles of sonication on ice using a probe - tip
sonicator .
one fraction ( protocol-2a ) was incubated at 95 c
for 1 h at atmospheric pressure in a sand bath .
the second fraction
( protocol-2p ) was incubated at 95 c for 1 h at 40,000 psi using
the barocycler instrument .
a 750 m section of matched fresh - frozen
liver was cut with a razor blade and homogenized in 22.5 ml of eb2
by sonication as described above .
proteins were extracted by incubating
the homogenate at 95 c for 3 min at atmospheric pressure using
a sand bath ( protocol-2fh ) .
all ffpe and matched
fresh - frozen mouse liver tissue extracts ( 40 g each ) were separated
by sds - page on precast nupage bis - tris 412% gels using 2-(n - morpholino)ethanesulfonic acid
the gels were stained using the silverquest silver staining kit and
documented using an epson v500 photoscanner and annotated in adobe
photoshop , version 7.1 . each gel lane
was then divided into 10 bands
and placed into microcentrifuge tubes . in - gel tryptic digestion was
carried out as previously described .
separation of the
digested peptides was performed using nanocolumns prepared in - house
( 75 m i.d . packed with jupiter c18 particles , 5 m , 300
) connected to an agilent 1100 nanoflow lc system , which
was used to deliver binary gradient solvents a ( 0.1% formic acid ( fa )
in water ) and b ( 0.1% fa in acetonitrile ) . reversed - phase chromatography
was performed by solubilizing the lyophilized tryptic peptides in
10 l of 0.1% trifluoroacetic acid and injecting 7 l
of sample per analysis .
after sample injection , a 20-min wash with
98% mobile phase a was used to remove any remaining salts from the
sample .
peptide elution was accomplished using a linear gradient of
2% solvent b to 42% solvent b over 45 min at a constant flow rate
of 250 nl / min .
the nanoflow reversed - phase lc column was coupled
online to a linear ion trap mass spectrometer ( ltq , thermoelectron )
using the manufacturer s nanoelectrospray source with an applied
electrospray potential of 1.75 kv and a capillary transfer tube temperature
of 185 c .
the ltq - ms was operated in a data - dependent mode where
each full ms scan was followed by seven tandem ms scans in which the
seven most abundant peptide molecular ions detected were dynamically
selected for ms / ms analysis using a normalized cid energy of 35% .
a dynamic exclusion of 60-s was applied to reduce redundant selection
of peptides .
the ms / ms spectra were analyzed using sequest ( thermoelectron )
against a combined uniprot nonredundant mouse proteome database containing
36,799 protein sequences . only peptides with conventional tryptic
termini ( allowing for up to two internal missed cleavages ) possessing
delta - correlation scores ( cn ) > 0.08 and charge state - dependent cross - correlation ( xcorr ) criteria as follows were considered as
legitimate identifications : > 1.9 for + 1 charged peptides , > 2.2
for
+ 2 charged peptides , and > 3.1 for + 3 charged peptides . a reverse - database
search , performed using the respective databases , resulted in a calculated
false - positive rate of < 2% for all samples analyzed .
ffpe
and matched fresh - frozen mouse liver tissue stored for 30 days were
extracted using protocol-1 .
the fresh liver tissue was completely
solubilized in the extraction buffer ( eb1 ) using extraction either
on ice ( protocol-1fi ) or at elevated temperature ( protocol-1fh ) .
the
quantity of protein solubilized in the buffer was designated as 100%
protein recovery in table 1 .
ffpe mouse liver
tissue extracted at atmospheric pressure ( protocol-1a ) resulted in
a protein recovery of only 17% , and a large plug of remaining tissue
was observed in the extraction vial . in contrast , when the extraction
was performed at 40,000 psi ( protocol-1p ) , the solubilized protein
increased to 77% and only a small amount of tissue residue remained
in the vial .
ffpe and matching fresh - frozen liver tissue stored for
1 year were extracted using protocol-2 .
the fresh liver tissue was
completely solubilized in the extraction buffer ( eb2 ) when extracted
at elevated temperature ( protocol-2fh ) .
the quantity of protein solubilized
in the buffer was designated as 100% protein recovery in table 1 .
the advantage of using elevated pressure was again
evident , as 79% of the ffpe liver protein was solubilized at elevated
hydrostatic pressure ( protocol-2p ) while only 18% was recovered at
ambient pressure ( protocol-2a ) .
ffpe mouse liver was homogenized
in extraction buffer and heated with or without elevated pressure .
fresh - frozen tissue was extracted either at atmospheric pressure using
the indicated extraction condition or on ice for 2.5 h. protocol used for protein extraction
( see materials and methods ) .
tissue was heated at 100 c
for 30 min ; then the temperature was lowered to 80 c for 2 h. the amount of protein extracted
from fresh frozen tissue was set to 100% .
previous studies using ffpe tissue surrogates containing
several
proteins indicated that failure to completely
solubilize the tissue surrogate led to extraction bias , such that
the composition of the solubilized protein solution differed significantly
from that of the original surrogate .
the extraction bias became less
significant as the percentage of total protein solubilized from the
tissue surrogate increased .
thus , the
4-fold improvement in protein solubilization realized when the extraction
was performed at elevated pressure is likely to lead to a protein
extract that more accurately represents the composition of the original
ffpe tissue .
further , the increased amount of recovered protein allows
a greater number of analytical techniques to be performed .
while most
published proteomic studies of ffpe tissue analyze only a few thousand
cells from microdissected tissue , the use of elevated pressure has the advantage of improving protein
extraction from whole tissue sections .
this ability is particularly
useful in instances where tissue microdissection is not practical
or when a more global proteomic analysis is desired .
unbiased protein
extraction and standardized protocols are particularly important with
techniques such as reverse phase protein arrays ( rppas ) , where the
protein components of cell signaling pathways are quantified to direct
clinical treatment of cancer .
the
30-day - old ffpe mouse liver extracted at 40,000 psi ( protocol-1p )
exhibited a number of well resolved high and low molecular weight
protein bands by sds - page , corresponding to 87% of those seen
in the matched fresh - frozen tissue extracted on ice ( figure 1 , lanes 2 and 1 , respectively ) .
the ffpe samples
extracted at ambient pressure ( protocol-1a ) contained relatively few
well - resolved protein bands equivalent to 25% of those seen
in frozen mouse liver ( figure 1 , lane 3 ) .
similar
results were seen with 1-year - old ffpe mouse liver extracted at 40,000
psi ( protocol-2p ) with well resolved high and low molecular weight
protein bands corresponding to 74% of those seen in the matched fresh - frozen
tissue ( not shown ) . when the extraction was performed ambient pressure
( protocol-2a ) this value
1d sds - page of fresh - frozen
and ffpe mouse liver extracts : lane
1 , fresh - frozen tissue ( protocol-1fi ) ; lane m , molecular weight marker ;
lane 2 , ffpe tissue extracted with heat at 40,000 psi ( protocol-1p ) ;
lane 3 , ffpe tissue extracted with heat alone ( protocol-1a ) . as top - down ms sequencing technology improves ,
the ability to extract
and analyze intact proteins from ffpe tissue will become more important .
top - down sequencing facilitates the measurement of combinations of
modifications , such as phosphorylation and glycosylation , and the
direct quantitation of specific protein isoforms and splice variants .
few of these measurements are directly obtainable using bottom - up
proteomic approaches in which proteins are digested into peptides .
the ability to extract intact proteins from the seemingly inexhaustible
source of ffpe tissues will increase the diagnostic and prognostic
efficacy of proteomic - based biomarker discovery by allowing biomarker
validation using orthogonal methods such as western blotting , immunohistochemistry ,
immunoassays , and structural and interaction proteomics . in this context ,
the > 3-fold increase in the recovery of intact proteins from ffpe
tissue achieved by using elevated pressure represents a significant
breakthrough .
ffpe
and matched fresh - frozen liver tissue stored for 30 days were extracted
using protocol-1 .
ffpe tissue was extracted at ambient pressure ( protocol-1a )
or 40,000 psi ( protocol-1p ) , while the matched fresh - frozen liver
tissue was extracted using protocol-1fi and protocol-1fh .
the solubilized
proteins were separated by 1d - page , and each gel lane was excised ,
digested with trypsin , desalted , and analyzed by lc - ms / ms .
the total
unique peptide and protein identifications for each tissue type and
extraction condition are shown in table 1 .
ffpe tissue extracted with heat alone resulted in the identification
of 5565 unique peptides and 3449 unique proteins .
the addition of
elevated hydrostatic pressure significantly improved both the number
of unique peptides ( 9621 ) and proteins ( 5192 ) identified .
the number
of proteins identified from the high pressure - extracted sample was
comparable to the number of unique proteins identified from fresh - frozen
tissue , which ranged from 4932 for tissue extracted on ice to 4451
for frozen tissue extracted with heat ( table 1 ) .
the ms results for the 30-day - old ffpe mouse liver extracted
under elevated pressure ( protocol-1p ) and the matched fresh - frozen
tissue extracted on ice ( protocol-1fi ) were searched using gominer ,
a gene ontology program .
the percentages of nuclear , membrane , intracellular ,
and extracellular proteins identified in fresh - frozen and ffpe liver
were virtually identical ( figure 2a ) , as were
the results for classification by biological function ( figure 2b ) .
gene ontology analysis of proteins identified by lc - ms / ms .
proteins
identified using fresh - frozen mouse liver ( protocol-1fi ) or ffpe liver
extracted with heat and elevated pressure ( protocol-1p ) were categorized
by subcellular localization ( a ) or biological process ( b ) , using gominer
gene ontology software . to address the effect of long - term storage of the
ffpe specimens
,
the mouse liver samples were investigated after an additional 11 months
of storage ( 1-year - old sample ) .
ffpe tissue was extracted at ambient
pressure ( protocol-2a ) or 40,000 psi ( protocol-2p ) , while the matched
fresh - frozen liver tissue was extracted at high temperature ( protocol-2fh ) .
the solubilized proteins were separated by 1d - page , and each gel lane
was excised , digested with trypsin , desalted , and analyzed by lc - ms / ms .
from the 1-d gel
, we were able to identify 3492 nonredundant proteins
in the 1-year - old ffpe liver extracted under elevated pressure , which
was comparable to the 3415 nonredundant proteins identified in the
matched fresh - frozen mouse liver extracted at high temperature .
in
contrast , only 107 unique proteins were identified in the ffpe tissue
extracted at ambient pressure .
figure 3 shows venn diagrams of the unique
and common ( overlapping ) proteins identified in mouse liver tissue
extracts prepared by different methods .
figure 3a compares 30-day - old fresh - frozen liver tissue extracted on ice
( protocol-1fi ) versus 100 c for 30 min followed by 80 c
for 2 h ( protocol-1fh ) . figure 3b compares
30-day - old ffpe liver tissue extracted under pressure ( protocol-1p )
figure 3c compares 1-year - old
ffpe liver tissue extracted under pressure ( protocol-2p ) versus matched
fresh - frozen liver extracted at elevated temperature ( protocol-2fh ) .
notably , the common proteins , expressed as a percentage of either
the ffpe or matched fresh - frozen mouse liver tissue , were 50%
for all three tissue pairs .
venn diagrams showing the number of unique and
common proteins
identified using lc ms / ms analysis : panel a , fresh - frozen tissue extracted
on ice ( protocol-1fi ) or with heat ( protocol-1fh ) ; panel b , fresh - frozen
tissue extracted with heat ( protocol-1fh ) and ffpe mouse liver extracted
with elevated pressure ( protocol-1p ) ; panel c , fresh - frozen tissue
extracted with heat ( protocol-2fh ) and ffpe mouse liver extracted
with elevated pressure ( protocol-2p ) .
figure 4a is a pie chart
showing the number
of unique proteins identified by two or more fully tryptic peptides
for 30-day - old ffpe mouse liver extracted under pressure ( protocol-1p ) .
the results reveal that 49% of the proteins were identified by two
peptides , 51% were identified by three or more peptides , and 21% of
the proteins were identified by five or more peptides .
figure 4b shows similar results for 1-year - old ffpe mouse
liver extracted under pressure ( protocol-2p ) , with 57% of the proteins
identified by two peptides , 41% by three or more peptides , and 15%
identified by five or more peptides .
these results are similar to
those for the matched fresh - frozen mouse liver tissue ( not shown ) .
the complete list of peptides identified in the fresh - frozen and high - pressure - recovered
ffpe tissue , with their corresponding xcorr values , can be found online
( supporting information table 1 ) .
total number
of unique proteins identified using lc - ms / ms by two
or more unique , fully tryptic peptides in ffpe mouse tissue extracted
with heat and elevated pressure ( 40,000 psi ) : a , 30-day - old ffpe mouse
liver ( protocol-1p ) ; b , 1-year - old ffpe mouse liver ( protocol-2p ) .
virtually all protocols reported in the
literature for the extraction
of proteins from ffpe tissue use a variation of the heat - induced antigen
retrieval technique developed by shi and taylor for the recovery of antigenicity in immunohistochemical
studies .
this method involves exposing ffpe tissue sections to a buffer
solution containing a detergent and/or protein denaturant and elevated
temperatures of 90120 c for a period of 1030
min .
optimal antigen recovery varies
with the host ffpe tissue , with each type requiring different buffers ,
ph values , buffer additives , and incubation temperatures .
although the ffpe proteomic literature is still quite limited , it
is not unreasonable to propose that the same situation applies to
the proteomic analysis of ffpe tissues .
elevated hydrostatic pressure
is not a technique unto itself but rather an adjuvant method that
can be applied to existing or future ffpe protein extraction protocols .
while it remains to be shown that this method is useful with every
tissue fixation and protein extraction protocol , elevated pressure
acts through purely physical means and
should be compatible with ffpe protein extraction buffers of any ph
and containing any detergent , protein denaturant , or other additive .
this should allow its integration into a wide
range of protein extraction protocols for ms - based proteomics with
little to no alteration to downstream sample preparation and analysis .
this was demonstrated in this report by the successful application
of elevated pressure to two very different published ffpe protein
extraction protocols .
an increase in pressure to 40,000 psi , to augment
heat treatment , improved protein extraction efficiency from ffpe mouse
liver tissue by approximately 4-fold and increased the number of unique
proteins identified by up to 30-fold over the published methods used
at ambient pressure .
further , the tryptic digests of these pressure - extracted
tissues resulted in protein profiles that more closely resembled those
from matched fresh - frozen tissue when analyzed by lc / ms than did those
extracted with heat alone , while maintaining a false - identification
rate of < 2% .
the ability of elevated pressure to significantly
improve the recovery of intact proteins from ffpe tissues over the
use of heat alone has great potential for broad application to top - down
proteomic studies for the identification of disease biomarkers .
|
formaldehyde - fixed , paraffin - embedded ( ffpe ) tissue repositories
represent a valuable resource for the retrospective study of disease
progression and response to therapy .
however , the proteomic analysis
of ffpe tissues has been hampered by formaldehyde - induced protein
modifications , which reduce protein extraction efficiency and may
lead to protein misidentification . here , we demonstrate the use of
heat augmented with high hydrostatic pressure ( 40,000 psi ) as a novel
method for the recovery of intact proteins from ffpe mouse liver .
when ffpe mouse liver was extracted using heat and elevated pressure ,
there was a 4-fold increase in protein extraction efficiency , a 3-fold
increase in the extraction of intact proteins , and up to a 30-fold
increase in the number of nonredundant proteins identified by mass
spectrometry , compared to matched tissue extracted with heat alone .
more importantly , the number of nonredundant proteins identified in
the ffpe tissue was nearly identical to that of matched fresh - frozen
tissue .
|
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] |
eligibility requirements included body mass index > 18.5 kg / m ( mean 24.8sd 4.5 kg / m ) , age 1850 years ( 267.5 ) , no current use of antioxidant dietary supplements or a willingness to refrain from their use three weeks prior to and during the study , and self - reported smoking of 3 cigarettes / day ( 11.05.1 ) for 1 year ( 8.06.3 ) .
smoking status was confirmed by a saliva sample analyzed for cotinine by salimetrics , llc , state college , pa ( 189.3121.0 ng / ml ) .
( 21 ) , was 50.528.2 m , which was between the 59.1 m detected in passive smokers and the 40.3 m detected in persons smoking 15 cigarettes per day ( 22 ) .
half of the participants consumed gj in the first experimental session and placebo in the second ; the other half followed the opposite order .
most participants three sessions were on the same day of the week over the course of three consecutive weeks .
each session consisted of two visits to the laboratory a lunch visit and a diet recall visit the following day .
the treatments were 10 ml / kg body weight of a placebo beverage or 100% concord gj containing 2,100 mg / l total phenolics as gallic acid equivalents ( beverages and data supplied by welch foods , inc .
the placebo was formulated to match concord gj for energy , fructose and glucose content , acidity , taste , color and aroma .
participants were instructed to eat a similar breakfast on each treatment day ( which participants reported during the following day 's diet recall ) and not to consume any food or energy - containing beverage within three hours of arrival at the laboratory .
participants were also instructed not to consume any additional gj or red wine for the entire day . for the lunch visit
, participants arrived at the laboratory between 12:00 and 13:00 hours on weekdays and completed a profile of mood states questionnaire ( poms ) ( multi - health systems inc .
, north tonawanda , ny ) and an affect grid ( ag ) ( 23 ) .
next , participants completed the first of four word fragment completion ( wfc ) tasks ( see description below ) , followed by an appetite assessment .
appetite ratings were provided on a personal digital assistant ( pda ) programmed to record marks made by participants on visual analog scales .
next , participants had up to 30 minutes to consume a lunch of as much kraft easy mac ( kraft foods inc . ,
northfield , il ) as desired , one 99 g hunt 's snack pack butterscotch pudding cup ( conagra foods inc . , omaha , ne ) , and the treatment beverage .
after lunch , participants provided appetite ratings and completed a second poms and ag . then , they performed a second wfc task . immediately afterward , another ag was completed , followed by a third wfc task and another ag .
finally , a fourth and final wfc task was completed , followed by a third set of appetite ratings , the final poms and the final ag . during all wfc tasks , participants wore noise - reducing headphones to reduce auditory distractions ( maxell noise cancellation headphone , maxell corporation of america , fair lawn , nj ) . after leaving the laboratory ,
participants provided hourly appetite ratings on the pdas and continued recording their food intake for the remainder of the day .
the following day , participants returned their pda to the laboratory and participated in a computer - aided 24-hour diet recall interview with nutrition data system for research software ( nds - r , 2005 and ndsr , 2006 nutrition coordinating center , university of minnesota , minneapolis , mn ) . due to the nature of a within - subject study design and the desire not to arouse participants out of the post - lunch dip by administering a novel test of mental function ,
changes in mental ability were assessed in a covert manner on a repetitive task using a modified test of implicit memory .
this test involved a wfc task where participants were shown a fragment ( e.g. _ ha_ad _ ) and were asked to fill in the missing spaces to make a word ( e.g. charade ) . for assessing implicit memory ,
this task usually involves prior presentation of the unfragmented words in a long list ( 24 ) .
later , fragments of those words are presented along with fragments of new words , and participants are asked to complete the fragments ( 24 ) .
the increased likelihood of participants completing the fragments with previously studied words is considered priming ( 25 ) . in the present study ,
due to its monotonous nature , this task was not only unlikely to arouse participants out of the post - lunch dip , but may have augmented its development .
fragments were considered incomplete if there was no response , if the response was spelled incorrectly or if the response was not a primed word .
six of the fragments in the first task were repeated in the final task to serve as the test of implicit memory , and only the proportion of these words completed correctly is reported .
thirty - six words were used as priming words , divided into six sets of six words that were counter - balanced among the thirty - six participants and three sessions in order to avoid confounding by word set .
as there were not enough fragments in the source used ( 26 ) , some additional distraction fragments were introduced by the researchers .
an affective post - lunch dip state was expected to be induced and maintained after 30 minutes of performing the wfc tasks based on a previous study attempting to induce the post - lunch dip through a letter cancellation task ( 27 ) .
the final fragment completion task was performed by participants approximately 4767 minutes after finishing lunch , so they were within the expected post - lunch dip time both by the previous study and by indications that the post - lunch dip occurs one hour after beginning lunch ( 28 ) .
further , because debate exists regarding whether the post - lunch dip is related to consuming lunch or circadian rhythms , the study was designed to administer the final fragment completion task between approximately 13:30 and 14:45 hours , depending on when participants arrived and how quickly they consumed lunch .
thus , the timing of this study was designed so that participants completed the fourth fragment completion task both at the appropriate time of day and period of time after consuming lunch for the post - lunch dip to occur .
further , seeking effects between 45 and 65 minutes after consuming the juice coincides with other research documenting acute effects of grape polyphenol consumption .
these acute effects include improved flow mediated dilatation from dealcoholized red wine as compared to regular red wine in coronary artery disease patients ( 29 ) and increased serum total antioxidant capacity ( 30 ) . finally ,
as several participants requested , but were denied , a cigarette smoking break upon completion of lunch , participants may have experienced further deficits due to forced smoking abstinence during this timeframe .
thus , participants were tested with the final wfc task just as the gj polyphenols were expected to begin inducing physiological effects , and the post - lunch dip , smoking abstinence and tedium of a repetitive task were likely to impair their ability / motivation .
participants were compensated financially for participation , and the study protocol was approved by the purdue university institutional review board using signed informed consent .
upper and lower outliers were removed if they were greater or lower than the value of the third or first quartile plus one and one - half times the interquartile range , respectively ( 31 ) .
the proportion correct on wfc tasks ( by - person and by - word ) and the post - lunch dip affective state were analyzed for a main effect of time and a time - by - juice interaction using repeated measures analysis of variance .
food intake and appetite ratings were analyzed for differences between treatments using the wilcoxon signed ranks test .
upper and lower outliers were removed if they were greater or lower than the value of the third or first quartile plus one and one - half times the interquartile range , respectively ( 31 ) .
the proportion correct on wfc tasks ( by - person and by - word ) and the post - lunch dip affective state were analyzed for a main effect of time and a time - by - juice interaction using repeated measures analysis of variance .
food intake and appetite ratings were analyzed for differences between treatments using the wilcoxon signed ranks test .
thirty - three participants provided saliva samples , and one individual had a cotinine concentration below the 713 ng / ml cutoff recommended for identifying smokers ( 32 ) so was not included in further analyses .
the by - person and by - word analyses , the proportion correct for word fragments before lunch was unexpectedly and significantly higher in the gj treatment : f(1 , 27 ) = 7.01 , p=0.01 and f(1 , 35 ) = 4.84 , p=0.04 , respectively ( table 1 ) .
there was no time - by - juice interaction or main effect of time .
mean ( standard deviation ) for repeated word fragment completion ( wfc ) , hunger , fullness and energy intake * significantly higher than placebo ( p<0.05 ) .
for the ag ( table 2 ) , there were no time - by - juice interactions , but there was a significant overall effect of time for both pleasure - displeasure , f(3.6 , 115.0 ) = 2.89 , p=0.03 , and arousal - sleepiness , f(3.0 , 97.9 ) = 12.52 , p<0.01 . for pleasure - displeasure ,
the final two post - lunch measurements were significantly lower than baseline . for arousal - sleepiness ,
affect grid ratings , mean ( standard error ) time effects * significantly different from baseline measurement ( p<0.05 ) .
for the poms questionnaire ( table 3 ) , there were no time - by - juice interactions . however , time had an overall significant effect for confusion - bewilderment , f(1.9 , 59.0 ) = 4.14 , p=0.02 ; fatigue - inertia , f(1.4 , 41.2 ) = 15.37 , p<0.01 ; total mood disturbance ( tmd ) , f(1.7 , 50.6 ) = 10.03 , p<0.01 ; and vigor - activity , f(2.0 , 66.0 ) = 34.78 , p<0.01 . for confusion - bewilderment , fatigue - inertia and tmd , the second rating was not different from baseline , but the third rating was significantly higher than baseline . for vigor - activity ,
profile of mood state ( poms ) , ratings mean ( standard error ) time effects * significantly different from baseline measurement ( p<0.05 ) . for the appetite ratings , during the free - living period after lunch , at least 50% of participants provided entries hourly from 15:00 to 21:00 hours . the data from those seven time points , along with the two in - laboratory entries provided after lunch , were used to determine mean , peak and nadir ratings .
there were no significant differences in hunger , fullness or energy intake ( table 1 ) .
for the cognitive component , this study mirrored a recommendation from research on cocoa beverages to test participants in a state of fatigue .
this augments identification of treatment effects ( 12 ) as it allows for measurement of revival of function or protection from a performance decline ( 1 ) .
this study did so through use of the post - lunch dip , which is the deterioration in mental function that often occurs after consuming the mid - day meal ( 33 ) .
all measures of mood were significantly modified over time in the direction expected to indicate the presence of a post - lunch dip by the time of the final mood assessment .
however , the lack of time - by - juice interactions indicates beverage type had no effect on subjective feelings of the post - lunch dip . to further enhance measurement sensitivity , participants with likely increased risk for oxidative stress
this was accomplished by recruiting individuals who smoked at least three cigarettes daily for at least one year ( 34 ) .
this sub - group may be more likely to benefit from antioxidant treatment and have been used previously in research on grape products as a model of oxidative stress ( 35 ) .
smokers also tend to have low antioxidant vitamin intake ( 36 ) . as wfc performance
was not significantly changed over time for either treatment , it appears gj does not affect implicit memory and/or that performance on individual words is too stable to be affected .
also , wfc rates before lunch were consistently higher in the gj treatment , which may have predisposed the results toward non - significance or favoring the placebo as there was less freedom to improve over time in the gj treatment .
this study failed to detect effects of acute gj consumption by smokers on measures of appetite / food intake , mood and implicit memory .
future research should explore the effects of a chronic dosing regimen on a wider variety of cognitive tasks , physiological measures and possibly a larger and different subset of participants ( e.g. nonsmokers ) .
5 p50 at00477 from the national center for complimentary and alternative medicine ( ncaam ) and the office of dietary supplements and a hatch grant from the usda # ind 084055h .
|
backgroundanimal experiments document effects of grape juice on cognitive performance and motor skills , and observational studies in humans suggest an inverse association between flavonoid intake and cognitive decline .
these effects may be related to the antioxidant properties of polyphenols .
juice consumption and flavonoid intake may also affect appetite.objectiveto study the acute effects of grape juice consumption on appetite , mood and implicit memory during a time of increased lethargy
the post - lunch dip.designthirty-five participants with a mean age of 26 years who smoked a mean of 11 cigarettes / day for 8 years were included in the study .
it included a practice session and two treatment sessions .
all sessions involved consumption of grape juice or an energy - matched placebo with lunch followed by assessments of mood , implicit memory , appetite and food intake.resultsmood decreased over time for both treatments , but there were no differences after lunch between grape juice and placebo for any measure.conclusionthis study did not document any acute effects of grape juice consumption on mood , implicit memory , appetite or food intake in smokers .
|
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] |
langerhans cell histiocytosis ( lch ) refers to a clonal , neoplastic expansion of langerhans cells .
stimulation by tumor necrosis factor and granulocyte - monocyte colony - stimulating factor has been implicated in the pathogenesis ( as has human herpesvirus 6 ) .
it most commonly affects children between the age of 1 and 4 , although cases have been described in all age groups ; the incidence in pediatrics has been estimated at 25 per million / year .
it is exceptional for lch to present with brain involvement as the sole presenting symptom .
this complication is reported as occurring in 14% of patients with multisystem disease , typically 520 years from the initial diagnosis .
he developed depression , anxiety and agitation , which led to his retirement from construction work .
shortly thereafter , he developed episodes described as hyperventilation , lasting from seconds to minutes , mostly in the morning , which were followed by confusion and a severe pounding headache affecting the whole head , both of which lasted for hours .
one year prior to presentation , his family described personality changes ( it was hard for them to characterize this further ) and worsening confusion , particularly over the preceding 3 months .
three months before presentation , he developed weakness of the right leg , which had been slowly progressing since .
two weeks prior , it became so bad that he needed help walking . at this time
, he also noticed weakness of the right arm and slurring of speech , which led him to seek medical help . he had been diagnosed with peripheral arterial disease ( claudication in his left leg ) 4 months prior to presentation , and paroxysmal atrial fibrillation 1 month prior
his initial review of systems revealed that he was anorexic and had lost 30 lb over the last 2 years .
he also complained of a loss of libido and an occasional shortness of breath when walking . on initial exam ,
he was unable to sustain upward gaze ; vertical saccades were followed by a slow downward drift , then a compensatory saccade .
he had a weakness of the right side of his face , sparing the forehead .
he was weak throughout , with strength of 4/5 on his right side in an upper motor neuron pattern with signs of spasticity ; babinski 's reflex was present on this side .
his initial complete blood count , comprehensive metabolic panel , serum protein electrophoresis and folate were normal apart from a white cell count of 13 10/l ( 95% neutrophils ) and platelets of 570 10/l .
his sodium was initially normal , but , during his admission , it was generally in the range of 145160 mmol / l .
mg / l ( < 10 ) , and the erythrocyte sedimentation rate 60 mm / h ( < 20 ) .
he was hypothyroid with a thyroid - stimulating hormone ( tsh ) level of 5.6 iu / ml ( < 4.9 ) and a free t4 of 0.6 ng / dl ( > 0.7 ) .
he had hypogonadism ( presumably secondary ) with total testosterone of < 20 ng / dl ( > 200 ) .
a lumbar puncture was done shortly after admission , showing 4 red cells , 9 white cells ( 56% lymphocytes , 43% macrophages ) , and a protein level of 45 mg / dl ( < 35 ) .
oligoclonal bands were absent and cytology showed no signs of malignancy . shortly after admission , a complete serum paraneoplastic panel was sent ; this was negative .
the antibodies reported were : anti - neuronal nuclear types 13 , anti - glial nuclear type 1 , purkinje cell types 12 and type tr , amphiphysin , crmp-5-igg , striatal , t / q and n - type calcium channels , ach receptor - binding , ach receptor ganglionic and neuronal voltage - gated k channels .
mri is notable for the presence of t2-hyperintense lesions in the left frontal lobe , the right mesial temporal lobe and the brainstem , which were minimally enhanced .
an mr perfusion scan of the brain showed no significant increase in relative blood volume or relative blood flow in these lesions .
a ct of the thorax , abdomen and pelvis showed 5 small lung nodules ( maximum diameter 6 mm ) and a pneumatocele in the left lower lobe .
as there was nothing further on history , exam or initial investigations to suggest cancer anywhere else , a pet / ct was not performed .
immunohistochemistry shows the lesion to be cd1a- and s100-positive , which is characteristic for histiocytosis .
initially , we were unsure of the cause of his right temporal lesion . given the rarity of lch with onset in the central nervous system ( cns ) in adults , the possibility of another tumor was raised , and so a second biopsy was performed .
in retrospect , it would have been better to avoid performing this , as the diagnosis was already clear and his other lesions were attributable to lch .
furthermore , the procedure precipitated status epilepticus and led to preventable complications which ultimately contributed to his death .
this illustrates mild edema , hypercellularity and reactive changes , all of which are consistent with the edge of an inflammatory process .
septic shock ( likely due to ventilator - associated pneumonia ) and a pulmonary embolism complicated his course thereafter .
histiocytosis affecting his skull was thought to be in remission as a result of the treatment , as there was no sign of new or enlarging lytic lesions .
after discharge from the icu , he showed signs of critical illness myopathy / neuropathy .
there was widespread muscle atrophy and generalized weakness . he was unable to stand without support and his tracheostomy remained in place .
given the suspicion of paraneoplastic brainstem inflammation , he was started on intravenous immune globulin ( ivig ) 400 mg / kg 5 days .
two weeks after the initial treatment , the brainstem showed some improvement in the degree of flair hyperintensity ( fig .
an mri 1 month after a second course of ivig showed these changes to be stable overall , with some increase in pontine enhancement ( fig .
he then developed recurrent pneumonia and septic shock , again requiring an icu admission . following this , his family elected a palliative approach to care and he died 6 months after his initial presentation .
our patient 's episodes of hyperventilation followed by confusion are typical for seizures involving the right mesial temporal lobe ; the similarity to panic attacks has previously been identified in a case series .
the development of status epilepticus following biopsy of this region lends support to this being an epileptogenic focus . in
his cerebrospinal fluid ( csf ) , the finding of a high proportion of macrophages is striking .
we were unable to find literature specifically addressing the significance of this finding ; classically it is said to be associated with meningitis due to fungi or tuberculosis .
none of the other case reports we studied , including one with langerhans cells in the csf , reported this finding .
one series has shown elevations of neurofilament protein light chain , glial fibrillary acid protein and total tau protein in the csf of patients with lch affecting the brain .
these advise the use of cladribine for patients such as ours with multisystem and cns involvement ( cytarabine is thought be a reasonable alternative ) .
myelosuppression is the main dose - limiting toxicity ; this has best been studied in the setting of hairy cell leukemia where neutropenia was reported in 70% , anemia ( hb < 8 g / dl ) in 35% and thrombocytopenia in 10% of patients .
the most recent and thorough description of the neuropathology of lch comes from a series of 12 cases from grois et al . in 2005
the authors divide the pathology into 3 categories : ( 1 ) the typical circumscribed granulomas found in other organs ; ( 2 ) infiltrating granulomas with profound t - cell dominated inflammation and severe neurodegeneration ; ( 3 ) neurodegenerative lesions lacking langerhans cells , typically affecting the brainstem and cerebellum and resembling paraneoplastic encephalitis .
it appears that the temporal and frontal lesions in our patient were of the second type ; it is probable that the biopsy came from the neurodegenerative part of the lesion rather than that containing langerhans cells .
the mri appearances of the brainstem in our patient are typical of the third type .
the presence of inflammation without langerhans cells has led to the view that these lesions are paraneoplastic in origin and that the neurodegeneration seen is thus autoimmune .
the langerhans cells are thought to take up normal neuronal antigens , provoking an immune response to these ; alternatively , the immune system may target antigens shared by langerhans cells and microglia . if an antigen is involved , the pathology reported by grois et al .
the antigens responsible for this phenomenon have not been identified and thus it was to be expected that our patient 's paraneoplastic panel was negative . using multiple immunosuppressants
langerhans cells in their normal form are thought to have a lifespan of months , and lch is characteristically a slowly progressive disorder .
by contrast , cytotoxic t - cells typically have a half - life of 2448 h when activated .
when used in the treatment of leukemia and lymphoma , it is typically given every 4 weeks .
thus , the initial doses of cladribine would have been expected to lead to the death of a majority of neoplastic histiocytes while the t - cell - mediated inflammation would have been expected to resume within 34 weeks .
ivig would not have been expected to affect the viability of langerhans cells , but should have reduced t - cell activity for 34 weeks .
it leads to a selective decrease in t - cells overall and an increase in regulatory t - cells . in children with lch
, ivig has been reported to contribute to a stabilization of inflammation in 4/5 patients treated and to enable long - term remission in 1 case [ 11 , 12 ] .
it has been suggested that ivig be given prophylactically to prevent the development of cns complications of lch , particularly diabetes insipidus , which tends to be irreversible .
alternative agents reported to treat the paraneoplastic inflammation in lch include vincristine / cytarabine and all - trans - retinoic acid .
the use of steroids in our patient would not have been expected to have had long - lasting effects .
bearing the above in mind , as well as the results of treatment in other t - cell - mediated paraneoplastic conditions , strong consideration should be given to agents that primarily target t - cells : steroids , ivig , plasma exchange and mycophenolate mofetil or cyclophosphamide .
other paraneoplastic disorders recognized in patients with lch include cancer - associated retinopathy and spinocerebellar degeneration .
brain imaging should always be undertaken in an older patient with episodes suggestive of seizures , diabetes insipidus of central origin or symptoms suggestive of pituitary dysfunction .
once a diagnosis of lch is made , a brain biopsy is not essential if the mri appearances are typical for the inflammatory processes that accompany this condition .
once lch appears stable , prolonged treatment may be needed to prevent recurrence of inflammation .
ivig appears to be a reasonable adjunct to cladribine , particularly in a case such as this where cytopenias or infections limit the use of conventional chemotherapy .
|
we report a case of langerhans cell histiocytosis in a 64-year - old male who presented with symptoms and signs of brain involvement , including seizures and hypopituitarism .
the diagnosis was confirmed with a biopsy of a lytic skull lesion .
the disease affecting the bone showed no sign of progression following a short course of cladribine .
signs of temporal lobe involvement led to an additional biopsy , which showed signs of nonspecific neurodegeneration and which triggered status epilepticus .
lesions noted in the brainstem were typical for the paraneoplastic inflammation reported in this condition .
these lesions improved after treatment with cladribine .
they remained stable while on treatment with intravenous immune globulin .
|
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studies have shown the incidence of injury from squash to range from 35.5 to 80.9 per 100,000 players .
trauma to the head ( including the eyes ) is the most common cause of squas - related hospital presentation , comprising 48.7% of all emergency department presentations .
craniofacial and spinal fractures typically result from high - energy blunt force to the skeleton , as seen in motor vehicle accidents .
this is the first case reported of a patient sustaining such extensive head , neck and spinal injuries from a squash - related injury .
a 55-year - old man was brought in by ambulance after colliding with the corner of the wall and floor of a squash court , without the use of protective eye - glasses or mouth - guard .
he was chasing a ball toward the corner of the court when he fell and hit the junction of the wall and floor with his head .
on examination in the emergency department , the patient had a glasgow coma scale ( gcs ) of 14 , with confusion to time and place .
initially , the patient reported diplopia at primary position but examination by the surgical team elicited normal eye movements with nil complaint of diplopia .
there was significant boggyness to palpation of the forehead with a palpable defect of the frontal bone .
the cervical spine was slightly tender over / c1 - 2 and c5 - 6 .
upper limb examination was limited by pain maximal over the distal radii but bilateral arm weakness was noted with nil sensory dysfunction .
primary and secondary surveys did not elicit any further findings . a non - contrast computed tomography ( ct )
imaging of the facial bones [ figure 1 ] showed a le fort type ii fracture , with comminuted fractures of the roofs of the orbit bilaterally and a depressed fracture of the left orbital floor .
there was also fracture of the right maxilla at the junction of the maxilla and zygomatic arch , extending towards but not involve the right orbital floor . on the left , there was fracture of the anterior part of the left zygomatic arch which extended into the anterolateral wall of the left maxillary sinus .
a large , depressed fracture of the frontal bone was also evident , extending superiorly from the superior lateral wall of the right orbit and extending across the frontal bone and inferiorly toward the mid - portion of the roof of the left orbit .
the fracture involved the inner and outer tables of the frontal bone and was depressed by at least 3.5 mm .
there was also a comminuted fracture of the nasal bone , which was not significantly displaced .
anterior and anterolateral views of three - dimensional ct reconstructions demonstrating lefort type ii , frontal , orbital and zygomatic fractures ct brain showed tiny subarachnoid or intraparenchymal haemorrhages but there was no evidence of extensive intra- or extra - axial haemorrhage .
imaging of the cervical spine [ figure 2 ] showed an undisplaced fracture of the posterior arch of c1 on the right side and a type 3 dens fracture with posterior displacement of 4 mm but no contact with the spinal cord .
anterolateral view 3 dimensional ct reconstruction demonstrating type 3 dens fracture with posterior displacement and fracture of c7 spinous process subsequent ct of the thoracic spine showed a comminuted t6 vertebral body fracture with loss of vertebral height and extension into the left costovertebral joint .
there was a small bony fragment projecting posteriorly into the spinal canal and making contact with the cord , but there was nil evidence of compression .
associated with this finding were two to three hyperdense foci within the spinal cord likely to represent small contusions .
the above findings were confirmed on magnetic resonance imaging ( mri ) of the spine , which also demonstrated a small cortical buckle at the t6 level without significant cord impingement .
the patient underwent craniotomy , obliteration of the frontal sinus and open reduction internal fixation ( orif ) of the frontal bone .
orif of the maxilla bilaterally , orif of the infra - orbital rim and supra - orbital rim bilaterally and inter - maxillary screw fixation was also performed .
the patient was admitted to the neurosurgical intensive care unit post - operatively and underwent cervical fusion with odontoid screw fixation of the c2 fracture one week after surgical correction of the head injuries .
post - operative ct scans demonstrated complete bony union in the c2 fracture site [ figure 3 ] and uncomplicated fixation of the facial bone fractures [ figure 4 ] .
post - operative lateral x - ray of the cervical spine in extension demonstrating union of dens fracture with overall alignment in - tact post - operative coronal ct demonstrating surgical correction of multiple craniofacial fracture sites
our patient suffered injuries to multiple sites in the cranium , facial bones and the vertebrae that have previously not reported in the literature .
the thinnest and weakest segment of the orbit is the floor , which is the most common site of fracture .
our patient suffered a le fort type ii fracture , which is pyramid shaped and passes through the posterior alveolar ridge , lateral walls of maxillary sinuses , inferior orbital rim and nasal bones .
he also suffered a depressed frontal bone fracture involving the inner and outer tables , an extremely rare complication of sport seen in only 1.3% of sports related craniofacial fractures .
spinal injury is associated with sports in approximately 8.7% of cases , usually in relation to contact sports such as wrestling .
our patient had a fracture of the posterior arch of c1 on the right side and a type 3 dens fracture , which required cervical fusion .
there was also fracture of the c7 cervical process , from forceful flexion of the cervical spine .
we report the first case of extensive head and spinal injury resulting from collision with the wall of a squash court .
the injuries ranged from the frontal bone to the thoracic vertebra and are usually seen in high velocity trauma .
|
squash is a popular racquet sport not usually associated with severe head or spinal injury .
the incidence of squash - related injury ranges from 35.5 to 80.9 per 100,000 players , with the most common sites being the lower - limbs and eyes .
we present a case of extensive traumatic craniomaxillofacial and vertebral injury resulting from collision on a squash court , without use of protective gear .
the patient sustained fractures of the frontal bone , orbits , maxillae , zygomas , the first and second cervical vertebrae and the spinous process of the seventh cervical vertrebra .
this is the first case of squash - related injury with such extensive craniofacial and vertebral involvement .
this unique case required multiple surgical procedures as well as an extensive admission to the intensive care unit and highlights the risk of significant craniomaxillofacial trauma in sports not usually associated with such injuries .
|
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the data file of recommendations by pbac was created by abstracting data from the pbac web site ( http://www.health.gov.au/internet/main/publishing.nsf/content/public-summary-documents-by-product ) .
data were taken from recommendations made from july 2005 through november 2009 . in the data
if a product had more than one submission for the same indication , more than one record was created under the same unique identification number . however ,
if a product had multiple submissions that included a different indication , a new unique identification number was created for that product and indication .
the data file included pbac 's recommendation ( to recommend a listing with or without restrictions , not to recommend a listing , or to defer ) , the incremental cost - effectiveness ratio ( cost per quality - adjusted life - year [ qaly ] ) , and the highest value of the financial impact presented in the summary document for each product .
in addition , other variables were abstracted that have been shown to be associated with pbac 's reimbursement recommendations in previous studies ( 12;13 ) ( supplementary table 1 , which can be viewed online at www.journals.cambridge.org/thc2013079 ) .
the four categories of outcomes abstracted were combined to create a binary variable with categories recommended ( yes , restricted yes ) , and not recommended ( no , deferred ) .
the predictors of the pbac recommendation that were tested in the analyses were either abstracted or derived variables that were classified as either economic variables or clinical variables as seen in previous studies ( 1214 ) .
economic variables included the following : ( i ) population size , created from reported patient population size treated per year : low ( < 10,000 ) , medium ( 10,00050,000 ) , and high ( > 50,000 ) .
( ii ) estimated financial impact per annum , using the upper bound value presented and estimated for the impact on only the drug budget : a$0 , > a$0 to < a$10 million , a$10 million to a$30 million , and > a$30 million .
a categorical variable was used for the analysis because the pbac summaries presented financial impact as an upper bound or range rather than a continuous variable for most of the submissions .
( iii ) cost per qaly , using the upper bound base case analyses : > a$0 to a$45,000 , > a$45,000 to a$75,000 , and > a$75,000 as used in the chim and colleagues study ( 12 ) of the impact of cost - effectiveness on reimbursement decisions .
a fourth category , no cost - effectiveness analysis presented , was assigned to those submissions that used a cost - minimization approach for the economic evaluation .
clinical variables included the following : ( i ) active comparator ( yes or no ) that indicated whether an active comparator was used as the comparison group in at least one of the pivotal studies ; ( ii ) manufacturer claim for the clinical benefits of the new product : noninferior or equivalent or superior ; ( iii ) comparative clinical evidence available from randomized clinical trials only ( rct ) or from rct data plus a meta - analysis or indirect comparison analysis ( rct plus meta - analysis or indirect comparison analysis ) ; ( iv ) disease category ( oncology or other ) , as a proxy measure of likelihood of reduced life expectancy and the dread factor associated with the disease ( 12 ) ; and ( v ) surrogate end point ( yes or no ) , derived from a review of the end points in the submission .
the unit of analysis for all analyses was the unique drug and indication submission after july 2005 . only the first observed submissions of the unique drug and indication combination within our database
were included in the univariate and initial multivariable logistic analyses because subsequent resubmissions were correlated with the first observed submission .
a test result was declared statistically significant if p value was < .05 and marginally statistically significant if p value was > .05 but .1 .
first , a univariate analysis was performed to explore the association between the pbac recommendation and the variables described previously .
next , a multivariable logistic regression was performed to evaluate the relationship between the probability of a positive recommendation and the categorical financial impact , while adjusting for other factors .
the variables included in the logistic model were those that had an association with the recommendation with a p value .30 in the univariate analysis ( 15 ) .
a discrete time - to - event analysis was performed , including all extracted data : both the first observed submission data and all resubmission data to determine the relationship between multiple submissions and pbac recommendations while accounting for the correlations between repeated submissions and to determine the impact of the omission of the resubmissions on our estimates for financial impact .
we performed the analysis using the logistic model as described in allison ( 16 ) .
total submission count was determined by counting the number of times the same drug plus indication was submitted .
only nine records had a total count of four or more , and these records were omitted from the analysis .
the variable time since previous submission was included because the resubmissions happened at irregular intervals . a submission or resubmission for a drug plus indication that happened once or more than once but that was not recommended for reimbursement
also , submissions could be left censored if data for the first observed submission indicated that previous submissions had occurred before july 2005 .
finally , a decision tree analysis was performed using the recursive partitioning algorithm in jmp analysis software ( sas , cary , nc ) .
recursive partitioning is a nonparametric classification technique that splits into subsets , called nodes , observations with similar response values for predictor variables ( 17 ) . in each node ,
the predictor variable with the strongest association to the outcome variable is chosen for splitting the node .
for a categorical outcome , the recursive partitioning algorithm in jmp analysis software uses log10 ( p value ) , also called logworth , where the p value is an adjusted p value given by a likelihood ratio test . to avoid overfitting , only partitions that had a logworth value 1.12 , which corresponded to log10 ( 0.05 ) ,
because this is a nonparametric method , it has an advantage over logistic regression by not assuming any functional form for the association between predictors and outcome .
furthermore , recursive partitioning has the advantage of detecting possible complex interactions between predictors that may not have been detected by the logistic regression , and because it is easy to visualize and interpret , it is suitable for a decision - making process ( 18 ) .
moreover , the relative importance of the predictors can be inferred by the order in which they partition the data ( i.e. , the earlier the predictor is used by the partition algorithm , the more important it is ) .
the logistic regression and decision tree analyses were also performed using only those submissions with a reported cost per qaly to assess the importance of financial impact in this subset of the total submissions .
a total of 260 submissions , representing 214 unique drug plus indication combinations and 46 resubmissions during the data abstraction time period , were extracted from the pbac web site .
most , or 170 of the 214 ( 79.5 percent ) unique drug plus indication combinations , were not submitted before july 2005 ; 27 ( 12.6 percent ) were submitted once before july 2005 ; and 17 ( 7.9 percent ) were submitted multiple times before july 2005 .
of the 260 submissions , 106 ( 40.8 percent ) were recommended for reimbursement , 47 ( 18.1 percent ) were partially recommended for reimbursement ( i.e. , recommended with restrictions ) , 100 ( 38.4 percent ) were not recommended for reimbursement , and 7 ( 2.7 percent ) were deferred .
therefore , the binary variable pbac recommendation for reimbursement had 153 submissions ( 58.9 percent ) that were recommended and 107 submissions ( 41.1 percent ) that were not recommended .
five variables financial impact , cost per qaly , manufacturer 's claim , active comparator , and disease category had a statistically significant association with the pbac recommendation . with the exception of the financial impact category
> a$30 million , the percentage of submissions that were recommended decreased as the financial impact and the cost per qaly increased .
in addition , 74.3 percent of the submissions that did not report a cost per qaly were recommended as compared with 40.0 percent for those submissions with a reported cost per qaly .
the percentage of recommendations was higher for entries that claimed noninferiority or equivalence , that had used an active control as the comparator in at least one pivotal clinical trial , and that were not in the oncology category .
the other included variables ( population size , comparative clinical evidence , and surrogate end points ) did not have a statistically significant association with the pbac recommendation . based on the univariate analyses , six possible predictors of pbac recommendations with a univariate p value .30
table 1.univariate association between pbac recommendations and predictorsvariableno . in each variable categorypercentage in each variable category recommended for reimbursement by pbacp value for difference between categoriesfinancial impact ( million a$)>302245.5%<.000110 to 304235.7%>0 to < 1010154.5%03992.3%cost per quality - adjusted life - year ( thousand a$)>751915.8%<.0001>45 to 752733.3%>0 to 455950.9%none10974.3%population sizehigh4047.5%.3671medium5259.6%low12259.8%manufacturer claimsuperior or advantages10843.5%<.0001noninferior or equivalent10671.7%comparative clinical evidencerandomized controlled trial9953.5%.2792rct + meta - analysis or indirect comparison analysis11560.9%active comparatorno5232.7%<.0001yes16265.4%disease categoryoncology3740.5%.0219other17761.0%surrogate end pointyes15058.0%.5846no5453.7% percentages were calculated out of the available data for the respective variable category .
p value was calculated using pearson 's chi - square test for difference between the variable categories.pbac , pharmaceutical benefits advisory committee .
univariate association between pbac recommendations and predictors percentages were calculated out of the available data for the respective variable category .
p value was calculated using pearson 's chi - square test for difference between the variable categories .
figure 1 and supplementary table 2 , which can be viewed online at www.journals.cambridge.org/thc2013079 , present the results of the multivariable logistic analyses .
only the effect of financial impact ( p = .0242 ) , cost per qaly ( p = .0235 ) , and active comparator ( p = .0365 ) were statistically significant .
after adjusting for the other factors in the model , the only statistically significant odds ratios for financial impact were for comparing either category a$10 million to a$30 million ( 0.12 ; 95 percent confidence interval [ ci ] : 0.030.51 ) or > a$0 to < a$10 million ( 0.16 ; 95 percent ci : 0.040.60 ) with the category a$0 .
the odds ratio for the financial impact category > a$30 million compared with the category a$0 was not significant ( 0.25 ; 95 percent ci : 0.051.34 ) .
for cost per qaly , the only statistically significant odds ratios were for comparing the category > a$75,000 with either the category > a$0 to a$45,000 ( 0.11 ; 95 percent ci : 0.020.55 ) or to the category none ( 0.06 ; 95 percent ci : 0.010.40 ) .
the odds of recommending a drug submission that used an active comparator were 2.49 ( 95 percent ci : 1.065.85 ) times the odds of recommending a drug submission that used placebo as the primary comparator .
figure 1 presents a plot of selected odds ratios obtained from the first logistic model .
financial impact was not statistically significant ( p = .1801 ) when the same model was run using only submissions that reported a cost per qaly ( n = 103 ) .
cost per qaly was the only statistically significant factor ( p = .0158 ) in this model .
figure 1.multivariable logistic regression results ( n = 204 ) : odds ratios with 95% ci plots .
ci , confidence interval ; qaly , quality - adjusted life - year ; rct , randomized controlled trial .
multivariable logistic regression results ( n = 204 ) : odds ratios with 95% ci plots .
ci , confidence interval ; qaly , quality - adjusted life - year ; rct , randomized controlled trial .
figure 2 and supplementary table 2 present the results of the analysis of discrete - time survival data , using multivariable logistic analysis .
the statistically significant effects were total submission count ( p = .0029 ) , financial impact ( p = .0021 ) , cost per qaly ( p = .0135 ) , and an active comparator ( p = .0229 ) .
the odds ratio estimates for financial impact , cost per qaly , and an active comparator were very similar to the corresponding odds ratios obtained in the first model .
after adjusting for other factors in the model , the odds of recommending a drug that was submitted a third or second time were 9.62 ( 95 percent ci : 2.4737.42 ) or 3.66 ( 95 percent ci : 1.359.96 ) times the odds of recommending a drug submitted the first time , respectively .
the odds of recommendation were 0.83 lower for a 1-trimester increase in time since the previous submission .
figure 2 presents the odds ratio plots for the second logistic model . when the same model was run only using submissions ( n = 126 ) with a reported cost per qaly , financial impact was still statistically significant ( p = .0223 ) .
figure 2.multivariable logistic regression results for discrete time - to - event data ( n = 238 ) : odds ratios with 95% ci plots .
ci , confidence interval ; qaly , quality - adjusted life - year ; rct , randomized controlled trial .
multivariable logistic regression results for discrete time - to - event data ( n = 238 ) : odds ratios with 95% ci plots .
ci , confidence interval ; qaly , quality - adjusted life - year ; rct , randomized controlled trial .
in addition to the variables used in the logistic analyses , population size and surrogate end point were also included .
categorical financial impact was the most important factor to make the first partition ( logworth = 7.18 ) by grouping the three financial impact categories , > a$0 to < a$10 million , a$10 million to a$30 million , and > a$30 million , into a single category and comparing it with a$0 .
cost per qaly , active comparator , and disease category were the next predictors selected for recursive partitioning .
for cost per qaly , the categories none and > a$0 to a$45,000 were combined by the model program and compared with > a$45,000 to a$75,000 and > a$75,000 .
the results of the recursive partitioning model indicated that the chance of being recommended for reimbursement for drug submissions with a financial impact a$0 was 91.4 percent , and the chance for drug submissions with a financial impact > a$0 and a cost per qaly either not estimated or a$0 to a$45,000 was 57.5 percent as compared with 24.4 percent for submissions with a financial impact > a$0 and cost per qaly > a$45,000 .
lower chances for reimbursement recommendation were estimated for drug submissions with a financial impact > a$0 , a cost per qaly either not calculated or > a$0 to a$45,000 , and without an active comparator ( 29.6 percent ) . when a new tree was constructed considering only submissions with a reported cost per qaly , the cost per qaly was the only important factor in the partitioning analysis .
the results of the analyses presented in this study indicate that the estimated financial impact of a drug on the australian drug budget is a predictor of the pbac reimbursement recommendation , even when controlling for the cost - effectiveness ratio and other confounding variables . in the descriptive analysis
, there was a gradient in probability of reimbursement , with the highest probability for drugs that were estimated to be cost - saving and the lowest probability for drugs that were estimated to increase annual costs between a$10 million and a$30 million . however , probability of recommendation was higher for those submissions with a financial impact of > a$30 million compared with those with a financial impact of a$10 to a$30 million .
the logistic analyses demonstrated that this pattern was similar even when controlling for the cost - effectiveness ratio and other confounding variables and the number of submissions and even when only including those submissions that presented a cost per qaly estimate .
a review of the submissions with an estimated financial impact > a$30 million ( 22 submissions ) found that products in this category that were recommended for reimbursement either had cost per qaly estimates in the lower end of the a$0 to a$45,000 range , or very favorable clinical benefits , or indications where there were no alternative treatments that might explain this seemingly anomalous result .
the impact of multiple submissions on the probability of recommendation for reimbursement was significant with an odds ratio of 9.62 for a third and 3.66 for a second observed submission compared with the first observed submission .
supplementary table 3 , which can be viewed online at www.journals.cambridge.org/thc2013079 , presents a summary of the changes in the categorical values among the 29 multiple submissions .
a review of these changes indicated that , although reductions in price were likely key factors in obtaining a positive reimbursement recommendation in many cases , changes in the clinical data submitted were also influential in obtaining a positive recommendation . finally , the recursive partitioning decision analysis supported the importance of a positive financial impact for the reimbursement decision with the full database because the variable with the greatest discriminative power for reimbursement recommendations was shown to be a positive financial impact of any magnitude , followed by the cost per qaly .
however , the results for the subset of submissions that report a cost per qaly , all of which had a positive financial impact , indicated that the cost per qaly variable had the greatest discriminatory power .
a threshold value of a$10 million was used for the financial impact analysis because full approval by the cabinet of the federal government was needed for drugs when their annual financial impact was expected to exceed a$10 million in any 12-month period within the first 4 full years of product listing .
having mandated that a multiyear financial impact analysis be included in submissions has allowed the pbs to put in place price - volume contracts for those drugs that exceed the a$10 million per year limit ( 19 ) , although the specific agreements are confidential .
the results of our analyses are supported by and extend those from three previous analyses of pbac data ( 1214 ) .
all three studies included financial impact on the healthcare sector as an explanatory variable . in the supplemental appendix of clement et al .
( 13 ) , in a descriptive analysis of submissions not needing a cost - per - qaly estimate , the probability of reimbursement in australia was much higher ( 81 percent ) than for those needing a cost - per - qaly estimate ( 44 percent ) ( 13 ) . in the harris et al .
( 14 ) study , for a sample of the decisions that also included an estimate of the cost - per - qaly gained , harris and colleagues ( 14 ) estimated that for each increase of a$5 million in financial impact above the mean value , the probability of a pbac recommendation for reimbursement would decrease by 0.03 .
however , by excluding any submissions that estimated financial cost - savings and many that estimated a budget impact < a$10 million , they may have underestimated the importance of the financial impact for reimbursement decisions , especially for drugs with multiple submissions .
finally , chim and colleagues ( 12 ) compared the impact on reimbursement of two financial impact categories : < a$10 million and a$10 million . in a logistic analysis
, they estimated a statistically significant odds ratio of reimbursement of 0.46 for submissions with a financial impact of a$10 million compared with those with a financial impact of < a$10 million .
the strengths of our analysis included the use of multiple analytic techniques that all provided similar results .
the use of the survival analysis allowed us to include data from multiple submissions as well as right- and left - censored submissions , appropriately accounting for the correlation among repeat submissions of the same drug / indication pair .
the use of the recursive partition decision analysis estimation technique is important because the results provided a simple stepped process for predicting whether a submission will be recommended for reimbursement in australia .
the major limitation is that , in many cases , the financial impact on the drug budget or the cost per qaly was provided in the public summary document only as an inequality or range .
thus , it was not possible to enter financial impact into the model as a continuous variable . instead , we created four financial impact categories . also , the sample size was relatively small ( n = 260 ) , limiting our ability to include a high number of explanatory variables or more than four categories of financial impact .
there was also the possibility of multicollinearity among the independent variables used in the analysis . to test for this , we estimated tolerance
scores for each independent variable and found that all variable scores were above 0.40 , values not considered to be of concern for multicollinearity ( 16 ) .
supplementary table s4 , which can be viewed online at www.journals.cambridge.org/thc2013079 , presents the regression results .
the choice of oncology as a proxy for disease severity was also a source of uncertainty because other diseases may also be associated with very limited life expectancy and dread .
the implications of the findings in this study are that , in australia , the financial impact on the drug budget is an important determinant of whether a new drug is recommended for reimbursement with the strongest positive impact for products that have a zero or negative financial impact .
our findings are consistent with the conclusions of the recent published analyses on the possibility that financial impact plays a role in the hta agency reimbursement recommendations , although not specifically listed as a criterion ( 68 ) .
in addition , the explicit instructions provided in the pbac submission guidelines for performing financial impact analysis indicate the potential importance of these results for the pbac review . although financial impact is a specific part of the decision making for reimbursement in australia ,
this is not necessarily the case in other countries . whether financial impact influences reimbursement decisions in these countries is an empirical question that can be resolved only by performing similar analyses in these jurisdictions .
supplementary table 1 : www.journals.cambridge.org/thc2013079 supplementary table 2 : www.journals.cambridge.org/thc2013079 supplementary table 3 : www.journals.cambridge.org/thc2013079 supplementary table 4 : www.journals.cambridge.org/thc2013079
josephine mauskopf , phd , vice president health economics ( [email protected] ) , costel chirila , phd , senior statistician , biometrics , catherine masaquel , mph , associate director in the market access and outcomes strategy , rti health solutions , po box 12194 , research triangle park , nc , 27709 kristina s. boye , phd senior director global health outcomes , diabetes , lee bowman , phd senior director , global health outcomes oncology , julie birt , pharmd senior research scientist , global health outcomes , health technology assessment center of expertise , david grainger , bs global public policy director , lilly and company , indianapolis , indiana , united states
josephine mauskopf , catherine masaquel , costel chirila report payment to their institution by a contract from eli lilly and company for leading the research reported in the manuscript .
|
objectives : the aim of this study was to estimate the relationship between the financial impact of a new drug and the recommendation for reimbursement by the australian pharmaceutical benefits advisory committee ( pbac).methods : data in the pbac summary database were abstracted for decisions made between july 2005 and november 2009 . financial impact the upper bound of the values presented in the pbac summary database was categorized as a$0 , > a$0 up to a$10 million , a$10 million up to a$30 million , and > a$30 million per year .
descriptive , logistic , survival , and recursive partitioning decision analyses were used to estimate the relationship between the financial impact of a new drug indication and the recommendation for reimbursement .
multivariable analyses controlled for other clinical and economic variables , including cost per quality - adjusted life - year gained.results : financial impact was a significant predictor of the recommendation for reimbursement . in the logistic analysis ,
the odds ratios of reimbursement for drug submissions with financial impacts a$10 million to a$30 million or > a$0 to < a$10 million compared with a$0 were 0.12 ( 95 percent confidence interval [ ci ] : 0.030.51 ) and 0.16 ( 95 percent ci : 0.040.60 ) , respectively . in the recursive partition decision analysis ,
the first split of the data was for submissions with a positive financial impact compared with those with a zero or negative financial impact.conclusions : in australia , financial impact on the drug budget is an important determinant of whether a new drug is recommended for reimbursement when cost - effectiveness estimates and other clinical and economic variables are controlled .
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we administered intravitreal bevacizumab injection to eight eyes of eight patients of various etiologies ranging from retinal vein occlusion to diabetic retinopathy , age related macular degeneration amd , and choroidal neovascular membrane cnvm .
the best corrected visual acuity of seven out of eight patients was poor and ranged from 20/120 to finger counting .
the details of the procedure , complications were discussed with the patients , and written consent taken .
injection bevacizumab was procured by a patient five days prior to the date of appointment and stored under recommended conditions ( i.e. , below 4c ) . on the day of injection , under aseptic precautions
, the vial was opened in operation theatres , hood was not used due to its nonavailability .
the syringe was then capped with a 30-gauge needle and kept on a sterile tray .
best corrected visual acuity of patients and indication for intravitreal injection the eye of each patient was prepared following standard aseptic procedures ( i.e. , lids cleaned sequentially first with spirit and then with 10% povidone - iodine ) .
fornices were flushed with normal saline and 1 drop of 10% povidone - iodine was instilled 2 min before the procedure .
intravitreal bevacizumab injection was administered into the superotemporal quadrant , 4 mm from the limbus .
different needles were used each time after cleaning the surface of vial with spirit , thereby administering multiple pricks in the vial .
all of them were asked to come next week for follow - up or earlier if patient experienced severe discomfort .
four of the eight patients reported to the hospital on the 3rd day after injection with complaints of pain , watering , and diminution of vision .
two patients who did not report by the 4th day were contacted and recalled for an examination .
six out of eight patients had absent fundal glow along with presence of cells and flares [ table 2 ] .
clinical findings in patients following intravitreal injection of bevacizumab these six patients were clinically diagnosed to have endophthalmitis and were administered intravitreal antibiotics ( injection ceftazidime 2.25 mg in 0.1 ml and injection vancomycin 1 mg in 0.1 ml ) on the same day of presentation .
vitreous samples and drug vial were sent for culture sensitivity in two different laboratories which turned out to be sterile .
intravitreal antibiotics were repeated after 48 h. all patients were closely followed up and remaining drug was discarded .
while pegaptanib and ranibizumab are labeled for intravitreal use , bevacizumab is labeled for use in cancer therapy and is currently being used off - label for the treatment of ocular neovascular diseases .
because of its off - label use , bevacizumab is supplied in much larger volumes than those needed for single intravitreal injection .
thus , hospitals and compounding pharmacies must divide the larger volume of bevacizumab into smaller units suitable for single - use , individual doses .
contaminants could possibly be introduced during the compounding process and compromise the sterility of the aliquoted drug . multiple pricks ( procedure common in india ) were made in vial to prepare administrating dose for eight patients .
an alternative protocol suggested is that small aliquot of drug should be prepared using single prick technique , i.e. , 0.5 in .
26 gauge needle should be inserted into rubber cap of vial and drug should be drawn into different 1 ml syringes , every time changing only the syringe , leaving the needle in place .
next group should be administered injection after one week , i.e. , after the first follow - up of the previous batch . in this way
, we would be able to minimize incidence of cluster endophthalmitis and detect possible contamination in the compounded aliquoted drug before it is administered to the next batch .
if required , each eye should be injected using drug from different lots under sterile conditions .
six out of eight patients had endophthalmitis , remaining two patients though belonging to different age groups ( 50 and 72 years ) , did not develop endophthalmitis .
the possible reason for this could be the inherent immunity against the causative organism or the quantity of causative organism in the inoculums could have been below the threshold required for endophthalmitis .
presentation of cases with signs and symptoms of endophthalmitis and response with intravitreal injection of antibiotics led us to assume infective pathology despite the negative culture report .
the possibility of tass syndrome in these patients was considered , but review of literature suggests that series of patients , who developed tass syndrome in canada , had reported as early as 24 h. the final visual outcome was poor even with aggressive treatment . all patients had worse visual acuity at the end of follow - up than on injection day .
four patients in our study presented on day 3 , while two patients reported on day 4 , after intravitreal injection .
three out six patients with endophthalmitis showed improvement in visual acuity with intravitreal antibiotic therapy , as compared to pretreatment level .
visual acuity remained same in two cases , while it deteriorated drastically in one case even after aggressive treatment [ table 3 ] .
as the approval of intravitreal use of bevacizumab and its subsequent availability in the market in single dose ( 0.05 ml ampoules ) is still awaited , using the single - dose vial and aliquoting into smaller doses for multiple uses , is the call of the day .
however , the present incident highlights the risks of microbial contamination and the need to stay vigilant against preexisting contamination within the vial or its access to the drug via multiple pricks . the alternative protocol , as described previously , is recommended to increase the safety margin of the intravitreal injection of bevacizumab .
|
the risk of endophthalmitis is always a concern when an intraocular procedure is performed .
intravitreal injection is a frequently used method for therapeutic management of many diseases , affecting the posterior segment of the eye .
hence , it is important to assess the risk of complications , especially endophthalmitis .
most studies conducted concentrate on risk assessment from single use from single drug vial .
the present article reports the occurrence of cluster endophthalmitis following multiple intravitreal bevacizumab injections from a single vial .
intravitreal injection of bevacizumab was administered to eight eyes of eight patients .
administered dose was prepared from single 4-ml vial of bevacizumab and was injected in the eye , after patient preparation and under aseptic conditions .
the procedure was repeated for the remaining patients , thereby imparting multiple pricks in the same vial .
four of the eight patients reported to the hospital on the 3rd day after injection with complaints of pain , watering , and diminution of vision .
two patients reported the following day with similar complaints .
two patients who did not report by the 4th day were contacted and recalled for an examination .
all the patients were thoroughly examined using slit lamp biomicroscopy and indirect ophthalmoscopy .
six out of eight were clinically diagnosed to have endophthalmitis and were administered intravitreal antibiotics .
the present report highlights possibility of microbial contamination of the drug vial or during compounding process .
however , from the present incident , we are encouraged to stay vigilant and wary of contamination
|
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membrane proteins ( mps ) constitute about 30% of all the proteins encoded in the currently known genomes , and play critical roles in cell signaling , ion transport , and cell - cell communications , as well as assist the folding of other mps ( 1 ) . because of these biological significance
, mps represent the most important class of drug targets about 50% of current molecular targets are membrane - bound ( 2 ) .
however , only about 2% ( 518 of 25,176 ) of the 3d structures deposited in the protein data bank ( pdb ; ref .
3 ) are for mps . and the number of high - resolution structures ( from x - ray diffraction and more recently from nmr ) remains even smaller , largely because of the difficulties in crystallizing mps .
recently , some new ideas and experimental approaches have been introduced in the area of mp crystallization ( 4 ) , all of which exploit the spontaneous self - assembling properties of lipids and detergent as vesicles ( vesicle - fusion method ) , discoidal micelles ( bicelle method ) , and liquid crystals or mesophases ( in meso or cubic - phase method ) . despite these promising new methods ,
the current gap between need and supply of mp 3d structures makes prediction algorithms important and essential .
mps come in a variety of sizes and shapes , though the available 3d structure principles are far less diverse than those of the globular proteins . from a structural point of view
one is the -helix bundle protein , in which one or several -helices span the membrane ; and the other is -barrel protein , in which eight or more antiparallel tm -strands form a closed barrel 5 . , 6 .. two recent examples 7 .
since jhnig and edholm in 1992 presented one of the first methods using secondary structure prediction to build suitable model structures as initial conformations for molecular dynamic studies ( 9 ) , several groups have tried computational approaches to elucidate mp structures . in 1993 , milik and skolnick presented a method based on the combination of a hydropathy scale for the prediction of trans - bilayer fragments with dynamic monte carlo simulation techniques ( 10 ) . in 1994 , taylor et al .
adapted some programs originally developed for the prediction of globular protein structures to derive a method for the prediction of integral mp structures ( 11 ) .
each step in the method is fully automated , from the initial sequence data bank searches to the final construction of 3d models .
consequently , estimates for prediction accuracy are perhaps overly optimistic . here , we summarize recent attempts within the field of computational biology and bioinformatics to predict an mp s structure .
most current methods of theoretical mp structure prediction do not actually deal with predicting the 3d structure , but rather try to predict the most likely topology of the protein , that is to say , the in / out location of the n and c termini relative to the membrane , and the number and position of transmembrane ( tm ) segments . a high - quality model of secondary structure and topology
is a prerequisite for experimental structure - function studies , and can be a starting point for attempts to model the 3d structure before molecular dynamics or simulated annealing simulations . in recent years , various accurate methods
because the number of high - resolution structures of -barrel proteins is less than that of the -helix proteins , the neural network has been more frequently adopted in the -strand topology prediction .
the details of some methods based on hidden markov models ( hmms ) are listed in table 2 .
many secondary structure prediction methods are based on statistical methods , physicochemical methods , sequence pattern maching , and evolutionary conservation ( 12 ) .
the main methods for identifing tm helices are on the basis of their hydrophobicity and known minimum length ( at least 15 residues ; ref .
membrane propensities were defined by a statistical analysis carried out on a set of 640 tm helices , belonging to 133 mps extracted from swiss - prot ( 14 ) that have experimentally defined topologies .
the five widely used prediction methods for predicting the topology of -helix bundle mps are tmhmm ( 15 ) , hmmtop ( 16 ) , memsat ( 17 ) , phdhtm ( 18 ) , and toppred ( 19 ) .
tmhmm , hmmtop , and memsat are all based on hmms with 5~7 types of structural states .
toppred was the first topology prediction method that combined hydrophobicity analysis and the positive - inside rule .
generally , these sequence - based methods for predicting the number and approximate location of tm helices within mps have about 85% accuracy . in 2003 , karin meln et al . tried to construct useful reliability scores for these methods ( 20 ) .
21 ) algorithm can provide a solution to the problem that non - transmembrane query sequences may give false positive hits ( 20%-30% ) in the prediction process .
the upgraded and modified version of the das - prediction method , das - tmfilter algorithm , has been distributed ( 22 ) .
the new algorithm is designed to make distinction between protein sequences with and without tm helices at a reasonably low rate of false positive prediction ( ~1 among 100 unrelated queries ) while the high efficiency of the original algorithm locating tm segments in queries is preserved ( sensitivity of ~95% among documented proteins with helical tm regions ) . in 2003 ,
xia and colleagues presented a new approach , conpred_elite ( 23 ) , that can predict the whole topology with accuracies of 98% for prokaryotic and 95% for eukaryotic proteins as they reported .
besides the tm helix , another tm segments type is -barrel , which consists of several tm strands . unlike -helical mps , there are no simple low - resolution experiments that yield large amounts of data for -barrel mps .
in fact , the overall hydrophobicity for -barrel mps is similar to that of soluble proteins ( 13 ) .
gromiha and colleagues combined amino acid preferences for -strands with the surrounding hydrophobicity of the respective residues to predict -strands ( 24 ) .
diederichs and colleagues proposed to use a neural network to predict the topology of the bacterial outer membrane -strand proteins and to locate residues along the axes of the pores ( 25 ) .
jacoboni and colleagues applied a method combining neural networks and dynamic programming to predict the location of membrane strands ( 26 ) .
the authors estimated that their system correctly predicted about 93% of all known membrane strands .
more recently , martelli et al . developed a sequence - profile - based hmm model that can predict the topology of -barrel mps cyclicing with 6 types of states ( 27 ) .
they reported that the accuracy of per residue of the model was about 83% . lately the following protocol starting from secondary structure prediction and tm segments topology prediction are often used .
secondary structure prediction followed by tm segments identification along with prediction of loops connecting the segments , and molecular dynamics or simulated annealing simulations , may be finally used to refine these primal models . during the last refinement step
, the protein is often inserted into a water / lipid bilayer / water or a water / n - octane / water environment to take into account the presence of the cell membrane .
charmm , gromos , amber , and cvff - insight are some widely used force fields in molecular dynamics calculation .
the slow dynamics of lipid molecules in the bilayer might bring the difficulties in equilibrating the system ( 28 ) .
for globular proteins , the major successful methods for structure prediction include homology modeling , threading , and ab initio folding . along with lucubrating the mechanism of mp folding and increasing the number of high - resolution mp structures , these methods will been applied to the direct prediction of whole mp 3d structures .
the question of how the controlled integration of an mp into the lipid bilayer takes place is still not fully worked out , and there are certainly aspects of mp structures that will probably not be fully appreciated until this step has been accomplished .
some pursuers educed the viewpoint that the prediction of mp structures from amino acid sequences was , in large measure , a problem of physicochemistry ( 29 ) . physical influences that shape mp structures include interactions of the polypeptide chains with water , bilayer hydrocarbon core , bilayer interfaces , and cofactors .
studies on the mechanism of insertion and folding of mps into membranes are relatively rare and have been mostly performed with two model proteins : bacteriorhodopsin ( br ; ref .
while br is a representative -helical bundle protein , ompa belongs to the class of -barrel protein .
homology modeling constructs structures ( targets ) that are homologous to other protein(s ) whose 3d structure is known ( templates ) .
because few high - resolution mp 3d structures are available to be used as templates , and the modeling can be unreliable when the sequence identity between the template and target proteins falls below 20%-30% , the applicability of homology modeling is limited .
an ab initio method was presented ( 32 ) , whose knowledge - based technique added a membrane potential to the energy terms ( pairwise , solvation , steric , and hydrogen bonding ) .
the method is based on the assembly of supersecondary structural fragments taken from a library of highly resolved protein structures using a standard simulated annealing algorithm .
results obtained by applying the method to small mps of known 3d structures showed that the method is able to predict , at a reasonable accurate level , both the helix topology and the conformations of these proteins .
the structure prediction of membrane proteins still remains an interesting scientific problem . because of the physical difference between mps and gps ( globular proteins )
current segment accuracy of reported algorithms are pretty high ( above 90% ) , while the overall accuracy are still around 50%-60% , which gives birth to hand - raising methods to combine the reports from several other algorithms .
the lack of both high - resolution and low - resolution experimental data of mp structures makes the algorithm development and their evaluation difficult , but the fact that most mp sequences are used as space blocks to get through the membrane bilayer that has predefined thickness makes the structure prediction of mps simple on functional aspects .
new algorithms will emerge and reported algorithms will be refined to give a better answer to this problem .
|
there is a large gap between the number of membrane protein ( mp ) sequences and that of their decoded 3d structures , especially high - resolution structures , due to difficulties in crystal preparation of mps .
however , detailed knowledge of the 3d structure is required for the fundamental understanding of the function of an mp and the interactions between the protein and its inhibitors or activators . in this paper ,
some computational approaches that have been used to predict mp structures are discussed and compared .
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subjects for this nested case - controlled study were selected from ongoing clinical investigations of skeletal muscle insulin resistance . only female subjects were included to provide a sex - controlled analysis .
women aged 6075 years were recruited through advertisements in the pittsburgh , pa , area . a brief phone screening initially determined eligibility for study participation .
potential subjects who were determined to be sedentary ( exercise 1 day / week ) , weight stable ( 3 kg in the previous 6 months ) , with a bmi > 30 kg / m , and nonsmokers were further evaluated at the clinical translational research center ( ctrc ) at the university of pittsburgh .
exclusionary criteria included uncontrolled hypertension ( > 150/95 mmhg ) , anemia ( hematocrit < 34% ) , elevated liver enzymes ( 25% above normal ) , proteinuria , or hypothyroidism ( sensitive thyroid - stimulating hormone [ tsh ] 8
potential subjects were also excluded if they reported taking chronic medications known to affect glucose homeostasis .
after the medical screening , potential subjects then completed a 2-h 75-g oral glucose tolerance test to determine glucose tolerance status .
subjects with type 2 diabetes determined by fasting glucose > 126 mg / dl were excluded .
all subjects gave written consent to the protocol , which was approved by the university of pittsburgh 's institutional review board .
insulin sensitivity was determined by a 4-h hyperinsulinemic ( 40 mu m min ) euglycemic clamp and [ 6,6-h2]glucose infusion as previously described ( 28 ) .
briefly , subjects reported to the ctrc the evening before the clamp and after a 48-h period , in which they refrained from any vigorous physical activity .
a continuous intravenous infusion of insulin ( humulin ; eli lilly , indianapolis , in ) was begun at 7:00 a.m. , and euglycemia ( target 90 mg / dl ) was maintained using an adjustable infusion of 20% dextrose . to measure rate of insulin - stimulated glucose disposal and residual endogenous glucose production , a primed ( 0.22 mol / kg ) continuous ( 17.6 mol kg min ) infusion of [ 6,6-h2]glucose was administered ( 29 ) .
[ 6,6-h2]glucose enrichment in plasma was determined by gas chromatography / mass spectrometry ( 6890 network/5973 series ; agilent , santa clara , ca ) as previously described ( 30 ) .
plasma glucose was measured by the glucose oxidase method ( 2300 stat plus ; yellow springs instruments , yellow springs , oh ) .
fasting plasma insulin concentration was determined by an enzyme - linked immunosorbent assay ( millipore , billerica , ma ) .
rate of glucose disposal ( rd ) was calculated using the tracer : tracee method with a modified version of the steele et al .
percutaneous muscle biopsy samples were obtained in the fasted state before the glucose clamp , as described previously ( 32 ) .
briefly , muscle biopsies were obtained from the vastus lateralis , 15 cm above the patella .
a portion of the specimen ( 30 mg ) was snap - frozen in liquid nitrogen and stored at 80c for sphingolipid and diacylglycerol analysis . a second portion ( 20 mg ) was snap - frozen and stored at 80c for gene expression analysis .
a third portion of the specimen for histochemistry was mounted on a small piece of cork with mounting medium ( shandon cryochrome ; thermo electron , pittsburgh , pa ) , frozen in isopentane cooled with liquid nitrogen for 23 min ( 160c ) , and then placed into liquid nitrogen .
briefly , liquid nitrogen frozen samples ( 30 mg ) were homogenized in ice - cold buffer ( 250 mmol / l sucrose , 25 mmol / l kcl , 50 mmol / l tris , and 0.5 mmol / l edta , ph 7.4 ) .
total content as well as the various molecular species of diacylglycerol and sphingolipid were measured by high - performance liquid chromatography tandem mass spectrometry as previously described in detail ( 33 ) .
plasma free fatty acid concentrations were determined by gas chromatography with flame ionization detection ( 6890n ; agilent , santa clara , ca ) using heptanoic acid as an internal standard .
free fatty acids were derivatized with dimethylamine using deoxo - fluor reagent ( sigma - aldrich , st .
histochemical analysis was performed on serial sections using a modified version of methods previously used in our laboratory ( 32 ) .
briefly , mounted biopsy samples were sectioned ( 10 m ) on a cryostat ( cryotome e ; thermo shandon , pittsburgh , pa ) at 20c and placed on a glass slide .
sections were then stained in a filtered solution of oil red o ( 300 mg / ml in 36% triethylphosphate ) for 30 min at room temperature .
thereafter , sections were incubated with primary antibodies for anti - human myosin heavy chain ( myh)-7 ( type i myocytes ) and myh2 ( type iia myocytes ) overnight at room temperature and subsequently incubated with fluorescein ( fitc ) ( type iia myocytes ) and rhodamine ( type i myocytes ) conjugated secondary antibodies ( santa cruz biotechnology , santa cruz , ca ) .
images were visualized using a leica microscope ( leica dm 4000b ; leica microsystems , bannockburn , il ) , digitally captured ( retiga 2000r camera ; q imaging , surrey , canada ) , and analyzed using specialized software ( northern eclipse , v6.0 ; empix imaging , cheektowaga , ny ) .
oil red o staining intensity and cross - sectional area were determined in type i and type ii myocytes .
gene expression analysis was carried out in a subset of subjects from each group , based on availability of vastus lateralis biopsy specimen ( six ir and six is ) .
isolated rna was then further purified using a silicon - membrane spin column including an on - column dnase i treatment ( qiagen , valencia , ca ) .
total rna was reverse - transcribed using moloney murine leukemia virus ( mmlv ) reverse transcriptase ( sabiosciences , frederick , md ) .
gene expression was determined by quantitative real - time pcr ( qrt - pcr ) using sybr green - based custom rt profiler pcr arrays ( sabiosciences , frederick , md ) .
qrt - pcr was performed on an abi prism 7900 ( applied biosystems , foster city , ca ) .
baseline , threshold cycle ( ct ) and threshold were determined and set for all samples .
the relative level of mrna expression for each gene in each sample was normalized to the average expression of 2-microglobulin , hypoxanthine phosphoribosyltransferase ( hprt1 ) , glyceraldehydes-3-phosphate dehydrogenase ( gapdh ) , and -actin ( actb ) and expressed as 2 .
total body fat and lean mass were assessed using dual - energy x - ray absorptiometry ( ge lunar prodigy and encore 2005 software v9.3 ) .
subjects performed a vo2 peak test on an electronically braked cycle ergometer to determine aerobic fitness ( sensor medics , yorba linda , ca ) as previously described ( 34 ) .
insulin sensitivity was determined by a 4-h hyperinsulinemic ( 40 mu m min ) euglycemic clamp and [ 6,6-h2]glucose infusion as previously described ( 28 ) .
briefly , subjects reported to the ctrc the evening before the clamp and after a 48-h period , in which they refrained from any vigorous physical activity .
a continuous intravenous infusion of insulin ( humulin ; eli lilly , indianapolis , in ) was begun at 7:00 a.m. , and euglycemia ( target 90 mg / dl ) was maintained using an adjustable infusion of 20% dextrose . to measure rate of insulin - stimulated glucose disposal and residual endogenous glucose production , a primed ( 0.22 mol / kg ) continuous ( 17.6 mol kg min ) infusion of
[ 6,6-h2]glucose enrichment in plasma was determined by gas chromatography / mass spectrometry ( 6890 network/5973 series ; agilent , santa clara , ca ) as previously described ( 30 ) .
plasma glucose was measured by the glucose oxidase method ( 2300 stat plus ; yellow springs instruments , yellow springs , oh ) .
fasting plasma insulin concentration was determined by an enzyme - linked immunosorbent assay ( millipore , billerica , ma ) .
rate of glucose disposal ( rd ) was calculated using the tracer : tracee method with a modified version of the steele et al .
percutaneous muscle biopsy samples were obtained in the fasted state before the glucose clamp , as described previously ( 32 ) .
briefly , muscle biopsies were obtained from the vastus lateralis , 15 cm above the patella .
a portion of the specimen ( 30 mg ) was snap - frozen in liquid nitrogen and stored at 80c for sphingolipid and diacylglycerol analysis . a second portion ( 20 mg ) was snap - frozen and stored at 80c for gene expression analysis .
a third portion of the specimen for histochemistry was mounted on a small piece of cork with mounting medium ( shandon cryochrome ; thermo electron , pittsburgh , pa ) , frozen in isopentane cooled with liquid nitrogen for 23 min ( 160c ) , and then placed into liquid nitrogen .
briefly , liquid nitrogen frozen samples ( 30 mg ) were homogenized in ice - cold buffer ( 250 mmol / l sucrose , 25 mmol / l kcl , 50 mmol / l tris , and 0.5 mmol / l edta , ph 7.4 ) .
total content as well as the various molecular species of diacylglycerol and sphingolipid were measured by high - performance liquid chromatography tandem mass spectrometry as previously described in detail ( 33 ) .
plasma free fatty acid concentrations were determined by gas chromatography with flame ionization detection ( 6890n ; agilent , santa clara , ca ) using heptanoic acid as an internal standard .
free fatty acids were derivatized with dimethylamine using deoxo - fluor reagent ( sigma - aldrich , st .
histochemical analysis was performed on serial sections using a modified version of methods previously used in our laboratory ( 32 ) .
briefly , mounted biopsy samples were sectioned ( 10 m ) on a cryostat ( cryotome e ; thermo shandon , pittsburgh , pa ) at 20c and placed on a glass slide .
sections were then stained in a filtered solution of oil red o ( 300 mg / ml in 36% triethylphosphate ) for 30 min at room temperature .
thereafter , sections were incubated with primary antibodies for anti - human myosin heavy chain ( myh)-7 ( type i myocytes ) and myh2 ( type iia myocytes ) overnight at room temperature and subsequently incubated with fluorescein ( fitc ) ( type iia myocytes ) and rhodamine ( type i myocytes ) conjugated secondary antibodies ( santa cruz biotechnology , santa cruz , ca ) .
images were visualized using a leica microscope ( leica dm 4000b ; leica microsystems , bannockburn , il ) , digitally captured ( retiga 2000r camera ; q imaging , surrey , canada ) , and analyzed using specialized software ( northern eclipse , v6.0 ; empix imaging , cheektowaga , ny ) .
oil red o staining intensity and cross - sectional area were determined in type i and type ii myocytes .
gene expression analysis was carried out in a subset of subjects from each group , based on availability of vastus lateralis biopsy specimen ( six ir and six is ) .
isolated rna was then further purified using a silicon - membrane spin column including an on - column dnase i treatment ( qiagen , valencia , ca ) .
total rna was reverse - transcribed using moloney murine leukemia virus ( mmlv ) reverse transcriptase ( sabiosciences , frederick , md ) .
gene expression was determined by quantitative real - time pcr ( qrt - pcr ) using sybr green - based custom rt profiler pcr arrays ( sabiosciences , frederick , md ) .
qrt - pcr was performed on an abi prism 7900 ( applied biosystems , foster city , ca ) .
baseline , threshold cycle ( ct ) and threshold were determined and set for all samples .
the relative level of mrna expression for each gene in each sample was normalized to the average expression of 2-microglobulin , hypoxanthine phosphoribosyltransferase ( hprt1 ) , glyceraldehydes-3-phosphate dehydrogenase ( gapdh ) , and -actin ( actb ) and expressed as 2 .
total body fat and lean mass were assessed using dual - energy x - ray absorptiometry ( ge lunar prodigy and encore 2005 software v9.3 ) .
subjects performed a vo2 peak test on an electronically braked cycle ergometer to determine aerobic fitness ( sensor medics , yorba linda , ca ) as previously described ( 34 ) .
ten insulin - sensitive ( is ) and 12 insulin - resistant ( ir ) women were matched for age , body mass , bmi , and physical fitness ( vo2 peak ) ( table 1 ) .
there was no significant group difference in fasting plasma glucose , insulin , or free fatty acid levels .
however , by design , rd during the hyperinsulinemic - euglycemic clamp was greater ( p < 0.01 ) in the is group when compared with the ir group ( table 1 ) .
the 2-h plasma glucose levels during the oral glucose tolerance test were also significantly higher in the ir group compared with the is group ( p = 0.016 ) .
the ir ( n = 6 ) and is ( n = 6 ) subgroups for gene expression analysis had similar characteristics as their respective groups overall ( data not shown ) .
type i and ii myocyte content and myocyte average cross - sectional area are presented in table 2 .
the is group had a higher proportion of type i myocytes ( p = 0.002 ) and a lower proportion of type iia ( p = 0.014 and type iix ( p = 0.02 ) than the ir group .
when data from both groups were combined , there was a higher proportion of type iia compared with type iix myocytes ( p = 0.008 ) .
both groups combined , there was a significant positive correlation between the proportion of type i myocytes and insulin sensitivity and a negative correlation between the proportion of type ii myocytes ( type iia and iix combined ) and insulin sensitivity ( table 3 ) .
muscle fiber type distribution and surface area in vastus lateralis muscle * significantly different from is group , p < 0.05 .
data generated from analysis of subgroup ( ir , n = 10 ; is , n = 5 ) .
correlation of rate of glucose disposal ( ml kg fat - free mass min ) with other study variables when data from both ir and is groups were combined * correlation is significant at the level p < 0.01 .
correlation is significant at the level p < 0.05 . the data for muscle content of sphingolipid and diacylglycerol species is presented in fig .
we observed higher total levels of ceramide ( 555 53 vs. 293 54 pmol / mg protein , p = 0.004 ) , total unsaturated ceramide ( 198 29 vs. 114 21 pmol / mg protein , p = 0.034 ) , and total saturated ceramide ( 361 29 vs. 179 34 pmol / mg protein , p = 0.001 ) in the ir group compared with the is group .
we also observed higher levels of specific ceramide species containing the following fatty acids : myristic ( c14:0 ) ( 4.0 0.8 vs. 1.9 0.4 pmol / mg protein , p = 0.044 ) , palmitic ( c16:0 ) ( 58 14 vs. 18 5 pmol / mg protein , p = 0.023 ) , and stearic ( c18:0 ) ( 72 10 vs. 40 7 pmol / mg protein , p = 0.02 ) in ir compared with is muscle
. there also tended to be greater content of sphingolipids containing palmitoleic ( c18:1 ) ( 43 11 vs. 19 3 pmol / mg protein , p = 0.077 ) and lignoceric ( c24:0 ) ( 201 25 vs. 123 29 pmol / mg protein , p = 0.06 ) acids and sphingosine ( 3.4 0.6 vs. 2.2 0.3 pmol / mg protein , p = 0.08 ) in ir muscle . when data were combined from both groups
, we observed a significant negative correlation between total unsaturated , total saturated , and total ceramide content and insulin sensitivity ( table 3 ) .
total ceramide ( r = 0.517 , p < 0.05 ) and total saturated ceramide ( r = 0.523 , p < 0.05 ) were positively correlated with type ii myocyte percentage .
however , no significant differences in total or individual diacylglycerol species were observed between is and ir subjects .
sphingolipid ( a ) and diacylglycerol ( b ) content in vastus lateralis of ir and is subjects .
ir data are expressed relative to is group , which was set to a value of 1 . * significantly different from is group , p < 0.05
total muscle imtg was greater in the ir group compared with the is group ( p = 0.009 ) .
type i myocyte specific imtg content was also greater in the ir group compared with the is group ( p = 0.005 ) .
specific imtg content between is and ir groups , nor was imtg content different in ir within subtypes of type iia and type iix myocytes .
when both groups were combined , we observed a significant negative correlation between type i myocyte imtg content and insulin sensitivity , whereas there was no relationship between insulin sensitivity and type ii myocyte ( type iia and iix combined ) imtg content ( fig .
e : imtg content in type i myocytes , type ii myocytes , and total imtg in vastus lateralis from ir and is groups . * significantly different from is , p < 0.05 .
( a high - quality color digital representation of this figure is available in the online issue . )
expression levels of key genes , including facilitated glut member 4 ( glut4 ) , insulin receptor substrate ( irs)-1 , and irs2 were lower in the ir group , indicating insulin resistance in this group at the tissue level in accord with their insulin resistance assessed in vivo .
the expression of genes relating to oxidative phosphorylation transcription factors , fatty acid metabolism , ceramide metabolism , and lipid droplet associated proteins were examined .
we found lower expression of the transcription factors peroxisome proliferator activated receptor ( ppar)- and ppar- in the ir group compared with the is group .
also , expression of ppar- coactivator 1 ( pgc-1 ) and nuclear respiratory factor 1 ( nrf1 ) tended to be lower in ir muscle .
furthermore , we observed a lower expression of carnitine palmitoyltransferase 1b ( cpt1b ) and malonyl - coa decarboxylase ( mlycd ) , genes that are related to fatty acid metabolism . gene expression of lipid droplet associated proteins hormone - sensitive lipase ( hsl ) , adipose differentiation related protein ( adrp ) , adipose triglyceride lipase ( atgl ) , and synaptosomal - associated protein 23 ( snap23 ) was lower in the ir group compared with the is group .
interestingly , the expression of genes relating to ceramide metabolism was not different between groups .
the expression of tumor necrosis factor ( tnf)- tended to be higher in the ir group compared with the is group ( p = 0.09 ) . gene expression in vastus lateralis of ir and is subgroups .
ir gene expression is expressed relative to is group , which was set to a value of 1 .
abhd5 , abhydrolase domain containing 5 ; acac , acetyl - coa carboxylase ; asah , n - acylsphingosine amidohydrolase ; cerk , ceramide kinase ; cpt , carnitine palmitoyltransferase ; mlycd , malonyl - coa decarboxylase ; nrf , nuclear respiratory factor ; s3 - 12 , plasma membrane associated protein kiaa1881 ; sptlc , serine palmitoyltransferase , long - chain base . * significantly different from is group , p < 0.05 .
type i and ii myocyte content and myocyte average cross - sectional area are presented in table 2 .
the is group had a higher proportion of type i myocytes ( p = 0.002 ) and a lower proportion of type iia ( p = 0.014 and type iix ( p = 0.02 ) than the ir group .
when data from both groups were combined , there was a higher proportion of type iia compared with type iix myocytes ( p = 0.008 ) .
both groups combined , there was a significant positive correlation between the proportion of type i myocytes and insulin sensitivity and a negative correlation between the proportion of type ii myocytes ( type iia and iix combined ) and insulin sensitivity ( table 3 ) .
muscle fiber type distribution and surface area in vastus lateralis muscle * significantly different from is group , p < 0.05 .
data generated from analysis of subgroup ( ir , n = 10 ; is , n = 5 ) .
correlation of rate of glucose disposal ( ml kg fat - free mass min ) with other study variables when data from both ir and is groups were combined * correlation is significant at the level p < 0.01 .
we observed higher total levels of ceramide ( 555 53 vs. 293 54 pmol / mg protein , p = 0.004 ) , total unsaturated ceramide ( 198 29 vs. 114 21 pmol / mg protein , p = 0.034 ) , and total saturated ceramide ( 361 29 vs. 179 34 pmol / mg protein , p = 0.001 ) in the ir group compared with the is group .
we also observed higher levels of specific ceramide species containing the following fatty acids : myristic ( c14:0 ) ( 4.0 0.8 vs. 1.9 0.4 pmol / mg protein , p = 0.044 ) , palmitic ( c16:0 ) ( 58 14 vs. 18 5 pmol / mg protein , p = 0.023 ) , and stearic ( c18:0 ) ( 72 10 vs. 40 7 pmol / mg protein , p = 0.02 ) in ir compared with is muscle
. there also tended to be greater content of sphingolipids containing palmitoleic ( c18:1 ) ( 43 11 vs. 19 3 pmol / mg protein , p = 0.077 ) and lignoceric ( c24:0 ) ( 201 25 vs. 123 29 pmol / mg protein , p = 0.06 ) acids and sphingosine ( 3.4 0.6 vs. 2.2 0.3 pmol / mg protein , p = 0.08 ) in ir muscle . when data were combined from both groups
, we observed a significant negative correlation between total unsaturated , total saturated , and total ceramide content and insulin sensitivity ( table 3 ) .
total ceramide ( r = 0.517 , p < 0.05 ) and total saturated ceramide ( r = 0.523 , p < 0.05 ) were positively correlated with type ii myocyte percentage .
however , no significant differences in total or individual diacylglycerol species were observed between is and ir subjects .
sphingolipid ( a ) and diacylglycerol ( b ) content in vastus lateralis of ir and is subjects .
ir data are expressed relative to is group , which was set to a value of 1 . *
total muscle imtg was greater in the ir group compared with the is group ( p = 0.009 ) .
type i myocyte specific imtg content was also greater in the ir group compared with the is group ( p = 0.005 ) .
specific imtg content between is and ir groups , nor was imtg content different in ir within subtypes of type iia and type iix myocytes .
when both groups were combined , we observed a significant negative correlation between type i myocyte imtg content and insulin sensitivity , whereas there was no relationship between insulin sensitivity and type ii myocyte ( type iia and iix combined ) imtg content ( fig .
myocyte - specific imtg content in vastus lateralis of ir and is groups . a : representative image of oil red o stain ( 20 objective ) .
e : imtg content in type i myocytes , type ii myocytes , and total imtg in vastus lateralis from ir and is groups . * significantly different from is , p < 0.05 .
( a high - quality color digital representation of this figure is available in the online issue . )
3 . expression levels of key genes , including facilitated glut member 4 ( glut4 ) , insulin receptor substrate ( irs)-1 , and irs2 were lower in the ir group , indicating insulin resistance in this group at the tissue level in accord with their insulin resistance assessed in vivo .
the expression of genes relating to oxidative phosphorylation transcription factors , fatty acid metabolism , ceramide metabolism , and lipid droplet associated proteins were examined .
we found lower expression of the transcription factors peroxisome proliferator activated receptor ( ppar)- and ppar- in the ir group compared with the is group .
also , expression of ppar- coactivator 1 ( pgc-1 ) and nuclear respiratory factor 1 ( nrf1 ) tended to be lower in ir muscle .
furthermore , we observed a lower expression of carnitine palmitoyltransferase 1b ( cpt1b ) and malonyl - coa decarboxylase ( mlycd ) , genes that are related to fatty acid metabolism . gene expression of lipid droplet associated proteins hormone - sensitive lipase ( hsl ) , adipose differentiation related protein ( adrp ) , adipose triglyceride lipase ( atgl ) , and synaptosomal - associated protein 23 ( snap23 ) was lower in the ir group compared with the is group .
interestingly , the expression of genes relating to ceramide metabolism was not different between groups .
the expression of tumor necrosis factor ( tnf)- tended to be higher in the ir group compared with the is group ( p = 0.09 ) . gene expression in vastus lateralis of ir and is subgroups .
ir gene expression is expressed relative to is group , which was set to a value of 1 .
abhd5 , abhydrolase domain containing 5 ; acac , acetyl - coa carboxylase ; asah , n - acylsphingosine amidohydrolase ; cerk , ceramide kinase ; cpt , carnitine palmitoyltransferase ; mlycd , malonyl - coa decarboxylase ; nrf , nuclear respiratory factor ; s3 - 12 , plasma membrane associated protein kiaa1881 ; sptlc , serine palmitoyltransferase , long - chain base .
despite a preponderance of evidence from cell culture and animal models suggesting a role for both ceramide and diacylglycerol in skeletal muscle insulin resistance ( 7,8 ) , there is actually no consensus as to whether this is relevant to human insulin resistance ( 12,13,15,17,19 ) .
this study was conducted for three purposes : 1 ) to determine whether diacylglycerol or sphingolipid species are elevated in ir human skeletal muscle , 2 ) to investigate whether the associations between intramyocellular lipids are dependent on muscle fiber type , and 3 ) to explore whether the skeletal muscle expression of several key genes relating to lipid metabolism are altered in ir muscle corresponding to intramyocellular lipid content .
a primary finding of our study was that total as well as saturated and unsaturated ceramide content was approximately twofold higher in ir skeletal muscle in human subjects independent of obesity , physical fitness , and plasma free fatty acid concentration .
( 13 ) , who observed elevated ceramide content in skeletal muscle of obese subjects .
( 12,14 ) also found that ceramide accumulation in skeletal muscle was inversely related to insulin sensitivity .
they also suggested that higher ceramide content in lean offspring of patients with type 2 diabetes was due to decreased ceramide degradation , whereas ir of obesity was caused by an increase in de novo ceramide synthesis ( 12,14 ) .
not all studies , however , have observed an association between muscle ceramide content and insulin resistance ( 18,19 ) .
these discrepant results could be attributed to differences in the subject population studied , e.g. , age , sex , physical activity , and methodological differences , in assessing ceramide content and insulin sensitivity ( 11 ) .
thus , our study provides important new information about the relationship between elevated ceramide content and muscle insulin resistance .
this is the first to a comprehensive profile of the content of the various molecular species of ceramide in human ir muscle using tandem mass spectrometry while controlling for obesity , physical fitness , and age .
this is the first study to demonstrate elevated levels of both saturated and unsaturated ceramides , as well as specific ceramides containing c14:0 , c16:0 , and c18:0 fatty acids in ir human muscle .
none of the prior studies examining ceramide in human muscle have accounted for heterogeneity in muscle type that could account for differences in insulin sensitivity .
our novel finding that ceramide content was associated with a higher percentage of type ii myocytes suggests that ceramide accumulation may be fiber - type dependent .
in addition , the complementary observations that both the imtg content in type i myocytes and the lower proportion of type i myocytes are related to insulin resistance provide insight into muscle heterogeneity in lipid metabolism in insulin resistance .
these data suggest a possibility that type i myofibers may have a greater capacity to buffer increases in free fatty acids by partitioning to lipid droplet imtg , whereas type ii fibers have a reduced capacity to incorporate excess fatty acids into imtg and are more inclined to partition to lipotoxic mediators such as ceramide species .
an impairment of skeletal muscle oxidative capacity has been suggested to initiate or potentiate insulin resistance ( 35 ) .
our data indicate that the proportion of type i and type ii myocytes is associated with higher ceramide content and insulin resistance and that the expression levels of several genes related to mitochondrial biogenesis and fatty acid metabolism are lower in insulin resistance .
therefore , our complementary results support the concept that a low oxidative capacity of muscle may be associated with ceramide accumulation and insulin resistance .
this is consistent with previous reports that the increase in whole muscle imtg content in obesity or type 2 diabetes is primarily due to increased imtg in type i myocytes ( 25 ) and that differences in fiber type proportion also play a role in insulin resistance of obesity and type 2 diabetes ( 36,37 ) .
these observations are supported by a number of reports suggesting that type i myocytes are more insulin responsive than type ii myocytes ( 26,27,38 ) .
yet , differences in fiber type proportion between obese subjects either with or without type 2 diabetes have been reported in some ( 36,37 ) , but not all previous studies ( 22,24 ) . despite evidence in ir animal models supporting a role for dag in insulin resistance ( 8,9 )
, we did not observe an increased level of dag in insulin - resistant muscle .
( 12 ) have also reported elevated levels of ceramide , but not dag , in obese impaired glucose tolerant versus obese normal glucose tolerant ( ngt ) subjects . a possible reason for lipid - induced insulin resistance by ceramide and not dag , and visa versa ,
may be that higher levels of palmitate result in greater ceramide accumulation , while unsaturated fatty acids partition toward dag ( 39 ) .
thus , both ceramides and dag can potentially induce insulin resistance depending on the degree of saturation of the lipid overload .
our finding that c16:0 ceramide was more than threefold higher in ir muscle is consistent with this .
thus , it is plausible that basal levels of ceramide and dag in muscle are influenced by the degree of saturation and palmitate content in the diet of obese individuals .
we also examined the differential expression of several other genes relating to lipid metabolism , which could underlie accumulation of imtg and other bioactive species , i.e. , dag and ceramide .
we found that many genes involved in ceramide synthesis and degradation were not differentially expressed in ir muscle with increased ceramide content .
a recent report suggests that over expression of acid ceramidase prevents the inhibitory effect of saturated fatty acids on insulin signaling ( 40 ) .
also , inhibition of serine palmitoyltranseferase in zucker diabetic fatty ( zdf ) rats , by treatment with the amino acid antibiotic myriocin , prevented the onset of type 2 diabetes and inhibited an increase in ceramide content in muscle , when compared with nontreated zdf rats ( 41 ) .
our results suggest that factors other than increased enzyme content , possibly increased enzyme activity or fatty acid flux , contribute to ceramide accumulation in ir muscle . in support of this , straczkowski et al .
( 12 ) demonstrated that the activity of neutral sphingomyelinase , the enzyme that converts sphingomyelin to ceramide , is increased in muscle of obese impaired glucose tolerant subjects compared with obese normal glucose tolerant subjects , concomitant with higher ceramide content .
finally , the observation that gene expression of tnf- tended to be elevated in ir muscle , albeit statistically nonsignificant , concomitant with elevated ceramide content is consistent with data demonstrating that tnf- can stimulate de novo ceramide biosynthesis ( 42 ) . to further characterize intramyocellular lipid content and metabolism in ir skeletal muscle , we determined the expression of selected genes coding for lipid droplet proteins and proteins involved in lipid droplet hydrolysis .
atgl and hsl expression / activity have been demonstrated to contribute to adipose tissue and skeletal muscle triacylglycerol lipolysis , elevated plasma fatty acid levels , and accumulation of ectopic lipid deposits associated with type 2 diabetes ( 43 ) .
our observation that atgl and hsl mrna are lower in ir muscle is in line with reports of lower expression of these lipases in obesity and/or insulin resistance ( 44,45 ) .
consistent with this , phillips et al . showed that skeletal muscle protein expression of adrp was upregulated in response to improvements in insulin action induced by weight loss and insulin sensitizer pharmacotherapy ( 46 ) .
furthermore , atgl and adrp have been reported to be more highly expressed in type i muscle fibers ( 47,48 ) .
the fiber type specific expression of atgl and adrp , and our observation of reduced type i fibers in obese ir subjects , potentially explains the lower level of atgl and adrp observed in whole muscle in the present study .
it is tempting to speculate the same may be true for other lipases and that fiber type composition plays a greater role in insulin resistance than previously thought .
furthermore , the trend toward higher tnf- gene expression in ir muscle is in line with reports that tnf- represses atgl expression ( 49 ) .
we also observed for the first time in human muscle that the expression of snap23 , a member of the snare family of proteins whose function is to mediate intracellular vesicle fusion events including glut4 transport to the plasma cell membrane and lipid droplet fusion ( 50 ) , is lower in ir muscle .
this novel finding potentially linking intramyocellular lipid droplets and glucose transport is supported by others describing an association between snap23 and fatty acid induced insulin resistance ( 50 ) .
future studies should be conducted to examine whether or not interventions such as exercise or weight loss affect oxidative capacity , dag , ceramide , and insulin sensitivity to better understand these associations in humans .
although we substantiated our insulin resistance phenotype with expression of key genes involved in insulin signaling and glucose transport , additional studies are needed to link intramyocellular lipids , fiber type , oxidative capacity , and activation of specific insulin - signaling proteins .
taken together , however , these results suggest a role for muscle fiber type proportion and/or the oxidative capacity of muscle in insulin resistance , possibly through partitioning muscle lipids into either triglycerides or ceramides . in summary ,
ceramide but not diacylglycerol is elevated in ir human skeletal muscle independent of obesity , physical activity , or aerobic fitness .
moreover , muscle fiber type as well as fiber type specific lipid content is associated with insulin resistance in muscle .
importantly , key genes associated with fatty acid metabolism and lipid droplet regulation are decreased in ir muscle .
together , these data suggest that mechanisms underlying the associations between intramyocellular lipids and insulin resistance likely involve ceramide , which in turn may depend on the muscle fiber type or its oxidative capacity .
|
objectivewe tested the primary hypotheses that sphingolipid and diacylglycerol ( dag ) content is higher within insulin - resistant muscle and that the association between intramyocellular triglycerides ( imtg ) and insulin resistance is muscle fiber type specific.research design and methodsa nested case - control analysis was conducted in 22 obese ( bmi > 30 kg / m2 ) women who were classified as insulin - resistant ( ir ; n = 12 ) or insulin - sensitive ( is ; n = 10 ) , determined by hyperinsulinemic - euglycemic clamp ( > 30% greater in is compared with ir , p < 0.01 ) .
sphingolipid and dag content was determined by high - performance liquid chromatography tandem mass spectrometry .
fiber type
specific imtg content was histologically determined .
gene expression was determined by quantitative pcr.resultstotal ( 555 53 vs. 293 54 pmol / mg protein , p = 0.004 ) , saturated ( 361 29 vs. 179 34 pmol / mg protein , p = 0.001 ) , and unsaturated ( 198 29 vs. 114 21 pmol / mg protein , p = 0.034 ) ceramides were higher in ir compared with is .
dag concentrations , however , were similar .
imtg content within type i myocytes , but not type ii myocytes , was higher in ir compared with is subjects ( p = 0.005 ) .
insulin sensitivity was negatively correlated with imtg within type i myocytes ( r = 0.51 , p = 0.026 ) , but not with imtg within type ii myocytes .
the proportion of type i myocytes was lower ( 41 vs. 59% , p < 0.01 ) in ir subjects .
several genes involved in lipid droplet and fatty acid metabolism were differentially expressed in ir compared with is subjects.conclusionshuman skeletal muscle insulin resistance is related to greater imtg content in type i but not type ii myocytes , to greater ceramide content , and to alterations in gene expression associated with lipid metabolism .
|
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a 50-year - old , 168 cm , 68 kg man without significant past medical history , was admitted to emergency room following a slip - down accident . on admission ,
neurologic examination confirmed a glasgow coma scale ( gcs ) of 15 , and the pupils were symmetrical and reactive . emergent skull x - ray series , facial computerized tomography ( ct ) and forearm x - ray revealed fracture of the frontal sinus , skull base , and right distal radius fracture . computed tomography ( ct ) of the brain showed multiple small subdural pneumocephalus ( fig .
emergent lumbar drainage was performed to manage csf rhinorrhea and empirical prophylactic antibiotic treatment was given .
the patient 's post - traumatic symptoms improved gradually in the surgical intensive care unit . at post - trauma 8 days , following trauma , the patient underwent orthopedic surgery for distal radius fracture under general anesthesia .
anesthesia was induced with intravenous thiopental 300 mg and 0.5 g / kg / min remifentanil infusion .
endotracheal intubation was performed after administering 50 mg rocuronium injection with 100% oxygen mask ventilation for 3 min without any difficulties .
anesthesia was maintained with oxygen ( 2 l / min)-air ( 2 l / min)-sevoflurane ( 1 - 1.5 vol% ) , continuous infusion of 0.125 - 0.5 g / kg / min remifentanil and 10 - 15 mg / h of rocuronium .
tidal volume and respiratory rate were adjusted to maintain end - tidal carbon dioxide tension at 30 - 35 mmhg .
following surgery , the patient was extubated in the operation room after gaining consciousness and spontaneous ventilation .
he was then transferred to the post - anesthesia care unit where he recovered fully without complications .
ten hours later , the patient complained of severe , increasing , headache and exhibited abrupt deterioration in mental state . in view of his clinical aggravation ,
neurologic examination demonstrated a gcs 11 and emergent brain ct showed large quantity of both frontal and right occipital pneumocephalus ( fig .
high flow ( 8 l / min ) oxygen via a partial non - rebreathing mask was administered in conjunction with supportive care for 2 days .
no infectious neuromeningeal complications occurred during hospitalization , and the patient was discharged at post - trauma day 32 .
it develops mostly from acute and subacute complications of head trauma , or as late complications during the recovery phase of head injury .
subarachnoid or subdural air accumulation is the most common form of pneumocephalus , and intracerebral pneumocephalus can occur albeit rarely .
intracranial air is accumulated as a result of communications between paranasal sinuses , mastoid air cells and the cranial fossa , associated with dural tear .
the other mechanism of air penetration is a ball - valve effect like sudden increase of nasopharyngeal pressure , such as during coughing and sneezing , forcing air into the cranial cavity which subsequently remained trapped .
injuries of the paranasal sinuses , which are closely contiguous to bony structures , the dura mater and the brain frequently accompany head traumas .
75 - 90% of pneumocephalus are posttraumatic and the incidence of pneumocephalus secondary to head injury range from 0.5% to 13.2% .
other causes include facial tumors that eroded through the skull , gas - forming bacterial infections , paranasal sinusitis and neurosurgical and facial procedures .
pneumocephalus is mostly asymptomatic and self - limiting , because gas is not irritating to the surrounding brain .
intracerebral pneumocephalus have usually become sizable when it is confirmed by simple skull x - ray or brain ct .
tension pneumocephalus caused by large amounts air can evoke sudden or insidious mass effects capable of shifting midline structures , causing major neurological deficits .
representable iatrogenic pneumocephalus are related to positive pressure ventilation in the upper airway , such as nasal continuous positive airway pressure , face mask ventilation and even nasal oxygen cannulae .
it is well known that positive airway pressure can result in pneumothorax , pneumomediastinum and subcutaneous emphysema .
it is difficult to predict which head trauma patients who do not initially develop pneumocephalus will experience iatrogenic pnemocephalus during positive pressure oxygen therapies .
the mechanism of air delivered via manually operated mask tracking into the cranial cavities appears to be identical to that in traumatic pneumocephalus . because csf leakage is usually a result of osteomeningeal fistula , csf rhinorrhea is commonly associated with pneumocephaus , as demonstrated in this patient .
his initial primary pneumocephalus accompanied by rhinorrhea was completely subsided following lumbar drainage and general supportive care , but post - anesthetic secondary pneumocephalus probably developed during anesthesia induction using ppmv .
therefore , avoidance of excessive positive pressure during mask ventilation is recommended to prevent pneumocephalus . because most mild pneumocephalus usually recover spontaneously within one week
, its management includes strict bed rest in the semi - fowler position , analgesia and antibiotics .
prevention of intracranial pressure elevation by avoiding the valsalva maneuvers , coughing , sneezing , nose blowing , and excessive physical activities is important to avoid pneumocephalus aggravation . because severe headache , personality change , disorientation , and mass effect associated with midline structure shift are the symptoms and signs of tension pneumocephalus , emergent surgical procedures , such as subdural drainage to decrease intracranial pressure , should be considered immediately after confirming the diagnosis with x - rays or brain ct .
when the patient experienced severe headache and became rapidly lethargic , analgesia , antibiotic and high flow , high fio2 oxygen via a partial non - rebreathing reservoired mask were administered under a presumed diagnosis of impending tension pneumocephalus .
the management principle is as follows : high fio2 oxygen reduces blood and brain tissue nitrogen concentration , thereby increases the nitrogen concentration gradient that promotes pneumocephalus gas absorption . to avoid tension pneumocephalus in head trauma patients , lma or tracheostomy tube intubation
in addition , anesthesiologists should be aware of potential complications caused by low intracranial pressure during anesthesia in head trauma patients . in theory ,
hypotension , hypovolemia , excessive hypercapnia and head - up position during anesthesia can reduce intracranial pressure that facilitates air influx through open osteomeningeal fistulae secondary to head trauma , causing pneumocephalus . in conclusion
, anesthesiologists and surgeons should be aware of pneumocephalus as a potential complication during positive pressure mask ventilation for facial trauma , secondary to air invasion through osteomeningeal fistulae .
close monitoring for pneumocephalus development should be mandatory following general anesthesia or during respiratory care with continuous positive airway pressure in head trauma patients .
|
pneumocephalus is a condition which usually results from head trauma .
it has been known that iatrogenic pneumocephalus can occur as a complication of positive pressure mask ventilation during induction of anesthesia or ventilatory care for head trauma patients .
we report a case of mask ventilation during anesthesia induction in a 50-year - old male patient with head trauma .
initial pneumocephalus associated with cerebrospinal fluid leakage was diagnosed immediate following head injury involving facial sinuses .
he was managed with emergent lumbar drainage and supportive care .
pneumocephalus recurred following positive pressure mask ventilation ( ppmv ) during anesthesia induction for surgery on the right arm .
recurred pneumocephalus was managed with high flow oxygen and supportive care .
anesthesiologists should be aware of pneumocephalus as a potential complication of ppmv in head trauma patients , even after resolution of previous pneumocephalus .
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the online version of this article ( doi:10.1007/s13555 - 015 - 0084 - 3 ) contains supplementary material , which is available to authorized users .
finasteride 1 mg is used successfully all over the world for the treatment of androgenetic alopecia in male pattern baldness .
various clinical trials have established finasteride s efficacy in adult men with predominant vertex , anterior and midscalp region [ 2 , 3 ] . on average ,
common side effects are loss of libido , erectile dysfunction and decreased ejaculate volume . these side effects were found in less than 0.5% of patients . to assess the huge difference [ 2 , 11 ] in reporting the side effects
, we conducted a 2-year randomized , questionnaire - based research study on patients receiving finasteride 1 mg tablets for androgenetic alopecia .
for this , we used a questionnaire based on the international index of erectile function ( iief ) ( supplementary table 1 ) .
this checks five domains : erectile function , orgasmic function , sexual desire , intercourse satisfaction and overall sexual satisfaction .
a higher iief score indicates better sexual function and low score ( below 25 ) indicates erectile dysfunction .
the aim of the present study was to evaluate an age - matched comparison and evaluation of sexual function in patients treated by finasteride 1 mg for early androgenetic alopecia by using iief .
inclusion criteria were age 1840 years , who came for an outpatient consultation for male pattern androgenetic alopecia , which was diagnosed using the norwood
hamilton s grading of male pattern alopecia with grade 3 to 5 on treatment with finasteride 1 mg , with good physical health .
exclusion criteria were patients being treated for various other causes of hair loss other than androgenetic alopecia .
those selected patients were briefed about the iief questionnaire and , with their consent , the questionnaire was provided for completion .
a mean duration of approximately 16 weeks of medication was consumed by the patients at the time of answering the questionnaire .
our controls were 586 patients who attended outpatient consultation for various other non - hair related diseases such as psoriasis vulgaris , lichen planus , allergic contact dermatitis , irritant contact dermatitis , nail disorders , etc .
the resultant scores of erectile function domain and iief domain of both the study group taking finasteride 1 mg and their age - wise control group were analyzed with the ibm software package ( spss statistics , ny , usa ) . all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1964 , as revised in 2013 .
both the study group and controls were asked to answer the iief questionnaire anonymously to avoid any identity on sexual weakness .
the results were compared in an age - wise manner . comparing the age - wise finasteride - consuming patients and the control group ,
there was no statistical difference between the groups regarding their sexual function in total iief score or in any of the five domains mentioned .
1 ) show the scores in the erectile function domain ( a ) and international index of erectile function domain ( b ) in patients on finasteride and their controls.fig .
1age controlled 3d graphs showing the scores of domain in erectile function ( a ) and international index of erectile function domain ( b ) in patients on finasteride and their controls with age and scores both depicted in y axis age controlled 3d graphs showing the scores of domain in erectile function ( a ) and international index of erectile function domain ( b ) in patients on finasteride and their controls with age and scores both depicted in y axis
the main mechanism in androgenetic alopecia is miniaturization of hair follicles from terminal to vellus hair due to the effect of androgen , especially dihydrotestosterone .
testosterone is converted by 5 alpha reductase to 5 alpha dihydrotestosterone , which is five times more potent than testosterone .
this dihydrotestosterone binds to the androgen receptors of the genetically marked hair follicle and leads to miniaturization of that hair follicle .
finasteride is an azasteroid type 2 isoenzyme , 5 alpha reductase inhibitor , which inhibits dihydrotestosterone conversion from testosterone .
this type 2 isoenzyme is present in the hair follicles and its activity is important in controlling the end organ hyper - reactivity causing androgenetic alopecia .
observation of eunuchs and prepubertal boys highlighted the role of testosterone in the induction of androgenetic alopecia .
therefore , it is evident that finasteride should not cause any side effects , relating to sexual health , as there will be no decrease in the quantity or quality of the testosterone level .
however , clinical trials conducted by leyden et al . and kaufman et al .
resulted in 2% of patients experiencing sexual side effects such as the loss of libido , erectile dysfunction and decreased ejaculate volume . in this study
, patients taking finasteride 1 mg for androgenetic alopecia and the control group were provided with the iief questionnaire and their responses analyzed .
the results indicated that erectile and sexual function was not reduced compared to the controls .
so , not only theoretically , finasteride was found to be a comparatively safe drug in this indian male population .
this study was conducted in the indian context on the basis of the iief and the european study by tosti et al . .
further studies should be conducted in a multi - center , double - blind manner .
in both our study and the study conducted by tosti et al . , sexual and erectile function of those subjects who were orally treated with finasteride was not reduced compared with their age - related controls .
supplementary material 1 ( pdf 301 kb ) supplementary material 2 ( docx 19 kb )
all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1964 , as revised in 2013 .
this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made .
|
introductionfinasteride is one of the most common drugs used in androgenetic alopecia .
the literature discusses the sexual side effects of the drug ; however , in practice there is little evidence to support this .
the aim of the present study was to investigate the sexual dysfunction in patients taking finasteride 1 mg for androgenetic alopecia.methodsa questionnaire , based on the international index of erectile function , was given to approximately 586 patients with androgenetic alopecia who were being treated with finasteride 1 mg for an average of 16 weeks .
these patients were compared to an age - related control group who were attending the dermatology out patients department for various other skin ailments not related to hair disorders.resultsstatistical analysis of the results showed no significant difference in the scores between patients taking finasteride and the control group.conclusionanalysis showed no significant difference in sex - related problems with that of patients taking finasteride and age matched controls , suggesting that sex - related issues are not a side effect of finasteride.electronic supplementary materialthe online version of this article ( doi:10.1007/s13555 - 015 - 0084 - 3 ) contains supplementary material , which is available to authorized users .
|
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neoadjuvant chemotherapy ( nac ) has generally been used in treatment of locally advanced and inflammatory breast cancer , but its use is increasing for earlier stages of the disease [ 1 - 3 ] .
the number of patients who are candidates for breast conserving treatment ( bct ) increases with the use of nac , which downsizes tumors , facilitating bct in patients who would otherwise require mastectomy .
several clinical trials have reported equivalent impacts of neoadjuvant versus adjuvant chemotherapy on survival . the locoregional recurrence ( lrr ) rate was also acceptably low in patients treated with nac followed by breast conserving surgery ( bcs ) and radiotherapy ( rt ) .
dna microarray analysis of gene expression profiles has divided breast cancer into distinct molecular subtypes with different clinical outcomes and responses to treatment , including estrogen receptor ( er)positive / luminal , basal - like , and her2-positive subtypes . however , dna microarray analysis has challenges for wide use in routine clinical care , determination of molecular subtypes based on clinically available immunohistochemical ( ihc ) markers such as hormone receptor ( hr ) and her2 status has been considered and validated as a more practical approach to identification of the corresponding subgroups based on gene expression profiling .
it has been demonstrated that different molecular subtypes can predict lrr in addition to survival and distant metastasis ( dm ) in the adjuvant setting [ 10 - 12 ] .
however , the impact of molecular subtypes on ipsilateral breast tumor recurrence ( ibtr ) and lrr in patients who undergo nac warrants further investigation .
therefore , the current study was conducted to evaluate whether molecular subtypes can identify patients at high risk for ibtr and lrr following nac and bct .
this was a single - institution retrospective review of an institutional review board approved prospective breast cancer database .
a total of 335 consecutive patients with non - metastatic breast cancer who underwent nac followed by bcs and rt from 2002 to 2009 were identified . before initiation of nac
, all patients had been clinically staged according to the sixth edition of the american joint committee on cancer ( ajcc ) guidelines .
clinical stages were evaluated by physical examination , ultrasonography , fluorodeoxyglucose - positron emission tomography ( pet)/computed tomography ( ct ) , and chest ct .
clinicopathological data were recorded , including age , menopause status , ct stage , cn stage , pathological tumor size , number of lymph nodes ( lns ) identified pathologically , histological type , histological grade , er , progesterone receptor ( pr ) , her2 , and ki-67 status .
nac consisted of anthracycline - based ( doxorubicin 60 mg / m and cyclophosphamide 600 mg / m every 3 weeks for four cycles , n=150 ) , taxane - based ( docetaxel 75 mg / m and capecitabine 1,000 mg / m orally twice daily on days 1 - 14 every 3 weeks for four cycles , n=85 ; paclitaxel 80 mg / m followed by gemcitabine 1,200 mg / m on days 1 and 8 every 3 weeks for four cycles , n=27 ) , or combined anthracycline - taxane based therapy ( doxorubicin 60 mg / m and cyclophosphamide 600 mg / m every 3 weeks for four cycles followed by docetaxel 100 mg / m every 3 weeks for four cycles , n=73 ) .
a total of 245 patients ( 73.1% ) were treated in one of the two prospective institutional clinical trials .
nac regimen for the remainder was chosen at physician s discretion . in the bcs procedure ,
residual primary tumors were excised , and clear margins to healthy tissues were determined from frozen biopsy specimens . however , five patients with persistent positive resection margins in the final pathology reports declined further surgical resection .
no further revision surgery was attempted in 30 patients with close resection margins ( < 2 mm ) .
standard level i and ii axillary ln dissections were performed in all except 14 patients who underwent sentinel ln biopsy without axillary dissection .
no residual tumor or only carcinoma in situ in both primary breast tumor and lns was considered pathologic complete response ( pcr ) . following bcs ,
rt was performed with tangential fields at a median dose to the breast of 50.4 gy in 28 fractions over 5.5 weeks in all patients .
all patients received an electron boost to the tumor bed with a median dose of 10 gy in five fractions .
supraclavicular nodal rt was delivered in 318 patients ( median dose , 45 gy in 25 fractions ) .
internal mammary nodal rt was administered at a median dose of 55 gy to only seven patients with pre - nac initial pet - positive internal mammary lns .
adjuvant hormone suppression therapy was offered to all patients with er - positive or pr - positive tumors .
some patients showed changes in er and pr expression before and after nac , but hormonal suppression therapy was administered to all patients whose tumors were er- or pr - positive in one or more tests . following rt ,
an ihc assay was used to evaluate the expression of the er , pr , her2 , and ki-67 markers in pretreatment core biopsies . er and pr positivity were defined using the allred score when strong nuclear staining was observed in at least 3/8 tumor cells examined . er and pr status were categorized as hr - positive when er or pr staining was positive , and as hr - negative when er and pr staining were negative .
immunostaining for her2 was considered positive in the case of strong ( 3 + ) membranous staining in at least 10% of tumor cells , or in the case of 2 + with unequivocal amplification by fluorescence in situ hybridization . for evaluation of ki-67 ,
areas with the highest ki-67 staining were examined ; 15% was used as the cut - off value for ki-67 to dichotomize the patients . according to the ihc features on core biopsies before nac ,
patients were classified according to ihc - based molecular subgroups as follows : luminal a ( hr+/her2/ki-67 < 15% ) , luminal b1 ( hr+/her2/ki-67 15% ) , luminal b2 ( hr+/her2 + ) , her2 ( hr/her2 + ) , and triple negative ( tn ) ( hr/her2 ) .
in this study , the her2 group , which is known as an unfavorable feature , was divided into two subtypes based on the use of trastuzumab .
the final six groups were as follows : luminal a , luminal b1 , luminal b2 , her2 with trastuzumab ( her2[t+ ] ) , her2 without trastuzumab ( her2[t ] ) , and tn .
lrr was defined as recurrent disease in the ipsilateral breast , chest wall or axillary , supraclavicular , infraclavicular , or internal mammary lns .
all ibtrs and lrrs were considered events , regardless of whether they were the first site of failure versus occurred with or after dm .
patients who did not experience ibtr or lrr were censored at the last follow - up or at the time of death .
distributions of the clinical factors among groups were compared using the kruskal - wallis test for continuous variables and the chi - square test for categorical variables .
actuarial rates of ibtr and lrr were calculated using the kaplan - meier method , and differences between groups were compared using the two - sided log - rank test .
logistic regression was used to evaluate the association between covariates of interest and the probability of ibtr or lrr .
significant differences in the distribution of histological type , histological grade , resection margin status , and response to nac were observed among subtypes . in evaluation of the response to nac
, we noted a difference ( p < 0.01 ) in pcr rates with a lower percentage of patients in the luminal a ( 10.6% ) and b1 ( 6.1% ) subgroups compared with patients in the her2(t ) ( 35.5% ) , and tn ( 23.0% ) subgroups .
the median follow - up period was 7.2 years ( range , 0.7 to 11.6 years ) .
twenty - six ibtrs , 15 regional recurrences , 67 dms , and 56 deaths occurred during follow - up ( table 2 ) .
the 5-year lrr - free survival rates in the subtypes were as follows : luminal a , 96.4% ; b1 , 93.9% ; b2 , 90.3% ; her2(t+ ) , 92.9% ; her2(t ) , 78.3% ; and tn , 79.6% ( fig .
compared with the luminal a subtype , significantly higher lrr rates were observed for the luminal b2 , her2(t ) , and tn subtypes ( p=0.02 , p < 0.01 , and p < 0.01 , respectively ) .
the 5-year ibtr - free survival rates in the subtypes were as follows : luminal a , 97.2% ; b1 , 93.9% ; b2 , 92.8% ; her2(t+ ) , 92.9% ; her2(t ) , 89.1% ; and tn , 84.6% ( fig .
the her2(t ) and tn subtypes had significantly higher rates of ibtr compared with the luminal a subtype ( p=0.04 and p < 0.01 , respectively ) . despite the same unfavorable molecular markers with her2(t ) , her2(t+ ) subtype showed no difference in ibtr and lrr rates compared with the luminal a subtype . the 5-year dm - free and disease - free survival ( dfs )
rates were as follows : luminal a , 90.2% ; b1 , 75.6% ; b2 , 83.0% ; her2(t+ ) , 85.7% ; her2(t ) , 76.6% ; and tn , 75.4% ( fig .
1c ) and luminal a , 88.4% ; b1 , 75.6% ; b2 , 81.9% ; her2(t+ ) , 85.7% ; her2(t ) , 70.0% ; and tn , 72.1% ( fig .
the clinicopathological variables associated with ibtr and lrr were analyzed by univariate and multivariate analyses ( table 3 ) . in univariate analysis ,
the factors affecting ibtr development included the tn subtype ( p < 0.01 ) , poorly differentiated tumors ( p=0.03 ) , and clinical t3 - 4 stage ( p < 0.01 ) .
luminal b2 subtype ( p=0.03 ) , her2(t ) subtype ( p < 0.01 ) , tn subtype ( p < 0.01 ) , poorly differentiated tumor ( p=0.01 ) , and clinical t3 - 4 stage ( p < 0.01 ) were also associated with lower lrr - free survival rates . in the multivariate model , the her2(t )
subtype , tn subtype , and clinical t3 - 4 stage affected the development of both ibtr and lrr . compared with the luminal a subtype , the her2(t ) and
tn subtypes were potent factors affecting ibtr / lrr , with hazard ratios of 4.2 ( p=0.04)/7.6 ( p < 0.01 ) and 6.9 ( p=0.01)/8.1 ( p < 0.01 ) , respectively ( table 3 ) .
notably , a pcr after nac was not associated with the development of ibtr ( p=0.39 ) or lrr ( p=0.65 ) .
patients of the her2(t+ ) subtype had significantly lower hazard ratios for ibtr and lrr compared with her2(t ) patients . in the analysis of ibtr and lrr according to pcr versus non - pcr after nac , patients of the tn subtype who failed to achieve pcr showed a significantly higher lrr ( p=0.03 ) ( fig .
however , among patients of the non - tn subtypes , including her2(t ) , no significant effect of a pcr on either lrr ( p=0.52 ) ( fig .
breast cancer is now regarded as a biologically heterogeneous disease comprising different molecular subtypes , each with a different prognosis and response to treatment [ 10 - 12 ] .
these subtypes , including luminal , her2 , and basal - like , can be defined by gene expression profiling or approximations to this classification using ihc .
clinicians should consider these features for proper assessment of the relevant evidence and decide on an appropriate therapeutic course of action . in a series of women with clinical stage ii - iii breast cancer who underwent nac and bct
, we found that molecular subtypes showed correlation with different rates of ibtr and lrr .
the tn and her2(t ) subtypes had worse outcomes with significantly higher ibtr and lrr rates than those of other subtypes despite excellent tumor responses to nac .
several authors have examined the impact of molecular subtype on lrr in different patient populations .
nguyen et al . evaluated 793 patients treated with bct as a first - line intervention .
after a median follow - up period of 70 months , the 5-year lrr rate was 0.8% for luminal a , 8.4% for her2 , and 7.1% for basal subtypes .
also evaluated differences in lrr according to subtype in patients undergoing bct as initial treatment .
these patients were classified based on receptor status as well as nuclear grade , with subgroups defined as luminal a ( hr+/her2/grade 1 - 2 ) , luminal b ( hr+/her2/grade 3 ) , luminal her2 ( hr+/her2 + ) , her2 ( hr/her2 + ) , and tn ( hr/her2 ) . the 5-year lrr rates were 0.8% for luminal a , 10.8% for her2 , and 6.7% for tn subtypes .
in contrast to our study , both of these studies were limited to patients undergoing initial surgery .
after a median follow - up period of 43 months , 5-year lrr rates were 3.8% , 1.3% , and 4.2% for luminal a , her2 , and basal subtypes , respectively .
the molecular subtype and pcr predicted dm , dfs , and overall survival ( os ) .
only patients who received nac were included ; however , patients underwent bct or mastectomy .
after a median follow - up period of 55 months , a higher rate of lrr in patients with basal ( 14% ) versus luminal ( 4% ) or her2 ( 5% ) tumors was reported . by evaluating only the 49 patients who underwent bct ,
no lrr events were observed in the luminal or her2 groups , while 8% of the basal group developed lrr .
most recently , caudle et al . analyzed the clinicopathological data from 595 patients who received nac and bct .
after a median follow - up period of 64 months , the 5-year lrr - free survival rates were found to vary by subtype : hr+/her2 , 97.0% ; hr+/her2 + , 95.9% ; hr/her2 + , 86.5% ; and hr/her2 , 89.5% ( p=0.001 ) .
first , our data encompassed a homogeneous group of patients with clinical stage ii - iii breast cancer who underwent nac followed by bct at a single institution , compared with the results from patients treated with nac followed by bct or mastectomy .
to the best of our knowledge , the current study is unique in its analysis of the impact of molecular subtypes on ibtr and lrr in patients who underwent nac followed by only bct , which could be associated with the concerns regarding a higher lrr rate compared with mastectomy .
most previous studies have focused on dfs , os , or lrr alone [ 23,26 - 28 ] .
inclusion of patients from previous treatment eras may yield higher rates of lrr compared with those treated more recently due to several factors .
the evolution of systemic therapy has resulted in better local control and better outcomes on systemic recurrence .
the use of modern radiation techniques and the evolution of breast imaging may have an impact on the rates of ibtr and lrr .
third , we included 36 patients treated with trastuzumab , of whom 14 her2(t+ ) patients had a better local outcome compared with her2(t ) patients .
five - year ibtr- and lrr - free survival rates were 92.9% versus 89.1% and 92.9% versus 78.3% in her2(t+ ) versus her2(t ) , respectively .
this result suggests that the use of trastuzumab could alter the impact of the molecular subtype on local outcome in her2 subtype patients .
last , we found that a pcr to nac had no impact on locoregional outcomes in any patients of non - tn groups . in tn patients , however , a pcr was associated with excellent ibtr and lrr control .
the association between the extent of response to nac and prognosis has been examined [ 3,5,23,25 - 27 ] .
the best relative dfs , as well as dm - free survival , and os was observed in those who achieved a pcr .
reported that a pcr to nac did not affect lrr or ibtr regardless of subtype , while caudle et al . reported that patients achieving a pcr had similar lrr rates among subtypes
a second limitation was the modest number of patients evaluated ; categorization according to the six subtypes resulted in a small number of patients in some subtypes , including her2(t+ ) , luminal b1 , and her2(t ) patients .
therefore , these findings should be confirmed in a larger prospective study in the future .
in conclusion , we demonstrated that the tn and her2 subtypes predicted higher rates of ibtr and lrr after nac followed by bct . among the non - tn
subtype patients , pcr was not predictive of better ibtr or lrr . however , among the tn subtype patients , a pcr to nac was a predictor of better lrr control .
taken together , a novel locoregional treatment strategy to decrease ibtr and lrr such as mastectomy instead of bct in tn subtype patients with non - pcr to nac deserves further investigation .
improvements in systemic therapy , investigation of radiosensitizing agents , radiation dose escalation , and other new techniques may prove to be important .
|
purposethe purpose of this study is to determine whether breast cancer subtype can affect locoregional recurrence ( lrr ) and ipsilateral breast tumor recurrence ( ibtr ) after neoadjuvant chemotherapy ( nac ) and breast - conserving therapy ( bct).materials and methodswe evaluated 335 consecutive patients with clinical stage ii - iii breast cancer who received nac plus bct from 2002 to 2009 .
patients were classified according to six molecular subtypes : luminal a ( hormone receptor [ hr]+/her2/ki-67 < 15% , n=113 ) , luminal b1 ( hr+/her2/ki-67 15% , n=33 ) , luminal b2 ( hr+/her2 + , n=83 ) , her2 with trastuzumab ( her2[t+ ] ) ( hr/her2+/use of trastuzumab , n=14 ) , her2 without trastuzumab ( her2[t ] ) ( hr/her2 + ,
n=31 ) , and triple negative ( tn ) ( hr/her2 , n=61).resultsafter a median follow - up period of 7.2 years , 26 ibtrs and 37 lrrs occurred .
the 5-year lrr - free survival rates were luminal a , 96.4% ; b1 , 93.9% ; b2 , 90.3% ; her2(t+ ) , 92.9% ; her2(t ) , 78.3% ; and tn , 79.6% .
the 5-year ibtr - free survival rates were luminal a , 97.2% ; b1 , 93.9% ; b2 , 92.8% ; her2(t+ ) , 92.9% ; her2(t ) , 89.1% ; and tn , 84.6% . in multivariate analysis , her2(t )
( ibtr : hazard ratio , 4.2 ; p=0.04 and lrr : hazard ratio , 7.6 ; p < 0.01 ) and tn subtypes ( ibtr : hazard ratio , 6.9 ; p=0.01 and lrr : hazard ratio , 8.1 ; p < 0.01 ) were associated with higher ibtr and lrr rates . a pathologic complete response ( pcr )
was found to show correlation with better lrr and a tendency toward improved ibtr controls in tn patients ( ibtr , p=0.07 ; lrr , p=0.03).conclusionthe tn and her2(t ) subtypes predict higher rates of ibtr and lrr after nac and bct .
a pcr is predictive of improved ibtr or lrr in tn subtype .
|
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coltheart , langdon , and mckay ( 2007 ) extended a two - factor explanation of capgras ' delusion to somatoparaphrenic delusions and from there to paranoid delusions in schizophrenia .
the authors argued that abnormal experiences or data give rise to delusional hypotheses which are not rejected because of damage to a belief evaluation system located in the right frontal cortex .
's attempt to bring all delusions under a single theory represents an important advance on theories of psychosis that neglect somatoparaphrenic symptoms and/or focus on cognitive bias to underwrite the transition from abnormal experiences to delusions ( see , for example , broome et al . , 2005 ) .
metcalf , langdon , and coltheart ( 2007 ) and turner and coltheart ( 2010 ) then applied the two - factor theory to inform the relationship between confabulation and delusion .
essentially , the authors argued , first , that mnemonic factors in confabulation and experiential factors in delusions operate at the same first - factor level , and second , that confabulations and delusions share common evaluative and monitoring second - factor processes .
the validity of these claims rests on crucial assumptions about the first - factor aetiology of confabulation and the original justification for relying on second - factor defective belief evaluation in a theory of delusions .
the starting point for this paper is the observation that theories that rely on abnormal experience and defective belief evaluation and/or cognitive bias have difficulty explaining how complex delusional hypotheses are generated .
it will be proposed that the solution can be found by reflecting on the aetiology of somatopsychotic symptoms and also on the work of johnson ( 1988 , 1991 ; johnson , o'connor , & cantor , 1997 ; johnson & raye , 2000 ) , and involves replacing the defective belief evaluation and/or cognitive bias factor with confabulation .
it will be argued that the resultant two - factor theory clarifies , first , the aetiological role of autonoetic agnosia ( frith & done , 1989 ; keefe , 1998 ) , and , second , the relationship between confabulation and delusion .
the latter will be achieved through a discussion of the aetiology of confabulation which allows defective evaluation to be reintroduced in its proper , more restricted context .
it will then be suggested that the new theory provides a natural explanation of why individuals with schizophrenia confabulate , and can inform the understanding the relationship between confabulation and thought disorder ( lorente - rovira , pomerol - clotet , mccarthy , berrios , & mckenna , 2007 ; nathaniel - james & frith , 1996 ) in a way that gives an early indication of how the gap between research on delusions and research on language impairments in schizophrenia might be bridged .
the existence of pseudohallucinations or subjective sensory experiences which are the consequence of functional psychiatric disorders and which are interpreted in a non - morbid way by the patient
( hare , 1973 , p. 474 ) , coupled with the observation that delusions can arise from thematically related abnormal experiences , suggest that theories that explain delusional content primarily in terms of abnormal experience are likely to be correct that this should be the first factor in a two - factor explanation of psychotic symptoms . strictly speaking
, the nature of the first factor will need qualifying when somatopsychotic symptoms and autonoetic agnosia are discussed later in the paper .
the contentious part of empiricist theories , as campbell ( 2001 ) refers to them , is the account they give of how abnormal experiences are converted into delusions . in an important paper ,
2007 ) argue that there are two requirements on an adequate explanation of this transition : ( 1 ) to explain how ( often complex ) delusional content arises from a mere experience which has become known as the abductive inference problem
( bayne & pacherie , 2004 ; coltheart , menzies , & sutton , 2010 ) ; and ( 2 ) to explain why delusional content , once generated , is not simply rejected as false .
the most parsimonious approach is to address ( 1 ) and ( 2 ) simultaneously by suggesting that delusions are abnormal experiences that are somehow endorsed as accurate representations of reality ( bayne & pacherie , 2004 , p. 3 ) .
the difficulty with subsuming all delusions under this theory , however , is that it is prima facie unlikely that the content of complex delusions ( even nonclassical capgras ' delusions such as cutting 's patient who thought her common - law husband was the son of adam and eve [ 1997 , p. 141 ] ) is exclusively experiential .
most empiricist theories acknowledge this , and argue that delusional content arises , in part , out of an attempt to explain abnormal experiences .
( 2005 ) , for example , arguing that data - gathering and attribution biases result in the delusional interpretation of abnormal experiences .
however , we will not discuss this proposal in detail since not only do not all psychotic individuals exhibit cognitive bias ( kemp , chua , mckenna , & david , 1997 ) , but delusions can be
it is perhaps worth adding , that the analogous but more familiar role that cognitive bias plays in two - factor explanations of somatisation disorder ( turner , 2006 , pp .
2930 ) suggests that a two - factor theory which relies on it will in any case struggle to account for complex delusional abductive inferences .
( 2007 ) share the view that delusional content results from an attempt to explain abnormal experiences , but the authors continue to rely on abnormal experience to do most of the content generating work . according to coltheart et al .
( 2010 ) , for example , the delusional hypothesis provides a much more convincing explanation of the highly unusual data [ i.e. , abnormal experience ] than the nondelusional hypothesis ; and this fact swamps the general implausibility of the delusional belief ( p. 278 ) .
if this approach is extended to complex delusions , it suggests that david 's ( 1990 , p. 804 ) patient 's delusional hypothesis that he had an actual power station , complete with labourers , machinery , and cooling towers inside him is exhibiting a perfectly rational response to very abnormal data ( coltheart , menzies , & sutton , 2010 , p. 281 ) .
furthermore , by not employing a second content generating mechanism , which could contribute to the explanation of why delusional hypotheses are not rejected , coltheart et al .
( 2007 ) have no way of avoiding the introduction of a second aetiological factor the sole theoretical purpose of which is to fulfil this role . with this in mind ,
postulate that there is a region of the right frontal cortex which , when damaged , prevents the correct evaluation of delusional hypotheses .
( 2010 ) articulate defective belief evaluation as irrationally ignor[ing ] or discount[ing ] the evidence on the basis of its incompatibility with the hypothesis to which
they have become committed ; so the delusional belief persists ( p. 281 ) .
now , we have already suggested that there are difficulties relying on cognitive bias to generate content , and the same considerations are likely to apply if the process is reintroduced to explain defective belief evaluation . on the other hand , if defective belief evaluation is understood as a neuropsychological deficit , as coltheart et al .
( 2007 ) imply , then this , as we will see in the next section , is difficult to reconcile with a point made by the philosopher gareth evans that when a subject wants to make absolutely sure that his judgment is correct , he gazes again at the world ( thereby producing or reproducing , an informational state in himself ) ( 1982 , p. 227 , emphasis in original ) .
we will return to defective belief evaluation when discussing the relationship between delusions and confabulations , but the natural solution to the problems caused by complex delusional hypotheses is to construct a theory that relies on a content generating mechanism which can both make a substantial contribution to content and simultaneously explain why delusional hypotheses are not rejected .
. 's ( 2007 ) extension of their theory of capgras ' syndrome to somatoparaphrenic delusions .
( 2007 ) consider the development of a delusion of nonownership of a paralysed limb in an individual with a left hemiplegia following a right - sided brain insult .
the authors then ask , what could have happened to the hemiplegic individual for this delusion to arise ?
the answer , they suggest , is that the abnormal experience of paralysis requires an explanation and the delusion of nonownership of the limb provides this .
then argue that because many hemiplegic individuals do not develop delusions , a second aetiological factor is required and , in view of the typically intact left hemisphere , this must have something to do with the right hemisphere .
we will refer to this as assumption 2 . in terms of assumption 1 , it is difficult to understand why not being able to move a paralysed limb would lead an individual to hypothesise that their limb belonged to someone else .
why , especially in the absence of significantly disturbed thinking , is the paralysed individual 's first thought not i am paralysed ?
this concern is compounded by reports of cases that involve recognition of paralysis but not of ownership . cutting ( 1997 , p. 273 ) , for example , mentions von anygal and frick 's ( 1941 ) patient who claimed his paralysed side was his paralysed brother .
( 2010 , p. 283 ) later recognise that such cases represent an objection to their theory , but it is the reversal of the direction of causation between limb ownership and paralysis at the point of theory construction which gives rise to the difficulties .
the most likely explanation for such cases is that the failure to recognise one 's paralysis , i.e. , anosognosia , is a manifestation of difficulties with bodily representation or
, as devinsky and d ' esposito ( 2004 , p. 77 ) refer to it , which in their most severe form amount to hemisomatagnosia , which involves a failure to recognise the limb(s ) as one 's own . as in von anygal and frick 's ( 1941 )
paralysed brother case , this does not preclude implicit awareness of the existence of a paralysis , and it is well recognised individuals use personification and other metaphors such as a piece of rusty machinery or dead wood indicating knowledge that something is amiss ( weinstein , 1991 , p. 242 ) . however , it is the underlying absence of experience , what bisiach and germiniani ( 1991 ) refer to as a representational lacuna ( p. 31 ) , and what we might refer to as the experiential abnormality rather than an abnormal experience of paralysis , that is the natural basis of delusions of nonownership .
hemisomatagnosia interferes with the capacity to form a correct judgement and , in doing so , makes the introduction of a separate process of defective belief evaluation , at least in the sense intended in coltheart et al .
in other words , a delusion is not something that results from an experiential abnormality which is then incorrectly evaluated .
it results ( in part ) from an experiential abnormality which itself involves an interference with the capacity to form correct judgements .
's second - factor process of defective evaluation and this is where we see the importance of evans 's ( 1982 ) point .
when a subject wants to make absolutely sure that his judgement is correct ( i.e. , evaluate it ) , he gazes not at his experiential abnormality , but rather through it , and this will only reproduce the original information state in himself .
the difficulty , however , is that we still require an explanation of how complex somatopsychotic delusions , such as those about
we need to postulate a further cognitive process which creates , as weinstein puts it , a
this process is recognised to be confabulation , and , unlike defective belief evaluation and cognitive bias , it is a familiar consequence of brain damage that has clear content - generating capabilities .
furthermore , and turning to coltheart et al . 's ( 2007 ) assumption 2 , although confabulation is likely to arise out of an attempt to make sense of the experiential abnormalities originating in the damaged right hemisphere , the evidence from split brain or corpus callosotomy patients supports the hypothesis that it requires left hemispheric interpretative activity in the presence of disrupted interhemispheric relations ( phelps & gazzaniga , 1992 ) . in other words ,
the generation of psychotic symptoms is likely to require the constructive activity of an intact left hemisphere as much as the lack of activity associated with a damaged right hemisphere . by identifying the difficulties with coltheart et al
. 's ( 2007 ) account of somatopsychotic symptoms , we have arrived at a two - factor theory that relies on confabulation and a broader experiential factor , which includes agnosia , i.e. , an experiential abnormality rather than an abnormal experience . the latter distinction may appear semantic , but empiricism 's overly narrow construal of the experiential factor at the theory construction stage obscured the theoretical availability of confabulation , notwithstanding that it is phenomenologically related to the experiential deficits that the theory maintains underlie delusions , and can perform the content generating work explaining the complex somatopsychotic delusional hypotheses in a way that obviates the need to rely on coltheart et al .
. 's ( 2007 ) proposal that it recognises the pivotal theoretical importance of somatopsychotic symptoms to formulating a general theory of delusions .
however , the neglect of somatopsychotic symptoms by earlier empiricist theories ( bayne & pacherie , 2004 ) drives an aetiological wedge through the reduplicative delusions ( i.e. , capgras ' and somatopsychotic ) and removes the means to explain even complex versions of the very capgras ' delusions that empiricist theories were originally formulated to address .
there are a number of reasons why empiricist theories have failed to appreciate the relevance of confabulation to the aetiology of delusions .
maher 's ( 1974/1988 ) original emphasis on experientially generated content exerts a continuing influence , as does the continuing tendency to attribute confabulation to memory dysfunction .
we will examine the relationship between confabulation and memory dysfunction later , but it is important to notice that even if one focuses on this , it has long been recognised that there are two cognitive processes that generate false memories ; as kopelman ( 1987 ) put it , two types of confabulation . bonhoeffer ( 1904 ) originally recognised the difference between provoked confabulation , involving minor distortions in recall , and spontaneous confabulation , involving
an alternative autobiographical or semantic narrative , which may or may not accompany genuine lacunae in the
provoked confabulation is essentially a form of cognitive bias on which empiricist theories rely to act as their second factor , either in isolation or as part of the mechanism that leads to defective belief evaluation .
however , recalling weinstein 's ( 1991 ) description of the cognitive deficit in somatopsychosis , it is spontaneous confabulation that has the theoretical potential to explain complex content .
this realisation can be attributed to johnson who , in a series of papers , developed a reality monitoring theory of confabulation and then extended this to explain delusions . according to johnson , confabulations are false statements that are not made to deceive ( 1991 , p. 187 ) and which occur when people confuse the origin of information , misattributing something that was reflectively generated to perception ( p. 180 ) . by reflectively generated , johnson means produced by the imagination and this indicates that confabulations are essentially imaginations mistaken for memories .
johnson refers to these errors as reality monitoring failures and suggests that they are more likely to occur when imaginations are rich in perceptual information and/or poor in cognitive operations information , as these make it more difficult to identify the source of information .
however , johnson 's difficulty is that reality - monitoring failure does not explain how often - complex imaginations arise in the first place .
johnson et al . ( 1997 ) solve this problem by hypothesising that confabulators have a propensity towards detailed imaginations
( p. 203 ) and conclude that confabulation is due to an interaction between detailed imagination , defective source monitoring , and problems with the systematic retrieval of memories .
we will argue later that confabulation does not require memory dysfunction and that the propensity to detailed imaginations and source monitoring problems form the basis of an adequate two - factor theory of confabulation . in the remainder of this section we will concentrate on johnson 's extension of her work on confabulation to explain delusions .
one way of understanding johnson 's strategy is to view her as , first , separating off the mistaking of ongoing imaginations for perceptions in order to explain hallucinations , and , second , arguing that delusions arise when specific imaginations are subject to reality monitoring failures .
the difficulty is that most delusional content can not be understood in terms of mistaking imaginations for memories and this suggests that reality - monitoring failures may account for only a subset of delusions , i.e. , delusional memories .
interestingly , kopelman ( 2009 ) recently emphasised the similarity between confabulation and delusional memories , whilst concluding that subsuming confabulation and delusion under a single theory has limited explanatory utility .
in fact , many imaginations seem to be accepted as they are imagined for the first time and this suggests that the reliance on detailed imaginations will have to be supplemented with an account of how ongoing imaginations can be mistaken for beliefs .
now , whilst johnson herself does not articulate the issues this way , her ( 1988 ) treatment of hallucinations as reality testing failures
( p. 35 ) contains crucial insights which can be generalised to produce the two - factor theory of delusions , which is consistent with the two - factor theory of somatopsychotic symptoms developed earlier . in considering how hallucinations arise ,
johnson 's first argument is that imaginations are so similar to perceptions that an individual could easily become confused .
however , since imaginations have insufficient perceptual information and too much cognitive operation information to be misclassified , johnson recognises that this will not explain hallucinations .
in order to address the perceptual information requirement , johnson invokes abnormal experience through a discussion of horowitz 's suggestion that hallucinations may be elaborated from elementary sensations in the retinal ganglionic and postretinal neural network and/or from anatomic bodies within the eyeball
unfortunately , even when abnormal experience relieves the imagination of responsibility for some content generation , it takes a considerable amount of imaginative work to interpret muscae volitantes as rats , or the visual consequences of a partially detached retina as the blood of christ ( johnson , 1988 , pp .
johnson 's solution , which we can infer from the remark that loss of control makes a self - generated event seem like a perceptual event ( p. 53 ) , is to hypothesise that imaginations have little cognitive operations information attached to them .
if a confabulation is reasonably detailed and does not have strong cognitive - operations information associated with generating it , then it would be judged
( p. 57 ) . if we now generalise this interpretation of johnson 's ( 1988 ) proposals to delusions , we find that a combination of abnormal experience and detailed imaginations , which have reduced cognitive operations information attached to them , explains how content arises and is not rejected .
this proposal has the advantages of , first , embodying the main insight of empiricism by acknowledging the importance of abnormal experience , and , second , avoiding the need to introduce coltheart et al . 's ( 2007 ) notion of defective evaluation , by ensuring that the two delusional hypothesis - generating factors contribute to the defective source monitoring , which explains why these hypotheses are not rejected : ( 1 ) experiential abnormalities , by furnishing thoughts with the perceptual information , and ( 2 ) confabulation , by reducing cognitive operations information attached to psychotic content .
it is worth emphasising the fundamental difference between johnson 's ( 1988 ) notion of defective monitoring , which focuses on the process , which gives rise to information , and coltheart et al . 's ( 2007 ) notion of defective evaluation , which focuses on the content of information .
originally intended by defective evaluation , and in fact has more in common with the defective monitoring of actions which will be discussed in the next section .
however , since johnson 's defective monitoring contributes to the explanation of how individuals make false statements without being aware that they are false , it is partly responsible for delivering what coltheart et al .
were trying to achieve by introducing defective evaluation , albeit without having to elevate this to the status of a factor in a theory of delusions . returning to the thread of the discussion ,
johnson and raye ( 2000 ) argue that perceptual information plays a significant role in heuristic ( or nondeliberative ) source monitoring and that cognitive operations information is important to systematic ( or deliberative ) source monitoring , which they suggest may require interhemispheric cooperation ( p. 62 ) .
we will argue later that the defective monitoring that constitutes the second factor in a theory of confabulation is due to imaginations lacking cognitive operations information , and then relate this to problems with interhemispheric communication .
however , for now the important point is that by reflecting on johnson 's ( 1988 ) work , we arrived at a two - factor theory of visual hallucinations which is consistent with our two - factor theory of somatopsychotic symptoms and , in virtue of relying on a propensity to detailed imaginations , is capable of being seamlessly extended to explain even complex allopsychotic delusional hypotheses .
autonoetic agnosia forms the basis of an entirely separate theory of psychosis ( frith , 1992 ; frith & done , 1989 ; keefe , 1998 ; lari , barr , & keefe , 2004 ) , a clarification of which will offer an opportunity to further underline the need for both confabulation and for a broader construal of the experiential factor than empiricist theories of delusions strictly allow .
the approach is based on the idea that when , in ordinary circumstances , a motor instruction is sent , a corollary message or reafference copy of this signal is also sent to an internal monitoring system .
the purpose of the reafference copy is to allow for an internal prediction of what will occur and , by comparing this prediction with perceived outcome , behaviour can be adjusted to meet the changing demands of complex tasks .
the system is a monitoring system that allows identification of self - generated stimuli ( cahill & frith , 1996 , p. 284 ) , and , according to cahill and frith 's ( 1996 ) version of the theory , psychotic symptoms occur when an individual executes an action without a reafference copy of the motor instruction being available for internal monitoring , with the consequence that the individual is unable to recognise that the action was caused by himself .
this approach to the monitoring of action is clearly well suited to explaining certain auditory hallucinations ( failure to monitor the initiation of inner speech and thought ) and passivity experiences such as delusions of control ( failure to monitor intentions to act ) ( p. 285 ) . however , there are two sets of symptoms that are resistant .
the first includes symptoms the content of which can not be traced to the failure to identify a normal internal event .
cahill and frith ( 1996 ) acknowledge these cases when they indicate that it is not clear the account can explain visual hallucinations .
however , they do not mention equally problematic cases in which the content is in some sense externally generated , such as in psychotic illusions .
the second set of symptoms that can not be fully explained by autonoetic agnosia are complex delusions
. indeed , even complex auditory hallucinations and passivity phenomena are problematic , in that it is one thing to believe that one 's thought is a voice , but quite another to attribute it to evil spirits ( to use one of cahill and frith 's own case examples ) . the only way of salvaging an aetiological contribution for autonoetic agnosia is for cahill and frith ( 1996 ) to acknowledge the point made by johnson and raye ( 2000 , p. 62 ) that more complex delusions are likely to involve additional factors , and to add confabulation to his theory .
the modified version of the theory will then explain complex delusions and complex forms of symptoms which autonoetic agnosia was originally directed at .
however , notice that visual hallucinations remain recalcitrant , since these ( as johnson and frith have effectively concluded from opposite directions ) require the additional postulation of abnormal experience ( johnson & raye , 2000 ; cahill & frith , 1996 ) .
this suggests that we will have to allow that abnormal experience in visual hallucinations plays an equivalent aetiological role to that played by autonoetic agnosia in auditory hallucinations and passivity phenomena .
it is interesting that empiricist theories have effectively recognised this , although their attempts to bring auditory hallucinations and passivity phenomena under their own theory causes them to construe autonoetic agnosia as abnormal experience .
coltheart and davies suggest that a cognitive abnormality , either in the generation of the efference copy and feedback information , might give rise to abnormal experiences of action [ which if accepted ] as veridical would be a step on the way to the delusion of alien control ( 2000 , p. 37 ) .
this approach has an advantage over frith 's theory because it brings out that autonoetic agnosia is really just an experiential abnormality , thereby facilitating its assimilation into a two - factor theory .
the difficulty , however , is that the implicit removal of the agnosia from autonoetic agnosia is a further example of the experiential abnormality being construed too narrowly at the theory construction stage .
the neglect of confabulation is less obvious in this situation because it results from a distancing of autonoetic agnosia from the agnosias underlying the somatoparaphrenic delusions that empiricist theories prior to coltheart et al .
this is perhaps why bayne and pacherie ( 2005 , p. 176 , note 7 ) detect frith 's difficulties with complex delusions , whilst not perceiving that their own version of empiricism is susceptible to the same criticism .
the result , in any case , is that we have again arrived at our original two - factor theory , only now , assisted by frith 's contribution , the theory has been further refined by an understanding that the experiential abnormality underlying a further subset of psychotic symptoms , i.e. , auditory hallucinations and passivity phenomena , is autonoetic agnosia .
johnson and raye ( 2000 ) argue that the distinction between the delusion and confabulation is governed by whether or not a particular false claim is made in the presence of brain damage or psychopathology and that an aetiology - based distinction between delusions and confabulations [ is ] questionable ( p. 37 ) . however , because johnson 's work antedated the contributions that imposed the current structure on the debate about the aetiology of delusions ( bayne & pacherie , 2004 ; campbell , 2001 ; currie , 2000 ; langdon & coltheart , 2000 ) , a two - factor theory that included confabulation was not clearly articulated . on the other hand ,
theorists who have taken maher 's proposals as a starting point have emphasised the experiential basis of monosymptomatic delusional content to an extent that the relevance of confabulation did not , at least until recently , come into view .
( 2007 ) and turner and coltheart ( 2010 ) constitute an important an attempt to progress beyond these obstacles by relating confabulation to a two - factor theory of delusions .
however , before we try to show that these authors ' reliance on a common second factor failure to reject unsubstantiated thought
( turner & coltheart , 2010 , p. 357 ) inherits the original difficulties caused by focusing on evaluation at the expense of content generation , we need to lessen the resistance to the current proposals , by clarifying the relationship between confabulation and memory disorder .
this is particularly important , because whilst turner and coltheart ( 2010 ) focus primarily on a common second factor shared by confabulations and delusions , and could , strictly speaking , remain neutral on the first - factor process that gives rise to confabulations , the current proposals rely on a component of confabulation ( detailed imaginations ) to produce delusional content and as such confabulation can not be due to memory dysfunction .
hirstein ( 2005 ) refers to studies of patients with frontal lobe lesions ( shimamura , janowsky , & squire , 1990 ) and post anterior communicating aneurysm surgery ( vikki , 1985 ) who have memory impairment but do not confabulate ; and second , studies of korsakoff 's syndrome where memory impairment persists whilst confabulation improves ( stuss , alexander , lieberman , & levine , 1978 ) . the more difficult question is whether memory impairment is necessary for confabulation .
turner , cipolotti , yousry , and shallice ( 2008 ) concluded that it does seem to be , although since some of their confabulating patients scored in the normal range on memory tests , and they could find no single measure of memory on which all of their sample were impaired , their findings do not exclude the possibility that memory impairment and confabulation involve damage to two different closely associated neural systems ( stuss et al . , 1978 ) .
if one then considers that confabulation also occurs in hemisomatagnosia , corpus callosotomy , and schizophrenia in the absence of memory impairment ( on schizophrenia see lorente - rovira et al .
, 2007 ; nathaniel - james & frith , 1996 ) , it appears likely that memory impairment is not necessary for confabulation .
this does not exclude the possibility of coexisting memory dysfunction , and indeed cognitive impairment more generally , exacerbating confabulation ( dalla barba , 1993 ) , and it is perhaps worth noting the situation in which amnesia is associated with metamemory difficulties ( i.e. , a representational lacuna in the domain of memory ) .
there is no reason why a representational lacuna relating to the memory could not function like a representational lacuna relating to the body and give rise to an experiential abnormality that would influence confabulatory content . in such cases
, the resultant mnemonic confabulation would be what the theory proposed in the paper regards as a delusion , thereby giving substance to kopelman 's point about the relationship between confabulations and delusional memories .
however , this is a special case that does not lend any more support than does hemisomatagnosia for a paralysed limb to the claim that confabulation in general is due to memory dysfunction .
the absence of a primary aetiological role for memory dysfunction must be accommodated by any attempt to relate confabulations and delusions and this brings us back to metcalf et al . 's ( 2007 ) extension of their two - factor theory of delusions to confabulations .
essentially , metcalf et al . suggest that the first factor in confabulation is operating at the same level as the first factor in delusion , although in the former it is mnemonic and in the latter perceptual or affective .
this is somewhat difficult to reconcile with turner and coltheart 's ( 2010 ) comment that a confabulation by a patient that he had met a woman who had a bee 's head did not involve distortion of true memory ; instead it appeared to involve imagination
furthermore , if , as this example suggests , memory dysfunction is not essential to confabulation , metcalf et al .
have identified a first factor that does not have a role in a theory of confabulations with a first factor that most authors agree does have a role in a theory of delusions .
the importance of turner and coltheart 's ( 2010 ) comments about the role of the imagination in the bee 's head confabulation now becomes apparent in that we require another first factor in a theory of confabulation and , according to the current proposals , it is this factor that contributes to content generation in both confabulations and delusions .
's account of the aetiology of somatopsychotic symptoms and argue that since these involve confabulation , the first factor in confabulation must be experiential , as this places empiricism on the horns of a dilemma : either deny that somatopsychotic and allopsychotic symptoms should be subsumed under the same theory of delusions , or accept that delusions , at least in complex cases , are not primarily experiential .
the experiential basis of delusional content is closely associated with the claim that delusions are beliefs and together these represent the central tenets of the empiricism that coltheart et al . 's original approximation of somatopsychotic and allospsychotic symptoms was intended to protect .
the possibility of filling the first factor gap left by the absence of memory dysfunction by extending the first factor experiential abnormality is almost equally unattractive to the current proposals as this would involve completely absorbing the concept of confabulation into the concept of delusion .
the problem with this is that corpus callosotomy patients have neither memory nor experiential deficits in the sense required by the two - factor theory of delusions developed here , and yet they confabulate .
so , providing we want to allow that this is essentially the same phenomenon as that which generates delusional content , which seems intuitively plausible , we must look to interhemispheric communication for the first factor in a theory of confabulation .
what corpus callosotomy cases indicate is that when the left hemisphere is deprived of information from the right hemisphere it generates interpretations and it is these that constitute confabulatory content .
however , for this to occur , it may only be necessary that a portion of the corpus callosum or crucial adjacent structures are damaged .
this could occur with a circumscribed lesion in the inferior medial prefrontal system ( turner et al . ,
2008 ) , which would be consistent with evidence of dissociation between frontal executive dysfunction ( the traditional alternative aetiology to memory dysfunction ) and confabulation , even in schizophrenia ( lorente - rovira et al . , 2007 ) .
let us suggest , then , that such a lesion is the cause of the first factor in a theory of confabulation , johnson 's propensity to detailed imaginations , which when combined with the defective monitoring to be discussed in the next section , constitutes the second factor in the proposed theory of delusions .
this suggestion is consistent with evidence of damage to the corpus callosum in schizophrenia ( downhill et al . , 2000 ) , but must be reconciled with delusions where there are first - factor experiential abnormalities due to a posterior lesion . here
one can envisage two possible routes to the propensity to detailed imaginations : first , through coexisting damage by a second lesion in the inferior medical prefrontal region , or , second , because the posterior lesion causing the experiential abnormalities is sufficiently extensive that it involves relevant structures .
( see feinberg , venneri , simone , fan , & northoff , 2010 , for evidence that somatoparaphrenic patients have greater orbitofrontal damage than hemisomatagnosic patients . )
irrespective of which of these pertains , they entail that the first factor in delusions is not the first factor in confabulation per se , a proposal that firms up the distinction between hemisomatagnosic claims due to right - sided representational problems , and complex delusional content , such as in von anygal and frick 's ( 1941 ) paralysed brother case , due to inferior medial prefrontal damage .
we are now in a position to address the more complex problems associated with turner and coltheart 's ( 2010 ) claim that confabulation and delusion share a common second factor . with this in mind
, we have seen that empiricist theories in general share a common reliance on experience to generate delusional content and that they were originally formulated to explain monosymptomatic delusions , specifically capgras ' delusion , and it was suggested that this approach has difficulties explaining even complex capgras ' delusions . however , it was only when coltheart et al .
( 2007 ) generalised their account to somatopsychotic symptoms ( without attempting to address complex delusions ) that the precise nature of the difficulties became apparent .
in order to bring out these difficulties , we need to revisit the key elements of coltheart 's et al . 's ( 2007 ) arguments : suppose we construct a general theory meant to apply to the explanation of all kinds of monothematic of delusion : to somatoparaphrenia , for example what distinguishes left - hemiplegic people with somatoparaphrenia from these left - hemiplegic non deluded others .
whatever this is , it is something to do with the right hemisphere because the left hemisphere is typically intact the function of this [ damaged ] region of the right hemisphere is , therefore , belief evaluation .
suppose we construct a general theory meant to apply to the explanation of all kinds of monothematic of delusion : to somatoparaphrenia , for example what distinguishes left - hemiplegic people with somatoparaphrenia from these left - hemiplegic non deluded others .
whatever this is , it is something to do with the right hemisphere because the left hemisphere is typically intact the function of this [ damaged ] region of the right hemisphere is , therefore , belief evaluation .
is that by not recognising the need to accommodate complex delusional contents , and then not building confabulations into a theory of delusions , coltheart et al . had no alternative but to direct evaluation at the first factor in their theory of delusions . in other words ,
evaluation is directed at the representational problems that underlie psychotic symptoms , but which , according to the current proposals , are not susceptible to evaluation because experiential abnormalities themselves interfere with the ability to form correct judgements . instead
, it is the interpretations generated by the left hemisphere ( which , as mentioned earlier , corpus callosotomy patients show us can be intact in confabulation ) that provide the natural target for defective evaluation .
thus , when turner and coltheart ( 2010 ) apply coltheart et al . 's ( 2007 ) theory of delusions to confabulations and postulate a common set of evaluative and monitoring processes ( the second factor )
( p. 357 ) , this involves an inter - level identification between a factor that we have suggested has no role in a theory of delusions , i.e. , defective belief evaluation , and a factor which johnson holds has a central role in the aetiology of confabulation , i.e. , defective monitoring . as a result
the question of whether a theory of delusions can do without defective belief evaluation , which we have argued it can and should , is conflated with that of whether a theory of confabulation ( and , by proxy , of delusions ) can do without defective monitoring .
we have already acknowledged that it can not when discussing johnson 's reliance on cognitive operations information to explain why detailed imaginations are not rejected or , perhaps more accurately , correctly categorised ( johnson & raye , 2000 ) .
we will return to cognitive operation information shortly , but having concluded that experiential abnormalities interfere with the ability to make correct judgements in a way that obviates the need for defective evaluation of first - factor content in delusions , could we not simply redeploy coltheart et al . 's ( 2007 ) defective evaluation against detailed imaginations ?
after all , it would seem that an individual 's failure to detect that his unsubstantiated hemisomatagnosic claims are false must at least in part be due to the right - sided representational lacuna itself .
hirstein ( 2005 ) has this possibility in mind , when he writes : first , a false claim will be generated because the area responsible for that knowledge domain is damaged and some other much less competent area has generated the answer .
then the answer can not be properly checked because the only area that can do this is damaged .
( p. 147 )
first , a false claim will be generated because the area responsible for that knowledge domain is damaged and some other much less competent area has generated the answer .
then the answer can not be properly checked because the only area that can do this is damaged .
's ( 2007 ) claim that the defective evaluation of evidence for an unsubstantiated claim plays a role in the failure to reject unsubstantiated delusional content .
however , if we understand this as a higher level right - sided process defectively evaluating the content arising from a lower level right - sided process , then we will conflate hemisomatagnosic claims with complex delusional contents . alternatively
, if we understand the claim in terms of a higher level right - sided process defectively evaluating left hemispheric detailed imaginations , then since these would not arise without the presence of the first factor , there is no conceptual space for nondefective evaluation . in other words ,
the interference with the capacity to form correct judgements involves an interference with the capacity to evaluate the judgements thus formed , even if they are delivered by the left hemisphere .
the delusion that one 's paralysed side is one 's brother surely can not in any meaningful sense be attributed to a failure to evaluate a false claim .
in other words , it seems counterintuitive to claim that an individual is able to check , as it were , whether their arm is their brother or indeed , whether they have a power station inside them .
defective monitoring , however , is an important notion and this brings us back to the second factor in confabulation , and to johnson and raye 's ( 2000 ) discussion of cognitive operations information and its relationship to interhemispheric relations .
now , we have previously attributed detailed imaginations to problems with the right to left transfer of information due to damage in the inferior medial prefrontal region .
however , interhemispheric traffic is not one way , and this opens up the possibility that problems with the left to right transfer are responsible for detailed imaginations not having cognitive operations information attached to them .
turner and coltheart ( 2010 ) articulate defective evaluation / monitoring as not tagging for doubt , but the introduction of the notion of a tag when viewed in light of the following comment suggests a move away from coltheart et al . 's ( 2007 ) original construal of defective belief evaluation : in the absence of a tag , fragments of ideas , or ideas purely derived from the imagination , would acquire the same status as fully formed beliefs ( p. 359 ) .
this formulation is consistent with the theory developed in this paper , providing that we understand
tag in terms of cognitive operations information , and therefore defective monitoring as not tagging for source .
this avoids both the intuitive and theoretical difficulties associated with the notion of evaluating the content of detailed imaginations whilst successfully accounting for why they are not correctly categorised .
note also that the existence of pseudologia fantastica is evidence of conceptual room for the nondefective monitoring which is not possible if the process is directed at first - factor contents . in conclusion , then , the second factor in a theory of confabulations ( and the second part of the second factor in a theory of delusions ) is defective source monitoring , caused by the very same damage in the inferior medial prefrontal system that causes the first factor .
finally , it is worth adding that currie ( 2000 ) proposes a theory of delusions according to which detailed imaginations are not correctly categorised because of defective source monitoring explicated in terms of a frithian autonoetic agnosia for the imagination .
( currie & ravenscroft , 2002 , p. 11 ) , currie 's theory of delusions approximates to a theory of confabulation .
in the course of examining currie 's proposals , bayne and pacherie ( 2005 ) have noticed that
it may be more plausible to describe delusional patients as suffering from an impairment in the control of rather than the monitoring of imagination
this paper has tried to show that , in fact , an adequate theory of confabulation will include impairments in the control and not just the monitoring of the imagination , and that an adequate theory of delusions will include this and underlying experiential abnormalities .
according to the current proposals , two separate aetiological processes come together to give rise to psychotic symptoms .
if this is correct , then evidence of these processes coming apart and operating independently of one another would provide significant support for the theory . with this in mind , the presence of experiential abnormalities in schizophrenia is a relatively uncontroversial matter with cutting ( 1997 ) , for example , referring to gross and huber 's case of a female with schizophrenia who reported that the left side of her husband 's face suddenly seemed so sad and serious as if he were split into two parts ( p. 109 ) .
it is difficult to conceive of a reason for doubting that such symptoms tell us something important about the aetiology of psychotic symptoms .
however , it is the relationship between confabulation and schizophrenia , which has only recently started to attract empirical attention ( nathaniel - james & frith , 1996 ) , that is more interesting .
it has long been recognised that patients with schizophrenia confabulate and , although these will naturally present as provoked in experimental settings , the historical literature contains unequivocal accounts of spontaneous confabulation .
lorente - rival et al . ( 2007 ) refer to kraepelin 's account of extraordinary stories
( p. 1 ) and fish ( 1974 ) points out that some schizophrenics confabulate , producing detailed descriptions of fantastic events which have never happened ( p. 63 ) .
the current proposals provide both a natural way of understanding the presence of confabulation in schizophrenia and a potential explanation of aspects of the phenomenology , such as the fact that delusions tend [ sic ] to be fixed , compared with the shifting and changing nature of many confabulations ( kopelman , 1999 , p. 202 ) . in other words ,
perhaps psychotic individuals have a general tendency to confabulate and coexisting experiential abnormalities anchor a subset of confabulations to produce ( two - factor ) delusions .
the more complicated question is whether the current proposal can advance the understanding of empirical correlates of confabulation is schizophrenia , and in particular nathaniel - james and frith 's ( 1996 ) and lorente - rovira et al
the difficulty is , how do we understand this relationship and both authors question whether clinicians and researchers could be describing the same phenomenon in different ways .
( 2007 ) perceive to this possibility is that the speech of neurological patients with confabulation is understandable it is only the factual content which strikes the listener as odd
the authors go on to make the point that if it is accepted that confabulation in schizophrenia is different from the neurological form of the symptoms , then some phenomenological overlap with thought disorder might become a more viable option
( 2007 ) point out that empirical findings on confabulation in schizophrenia , namely the reorganising and restructuring of ideas in a story and the use of approximate and new words , resemble the manifestations of thought disorder .
after referring to one of chaika 's ( 1974 ) patients who said his mother 's name was bill and that st .
valentine 's day was the start of the official breeding season for birds , lorente - rovira et al .
conclude that while episodic confabulation , as seen in neurological disorders , and thought disorder , are not the same thing , conceptualising some aspects of thought disorder as semantic confabulation may be an idea with some heuristic value
( 2007 ) do not , however , consider the possible role of confabulation in the aetiology of delusions and this both reintroduces the distinction between two types of confabulation
( albeit in a different form to that proposed by bonhoeffer , 1904 , and kopelman 1987 ) and drives an aetiological wedge between somatopsychotic and allopsychotic delusions .
what is required is an interpretation of association between confabulation and thought disorder in schizophrenia that is informed by its proposed role in the aetiology of delusions , and there are two possibilities : first , confabulation and thought disorder are coexisting processes that have a joint impact on the use of language ; second , confabulation and thought disorder are different manifestations of the same process .
the first interpretation is an extension of arguments about the nonessential relationship between memory / frontal executive dysfunction and confabulation . if confabulations are part of delusions , then the correlation between confabulation and thought disorder could be due to an underlying correlation between delusions and thought disorder .
this interpretation would need to accommodate the phenomenological overlap between thought disorder and confabulation , but this could be explained by former being superimposed on the latter .
accordingly , nathaniel - james and firth ( 1996 ) suggest that thought disorder may be a contributor to the severity of confabulation rather than its presence ( p. 397 ) .
one potential difficulty is that the current theory predicts a correlation between confabulation and delusions , and nathaniel - james and frith did not find this
. the second interpretation ( favoured by lorente - rovira et al . , 2007 , but more difficult to reconcile with neuroanatomical considerations ) is that confabulation and thought disorder are on a semantic syntactic continuum between unsubstantiated claims and incoherent speech .
the theory developed in this paper offers a way of reconciling this interpretation with campbell 's ( 2001 ) underexplored insight that the really key question about the deluded subject is how the use that she makes of the terms in which she frames her delusion relates to her knowledge of the meaning of those terms ( p. 95 ) .
empiricist theories , on the other hand , are more or less committed to the preservation of meaning in delusions ( bayne & pacherie , 2004 , p. 2 ) .
the question is whether , given the requirement to explain both complex delusions and thought disorder in light of the association between confabulation and thought disorder , they can afford to be .
this paper has argued that theories that rely on defective belief evaluation and/or cognitive bias can not explain complex delusions , and that replacing these with confabulation solves this problem in a way that brings somatopsychotic and allopsychotic symptoms under one theory of delusions .
the development of this theory yielded a two - factor theory of confabulation consisting of the propensity to detailed imagination and defective source monitoring .
it was suggested that the two factors that lead to confabulation are due to reciprocal problems with interhemispheric communication consequent upon damage in inferior medial prefrontal region and that , as with delusions , memory and frontal executive dysfunction are not of primary aetiological relevance .
it was argued that the relationship between confabulation and language impairments may also be nonessential , but that confabulation may be on a continuum with thought disorder and , if this were the case , the inclusion of confabulation in a theory of delusions provides a possible starting point for relating research on delusions to research on language impairments in schizophrenia .
|
introductiontheories of delusions which rely on a combination of abnormal experience and defective belief evaluation and/ or cognitive bias are the subject of an emerging consensus .
this paper challenges the validity of these theories and constructs a two factor alternative.methodsthe paper starts by identifying the difficulty the current theories have explaining the complex delusions of schizophrenia and then , by considering , first , the aetiology of somatopsychotic symptoms , and second , the literature on the relationship between confabulation and allopsychotic symptoms , demonstrates that the natural solution is to retain the experiential factor whilst replacing the second factor with confabulation.resultsthe paper is then able to demonstrate that the resultant two - factory theory can clarify recent work on the aetiological role of autonoetic agnosia and on the relationships between confabulation , delusion , and thought disorder.conclusionsthe theory supersedes currently available theories in terms of its simplicity , fruitfulness , scope and conservatism and represents an advance in the search for unified theory of psychosis .
|
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mapping nucleobases
in dna or rna modified by alkylation or oxidation
often relies on the ability to specifically cleave the oligonucleotide
at the site of modification , followed by gel electrophoresis to develop
the map . a method established by maxam and gilbert
utilizes base - specific
alkylation followed by hot piperidine cleavage as the key steps in
sequencing the dna bases .
the success
of this method lies in the ability to specifically alkylate the dna
bases , as well as having high yields in the hot piperidine cleavage
step .
a similar base - specific labeling protocol followed by chemical
cleavage has been advanced for sequencing rna ; however ,
the inherent alkaline instability of rna requires substitution of
piperidine at ph 11 with aniline at ph 4.5 .
these methodologies developed
a framework for identifying other labile sites of modification in
dna and rna , for example , those resulting from base oxidation .
site - specific oxidation of nucleobases , if left unrepaired , can
lead to mutations that are proposed to cause a wide range of diseases
such as alzheimer s disease , melanoma , prostate cancer , and
diabetes .
many research efforts have focused
on understanding the mechanisms and products that result from oxidation
of the nucleobases in dna oligomers to develop hypotheses that explain
cellular mutagenesis .
the nucleobase guanine stands out as a prominent
site of oxidation
because it has the lowest standard reduction potential . in dna ,
oxidation of 2-deoxyguanosine
( dg ) is modulated by the context of the reaction ( i.e. , single - stranded ,
duplex , or g - quadruplex contexts ) . moreover , in duplex
dna , the reactivity of dg toward oxidation shows a subtle sequence
context effect . locating site - specific
oxidation at dg nucleotides in dna oligomers is determined by hot
piperidine work up followed by gel electrophoresis to read the damaged
sites in comparison to a control ladder . another approach to identifying
damage sites
utilizes the base excision repair enzyme fpg for cleavage
at the lesion . in rna ,
oxidation of guanosine
( rg ) has been studied , but to a lesser degree than dna oxidation . for the chemical cleavage methods to be successful at mapping oxidation
sites , it is critical to know the rates and extent to which scission
of the strand occur for different oxidation products of dg and rg .
oxidation of the guanine heterocycle yields classes of products
based on the extent of oxidation .
two - electron oxidation of dg yields
8-oxo-7,8-dihydro-2-deoxyguanosine ( dog ) and 5-carboxamido-5-formamido-2-iminohydantoin-2-deoxyribonucleoside
( d2ih ) , while four - electron oxidation of dg yields two diastereomers of
spiroiminodihydantoin-2-deoxyribonucleoside ( dsp ) , two
diastereomers of 5-guanidinohydantoin-2-deoxyribonucleoside
( dgh ) , and 2,5-diaminoimidazolone-2-deoxyribonucleoside ( diz )
that readily hydrolyzes to 2,2,4-triamino-2h - oxazol-5-one-2-deoxyribonucleoside
( dz ) .
the six - electron oxidation of dg yields dehydroguanidinohydantoin-2-deoxynucleoside
( dgh ) , a compound of limited stability that ultimately
decomposes to a ribosyl - urea lesion .
another product that is formed during certain oxidations , but is
not formally oxidized , is 2,6-diamino-4-hydroxy-5-formamidopyrimidine-2-deoxyribonucleoside
( fapydg ) , a ring - opened hydrolysis product of dg ( scheme 1 ) .
many of these lesions resulting
from dg oxidation have been observed in genomic samples of biological
origin , except d2ih and dgh , whose cellular existence is awaiting
confirmation . in cellular rna samples , rog is the only lesion that
has been characterized thus far .
detection
of these lesions in cellular samples typically occurs by nuclease
digestion that leads to complete loss of sequence information , followed
by mass spectrometry .
therefore , in vitro model studies with dna or rna
oligomers allow understanding the sequence context effects on lesion
formation .
the standard approach for identification of lesion sites
requires strand cleavage by piperidine ( dna ) or aniline ( rna ) followed
by gel electrophoresis ; another method utilizes esi - ms , but
this approach is limited to short oligomers with only one possible
site of modification .
oxidations conducted on dna
oligomers of known sequences have yielded
a wealth of information concerning the sequence context - dependent
reactivity of dg toward oxidants and the products . for interpretation of these data from the point of view of biology ,
the oxidation reactions are conducted under low product conversion
( i.e. , single - hit chemistry ) .
typically ,
the reaction yield is determined by hot piperidine cleavage of the
oligomers using a standard protocol ( 0.21 m piperidine , 30
min , 90 c ) ; however , the yields
and rates for cleavage of the consistently observed dg oxidation products
have not been established under identical reaction conditions .
ligation - mediated pcr is an approach for determining lesion sites
in genomic samples that relies on the ability to site - specifically
cleave the lesion for ligation on a primer sequence used to identify
the original lesion location . in this
method , chemical cleavage is one approach for effecting strand scission
at the lesion site . in rna , cleavage at sites of base modification
sites employs aniline following a standard procedure ( 0.21
m , ph 4.5 , 60 c , 20 min ) . as for
dna ,
strand cleavage efficiencies are not known for rg oxidation products
in rna oligomers . because rates of chemical strand scission for dg
and rg lesions have not been established , the extent of an oxidation
reaction on dna or rna polymers is inaccurately determined . in the
present study ,
the rates of cleavage for og , sp , gh , 2ih , and z were
determined in dna oligomers treated with piperidine , and in rna oligomers
treated with aniline under standard reaction conditions .
these data
determined that lesion cleavage rates using typical reaction conditions
are quite variable , and some are not significantly cleaved , even at
long incubation times .
these data will be paramount for researchers
that oxidize oligomers of dna or rna with known sequences and then
interpret the reaction yields from a biological prospective , for which
oxidation events on these polymers are a rare occurrence .
the dg oxidation
products dog , ( s)-dsp , ( r)-dsp , and a mixture of the dgh diastereomers were synthesized in the 18-mer
dna oligomer 5-tca tgg gtc xtc ggt ata-3
( where x = lesion site ) .
the dna oligomer was synthesized
via solid - phase synthesis by the dna / peptide core facility at the
university of utah with a dog phosphoramidite ( glen research , sterling ,
va ) at the position of x following standard methods .
analytical ion - exchange hplc - purified samples provided the dog - containing
dna oligomer for the piperidine studies .
syntheses
of the dsp diastereomers and dgh diastereomers were conducted following
literature protocols .
the dsp diastereomers
were individually purified while the diastereomers of dgh were studied
as a mixture because they readily interconvert .
syntheses of dz and the ( r)- and ( s)-d2ih diastereomers were
achieved in a 15-mer dna oligomer containing a single dg site ( 5-aat
cca cga cac ctc-3 ) following literature methods . all product oligomers were purified using an analytical ion - exchange
hplc column that resolves diastereomeric products ( figure s1 ) .
the dna oligomer products were characterized by
esi - ms ( dog : calcd 5560.6 , found 5560.8 ; mixture of the dsp diastereomers :
calcd 5576.6 , found 5576.4 ; mixture of the dgh diastereomers : calcd
5550.6 , found 5550.2 ) , or maldi - ms was used to characterize a mixture
of the d2ih diastereomers as well as for dz ( d2ih : calcd 4498.8 , found
4498.2 ; dz : calcd 4445.8 , found 4445.3 ) .
the dz - containing oligomer
was found to have limited stability due to further decomposition of
dz to guanidinoformimine-2-deoxyribonucleoside ( dgf ) .
the dgf - containing strand was characterized
by maldi - ms ( dgf : calcd 4398.8 ; found 4398.2 ) .
because of the limited
stability of dz in the dna oligomer leading to dgf , the dz sample
was studied immediately after purification and a 2 h dialysis to remove
the purification salts .
the rg - oxidation products rog , ( r)-rsp , ( s)-rsp , rz , ( r)-r2ih , ( s)-r2ih , and rgh diastereomers were synthesized
in the 7-mer rna oligomer 5uuuguuu-3.
selective conversion of the single rg to the rsp diastereomers , rgh
diastereomers , r2ih diastereomers , and dz was achieved via literature
protocols .
these strands were then purified
by the same method as stated for the lesions in the dna oligomers
( figure s2 ) .
the 7-mer containing rog was
synthesized using the phosphoramidite of rog ( chem genes , wilmington ,
ma ) following the manufacturer s protocol .
the rna oligomer
products were characterized by maldi - ms ( rog : calcd 2136.3 , found
2136.7 ; ( s)-rsp and ( r)-rsp diastereomer
mixture : calcd 2152.3 , found 2153.0 ; rgh diastereomer mixture : calcd
2126.3 , found 2126.1 ; rz : calcd 2099.3 , found 2099.4 ; ( r)-r2ih and ( s)-r2ih diastereomer mixture : calcd
2154.4 , found 2154.3 ) . to monitor the cleavage reactions ,
the lesion - containing dna oligomers
were 5-labeled with p for visualization by gel
electrophoresis followed by phosphorimager autoradiography .
piperidine cleavage reactions for lesion - containing
dna oligomers were conducted by mixing 100 pmol of unlabeled strand
with 20 000 cpm of p - labeled strand in 50 l
of ddh2o . for each lesion ,
nine tubes were prepared for
taking time points at 0 , 5 , 10 , 20 , 30 , 45 , 60 , 90 , and 120 min . to
these samples
were added 50 l of freshly prepared aqueous piperidine
( 2 m ) and -mercaptoethanol ( bme , 0.5 m ) to give a final concentration
of 1 m dna with 1 m piperidine and 0.25 m bme .
next , the samples
were incubated at 90 c , and at each time point , a sample was
removed from the heat source .
the samples were lyophilized to dryness
and resuspended in 10 l of gel loading dye ( 0.25% xylene cyanol ,
0.25% bromophenol blue in an aqueous 30% v / v glycerol solution ) . to
a 20% denaturing polyacrylamide gel electrophoresis ( page ) , 5 l
of sample in loading dye was added .
the samples were electrophoresed
at 45 w for 2 h , after which the gel was loaded into a phosphor screen
and visualized by phosphorimager autoradiography after an 18 h exposure .
one reaction was also monitored by ion - exchange hplc on
a sample that was not p labeled that allowed observation
of all species in solution during the reaction .
the method utilized
was the same as the one used for product purification . to monitor
the cleavage reactions ,
the lesion - containing rna oligomers
were 5-labeled with p for visualization by gel
electrophoresis followed by phosphorimager autoradiography .
aniline cleavage reactions for lesion - containing
rna oligomers were conducted by mixing 100 pmol of unlabeled strand
with 20 000 cpm of p - labeled strand in 50 l
of ddh2o . for each lesion ,
seven tubes were prepared for
taking time points at 0 , 10 , 20 , 30 , 40 , 60 , and 90 min . to these
samples
was added 50 l of freshly prepared aniline ( ph 4.5 ,
2 m ) to furnish a final concentration of 1 m rna and 1 m aniline .
the aniline at ph 4.5 was prepared by taking a solution of reagent
grade aniline and adjusting the ph to 4.5 with glacial acetic acid
to obtain a 2 m stock solution .
next , the samples were incubated at
60 c in the dark , and at each time point , a sample was removed
from the heat source .
after completion of the reactions , the samples
were processed in an identical fashion as previously described for
the lesion - containing dna oligomers .
scheme 2a shows the proposed mechanism for
piperidine - induced strand scission at the site of a dna lesion . in the first step ,
this
intermediate increases the acidity of h2 that accelerates
-elimination of the 3-phosphate terminus in the second
step . in the third step , -elimination of
the 5-phosphate
terminus occurs to yield complete cleavage at the site of the lesion .
the correct kinetic rate equation for this complex reaction should
take into account all intermediate reactions ; however , in practice ,
piperidine cleavage reactions are followed by page , in which only
the intact strand and the cleaved strand are observed . by monitoring
loss of the starting material and the appearance of the product ,
the
rate equation can be simplified to a pseudo - first - order rate equation .
the assumptions in this simplified approach are that the piperidine
concentration does not change , as is validated by the large excess
of piperidine used to effect strand scission ( 10-fold
excess ) .
the second simplifying assumption was the use of the steady - state
approximation , in which the concentrations of the reaction intermediates
were assumed to reach a steady state , and their concentrations were
limited by the rate of the first reaction step .
verification of this
assumption was applied to a test reaction that monitored the piperidine
cleavage of a ( r)-dsp - containing dna oligomer by
hplc .
this approach allowed inspection of all species in solution ,
which is not possible by page . in this analysis , the intact strand ,
cleavage products ( 5 and 3 fragments ) , and intermediates
the intermediates were not characterized due to their
instability . in support of the steady - state approximation ,
the intermediate
peak ratios did not significantly change during the 90 min reaction
profile , while the starting material decreased and the product strands
increased ( figure s3 ) . to monitor cleavage
reactions for all lesions , the traditional approach of monitoring
these reactions by page
was selected , and a pseudo - first - order reaction
rate equation was applied for mathematically modeling .
the rate - limiting
step was assumed to be formation of the schiff - base intermediate .
thus , plots of the ln [ intact strand ] vs reaction time provided linear
data points that were described by eq 1 , where
the slope of the line provides the reaction rate constant.1 monitoring the time - course cleavage for the guanine oxidation
products
studied in a dna oligomer by page gave a wide distribution of reaction
course profiles ( figure 1a and figure s4 ) . for example , the dog lesion was not
significantly cleaved ( < 1% ) by hot piperidine during the 2 h reaction
( figure 1a ) .
next , the dsp diastereomers were
found to have undergone slow hot piperidine strand scission ( figure 1a ) , with rate constants for the isomers to be nearly
identical values of ( 9.5 1.5 ) 10 and ( 9.5 1.2 ) 10 min for the r and s isomers , respectively
( table 1 ) .
for dgh , strand scission by hot
piperidine was faster , with a cleavage rate constant of ( 22
3 ) 10 min ( table 1 ) , while the dz lesions were cleaved with a rate
constant of ( 51 5 ) 10 min ( table 1 ) .
finally , the diastereomers of
d2ih cleaved the quickest with rate constants of ( 100 10 )
10 and ( 110 11 ) 10 min for the r and s isomers , respectively ( table 1 ) .
these studies
demonstrate that the dg oxidation products , other than dog , cleave
with hot piperidine at variable rates , and near - quantitative reaction
yields can be obtained if the reactions are allowed to progress long
enough .
the pseudo - first - order kinetic rate constants allow
determination
of the half - life ( t1/2 ) of each lesion
under the reaction conditions . these values will allow researchers
to calculate the reaction time required to achieve the yield they
would desire . the dog lesion in dna
can not be found by piperidine
cleavage reactions , and this point is further discussed below . as
for the other dg lesions in dna , the t1/2 values were 73 min for the dsp diastereomers , 32 min for the dgh
diastereomers , 14 min for dz ,
6.8 min for ( r)-d2ih ,
and 6.0 min for ( s)-d2ih ( table 1 ) . on the basis of these results , all dg - lesions , besides
dog , can be cleaved in high yield as long as the reactions are allowed
to progress for a sufficiently long time period .
plots of ln[intact strand ]
vs time for dg oxidation products in
dna ( a ) and rg oxidation products in rna ( b ) oligomers .
( a ) time - dependent
cleavage yields for the dna lesions , dog , ( r)-dsp ,
( s)-dsp , dgh , dz , ( r)-d2ih , and
( s)-d2ih are plotted .
the reaction yields were monitored
on 5-p - labeled strands that were separated by
page and quantified by phosphorimager autoradiography .
cleavage reactions
for these damaged dna oligomers were conducted with fresh , aqueous
piperidine ( 1 m ) and bme ( 0.25 m ) at 90 c .
( b ) time - dependent
cleavage yield for the rna lesions , rog , ( r)-rsp ,
( s)-rsp , rgh , rz , ( r)-r2ih , and
( s)-r2ih are plotted .
the reactions were monitored
in the same fashion as those for the dna oligomers .
cleavage reactions
for the damaged rna oligomers were conducted with fresh , aqueous aniline
( ph 4.5 ) at 60 c in the dark .
scheme 2b shows the
proposed mechanism for
aniline - induced strand scission at the site of a rna lesion .
this intermediate increases the
acidity of h2 that accelerates -elimination of the
3-phosphate terminus .
finally , -elimination of the
5-phosphate terminus occurs to yield complete cleavage at
the site of the lesion .
because of the similarity in the cleavage
mechanisms , and the fact that the same concentration of aniline was
used to cleave rna lesions as was used for piperidine to cleave dna
lesions , the same assumptions were used to fit the data to determine
the pseudo - first - order reaction rate constants ( eq 1 ) .
the rates of aniline - mediated cleavage ( 1 m , ph 4.5 ,
60 c ) for all rg lesions were assayed by following strand cleavage
via page analysis . under these reaction conditions , rog showed < 1%
cleavage after a 90 min reaction ( figure 1b ) .
the diastereomers of rsp gave slightly more cleavage than rog with
< 10% strand scission .
the fitting equations to these data determined
the cleavage rate constants for the r and s diastereomers of rsp to be ( 1.2 0.2 ) 10 and ( 1.4 0.2 ) 10 min , respectively ( table 1 ) .
next , the diastereomers of rgh were found to give more strand
scission under these conditions ( figure 1b ) .
for rgh , the aniline cleavage rate constant was ( 6.9 0.5 )
10 min ( table 1 ) .
the rz reaction preceded more quickly giving a reaction
rate constant of ( 51 6 ) 10 min ( table 1 ) .
finally , the r2ih
diastereomers cleaved the fastest ( figure 1b ) with the r and s isomers having
reaction rate constants of ( 61 7 ) 10 and ( 63 7 ) 10 min , respectively ( table 1 ) .
these studies demonstrate
that site specific cleavage of rg oxidation lesions in rna were slower
than the analogous cleavage reactions in dna with piperidine . the pseudo - first - order reaction rate constants for cleavage of
rg lesions in rna allow calculation of the t1/2 values for these lesions by aniline . on the basis of these
rate constants ,
the t1/2 for the rsp diastereomers
were calculated to be 580 and 500 min for the r and s isomers , respectively ( table 1 ) .
these lifetimes are too long to be usable for detection of rsp lesions
in rna , and therefore , support rsp as not being cleavable by aniline
in any practical applications . for the other rg lesions , the t1/2 values were determined to be 100 min for
rgh , 14 min for rz , and 11 min for the diastereomers of r2ih .
in the current studies , dog was not piperidine labile ,
an observation that has stirred some debate in the literature .
the bme added during the reaction is hypothesized
to quench unwanted oxidation of dog .
after a 2 h incubation ( 1 m piperidine , 90 c ) , 10% of
the dog - containing strand cleaved based on page analysis , thus supporting
the hypothesis that dog can be oxidized during the cleavage reaction ,
albeit slowly .
analysis of a test reaction by hplc confirmed dog to
slowly oxidize to dsp under the hot piperidine reaction conditions
( figure s5 ) , an expected result because
at high ph oxidation of dog yields dsp . because dog is poorly cleaved by hot piperidine ,
one approach for
sequencing this lesion via chemical cleavage has been developed by
our laboratory . in this method ,
dog is
further oxidized to dgh with a selective and mild one - electron oxidant ,
such as na2ircl6 , and the corresponding secondary
hydantoin lesion is much more labile to piperidine ( figures 1a and table 1 ) .
the hydantoins dsp and dgh were both found
to be labile to piperidine , but with considerably different rate constants
( dsp = 9.5 10 min ;
dgh = 22 10 min ; table 1 ) .
further , the r and s isomers of dsp were studied individually and shown to give similar
cleavage rates with hot piperidine ( figure 1a and table 1 ) .
the standard piperidine reaction
time is 30 min that will give 25% and 48% strand scission for the
diastereomers of dsp and dgh , respectively ( figure 2a ) ; the incomplete strand scission currently observed for
these lesions after a 30 min piperidine reaction is consistent with
previous literature reports . to effect strand scission
in a yield >
90% for the hydantoins , the piperidine reaction times
must be > 242 min and > 106 min for dsp and dgh , respectively
( figure 3a ) .
as for dz after a 30 min reaction ,
the cleavage
yield would be 78% , an observation that is nearly identical to literature
reports ( 80% cleavage after 30 min ) .
furthermore ,
extension of the reaction time to > 46 min will lead to > 90%
cleavage
at dz sites ( figure 3a ) .
( figure 2a ) , and the standard 30 min piperidine cleavage
reaction time will sufficiently identify d2ih sites ( figure 3a ) .
only one sequence context was studied for each
g lesion in dna , and sequence context effects on reaction rates may
exist ; however , it is anticipated that these effects will be minimal
at ph 11 and 90 c .
other reagents that cleave dna lesions at
lower ph have been utilized , such as n , n-dimethylethylenediamine ( 0.1 m ) at ph 7.4 and 37 , 65 , or
90 c .
studies were conducted to
determine if this reagent could cleave dog , dsp , or dgh under the
reported conditions ( 0.1 m , ph 7.4 , and 65 c ) .
these reactions
were monitored by hplc and found that n , n-dimethylethylenediamine was incapable of cleaving all three
lesions at reaction times < 2 h ( figure s6 ) .
cleavage efficiency
for dg lesions ( a ) and rg lesions ( b ) following
the standard chemical cleavage protocols .
( a ) the dg lesion - containing
oligomers were incubated following the standard cleavage recipe that
includes fresh , aqueous 1 m piperidine and 0.25 m bme at 90 c
for 30 min .
( b ) the rg lesion - containing oligomers were incubated
following the standard cleavage recipe that includes fresh , aqueous
1 m aniline ( ph 4.5 ) at 60 c for 20 min in the dark .
chemical cleavage of rg lesions in rna was determined
to have lower
rate constants for all lesions compared to dg lesions in dna . for
example
, the reaction rate for aniline cleavage of the rsp diastereomers
was very slow , and almost undetectable ; these reaction rate constants
determined that after a standard 20 min aniline reaction time , 3%
of the rsp diastereomers would cleave ( figure 2b ) .
the aniline reaction time would need to be increased to 1900
and 1700 min for the r and s diastereomers
of rsp , respectively , to effect > 90% strand scission ( figure 3b ) .
these reaction times are not practically useful ,
effectively causing the rsp isomers to be resistant to aniline treatment .
in comparison
, the rgh isomers will cleave 13% after the standard
aniline reaction time ( figure 2b ) , and the
reaction time would need to be extended to > 330 min to effect > 90%
strand scission yield at these sites ( figure 3b ) .
next , rz would cleave by 64% under the standard reaction time
( figure 2b ) , and extension of the cleavage
reaction to > 47 min will facilitate > 90% cleavage at these sites
( figure 3b ) .
finally , 71% of the r2ih diastereomers
will
cleave under the standard reaction time ( figure 2b ) ; for these lesions , extending the reaction time to > 37 min
will
lead to > 90% strand scission at these sites ( figure 3b ) .
in contrast , rog did not give detectable amounts
of strand scission
with aniline , even after a 90 min reaction .
the only rg lesion detected in vivo so far is rog , an observation that validates rg
oxidation occurrence in the cell .
this is particularly
striking because there exists 3 times more rna in a cell than
dna , and rna takes on a variety of secondary structures that expose
the bases to solvent .
these exposed bases will be much more prone
to oxidation than those in duplex structures , and a major rg oxidation product will likely be rsp .
however , the use of rna oligomers and chemical cleavage agents will
not provide an accurate measure of the sites and yields for the rg
oxidation product rog , and likely rsp .
therefore , to study the formation
of these lesions in rna will require a different assay .
either a polymerase
stop assay can be performed , as has been previously shown , or rog can be further oxidized with a mild oxidant
( na2ircl6 ) to yield a hydantoin ( e.g. , rgh )
that is labile to aniline , an approach that has also been previously
proposed .
these studies in rna were only
conducted in one sequence context ; however , at ph 4.5 and 65 c ,
it is anticipated that sequence context effects will be minimal .
minimum
reaction time to achieve > 90% cleavage at dg lesions in
dna oligomers ( a ) and at rg lesions in rna oligomers ( b ) .
the chemical
cleavage of lesion - bearing dna oligomers was conducted with freshly
prepared , aqueous piperidine ( 1 m ) and bme ( 0.25 m ) at 90 c ,
and the lesion - bearing rna oligomers was conducted with freshly prepared ,
aqueous aniline ( ph 4.5 ) at 60 c in the dark .
the reaction times
were computed from the strand scission rate constants values for these
lesions provided in table 1 . * these lesions
the
current research determined the chemical cleavage rate constants
for many lesions derived from oxidation of the nucleobase guanine
in dna and rna oligomers . in these studies , the base damages og , sp ,
gh , z , and 2ih were site - specifically synthesized in dna and rna oligomers .
these strands were then subjected to the standard chemical cleavage
reagents for dna ( 1 m piperidine , ph 11 , 90 c ) or rna ( 1 m aniline ,
ph 4.5 , 60 c ) , and the pseudo - first - order rate constants for
stand scission were determined . in dna
, dog was not cleaved , and the
rate for the dsp diastereomers was found to be slow ( table 1 ) .
the rates of strand scission for dz , dgh , and
d2ih were faster , and only d2ih will cleave in a > 95% yield after
the standard reaction time of 30 min ( figure 2a ) .
the current results highlight that most lesions may require long
reaction times to obtain high strand scission yields ( figure 3a ) .
these results taken together identify a great
variability in the cleavage yield for the dg lesions .
the alternative
approach for identifying lesion sites in dna utilizes a base excision
repair enzyme , such as fpg ; however , fpg cleavage also suffers from
lesion - dependent cleavage kinetic differences .
in addition ,
in rna strands , all lesions were found to cleave at a slower rate
than their analogous dna counterparts ( table 1 ) .
more specifically , rog and rsp gave insignificant cleavage with
aniline at long reaction times ( figure 1b ) ,
while rgh and rz could be cleaved at very long reaction times ( figure 3b ) .
finally , these results identify og and sp lesions
in dna and rna strands to be the most stable ( table 1 ) , and they support a hypothesis that these lesions in the
genome or transcriptome would be much longer lived than the other
guanine base lesions .
|
the nucleobase guanine in dna ( dg )
and rna ( rg ) has the lowest
standard reduction potential of the bases , rendering it a major site
of oxidative damage in these polymers .
mapping the sites at which
oxidation occurs in an oligomer via chemical reagents utilizes hot
piperidine for cleaving oxidized dna and aniline ( ph 4.5 ) for cleaving
oxidized rna . in the present studies , a series of time - dependent cleavages
of dna and rna strands containing various guanine lesions were examined
to determine the strand scission rate constants .
the guanine base
lesions 8-oxo-7,8-dihydroguanine ( og ) , spiroiminodihydantoin
( sp ) , 5-guanidinohydantoin ( gh ) , 2,2,4-triamino-2h - oxazol-5-one ( z ) , and 5-carboxamido-5-formamido-2-iminohydantoin
( 2ih ) were evaluated in piperidine - treated dna and aniline - treated
rna .
these data identified wide variability in the chemical lability
of the lesions studied in both dna and rna .
further , the rate constants
for cleaving lesions in rna were generally found to be significantly
smaller than for lesions in dna .
the og nucleotides were poorly cleaved
in dna and rna ; sp nucleotides were slowly cleaved in dna and did
not cleave significantly in rna ; gh and z nucleotides cleaved in both
dna and rna at intermediate rates ; and 2ih oligonucleotides cleaved
relatively quickly in both dna and rna .
the data are compared and
contrasted with respect to future experimental design .
|
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physical fitness is the ability that helps people to adapt with their environmental conditions and plays an important role in daily activities .
it is highly associated to one 's health status and has a striking effect on the quality of life , learning and working efficiency , and physical activity contributions .
physical fitness is composed of performance - related physical fitness and health - related physical fitness .
indeed , genetic somehow determines physical fitness , but environmental factors including physical activity levels , socioeconomic status , television viewing , and anthropometric factors highly modify it . since fitness level drops with aging , physical involvement should be highly suggested .
however , inactive life affects body muscle and functioning ability negatively , and people are vulnerable to chronic diseases as consequences .
a higher quality of life can be obtained through moderate exercises to develop or maintain physical fitness .
lamb declared that individuals can successfully cope with challenges in their life via optimal physical fitness .
physical training program can be effective for athletes and nonathletes in different ages to improve aerobic power , muscle strength , anaerobic power , jumping ability , and local muscle endurance .
additional strength training and plyometric training in 912-year - old soccer players for 2 years showed a significant improvement of motor performance factors .
mini - trampoline exercises may need higher levels of involvement in the muscles of lower extremities .
since mini - trampoline exercises comprise a multicomponent approach , numerous physical factors including strength , body stability , muscle coordinative responses , joint movement amplitudes , and spatial integration can be affected positively .
. indicated not only balance and strength improvement but also muscular imbalances equalization of the two limbs due to mini - trampoline training .
. showed improvement in motor performance of participants with intellectual disabilities after applying trampoline training intervention .
however , rebounding exercise for 11 weeks revealed minimal cardiorespiratory fitness improvement and no significant body composition changes in 17 sedentary women .
very few studies were conducted to detect specific contributions derived from trampoline exercise as a different training model approach .
therefore , the purpose of this study was to examine the effect of trampoline training intervention on anthropometric variables and physical fitness components of 1114-year - old male students , iran .
thirty - eight healthy school - aged male students ( mean age : 12.6 years , standard deviation : 2.1 ) were selected randomly from the junior high schools of ardabil , iran .
they were divided into two groups of 19 students based on gender , residential school , and no trampoline training experience .
the first five students were omitted as they had prior experience , and the second five students were excluded because they had failed to attend the exercise sessions consistently [ figure 1 ] .
the study protocol received approval from the ethics committee of the university of urmia , and it was in accordance with the declaration of helsinki ( last modified in 2000 ) .
flow chart of the progress through the phases of the study according to the consort statements evaluation process of participants was conducted within a 2-day period by the same researcher .
the 1 day was assigned to anthropometric measurement and the 2 day for motor ability tests .
anthropometry and motor ability of the participants were measured before and after the 20 weeks trampoline training intervention .
, crymych , dyfed , uk ) and weight ( model ds-410 , seiko , tokyo , japan ) were assessed to the nearest 10 mm and 0.1 kg respectively , and body mass index was calculated using following formula : weight ( kg)/(height [ m ] ) .
moreover , lower extremity girths including thigh and calf were measured on the right side of each participant .
skinfolds thickness was measured at three body sites ( triceps , subscapula , and suprailiac ) with a harpenden skinfold caliper .
participants in experimental group trained 20-week , 1.5 h / session with 10 min rest , in 4 times trampoline training programs per week .
although none of the students had balance problems , some important safety guidelines were considered to prevent the possibility of injury .
all participants were instructed to perform variety of movements with eyes opened including one foot and two - foot jump , hop with rotation in different directions , hand and toe contacts in the air , and alternate sit and stand movement . the instructor started to teach forward and backward somersault when participants were familiar with spatial awareness , spatial orientation , and body control after performing all of the mentioned movements . finally , students were asked to combine individual movements and execute them as a routine throughout the training sessions .
however , participants in the control group only participated in school physical education classes 2 times / week for 45 min .
motor ability of all participants was assessed after a short period of warm up using standing long jump and vertical jump based on eurofit test battery .
each participant standing reach height , tip of middle finger in the hand closest to the wall while participant stood side on to the wall , was marked .
then , participant used both arms and legs contributions to project the body as high as possible while standing away from the wall . the standing reach height and the jump height difference were the score .
each participant stood behind the marked line on the ground with swinging arms and bending knees .
the take - off was done when the arms reached forward to lead participant as far as possible .
a two - way repeated measures analysis of variance ( anova ) ( condition trial ) with repeated measures on both factors was used . to further analyze the responses of the groups
thirty - eight healthy school - aged male students ( mean age : 12.6 years , standard deviation : 2.1 ) were selected randomly from the junior high schools of ardabil , iran .
they were divided into two groups of 19 students based on gender , residential school , and no trampoline training experience .
the first five students were omitted as they had prior experience , and the second five students were excluded because they had failed to attend the exercise sessions consistently [ figure 1 ] .
the study protocol received approval from the ethics committee of the university of urmia , and it was in accordance with the declaration of helsinki ( last modified in 2000 ) .
flow chart of the progress through the phases of the study according to the consort statements
the 1 day was assigned to anthropometric measurement and the 2 day for motor ability tests .
anthropometry and motor ability of the participants were measured before and after the 20 weeks trampoline training intervention .
, crymych , dyfed , uk ) and weight ( model ds-410 , seiko , tokyo , japan ) were assessed to the nearest 10 mm and 0.1 kg respectively , and body mass index was calculated using following formula : weight ( kg)/(height [ m ] ) . moreover , lower extremity girths including thigh and calf were measured on the right side of each participant .
skinfolds thickness was measured at three body sites ( triceps , subscapula , and suprailiac ) with a harpenden skinfold caliper .
participants in experimental group trained 20-week , 1.5 h / session with 10 min rest , in 4 times trampoline training programs per week .
although none of the students had balance problems , some important safety guidelines were considered to prevent the possibility of injury .
all participants were instructed to perform variety of movements with eyes opened including one foot and two - foot jump , hop with rotation in different directions , hand and toe contacts in the air , and alternate sit and stand movement . the instructor started to teach forward and backward somersault when participants were familiar with spatial awareness , spatial orientation , and body control after performing all of the mentioned movements .
finally , students were asked to combine individual movements and execute them as a routine throughout the training sessions .
however , participants in the control group only participated in school physical education classes 2 times / week for 45 min .
motor ability of all participants was assessed after a short period of warm up using standing long jump and vertical jump based on eurofit test battery .
each participant standing reach height , tip of middle finger in the hand closest to the wall while participant stood side on to the wall , was marked .
then , participant used both arms and legs contributions to project the body as high as possible while standing away from the wall . the standing reach height and the jump height difference were the score .
each participant stood behind the marked line on the ground with swinging arms and bending knees .
the take - off was done when the arms reached forward to lead participant as far as possible .
. a two - way repeated measures analysis of variance ( anova ) ( condition trial ) with repeated measures on both factors was used . to further analyze the responses of the groups
anthropometric and physical fitness variables of the two groups are reported in tables 1 and 2 .
the anova repeated measurement test showed a statistically significant main effect of time in calf girth p = 0.001 , fat% p = 0.01 , vertical jump p = 0.001 , and long jump p = 0.001 .
the anova repeated measurement test revealed a statistically significant main effect of group in fat% p = 0.001 .
characteristics of the study participants differences between pre- and post - training of anthropometric and physical fitness indicators in the trampoline and control groups post hoc paired t - tests indicated statistical significant differences in trampoline group between the two measurements about calf girth ( t = 4.35 , p = 0.001 ) , fat% ( t = 5.87 , p = 0.001 ) , vertical jump ( t = 5.53 , p = 0.001 ) , and long jump ( t = 10.00 , p = 0.001 ) .
post hoc paired t - tests showed statistical significant differences in control group between the two measurements concerning calf girth ( t = 5.67 , p = 0.001 ) , vertical jump ( t = 3.88 , p = 0.002 ) , and long jump ( t = 4.22 , p = 0.001 ) .
the analysis of independent t - test ( pre- to post - subtraction between two groups ) indicated significant differences in fat% p = 0.001 , high jump p = 0.02 , and long jump p = 0.02 .
this study investigated whether anthropometric measures and motor performance components are changed via trampoline training considering that this is the first study in a relatively longer period . since athletes increase metabolism through practice , our finding indicated significant changes of body fat% as a result of trampoline training that is not consistent with the findings of edin et al .
indeed , no significance changes of body composition variables in the study of edin et al ( 1990 ) may be attributed to the 11 weeks exercise that is limited in terms of training period compared to our study .
although trampoline exercises are not basically aerobic , long - term training program made significant changes in body fat% between the two groups .
lower limb characteristics such as thigh girth and calf girth were improved considering pre - test and post - test differences .
however , we found no significant difference between experimental and control group in this regard .
the improved lower limb muscle sizes are related to participant 's growth process , and trampoline training has no significant effect on the hypertrophy of lower limb muscles .
( 2011 ) reported balance improvement due to mini - trampoline exercises in elderly participants .
( 2013 ) indicated improvement in motor performance of participants with intellectual disabilities after applying trampoline training intervention .
our findings are in agreement with those studies in this regard and showed significant improvement of jumping ability in healthy trampoline participants compared to control group .
this improvement can be attributed to the higher amount of biomechanical stimuli with jumping on a trampoline compared to other forms of activity like running .
furthermore , body segments coordination due to training programs can help participants to use the ideal motor patterns for performing jumping tests .
trampoline training stimulates proprioceptive sense that can facilitate motor performance and prevent musculoskeletal injuries as a result of sudden fall in life .
since the nature of this training intervention is recreational , people in different ages can be involved to promote the quality of their life .
based on these finding , we can conclude that a 20-week trampoline training with four physical activity sessions / week in 1114-year - old students seem to have a significant effect on body fat% reduction and effective results in terms of anaerobic physical fitness .
therefore , it is suggested that different training model approach such as trampoline exercises can help students to promote the level of health and motor performance .
|
background : physical exercises can influence some anthropometric and fitness components differently .
the aim of present study was to evaluate how a relatively long - term training program in 11 - 14-year - old male iranian students affects their anthropometric and motor performance measures.methods:measurements were conducted on the anthropometric and fitness components of participants ( n = 28 ) prior to and following the program .
they trained 20 weeks , 1.5 h / session with 10 min rest , in 4 times trampoline training programs per week .
motor performance of all participants was assessed using standing long jump and vertical jump based on eurofit test battery.results:the analysis of variance ( anova ) repeated measurement test showed a statistically significant main effect of time in calf girth p = 0.001 , fat% p = 0.01 , vertical jump p = 0.001 , and long jump p = 0.001 .
the anova repeated measurement test revealed a statistically significant main effect of group in fat% p = 0.001 .
post hoc paired t - tests indicated statistical significant differences in trampoline group between the two measurements about calf girth ( t = 4.35 , p = 0.001 ) , fat% ( t = 5.87 , p = 0.001 ) , vertical jump ( t = 5.53 , p = 0.001 ) , and long jump ( t = 10.00 , p = 0.001).conclusions : we can conclude that 20-week trampoline training with four physical activity sessions / week in 1114-year - old students seems to have a significant effect on body fat% reduction and effective results in terms of anaerobic physical fitness .
therefore , it is suggested that different training model approach such as trampoline exercises can help students to promote the level of health and motor performance .
|
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] |
derangements of the condyle - disc complex arise from a breakdown of the normal rotational movement of the disc on the condyle .
the thinning of the posterior border of the disc can cause the disc to be displaced in a more posterior position . with the condyle resting on a more posterior portion of the disc or retrodiscal tissues , an abnormal translatory shift of the condyle over the posterior border of the disc can occur during the opening .
a click is associated with the abnormal condyle - disc movement and may be initially felt just during opening ( single click ) but later may be felt during opening and closing of the mouth ( reciprocal clicking).1 molinari et al.2 reported that occasionally a second clicking sound is heard during mouth closure ( reciprocal click ) , because the posterior band of the disc slips forward off the condyle .
other clicking sounds can also be produced by irregularities or defects in the surface of the disc or by changes in the convexity of the condylar and/or articular eminence .
they are also found at the same point of the temporomandubular joint ( tmj ) traslator movement rather than at different points , as occurs with reciprocal clicking .
clicking and crepitation should be considered signs of morphological alterations , being indicative of anterior disk displacement with reduction3 and arthrosis , respectively .
electrovibratographic records and macroscopic examinations of articulations of corpses showed that 20% of the tmjs with clicking had the disk displaced anteriorly and 22% of the tmjs with crepitation had arthrosis or disk perforation.4 later recapture of the disk causes clicking at the end of mouth opening and indicates that the bilaminar zone is more affected.5 the microscopic aspects of the disk surface can also be altered.6 qualitative and semi - quantitative methods have been developed for tmj sound classification , but the criteria presented are completely inhomogeneous.7 - 12 thus , to develop more objective criteria for defining tmj sounds , electroacoustical systems have been developed.7 - 9 , 11 - 15 we used joint vibration analysis ( jva ) in the biopak system ( bioresearch inc . , milwaukee , usa ) as the electrovibratography , and jaw tracker ( jt)-3 device in the biopak system ( bioresearch inc . ,
milwaukee , usa ) . using jt-3 deivce allowed the computer to estimate where a joint vibration occurs in the open / close cycle and let us distinguish tooth contact from joint sound precisely .
ishigaki et al.17 reported a disc displacement with reduction generates a " click " in the lower frequencies ( under 300 hz ) and a degenerative condition generates " crepitus " in the higher frequencies ( over 300 hz ) .
in the previous study , we found that in an integral > 300 hz / < 300 hz ratio it is conceivable that the higher the integral > 300 hz / < 300 hz ratio number , a more advanced degenerative condition exists .
gallo et al.16 reported that tmj clicking was subjectively and objectively stable over a period or 10 days .
we found few studies about long term follow - up based on the frequency spectrum patterns associated with the integral > 300 hz / < 300 hz ratio .
the aim of this study was to examine the tmj sounds with repect to frequency spectra patterns and the integral > 300 hz / < 300 hz ratios via six - months follow - up .
twenty dental school students ( 18 males and 2 females : age range 25 - 34 years old ; mean age = 22.4 years old ) participated in the before ( control group ) and after ( experimental group ) the six - months joint sound recordings .
group i ( 8 males and 2 females ) was composed of the subjects that showed anterior disk displacement with reduction .
they were selected by means of clinical examinations . as inclusion criteria , all subjects had clicking in both tmjs upon mouth opening and/or closing and a normal range of jaw movement during opening and/or pain at palpation ( any of the masseter , temporalis , pterygoid , digastric muscles ) and jaw movement during chewing .
group ii ( 10 males ) was composed of subjects that showed a normal state of tmj .
they showed absence of tmj noises , pain at palpation ( any of the masseter , temporalis , pterygoid , digastric muscles ) and jaw movement or chewing . in each subject ,
a magnet was attached to the labial surface of mandibular incisors of the subjects in order to bring the midline of the magnet to the labial frenum and to locate the groove of the magnet to the left side of the subjects . if the subject tended to have a deep bite so that it is impossible to attach the magnet , it was attached to the labial gingival surface or lingual tooth surface .
one transducer was placed on the skin over the right tmj , and the other over the left tmj .
once the horizontal and vertical standard points were set , we controlled them to fit with the subjects'heads .
the bar of the front side was kept parallel to the interauditory axis and the lateral side to the frankfort horizontal plane . the accessory bar for approaching the magnet
was fixed temporally and operated in order to set the exact midline . as the subject performed metronome - guided maximun active opening / closing with the jva , the condyles rubbed against the various surfaces in the joint , creating characteristic vibrations which are then , in turn , detected by the accelerometers , which convert those specific vibrations into an electronic signal .
the signal from the accelerometers is amplified by a small , light - weight amplifier which is placed around the patient 's neck .
the amplified signals are then transmitted to a pc computer where they are recorded and analyzed with a software program , then displayed on a crt .
after the best recording was selected from three , vibrations showing the highest amplitude were screened priorly .
when we excluded tooth contact precisely , reproducible joint sound was analyzed for each opening & closing cycle .
after subject selection , the largest vibration amplitude consistently occurring in each joint recording was used to calculate frequency spectrum computed by the fast fourier transform ( fft ) algorithm .
the numeric values that are calculated and displayed in the jva summary view are based on the absolute frequency spectra . the frequency spectra view plots amplitude ( vertical axis ) versus frequency ( horizontal axis ) .
the height of the curve is directly proportional to the energy of the spectrum at each frequency .
two spectra are plotted for each side : the smaller of the two represents the absolute magnitude of the vibrations'spectra as recorded ( n / m ) , the larger one has been scaled to the maximum range ( at the recorded amplification ) and is known as the relative plot .
the relative plot accentuates features that may not be visible in the absolute plot ( fig .
the integral > 300 / < 300 ratios in group i and group ii listed in table i and ii show variations before and after the six - months recordings respectively . in group ii ,
17 - 20 showed integral > 300 / < 300 ratios disappeared six - months later .
the integral > 300 / < 300 ratios and the frequency spectra were analyzed in all subjects .
the frequency spectra in some subjects showed similar patterns while the others showed varied patterns . by the comparative study between the integral > 300 / < 300 ratios and the frequency spectrums , it was conceivable that the frequency spectrums showed similar patterns at the same location that the joint sound occurred between before and after the six - months recordings . while the frequency spectra showed varied patterns at the different location that the joint sound occurred in before and after the six - months recordings ( fig . 2 , 3 ) .
disc displacement is characterized by a normal range of jaw movement during opening and eccentric movements .
when reciprocal clicking is present , the two clicks normally occur at different degrees of mouth opening , with the closing click usually occuring near the intercuspal position.1 although physiological changes occur in the disc , its ability to remodel is lower than that of other tissues of the tmj , such as the capsule , capsular ligaments , and retrodiscal tissues .
decreased vascularity and extensive fibrous transformation have been reported in the retrodiscal tissue for continuous compression and shear .
these adaptative changes can also have mechanical implications on the behavior of the articular disc .
however , as long as the system preserves the ability to adapt to the new functional status , the altered mechanical loading is compensated for by the structural modeling of the tmj . although the coordination of the disc - condyle complex may be lost in this stage , the patient is usually asymptomatic.2 garcia et al.19 reported that some patients present alterations in the structure of the arcitular disk located in several areas .
small vibrations in the position of the condyle may induce unstable areas with production of articular vibrations .
the frequencies ( in hertz ) , as well as the amplitude of the vibration can be expressed mathematically .
the precise moment of the sound generated in the opening and closing cycles . in this study group
i and group ii showed varied integral > 300 / < 300 ratios before and the after six - months recordings . also , by the comparative study between the integral > 300 / < 300 ratios and the frequency spectrums , it was conceivable that the frequency spectrums showed similar patterns at the same location that the joint sound occurred before and after the six - months recordings .
while the frequency spectrums showed varied patterns at the different locations that the joint sound occurred before and after the six - months recordings , it would possibly be due to the differences in the degree of internal derangement and/or in the shape of the disc .
it is suggested that clinicians consider the integral > 300 / < 300 ratios as well as the frequency spectrums to decide the starting - point of the treatment for tmj sounds .
therefore jva will provide the clinician with the visible patterns of tmj sounds for patient management .
|
statement of problemqualitative and semi - quantitative methods have been developed for tmj sound classification , but the criteria presented are completely inhomogeneous . thus , to develop more objective criteria for defining tmj sounds , electroacoustical systems have been developed .
we used joint vibration analysis in the biopak system ( bioresearch inc . ,
milwaukee , usa ) as the electrovibratography.purposethe aim of this study was to examine the tmj sounds with repect to frequency spectra patterns and the integral > 300 hz / < 300 hz ratios via six - months follow-up.material and methodsthis study was done before and after the six - months recordings with 20 dental school students showed anterior disk displacement with reduction .
joint vibrations were analyzed using a mathematical technique known as the fast fourier transform.resultsin this study group i and group ii showed varied integral > 300 / < 300 ratios before and after the six - months recordings . also , by the comparative study between the integral > 300 / < 300 ratios and the frequency spectrums , it was conceivable that the frequency spectrums showed similar patterns at the same location that the joint sound occurred before and after the six - months recordings .
while the frequency spectrums showed varied patterns at the different locations that the joint sound occurred before and after six - month recordings , it would possibly be due to the differences in the degree of internal derangement and/or in the shape of the disc.conclusionsit is suggested that clinicians consider the integral > 300 / < 300 ratios as well as the frequency spectrums to decide the starting - point of the treatment for tmj sounds .
|
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] |
a malignant peripheral nerve sheath tumor ( mpnst ) is a sarcoma that arises from peripheral nerves or cells of the associated nerve sheath ( schwann cells , perineural cells , fibroblasts ) .
the term " mpnst " has substituted previously used terms such as malignant schwannoma , neurofibrosarcoma , and neurogenic sarcoma1,2 ) .
the mpnsts comprise approximately 5% to 10% of all soft tissue tumors and have an incidence of 0.001% in the general population and the etiology is unknown .
however , more than half of mpnst cases develop in patients with neurofibromatosis type-1 ( nf-1 ) and there is a higher incidence in patients that have undergone prior radiotherapy1,2,5,8,13 ) .
mpnsts are most likely to metastasize to the lungs , followed by the bone and finally the pleura , but , spinal cord and brain metastasis is very rare in non - nf-1 mpnst3,7 ) .
the authors report a very rare case of spinal cord and brain metastasis of mpnst in the lumbar spine that occurred in a 18-year - old man without nf .
an 18-year - old man presented initially with a 6-month history of low back pain that worsened over time .
one month prior to admission , the patient developed radiating pain to his anterior thigh .
neither caf au lait spots nor neurofibromas were seen and his family history was absent of nf-1 .
the neurologic examination showed normal motor power in his lower extremities . magnetic resonance imaging ( mri ) showed a paraspinal mass that extended from the central space of l2 to the right psoas muscle through right l2 - 3 foraminal space .
it was an asymmetrical dumbbell shaped mass and the thecal sac was displaced left by the mass .
the mass showed iso - signal intensity on t1-weighted images and mixed signal intensity on t2-weighted images .
a paramedian incision was made from the l1 to l4 and we exposed the right l2 and l3 lamina with transverse process .
right l2 hemilaminectomy was performed using a high speed drill and kerrison rongeur to expose the thecal sac .
the margin of mass was relatively distinct from neural tissue , thus gross total resection was possible under microscopic view . to remove the mass extended to the psoas muscle ,
the right transverse process of l2 and l3 was resected and the mass was visible .
after the operation , his symptom had improved . however , a few days later , his confirmed pathologic report was presented as mpnst ( fig .
the contrast - enhanced mri was followed at 1 month after the operation and revealed a total removal of tumor ( fig .
however , the patient declined the external beam radiation due to the fear of potential radiation risk .
spine and brain mri showed that the tumor recurrence with leptomeningeal , multiple spinal cord and brain metastases ( fig .
4 ) . complete surgical clearance of the tumor was not technically feasible , owing to the multiple metastases .
therefore , the patient was referred for adjuvant chemotherapy . in addition , intrathecal chemotherapy was performed .
after 4 months of chemotherapy , he was referred to the hospice hospital and died within a month .
an 18-year - old man presented initially with a 6-month history of low back pain that worsened over time .
one month prior to admission , the patient developed radiating pain to his anterior thigh .
neither caf au lait spots nor neurofibromas were seen and his family history was absent of nf-1 .
the neurologic examination showed normal motor power in his lower extremities . magnetic resonance imaging ( mri ) showed a paraspinal mass that extended from the central space of l2 to the right psoas muscle through right l2 - 3 foraminal space .
it was an asymmetrical dumbbell shaped mass and the thecal sac was displaced left by the mass .
the mass showed iso - signal intensity on t1-weighted images and mixed signal intensity on t2-weighted images .
a paramedian incision was made from the l1 to l4 and we exposed the right l2 and l3 lamina with transverse process .
right l2 hemilaminectomy was performed using a high speed drill and kerrison rongeur to expose the thecal sac . for removal of intradural portion
the margin of mass was relatively distinct from neural tissue , thus gross total resection was possible under microscopic view . to remove the mass extended to the psoas muscle ,
the right transverse process of l2 and l3 was resected and the mass was visible .
after the operation , his symptom had improved . however , a few days later , his confirmed pathologic report was presented as mpnst ( fig .
the contrast - enhanced mri was followed at 1 month after the operation and revealed a total removal of tumor ( fig .
however , the patient declined the external beam radiation due to the fear of potential radiation risk .
spine and brain mri showed that the tumor recurrence with leptomeningeal , multiple spinal cord and brain metastases ( fig .
4 ) . complete surgical clearance of the tumor was not technically feasible , owing to the multiple metastases .
in addition , intrathecal chemotherapy was performed . however , no significant effect was observed and the patient 's condition deteriorated gradually .
after 4 months of chemotherapy , he was referred to the hospice hospital and died within a month .
non - nf-1 mpnsts are very rare and the occurrence of mpnsts occurring in adolescents is very rare
. mpnsts generally occur in adulthood typically between the ages of 20 and 50 years of age .
approximately 10 - 20% of cases have been reported to occur in the first 2 decades of life , with occasional cases involving infants as young as 11 months of age5,6 ) .
hruban et al . described 43 patients with mpnst and of these patients , 28 ( 65% ) had distant metastasis3 ) .
sites of metastasis included the lungs ( 22 cases ) , bone ( 9 cases ) , pleura ( 6 cases ) , retroperitoneum ( 4 cases ) , diaphragm ( 3 cases ) , inguinal lymph nodes ( 2 cases ) , liver ( 2 cases ) , chest wall ( 2 cases ) , soft tissue ( 2 cases ) , pulmonary hilum , pericardium , thyroid gland , and adrenal gland ( 1 case each ) .
described 11 distant metastasises in 54 patients with mpnst that included metastases to lymph nodes , pleura , lung , liver , adrenal gland , leptomeninges and brain7 ) .
william et al.4 ) reported a non - nf-1 mpnst case of metastasis to the spinal cord4 ) .
our case showed that leptomeningeal , spinal cord and brain metastasis of non - nf-1 mpnst for relatively short period after tumor removal in young male patient , reflecting aggressive nature of this malignancy .
the outcome with respect to both local recurrence and distant metastasis largely depends on grade of surgical excision .
radiation therapy combined with wide surgical excision offers statistical significant reduction in the rates of local disease recurrence .
however , it has not had a meaningful reduction in either rate of distant metastasis or overall survival15 ) .
benefit of chemotherapy is unproven and its application is limited to high grade metastatic disease3 ) .
the italian and german soft tissue sarcoma cooperative group reported an overall pediatric response rate of 45% , with the highest response noted within the ifosfamide group9,11,12 ) .
the recurrence rate for mpnsts has been reported to range from 40 - 68%7,10,14 ) .
the 5-year survival rate has been reported at 16 - 52% and a favorable prognosis has been related with complete surgical excision of the tumors that are sized less than 5 cm7,10 ) .
historically , mpnsts have been difficult tumors to treat due to their inherently aggressive nature and dismal prognosis .
our patient underwent complete surgical excision , but the patient was not treated with radiotherapy .
follow - up images revealed multiple spinal cord and brain metastases . the patient was managed with chemotherapy , but expired several months later . despite complete surgical excision ,
we reported a case of mpnst that had metastasized to the brain and spinal cord .
because the mpnst showed both rapid and aggressive progression , patients should be followed carefully to identify local recurrence or metastasis and be mandatory an adjuvant radiotherapy .
|
a malignant peripheral nerve sheath tumor ( mpnst ) is a type of sarcoma that arises from peripheral nerves or cells of the associated nerve sheath .
this tumor most commonly metastasizes to the lung and metastases to the spinal cord and brain are very rare .
we describe a case of young patient with spinal cord and brain metastases resulting from mpnst .
an 18-year - old man presented with a 6-month history of low back pain and radiating pain to his anterior thigh . magnetic resonance imaging showed a paraspinal mass that extended from the central space of l2 to right psoas muscle through the right l2 - 3 foraminal space .
the patient underwent surgery and the result of the histopathologic study was diagnostic for mpnst .
six months after surgery , follow - up images revealed multiple spinal cord and brain metastases .
the patient was managed with chemotherapy , but died several months later . despite complete surgical excision ,
the mpnst progressed rapidly and aggressively .
thus , patients with mpnst should be followed carefully to identify local recurrence or metastasis as early as possible .
|
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giving birth is one of the most amazing physiologic events in human life and it can be safe and pleasant in many circumstances , but in some situations it might end with known difficulties and complications for mother and her fetus .
it means regular uterine contractions with adequate frequency and intensity resulting in giving birth before 37 completed weeks of pregnancy .
preterm birth is the major reason of the neonatal death and it is responsible for about one third of neonatal mortality in the world [ 1 , 2 ] . who publication mentions that each year more than 1 million babies die from complication of preterm delivery and the annual numbers of premature newborns are about 15 millions and this number is rising each year . based on who publications , un millennium development goal 4 is reduction of children mortality in two - thirds till 2015 by special attention to neonatal deaths in countries where the neonatal mortality is higher than other places . a great number of the global neonatal deaths happen in asia . finding the ways to prevention of preterm birth is a very important issue for health care providers .
there are a lot of modalities to achieve this goal but none of them has been introduced as the best way and the role of different methods in preventing preterm labor varies in different places .
magnesium sulphate ( mgso4 ) as the major tocolytic agent has been used by many medical centers as the first line therapy to inhibition of preterm labor .
however , women receiving mgso4 should be closely monitored during treatment ; also the severe pain and discomfort in injection site in case of i m administration is a problem with this item . on the other hand
, some studies have mentioned that mgso4 can not be so effective in suppression of preterm labor .
prostaglandin producing by the decidua and fetal membranes might be one of the essential events of parturition that is followed by initiation of the uterine contraction .
it seems likely that prevention of producing prostaglandins or suppression of their effects can stop preterm uterine contraction .
so in theory inhibitors of prostaglandin synthesis ( including cyclooxygenase inhibitors such as celexib ) are capable of arresting uterine contractility and they can prevent preterm birth .
celecoxib is a sulfa nonsteroidal anti - inflammatory drug ( nsaid ) and selective cox-2 inhibitor .
it is known under the brand name celebrex or celebra and onsenal used in the treatment of inflammatory diseases and painful menstruation , menstrual symptoms , and so forth .
labor was arrested for 48 h in 42 ( 81% ) and 45 ( 87% ) of the patients in the celecoxib and magnesium sulfate groups , respectively ( p-0.298 ) .
we aimed to use celecoxib as a cyclgenase inhibitor to suppression of preterm labor instead of magnesium sulfate ( mgso4 ) to prevent preterm labor .
it was a single blinded randomized clinical trial study which had been done on 600 pregnant women with gestational age > 24 weeks and < 34 weeks ; those had referred to ahvaz educational hospitals from march to august 2013 .
these criteria were as follows : at least 4th uterine contractions during 20 minutes or 8th uterine contractions during 1 hour followed by progressive changes in the cervical dilatation and effacement , the cervical dilatation of more than 2 cm , the cervical dilatation of more than 80% at the beginning .
at least 4th uterine contractions during 20 minutes or 8th uterine contractions during 1 hour followed by progressive changes in the cervical dilatation and effacement , the cervical dilatation of more than 2 cm , the cervical dilatation of more than 80% at the beginning .
informed written consent was obtained from all subjects and they received preterm labor routine and maintenance therapy including bed rest and hydration ( bolos infusion of 500 cc ringer lactate during half and over , corticosteroid therapy ) and if the uterine preterm contractions did not stop with these modalities , all patients were divided in two groups by simple random sampling ; the first group has taken 4 gr of mgso4 intravenously as loading dose and 1 gr per hour as maintenance dose till suppuration of the uterine contractions or maximum 48 hours and in this period the patients were observed for signs of mgso4 toxicity by control of the urine output , deep tendon reflex , and respiratory rate .
conditions of the uterine contractions were checked every 2 hours in 20-minute periods . also the patients were observed for symptoms of headache ; the patients ' vital signs were checked as routine protocol .
the second group had taken 100 mg celecoxib ( one capsule ) orally every 12 hours till suppuration of the uterine contractions or for maximum duration of 48 hours .
the patients in this group were checked for nausea and vomiting and skin rush and headache . in all subjects
if the uterine preterm contractions did not stop with those medications , so immediatry drug has been stopped .
gestational age 24 weeks and 34 weeks with diagnosis of preterm labor based on upper criteria .
gestational age 24 weeks and 34 weeks with diagnosis of preterm labor based on upper criteria .
existing problems that interfere with normal labor process and might need early termination of pregnancy : premature preterm rapture of membrane ( pprom ) , evidences of placental abruption in clinical and ultrasound examination , and ultrasound , maternal fever chorioamnionitis , none reassuring fetal heart rate ( fhr ) patterns.contraindication for stopping preterm labor uterine contraction : some maternal medical conditions such as preeclampsia , any signs of vaginal bleeding , fetus anomaly , intrauterine fetal death ( iufd ) , and fetal growth restriction ( fgr).issues related to mediations : history of being allergic to nonsteroidal anti - inflammatory drugs ( nsaids ) , renal or hepatic dysfunction , concurrent use of other medications except supplements , and history of peptic ulcer.the patients ' disagreement to enter the study.stopping the uterine preterm contractions after initial preterm labor maintenance therapy .
existing problems that interfere with normal labor process and might need early termination of pregnancy : premature preterm rapture of membrane ( pprom ) , evidences of placental abruption in clinical and ultrasound examination , and ultrasound , maternal fever chorioamnionitis , none reassuring fetal heart rate ( fhr ) patterns .
contraindication for stopping preterm labor uterine contraction : some maternal medical conditions such as preeclampsia , any signs of vaginal bleeding , fetus anomaly , intrauterine fetal death ( iufd ) , and fetal growth restriction ( fgr ) .
issues related to mediations : history of being allergic to nonsteroidal anti - inflammatory drugs ( nsaids ) , renal or hepatic dysfunction , concurrent use of other medications except supplements , and history of peptic ulcer .
the patients ' disagreement to enter the study . stopping the uterine preterm contractions after initial preterm labor maintenance therapy .
the study protocol was approved by ethics committee of ahvaz jundishapur university of medical sciences .
all patients provided written informed consent the data were entered and analyzed using spss 11 ( spss inc . ,
chi - square and independent t - test were used for statistical analysis . for all analyses ,
the finding of this study has shown that mean age of all subjects was 26.03 4.33 in mgso4 group and in celecoxib group was 26.6 4.01 , 25.8 4.63 , respectively . with using independent t - test
, there were no significant differences between mean age subjects in two groups ( p = 0.194 ) .
frequencies of gestational age at time of delivery in all subjects were 31.74 1.86 weeks and in mgso4 group and celecoxib group were 32.1 1.82 and 31.38 1.83 weeks , respectively .
the lowest gestational age was 27 weeks and the greatest gestational age was 34 weeks .
frequencies of nulliparity in all pregnant women were 350 ( 58.3% ) , in mgso4 group were 174 ( 58% ) , and in celecoxib group were 176 ( 58.7% ) .
frequencies of history of preterm delivery in celexib group were 12 ( 4% ) and in mgso4 group were 12 ( 4% ) .
there were no significant differences between two groups ( mgso4 group and celecoxib group ) in frequency of history of preterm labor ( p = 1 ) , frequencies of nulliparity ( p = 0.99 ) , duration of drug use and arrest contraction ( p = 0.29 ) , delivery before 48 hours ( p = 0.20 ) , and mean gestational age in lack of response to treatment ( p = 0.24 ) ( tables 1 and 2 ) .
there was significant association between two groups ( mgso4 group and celecoxib group ) in frequency of effacement ( p < 0.0001 ) , gestational age ( p
< 0.0001 ) , and dilation ( p = 0.06 ) ( table 3 ) .
about two - thirds of neonatal mortality are due to preterm birth , so preterm labor suppression is a big deal for health care providers . on the other hand decreasing childhood mortality
. a similar study has been done by borna and saeidi in 2007 and preterm labor was suppressed in 87% of mgso4 group and 81% of celecoxib group so their result was similar to our result .
they concluded that celecoxib is as effective as mgso4 in preterm labor suppression [ 8 , 10 ] . in another study by mcwhorter et al . in 2004
they concluded that there was no difference between oral rofecoxib and intravenous magnesium sulfate in arresting preterm labor . another study compared safety of indomethacin and celecoxib to arrest preterm labor .
they reported that safety of short - term celecoxib in women with preterm labor was superior to indomethacin . in this study 600 pregnant women with preterm labor criteria
mgso4 was used in one group for suppression of preterm labor and the other group took celecoxib instead of mgso4 .
80% of mgso4 group and 75% of celecoxib group showed positive response to these tocolytic agents and uterine contraction stopped at least for 48 hours and cervical dilatation and effacement did not progress in this period of time , but in this aspect ; statistical calculation did not show any significant differences between two groups . in our study
there were not any significant differences between cervical dilatation in two groups , but from the beginning , cervical effacement showed a significant difference between two groups ; we found that although celecoxib group already had more cervical effacement , celecoxib effectiveness was as the same as mgso4 group . in this study ,
cost effectiveness of celecoxib is more reasonable than mgso4 and it can play a role in expenses reduction in health centers .
no need for closes monitoring of the patients , and there are not invasive procedures such as injections and same effect of mgso4 .
|
we aimed to use celecoxib to suppress preterm labor instead magnesium sulfate ( mgso4 ) to prevent preterm labor . methods .
it was a randomized clinical trial study , which was done on 600 pregnant women .
all subjects were divided into two groups by simple random sampling .
one group was given 4 grams of mgso4 intravenously and second group was given 100 mg of celecoxib orally every 12 hours for at least 2 days .
the data were entered and analyzed using spss 11 and performed using t - test and chi - square test .
results .
the finding of this study has shown that preterm labor may be prevented in 75.7% of subjects who had received celecoxib and there were no significant difference between two groups in frequency of history of preterm labor ( p = 1 ) , frequencies of nulliparity ( p = 0.99 ) , duration of drug use and arrest contraction ( p = 0.29 ) , delivery before 48 hours ( p = 0.20 ) , and mean gestational age in lack of response to treatment ( p = 0.24 ) . conclusions .
result has shown that celecoxib was similar to mgso4 as a medication to prevent preterm labor ; it was recommended to be prescribe to prevent preterm labor , because it was cheaper than magnesium sulfate .
|
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depressive disorders affect 340 million people ( approximately 9% ) globally regardless of gender , culture , or ethnicity . from 1990 till present time
, depression has been rated one of the leading causes of years lived with disability ( ylds ) .
for instance , depression predicts future coronary events and cardiac deaths in healthy individuals and in those with established cardiovascular disease ( chd ) [ 3 , 4 ] .
depression has also been shown to be associated with several other physical diseases , including cancer and osteoporosis , as well as with severe psychiatric disorders such as schizophrenia .
furthermore , depression relates to low educational attainment and reduced work productivity . identifying factors that contribute to depression and developing tools that can be applied in early prevention of depression
even though some conflicting findings concerning the association between physical activity and depression exist [ 8 , 9 ] , prior evidence provides support for the value of physical activity in reducing depressive symptoms in both healthy and clinical populations .
literature has also indicated that even low doses of physical activity may protect against depression .
the relation between physical activity and depression may also be bidirectional , as physical activity may alleviate depressive symptoms , but these symptoms may also decrease the likelihood of initiating physical activity [ 8 , 11 ] .
physical activity has also been related to neurobiological functioning , which may preempt depressive mood .
in addition , physical activity has been shown to be associated with the development of individual qualities such as self - confidence , emotional self - regulation , and capacity of social bonding , which , in turn , may be protective against depression [ 13 , 14 ] .
several studies have indicated that regular physical activity associates with reduced risk of depression in all age groups from early childhood to late adulthood [ 8 , 10 , 15 ] .
there is also evidence that childhood physical activity may lead to maintenance of lifetime physical activity patterns conducive to well - being ( e.g. , ) .
many previous findings regarding the association between physical activity and depression derive from cross - sectional designs .
previous longitudinal or intervention designs cover relatively short follow - up phases of physical activity [ 10 , 18 ] .
prospective studies examining the association between lifelong physical activity and depressive symptoms are very rare .
it has been stated that more sophisticated methods are needed when studying the development and etiology of depression .
it has also been shown that people 's health behaviors may have various developmental tendencies instead of a one growth trajectory [ 20 , 21 ] , and some of these behavioral profiles may be more detrimental to health than others .
for instance , it is theoretically possible that physical activity 's decline towards later life may start earlier and/or be steeper among specific groups than in others , and thus the different physical activity profiles may associate with distinct health outcomes .
the need for examinations assessing the development and decline of physical activity has been acknowledged [ 17 , 22 ] .
it is also essential to study whether long - term physical activity contributes to health over and above the concurrent physical activity .
furthermore , identifying people , whose behavioral tendencies associate with decreased health , might be useful for professionals who aim to adjust lifestyle interventions to targeted groups .
it is also important to gain information of the behavioral determinants and contributors of good health .
growth mixture modeling provides an appropriate framework for studying developmental processes [ 20 , 24 ] .
the key advantage of such modeling is that it allows for the estimation of interindividual variability in intraindividual patterns of change over time [ 20 , 24 ] .
current approaches to growth modeling have also been regarded as flexible for instance in terms of including partially missing data , unequally spaced measurement points , nonnormally distributed or discretely scaled repeated measures , and linearly or nonlinearly shaped trajectories . these issues , or some of these , typically emerge in developmental research .
extensive adjustment for potential confounders has been lacking in many previous studies regarding physical activity and health outcomes [ 16 , 18 ] .
development of depression often depends on the interplay of multiple psychological and lifestyle related factors that ideally should be considered when examining associations between health behaviors and depression [ 2530 ] .
child 's early emotional experiences ( e.g. , negative emotionality ) may have effects on the development of depression later in life .
childhood family 's socioeconomic status has been shown to be associated with the development of health behaviors and well - being . in adulthood ,
it has been proven that depression is related to age , and women tend to report higher levels of depression than men .
adulthood socioeconomic position , experiences of social support , body mass index , and smoking status are also associated with depression in adulthood [ 26 , 29 , 30 ] .
we examined the potential heterogeneity in physical activity trajectories in relation to depressive symptoms over a 30-year period from childhood to adulthood in a population - based sample with six cohorts and including eight study waves .
to our knowledge , this is the first study assessing physical activity and depressive symptoms in such design .
physical activity measurements were performed during participants ' childhood and adulthood ( from the age of 9 to 49 ) , and depressive symptoms were assessed in participants ' adulthood ( participants aged from 35 to 50 ) . to gain a comprehensive picture of the association between physical activity and depressive symptoms ,
the association was studied cross - sectionally , with respect to change and with respect to long - term trajectories .
the objectives of this study were ( 1 ) to explore the potentially distinct trajectories of physical activity from childhood to adulthood , ( 2 ) to examine whether physical activity was associated with cross - sectional , short - term , and long - term changes in depressive symptoms in adulthood , and ( 3 ) to examine whether the physical activity trajectories contributed to depressive symptoms in adulthood to a greater degree than adulthood physical activity .
the study participants were from the ongoing prospective cardiovascular risk in young finns study that began in 1980 .
the original sample consisted of 3596 children and adolescents ( 83.2% of those invited , 1832 females and 1764 males ) from six birth cohorts ( aged 3 , 6 , 9 , 12 , 15 , and 18 ) . to obtain a representative sample ,
finland was divided into five areas based on the locations of universities with medical schools ( helsinki , kuopio , oulu , tampere , and turku ) , and the participants were randomly selected based on their social security numbers from nearby urban and rural areas .
informed consent was requested from each participant ( or from the parents of small children ) , and the study was approved by the local ethics committees .
the study was conducted according to declaration of helsinki and american psychological association 's ethical principles .
the sample was followed in 8 waves , 1983 , 1986 , 1989 , 1992 , 1997 , 2001 , 2007 - 2008 , and 2012 , in which medical , psychological , and physical activity studies were conducted .
physical activity from childhood to middle adulthood was assessed in 1980 , 1983 , 1986 , 1989 , 1992 , 2001 , 2007 , and 2011 , response rate ranging from 53.1% to 72.8% ( n = 19102619 ) of the original study participants ( table 1 ) .
depressive symptoms were measured in 2012 and 47.9% ( n = 1724 ) of the original study subjects participated in the examination ( table 1 ) .
previous studies of sample attrition have shown that there has not been systematic selection bias regarding study participants ' medical profiles or physical activity [ 31 , 32 ] , but some selective attrition with respect to personality and depressive symptoms exists [ 33 , 34 ] .
participants who were less self - directed and less agreeable and had higher levels of neuroticism as well as depressive symptoms had discontinued the study more often than others [ 33 , 34 ] .
information concerning 3- and 6-year - old children 's ( born in 1974 and 1977 ) physical activity levels is missing from this study , because they were not able to self - report their physical activity levels in 1980 .
participants , from whom physical activity was assessed , were aged from 9 to 18 in 1980 and from 34 to 49 in 2011 . from 1980 to 1989 , physical activity questionnaires consisted of five questions focusing on the intensity and frequency of participants ' leisure time physical activity , participation in sports - club training , participation in sports competitions , and participants ' usual way of spending leisure time . from 1992 till present time , questionnaires consisted of five questions as well , assessing the intensity and frequency of leisure time physical activity , hours spent on physical activity per week , average duration of a physical activity session , and participation in organized physical activity . from 1980 to 1992 , the answers for the questions were coded into 3 categories ( ranging from 1 to 3 ) , excluding the items that assessed participation in sports competitions ( 19801989 ) and participation in organized sports ( 1992 ) which had a response range from 1 to 2 . from 2001 to 2011 , all responses to the questions were coded into 3 categories ( response scale ranging from 1 to 3 ) . a sum score ( physical activity index ) of question responses
was created for each participant each year ( table 1 ) , higher scores reflecting higher physical activity level . the index has been found reliable and valid .
participants ' depressive symptoms were assessed with beck depression inventory ii ( bdi - ii ) .
these symptoms were measured in 2012 when the participants were aged from 35 to 50 .
bdi - ii consists of 21 symptoms with a severity range from 0 ( no symptoms ) to 3 ( severe level of depressive symptoms ) .
a sum score of all items was computed for each participant ( table 1 ) , and no missing items were allowed .
the reliability estimate ( cronbach 's ) for the depressive symptom scores was > 0.90 .
bdi - ii has demonstrated to be a valid instrument [ 3638 ] , and it has been regarded as an acknowledged standard in the measurement of depressive mood [ 3639 ] .
it is applicable in clinical and nonclinical contexts , including in general populations [ 3639 ] .
bdi - ii correlates highly with its earlier versions , including modified bdi [ 36 , 38 ] , which has also been considered as a valid measure for assessing depressive symptoms in general populations .
the instrument has been designed and also demonstrated to be a useful screening tool for potential depressed cases [ 36 , 38 ] .
childhood , adulthood , and general ( age , sex , and body mass index ) covariates were controlled for in this study ( table 1 ) [ 2530 ] .
participants ' negative emotionality was reported by the primary caretaker via six questions reflecting participants ' behavior in childhood ( e.g. , the child hits / kicks other children accidentally ) , on a scale from 1 ( true ) to 2 ( not true ) , and average of the items was calculated for each participant . as some of the participants were adolescents in 1980 , their caretakers responded to this question retrospectively .
participants ' parents ' socioeconomic status was assessed via two indices , educational and income levels .
parents ' educational level was determined via educational information collected from participants ' mothers ' and fathers ' ( 1 = below 9 years / comprehensive school , 2 = 9 to 12 years / secondary school , and 3 = over 12 years / academic education ) .
if parents ' educational information differed , we based parental educational status on the information collected from the parent with the higher educational level . if educational information was available for only one parent , family 's educational status was determined using his / her educational information .
family 's income level was rated in an 8-point scale [ 1 = < 15 000 marks ( ~2523 euros ) and 8 = > 100 000 marks ( ~16819 euros ) ] .
symptoms of participants ' adulthood depression were determined in 1992 , 1997 , 2001 , and 2007 via a modified version of beck depression inventory , referred to as a modified bdi [ 34 , 40 ] .
items of the measure were rated in a 5-point scale , and average of the items was computed each year for each participant .
participants ' socioeconomic status ( 2007 ) was determined via two indices ; education was assessed via a 3-category scale ( 1 = comprehensive school , 2 = secondary school , and 3 = academic level ) and income level via an 8-point scale ( 1 = < 10 000 euros and 8 = > 70 000 euros ) .
additionally , participants ' experiences of social support , body mass index , and smoking status measured in 2007 were controlled for [ 29 , 30 ] .
social support was determined via a 12-question inventory using a 5-point scale , and a mean score of the items was calculated for each participant .
participants ' smoking status was examined via a 5-category scale ( 1 = smokes a cigarette per day or more , 2 = smokes once in a week , 3 = smokes less than once in a week , 4 = has quitted smoking , and 5 = has never smoked ) .
physical activity questionnaires , which were designed for children and adolescents ( 19801989 ) and adults ( 19922011 ) differed slightly in their content . to assure that the findings of the present study were based on changes in physical activity and not due to a measurement artifact , a confirmatory factor model was used to examine whether the physical activity indices consisting of five indicator variables had measurement and structural invariance over time [ 42 , 43 ] .
weighted least squares means and variance adjusted ( wlsmv ) estimation was used for all analyses .
the goodness of fit for scalar invariance was assessed with comparative fit index ( cfi ) , tucker - lewis index ( tli ) , and root - mean square error of approximation index ( rmsea ) .
factor scores derived from this examination were used in subsequent analyses . within the growth mixture modeling framework , latent class growth analysis ( lcga )
lcga captures information about developmental processes at inter- and intraindividual levels , detecting subpopulations with distinct growth trajectories .
the determination of the number of subgroups for physical activity was based on akaike 's information criterion ( aic ) .
in addition , the determination of the groups was based on the classification quality estimations and practical considerations [ 20 , 45 ] . within the lcga model , the average temporal trajectories in the physical activity groups
were modeled by regression equations , in which both the linear and quadratic terms were tested for the independent variable ( time ) .
the associations between physical activity factor scores ( assessed from age 9 to 49 ) and depressive symptoms ( participants aged from 35 to 50 ) were first examined cross - sectionally and longitudinally with linear regression analyses . due to the potential multiple testing problem , bonferroni - corrected p values ( p < 0.003 )
thereafter , the associations between the physical activity trajectory groups and depressive symptoms measured in 2012 were examined with analyses of variance , and post hoc tests were also performed ( bonferroni 's method ) .
furthermore , we examined the longitudinal associations between physical activity levels assessed in participants ' adulthood ( 2007 , including participants aged from 30 to 45 ) and depressive symptoms ( 2012 ) using a linear regression . due to the number of missing values , the variance analyses and the regression analyses in which the adulthood physical activity ( 2007 ) was used as a predictor were performed in another dataset which was imputed using the expectation - maximization ( em ) algorithm .
analyses were performed in statistical software programs mplus ( version 7.1 and version 7.2 ) , ibm spss ( version 21 ) , and stata ( version 13 ) .
descriptives of the original sample ( n = 17243596 ) are shown in table 1 .
supplementary tables 1 and 2 ( see supplementary material available online at http://dx.doi.org/10.1155/2016/8947375 ) provide descriptives of the sample in the complete ( n = 648 ) and imputed ( n = 35643596 ) data , respectively . although the scalar invariance model for physical activity did not demonstrate strong factorial invariance over time , the fit for partial scalar invariance model was adequate ( cfi = 0.90 , tli = 0.90 , and rmsea = 0.047 ) , given partial invariance of the threshold parameters . for rmsea , values < 0.05 indicate a very close model fit , and cfi and tli values close to 0.90 denote an adequate fit . since the partial scalar invariance model was considered acceptable , factor scores
were predicted for each subject to be used in subsequent analyses ( supplementary table 3 ) .
lcga suggested that a three - factor ( group ) solution was the best fitting model for the data based on aic indices ( aic = 48915.44 , 48915.39 , 48901.43 , 48902.85 , and 48907.08 for 1 , 2 , 3 , 4 , and 5 groups , resp . ;
the classification quality of the three - factor model was also adequate based on the average probability estimates ( group 1 = 0.74 , group 2 = 0.86 , and group 3 = 0.71 ) . values > 0.70 delineate that the group consists of individuals with similar patterns of change .
linear parameter estimates were found for the physical activity groups ( supplementary table 4 ) .
figure 1 shows the central tendencies of the three groups , lightly ( n = 371 ) , moderately ( n = 3046 ) , and highly ( n = 147 ) physically active groups by age . based on the estimated marginal means , participants ' physical activity levels remained relatively unchanged from childhood to adulthood in each group , although these levels appeared to diminish minimally towards middle adulthood in all participants ( figure 1 ) .
the regression analyses performed in the original sample ( n = 2551467 ) indicated that low levels of physical activity factor scores were , in most examined ages , associated with higher levels of depressive symptoms in participants ' adulthood ( p < 0.05 ) , although some of the associations attenuated when bonferroni - corrected p values ( p <
the analyses of variance in the original sample ( n = 1722 ) indicated that the physical activity trajectory groups predicted symptoms of depression [ f ( 2,1719 = 8.12 , p < 0.001 , adjusted r = 0.01 ) ] .
post hoc tests showed that highly physically active group had lower levels of depressive symptoms than lightly active group ( mean difference = 3.26 ; p < 0.001 , 95%ci : 5.25 to 1.26 ) .
highly physically active participants had lower levels of depression in comparison to moderately active ones ( mean difference = 1.92 , p = 0.02 , 95%ci : 3.66 to 0.18 ) . also moderately active participants had lower levels of depressive symptoms than lightly active participants ( mean difference = 1.33 , p = 0.02 , 95%ci : 2.48 to 0.19 ) .
based on the analyses of variance after adjusting for the covariates [ 2530 ] , the associations attenuated to nonsignificance [ f ( 2 , 631 ) = 2.13 , p = 0.12 , adjusted r = 0.25 ] . in particular
the previous symptoms of depression measured in 1992 , 1997 , 2001 , and 2007 attenuated the associations ( for details , see supplementary table 5 ) .
we also examined the unadjusted associations in the data with full information on all study variables ( n = 648 ) , in which case the association between the physical activity trajectory groups and depressive symptoms became only marginally significant [ f ( 2,645 ) = 2.76 , p = 0.06 , adjusted r = 0.01 ] .
as the sample attrition might have affected this association ( i.e. , by reducing the statistical power ) , the analyses were also performed in a dataset which was imputed using em - algorithm ( n = 35643596 ) .
this method was applied as the little 's mcar test confirmed that the data were not missing completely at random ( = 3903.02 , df = 2923 , p < 0.001 ) . in the imputed dataset , physical activity groups predicted the symptoms of depression [ f ( 2,3561 ) = 16.74 , p < 0.001 , adjusted r = 0.01 ] .
post hoc tests indicated that highly physically active group had lower levels of depressive symptoms than lightly active group ( mean difference = 2.74 ; p < 0.001 , 95%ci : 3.91 to 1.56 ) ( figure 2 ) .
highly physically active participants had lower levels of depressive symptoms in comparison to moderately active ones ( mean difference = 1.59 , p = 0.001 , 95%ci : 2.60 to 0.57 ) ( figure 2 ) .
also moderately active participants had lower levels of depressive symptoms than lightly active participants ( mean difference = 1.15 , p < 0.001 , 95%ci : 1.81 to 0.49 ) ( figure 2 ) . when the covariates [ 2530 ] were adjusted for , the results became nonsignificant [ f ( 2,3547 ) = 0.53 , p = 0.59 , adjusted r = 0.47 ] . in particular
the previous symptoms of depression measured in 1997 and 2001 attenuated the associations ( for details , see supplementary table 5 ) .
thereafter , we examined the longitudinal associations between adulthood physical activity ( assessed in 2007 , participants ' aged from 30 to 45 ) and symptoms of depression ( assessed in 2012 ) . in the original sample ,
adulthood physical activity was associated with depressive symptoms ( b = 1.10 , p < 0.001 , 95%ci : 1.58 to 0.61 , adjusted r = 0.01 ) .
when the covariates [ 2530 ] were adjusted for , the association attenuated to nonsignificance ( b = 0.17 , p = 0.62 , 95%ci : 0.85 to 0.51 , adjusted r = 0.24 ) .
in particular the previous symptoms of depression assessed in 2001 and 2007 attenuated the association ( for details , see supplementary table 6 ) .
thereafter , the analyses were performed in a sample with full information on all study variables ( n = 648 ) , in which case the adulthood physical activity was not associated with depressive symptoms ( b = 0.55 , p = 0.16 , 95%ci : 1.31 to 0.21 , adjusted r = 0.002 ) . due to the sample attrition , the association was studied also in the imputed data ( n = 3596 ) , and the results showed that the adulthood physical activity was associated with decreased levels of depressive symptoms ( b = 1.07 , p < 0.001 , 95%ci : 1.36 to 0.78 , adjusted r = 0.01 ) . when the covariates [ 2530 ] were adjusted for , the association attenuated to nonsignificance ( b = 0.06 , p = 0.57 , 95%ci : 0.16 to 0.28 , adjusted r = 0.47 ) .
in particular the symptoms of depression assessed in 2001 and 2007 attenuated the association ( for details , see supplementary table 6 ) .
this study examined whether distinct trajectories of lifelong physical activity existed in the data and whether physical activity was related to depressive symptoms in adulthood .
we also studied whether the lifelong physical activity trajectories contributed to the outcome to a greater degree than adulthood physical activity .
this inspection was important , because the information whether lifelong trajectories contribute to depressive symptoms over and above the concurrent ( adulthood ) physical activity is lacking .
lcga revealed three distinct groups , the lightly , moderately , and highly physically active groups .
physical activity levels remained relatively similar from childhood to adulthood in each group , although the groups ' physical activity levels decreased slightly towards middle adulthood .
these results are in line with previous studies demonstrating the decline of physical activity with age .
high physical activity associated with lower level of depressive symptoms in adulthood , which is in accord with previous research [ 10 , 18 ] .
physical activity may also relate to enhanced self - confidence , emotion regulation skills , and social capacities that are associated with positive mood [ 13 , 14 ] .
the association between high physical activity and lower levels of depressive symptoms in adulthood , however , disappeared when covariates ( age , sex , childhood negative emotionality , parents ' socioeconomic status , participants ' previous symptoms of depression , participants ' socioeconomic status , social support , body mass index , and smoking status ) were taken into account .
the explanatory power of the fully adjusted models was substantially higher in comparison to the unadjusted ones ( 46% higher in the imputed data ) .
these same results were found when lifelong physical activity trajectories and adulthood physical activity ( assessed in 2007 ) were used as predictors for depressive symptoms in adulthood .
specifically , physical activity did not predict lower levels of depressive symptoms in adulthood when the experiences of previous depression were taken into account .
the bidirectional nature of the association between physical activity and depression has been documented [ 8 , 11 ] .
hence , it is theoretically possible that the childhood symptoms of depression preceding our first measurement have decreased the likelihood of initiating physical activity [ 8 , 11 ] or that of maintaining adequate level of physical activity .
summarizing , the study indicated that lifelong physical activity trajectories or adulthood physical activity levels were not associated with the progression of depressive symptoms in adulthood .
the study suggests that lifelong physical activity history does not contribute to the progression of the depressive symptoms to a greater degree than adulthood physical activity .
participants did not provide information regarding each variable across the measurement years , which diminished the complete - sample size considerably .
the measurements concerning the participants ' physical activity focused only on the self - reported leisure time physical activity , not total physical activity or energy expenditure . also the depressive symptoms were assessed with self - administered questionnaires , and thus the possibility of subjective bias can not be excluded . however , this is common in epidemiological studies by necessity . to our knowledge , comparably long follow - up studies using diagnostic interviews do not exist .
furthermore , early childhood depression was not assessed in the study , but this deficiency was retrieved to an extent by controlling for the childhood negative emotionality .
the strengths of the study were the prospective , population - based study design , relatively large sample size , use of lcga , and utilization of an extensive set of covariates .
in lcga , the main advantage is that it allows a researcher to model developmental processes at inter- and intraindividual levels .
such modeling has been shown to be especially useful regarding the development of health behaviors [ 20 , 21 ] , and the need for further studies has been recognized .
however , in the case of this study , the traditional cross - sectional and longitudinal studies yielded to very similar results .
this was not evident a priori , and therefore our study adds evidence regarding the etiology of depression by showing that physical activity trajectories appear not to play a special role over and above the adulthood physical activity . furthermore , depressive symptoms were studied with a well - validated instrument , bdi - ii [ 3639 ] .
we were able to study the whole variance of participants ' depressive symptoms instead of categorical diagnoses .
this is important when attempting to find preventive tools for depression , because functional impairment is much more strongly associated with symptom severity than with diagnostic symptom count .
also the people who have experienced symptoms of depression are at risk of getting a diagnosis .
this study identified three distinct physical activity groups , the lightly , moderately , and highly physically active ones .
each group 's physical activity levels remained relatively unchanged from childhood to adulthood , although the levels tended to diminish slightly towards later adulthood .
highly physically active participants from childhood to adulthood had lower levels of depressive symptoms in adulthood compared to lightly physically active ones .
furthermore , participants ' adulthood physical activity assessed in 2007 was associated with decreased levels of depressive symptoms in adulthood .
the associations between physical activity and depressive symptoms disappeared when the preexisting symptoms of depression were controlled for , indicating that physical activities did not associate with the progression of depressive symptoms .
thus , the study suggests that lifelong physical activity history does not contribute to the progression of the depressive symptoms to a greater degree than adulthood physical activity .
obtaining information of mental health history might benefit clinicians and other professionals in evaluating the role of physical activity in well - being .
|
this prospective , community - based study examined trajectories of physical activity from childhood to adulthood and whether these trajectories contributed to depressive symptoms in adulthood to a greater degree than adulthood physical activity .
participants ( n = 3596 ) were from the ongoing cardiovascular risk in young finns study which started in 1980 .
depressive symptoms were measured with beck depression inventory ( bdi - ii ) in 2012 , and physical activity was assessed from 1980 to 2011 with self - reports .
analyses were adjusted for age , sex , childhood negative emotionality , socioeconomic factors , previous depressive symptoms , social support , body mass index , and smoking status ( 19802007 ) .
highly , moderately , and lightly physically active trajectory groups were identified .
highly active participants reported lower levels of depressive symptoms compared to lightly active ones ( p < 0.001 ) and compared to moderately active ones ( p = 0.001 ) .
moderately active participants had less symptoms than lightly active ones ( p < 0.001 ) .
high levels of adulthood physical activity associated with lower levels of depressive symptoms ( p < 0.001 ) .
the findings did not withstand adjustment for previous depressive symptoms ( p > 0.05 ) .
lifelong physical activity trajectories or adulthood physical activity was not associated with the progression of depressive symptoms in adulthood .
thus , physical activity history does not contribute to the progression of the depressive symptoms to a greater degree than adulthood physical activity .
|
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in 2012 , an estimated 790,000 women in the united states were diagnosed with cancer , of whom 10% were of reproductive age ( american cancer society , 2012 , schover , 2005 ) .
[ nih surveillance ] ( national institute of health , 2010 , society of family planning , 2012 ) addressing the possibility of future pregnancy is an important aspect of survivorship and quality of life for patients undergoing cancer treatment , and several ways to preserve fertility during cancer therapy have been developed and studied ( letourneau et al . , 2012 ,
as background , 49% of pregnancies in the united states are unintended and unplanned ( the alan guttmacher institute ( agi ) , 2013 ) .
patients with medical co - morbidities , including those undergoing cancer treatment and/or surveillance , are among this group .
it is estimated the incidence of malignancy and pregnancy is approximately 1:1000 ( pavlidis , 2002 , smith et al . , 2003 ) . although rare , pregnancy in the setting of cancer treatment may create clinical and ethical dilemmas , with potential increased risks for both the patient and gestation .
these dilemmas are further compounded when a pregnancy in this setting is unplanned . among women who have undergone or are currently undergoing cancer treatment
, there is limited data regarding the rate of unintended pregnancy or patients ' impression of fertility .
it has been previously reported that childhood cancer survivors between 15 and 30 years old were more likely to terminate a pregnancy compared to age - matched controls ( green et al . , 2002 ) .
a survey of female cancer patients revealed that 55% believed that they could not become pregnant after cancer treatment , and 45% of the same group denied using any contraception method ( patel et al . , 2009 ) .
both of the aforementioned studies support the notion that we could improve counseling in this patient population regarding fertility potential and contraception planning . in gynecologic cancer patients , women with early stage cancers may be eligible for fertility sparing treatment , and therefore may be at risk for unplanned pregnancy after therapy if contraception is not addressed as well ( mclaren and bates , 2012 ) . for this reason
, we conducted a survey of current members of the society of gynecologic oncology ( sgo ) regarding reproductive counseling , practices , and experience with unplanned pregnancy in this population .
after obtaining approval from our institutional review board , a list of member email addresses was obtained from the sgo .
an email statement of confidentiality with the questionnaire link was emailed to all listed members . following the initial e - mail , members received two additional follow - up e - mails , two and four weeks respectively , after the initial contact .
the survey remained open for responses for two weeks following the third , and final , reminder .
survey monkey was utilized to create and administer a nineteen question survey regarding demographics , contraception counseling and use practices , referral patterns , and incidence of unplanned pregnancy .
demographic data queried were age , gender , region of country of current residence , medical specialty , level of training , description of clinical practice , and number of years caring for gynecologic oncology patients .
questions regarding consistency to which the provider addresses contraception and fertility concerns were answered using a likert - type scale ( i.e. , always , sometimes , rare , never , not applicable ) .
the remaining questions were answered using a yes / no or a multiple - choice format .
the multiple - choice questions specifically addressed the frequency of contraception methods counseling and administration , and number of patients experiencing unplanned pregnancy .
participants were asked to check all contraceptive methods that they counseled and administered / prescribed .
contraceptive methods specifically included were the oral contraception pill ( ocp ) , injectable , intrauterine device ( iud ) , or subdermal levonorgestrel implant .
prevalence of unplanned pregnancy was evaluated by asking practitioners to estimate prevalence of unplanned pregnancy among their patients ( during or after treatment ) in the previous one and five years respectively .
we excluded the responses of anyone who reported that he / she was retired or did not provide information on their reproductive counseling patterns .
logistic regression analysis was used to model separately the odds of always or sometimes offering fertility and/or contraceptive counseling .
all analyses were conducted using stata 13.0 ( statacorp , college station , texas ) .
questionnaires were completed by 261 respondents , yielding a response rate of 18% . among the respondents 21 ( 8% ) reported that they were retired and/or did not provide information regarding their reproductive counseling patterns and thus were excluded from the analysis .
overall , the respondents had a mean age of 47 years with a slight predominance of females ( 52% ) , which is consistent with sgo demographics ( mean age of male members 50 years old , mean age of female members 45 years old ) .
our survey had a higher proportion of fellow respondents ( 24% ) than sgo as a whole ( 9% ) [ gynecologic oncology 2015 : state of the subspecialty ] selected characteristics of respondents are presented by reproductive counseling status in table 2 . among respondents ,
34.6% reported sometimes or always providing counseling on fertility and contraception , 19.2% reported providing fertility counseling only , 15.8% reported providing contraception counseling only , while 30.4% reported not routinely providing counseling on either topic .
in general , providers who reported providing reproductive counseling on neither topic were more likely to be male and/or a fellow .
they also tended to be < 40 years of age , though this difference was not statistically significant .
physicians who reported not routinely counseling on either topic were less likely to report prescribing or administering contraception and/or were less likely to report referring to reproductive endocrinology .
in contrast , those who reported providing counseling on both fertility and contraception were more likely to be female and/or an attending .
they also were more likely to be older ; although that difference was not statistically significant . providers who reported sometimes or always providing counseling on both reproductive topics were also more likely to report providing or administering contraception and/or to refer to reproductive endocrinology . there were no notable differences in reproductive counseling status by u.s .
next , we examined what factors were associated with an increased odds of reporting providing fertility counseling sometimes or always ( table 3 ) .
after adjustment for gender , we found that the odds of reporting providing fertility counseling were nearly three times higher among attending physicians as compared to fellows [ aor = 2.72 ; 95% ci = ( 1.44 , 5.12 ) ] . in examining contraceptive counseling ( table 4 )
, we found that the odds of reporting providing contraceptive counseling was 2.8 times higher in women as compared to men and was 4.91 times higher in individuals age 50 + compared to those < 40 after adjustment for level of practice .
overall , 81.7% of providers reported counseling , prescribing , or administering contraception to their patients . among these individuals ,
the most frequently cited contraceptive that providers reported addressing with their patients included : oral contraceptive pills ( 81% ) , intrauterine devices ( 73% ) , depot medroxyprogesterone acetate ( dpma ) injections ( 56% ) , and contraceptive implants ( 21% ) .
the most frequently cited contraceptives that providers reported prescribing or administering included : oral contraceptive pills ( 80% ) , intrauterine devices ( 62% ) , dpma injections ( 48% ) , and contraceptive implants ( 9% ) .
the reported prevalence of unplanned pregnancy among the surveyed providers ' patients was relatively rare , perhaps because it is not routinely addressed .
most providers ( 95% ) reported 05 unplanned pregnancies among their patients in the last 5 years , with only 3 providers ( 1% ) reporting more than ten unplanned pregnancies .
unplanned pregnancy in the setting of cancer treatment or surveillance is a complicated issue , and may create clinical and ethical dilemmas for the patient , partner , family , and treatment team .
the majority of survey respondents appropriately acknowledged the risk of unplanned pregnancy among gynecologic cancer patients maintaining fertility potential .
however , only half reported addressing contraception planning in this population on a consistent basis ( 52% always addressing contraception , versus 35% sometimes ) . regarding the consistency with which contraception is addressed ,
these findings are consistent with a retrospective chart review completed at our institution where 45% of initial consultations documented a contraception plan in fertile patients of reproductive age , and 32% of follow - up visits for those that maintained fertility potential after cancer treatment ( crafton et al . , 2016 ) . depending on individual circumstances
patient 's reproductive goals should be determined in order to tailor fertility - sparing treatment , when possible , versus contraception planning .
unfortunately , only half of respondents reported always addressing fertility concerns with patients in this population .
this is consistent with the fact 59% of those surveyed routinely offer referral for preconception counseling for those patients considering fertility preservation options or planned pregnancy .
referral specifically for contraception counseling was reported less frequently , and even fewer reported follow up for plan establishment .
our study was unable to determine which patients or providers relied on the patient 's primary care physician or gynecologist for the establishment of a contraception plan .
the incidence of unplanned pregnancy in this population is reportedly rare , with 96% of responding physicians experiencing < 5 unplanned pregnancies in the previous five years .
this may be under - reported , as oncologists may be unaware of pregnancies , especially during disease surveillance . despite the rarity ,
eleven providers report experiencing an estimated 620 unplanned pregnancies during that time same . the opportunity to avoid even a fraction of those merits further acknowledgement of this topic .
it is reassuring that the large majority of providers reportedly counsel for and administer contraception methods , including 89% reportedly counseling for the iud and 77% employing its use .
however , it was not specified in this survey if the indication for oral contraceptive pills ( ocps ) or an iud was for contraception planning or cancer therapeutic purposes . of reported methods ,
ocps were the most frequent contraception method both counseled for and administered . when compared to implantable methods , both the iud and subdermal implant , both actual and ideal use of ocps
have a higher failure rate , and therefore more reliable methods should be considered first , barring contraindications ( centers for disease control and prevention , 2010 ) .
given our findings that the attending cohort was more likely to provide contraception and/or fertility counseling sometimes or always compared to the fellow respondents ( table 2 , table 3 ) , there seems to be an opportunity to improve education for fellows regarding fertility preservation options for these patients . as seen in other survey - based studies ,
the primary limitation of the study is the modest response rate , despite multiple recruitment attempts and limiting the length of the survey ( cunningham et al . , 2015 ) .
given the response rate , the potential for selection and survey content bias ( i.e. , only those who were interested in the topic responded ) is present thus the results may have limited external validity .
however , as aforementioned , our respondent cohort is comparable to the data published in the 2015 society of gynecologic oncology : state of the subspecialty .
inclusion of both fellows in training and retired members also limits the external validity of the survey regarding current members , but we felt inclusions of these populations were interesting and important in order to compare potential generational differences .
many gynecologic cancer diagnoses are made in peri - menopausal or postmenopausal women ; therefore assessing what portion of a provider 's practice is of reproductive age could aid discussion and interpretation of results .
likewise , as the type of cancer and respective treatment options may change the potential for or etiology of infertility , assessing providers ' experience with specific disease processes , such as gestational trophoblastic neoplasia , also could aid discussion , and intervention .
we acknowledge that a contraception plan may not appropriate for all patients undergoing cancer care , especially for who decline fertility sparing treatment , have been previously sterilized , or are actively attempting conception / currently pregnant .
however , routine recognition of reproductive goals should be addressed by providers to alleviate potential biases or assumptions regarding patients ' reproductive goals or sexual activity .
previous literature has reported providers ' lack of counseling and patients ' misunderstanding of reproductive potential after cancer treatment , and therefore a patient survey would be as valuable as the provider 's impression we report ( patel et al . , 2009 , karaoz et al . , 2010 ) .
as life expectancy following cancer treatment diagnosis and treatment improves , and quality of life of survivors is emphasized , helping women meet their reproductive goals should remain an important focus .
comprehensive reproductive counseling should be emphasized , including both fertility sparing options and contraception planning , with the ultimate goal of decreasing unplanned pregnancy .
|
we sought to identify how gynecologic oncologists approach reproductive counseling for their fertile , reproductive age patients , and their experience with unplanned pregnancies.members of the society of gynecologic oncology ( sgo ) were surveyed electronically regarding consistency of counseling patterns of contraception and fertility concerns , most and least common contraceptive methods utilized , referral patterns , and incidence of unplanned pregnancy . of the 1424 sgo members identified , 261 participated in the questionnaire , yielding a response rate of 18% .
eighty - two percent of respondents agreed unplanned pregnancy is a potential problem , but only 57% believed their patients understood unplanned pregnancy is possible during treatment .
half of respondents report always in terms of frequency that contraception is addressed among their high - risk patients . after adjustment for gender
, we found that the odds of reporting providing fertility counseling were nearly three times higher among attendings as compared to fellows [ aor = 2.72 ; 95% ci = ( 1.44 , 5.12 ) , three times higher in women as compared to men [ aor = 2.80 ; 95% ci = ( 1.46 , 5.38 ) ] , as well as in individuals 50 + years as compared to those < 40 years old [ aor = 4.91 ; 95% ci = ( 2.05 , 11.74 ) ] .
ninety - six percent reported < 5 unplanned pregnancies , to their knowledge , in the previous five years of clinical practice .
most providers acknowledge that unplanned pregnancy is a potential risk in fertile gynecologic oncology patients , but only half believe their patients understand an unplanned pregnancy is possible .
an opportunity exists to provide more directed counseling regarding fertility during and after cancer therapy , and to educate patients and providers regarding more reliable , long acting contraceptive methods .
|
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neonatal sprague - dawley rats ( seven days old ) together with their maternal rats were obtained from a commercial breeder ( orient co. , seoul , korea ) .
the experimental procedures were performed in accordance with the animal care guidelines of the national institutes of health ( nih ) and the korean academy of medical sciences .
each animal was housed under controlled temperature ( 232 ) and lighting ( 08:00 - 20:00 ) conditions with free feeding .
the neonatal rats were randomly divided into five groups ( n=15 in each group ) : a control group , and four cataract - induction groups , treated with either 0 , 50 , 100 , or 200 mg / kg catechin .
the rats in the catechin - treatment groups received catechin ( sigma chemical co. , st .
louis , mo , usa ) orally once a day for ten consecutive days at the respective doses , starting five days after cataract - induction .
the rats in the control group and in the cataract - induction groups received an equal amount of distilled water for the same duration .
cataracts were induced using a previously described procedure . in brief , at ten days of postnatal age , the neonatal rats received 100 mg / kg n - methyl - n - nitrosourea ( mnu , sigma chemical co. ) intraperitoneally . just before use , mnu was dissolved in physiological saline containing 0.05% acetic acid .
slit - lamp biomicroscopic examination was performed on each eye to provide a morphological assessment of the degree of opacification at 15 days after cataract induction .
prior to the examination , mydriasis was achieved using a topical ophthalmic solution containing tropicamide with phenylephrine hydrochloride ( santen pharmaceutical , osaka , japan ) .
one drop of the solution was instilled in each eye every 30 minutes for 2 hours , while the animals were in a dark room .
after 2 hours , the eyes were examined by slit - lamp biomicroscopy at 12magnification .
the rats were sacrificed immediately after determination of cataract formation with slit - lamp biomicroscopy ( 15 days after cataract induction ) .
the animals were anesthetized using zoletil 50 ( 10 mg / kg , i.p . ; vibac laboratories , carros , france ) . at necropsy ,
both lenses were quickly removed under a surgical microscope gl-99b - v7 ( davis , california , ca , usa ) ; a complete necropsy was performed on all animals .
the lenses were fixed in 4% paraformaldehyde , dehydrated in graded ethanol , treated in xylene , and infiltrated and embedded in paraffin .
coronal sections of 5 m thickness were made using a paraffin microtome ( leica , nussloch , germany ) and were mounted on coated slides , then dried at 37 overnight on a hot plate .
the lenses were collected and immediately frozen at -70. the tissues were homogenized with lysis buffer containing 50 mm tris - hcl ( ph 8.0 ) , 150 mm nacl , 10% glycerol , 1% triton x-100 , 1.5 mm mgcl26h2o , 1 mm egta , 1 mm pmsf , 1 mm na2vo4 , and 100 mm naf , and then ultracentrifuged at 50,000 rpm for 1 hours .
protein content were measured using a bio - rad colorimetric protein assay kit ( bio - rad , hercules , ca , usa ) .
forty micrograms of protein was separated on sds - polyacrylamide gels and were transferred onto a nitrocellulose membrane .
mouse antibodies against actin ( 1:2000 ; santa cruz biotech , santa cruz , ca , usa ) , bax ( 1:1000 ; santa cruz biotech ) , and bcl-2 ( 1:1000 ; santa cruz biotech ) were used as primary antibodies .
horseradish peroxidase - conjugated anti - mouse antibodies for bax and bcl-2 ( 1:2000 ; amersham pharmacia biothech gmbh , freiburg , germany ) were used as secondary antibodies .
the experiment was performed in normal lab conditions at room temperature , with the exception of membrane transfer .
membrane transfer was performed at 4 with a cold pack and a pre - chilled buffer .
band detection was performed using an enhanced chemiluminescence ( ecl ) detection kit ( santa cruz biotech ) .
detected bands were calculated densitometrically using molecular analyst version 1.4.1 ( bio - rad ) , in order to compare the relative expressions of proteins .
tunel staining was performed using an in situ cell death detection kit ( roche , mannheim , germany ) according to the manufacturer 's protocol , in order to visualize dna fragmentation , a marker of apoptotic cell death .
the epithelial cells were suspended in 10 mm tris - hcl buffer , ph 8.0 , containing 1 mm edta , through incubation at 55 for 30 minutes .
sections were then incubated with proteinase k ( 100 g / ml ) , rinsed , incubated in 3% h2o2 , permeabilized with 0.5% triton x-100 , rinsed again , and incubated in tunel reaction mixture .
the sections were rinsed and visualized using converter - pod with 0.03% 3,3'-diaminobenzidine ( dab ) and then mounted onto gelatin - coated slides .
the slides were air dried overnight at room temperature , and coverslips were mounted using permount. to visualize caspase-3 expression , we performed caspase-3 immunohistochemistry using a previously described method .
sections were drawn from each lens and incubated overnight with mouse anti - caspase-3 antibody ( 1:500 ; santa cruz biotech ) and then for another 1 h with biotinylated mouse secondary antibody ( 1:200 ; vector laboratories , burlingame , ca , usa ) . bound secondary antibodies were then amplified with a vector elite abc kit ( 1:100 ; vector laboratories ) .
the antibody - biotin - avidin - peroxidase complexes were visualized using 0.03% dab , and the sections were finally mounted onto gelatin - coated slides .
the slides were air dried overnight at room temperature , and coverslips were mounted using permount. to compare relative expressions of proteins , we examined detected bands densitometrically using molecular analyst version 1.4.1 ( bio - rad ) .
the area of the lens epithelium region in each slice was measured using the image - pro plus computer - assisted image analysis system ( media cybernetics inc .
, silver spring , md , usa ) attached to a light microscope ( olympus , tokyo , japan ) .
the tunel - positive and caspase-3-positive cells within each lens epithelium region were counted through the light microscope .
statistical analysis was performed using one - way anova followed by duncan 's post - hoc test .
neonatal sprague - dawley rats ( seven days old ) together with their maternal rats were obtained from a commercial breeder ( orient co. , seoul , korea ) .
the experimental procedures were performed in accordance with the animal care guidelines of the national institutes of health ( nih ) and the korean academy of medical sciences .
each animal was housed under controlled temperature ( 232 ) and lighting ( 08:00 - 20:00 ) conditions with free feeding .
the neonatal rats were randomly divided into five groups ( n=15 in each group ) : a control group , and four cataract - induction groups , treated with either 0 , 50 , 100 , or 200 mg / kg catechin .
the rats in the catechin - treatment groups received catechin ( sigma chemical co. , st .
louis , mo , usa ) orally once a day for ten consecutive days at the respective doses , starting five days after cataract - induction .
the rats in the control group and in the cataract - induction groups received an equal amount of distilled water for the same duration .
cataracts were induced using a previously described procedure . in brief , at ten days of postnatal age , the neonatal rats received 100 mg / kg n - methyl - n - nitrosourea ( mnu , sigma chemical co. ) intraperitoneally . just before use , mnu was dissolved in physiological saline containing 0.05% acetic acid .
slit - lamp biomicroscopic examination was performed on each eye to provide a morphological assessment of the degree of opacification at 15 days after cataract induction . prior to the examination
, mydriasis was achieved using a topical ophthalmic solution containing tropicamide with phenylephrine hydrochloride ( santen pharmaceutical , osaka , japan ) .
one drop of the solution was instilled in each eye every 30 minutes for 2 hours , while the animals were in a dark room .
after 2 hours , the eyes were examined by slit - lamp biomicroscopy at 12magnification .
the rats were sacrificed immediately after determination of cataract formation with slit - lamp biomicroscopy ( 15 days after cataract induction ) .
the animals were anesthetized using zoletil 50 ( 10 mg / kg , i.p . ; vibac laboratories , carros , france ) . at necropsy ,
both lenses were quickly removed under a surgical microscope gl-99b - v7 ( davis , california , ca , usa ) ; a complete necropsy was performed on all animals .
the lenses were fixed in 4% paraformaldehyde , dehydrated in graded ethanol , treated in xylene , and infiltrated and embedded in paraffin .
coronal sections of 5 m thickness were made using a paraffin microtome ( leica , nussloch , germany ) and were mounted on coated slides , then dried at 37 overnight on a hot plate .
the lenses were collected and immediately frozen at -70. the tissues were homogenized with lysis buffer containing 50 mm tris - hcl ( ph 8.0 ) , 150 mm nacl , 10% glycerol , 1% triton x-100 , 1.5 mm mgcl26h2o , 1 mm egta , 1 mm pmsf , 1 mm na2vo4 , and 100 mm naf , and then ultracentrifuged at 50,000 rpm for 1 hours .
protein content were measured using a bio - rad colorimetric protein assay kit ( bio - rad , hercules , ca , usa ) .
forty micrograms of protein was separated on sds - polyacrylamide gels and were transferred onto a nitrocellulose membrane .
mouse antibodies against actin ( 1:2000 ; santa cruz biotech , santa cruz , ca , usa ) , bax ( 1:1000 ; santa cruz biotech ) , and bcl-2 ( 1:1000 ; santa cruz biotech ) were used as primary antibodies .
horseradish peroxidase - conjugated anti - mouse antibodies for bax and bcl-2 ( 1:2000 ; amersham pharmacia biothech gmbh , freiburg , germany ) were used as secondary antibodies .
the experiment was performed in normal lab conditions at room temperature , with the exception of membrane transfer .
membrane transfer was performed at 4 with a cold pack and a pre - chilled buffer .
band detection was performed using an enhanced chemiluminescence ( ecl ) detection kit ( santa cruz biotech ) .
detected bands were calculated densitometrically using molecular analyst version 1.4.1 ( bio - rad ) , in order to compare the relative expressions of proteins .
tunel staining was performed using an in situ cell death detection kit ( roche , mannheim , germany ) according to the manufacturer 's protocol , in order to visualize dna fragmentation , a marker of apoptotic cell death .
the epithelial cells were suspended in 10 mm tris - hcl buffer , ph 8.0 , containing 1 mm edta , through incubation at 55 for 30 minutes .
sections were then incubated with proteinase k ( 100 g / ml ) , rinsed , incubated in 3% h2o2 , permeabilized with 0.5% triton x-100 , rinsed again , and incubated in tunel reaction mixture .
the sections were rinsed and visualized using converter - pod with 0.03% 3,3'-diaminobenzidine ( dab ) and then mounted onto gelatin - coated slides .
the slides were air dried overnight at room temperature , and coverslips were mounted using permount.
sections were drawn from each lens and incubated overnight with mouse anti - caspase-3 antibody ( 1:500 ; santa cruz biotech ) and then for another 1 h with biotinylated mouse secondary antibody ( 1:200 ; vector laboratories , burlingame , ca , usa ) . bound secondary antibodies were then amplified with a vector elite abc kit ( 1:100 ; vector laboratories ) .
the antibody - biotin - avidin - peroxidase complexes were visualized using 0.03% dab , and the sections were finally mounted onto gelatin - coated slides .
the slides were air dried overnight at room temperature , and coverslips were mounted using permount.
to compare relative expressions of proteins , we examined detected bands densitometrically using molecular analyst version 1.4.1 ( bio - rad ) .
the area of the lens epithelium region in each slice was measured using the image - pro plus computer - assisted image analysis system ( media cybernetics inc . , silver spring , md , usa ) attached to a light microscope ( olympus , tokyo , japan ) .
the tunel - positive and caspase-3-positive cells within each lens epithelium region were counted through the light microscope .
statistical analysis was performed using one - way anova followed by duncan 's post - hoc test .
the degree of lenticular opacification was enhanced in the cataract - induction rats and was reduced by catechin treatment .
2 . the number of tunel - positive cells was 8.302.78/section in the control group , 157.5010.26/section in the cataract - induction group , 143.308.94/mm in the cataract - induction and 50 mg / kg catechin group , 136.4011.36/section in the cataract - induction and 100 mg / kg catechin group , and 97.106.98/section in the cataract - induction and 200 mg / kg catechin group .
these results show that mnu injection enhanced apoptotic cell death in the lens epithelium , and catechin treatment significantly suppressed cataract - induced apoptosis in a dose - dependent manner .
3 . the number of caspase-3-positive cells was 6.202.23/section in the control group , 103.206.78/section in the cataract - induction group , 101.203.67/mm in the cataract - induction and 50 mg / kg catechin group , 103.003.10/section in the cataract - induction and 100 mg / kg catechin group , and 84.202.73/section in the cataract - induction and 200 mg / kg catechin group .
these results show that mnu injection enhanced caspase-3 expression in the lens epithelium , and that catechin ( 200 mg / kg ) treatment significantly suppressed cataract - induced caspase-3 expression . to verify cataract - induced apoptosis
, we ascertained the relative protein expressions of bax and bcl-2 related to apoptosis , and levels of bax and bcl-2 are presented in fig .
the level of bax ( 24 kda , the pro - apoptotic factor ) in the control group was set at 1.00 . in the cataract induction group ,
the levels of bax were 3.110.28 in the 0mg / kg catechin group , 2.790.28 in the 50 mg / kg catechin group , 1.980.18 in the 100 mg / kg catechin group , and 1.970.26 in the 200 mg / kg catechin group .
in contrast , catechin administration reduced the expression of bax in a dose - dependent manner .
specifically , 100 mg / kg and 200 mg / kg catechin significantly suppressed the expression of bax protein .
the level of bcl-2 ( 26~29 kda , the anti - apoptotic factor ) in the control group was set at 1.00 . in the cataract induction groups ,
the levels of bcl-2 were 1.430.04 in the 0mg / kg catechin group , 1.550.03 in the 50 mg / kg catechin group , 1.430.05 in the 100 mg / kg catechin group , and 1.630.03 in the 200 mg / kg catechin group .
cataract formation increased bcl-2 expression , while catechin ( 200 mg / kg ) administration slightly enhanced the expression of bcl-2 .
we calculated the ratio of bax to bcl-2 , one of the crucial factors determining if cells will undergo apoptosis .
the ratio of bax to bcl-2 in the control group was set at 1.00 . in the cataract induction groups ,
the ratios of bax to bcl-2 were 2.170.20 in the 0mg / kg catechin group , 1.800.18 in the 50 mg / kg catechin group , 1.380.13 in the 100 mg / kg catechin group , and 1.210.16 in the 200 mg / kg catechin group .
cataract enhanced the expression of both bax and bcl-2 , but bax expression increased much more than did increased bcl-2 expression . on the other hand , administration of catechin significantly suppressed the expression of bax protein . as a result
, the ratio of bax to bcl-2 was increased by cataract formation , representing ongoing apoptotic cell death in the lens epithelium .
catechin treatment suppressed the ratio of bax to bcl-2 , representing inhibition of apoptotic cell death in the lens epithelium .
the degree of lenticular opacification was enhanced in the cataract - induction rats and was reduced by catechin treatment .
2 . the number of tunel - positive cells was 8.302.78/section in the control group , 157.5010.26/section in the cataract - induction group , 143.308.94/mm in the cataract - induction and 50 mg / kg catechin group , 136.4011.36/section in the cataract - induction and 100 mg / kg catechin group , and 97.106.98/section in the cataract - induction and 200 mg / kg catechin group .
these results show that mnu injection enhanced apoptotic cell death in the lens epithelium , and catechin treatment significantly suppressed cataract - induced apoptosis in a dose - dependent manner .
3 . the number of caspase-3-positive cells was 6.202.23/section in the control group , 103.206.78/section in the cataract - induction group , 101.203.67/mm in the cataract - induction and 50 mg / kg catechin group , 103.003.10/section in the cataract - induction and 100 mg / kg catechin group , and 84.202.73/section in the cataract - induction and 200 mg / kg catechin group .
these results show that mnu injection enhanced caspase-3 expression in the lens epithelium , and that catechin ( 200 mg / kg ) treatment significantly suppressed cataract - induced caspase-3 expression .
to verify cataract - induced apoptosis , we ascertained the relative protein expressions of bax and bcl-2 related to apoptosis , and levels of bax and bcl-2 are presented in fig .
4 . the level of bax ( 24 kda , the pro - apoptotic factor ) in the control group was set at 1.00 . in the cataract induction group ,
the levels of bax were 3.110.28 in the 0mg / kg catechin group , 2.790.28 in the 50 mg / kg catechin group , 1.980.18 in the 100 mg / kg catechin group , and 1.970.26 in the 200 mg / kg catechin group .
in contrast , catechin administration reduced the expression of bax in a dose - dependent manner .
specifically , 100 mg / kg and 200 mg / kg catechin significantly suppressed the expression of bax protein .
the level of bcl-2 ( 26~29 kda , the anti - apoptotic factor ) in the control group was set at 1.00 . in the cataract induction groups ,
the levels of bcl-2 were 1.430.04 in the 0mg / kg catechin group , 1.550.03 in the 50 mg / kg catechin group , 1.430.05 in the 100 mg / kg catechin group , and 1.630.03 in the 200 mg / kg catechin group .
cataract formation increased bcl-2 expression , while catechin ( 200 mg / kg ) administration slightly enhanced the expression of bcl-2 .
we calculated the ratio of bax to bcl-2 , one of the crucial factors determining if cells will undergo apoptosis .
the ratio of bax to bcl-2 in the control group was set at 1.00 . in the cataract induction groups ,
the ratios of bax to bcl-2 were 2.170.20 in the 0mg / kg catechin group , 1.800.18 in the 50 mg / kg catechin group , 1.380.13 in the 100 mg / kg catechin group , and 1.210.16 in the 200 mg / kg catechin group .
cataract enhanced the expression of both bax and bcl-2 , but bax expression increased much more than did increased bcl-2 expression . on the other hand , administration of catechin significantly suppressed the expression of bax protein . as a result
, the ratio of bax to bcl-2 was increased by cataract formation , representing ongoing apoptotic cell death in the lens epithelium .
catechin treatment suppressed the ratio of bax to bcl-2 , representing inhibition of apoptotic cell death in the lens epithelium .
several animal species experience spontaneously occurring cataract of known inheritance and offer valuable model for studying human cataract .
various chemicals are known to contribute to the development of cataract in animals . among these chemicals ,
mnu , a direct - acting alkylating agent that does not require metabolic activation , is known as a cataractogenic agent in rats .
in addition , young animals are reported to be more susceptible to mnu than are adult animals .
therefore , in this study , a cataract model was constructed using a single intraperitoneal injection of nmu in rats at postnatal day 10 .
lens epithelial cells play a vital role in the metabolic homeostasis and maintenance of transparency in the lens , and damage to lens epithelial cells potently contributes to cataractogenesis .
moreover , apoptosis of lens epithelial cells has been reported to be the earliest event in the experimental formation of cataracts , such as those inducted by hydrogen peroxide and mnu . in human studies ,
the number of tunel - positive cells is above 50% in the lens epithelium after cataract surgery .
in addition , caspase-3 is up - regulated and activated in the early stages of apoptosis following cataractogenesis .
we found that the numbers of tunel - positive and caspase 3-positive cells in the lens epithelium were significantly higher following cataract induction .
these findings indicate that mnu injection - induced cataracts increased apoptosis in the lens epithelium . the bcl-2 family of proteins - including bcl-2 and bcl - xl - plays an important role in the regulation of apoptosis in the nervous system .
however , bcl-2 and bcl - xl form heterodimers with the main pro - apoptotic member bax , which can incapacitate their protective functions .
the bcl-2/bax balance is one of the crucial factors determining if cells undergo apoptosis , and the balance can change during cataract formation .
however , cataract formation increased bax expression much more than it increased bcl-2 expression . in the lens epithelium of the cataract ,
lens cell death occurs by apoptosis , and inhibition of apoptosis can delay cataract formation .
it has been reported that catechins can modulate apoptosis by altering the expressions of anti - apoptotic and pro - apoptotic genes . among the constituents of catechin
, egcg is known to protect against oxidative stress - induced and chronic glutamate - induced apoptosis in several human cells .
yao et al . reported that catechin protects against mitochondria - mediated apoptosis induced by h2o2 in human lens epithelial cells through the modulation of caspases and the mapk and akt pathways .
we observed that catechin significantly suppressed both cataract - induced increases in dna fragmentation and caspase-3 expression in the lens epithelium in dose - dependent manners .
many studies have shown that egcg , the main component of catechin , inhibits the expression of pro - apoptotic genes such as bax , bad , and mdm2 , and that egcg increases the expression of anti - apoptotic genes such as bcl-2 , bcl - w , and bcl - xl . in the present study ,
however , cataracts induced apoptosis by increasing the bax expression much more than it did the bcl-2 expression .
administration of catechin suppressed the expression of bax protein in a dose - dependent manner , but only slightly enhanced bcl-2 expression .
the ratio of bax to bcl-2 was increased by cataract formation . in contrast , catechin suppressed the ratio of bax to bcl-2 , showing that apoptosis was inhibited by catechin treatment . taken together , our results demonstrate that catechin alleviated cataract - induced apoptosis in lens epithelial cells .
catechin could potentially be used to delay cataractogenesis through the suppression of apoptotic cell death in the lens epithelium .
|
purposeto investigate the effect of catechin on apoptotic cell death in the lens epithelium of rats with cataract.methodscataract was induced by intraperitoneal injection of 100 mg / kg n - methyl - n - nitrosourea ( mnu ) to ten day - old sprague - dawley rats .
the neonatal rats were randomly divided into five groups ( n=15 in each group ) : a control group , and four cataract - induction groups , treated with either 0 , 50 , 100 , 200 mg / kg catechin .
we performed slit - lamp biomicroscopic analysis , terminal deoxynucleotidyl transferase - mediated dutp nick end labeling ( tunel ) assay , western - blot for bcl-2 and bax , and immunohistochemistry for caspase-3.resultsapoptotic cell death in lens epithelial cells that increased following cataract formation in rats was suppressed by cathechin.conclusionscatechin inhibited cataract - induced apoptotic cell death in the lens epithelium and may prove useful for the prevention of cataract progression .
|
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therapeutic drug monitoring ( tdm ) of antiepileptic drugs ( aeds ) is a common practice and is usually done to optimize their dosing regimens , efficacy and in investigating therapeutic failure or patient compliance and avoid toxicity .
as seizures occur irregularly , sometimes with long gaps in between the episodes , long - term administration and therefore observation of aed therapy becomes necessary to assess the patient compliance and its clinical benefit .
some aeds produce adverse effects which are difficult to distinguish from underlying neurologic disease and finally , there are no laboratory tests or diagnostic procedures available that can easily assess the clinical efficacy of the aeds .
patient demographics such as age , gender , and bodyweight play a key role in the pharmacokinetic variability of these drugs . measuring serum levels
is also indicated in patients with conditions such as hepatic or renal impairment and pregnancy which alter their pharmacokinetic characteristics so as to maintain there effective drug levels .
on the other hand , it is also a fact that if pharmacokinetic parameters of a drug are consistent and predictable , then dosing of that drug can often be done without the need for tdm which is usually seen with newer aeds .
levetiracetam ( lev ) is one of the newer aed which became available in the year 2000 .
although there exists a linear relationship between the dose and the serum levels of lev , some studies have suggested that serum levels of lev can get affected by a number of parameters .
reference ranges ( levels ) for the newer aeds like lev have been difficult to establish .
ideally , tdm guides physicians toward that serum concentration of the drug which would optimize the seizure control , while avoiding or at least minimizing their toxic effects .
however , it has been seen that a particular individual may show a good clinical response at aed concentrations even outside the reference range for that drug .
based on this understanding , serum levels of lev were measured and correlated with the patient 's demographics and clinical response , based on the assumption that the clinical effects for certain diseases correlate better with drug concentrations than with the dose given .
while tdm is an established method to individualize the dosage of aeds since many years , its impact on clinical course in patients with epilepsy has rarely been assessed especially with new aeds in a systematic manner .
patients suffering from epilepsy ( any seizure semiology ) and started on lev were enrolled for the study , after obtaining their written informed consent , from the outpatient department of neurology of a tertiary care medical college and hospital of north india from july 1 , 2014 to august 31 , 2015 , after approval from institutional ethics committee .
as this was a pilot study , 31 patients above 18 years of age and started on lev were selected but only 29 completed the study [ figure 1 ] .
patients who were pregnant , having a history of renal insufficiency , liver disease , or any other comorbid condition were excluded .
flow chart of patient recruitment patient demographics such as age , gender , height , and bodyweight along with clinical characteristics such as seizure frequency , detailed history of aeds intake both past and present and other concomitant treatment received were recorded .
the blood samples of these patients were collected for estimation of serum levels of lev ( after dose stabilization ) , creatinine , alanine transaminase ( alt ) , aspartate transaminase ( ast ) , and albumin .
serum levels of lev in these patients were measured using shimadzu 's high - performance liquid chromatography ( hplc ) system ( lc-2010 aht / cht ) .
c18 column was used as stationary phase and triethylamine buffer ( 10 ml triethylamine in 1000 ml water ) adjusted to ph 6.5 0.2 by phosphoric acid and acetonitrile in the ratio of 85:15 respectively was used as mobile phase .
flow rate of the mobile phase was 0.8 ml / min with a run time of 8 min .
unknown patient serum samples were run against the standard calibration curve of lev prepared which was linear with a correlation coefficient ( r ) of 0.999 .
the limit of detection and quantification of this assay method were 0.01 and 0.03 g / ml , respectively .
the determined serum levels of lev were correlated with age , gender , bodyweight , dose used , formulation , compliance , therapeutic effect , suspected toxicity , suspected drug interactions , and adverse drug reactions ( adrs ) in the patients .
figure 1 shows the recruitment of patients in a flow chart . among the 29 patients who were included in the study , 15 were males and 14 were females .
serum levels of creatinine , alt , ast , and albumin were within normal ranges for these patients .
other than lev , patients were also on clobazam , valproic acid ( va ) , oxcarbazepine , carbamazepine , phenytoin , phenobarbitone , lamotrigine , and topiramate in various combinations
. demographic characteristics of the study subjects the mean dose given to the patients in the study was 26.45 10.76 mg
/ kg / day with a range of 7.5750.00 mg / kg / day whereas the median total daily dose of lev given was 1500 mg ( range 5004000 mg ) .
the median serum level of lev obtained at different time intervals from the drug intake and blood sampling was 17.8 g / ml with a range of 0.4102.2 g / ml as shown in table 2 .
figure 2 illustrates the range of serum lev concentration at total dose given per day in milligrams .
it shows that there is no increase in the serum lev levels with an increase in doses .
figure 3 shows the distribution of serum lev levels of patients versus time gap since blood sample collection after the last dose in semi - logarithmic scale .
table 3 shows the serum lev levels with respect to age , gender , bodyweight and formulation . for making comparisons , patients with similar doses and blood sampling time since the last dose were taken . for comparing the effects of age and bodyweight
, patients were divided into below and above 40 years of age and 60 kg bodyweight , respectively . correlation ( r ) between serum lev concentration with dose administered ( mg / kg / day ) , age and bodyweight
all patients were on lev tablets except one each ( elderly at 73 and 65 years age ) on syrup and intravenous injection .
dosage and serum concentration of levetiracetam in the study subjects total levetiracetam dose versus serum concentration levetiracetam concentration versus time comparison of serum levetiracetam levels with respect to age , gender , bodyweight , and formulation all patients were compliant as they had detectable levels of lev in their serum but whether they were strictly adherent to drug therapy can not be commented upon as the levels show wide variations .
22 out of 29 ( 75.86% ) patients showed > 50% reduction in seizure frequency / week out of which 18 were completely seizure free .
two showed 50% reduction , three showed no change , and one showed 50% increase in seizure frequency / week as shown in figure 4 .
the patient with increased frequency of seizure episodes was suffering from absence seizures and was diagnosed with bilateral temporal lobe due to epilepsy .
one patient suffered from frequent oral twitching due to post hypoxic brain injury , but there was no record of the frequency of these twitchings .
table 4 shows serum lev concentrations with and without aed comedication whereas table 5 shows a comparison of serum levels of two patients each , on similar doses of lev while on concomitant use of other aeds .
although taken at different time intervals since drug intake , the serum levels of lev were 7.8% higher in patients on concomitant drug va and 56.8% lower in patients on concomitant inducer drugs ( carbamazepine , phenobarbitone , phenytoin ) as compared to those in patients on concomitant neutral drugs ( clobazam , lamotrigine , oxcarbazepine , topiramate ) or lev monotherapy .
serum lev levels of these patients were within or below the normal trough levels of serum lev and had no correlation with these adrs .
change in seizure frequency versus serum levetiracetam concentrations serum concentration of levetiracetam with and without antiepileptic drug comedication in the study subjects antiepileptic drug interactions among the study subjects
in this study , the serum lev levels ( nontrough ) showed a negligible negative correlation with the dose of lev given per kilogram ( kg ) bodyweight .
( n = 69 ) obtained a negligible positive correlation ( r = 0.29 ) while may et al .
( n = 297 ) obtained a moderately positive correlation ( r = 0.67 ) between trough serum lev concentration and dose per kg bodyweight .
although there exists a linear relationship between the dose and the trough serum levels of lev over a dose range of 5005000 mg , some studies have however suggested that serum levels of lev can get affected by a number of parameters such as age , bodyweight , comedication , hepatic , or renal insufficiency .
furthermore , the lev concentrations depend on the time interval from the last dose taken , and because of short half - life ( 68 h ) , large fluctuations are expected during the day ; as a result , nontrough levels of lev could not be well correlated with the dose .
serum levels of lev showed negligible positive correlation with the age of the patients as also seen by mathew et al .
this is in conjunction with a study conducted by may et al . according to which older patients need a lower lev dose per bodyweight than young adults to achieve comparable lev levels .
it has been concluded that the older adults have lower clearance ( cl ) than younger adults and therefore require a mean 40% lower dose of lev to achieve the same serum level .
age affect the apparent clearance of lev to the largest extent as shown by a 40% reduction in the elderly compared with the adults .
this difference in the serum concentration of lev can be due to the difference in the clearance rate of the drug from the kidney with age .
three studies done on the correlation of serum lev levels with gender came out with a conclusion that gender had no significant effect on the serum levels , and/or clearance of lev but another study found that females had 12% higher lev exposure than males . in the present study , only two patients ( one male and one female ) could be compared as blood samples for them were collected at the same time gap ( 3.5 h ) , where the female patient showed a higher concentration of lev as compared to the male patient .
this could be due to the differences in the bodyweight between the two genders ( males are heavier ) , but due to limited number of comparable subjects , no significant conclusions could be drawn as in regard to the effect of gender on serum lev levels .
radtke in his study ( n = 391 ) showed that any differences in the pharmacokinetic parameters ( if present ) are likely related to the differences in bodyweight of both the genders and show no differences when normalized for bodyweight . according to pigeolet et al .
( n = 524 ) bodyweight has a statistically significant effect on apparent plasma clearance of lev and its volume of distribution .
decreasing bodyweight from 70 kg to 40 kg increased lev exposure by 16% . in this study also , the serum levels were found to be higher in 60 kg patients as compared to > 60 kg patients , and the correlation between serum lev levels and bodyweight was negligibly negative against that seen by mathew et al .
( r = 0.12 ) in a study ( n = 24 ) , it was found that all pharmacokinetic parameters of both oral and liquid formulations of lev were bioequivalent and therefore no adjustment in dosage is necessary if a patient is switched from one formulation to another . in this study , all patients except two were on oral tablets .
the only patient on syrup was elderly ( 73 years ) and showed a higher concentration of lev which could be due to the age .
on the other hand , other patient ( age 65 years ) on intravenous injection had a high concentration which could also be due to the age or a higher dose [ table 3 ] .
therefore , no clear conclusion could be drawn of the effect of formulations on the serum concentration of lev .
all patients showed compliance to drug therapy as they had detectable levels of lev in their serum , but they may or may not be strictly adherent to the dosage schedule as these levels were measured at different time gaps from the drug intake and blood sample collection and vary widely with a range of 0.4102.2 g / ml . the values on the lower side of this range are well below the established lower trough range of lev ( before the scheduled next dose ) in the western studies ( 12 g / ml ) while patients still showing improvement in their seizure frequency .
there was no observed relationship of the clinical response seen in our patients with the serum concentration of lev .
this was similar to what was seen in the study conducted by sheinberg et al .
, ( n = 50 ) where 95% of the patients had more than a 50% reduction of seizure frequency ; only one patient had increased seizure frequency , and two had no change in seizure frequency after start of lev .
coadministration of enzyme inducer aeds reduced the serum concentration of lev as compared to the coadministration of neutral aeds whereas coadministration of va increased the lev concentration [ table 4 ] .
decrease in serum levels of lev occurs due to the increase in its clearance ( cl / f ) by concomitant use of enzyme inducer aeds which have an inducible effect on the enzymes ( plasma esterases , hydrolases ) involved in the lev metabolism . on the other hand , fat gain related to increased food consumption
increase in serum levels of lev with va occurs due to change in body water composition as lev dissolves in water .
the volume of distribution ( v / f ) of lev is decreased by va .
( n = 629 ) showed that drowsiness and psychiatric / behavioral effects were the most common adverse effects associated with lev use .
sheinberg et al . found that there was no relationship between serum concentrations of lev and adverse events . in this study
also , we found that the most common adrs were somnolence and aggressiveness and that they could not be correlated with the serum concentrations of lev .
there is no cut off limit of serum levels of lev in the literature above which toxicity appears .
only three patients showed serum levels of lev above upper limit of trough therapeutic range ( 46 g / ml ) .
as these levels have been obtained at 3 h ( 87.1 g / ml ) , 3.5 h ( 73.6 g / ml ) , and 14 h ( 102.2 g / ml ) gap of drug intake and blood sample collection , they may not suggest toxic levels even otherwise . despite being a pilot study with a small sample size
, the present study has shown that the serum lev levels are affected by the age , bodyweight and use of comedications and can also predict patient compliance . a study with a larger sample size in the future
although the purpose of the present project was to study the ranges of serum lev concentrations irrespective of the time gap between the last dose taken and the blood sample collection , a future study with blood samples collected at the same time after the drug intake , preferably the trough samples just before the next morning dose of lev , should be preferably done as lev shows variations in its serum levels throughout the day .
better comparisons can also be made if patients are enrolled with same total doses instead of different doses .
despite being a pilot study with a small sample size , the present study has shown that the serum lev levels are affected by the age , bodyweight and use of comedications and can also predict patient compliance . a study with a larger sample size in the future
although the purpose of the present project was to study the ranges of serum lev concentrations irrespective of the time gap between the last dose taken and the blood sample collection , a future study with blood samples collected at the same time after the drug intake , preferably the trough samples just before the next morning dose of lev , should be preferably done as lev shows variations in its serum levels throughout the day .
better comparisons can also be made if patients are enrolled with same total doses instead of different doses .
aed therapy is prescribed quite commonly at all levels of patient care , i.e. , both rural and urban clinical practice .
using newer aeds is quite costly but nevertheless necessary as regard to their advantages over the older well - established counterparts .
however , guided administration of these drugs is quite imperative for the purpose of rational prescribing and long - term patient care .
this study was done to find the lev concentrations irrespective of the timing of last doses for the purpose of population pharmacokinetic studies .
the study emphasizes that the tdm of newer aeds like lev should be done in routine clinical practice so as to adjust the dose according to age , bodyweight , and comedication and prevent its adverse effects .
more studies on tdm of newer aeds is required on a large number of indian patients to develop a pooled database for generating the pharmacokinetic data ranges for our population .
newer aeds should be prescribed based on the tdm data available so as to prevent the irrational practice of medicine , complications of adrs , toxicity , and various drug - drug interactions .
|
introduction : with the advancement of therapeutics , newer antiepileptic drugs ( aeds ) like levetiracetam ( lev ) , with good therapeutic efficacy and tolerability are available .
but unfortunately , therapeutic drug monitoring is not routinely done in india for these drugs.objectives:the objective of this study is to determine the range of serum levels of lev in patients at stabilized doses and correlate them with their clinical course.materials and methods : patients with epilepsy and started on lev were enrolled from the neurology department after the ethics committee approval .
serum levels of lev were estimated using high - performance liquid chromatography and correlated with patient demographics , dosage , dosage forms , concomitant aeds , compliance of the patient , therapeutic effect , adverse drug reactions ( adrs ) , and suspected toxicity.results:serum levels of lev ranged from 0.4 to 102.2 g / ml at different time points and demonstrated a negligible positive correlation with age of the patients ( r = 0.12 ) but negligible negative correlation with bodyweight ( r = 0.19 ) .
no conclusive relationship could be established for dose , gender , dosage forms , clinical efficacy ( seizure frequency ) , adrs , and toxicity .
compliance was verified in all the patients .
levels were found to reduce with the use of concomitant enzyme inducer drugs ( 56.78% ) whereas increase with valproic acid ( 7.8%).conclusion : these findings emphasize the need for monitoring the serum levels of newer aeds like lev considering the various parameters studied here , so as to maintain the therapeutic efficacy by preventing under or over dosage and to generate a broader database of serum levels of lev in the indian population to help appropriate prescribing with more confidence .
|
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uremia related , non -traditional risk factors , such as inflammation , oxidative stress , dyslipidemia , vascular calcification alterations in calcium and phosphorus ( p ) metabolism , have been proposed to play a central role ( 2 ) .
elevated plasma beta2 microglobulin ( 2 m ) , is a well - known characteristic of chronic renal failure .
predialysis serum 2 m level predicted mortality and increase of 2 m clearance during hd was associated with improved outcomes ( 3 ) .
the source of the elevated serum 2 m in hemodialysis patients , has not been explained absolutely .
there is controversy as to whether elevated levels are caused predominantly by increased synthesis , the use of membranes in hemodialysis with different clearance capacities , or diminished renal elimination .
use of middle and high - flux biocompatible membranes was shown to be associated with a notable reduction 2 m ( 4 ) .
the surface of lymphocytes and monocytes are particularly rich in 2 m , the latter being synthesized to large amounts by lymphocytes and regulated by interferons and proinflammatory cytokines ( 5 ) , which might explain the pathophysiological role in atherosclerosis . but remains to be further clarified if 2 m is solely a marker of inflammation or if it has a direct pathogenic effect and if other yet unknown confounders may influence 2 m levels ( 6 ) .
the uremic state is associated with an altered immune response , and intermittent stimulation by endotoxins originating from the dialysis water supply and artificial vein grafts or bio incompatibility caused the increased circulating inflammatory proteins , such as c - reactive protein ( crp ) ( 7 ) , produced by the liver , mainly in response to interleukin -6 ( il-6 ) .
hemodialysis patients , exhibit significant alterations in lipoprotein metabolism , which in their most advanced form result in the development of severe dyslipidemia .
lipid disorders stem largely from dysregulation of high density lipoprotein ( hdl ) and triglyceride - rich lipoproteins metabolism .
the down regulation of the expression of several genes along with the changes in the composition of lipoprotein particles and the direct inhibitory effect of various uremic
toxins on the enzymes involved in lipid metabolism , represent the most important pathophysiological mechanisms underlying the development of hypertriglyceridemia ( 8) .
several mechanisms , working in concert , may underlie the reduction in hdl levels , which is usually indicative of impaired reverse cholesterol transport .
thus , uremic patients exhibit decreased levels of apolipoproteins ai and aii , diminished activity of lecithin - cholesterol acyl - transferase , as well as increased activity of cholesteryl ester transfer protein that facilitates the transfer of cholesterol esters from hdl to triglyceride - rich lipoproteins , thus reducing the concentration of hdl ( 8) .
lipoprotein(a ) [ lp(a ) ] is an independent risk factor for clinical events attributed to atherosclerotic cardiovascular disease in chronic hemodialysis .
lp(a ) levels are frequently elevated in hd patients ( 9 ) and are considered a major risk factor for cardiovascular disease ( 10 ) .
secondary hyperparathyroidism , is one of the major complications of patients in chronic hemodialysis ( 11 ) .
parathyroid hormone ( pth ) starts to rise very early in the course of kidney disease .
as disease progresses , plasma levels of vitamin d and calcium begin to decline , thus contributing to greater secretion of pth . in addition
, the retention of phosphate further increases pth secretion independent of calcium and vitamin d levels .
previous studies have supported the view that high pth serum levels in uremic patients may cause deleterious effects in myocardium metabolism and function ( 12 ) .
the aim of this study is to investigate the association of 2-m with inflammatory markers , dyslipidemia and mineral disorders in high - flux membrane hemodialysis patients .
in study were included 40 patients , undergoing maintenance high - flux membrane hemodialysis treatment in the clinical centre in prishtina , for a period longer than 6 months .
a ) based in patient s history , angina , possible myocardial infarction , cerebrovascular events , infective diseases and cancer , were excluded . in all patients , 2-m , crp , il-6 , triglycerides , cholesterol , ldl , hdl , lp(a ) , pth , calcium ,
triglycerides , cholesterol , hdl , ldl , calcium , phosphorus and albumin were measured by biochemical analysis ; b ) the assay of -2 microglobulin is based on a latex enhanced immunoturbidimetric method .
c ) crp was determined by the turbidimetric method , il-6 with enzyme linked immunosorbent immunoassay ( elisa ) , lipoprotein ( a ) with immunoturbidimetric method depth with a chemiluminometric immunoassay ; d ) twenty - four healthy subjects ( 12 women and 12 men , aged 56.08 12.34 years ) served as controls .
serum concentration of 2 m , crp , il-6 , triglycerides , lp(a ) , p and pth in hemodialysis patients were significantly higher than in controls ( 19.84 2.23
mg / l vs 2.11 1.0 mg / l , p<0.001 ; 39.75 29.7 mg / l vs 16.25 3.78mg / l , p<0.001 ; 3.06 1.08 pg / ml vs 0.35 0.3 pg / ml , p < 0.001 ; 3.17 1.28
mmol / l vs 1.19 0.38 mmol / l , p<0.001 ; 40.25 12.98 mg / dl vs 24.33 8.51 mg / dl , p<0.001 ; 1.9 0.6
mmol / l , p<0.001 ; 137.35 50.7 pg / ml vs 46.56 28.64
the concentration of hdl and serum albumin was significantly lower ( 1.14 0.34 vs 1.52 0.35 mmol / l , p<0.001 ; 34.83 3.89
g / l vs 40.5 3.94 g / l , p<0.001 ( table 1 ) .
we did not find any difference in cholesterol , ldl and calcium levels between two groups ( 4.0 1.11mmol / l vs 4.26 0.16
mmol / l , p=0.16 ; 2.90 1.14 mmol / l vs 2.41 0.57
mmol / l , p<0.05 ; 2.2 0.24 mmol / l vs 2.26 0.25 mmol / l , p = 0.11 ( table 1 ) . biochemical parameters in hemodialysis patients and controls 2 m
was inversely associated with hdl , albumin and calcium concentration ( r = -0.73 , p < 0.001 ; r = -0.53 , p < 0.001 ; r = -0.50 , p < 0.01 ( table 2 ) , whereas positively was associated with triglycerides , p and pth ( r= 0.69 , p < 0.001 ; r= 0.53 , p < 0.001 ; r=0.58 , p < 0.05 ( table 2 ) .
the patients with high 2 m values , simultaneously had higher lp(a ) concentrations , but we did not observe significant correlation ( r=0.28 ( table 2 ) .
we also found low positive correlation of 2 m with age ( r=0.46 , p < 0.001 ( table 2 ) .
2 m levels were upper in patients with high crp levels , but there was no significant relationship between crp or il-6 and 2 m ( r= 0.11 , p <
0.001 ; r= 0.23 , p < 0.001 ( table 2 ) . there was no correlation of 2 m with cholesterol and ldl cholesterol ( r = 0.13 , p < 0.001 ; r = 0.18 , p < 0.001 ( table 2 ) .
positive correlation exists between crp and il-6 ( r=0.94 , p < 0.001 ( table 2 ) .
albumins correlated negatively with il-6 and lpa ( r = -0.60 , p < 0.001 ; r=-0.72 , p<0.005 ( table 2 ) .
correlation between serum 2 m levels with selected biochemical parameters during the follow - up period of three years , 6 out of 40 patients had died , from cardiovascular events .
significant correlation exists between b2 m and glomerular filtration rate even when renal function was only slightly impaired .
liabeuf , et al , reported plasma 2 m level to be a predictor of cardiovascular events and mortality in patients with different stages of chronic kidney disease ( 14 ) .
we examined the impact of 2 m on some cardiovascular risk factors in high - flux membrane hemodialysis patients . a correlation between an inflammatory response during hemodialysis and elevated serum 2 m has been described ( 15 ) .
compared with the controls , hd patients exhibited marked elevation of serum 2 m , crp and il-6 ( 19.84 2.23 mg
29.7mg / l vs 16.25 3.78mg / l , p<0.001 ; 3.06 1.08 pg / ml vs 0.35 0.3 pg / ml , p < 0.001 ( table 1 ) .
but we found no relationship between crp or il-6and 2 m ( r= 0.11 , p < 0.001 ; r= 0.23 , p < 0.001 ( table 2 ) when high - flux membranes are used .
( 16 , 17 , 18 ) . even in studies , where the low flux membranes were used
our findings suggested that plasma b2 m level is directly correlated with some metabolic and cardiovascular risk factors .
triglycerides start to increase in early stages of chronic kidney disease and show the highest values in dialysis patients .
hemodialysis patients presented increased triglycerides compared with controls ( 3.17 1.28 mmol / l vs 1.19 0.38 mmol / l , p<0.001 ( table 1 ) .
we find significant positive correlation between triglycerides and 2 m levels ( r= 0.69 , p < 0.001 ( table 2 ) .
hdl concentrations exhibited significant reduction in patients compared to controls ( 1.14 0.34 vs 1.52 0.35 mmol / l , p<0.001 ( table 1 ) .
serum 2 m concentrations were inversely associated with hdl ( r = -0.73 , p<0.001 ( table 2 ) .
supporting , linear regression analysis confirmed the negative impact of 2 m concentrations on hdl level .
lp(a ) start increasing early during the course of chronic kidney disease and becomes pronounced with increasing severity of disease .
lp(a ) levels were significantly higher in patients than in controls ( 40.25 12.98 mg / dl vs 24.33 8.51 mg / dl , p<0.001 ( table 1 ) , which confirm that kidney have an important role in lp(a ) metabolism .
the patients with high 2 m values , simultaneously had higher lp(a ) concentrations but we did not observe high correlation ( r=0.28 , p<0.05 ( table 2 ) .
the negative correlation of lp(a ) with albumin ( r=-072 , p<0.005 ( table 2 ) , suggests that the mechanism behind the increased lp(a ) levels may be related to the protein losses , perhaps via an increased synthesis rate of apolipoprotein ( a ) in the liver or via decreased lp(a ) catabolism in hd patients .
we found a significant indirect relationship between 2 m and albumin ( r=-0.53 , p<0.001 ( table 2 ) .
this correlation may be used to identify the patients at high risk , from cardiovascular disease ( 22 ) .
mineral and bone disorder is a growing health care concern associated with secondary hyperparathyroidism , mineral abnormalities , and increased risk of cardiovascular disease ( 23 ) . in hemodialysis patients , concentrations of pth and p
were significantly higher compared with controls ( 137.35 50.7 pg / ml vs 46.56 28.64
pg / ml , p<0.001 ; 1.9 0.6 mmol / l vs 1.26 0.33
calcium levels were lower in patients compare with controls , but with no significant difference ( 2.2 0.24
mmol / l vs 2.26 0.25 mmol / l , p = 0.11 ( table 1 ) .
mineral and bone disorders are associated with accelerated atherosclerosis ( 24 ) , which is an important cause of cardiovascular death in chronic hemodialysis ( 25 ) . in this study ,
serum concentration of 2 m positively correlated with p and pth ( r= 0.53 , p < 0.001 ; r=0.58 , p < 0.05 ( table 2 ) , whereas negatively with calcium ( r = -0.50 , p < 0.01 ( table 2 ) , which proves that 2 m has direct impact in mineral disorders and cardiovascular risk in hd patients . it has been suggested that the predictive value of serum 2 m concentration is superior to that provided by established prognostic factors for mortality , such as glomerular filtration , cystatin c and crp ( 26 ) . during the follow - up period of three years , 6 out of 40 patients had died from cardiovascular events .
even if there was no correlation of 2 m with inflammation , in high - flux membrane hemodialysis patients , our finding indicates that 2 m might have an important role in the development of cardiovascular diseases .
|
background : higher than expected cardiovascular mortality in hemodialysis patients , has been attributed to dyslipidemia as well as inflammation .
beta2-microglobulin ( 2 m ) is an independent predictor of outcome for hemodialysis patients and a representative substance of middle molecules.results:in 40 patients in high - flux membrane hemodialysis , we found negative correlation of 2 m with high density lipoprotein ( r=-0.73 , p<0.001 ) and albumin ( r= -0.53 , p<0.001 ) and positive correlation with triglycerides ( r=0.69 , p<0.001 ) , parathyroid hormone ( r=0.58 , p < 0.05 ) and phosphorus ( r= 0.53 , p<0.001 ) .
there was no correlation of 2 m with c- reactive protein ( crp ) and interleukin-6 ( il-6 ) . during the follow - up period of three years
, 6 out of 40 patients have died from cardiovascular events.conclusion:in high - flux membrane hemodialysis patients , we observed a significant relationship of 2 m with dyslipidemia and mineral bone disorders , but there was no correlation with inflammation .
|
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the experimental models of myocardial infarction have largely contributed to a better understanding of the pathophysiology of myocardial infarction . studies on large animal models revealed many mechanisms involved in myocardial injury and repair [ 1 , 2 ] . in the last few years , transgenic mice and gene targeting technologies allowed profound studies of the underlying pathomechanisms . for this purpose , we developed murine models of myocardial ischemia and reperfusion and used them to study the mechanisms involved in ischemic myocardial injury [ 3 , 4 ] .
we described pathological features of reperfused myocardial infarction in mice , that is , rapid formation of granulation tissue and subsequent development of a stable scar .
our studies also revealed an important role for macrophages and their associated inflammatory and remodeling - related mediators [ 5 , 6 ] .
a profound understanding of these mechanisms is crucial for projects utilizing novel cellular therapies , since the local microenvironment seems to exerts a strong influence on the cells applied in our model of myocardial cryoinfarction [ 79 ] .
this study compares the pathology and the sequence of cellular and molecular events in the two mechanistically different lesion models of myocardial cryoinfarction and reperfused infarction and reveals substantial differences in myocardial remodeling between them .
both models show a transient inflammatory response associated with induction of chemokines , cytokines , and remodeling - related mediators .
the cryolesioned heart showed a prolonged remodeling with postponed development of granulation tissue and scar formation , which was associated with persistent macrophage infiltration in the injured tissue when compared to reperfused infarction .
wild - type c57/bl6-mice ( charles river , sulzfeld , germany ) of 18 to 25 g weight and 8 to 10 weeks old were used in our infarction models .
group size in reperfused infarction model was n = 8 mice and in cryoinfarction n = 6 mice .
all experiments were performed in accordance with an animal protocol approved by the local governmental authorities . in an initial surgery
( 2.5 g / g ; merial , halbermoos , germany ) as previously described .
then , mice were intubated with a pe-90 tube ( becton dickinson , sparks , md , usa ) connected to a small animal respirator ( rodent ventilator mod .
millis , ma ) and ventilated at a frequency of 110/min with tidal volume of 0.25 ml .
pericardium was dissected and partially removed , and a prolene 8 - 0 suture ( ethicon , norderstedt , germany ) was placed around the left descending coronary artery ( lad ) .
suture ends were threaded through a sterile pe-10 tube ( becton dickinson ) of 3 mm of length , exteriorized through the thoracic wall , and then stored subcutaneously .
the thorax was closed with interrupted prolene 6 - 0 stiches , and the skin was closed with a running prolene 6 - 0 suture . at the end , metamizol ( 100 mg / kg ; novalgin ) was given for analgesia in a mixture with cefuroxim as antibiotic prophylaxis i.p .
( 100 mg / kg , zinacef ; bristol - myers squibb , munich , germany ) .
the skin was reopened , and the ends of the lad - ligature were connected to heavy metal picks . pulling on the ligature ends induced lad - occlusion , and this ischemia was maintained for 60 minutes .
after removal of the lad - ligature , the blood flow was restored in the reperfusion .
ecg monitoring of einthoven lead ii during and after lad - occlusion confirmed successful ischemia and reperfusion .
one - hour ischemia was followed by reperfusion for 6 hrs , 1 , 3 , 7 , and 14 days .
mice were euthanized using an overdose of pentobarbital i.p . ; hearts were excised and fixated in zinc - paraformaldehyde ( z - fix , 4% ; anatech , battle creek , mi , usa ) for histology or stored in rna - later solution ( qiagen , hilden , germany ) for mrna - studies .
animals were sedated by brief exposure to narcotic gas containing 50% o2 , 50% n2o , and 3 to 4 vol-% of isoflurane as previously described .
thereafter , mice were positioned on a temperature - controlled plate ( 37c ) in supine position , intubated , and ventilated using a small animal respirator ( harvard apparatus ) as described in the previous section .
an anterolateral skin incision was performed 5 mm above the costal margin , the anterolateral thoracic muscles were transected , and the thorax was opened in the fifth intercostal space . the pericardium was opened , and the apex of the heart was exposed
cryocoagulation was performed by placement of a copper probe ( 3 mm diameter , cooled in liquid nitrogen for 2 min ) to the free left ventricular wall ( 3 times for 20 seconds ) in order to achieve reproducible , large transmural myocardial lesion .
after induction of the cryoinfarction , a 22-g chest drain ( optiva , johnson & johnson , new brunswick , nj , usa ) was inserted into the left pleural cavity to prevent pneumothorax .
the thorax was closed with prolene 6 - 0 sutures , and air was drained using negative pressure on chest drain before extubation .
, the hearts were excised after the above - mentioned time periods and further processed for histological and molecular analysis . after excision , hearts were washed in cardioplegic solution containing 4 g nacl , 3.73 g kcl , 1 g nahco3 , 2 g glucose ( all from berlin chemie , berlin , germany ) , 3 g 2,3-butandion monoxime ( sigma - aldrich , munich , germany ) , 3.8 g ethylenglycol tetra acetic acid ( sigma ) , 0.2 mg nifedipine ( sigma ) , and 10 ml heparin ( 1000 iu / ml ; ratiopharm , ulm , germany ) , all of which were dissolved in 1 l of isotonic nacl ( berlin chemie ) .
hearts were fixated in zinc - paraformaldehyde for 24 hrs and further processed using standard paraffin embedding .
hearts where cut from basis to apex , and at every 250 m , a set of ten 5 m sections were mounted on glass slides .
myocardial sections below insertion of the papillary muscles were further used for histology and immunohistochemistry .
photographic images were recorded on a computer system equipped with a digital camera ( dp70 , olympus , hamburg , germany ) , and planimetric evaluation was performed using analysis software ( olympus ) .
picrosirius red staining of total collagen was used to evaluate development of fibrosis in the myocardial scar .
cell density was measured by cell count of immunohistochemically stained cells in infarcted myocardium and was expressed as number per mm .
we used the following primary antibodies for immunohistochemistry : alpha smooth muscle actin mouse monoclonal antibody ( clone 1a4 ; sigma , st .
louis , mo , usa ) for myofibroblasts , f4/80 rat monocyte / macrophage antibody ( serotec , duesseldorf , germany ) for macrophages and mca 771 g rat monoclonal antibody for neutrophils ( serotec ) .
furthermore , samples were stained for macrophage maturation marker osteopontin-1 using a goat polyclonal antibody ( p-18 , santa cruz , heidelberg , germany ) and for macrophage elastase using a rabbit monoclonal anti - mmp12 antibody ( abcam , cambridge , uk ) .
immunohistochemical staining was performed using appropriate vectastain elite abc kits and diaminobenzidine ( axxora , loerrach , germany ) .
excised hearts were cleared of large vessels and atria and placed in rna - later solution ( applied biosystems , foster city , ca , usa ) .
hearts were minced using a tissue tearor ( tissue tearor modell 398 ; biospec , bartlesville , ok , usa ) , and mrna was isolated using standard phenol / chloroform extraction method ( trizol , applied biosystems ) .
mrna was transcribed into cdna using high - capacity cdna transcription kit ( applied biosystems ) with random hexameric primers as described in the manufacturer 's protocol .
the mrna - expression was determined by taqman real - time quantitative pcr ( rt - qpcr , applied biosystems ) .
cdna was diluted 1/10 and then used for measurement of gene induction according to the manufacturer 's instructions on an abi prism 7900ht sequence detection system using sds2.2 software ( applied biosystems ) .
all murine primers were commercially available and measured with fam tamra chemistry using the relative standard curve method . at the end of rt - qpcr cycle , dissociation curve analysis was performed to ascertain the amplification of a single pcr product .
two - tailed , unpaired student 's t - test was used to determine a significant difference between two groups .
histopathological features of cryoinfarction and reperfused myocardial infarction in our model of cryoinfarction showed a transmural lesion with sharp infarction borders early after injury .
the cryoinjury presented after 3 days with extensive cellular infiltration and partial granulation tissue formation starting at the periphery of infarction ( figure 1(a ) ) .
the central part of the cryoinjury at the copper probe application site contained numerous dead cardiomyocytes and cellular debris .
the formation of loose granulation tissue was almost finished after 7 days ( figure 1(b ) ) .
the subsequent scar formation was largely completed after 14 days , but a substantial cellularity was still present in some parts of the cryoinjury whereas revascularization of large vessels was not observed ( figure 1(c ) ; arrow ) .
in contrast , the reperfusion of myocardial infarction in our closed - chest model led to a nontransmural lesion .
complete formation of granulation tissue was found after 3 days ( figure 1(d ) ) and followed by a compacted scar formation with low cellular content after 7 days reperfusion ( figure 1(e ) ) , as we published before .
the cryoinfarction showed large areas with loose collagen deposition after 7 days ( figure 1(f ) ) in contrast to compacted collagen - rich scar in reperfused infarction at the same time point ( figure 1(g ) ) .
this difference in scar formation was further underlined by a vast number of myofibroblasts in cryoinfarction after 7 days ( figure 1(h ) ) whereas only few myofibroblasts were found in the reperfused infarction ( figure 1(i ) ) .
the collagen was largely compacted in cryoinfarction after 14 days , but this was still associated with low myofibroblast persistence in the scar .
the cryoinjury led to prolonged scar formation probably due to a longer time period needed for debris clearance , which could only progress from the periphery of the injury due to completely cryodamaged vasculature in the scar .
in contrast , the reperfusion of myocardial infarction was associated with rapid scar formation due to an intact vascular network , which provided synchronous cell migration into the entire infarcted area .
immunohistochemical staining of neutrophils revealed a strong myocardial infiltration early after the injury , suggesting a comparable myocardial debris clearance in both models .
we found a significant difference in neutrophils density in infarcted area between the two models after 3 days ( figures 2(a ) and 2(b ) ) and slightly longer neutrophils persistence in cryoinfarctions after 7 days ( figure 2(c ) ) .
we found no difference in cellular density of neutrophils in noninfarcted remote myocardium in both models ( figure 2(d ) ) .
macrophage staining showed a persistent , strong infiltration of cryoinjured myocardium after 7 days whereas only few f4/80-positive cells were found after 7 days reperfusion of infarction , as previously published ( figures 3(a ) and 3(b ) ) .
we investigated macrophage activity using elastase staining after 7 days and found very strong signals in cryoinfarction , but no signals in reperfused infarction ( figures 3(c ) and 3(d ) ) .
interestingly , after 14 days , cryoinfarction showed much lower elastase activity , even though the total macrophage cell density was still high .
the macrophage cell density data strongly support a short , rapid course of tissue remodeling after reperfusion of murine infarction , in contrast to a persistent macrophage infiltration even 14 days after cryoinfarction ( figure 3(e ) ) . to our surprise ,
macrophage density was also significantly higher in the remote , noncryoinjured myocardium , thus suggesting a dysfunctional development of the infarction border zone in this model ( figure 3(f ) ) .
we further investigated the maturation of macrophages and found after 7 days a stronger infiltration of osteopontin-1-positive cells in cryoinfarcted hearts than in reperfused myocardial infarction ( figures 4(a ) and 4(b ) ) .
evaluation of osteopontin-1-positive cell density revealed a prolonged , but functional maturation of macrophages ( figure 4(c ) ) .
the cell density of osteopontin-1-positive cells in the noninfarcted area was also comparable between the models ( figure 4(d ) ) .
taken together , the course of cellular events involves a prolonged infiltration of cryoinfarction with inflammatory cells , thereby explaining the later development of a stable scar in this model .
in contrast , a transient , short infiltration of reperfused myocardial infarction with inflammatory cells is associated with a rapid resolution of myocardial remodeling .
the course of molecular events in cryoinfarction and reperfused infarction was assessed using mrna - expression measurements of specific inflammatory and remodeling - related mediators .
the myocardial damage leads to a rapid production of reactive oxygen species and induction of several scavenger enzymes , for example , heme oxygenase 1 , glutathione peroxidase , and so forth .
the expression of heme oxygenase 1 in reperfused myocardial infarction accompanied the transient short inflammatory reaction with a peak induction after 24 h of reperfusion and a rapid downregulation thereafter ( figure 5(a ) ) .
the cryoinfarction led to a significantly different expression pattern with prolonged induction of heme oxygenase 1 .
the cytokine tnf- , the next downstream mediator in the postischemic inflammatory cascade , presented with a significantly stronger mrna - induction early after reperfusion of infarction , which is probably triggering the rapid onset of inflammatory reaction ( figure 5(b ) ) .
tnf--expression decreased thereafter in reperfused infarction , in contrast to its maximal upregulation 3 days after cryoinfarction .
we measured the induction of the anti - inflammatory cytokine il-10 to investigate negative feedback regulation of proinflammatory response ( figure 5(c ) ) and found a comparable time course in both models with a significantly higher induction of it in cryoinfarction after 3 days .
based on our previous results regarding the role of chemokines in remodeling , we measured the expression of macrophage - related ccl2 and ccl4 , as well as neutrophils - related ccl3 . to our surprise
, we found a significantly higher induction of ccl2 in reperfused infarction whereas cryoinfarction did not lead to its significant upregulation at a later time point ( figure 5(d ) ) .
in contrast , the mrna - expression of ccl3 showed a significantly stronger early induction in cryoinfarction and a second maximum after 3 days when compared to reperfused infarction ( figure 5(e ) ) .
the chemokine ccl4 showed a comparable expression pattern between the models , but also significant differences at an early and later time point ( figure 5(f ) ) .
taken together , the expression of free radical scavenger enzymes , cytokines and chemokines , is directly influencing the course of cellular events in both models of infarction . still , the expression of chemokines shows a different time course , probably due to their additional role in collagen deposition during advanced stages of remodeling .
since osteopontin-1 is not only related to macrophage maturation but also is involved in extracellular matrix formation and remodeling , we measured its mrna - expression and found an earlier peak after 24 hrs in reperfused infarction whereas in cryoinfarction its maximum was reached after 3 days ( figure 6(a ) ) .
another marker of early remodeling , tenascin c , showed a significantly higher induction early after reperfusion of infarction , thus supporting the rapid granulation tissue formation ( figure 6(b ) ) .
tenascin c - expression was significantly higher after 3 days in cryoinjured myocardium , thus mediating the prolonged granulation tissue formation .
we also investigated the mrna - expression of tgf- and found a later induction of predominantly profibrotic acting tgf-1 and -2 isoforms ( figures 6(c ) and 6(d ) ) .
interestingly , we found a significantly higher induction of rather antifibrotic acting tgf-3 isoform 7 days after cryoinfarction , thus probably representing a strong signal for the resolution of myocardial remodeling ( figure 6(e ) ) .
these data reveal specific mediators responsible for the prolonged granulation tissue formation and delayed remodeling process after cryoinfarction .
our data also confirm our previously published findings on induction of tgf- isoforms in reperfused infarction and give us additional novel insights in osteopontin-1- and tenascin c - expression in the reperfused infarction model .
studies using large experimental models of myocardial infarction brought a detailed analysis of pathology and some insights into mechanisms involved in myocardial ischemia .
recent developments in transgenic mice and gene - targeting technologies provided tools for profound studies of the pathomechanisms and specific genes involved in cardiac repair .
we developed a chronic , closed - chest murine model of myocardial ischemia followed by reperfusion , which leads to infarction with rapid scar formation .
this closed - chest model has minimized the influence of the initial surgery trauma and is , therefore , particularly useful for studies of inflammatory response and early remodeling .
we used this model in cellular therapy experiments and found a very poor engraftment of the i.v .
injected whole bone marrow cells ( unpublished observation ) in contrast to a very good engraftment found in another model , that is , cryoinfarction .
therefore , we assumed differences in myocardial remodeling and specifically in local microenvironment of injured myocardium . in order to investigate these differences
, we compared the pathology and the course of cellular and molecular events during myocardial remodeling between the two models . in this study , we confirmed our previous findings on rapid development of granulation tissue and the time course of mediators involved in transient inflammatory reaction , as well as in subsequent remodeling [ 3 , 4 ] .
we found a transient , strong increase in proinflammatory cytokines and chemokines leading to a rapid tissue infiltration with neutrophils and macrophages , which was also described in large animal models .
in addition , we observed a transient induction of heme oxygenase 1 , which is caused by a massive production of reactive oxygen species during early reperfusion and precedes the cytokine release in the ischemic heart .
development of granulation tissue was associated with anti - inflammatory action of il-10 , with transient upregulation of early remodeling mediator tenascin c , and was followed by induction of macrophage maturation marker osteopontin-1 and , as previously published , upregulation of tgf- isoforms .
these mediators lead to differentiation of myofibroblasts and subsequent collagen deposition , thus resulting in the stable scar formation 7 days after reperfusion of murine infarction .
the timely resolution of myocardial remodeling is also confirmed by a lack of macrophage elastase and osteopontin-1 staining after 7 days reperfusion .
the pathology of cryoinfarction showed a similar course of events , but a longer duration of this process until formation of a stable myocardial scar is completed .
the basic histology revealed that cryoinfarction leads to an early , strong cellular infiltration of the damaged myocardium , which slowly decreased to a lower level until 14 days postinjury .
we observed a permanent damage and occlusion of the large vessels in cryoinjured myocardium , which mechanically prevented an evenly distributed cellular infiltration of the injured myocardium and thus rapid myocardial remodeling , as it is observed in reperfused infarction . in cryoinfarction ,
macrophages and neutrophils persist up to 14 days in the last area near the center of the epicardial copper probe application site .
this seems to be one of the main factors influencing the pace of remodeling in the cryoinfarction model .
interestingly , we found a significantly prolonged macrophage infiltration of the noninjured area , which may be associated with a dysfunctional border zone formation and infarction limitation in this model .
the concomitant appearance of elastase - producing macrophages and myofibroblasts 7 to 14 days postinjury represents an active myocardial remodeling process , which is further supported by not entirely compacted collagen in the scar . in consequence ,
this active myocardial remodeling is probably responsible for a good engraftment of the implanted cells in the cryoinfarction model .
the mrna data provide novel insights into the time course of the expression of several mediators involved in remodeling of cryoinfarcted myocardium .
the later induction of cytokines and remodeling - related mediators is leading to the prolonged remodeling in this model .
in particular , the relatively high expression of heme oxygenase 1 after 3 days represents persistent reactive oxygen species production from cell debris , while the high ccl2 and ccl4 expression characterize the inflammatory response associated with strong macrophage activity .
the concomitant upregulation of anti - inflammatory il-10 is acting towards resolution of the inflammatory reaction and formation of granulation tissue .
the significantly higher expression of neutrophils - related chemokine ccl3 seems to reveal a strong migration stimulus into the damaged area , which is limited by the lack of intact vasculature as discussed above .
chemokines , particularly the ccl2 , have also been associated with myofibroblasts activity and collagen production , which may provide additional explanation for its upregulation after 3 days in cryoinfarction . using transgenic mice
, we described a crucial role for the chemokine ccl2 and macrophages in timely resolution of myocardial remodeling and preservation of myocardial function .
the later induction of tenascin c and osteopontin-1 as well as the tgf- isoforms represent the prolonged extracellular matrix production and collagen deposition in myocardial remodeling .
this study shows substantial differences in local microenvironment and cellular and molecular events between the models of cryoinfarction and reperfused infarction . in a comparison between murine models of cryoinfarction and coronary ligation without reperfusion ,
a modest adverse remodeling was postulated for the cryoinfarction , and the cryoinfarction was suggested as a representative model for myocardial infarction encountered in clinical practice .
we do not completely share this opinion , since the main goal in treatment of acute infarction is early revascularization with reperfusion .
but for the experimental study purpose , the cryoinfarction indeed offers a very reproducible area at risk and infarct size whereas this is a major weakness of the reperfused infarction model due to the anatomical variability of coronary arteries .
the prolonged myocardial remodeling seems to be beneficial in cell transplantation studies , since the cellular engraftment after direct application into injury is very good .
our data provide novel insights into expression of cytokines and chemokines in cryoinfarction , where persistent proinflammatory milieu may be favorable for the cell engraftment . on the other side ,
the rapid resolution of inflammatory response in reperfused infarction may minimize the time span of suitable local environment for cell engraftment , but this concept still has to be further investigated . in respect to the ongoing clinical trials ,
the reperfused infarction model could be seen as a more relevant model , but it has not been widely used yet .
first , the findings are mainly of experimental interest since the model of reperfused myocardial infarction is comparable to a clinical situation in patients presenting with acute infarction , but it seems to be less favorable for the cell engraftment studies .
on the other hand , the cryoinfarction model provides good conditions for cell engraftment and it is very reproducible , but of a limited value to the clinical practice .
second , we did not measure infarct size and one could argue that a difference in infarct size may explain the observed differences in pathological and molecular events between the two models . based on previously published work , the model of reperfused infarction [ 4 , 5 ] affects mostly a larger portion of left ventricle than the cryoinfarction , but the reperfusion still leads to a faster resolution of inflammation and scar formation than the cryoinjury .
therefore , the infarct size does not reflect the quality and pace of myocardial remodeling , and we did not found it to be necessary for explanation of the findings in this study .
in conclusion , the cryoinfarction is associated with prolonged inflammatory response leading to a postponed granulation tissue formation and scar development , when compared to the reperfused myocardial infarction .
several inflammatory mediators and remodeling factors are involved in this process , and they all contribute to a specific , dynamic local environment in ischemic myocardium .
these substantial differences in remodeling may affect and even be favorable for cellular engraftment and should therefore be considered in cell therapy studies .
|
myocardial infarction is associated with inflammatory reaction leading to tissue remodeling .
we compared tissue remodeling between cryoinfarction ( cmi ) and reperfused myocardial infarction ( mi ) in order to better understand the local environment where we apply cell therapies .
models of closed - chest one - hour ischemia / reperfusion mi and cmi were used in c57/bl6-mice . the reperfused mi showed rapid development of granulation tissue and compacted scar formation after 7 days .
in contrast , cmi hearts showed persistent cardiomyocyte debris and cellular infiltration after 7 days and partially compacted scar formation accompanied by persistent macrophages and myofibroblasts after 14 days .
the mrna of proinflammatory mediators was transiently induced in mi and persistently upregulated in cmi .
tenascin c and osteopontin-1 showed delayed induction in cmi . in conclusion ,
the cryoinfarction was associated with prolonged inflammation and active myocardial remodeling when compared to the reperfused mi .
these substantial differences in remodeling may influence cellular engraftment and should be considered in cell therapy studies .
|
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the health of the elderly has elicited considerable social attention . with the social and economic development ,
health care conditions and living standards improve continuously , thus improving the physical health of the elderly and prolonging their life span . according to statistics from the chinese ministry of health , in 60 years of development ,
the average life span has increased by 39.5 years , reaching 74.8 years old in 2010 ( 1 ) . however , mental health problems of the elderly are increasingly becoming prominent .
serious psychological problems not only hurt physical and mental health and family happiness of the elderly , but also cause various social problems .
therefore , studying the mental health of the chinese elderly is important . since the reform and opening - up policy ,
china has sacrificed high environmental cost for rapid economic development , consequently bringing serious threats to public health .
approximately 300,000 to 500,000 people are killed early by indoor air pollution every year in china ( 3 ) .
however , controversy has arisen in the academe concerning the effect of air pollution on mental health .
the tradeoff between rapid economic development and environmental degradation has complicated this controversy ( 4 ) .
economic development improves the quality of living and social welfare , and thereby , improves the mental health of the public .
however , with the further worsening of environmental problems , its negative effects on people s mental health have gradually surfaced .
such a complicated relationship introduces difficulties in comprehending the influence of air pollution and other environmental problems on mental health . meanwhile , china is a country with an outstanding urban - rural dual structure .
significant differences occur between urban and rural regions in terms of medical insurance , living standards , social culture , and lifestyle .
whether those differences will be reflected on the mental health level of the elderly in urban and rural areas is an important question to be considered when studying the mental health of chinese elderly .
thus , this paper addresses three problems by using data from china , namely , 1 ) will air pollution influence the mental health of the elderly ?
2 ) is mental health different between the elderly from urban and rural regions ? in what ways ?
medical and sociological research reported that the so2 concentration in air would threaten physical health and even cause respiratory system diseases ( 5 ) .
being healthy is not only being free from diseases or weakness , but also the perfect combination of body , mental , and social adaptation ( 6 ) . hence , research on health also focuses on mental health .
clark et al . studied the 9 to 10-year - old british children and noted that air pollution ( no2 ) in schools will not influence their cognition and health .
after the no2 level was controlled , they found that aircraft noise and road traffic noise have significant different effects on cognition and health of children ( 7 ) . a study on 1,546 samples collected from salt lake county , utah
, usa found that higher so2 and no2 particles would increase risks of suicide ( 8) . from a medical perspective
, tiny particles in air pollution will hurt the nervus centralis in humans after entering the brain and may cause depression and apoplexy ( 9 ) . considering the physiological and psychological differences of the elderly with children and youth ,
the relationship between the mental health of the elderly and air pollution has elicited considerable attention in medical research attention .
studied the 699 elderly males and found the internal relation between pm2.5 and people s mood ( optimistic or pessimistic ) ( 10 ) .
however , yi et al . stated in a study of 732 old people ( 65 years old ) in boston that no correlation had been found between air pollution and depressive symptoms ( 11 ) .
these two studies report different results primarily because most medical researchers are based on individuals or non - random samples , which may incur selection bias .
therefore , we shall maintain a cautious attitude toward the corresponding research conclusions and policy implications .
scholars have also focused on the relationship between various social demographic characteristics and mental health .
small changes in smoking , drinking , sports , and diet would significantly affect both physical and mental health ( 12 ) . reducing smoking and increasing recreational sports
some scholars explored mental health from the perspective of social relations , and pointed out that social communication positively affected the health of the aged ( 14 , 15 ) .
some scholars studied the mental health of the elderly from the perspective of urban - rural difference .
a study on 404 old people ( 60 years old ) done in chinese rural and urban regions , found a significant difference in mental health between these regions ( 16 ) .
the urban old people have better mental health than do rural old people ( 17 ) .
however , mikael et al . offered the opposite conclusion , and they believed that rural old people had better mental health than urban old people had ( 18 ) .
studied 64,000 old people ( 65 years old ) in hong kong and revealed a significant correlation between depression and chronic diseases , such as cardiovascular disease and coronary artery disease ( 19 ) .
moreover , norstrand and xu analyzed the health conditions of chinese old people ( 65 years old ) from the perspective of social relations , and found a significant relationship between mental health of urban old people and social relations ( 20 ) . based on demography , mental health deteriorated greatly with aging , while physical health and income could improve mental health ( 21 ) . to sum up , although many research results concerning the mental health of the elderly have been achieved , mental health is characterized by complicated and diverse influencing factors .
these factors lead different research perspectives and thereby inconsistent conclusions , which may be caused by the lack of comprehensive and representative data .
moreover , only few studies on the relationship between mental health of chinese elderly and air pollution have been reported .
based on previous studies , this paper establishes a model to analyze comprehensively the influencing factors of the mental health of the elderly in china from the perspective of urban - rural difference and by combining local air quality , individual characteristics , and lifestyle .
this study also applies the representative data from china health and retirement longitudinal survey ( charls ) ( 22 ) and regional air pollution to conduct verify the established analysis model .
medical and sociological research reported that the so2 concentration in air would threaten physical health and even cause respiratory system diseases ( 5 ) .
being healthy is not only being free from diseases or weakness , but also the perfect combination of body , mental , and social adaptation ( 6 ) . hence , research on health also focuses on mental health .
clark et al . studied the 9 to 10-year - old british children and noted that air pollution ( no2 ) in schools will not influence their cognition and health .
after the no2 level was controlled , they found that aircraft noise and road traffic noise have significant different effects on cognition and health of children ( 7 ) . a study on 1,546 samples collected from salt lake county , utah
, usa found that higher so2 and no2 particles would increase risks of suicide ( 8) . from a medical perspective
, tiny particles in air pollution will hurt the nervus centralis in humans after entering the brain and may cause depression and apoplexy ( 9 ) . considering the physiological and psychological differences of the elderly with children and youth ,
the relationship between the mental health of the elderly and air pollution has elicited considerable attention in medical research attention .
studied the 699 elderly males and found the internal relation between pm2.5 and people s mood ( optimistic or pessimistic ) ( 10 ) .
however , yi et al . stated in a study of 732 old people ( 65 years old ) in boston that no correlation had been found between air pollution and depressive symptoms ( 11 ) .
these two studies report different results primarily because most medical researchers are based on individuals or non - random samples , which may incur selection bias .
therefore , we shall maintain a cautious attitude toward the corresponding research conclusions and policy implications .
scholars have also focused on the relationship between various social demographic characteristics and mental health .
small changes in smoking , drinking , sports , and diet would significantly affect both physical and mental health ( 12 ) . reducing smoking and increasing recreational sports could improve physical and mental health ( 13 ) .
some scholars explored mental health from the perspective of social relations , and pointed out that social communication positively affected the health of the aged ( 14 , 15 ) .
some scholars studied the mental health of the elderly from the perspective of urban - rural difference . a study on 404 old people ( 60 years old ) done in chinese rural and urban regions , found a significant difference in mental health between these regions ( 16 ) .
the urban old people have better mental health than do rural old people ( 17 ) .
however , mikael et al . offered the opposite conclusion , and they believed that rural old people had better mental health than urban old people had ( 18 ) .
studied 64,000 old people ( 65 years old ) in hong kong and revealed a significant correlation between depression and chronic diseases , such as cardiovascular disease and coronary artery disease ( 19 ) .
moreover , norstrand and xu analyzed the health conditions of chinese old people ( 65 years old ) from the perspective of social relations , and found a significant relationship between mental health of urban old people and social relations ( 20 ) .
based on demography , mental health deteriorated greatly with aging , while physical health and income could improve mental health ( 21 ) . to sum up ,
although many research results concerning the mental health of the elderly have been achieved , mental health is characterized by complicated and diverse influencing factors .
these factors lead different research perspectives and thereby inconsistent conclusions , which may be caused by the lack of comprehensive and representative data .
moreover , only few studies on the relationship between mental health of chinese elderly and air pollution have been reported .
based on previous studies , this paper establishes a model to analyze comprehensively the influencing factors of the mental health of the elderly in china from the perspective of urban - rural difference and by combining local air quality , individual characteristics , and lifestyle .
this study also applies the representative data from china health and retirement longitudinal survey ( charls ) ( 22 ) and regional air pollution to conduct verify the established analysis model .
the data were from the 2013 charls published by the national school of development of peking university .
the charls project covered 28 provinces , cities , and municipalities in china and investigated the health , economy , and family of the population ( 45 years old ) .
after missing and inappropriate data were deleted , 6,630 effective samples for final statistics were chosen , including 3,930 rural samples and 2,700 urban samples .
air pollution data of different regions were provided by china city statistical yearbook ( 23 ) .
chinese ministry of health announced that mental diseases , like depression and senile dementia , were the main threats to the health of the chinese elderly ( 24 ) .
surveys reported that the population aged 65 or more years has a 4.4% chance of having depression .
as a result , this paper evaluated the mental health degree of the elderly using cognition and depression data in the charls questionnaire . using the method of the center for epidemiological studies depression scale ( ces - d ) as reference ( 25 ) , charls designed a simple questionnaire of 10 problems , including feeling annoyed by trifles , difficulties in concentration , feeling down in the dumps , encountering difficulties in doing anything , being hopeful for the future , feeling frightened , experiencing poor sleep , feeling happy , feeling lonely , and encountering difficulties in continuing life .
each problem has four answers based on time span , namely , 0 for < 1d ; 1 for 1d < answer<2d ; 2 for 3d < answer<4d ; 3 for 5d < answer<7d . among these 10 questions ,
finally , scores of these 10 problems are added to identify cognition and depression degrees of respondents .
the higher the ces - d score is , the more depressed the elderly will be . based on the design principle of ces - d ,
therefore , the influencing factors of the psychological disorder of the elderly were further observed .
based on the processing method of yi et al . and zhao yaohui et al .
( 2,11 ) , respondents with more than 10 ces - d scores were determined to have depression symptoms , that is , having a psychological disorder .
( 26 ) , was measured by so2 discharge in cities and counties in which the respondents live .
the study area covered 123 cities and counties in 28 chinese provinces . to further analyze the effect of respondent background on mental health , this paper controlled demographic variables , including age , gender , marriage , education , and physical health . among the variables ,
physical health was measured with chronic disease and physical disability . meanwhile , the lifestyle of the elderly was introduced as a variable , including smoking , drinking , social activity participation , and employment . the definition of variables was shown in table 1 .
definition of variables included in the study note : 1 ) chronic disease data in charls included 12 common diseases of the elderly , namely , hypertension , hyperlipidemia , diabetes mellitus , cancer , chronic lung diseases , liver disease , heart disease , apoplexy , kidney disease , gastric disease or digestive system disease , arthritis or rheumatism , and asthma .
2 ) education background was divided into four groups , namely , illiteracy , primary school , middle school , as well as vocational education and junior college or above .
3 ) disability included five options , namely , physical disability , brain damage , blindness or half - blindness , deafness or half - deafness , and dumb or serious stammer . in this paper , a virtual variable
total ces - d score is the indicator of mental health of the elderly , which ranges between 0 and 30 . using the least square method
the basic model is :
yi*=+1rurali+2soi+3soi2+zixi+i
yi={yi*,0yi*300,yi*>30 or yi*<0
where yi is a potential variable .
yi is the total ces - d score of respondent i , which is used to assess their mental health .
so2 drainage has two effects on mental health of the elderly . on the one hand ,
so2 drainage amount reflects the industrial and economic development of cities , thereby affecting mental health of local residents through income level . on the other hand
therefore , the square of so2 drainage was added into the model analysis to determine the influence factor of air quality on mental health .
rurali is the urban - rural variable , 1 for urban regions and 0 for rural regions .
xi is the control variable , involving age , gender , marriage , education , physical health , smoking , drinking , social activity , and employment .
respondents will be evaluated depressed when yi ( ces - d score ) in equation increases to a certain extent ( 10 score ) .
a virtual variable di is defined , which represents if respondent i has psychological disorder ( yes:1 ; no : 0 ) , which is identified by the presence of depressive symptoms . di and yi are then correlated :
di{1,11yi*300,0yi*<11
considering that di is a 0 , 1 binary discrete variable , we chose the binary selection probit model to analyze the influencing factors of psychological disorder of the elderly .
the data were from the 2013 charls published by the national school of development of peking university .
the charls project covered 28 provinces , cities , and municipalities in china and investigated the health , economy , and family of the population ( 45 years old ) .
after missing and inappropriate data were deleted , 6,630 effective samples for final statistics were chosen , including 3,930 rural samples and 2,700 urban samples .
air pollution data of different regions were provided by china city statistical yearbook ( 23 ) .
chinese ministry of health announced that mental diseases , like depression and senile dementia , were the main threats to the health of the chinese elderly ( 24 ) .
surveys reported that the population aged 65 or more years has a 4.4% chance of having depression .
as a result , this paper evaluated the mental health degree of the elderly using cognition and depression data in the charls questionnaire . using the method of the center for epidemiological studies depression scale ( ces - d ) as reference ( 25 ) , charls designed a simple questionnaire of 10 problems , including feeling annoyed by trifles , difficulties in concentration , feeling down in the dumps , encountering difficulties in doing anything , being hopeful for the future , feeling frightened , experiencing poor sleep , feeling happy , feeling lonely , and encountering difficulties in continuing life .
each problem has four answers based on time span , namely , 0 for < 1d ; 1 for 1d < answer<2d ; 2 for 3d < answer<4d ; 3 for 5d <
finally , scores of these 10 problems are added to identify cognition and depression degrees of respondents .
the higher the ces - d score is , the more depressed the elderly will be . based on the design principle of ces - d ,
therefore , the influencing factors of the psychological disorder of the elderly were further observed .
based on the processing method of yi et al . and zhao yaohui et al .
( 2,11 ) , respondents with more than 10 ces - d scores were determined to have depression symptoms , that is , having a psychological disorder .
( 26 ) , was measured by so2 discharge in cities and counties in which the respondents live .
the study area covered 123 cities and counties in 28 chinese provinces . to further analyze the effect of respondent background on mental health , this paper controlled demographic variables , including age , gender , marriage , education , and physical health . among the variables ,
physical health was measured with chronic disease and physical disability . meanwhile , the lifestyle of the elderly was introduced as a variable , including smoking , drinking , social activity participation , and employment . the definition of variables was shown in table 1 .
definition of variables included in the study note : 1 ) chronic disease data in charls included 12 common diseases of the elderly , namely , hypertension , hyperlipidemia , diabetes mellitus , cancer , chronic lung diseases , liver disease , heart disease , apoplexy , kidney disease , gastric disease or digestive system disease , arthritis or rheumatism , and asthma .
2 ) education background was divided into four groups , namely , illiteracy , primary school , middle school , as well as vocational education and junior college or above .
3 ) disability included five options , namely , physical disability , brain damage , blindness or half - blindness , deafness or half - deafness , and dumb or serious stammer . in this paper , a virtual variable disability was designed .
total ces - d score is the indicator of mental health of the elderly , which ranges between 0 and 30 . using the least square method
the basic model is :
yi*=+1rurali+2soi+3soi2+zixi+i
yi={yi*,0yi*300,yi*>30 or yi*<0
where yi is a potential variable .
yi is the total ces - d score of respondent i , which is used to assess their mental health .
so2 drainage has two effects on mental health of the elderly . on the one hand , so2 drainage amount reflects the industrial and economic development of cities , thereby affecting mental health of local residents through income level . on the other hand
therefore , the square of so2 drainage was added into the model analysis to determine the influence factor of air quality on mental health .
rurali is the urban - rural variable , 1 for urban regions and 0 for rural regions .
xi is the control variable , involving age , gender , marriage , education , physical health , smoking , drinking , social activity , and employment .
respondents will be evaluated depressed when yi ( ces - d score ) in equation increases to a certain extent ( 10 score ) .
a virtual variable di is defined , which represents if respondent i has psychological disorder ( yes:1 ; no : 0 ) , which is identified by the presence of depressive symptoms . di and yi are then correlated :
di{1,11yi*300,0yi*<11
considering that di is a 0 , 1 binary discrete variable , we chose the binary selection probit model to analyze the influencing factors of psychological disorder of the elderly .
total ces - d score is the indicator of mental health of the elderly , which ranges between 0 and 30 . using the least square method
the basic model is :
yi*=+1rurali+2soi+3soi2+zixi+i
yi={yi*,0yi*300,yi*>30 or yi*<0
where yi is a potential variable .
yi is the total ces - d score of respondent i , which is used to assess their mental health .
so2 drainage has two effects on mental health of the elderly . on the one hand , so2 drainage amount reflects the industrial and economic development of cities , thereby affecting mental health of local residents through income level . on the other hand
therefore , the square of so2 drainage was added into the model analysis to determine the influence factor of air quality on mental health .
rurali is the urban - rural variable , 1 for urban regions and 0 for rural regions .
xi is the control variable , involving age , gender , marriage , education , physical health , smoking , drinking , social activity , and employment .
respondents will be evaluated depressed when yi ( ces - d score ) in equation increases to a certain extent ( 10 score ) .
a virtual variable di is defined , which represents if respondent i has psychological disorder ( yes:1 ; no : 0 ) , which is identified by the presence of depressive symptoms . di and yi are then correlated :
di{1,11yi*300,0yi*<11
considering that di is a 0 , 1 binary discrete variable , we chose the binary selection probit model to analyze the influencing factors of psychological disorder of the elderly .
the descriptive statistics of all samples , urban samples , and rural samples are listed in table 2 .
the average ces - d score of all samples is 8.09 , which is close to the critical value of depression in the simple ces - d questionnaire ( 10 ) .
the proportion of respondents with higher than 10 ces - d scores and evident depressive symptoms reaches as high as 27.8% .
however , no significant difference was observed between urban and rural samples in terms of age , gender , marriage , physical health indicators , and social activity .
the average age of the elderly was 67.66 years , and 67% old people had at least one chronic disease .
with respect to lifestyle , smoking and drinking respondents accounted for 46% and 41% of the total samples , respectively . in terms of educational background , urban samples present nearly 10 times higher proportions of vocational education or above than rural samples .
furthermore , deposit per capita of the urban samples was more than four times higher than that of the rural samples , but employment showed the opposite . a total of 63% rural samples had jobs , but only 28% of urban samples were employed .
urban and rural samples had both similarities and differences in terms of mental health and various influencing factors , which deserves further analysis .
1 ) reflects obvious differences among all samples , urban samples , and rural samples .
the ces - d distribution of rural samples inclines to the right end of all samples and urban samples because the average ces - d score of rural samples is 8.79 , which is higher than that of all samples ( 8.09 ) and urban samples ( 7.08 ) .
moreover , the ces - d score of female samples is at the right end of the male samples .
density distribution of ces - d this paper conducted a tobit regression on all samples , urban samples , and rural samples using the total ces - d score as dependent variable .
explanatory variables include air quality , urban - rural variable , demographic variables , and lifestyle variable .
tobit regression results on the influencing factors of the mental health of the chinese elderly 1).t statistics in parentheses . 2 ) .
* p<0.05 , * * p<0.01 , * * * p<0.001 . two - tailed test . table 3 shows that the regression result of urban - rural variable is negative ( the coefficient is 1.43 , p<0.001 ) , indicating that the ces - d score of urban old people is significantly lower than that of rural old people .
this result reflects that urban old people have better mental health than rural old people do .
so2 drainage has a significant negative effect on the mental health of all samples , urban samples , and rural samples ( p<0.001 ) .
this result reflects that so2 drainage lowers the ces - d score significantly , that is , reducing the depression degree of the elderly .
the square of so2 drainage is positive , which means that increasing so2 drainage will intensify the depression degree of the elderly ( p<0.001 ) .
the effect of so2 drainage on the mental health of the elderly presents a positive u - shaped
the regression results of age , gender , and marriage are the same . with the increase in age ,
the mental health of both urban and rural old people improves significantly ( the regression coefficients of all samples , urban samples , and rural samples are 0.062 , 0.07 , and 0.057 , respectively , ( p<0.001 ) .
the regression results of gender show that in both urban and rural areas , females have far better mental health than males ( p<0.001 ) .
the regression coefficients of all samples , urban samples , and rural samples are 1.77 , 1.38 , and 2.07 , respectively
different educational backgrounds affect urban and rural old people in similar ways but to various extents . compared with the control group , primary school , middle school , and vocational education significantly
however , only vocational education or higher educational background greatly improve the mental health of rural old people ( p<0.01 ) .
the regression coefficients of all samples , urban samples , and rural samples are 2.02 , 1.78 , and 2.13 , respectively .
viewed from all samples , smoking samples show poorer mental health than non - smoking samples .
regression coefficients of urban and rural samples have the same direction , but they have no statistical significance perhaps because old people struggling with mental problems often ease their mental problems by smoking . however , rural samples prefer drinking to smoking to alleviate mood .
therefore , drinking could significantly improve the mental health of rural samples ( p<0.01 ) .
deposit and employment are vital to the mental health of rural old people and are the main way for them to maintain life , which is why rural old people with more deposit and employment have better mental health ( p<0.001 ) .
by contrast , these two factors are insignificant to urban old people . the probit model estimation results of psychological disorder of all samples , urban samples , and rural samples are shown in table 4 . to better analyze the effect of explanatory variables on psychological disorder of the aged , table 4 presents the marginal effect of explanatory variables . according to the estimation result of all samples , rural old people are more likely to suffer psychological disorder than urban old people are ( the marginal effect is 0.074 , ( p<0.001 ) .
so2 drainage imposes a great effect , presenting a positive u - shaped variation ( p<0.001 ) , which reflects that air pollution affects the psychological disorder and mental health of the elderly in the same way .
probit model estimation results of psychological disorder of the chinese elderly 1 ) . z statistics in parentheses . 2 ) .
two - tailed test aging could greatly reduce the probability of psychological disorder ( p<0.001 ) . on the average marginal effect ,
the probability of psychological disorder of females is 11% higher than that of males , which is significant at 1% level .
the probability of psychological disorder of married samples is 4.9% lower than that of single samples ( p<0.001 ) .
viewed from all samples and urban samples , middle school and higher educational background could significantly reduce risks of psychological disorder ( p<0.001 ) .
however , only vocational education , junior college , and higher educational background could greatly reduce risks of psychological disorder of rural samples ( p<0.01 ) .
compared with physically healthy samples , disabled samples have 12.5% higher risks of having mental diseases ( p<0.001 ) .
however , social activity could reduce the risk of mental diseases by 6.1% ( p<0.001 ) .
drinking could significantly lower the probability of psychological disorder of rural samples ( p<0.01 ) , possibly because rural old people in china prefer to drink at parties .
compared with samples without chronic diseases , samples with one or more chronic diseases have 4.7% higher probability of psychological disorder and those with three or more chronic diseases have 10.6% higher probability of psychological disorder . for rural samples , deposit amount and employment are inversely proportional to the probability of psychological disorder ( the marginal effects are 0.002 and 0.043 , respectively )
the descriptive statistics of all samples , urban samples , and rural samples are listed in table 2 .
the average ces - d score of all samples is 8.09 , which is close to the critical value of depression in the simple ces - d questionnaire ( 10 ) .
the proportion of respondents with higher than 10 ces - d scores and evident depressive symptoms reaches as high as 27.8% .
however , no significant difference was observed between urban and rural samples in terms of age , gender , marriage , physical health indicators , and social activity .
the average age of the elderly was 67.66 years , and 67% old people had at least one chronic disease .
with respect to lifestyle , smoking and drinking respondents accounted for 46% and 41% of the total samples , respectively . in terms of educational background , urban samples present nearly 10 times higher proportions of vocational education or above than rural samples .
furthermore , deposit per capita of the urban samples was more than four times higher than that of the rural samples , but employment showed the opposite . a total of 63% rural samples had jobs , but only 28% of urban samples were employed .
urban and rural samples had both similarities and differences in terms of mental health and various influencing factors , which deserves further analysis .
1 ) reflects obvious differences among all samples , urban samples , and rural samples .
the ces - d distribution of rural samples inclines to the right end of all samples and urban samples because the average ces - d score of rural samples is 8.79 , which is higher than that of all samples ( 8.09 ) and urban samples ( 7.08 ) .
moreover , the ces - d score of female samples is at the right end of the male samples .
this paper conducted a tobit regression on all samples , urban samples , and rural samples using the total ces - d score as dependent variable .
explanatory variables include air quality , urban - rural variable , demographic variables , and lifestyle variable .
tobit regression results on the influencing factors of the mental health of the chinese elderly 1).t statistics in parentheses . 2 ) .
* p<0.05 , * * p<0.01 , * * * p<0.001 . two - tailed test . table 3 shows that the regression result of urban - rural variable is negative ( the coefficient is 1.43 , p<0.001 ) , indicating that the ces - d score of urban old people is significantly lower than that of rural old people .
this result reflects that urban old people have better mental health than rural old people do .
so2 drainage has a significant negative effect on the mental health of all samples , urban samples , and rural samples ( p<0.001 ) .
this result reflects that so2 drainage lowers the ces - d score significantly , that is , reducing the depression degree of the elderly .
the square of so2 drainage is positive , which means that increasing so2 drainage will intensify the depression degree of the elderly ( p<0.001 ) .
the effect of so2 drainage on the mental health of the elderly presents a positive u - shaped curve .
the regression results of age , gender , and marriage are the same . with the increase in age ,
the mental health of both urban and rural old people improves significantly ( the regression coefficients of all samples , urban samples , and rural samples are 0.062 , 0.07 , and 0.057 , respectively , ( p<0.001 ) .
the regression results of gender show that in both urban and rural areas , females have far better mental health than males ( p<0.001 ) .
the regression coefficients of all samples , urban samples , and rural samples are 1.77 , 1.38 , and 2.07 , respectively
different educational backgrounds affect urban and rural old people in similar ways but to various extents . compared with the control group , primary school , middle school , and vocational education
however , only vocational education or higher educational background greatly improve the mental health of rural old people ( p<0.01 ) .
the regression coefficients of all samples , urban samples , and rural samples are 2.02 , 1.78 , and 2.13 , respectively .
viewed from all samples , smoking samples show poorer mental health than non - smoking samples .
regression coefficients of urban and rural samples have the same direction , but they have no statistical significance perhaps because old people struggling with mental problems often ease their mental problems by smoking .
therefore , drinking could significantly improve the mental health of rural samples ( p<0.01 ) .
deposit and employment are vital to the mental health of rural old people and are the main way for them to maintain life , which is why rural old people with more deposit and employment have better mental health ( p<0.001 ) .
by contrast , these two factors are insignificant to urban old people . the probit model estimation results of psychological disorder of all samples , urban samples , and rural samples are shown in table 4 . to better analyze the effect of explanatory variables on psychological disorder of the aged , table 4 presents the marginal effect of explanatory variables .
according to the estimation result of all samples , rural old people are more likely to suffer psychological disorder than urban old people are ( the marginal effect is 0.074 , ( p<0.001 ) .
so2 drainage imposes a great effect , presenting a positive u - shaped variation ( p<0.001 ) , which reflects that air pollution affects the psychological disorder and mental health of the elderly in the same way .
probit model estimation results of psychological disorder of the chinese elderly 1 ) . z statistics in parentheses . 2 ) .
two - tailed test aging could greatly reduce the probability of psychological disorder ( p<0.001 ) . on the average marginal effect ,
the probability of psychological disorder of females is 11% higher than that of males , which is significant at 1% level .
the probability of psychological disorder of married samples is 4.9% lower than that of single samples ( p<0.001 ) .
viewed from all samples and urban samples , middle school and higher educational background could significantly reduce risks of psychological disorder ( p<0.001 ) .
however , only vocational education , junior college , and higher educational background could greatly reduce risks of psychological disorder of rural samples ( p<0.01 ) .
compared with physically healthy samples , disabled samples have 12.5% higher risks of having mental diseases ( p<0.001 ) .
however , social activity could reduce the risk of mental diseases by 6.1% ( p<0.001 ) .
drinking could significantly lower the probability of psychological disorder of rural samples ( p<0.01 ) , possibly because rural old people in china prefer to drink at parties .
compared with samples without chronic diseases , samples with one or more chronic diseases have 4.7% higher probability of psychological disorder and those with three or more chronic diseases have 10.6% higher probability of psychological disorder . for rural samples ,
deposit amount and employment are inversely proportional to the probability of psychological disorder ( the marginal effects are 0.002 and 0.043 , respectively ) .
this paper conducted a tobit regression on all samples , urban samples , and rural samples using the total ces - d score as dependent variable .
explanatory variables include air quality , urban - rural variable , demographic variables , and lifestyle variable .
tobit regression results on the influencing factors of the mental health of the chinese elderly 1).t statistics in parentheses . 2 ) .
* p<0.05 , * * p<0.01 , * * * p<0.001 . two - tailed test . table 3 shows that the regression result of urban - rural variable is negative ( the coefficient is 1.43 , p<0.001 ) , indicating that the ces - d score of urban old people is significantly lower than that of rural old people .
this result reflects that urban old people have better mental health than rural old people do .
so2 drainage has a significant negative effect on the mental health of all samples , urban samples , and rural samples ( p<0.001 ) .
this result reflects that so2 drainage lowers the ces - d score significantly , that is , reducing the depression degree of the elderly .
the square of so2 drainage is positive , which means that increasing so2 drainage will intensify the depression degree of the elderly ( p<0.001 ) .
the effect of so2 drainage on the mental health of the elderly presents a positive u - shaped curve .
the regression results of age , gender , and marriage are the same . with the increase in age ,
the mental health of both urban and rural old people improves significantly ( the regression coefficients of all samples , urban samples , and rural samples are 0.062 , 0.07 , and 0.057 , respectively , ( p<0.001 ) .
the regression results of gender show that in both urban and rural areas , females have far better mental health than males ( p<0.001 ) .
the regression coefficients of all samples , urban samples , and rural samples are 1.77 , 1.38 , and 2.07 , respectively
different educational backgrounds affect urban and rural old people in similar ways but to various extents . compared with the control group , primary school , middle school , and vocational education
however , only vocational education or higher educational background greatly improve the mental health of rural old people ( p<0.01 ) .
the regression coefficients of all samples , urban samples , and rural samples are 2.02 , 1.78 , and 2.13 , respectively .
viewed from all samples , smoking samples show poorer mental health than non - smoking samples .
regression coefficients of urban and rural samples have the same direction , but they have no statistical significance perhaps because old people struggling with mental problems often ease their mental problems by smoking .
therefore , drinking could significantly improve the mental health of rural samples ( p<0.01 ) .
deposit and employment are vital to the mental health of rural old people and are the main way for them to maintain life , which is why rural old people with more deposit and employment have better mental health ( p<0.001 ) .
the probit model estimation results of psychological disorder of all samples , urban samples , and rural samples are shown in table 4 . to better analyze the effect of explanatory variables on psychological disorder of the aged
, table 4 presents the marginal effect of explanatory variables . according to the estimation result of all samples , rural old people are more likely to suffer psychological disorder than urban old people are ( the marginal effect is 0.074 , ( p<0.001 ) .
so2 drainage imposes a great effect , presenting a positive u - shaped variation ( p<0.001 ) , which reflects that air pollution affects the psychological disorder and mental health of the elderly in the same way .
probit model estimation results of psychological disorder of the chinese elderly 1 ) . z statistics in parentheses . 2 ) .
two - tailed test aging could greatly reduce the probability of psychological disorder ( p<0.001 ) . on the average marginal effect ,
the probability of psychological disorder of females is 11% higher than that of males , which is significant at 1% level .
the probability of psychological disorder of married samples is 4.9% lower than that of single samples ( p<0.001 ) .
viewed from all samples and urban samples , middle school and higher educational background could significantly reduce risks of psychological disorder ( p<0.001 ) .
however , only vocational education , junior college , and higher educational background could greatly reduce risks of psychological disorder of rural samples ( p<0.01 ) .
compared with physically healthy samples , disabled samples have 12.5% higher risks of having mental diseases ( p<0.001 ) .
however , social activity could reduce the risk of mental diseases by 6.1% ( p<0.001 ) .
drinking could significantly lower the probability of psychological disorder of rural samples ( p<0.01 ) , possibly because rural old people in china prefer to drink at parties .
compared with samples without chronic diseases , samples with one or more chronic diseases have 4.7% higher probability of psychological disorder and those with three or more chronic diseases have 10.6% higher probability of psychological disorder . for rural samples , deposit amount and employment are inversely proportional to the probability of psychological disorder ( the marginal effects are 0.002 and 0.043 , respectively ) .
based on the effect of urban - rural difference on mental health and psychological disorder of the elderly , rural old people are facing more serious mental health problems due to poor living environment , entertainment culture conditions , and health care facilities .
for example , deposit amount and employment could significantly improve the mental health of rural samples , but only slightly influence the mental health of urban samples .
this result indicated that the aged in rural areas needed more assets to buy basic insurances , like medical service .
drinking could improve the mental health of rural samples , but did not influence urban samples .
the aged in urban areas paid more attention on their health and drank less , while the aged in rural areas enjoyed limited social activities and entertainment activities .
the effects of air pollution on mental health and disorder of the elderly confirm the opinion proposed early in this paper , i.e. , during the industrialization in china , so2 drainage facilitates urban economic development , improves people s living standard , enriches life of the aged , and thus improves the mental health and reduces risks of psychological disorder of the elderly .
however , with the further increase of so2 drainage , the negative effects of air pollution on the mental health of the elderly become more prominent .
the conclusion that the mental health of the elderly is improved with the increase of age agrees with the opinions of luan wenjing and li jianxin s studies ( 17 , 21 ) possibly because old people become introversive and easy - going with age , thus having better mental health conditions ( 28 ) .
the female elderly have far poorer mental health and more serious psychological disorders than do males .
this result revealed that in both urban and rural areas , chinese males have higher position in the family and additional social relationships , thus having better mental health .
higher educational background could improve mental health and greatly reduce the probability of psychological disorder ( the coefficient is increasing continuously ) .
this result implied that the elderly with higher educational background have richer and more diversified spiritual and cultural life , as well as pay more attention to knowledge on mental health . in terms of chronic diseases ,
this result showed the close relationship between mental health and physical health of the elderly .
hence , more attention must be paid on psychological changes of the elderly with poor physical health .
owing to the data limitations , these results , based on the cross - sectional micro - data , are more likely to be established within a specific period .
further research is necessary to analyze the change trend of the mental health of the elderly in china from the perspective of urban - rural difference when the data are available .
results show that air quality significantly influences mental health and psychological disorder of the aged , showing a positive u - shaped curve .
, local governments should consider the influence of air quality on the mental health of the aged .
specifically , regions with developed industries should adopt strict environmental protection measures to reduce effectively the emission of air pollutants .
moreover , local governments should be aware of the differences in mental health of urban and rural old people when making associated policies .
governments should reasonably allocate public resources , remove city - countryside dualization barrier , and accelerate urban - rural integration .
special attention should be paid to the psychological conditions of the female elderly and the aged with chronic diseases and physical disability .
great efforts should be made to enrich social activity of and increase employment opportunities for the elderly .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , and redundancy ) have been completely observed by the authors .
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background : china has become an aging society , and the mental health problem of the elderly is increasingly becoming prominent .
this paper aimed to analyze the effect of air pollution and rural - urban difference on mental health of the elderly in china.methods:using the data from the china health and retirement longitudinal survey ( charls , 2013 ) , after controlling the social demography variable via tobit and probit , a regression analysis of the effect of air pollution and rural - urban difference on mental health and psychological disorder was conducted on 6,630 old people ( 60 yr old ) of china from february to april 2015 .
mental health and psychological disorder of the elderly were measured by the ces - d score of respondents .
air pollution degree of counties and cities ( n=123 ) were measured by so2 emission.results:27.8% of old people had psychological disorders .
air pollution significantly influenced the mental health of the elderly , showing a positive u - shaped
curve ( p<0.001 ) . in china ,
the urban elderly had better psychological status than the rural elderly had .
the female elderly had more serious mental health problems .
marriage , education , and social activities had positive effects on the mental health of the elderly.conclusion:chinas local governments should consider the influence of air pollution on the mental health of the elderly during economic development .
this paper recommends paying attention to the difference in mental health between the urban and rural elderly when making public health policies .
governments could improve the mental health of the elderly by enriching social activities and increasing employment opportunities of the elderly .
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acromegaly is serious endocrinological derangement which , left untreated , reduces life expectancy and results in physiological derangements and complications that may negatively affect a patient 's quality of life .
those patients with residual or recurrent disease are often treated with medication to decrease growth hormone secretion or block its action on peripheral tissues .
these treatments are not universally effective for patients and are sometimes contraindicated . as an adjunct treatment , and sometimes as an alternative treatment , radiosurgery has proven to be an attractive therapy .
it is noninvasive , has few side effects , and is available in many centers internationally [ 13 ] . in the current paper , we first describe the pathophysiology of acromegaly , the existing surgical and medical treatments , and then introduce radiosurgical methods . in particular , we will focus on gamma knife radiosurgery ( gks ) since it has a broader base of supporting literature than alternative forms of stereotactic radiosurgery .
finally , the overall efficacy of gks is described , along with its reported morbidities .
acromegaly is a syndrome caused by elevated levels of circulating growth hormone ( gh ) .
the most common cause of the disorder , accounting for about 98% of cases , is a gh - secreting pituitary adenoma .
rare nonpituitary causes of acromegaly include diverse entities such as hypothalamic hamartomas , small - cell lung cancers , pheochromocytomas , and bronchial carcinoids .
among gh - secreting pituitary adenomas , roughly 60% are pure gh - secreting somatotrope adenomas , while the remainder are mixed mammosomatotropes , which secrete both gh and prolactin ( prl ) and sometimes thyroid - stimulating hormone ( tsh ) .
the symptoms of increased gh are mediated by both the direct effects of gh binding to the gh receptor , activating the jak / stat pathway , and indirectly via insulin - like growth factor 1 ( igf-1 ) .
the combined effects of gh and igf-1 on target tissues lead to bony and soft tissue growth , noted by a characteristic constellation of signs including frontal bossing , prognathism , widely spaced teeth ( due to mandibular growth ) , increased shoe or ring sizes , skin tags , carpal tunnel syndrome , and coarse facial features .
these externally recognizable signs in themselves are not as important to overall morbidity as internal changes , including cardiomegaly and visceromegaly [ 4 , 5 ] .
diabetes mellitus occurs in roughly 25% of patients , and cardiomyopathy with arrhythmia , hypertension , and diastolic dysfunction occurs in 30% .
additionally , colonic polyps are more frequent , as is sleep apnea which occurs in 60% of patients , presumably secondary to soft tissue expansion and macroglossia .
multiple studies through the 1920s to 1990s agreed on a prevalence of roughly 60 per million , a mean age of onset of 44 years , symptoms lasting on average 8 years before diagnosis , and no difference in incidence between men and women .
however , more recent studies place the prevalence at 86 per million , 124 per million , and a remarkably high 1034 per million . however , in addition to being geographically and demographically restricted ( primary care patients in germany ) , this last study was based on a biochemical definition of acromegaly ( elevated igf-1 and gh ) rather than a syndrome definition , and therefore will include many patients who would otherwise not seek treatment .
such a more liberal definition might be one method of discovering at - risk patients earlier in the course of their disease and permitting more time to mitigate the accruing morbidity .
surgery is typically the first line of treatment for acromegaly due to pituitary adenomas provided there are no surgical contraindications .
residual disease is then managed medically with somatostatin analogues , dopamine agonists , or gh receptor antagonists .
most adenomas are resected via the transsphenoidal approach ( transsphenoidal adenomectomy , tsa ) . using either a microscope or endoscope
, the surgeon enters the sphenoid sinus transnasally , then penetrates the sella , and finally debulks and removes the tumor , sparing as much of the normal pituitary as possible . for microadenomas ( < 10 mm in diameter ) , tsas lead to correction of gh levels in ~70% of patients .
only 50% of patients with macroadenomas achieve normalization . for those patients who do not experience normalization of gh levels after surgery , or who are not surgical candidates ,
the lar ( long - acting release ) formulation of octreotide uses polymeric microspheres and is injected monthly .
lanreotide atg ( autogel ) is the only formulation of lanreotide currently available in the united states and is suspended in aqueous solution in microsyringes for subcutaneous delivery by the patient .
the two formulations appear to be equally efficacious and normalize igf - i levels in 5060% of patients [ 1316 ] .
somatostatin analogues appear to induce tumor shrinkage in approximately 42% of patients , when data are pooled across studies .
interestingly , however , tumor shrinkage appears more pronounced in primarily treated patients ( 52% ) as opposed to patients receiving adjunctive , postsurgical treatment ( 21% ) .
dopamine agonists appear to work best in patients whose tumors also secrete prolactin . among dopamine agonists , cabergoline appears to be the most efficacious , resulting in normalization of igf - i levels in 34% of patients .
however , no dopamine agonist alone is as effective as a somatostatin analogue , although there might be synergistic effects when used in conjunction with somatostatin analogues [ 2124 ] .
pegvisomant is a pegylated growth hormone analogue that is subcutaneously injected by patients . in one study , treatment for 12 months led to normalized igf-1 levels in 97% of patients , and treatment for 24 months led to normalization in 76.3% of patients in a different study .
however , the side effects of pegvisomant include transaminitis , lipodystrophy at injection sites , and , most worrisome , possible tumor progression , due to blocking of the normal inhibitory feedback of growth hormone levels of the adenoma [ 2729 ] .
simply put , resective surgery is a means of removing unwanted tissue from the body .
radiosurgery ( rs ) achieves the identical goal but , rather than directly removing cells , induces cell death instead .
this cell death can be induced either directly , via necrosis or apoptosis , or indirectly , by damaging the tissue blood supply .
when inducing cell death , rs uses ionizing radiation , wherein charged particles or photons strip electrons from atoms and molecules , thereby damaging dna , proteins , and other molecules within cells and in the extracellular matrix . when this damage can not be repaired , cells undergo either apoptosis or necrosis [ 30 , 31 ] .
various types of radiosurgery are available , but each works by emitting charged particles or photons .
proton beams , for example , generated by particle accelerators and can be used to target structures deep within the cranium .
this is due , in part , to the characteristic peak and subsequent drop - off of radiation intensity in proton beams the bragg peak .
the depth of this peak can be modulated and is used to focus the effects of the radiation on particular structures , sparing healthy ones .
the drawback to proton therapy , however , is the high cost and relative scarcity of facilities capable of providing therapeutic proton beams .
the two major techniques are linear accelerators and radioactive isotopes . linear accelerators , like the cyberknife ( accuray inc . ,
sunnyvale , ca ) , produce photons which are then aimed toward deep structures within the brain or spine . to limit damage along the path of the beam
thus , the target of the beam is constant , always receiving radiation , but the intervening structures are exposed only briefly [ 3032 ] . the gamma knife ( elekta ab , stockholm , sweden ) , developed by lars leksell in 1968 ,
each source generates a beam of photons as the cobalt decays , and these beams are focused on a central target within the brain through the use of collimators .
because each individual beam is weak , intervening tissue is exposed to far less radiation than the central target , which is the common focus of all 201 beams .
the gamma knife has been around longer than most linear accelerator radiosurgical devices and is therefore a better - studied tool for use by neurosurgeons , though direct comparisons between the gamma knife and linear accelerators will undoubtedly change the prevailing practices in the future .
a large number of small case series have been carried out to evaluate the effects of gamma knife surgery ( gks ) on acromegaly ( table 1 ) .
most use remission criterion of a normal igf-1 level and many add the criterion gh < 1 to 2 ng / ml , but it should be noted that remission criteria vary across studies .
these criteria are roughly in line with the criteria set forth by the acromegaly consensus group on 2010 ( normal igf-1 and gh < 1 ng / ml ) .
also variable between studies is the follow - up time , along with radiation dose , targeting protocol , and , most critically , pre - gks therapy .
this last note is particularly important since most series include patients who have already received transsphenoidal surgical resection as an inadequate treatment .
unfortunately , the data are not presented in the reviewed papers in such a way as to separate out response rates in patients receiving prior treatment versus those who were treated primarily with gks .
overall , however , the results from these studies suggest that gks is an effective treatment for acromegaly . across the 29 studies and 964 cases examined , 43% of patients achieved remission . the time to remission is not
reliably reported , but most studies agree that the further out from rs patients are examined , the more likely they are to achieve a cure .
for example , vik - mo et al . showed an increase from 58% of patients with normal igh-1 levels to 86% of patients over the time span of 5 to 10 years after rs .
how exactly the effects of rs continue to evolve over such a protracted time is unknown , but consistent with how rs affects other diseases , like epilepsy and vestibular schwannoma .
interestingly , there is some ( still debated ) evidence that the use of antiacromegalic medicines prior to irradiation attenuates the effects of gks
. that is , using somatostatin analogues or gh receptor antagonists has been shown in select studies to decrease a patient 's chances of remission [ 3739 ] .
though this has not been thoroughly examined , the mechanism is believed to be suppression of tumor cell cycle , thereby making the cells less prone to radiation - induced damage .
there does not appear to be a correlation between radiation dose and rates of remission ( figure 1 ) .
however , the heterogeneity in study design , follow - up , and definition of remission makes such conclusions fraught .
the most common complication of gks for acromegaly is hypopituitarism , presumably from damage to the normal gland during irradiation , ranging from 0 to 43% ( table 2 ) .
however , the degree to which such damage is a pure result of gks versus prior surgery , if done , is unclear .
moreover , there is no discernible relationship between radiation dose and the incidence of this complication ( figure 2 ) , though these are very heterogeneous studies and it is possible that better - controlled or larger studies would uncover such relationships if they exist .
other complications are inadequately documented but include headache , epilepsy , carotid artery stenosis , and , more frequently , cranial nerve palsies or neuropathies ( including trigeminal neuralgia and visual decline ) ( table 2 ) .
the rate of visual disturbances seems to be , in the worst case , 11% [ 40 , 41 ] , though most trials either do not report these adverse events or show them to be on the order of 06% ( table 2 ) .
nevertheless , these complications should be viewed in light of the complications inherent to uncontrolled acromegaly or , if being used as an alternative to surgery , the morbidity of an endonasal neurosurgical procedure .
while surgical resection remains the first line of treatment , stereotactic radiosurgery is proving itself a feasible alternative therapy ( when surgery might be contraindicated ) and adjunct ( when surgical resection is unsuccessful in leading to complete disease remission ) .
ultimately , further studies are needed to delineate which patients are most likely to receive benefit from gks , along with ways to improve gks outcomes .
|
acromegaly is debilitating disease occasionally refractory to surgical and medical treatment .
stereotactic radiosurgery , and in particular gamma knife surgery ( gks ) , has proven to be an effective noninvasive adjunct to traditional treatments , leading to disease remission in a substantial proportion of patients .
such remission holds the promise of eliminating the need for expensive medications , along with side effects , as well as sparing patients the damaging sequelae of uncontrolled acromegaly .
numerous studies of radiosurgical treatments for acromegaly have been carried out .
these illustrate an overall remission rate over 40% .
morbidity from radiosurgery is infrequent but can include cranial nerve palsies and hypopituitarism .
overall , stereotactic radiosurgery is a promising therapy for patients with acromegaly and deserves further study to refine its role in the treatment of affected patients .
|
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oral melanoma is an extremely rare tumor arising from uncontrolled growth of melanocytes found in the basal layer of oral mucous membrane .
it occurs between 30 and 90 years of age , with a higher incidence in the 6th decade with a mean age of 56 years .
it is having a higher prevalence in yellows , blacks , japanese , and indians of asia due to more frequent finding of melanin pigmentation in oral mucosa of these races .
green et al . described criteria for diagnosis of primary oral melanoma which includes demonstration of melanoma in the oral mucosa , presence of junctional activity , inability to demonstrate extraoral primary melanoma .
a total of 80% to 90% of oral malignant melanoma arises in the mucosa of maxillary jaw with a majority occurring on the keratinized mucosa of hard palate and gingiva .
clinically , it is easy to diagnose them as these are pigmented ones and have irregular shape and outline .
these are mostly asymptomatic and detected only when there is ulceration or hemorrhage of the overlying epithelium
. the delayed detection may be the cause for the poor prognosis with a 5-year survival being between 15% and 38% .
the purpose of this article is to present a case of oral malignant melanoma , as well as to emphasize the necessity for early recognition and treatment of this lesion .
a 48-year - old male patient reported to the department of oral medicine and diagnosis with chief complaint of pain and swelling in the upper right gums .
the clinical examination revealed a large mass of 8 3 cm in dimension on buccal aspect of right maxillary alveolus involving marginal , attached , and interdental gingiva [ figure 1 ] .
anteriorly , it extends from the gingiva of mesial surface of 22 , to the gingiva in relation to 17 posteriorly .
13 was missing and 12 was displaced laterally ; while , 11 , 12 , and 21 exhibited mobility . primary oral malignant melanoma extending from 22 to distal aspect of 17 the palpatory findings revealed a firm consistency of lesion with mild pain .
a complete examination of the lesion was done and no other primary site of the lesion was found .
correlating all clinical features , diagnosis of primary malignant melanoma of oral cavity was made and the patient was referred for further investigations .
a computed tomography examination of neck , chest , abdomen , and bone scanning and ultrasounds of liver and kidney were normal excluding any diagnosis of distant metastasis .
an incisional biopsy was done for the lesion under local anesthesia and the specimen was sent for histopathologic examination .
the gross examination of tissue revealed a mass of 2 mm 3 mm 1 mm in size , which was black in color and firm in consistency .
the hematoxylin and eosin - stained section showed a melanin - producing tumor , consisting of atypical irregularly elongated spindle and oval - shaped melanocytes , exhibiting uniformly dark , enlarged and irregular nuclei [ figure 2 ] . in the superficial layers of the tissue , a junctional nevus with pigmentation
the diagnosis of an invasive melanoma arising most likely from a pre - existing junctional nevus was made and the patient was referred to the oral and maxillofacial surgery clinic for required therapy . as per the traditional approach , partial maxillectomy of the right side was performed . to reduce the defect and to reconstruct alveolus ,
microvascular fibula flap was used . the orbital floor near to maxilla was reconstructed with the help of premolded titanium mesh .
the histologic examination of the specimen confirmed the initial diagnosis of an invasive melanoma of the oral mucosa .
the patient has been followed - up with no evidence of recurrence or metastasis either clinically or radiographically , 11 months after the tumor 's resection .
the hematoxylin and eosin stained section shows melanoma with invasive pattern showing large cells with pleomorphic vesicular nucleus and brown pigment ( 40 ) the hematoxylin and eosin stained section shows stratified squamous keratinized epithelium with in situmelanotic pigment growth ( 10 )
it has no known predisposing factors and is difficult to diagnose and manage . differentiating it from a metastatic melanoma is often challenging .
the first symptoms of oral melanoma described by berthelsen were those of asymptomatic swelling and occasional bleeding , where he found that only 2 ( 14% ) patients had a pain .
because most of the melanomas are painless in their early stages , the diagnosis is often unfortunately delayed until symptoms resulting from ulceration , growth , or bleeding are noted .
the pain may be the later manifestation in melanoma as in our case that again could cause delay in seeking treatment . on gross appearance ,
the tumors on the palate are usually flat , with a varying degree of thickness .
microscopically , the tumor cells present themselves as densely packed , large epithelioid cells with eosinophilic cytoplasm . the melanin pigment , which is located intra or extracellularly , may be abundant , but may be sparse or even absent at the light microscopic level . the prognostic value of various levels of invasion , as established in the clark 's classification , does not apply for mucosal melanoma because of the absence of histologic landmarks , which are analogous to the papillary and reticular dermis . the staging systems that are applied to cutaneous melanoma are not applicable to mucosal melanomas . the american joint committee on cancer
the generally followed guideline is a clinical classification stage i clinically localized disease , stage ii regional lymph node disease , and stage iii - distant disease .
the tumor thickness is a reliable prognostic indicator for survival . in this case , the patient was in stage i. westbury describes a clinical classification as follows : 1only primary tumor present , and 2metastasis present , 2a adjacent skin involved , 2b adjacent lymph nodes involved , and 2ab adjacent skin and lymph nodes involved .
thus , according to these systems specification , our patient was in stage i. the etiology of malignant melanoma is essentially unknown
. tobacco use and chronic irritation from ill - fitting dentures have been considered as possible risk factors , but the evidence is weak .
the cause could also be related to our patient as he was having the history of smoking from last 15 years .
but most of the malignant melanomas arise de novo , from apparently normal mucosa , and about 30% are preceded by oral pigmentations for several months or even years .
some melanoma - associated antigens become expressed during transformation process from a benign melanocytic nevus to melanoma ; the majorities of these are related to the melanin production process and most are hla restricted .
a recent study demonstrates that the loss of heterozygosity at 12p13 and p27kip1 protein expression contributes to melanoma progression .
cytogenetic analysis and evaluation of melanocyte - specific gene-1 ( msg-1 ) appears to be very helpful for understanding the pathogenesis of oral malignant melanoma .
the current guidelines for the surgical management of primary cutaneous melanoma recommend a diagnostic excisional biopsy of the lesion followed by a wide local excision where the diagnosis is proved .
however , in oral cavity , the size of the lesion or anatomic limitations , particularly the presence of teeth , may preclude the taking of excisional biopsy .
younes et al . proposed to take an excisional biopsy with a 12 mm margin for small lesions in amenable locations , but incisional biopsy , through the thickest or the most suspicious part of the tumor , in case of a large lesion or a location in sites where an excisional procedure would involve extensive and militating surgery .
usually oral malignant melanoma can be diagnosed with confidence on hematoxylin and eosin stained sections .
if pigment is completely absent ( amelanotic melanoma ) , immunohistochemical stains are of significant help .
useful markers include s-100 protein , gp 100 ( hmb-45 ) , and mart-1 ( melan - a ) .
it has always been suggested that cutting into malignant neoplasm during incisional biopsy could result in accidental dissemination of malignant cells within adjacent tissues or blood or lymphatic stream with subsequent risk of local recurrence or regional or distant metastasis .
did find a somewhat reduced survival rate in patients with melanoma who had incisional biopsies but against the studies done by lederman and sober where they found no correlation in patient 's prognosis with incisional and excisional biopsies .
distant metastasis to the lungs , brain , liver , and bones are frequently observed .
the treatment of oral malignant melanoma is still controversial and there is no census regarding the best therapeutic approach .
data from several studies indicate radical resection of the primary as the treatment of choice .
regression in melanoma is a well - recognized phenomenon and may account for many of the cases of metastatic melanoma with occult primaries . partial regression of melanoma is relatively common , but complete regression is quite rare and relatively few cases are well documented .
a further feature of regression is its association with poor rather than a good prognosis .
the rather nonspecific features of regressed melanoma ( apparently inflammatory nodules , depigmented patches , and flat or slightly depressed scars ) are easily missed or discounted unless the patient had noticed the regression . from the prognostic point of view , clinical stage at presentation is probably the most important factor in determining the outcome .
it has been found by liu et al . that thickness of the tumor , cervical lymph node metastasis , presence or absence of ulceration , and the anatomic sites are all independent risk factors .
it has been calculated that nodal metastasis reduces the mean survival time from 46 to 18 months .
furthermore , a tumor thickness greater than 5 mm , presence of vascular invasion , necrosis , polymorphous tumor cell morphology , and inability to properly resect the lesion with negative margins have been associated with poor survival in patients with primary melanomas of head and neck region .
despite the improvement of surgical techniques and the introduction of new chemotherapeutic agents , prognosis of this malignancy remains poor .
the generally advanced stage of the tumor at initial diagnosis leads to a poorer survival of patients with mucosal melanomas as compared with patients with cutaneous melanomas and presence of vertical growth phase are associated with median survival rate .
analysis of published cases and recognition of new ones may be helpful in establishing definite classification and proposing clinical features that would facilitate its early diagnosis as a prerequisite for timely treatment and better prognosis of this rare pathology .
|
primary oral melanoma is a rare neoplasm of melanocytic origin , accounting for 0.5% of all oral malignancies .
the chameleonic presentation of a mainly asymptomatic condition , rarity of this lesion , poor prognosis , and the necessity of a highly specialized treatment are factors that should be seriously considered by the involved health provider .
here is a case report presenting a malignant melanoma of oral mucosa in 48-year - old male patient on maxillary gingiva .
the lesion was removed by partial maxillectomy and patient is disease free after 11 months of regular followup .
this case provides an example of how dental clinicians play a major role in the identification of pigmented lesions of oral cavity and also emphasize on the fact that any pigmented lesion detected in the oral cavity may exhibit potential growth and should be submitted to biopsy to exclude malignancy .
|
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rheumatoid arthritis ( ra ) is a chronic systemic inflammatory disease affecting predominantly joints , synovial membranes , articular cartilages , and subchondral bones .
disease progression is attributed to increases in reactive oxygen species ( ros ) and oxidative stress ( os ) in the lesion sites .
proinflammatory cytokines , such as tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and il-6 , regulate the inflammatory and immune responses and play a pivotal role in the disease .
overproduction of nitric oxide ( no ) , as a result of induction of inducible nitric oxide synthase ( inos ) due to enhanced production of these cytokines , is associated with persistent inflammation and tissue destruction in experimental arthritis models , including rheumatoid arthritis [ 4 , 5 ] . a number of inflammation stimuli , including tnf- , il-1 , il-6 , or ros , can activate proinflammatory pathways involved in ra pathogenesis , concerning predominantly nuclear factor-b ( nf-b ) , mitogen activated protein kinases ( mapks ) , or janus kinases / signal transducers and activators of transcription ( jak / stat1/3 ) [ 68 ] .
this results in translocation of relevant downstream transcription factors from the cytoplasm to the nucleus , where they activate messenger rna ( mrna ) expression of target genes , including il-1 , tnf- , inos , and 12/15-lipoxygenase ( lox ) , leading to overproduction of corresponding proteins .
cytokines released into the synovium reach also the systemic circulation and act in other tissues and organs such as lungs , vascular tissue , liver , and heart . several recent investigations reported damage of vital organs with various degrees of impairment , considered to be secondary complications of ra and a major predictor of mortality in ra patients .
increasing evidence is pointing to the critical role of the liver in modulating the immune response in autoimmune and chronic inflammatory diseases including ra [ 5 , 11 , 12 ] .
the hepatic biochemical and immunological alterations are associated with and influenced by changes in the oxidative state of liver cells .
adjuvant - induced arthritis ( aa ) in rats not only is an experimental model of polyarthritis but also induces pathological changes in a variety of other tissues , including the liver and spleen .
it is a useful tool to study immunopathologic processes , autoimmune chronic inflammation , and inflammatory cachexia in rodents .
in addition , at the molecular level , mrna profiling suggests that this model is also similar to human ra , particularly in tissue gene expression and in the activation of regulatory pathways [ 11 , 14 ] .
numerous studies reported natural polyphenols as potential therapeutic agents of diseases caused by os and inflammation [ 1517 ] .
n - feruloylserotonin ( n - f-5ht , n - feruloyl-5-hydroxy - tryptamine ) is a conjugated serotonin , a member of the indole hydroxycinnamic acid amides , with serotonin ( 5-ht ) and ferulic acid ( fa ) as representative components of its structure .
hydroxycinnamic acid amides of serotonin , synthesized by serotonin n - hydroxycinnamoyltransferase , are present in several vegetables and wild - growing plants whose seeds are used in herbal medicine in eastern countries [ 1820 ] . in cell - based studies , under short - term high - glucose conditions ,
n - f-5ht exerted an inhibitory effect on overproduction of mitochondrial superoxide by acting as scavenger of superoxide .
n - f-5ht attenuated the upregulation of mrna and proteins of ros - dependent adhesion ( vascular cell adhesion protein-1 ( vcam-1 ) ) and migration factors ( monocyte chemoattractant protein-1 ( mcp-1 ) ) , crucial in early atherosclerosis lesions in human aortic endothelial cells , and inhibited the activation of transcription factor nf-b .
furthermore , n - f-5ht showed a protective effect on ros - related neuronal damage by decreasing the activity of proapoptotic caspase-3 .
n - f-5ht isomers isolated from seeds of leuzea carthamoides were shown to inhibit protein kinase c / ii activation and decrease the oxidative burst of human whole blood and isolated neutrophils in vitro .
n - f-5ht was also found to have a protective effect against ldl oxidation and atherogenesis in experimental animals and in human studies [ 2426 ] .
methotrexate ( mtx ) , used as a standard drug in our study , represents the most frequently used pharmacotherapy of ra in clinical practice .
its administration is , however , limited due to its toxic side effects [ 27 , 28 ] .
yet application of a combination therapy of mtx with other potential immunomodulators , synthetic drugs or natural substances [ 3032 ] , might elevate the therapeutic efficacy : decrease the dose of mtx and thus its side effects . in our previous study , we showed that administration of n - f-5ht to mtx - treated arthritic rats lowered the dose of mtx for the required sustained antirheumatic impact . in this study , we focused on the therapeutic impact of n - f-5ht and mtx administered in monotherapy and on details of the inflammatory state in the arthritic rat liver with the aim to elucidate the molecular mechanisms of their effect .
one of the possible clarifying approaches is to study the mrna expression of key proinflammatory markers ( il-1 , tnf- , and inos ) in the liver of treated and untreated arthritic rats .
further , it is of particular interest to expand our knowledge on the effect of n - f-5ht and mtx in the aa model , which in turn should allow extrapolations of these results to ra patients .
to this aim we evaluated also conventional arthritic parameters ( hpv , arthritic score , body weight change , and weight of the liver ) along with changes in plasmatic levels of il-1 and crp and the activity of 12/15-lox in the liver .
adult male lewis rats weighing 160180 g were obtained from charles river wiga , germany .
the experimental protocol was approved by the ethics committee of the institute of experimental pharmacology and toxicology and by the slovak state veterinary and food administration in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes and was in line with slovak legislation . to induce a rat model of adjuvant arthritis ( aa ) ,
rats were intradermally injected with a suspension of heat - inactivated mycobacterium butyricum in incomplete freund 's adjuvant ( difco laboratories , detroit , mi , usa ) .
the third group comprised adjuvant arthritis rats treated with methotrexate ( methotrexat ebewe sol inj 20 mg/2.0 ml ) in oral dose of 0.4 mg / kg twice a week ( aa - mtx ) .
the fourth group comprised adjuvant arthritis rats treated with n - feruloylserotonin dissolved in suspension of methylcellulose tween 80 at a dose of 3 mg / kg / day orally ( aa - n - f-5ht ) .
drugs were administered orally by gastric gavage from day 0 ( the day of treatment ) to day 28 of the study .
blood for plasma preparation was taken by retroorbital puncture on day 14 and by cardiac puncture on day 28 under deep ketamine / xylazine anesthesia .
after the animals had been sacrificed under deep ketamine / xylazine anesthesia , tissues for liver and spleen homogenate preparation were taken at the end of the experiment ( day 28 ) .
blood in heparinized tubes for plasma preparation was centrifuged at 3000 rpm for 15 minutes at 4c .
fraction of four isomers of n - f-5ht ( table 1 ) was isolated from the seeds of leuzea carthamoides ( wild ) dc by solvent extraction .
this was then followed by column chromatography on silica gel and hplc separations under conditions previously reported [ 35 , 36 ] .
the hind paw volume ( hpv ) was recorded on days 14 , 21 , and 28 with the use of an electronic water plethysmometer ( ugo basile , comerio , varese , italy ) .
calculation of the increase in hind paw volume in ml assessed the intensity of the edema .
the arthritic score was measured as the total score of hpv ( ml , max .
points 5 ) + diameter of scab in the site of mb application , measured in parallel to the spinal column ( mm , max .
body weight change ( bwc ; g ) was measured on days 1 , 14 , 21 , and 28 .
bwc was calculated as the difference of the body mass measured on days 14 , 21 , and 28 to the body weight measured at the beginning of the experiment ( day 1 ) . for the determination of rat crp concentration in plasma ( g / ml ) , the elisa kit from immunology consultant laboratories , inc .
the reaction of secondary biotin - conjugated anti - rat crp antibody was evaluated by streptavidin - hrp .
the tetramethylbenzidine reaction with hrp bound to immune complex was measured at 450 nm ( microplate reader , labsystems multiskan rc ) .
for the determination of il-1 concentration in plasma , the elisa kit from r&d systems quantikine was used .
rat cytokine present in the samples binds to anti - rat cytokine antibodies absorbed in the microwells .
the reaction of secondary biotin - conjugated anti - rat cytokine antibody is evaluated by hrp .
the tetramethylbenzidine reaction with hrp bound to immune complex was measured at 490 nm in comparison with the reference wavelength of 620 nm ( microplate reader mrx ii ) .
concentration of proteins in liver homogenates was determined by using the bradford method and expressed in mg / ml of enzyme preparation ( cytosolic fraction from rat lung and liver tissues ) .
linoleic acid ( 99% , sigma - aldrich , usa ) was used as a substrate prepared in solubilized state as described in the concentration of 0.2143 100.7143 10 m. the assay of lox was monitored for 60 seconds as an increase in the absorbance at 234 nm , reflecting the formation of hydroperoxylinoleic acid . for the lox activity assay ,
an uv / vis spectrometer perkin - elmer lambda 35 ( usa ) was used .
the reaction medium contained a 50 mm tris - hcl buffer ( ph 7.0 ) , 2.5 l of the enzyme , and solubilized linoleic acid .
total rna was isolated from the rat liver and spleen using rnazol rt ( sigma - aldrich ) and converted into complementary dna ( cdna ) using the primescript rt reagent kit ( takara ) following the protocols of the manufacturers .
amplification and detection of cdna of reference and target genes were performed on a 7300 real - time pcr system ( applied biosystems ) using hot firepol evagreen qpcr mix plus ( rox ) ( solis biodyne ) .
relative mrna expressions of il-1 , tnf- , and inos were analyzed using the ct value method .
the sequences of the primers were designed and checked using primer3 and oligo analyzer 1.0.3 ( table 2 ) .
mean and sem values were calculated for each parameter in each group ( 810 animals in each experimental group ) .
statistically significant differences among treated , untreated , and control groups were tested using parametric analysis of variance ( anova ) .
post hoc tests ( tukey - kramer ( anova ) ) were applied in situations where differences among groups were significant at the level of significance = 0.05 .
after post hoc testing , the following significance levels were specified : extremely significant ( p < 0.001 ) , highly significant ( p < 0.01 ) , significant ( p < 0.05 ) , and not significant ( p > 0.05 ) .
antioxidant properties of polyphenols including n - f-5ht have been reported [ 21 , 40 , 41 ] .
nevertheless , the n - f-5ht impact on chronic inflammatory and os - inducing arthritis , which could widen the possibilities of the ra therapy , remains to be elucidated . in our previous study in the model of aa , n - f-5ht in the dosage of 15 mg / kg markedly potentiated the therapeutic effect of low - dose ( nontherapeutic dose ) mtx ( 0.3 mg / kg ) on arthritic ( hind paw volume and arthritic score ) and inflammatory parameters ( il-17 , mcp-1 , and crp ) , yet it resulted in insignificant effect in monotherapy . as data about the optimal n - f-5ht dose in the rat model
are scarce , we decided to study two doses of n - f-5ht : ( i ) when 15 mg / kg exceeded the physiologically acceptable concentration , we used 3 mg / kg , and ( ii ) when 15 mg / kg was too low to reach the maximal effect , we used 30 mg / kg .
unexpectedly , contrary to the lower dose of n - f-5ht , the higher dose exhibited minor effect on the parameters examined and/or these varied strongly among the animals .
for this reason , this report shows only the data evaluating the lower dose of n - f-5ht . in this study
, we used the therapeutic dose of mtx ( 0.4 mg / kg ) with the intention to compare each mechanism of action of mtx and n - f-5ht , both evaluated in monotherapy .
the significant rise in arthritic parameters , arthritic score , and hpv confirmed the arthritis in our model in rats .
the arthritic score showed an increase in the untreated arthritic group compared to the control group on all days monitored ( aa versus co , day 14 , p < 0.01 ; day 21 and day 28 , p < 0.001 ; table 3 ) . at the end of the experiment ,
the arthritic score was almost doubled in the aa group compared to controls . a trend toward reduction
was observed after administration of n - f-5ht to aa animals on day 28 , but the effect was not statistically significant .
the treatment with mtx significantly reduced the arthritic score on observation days 21 and 28 , compared to the untreated arthritic group , proving the therapeutic potential of the applied dose of mtx ( aa - mtx versus aa , day 21 , p < 0.05 ; day 28 , p < 0.01 ; table 3 ) .
similarly , the change in hpv showed an increase in the untreated arthritic group compared to the control group on days 21 and 28 ( aa versus co , day 21 , p < 0.01 ; day 28 , p < 0.05 ; table 3 ) .
the administration of n - f-5ht induced no modification of hpv of the arthritic animals on any day monitored .
mtx therapy significantly reduced the observed swelling on days 21 and 28 compared to the untreated arthritic group ( aa - mtx versus aa , day 21 and day 28 , p < 0.001 ; table 3 ) .
the muscle wasting condition due to high catabolic activity , known as rheumatoid cachexia , occurring in approximately two - thirds of all patients with ra , is mediated by tnf- and il-1 in ra .
papers published over the past years confirmed that oxidative metabolism was considerably enhanced in the liver of adjuvant - induced arthritis in rats [ 4346 ] .
rats used in this study revealed signs of cachexia ( table 3 ) . a significant decrease in body weight change ( bwc )
the bwc of the arthritic rats was 56% on day 14 , 19% on day 21 , and 27% on day 28 ( aa versus co , days 14 , 21 , and 28 , p < 0.001 ; table 3 ) of the bwc of healthy controls .
n - f-5ht treatment led to a significant increase of bwc on day 28 ( aa - n - f-5ht versus aa , p < 0.05 ; table 3 ) .
the administration of n - f-5ht in arthritic animals did not change these parameters on any of the days observed .
the liver weights were significantly lower ( aa - mtx versus aa , p < 0.05 ; table 3 ) only in the group of rats treated with mtx .
the reduced weight of the liver in mtx - treated rats was assumed to be the result of inhibition of the pathway of de novo dna synthesis by mtx [ 47 , 48 ] . in summary ,
the statistical significance of 3 mg / kg of n - f-5ht treatment was determined only for bwc . the arthritic score revealed a trend toward the positive effect increasing with time , indicating a late onset of n - f-5ht action ( table 3 ) .
as expected , significant differences were found in the arthritic score and hpv in the arthritic animals treated with the therapeutic dose of 0.4 mg / kg mtx compared to those treated with the low dose of 0.3 mg / kg mtx .
il-1 , a prototypic proinflammatory cytokine , is a major mediator of the inflammatory cascade in ra , which is involved in the mechanisms leading to progressive joint destruction . in the model of aa ,
the early phases of the disease seem to be characterized by a systemic increase of il-1 .
the plasmatic level of il-1 , a protein of multiorgan origin , was significantly increased in arthritic animals compared to the control group in the early phase of aa , on day 14 ( aa versus co , p < 0.001 ; figure 1(a ) ) , ascertaining the presence of inflammation .
administration of mtx did not lead to a significant change of plasmatic il-1 concentration ; only a trend toward reduction was observed on day 14 .
it is noteworthy that n - f-5ht treatment resulted in a significant decrease of il-1 level in plasma ( aa - n - f-5ht versus aa , p < 0.05 ; figure 1(a ) ) .
this result is interesting , as this molecule was reported to be relevant in driving the transition from the acute phase to the chronic irreversible phase of the disease and it has been suggested that it could be the target of early intervention to stop the course toward the chronic form of the disease .
the blocking il-1 protects bone and cartilage from progressive destruction in ra and its inhibition could be effective in the treatment of this disease .
activation of t and b cells , macrophages , and inflammatory mediators tnf- , il-1 , and il-6 aggravates the oxidative damage of the vital organs in rheumatoid arthritis , such as the liver .
the liver , in turn , influences the systemic inflammation via producing inflammatory cytokines and mediators such as tnf- , il-1 , il-6 , no , crp , and lox .
il-6 , il-1 , and tnf- promote the synthesis of crp in hepatocytes via stat3 [ 51 , 52 ] and nf-b pathways .
the level of the systemic inflammatory parameter crp in plasma , resulting from liver synthesis , was increased significantly in the group of arthritic animals compared with control animals in the chronic phase of the disease on experimental day 28 ( aa versus co , p < 0.001 ; figure 1(b ) ) .
administration of n - f-5ht and mtx significantly reduced the plasmatic levels of crp on day 28 compared to the untreated group of arthritic animals ( aa - n - f-5ht versus aa , p < 0.05 ; aa - mtx versus aa , p < 0.05 ; figure 1(b ) ) .
interaction of crp with fc - gamma receptors ( fcr ) fcri and fcriia is known to promote the production of proinflammatory cytokines , resulting in the amplification loop of inflammatory reaction .
these processes are initiated through the induction of the receptor activator of nuclear factor-b ligand ( rankl ) protein and direct stimulation of osteoclastogenesis , causing a loop between inflammation and bone destruction in ra .
crp enhances both the proinflammatory response and bone destruction . in the treatment of ra , a lowered crp level thus not only is a significant parameter in terms of disease progression elimination but also has a direct impact on decreasing the degree of bone destruction .
alterations in the oxidative state lead to the activation of nf-b and nf-b - dependent genes , such as lox .
the enzyme 5-lox catalyzes the conversion of arachidonic acid to leukotrienes , whose production has been associated with inflammation in arthritis .
suppression of 5-lox expression ameliorates clinical parameters in ra and aa [ 56 , 57 ] .
increased levels of nf-b in the lung and liver as well as increased activity of lox in the lung highlight the importance of extra - articular manifestations of aa . in our experiment ,
liver 12/15 lox activity increased in arthritic animals in comparison to healthy animals ( aa versus co , p < 0.001 ; figure 1(c ) ) .
the effect of n - f-5ht on the activity of 12/15-lox in liver homogenate was comparable with that of mtx .
after administration of mtx or n - f-5ht , a significant decrease to control levels was assessed in the liver of the aa group ( aa - n - f-5ht versus aa , p < 0.001 ; aa - mtx versus aa , p < 0.001 ; figure 1(c ) ) .
thus the anti - inflammatory effect of n - f-5ht in aa was supported by the ability of the molecule to inhibit 12/15-lox activity . similar to this result , recent observations also reported that several other flavonoids may act as lox inhibitors . in aa ,
the gene expression levels of tnf- and inos produced in the liver were reported to increase [ 60 , 61 ] .
also , in our study , the levels of tnf- and inos mrna expressions were significantly increased in arthritic animals ( both p < 0.001 , aa versus co ; figures 2(a ) and 2(b ) )
. it was proposed that these modifications in the liver of arthritic rats not only were a consequence of the metabolic alterations caused by the disease , especially the increased oxidative metabolism , but also depended on increased inflammatory parameters in the liver .
the same agents that increase oxidative metabolism , tnf- , il-1 , il-6 , and others , are responsible for increasing the activity of inos in several tissues .
an increase of inos activity as a consequence of elevated inos mrna expression was considered to play a dominant role in the pathogenesis of ra .
no generation by inos induced in chondrocytes in the initial stage of aa may play a key role in triggering the subsequent events in arthritis . in general , the use of nos inhibitors has been shown to exert beneficial effects in experimentally induced arthritis . however ,
which types of cells expressing inos are associated with the induction or progression of adjuvant - induced arthritis via no generation remains uncertain .
mrna expression of inos in rat liver was reduced following mtx ( aa - mtx versus aa , p < 0.001 ; figure 2(a ) ) and n - f-5ht treatment ( aa - n - f-5ht versus aa , p < 0.01 ; figure 2(a ) ) .
the effect of mtx treatment on tnf- protein and mrna expression differs among studies , depending on the conditions of the given study , concerning gender of patients , type of cell line , duration of treatment , mtx dose , and so forth . in our study in the rat aa model , administration of mtx
attenuated significantly the mrna expression of tnf- ( aa - mtx versus aa , p < 0.01 ; figure 2(b ) ) . in many patients , however , mtx treatment does not result in lower tnf- plasma concentration .
when mtx fails to produce an adequate response , newer therapies are used in combination with mtx .
blocking tnf- with anti - tnf- monoclonal antibodies significantly decreased the signs and symptoms of ra compared to placebo in ra patients with active disease receiving mtx [ 65 , 66 ] .
thus , the n - f-5ht - driven significant reduction of tnf- mrna expression ( a - n - f-5ht versus aa , p < 0.01 ; figure 2(b ) ) suggests an intriguing effect on ra treatment , calling for deeper investigation .
increase of mrna expression was observed for il-1 in the liver of arthritic animals ( aa versus co , p < 0.001 ; figure 3(a ) ) as expected .
administration of mtx did not lead to significant attenuation of il-1 transcription in the liver .
this is in concert with previous studies of mtx function in different types of cells ( e.g. , human peripheral blood mononuclear cells and murine peritoneal and splenic cells ) [ 67 , 68 ] . on the other hand
, mtx exhibits another mechanism of il-1 function inhibition , which involves blocking the binding of il-1 to il-1 receptor in the membrane of peripheral blood cells ( monocytes , lymphocytes , and granulocytes ) .
contrary to mtx , treatment with n - f-5ht led to a substantial inhibition of il-1 gene expression ( aa - n - f-5ht versus aa , p < 0.01 ; figure 3(a ) ) .
further , we examined il-1 mrna expression in the main immunocompetent organ , in the rat arthritic spleen , which has not been studied previously in terms of the aa model , related to il-1 expression .
we observed il-1 mrna expression activation comparable to that in the liver ( aa versus co , p < 0.001 ; figure 3(b ) ) .
interestingly , both mtx and n - f-5ht exhibited a significant and remarkably stronger inhibition of il-1 mrna expression in comparison to that in the liver ( aa - mtx versus aa , p < 0.01 ; aa - n - f-5ht versus aa , p < 0.001 ; figure 3(b ) ) .
in the spleen of n - f-5ht treated rats , the relative mrna expression decreased even to control level .
besides other events , mtx treatment leads to suppression of nf-b , a heterodimer consisting of two subunits p65 and p50 , one of the most prominent inflammatory transcription factors activated in ra .
this was confirmed in our previous work , along with the finding that also n - f-5ht ( 15 mg / kg ) suppressed the activation of nf-b ( p65 ) in the arthritic rat liver [ 21 , 33 ] .
interestingly , combination therapy ( mtx + n - f-5ht ) potentiated the effect of a single drug , suggesting different mechanisms leading to nf-b inhibition .
mtx driven reduction of cytokine transcription was attributed to abrogation of ib kinase activation and thereby suppression of ib ( nf-b inhibitor ) phosphorylation and degradation , resulting in retaining the inactive nf-b form in cytoplasm .
however , the contribution of n - f-5ht to nf-b pathway suppression needs to be further investigated .
studies of the proposed pathways involved in the transcription of tnf- , il-1 , and inos in ra could help evaluate the mechanism of action of these drugs [ 68 , 71 , 72 ] .
the gene expression of inos is mostly under the control of synergistically activating nf-b ( il-1 and tnf- stimulated ) and stat1 ( ifn- stimulated ) key proinflammatory signals in the liver .
in contrast to inos , tnf- does not contain the stat binding element in its promoter region .
the inhibition of tnf- and inos transcription observed in our study might be mostly attributed to the suppressed nf-b pathway for both mtx and n - f-5ht [ 33 , 64 , 70 ] .
however , the contribution of ap-1 to tnf- and stat1 for inos can not be excluded .
mtx - dependent suppression of nf-b was reported [ 33 , 70 , 73 , 74 ] , but in other cases mtx was not found to be effective in the attenuation of arthritic - increased mrna expression of il-1 [ 68 , 75 ] . taking into account our results , where mtx treatment did not lead to inhibition of il-1 mrna expression in the arthritic liver in contrast to the significant n - f-5ht impact , yet treatment of both mtx and n - f-5ht decreased the presumably nf-b - dependent lox activity and inos and tnf- transcription to a similar extent , the involvement of n - f-5ht in another pathway for transcription regulation of this cytokine in the arthritic liver should be considered .
after analysis of the reported pathways involved in the regulation of il-1 mrna expression , we hypothesized that tnf--driven ap-1 transcription factor activation or jak / stat3 pathway activated via il-6 or ifn- might play a role ( [ 7 , 8 , 71 , 72 , 76 , 77 ] , figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/7509653 ) .
papers reporting involvement of other polyphenols in anti - inflammatory regulation , for example , resveratrol , claim that these compounds exhibit their anti - inflammatory effect through suppression of nf-b and jak / stat signaling pathways [ 78 , 79 ] .
the enhanced influence of mtx and n - f-5ht on il-1 transcription in the spleen in comparison to the liver may be the consequence of different predominance of inflammatory pathways in this organ , presumably with a stronger nf-b contribution .
details about the relevance of these pathways and the role of n - f-5ht in the transcription regulation of il-1 , inos , and tnf- in the liver and other organs in ra are to be further elucidated .
the present study contributed additional evidence about the beneficial effect and mechanism of action of n - f-5ht and of mtx on a systemic inflammatory process in the liver and its association with the pathogenesis of adjuvant arthritis .
n - f-5ht treatment led to amelioration of inflammatory parameters tested ( plasmatic crp and il-1 protein levels , liver lox activity , and liver and spleen cytokine expression ) .
however , this did not result in a significant change of hpv , although a trend of improvement of the arthritic score was observed after 28 days .
a synergistic effect of tnf- and il-1 was shown to influence the balance between protein degradation and protein synthesis causing among others an increase in resting energy expenditure and net efflux of amino acids from muscle to liver .
the significant increase of bwc in n - f-5ht treated rats , probably sign of the partial improvement of rheumatoid cachexia , might be the result of lowered mrna expression of tnf- and il-1 determined in the arthritic liver . moreover , taking into account the reported association of weight loss with the il-1 production by splenic cells , the n - f-5ht mediated attenuation of increased il-1 mrna expression in the arthritic spleen might contribute to this complex process
. the contribution of the affected expression of tnf- and il-1 originating from other organs can not be excluded and is to be further elucidated .
unexpectedly , chronic daily treatment with a high concentration of n - f-5ht ( 30 mg / kg ) exhibited either a minor effect on the parameters examined and/or a strong variation among the animals ( not shown ) and that in contrast to a much lower concentration ( 3 mg / kg ) . since n - f-5ht possesses a serotonin ( 5-hydroxytryptamine , 5-ht ) moiety , the question if there might be some interplay between effects of these two molecules on ra pathogenesis is to be raised . since n - f-5ht inhibited the increase of cytosolic free ca concentration in rat vascular smooth muscle cells induced by serotonin mediated by 5-ht2 receptors , it was hypothesized that at a sufficient concentration n - f-5ht may act as a competitive antagonist , which displaces serotonin from its binding site .
intake of a high concentration of a 5-ht2 receptor antagonist may lead to a variety of effects : it may influence the receptor density , even enhance the effect of serotonin , or lead to desensitization and with time to receptor resistance ( through inhibitory feedback due to binding - induced enhanced production of serotonin ) .
interestingly , serotonin is known not only as a neurotransmitter . increasing but contradictory reports associate serotonin with immunoinflammatory pathways in the periphery .
serotonin , via its 5-ht2a , 5-ht2b , and 5-ht3 receptors , has been implicated to have both proinflammatory and anti - inflammatory roles in a number of studies of rheumatoid arthritis [ 8488 ] .
the reported effects of 5-ht receptor antagonist on macrophage - like synovial cells encourage the interest to study the effect of n - f-5ht from this point of view . to confirm this hypothesis ,
a precise characterization of interaction between n - f-5ht and 5-ht receptors is to be done . on comparing the effects of the two drugs , administration of mtx ( 0.4
mg / kg ) or n - f-5ht ( 3 mg / kg ) was found to lead to a decrease of the main plasma marker of systemic inflammation crp , the liver origin protein , and to inhibition of proinflammatory lox in the liver .
the impact of mtx and n - f-5ht on mrna expression of tnf- , il-1 , and inos in the liver and on the level of crp in plasma was mentioned at the conference .
mtx and n - f-5ht reduced the arthritis - increased transcription of tnf- and inos in the liver to a comparable extent . we suppose that the inhibition of tnf- and inos transcription might be mostly attributed to the suppressed nf-b pathway for the two drugs [ 21 , 33 , 70 ] .
as previously reported [ 67 , 68 ] and also proven by our study , mtx was not able to diminish the arthritic - induced il-1 mrna transcription in the liver .
this handicap might be compensated by coadministration of n - f-5ht , since this drug was shown to lower the level of proinflammatory cytokine il-1 in plasma in the acute phase of aa and to attenuate significantly the elevation of il-1 mrna expression in the arthritic rat liver and spleen in the chronic phase .
detailed studies are required to confirm the hypothesis that n - f-5ht might function through potentially different mechanisms of inhibition of the inflammatory pathway nf-b and not through mtx , as well as the possibility of an additional pathway influencing il-1 transcription under control of n - f-5ht but not mtx .
the confirmation would support n - f-5ht as a promising agent for the treatment of ra in combination therapy with mtx .
the positive effect was shown in our previous study , where n - f-5ht markedly potentiated the therapeutic effect of low - dose mtx .
as the therapeutic dose of mtx was used in this study and the purpose of combination study is to lower the mtx dose to decrease the side effects of this drug , the effect of combination therapy was not included
. oral daily intake of n - f-5ht could overcome the inconvenient administration and high costs of biological therapy using il-1 monoclonal antibody , which was shown in clinical trials to be superior to placebo in combination with mtx in reducing signs , symptoms , and radiographic progression in patients with advanced ra [ 91 , 92 ] .
future studies of n - f-5ht mechanisms of action should shed more light on the immunomodulatory function of this natural polyphenol .
it is to be expected that n - f-5ht is able to positively affect the activity of other markers of inflammation and oxidative stress not only in the liver and spleen but also in other organs ( lung , brain , etc . ) , a hypothesis to be tested by future work .
however , to establish the optimal dosing in light of the effects achieved is of primary importance .
|
rheumatoid arthritis ( ra ) is a chronic inflammatory disease , leading to progressive destruction of joints and extra - articular tissues , including organs such as liver and spleen .
the purpose of this study was to compare the effects of a potential immunomodulator , natural polyphenol n - feruloylserotonin ( n - f-5ht ) , with methotrexate ( mtx ) , the standard in ra therapy , in the chronic phase of adjuvant - induced arthritis ( aa ) in male lewis rats .
the experiment included healthy controls ( co ) , arthritic animals ( aa ) , aa given n - f-5ht ( aa - n - f-5ht ) , and aa given mtx ( aa - mtx ) .
n - f-5ht did not affect the body weight change and clinical parameters until the 14th experimental day .
its positive effect was rising during the 28-day experiment , indicating a delayed onset of n - f-5ht action .
administration of either n - f-5ht or mtx caused reduction of inflammation measured as the level of crp in plasma and the activity of lox in the liver .
mrna transcription of tnf- and inos in the liver was significantly attenuated in both mtx and n - f-5ht treated groups of arthritic rats .
interestingly , in contrast to mtx , n - f-5ht significantly lowered the level of il-1 in plasma and il-1 mrna expression in the liver and spleen of arthritic rats .
this speaks for future investigations of n - f-5ht as an agent in the treatment of ra in combination therapy with mtx .
|
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molecular hydrogen ( h2 ) is a zero - emission energy carrier and is one of the best alternative fuels for the future , especially in urban areas with stricter environmental requirements .
hydrogenase ( h2ase ) enzymes catalyze the bidirectional reaction of 2h + 2e h2 ( jugder et al . , 2013 ; kim & cha , 2013 ) and
thus have attracted interest for potential applications in h2 production in addition to their importance in biological sciences ( mertens & liese , 2004 ; cammack et al . , 2001 ; heinekey , 2009 ; tard & pickett , 2009 ; yang et al .
, 2011 ; fritsch et al . , 2013 ; simmons & artero , 2013 ; matsumoto et al . , 2013 ) .
while both diiron ( [ fefe ] ) and nickel iron ( [ nife ] ) h2ases possess high catalytic activity ( evans et al .
, 2013 ; armstrong , 2009 ; shafaat et al . , 2013 ; lubitz et al . ,
2014 ) , the latter are attractive practically since they exhibit greater o2-tolerance ( guiral et al . , 2006 ; evans et al . , 2013 ; lauterbach et al . , 2015
[ nife ] h2ase contains several fe s clusters and one nife active site , in which fe is coordinated by one co and two cn ligands ( kamali et al .
the ni is coordinated to the protein matrix by four cysteinyl thiolates ( s ) , two of which serve as bridging ligands to fe .
in addition to these two s ligands , there could be a third ligand to bridge the two metal atoms in several enzymatic states ; for example , a hydroxide ligand in the oxidized inactive forms ( ni a and ni b ) ( dole et al . , 1997 ; gu et al . , 2003 ; gastel et al .
, 2015 ) and a possible hydride ( ni h fe ) in the active forms ni c and ni
1(b
1)1(b
6 ) ] , and the possible mechanism for h2 binding and cleavage at the h2ases active site .
for example , via a density function theory ( dft ) calculation , the structures in figs .
1(b
5 ) and 1(b
6 ) are believed to be the most likely candidates in the real ni
information about the mechanism in turn could be useful for better producing h2 in the future . despite progress in characterization of h2ases by crystallography , infrared ( ir ) spectroscopy ( lubitz et al .
, 2014 ; fontecilla - camps et al . , 2007 ; de lacey et al . , 2007 ) and other x - ray spectroscopies ( wang et al .
, 2000 ; wang , patil , gu et al . , 2001 ; wang , patil , ralston et al . , 2001 )
, questions remain about the detailed molecular and electronic structure of various intermediates and inhibited species .
these highly debated questions include the nature or even the existence of a hydride bridge ( ni h fe ) in ni
recent high - resolution crystallography demonstrated that the electron density as a hydride was detected at the bridging position between ni and fe ( ogata , nishikawa & lubitz , 2015 ) .
r represents a special challenge for spectroscopic studies for a number of reasons : ( i ) it is epr ( electron paramagnetic resonance ) silent ; ( ii ) the raman spectroscopy presents a problem due to its photoreactivity ; and ( iii ) ir spectroscopy for hydride bands is extremely difficult to observe ( jayapal et al .
thus there has been no progress in traditional vibrational spectroscopy so far and the existence of ni h fe in ni
nuclear resonance vibrational spectroscopy ( nrvs ) scans an extremely monochromatic ( 1 mev ) x - ray beam through the nuclear resonance ( at 14.4 kev for fe ) , and measures the corresponding creation or annihilation of phonons ( seto et al . , 1995 ; sturhahn et al .
it is a relatively new x - ray spectroscopy that became available due to the development of third - generation synchrotron sources and advanced x - ray optics .
it has several distinguished advantages in comparison with traditional vibrational spectroscopic techniques such as ir and raman spectroscopies ( smith et al .
, 2005 ; kamali et al . , 2013 ; pelmenschikov et al . , 2011 ) . in the past ten years
, this technique has revealed / resolved fe s / p / cl and fe co / cn / no vibrational modes inside various inorganic complexes and iron enzymes and has become an excellent pin - point tool in recent years to study iron - specific inorganic and bioinorganic systems ( smith et al .
, 2005 ; cramer et al . , 2007 ; tinberg et al . , 2010
in addition , fe h - related nrvs features have also been resolved in several inorganic complexes ( bergmann et al .
via a series of careful and strenuous measurements , we have revealed the first direct spectroscopic evidence for ni
r form of desulfovibrio vulgaris miyazaki f ( or dvmf ) [ nife ] h2ase using fe - specific nrvs . while its biochemical science and theoretical simulations have been presented and discussed recently ( ogata , kramer et al .
, 2015 ) , the measurement details , especially the strenuous experimental journey leading to the successful observation of the weak ni
fe in a [ nife ] h2ase sample , and the task will face great difficulties due to the following two aspects : ( i ) a fe h - related bending or wagging mode has a much weaker nrvs signal than fe co ( pelmenschikov et al .
, 2011 ) while fe co in nife h2ase is already rather weak ( kamali et al . , 2013 ; ogata , kramer et al . , 2015
( ii ) as will be discussed in detail later , its real energy position is far from either model complexes or theoretical predictions and was virtually unknown before it was observed experimentally .
this article evaluates all the matters related to whether the feature is observable , where to search for a fe h - related feature , whether the observation is reliable , and how the experiments identify the observed 675 cm peak as a fe h - related feature .
r nrvs spectra were recorded at spring-8 beamlines bl09xu ( yoda et al . , 2001 ) and bl19lxu ( yabashi et al .
bl09xu is a dedicated nuclear scattering beamline , and its instrumentation and properties have been discussed in detail elsewhere ( yoda et al .
, it has a si(111 ) double - crystal high - heat - load monochromator ( hhlm ) producing 14.4 kev radiation with 1.0 ev resolution , followed by a high - energy - resolution monochromator ( hrm ) [ ge(422 ) 2si(975 ) ] producing 14.4 kev radiation with 0.8 mev resolution .
the beam flux was 1.4 10 photons s at 0.8 mev energy resolution .
the beam size was about 0.6 mm ( height ) 1 mm ( width ) .
bl09xu has a dedicated nrvs measurement system , including a 2 2 avalanche photodiode ( apd ) detector array , a set of data - acquisition electronics and a control computer with data - acquisition software .
it also has a dedicated liquid - helium ( lhe ) flow cryostat for measuring nrvs at a cryogenic temperature for air- or temperature - sensitive samples , such as h2ases .
however , due to the extremely short distance from the samples surface to the apd array , a cold - finger cryostat has to be used instead of a more efficient gas exchange cryostat ( dong et al .
the real sample temperatures were much higher than 69 k , e.g. 5070 k ( wang et al . , 2012 ) .
this standard nrvs setup has been used in many nrvs measurements in the past and their details are discussed elsewhere ( cramer et al .
, 2001 ) provides the fundamental beam in the 7.218 kev energy region , covering fe nuclear resonance at 14.4 kev . as shown in fig .
2(a ) , it has a 25 m - long undulator ( hara et al . , 2002 ) instead of the usual 5 m - long undulator at spring-8
. therefore its hhlm provides 2.5 10 photons s at 14.4 kev , i.e. about five times higher than the beam intensity at bl09xu ( 5 10 photons s after hhlm ) .
this beamline has many advanced applications and publications over a broad area from time - resolved measurements to weak feature observations ( tanaka et al .
it is also a good choice for nrvs measurements on weak vibrational features , which requires high beam intensity .
however , bl19lxu is not a dedicated nuclear scattering beamline and it does not have a fixed hrm and nrvs measurement station .
2b
) which consists of a hrm , a lhe cryostat , a 2 2 apd detector array , a rack of associated electronics , and a control computer with data - acquisition software .
while these items are duplicates of those used at bl09xu , they need to be integrated into bl19lxu s experimental hutch 1 within about 36 h ( including optimization time ) .
therefore , bl19lxu has similar hhlm and hrm as those at bl09xu , and provided 14.4 kev radiation with 0.8 mev resolution , which is suitable for nrvs measurement .
the beam size was also 0.6 mm ( height ) 1 mm ( width ) , the same as that at bl09xu .
after the hrm , a maximum 4.2 higher beam flux was recorded in comparison with that for bl09xu . however , owing to various practical limitations as well as using a moved - in hrm and nrvs apparatus , the ratio in nrvs counts per second ( cts / s ) for the two beamlines is 2.53.4 instead , while for bl09xu the cts / s is stable .
for example , in comparison with the bl09xu standard , the bl19lxu beam time for measuring the two ni
r in h2/h2o ( nir - h for short ) samples in this study has a bl19/bl09 cts / s ratio of 2.6 , while that for measuring the ni
r in d2/d2o ( nir - d for short ) has a ratio of 3 .
, 2007 ; seto et al . , 1995 ; sturhahn et al . , 1995 ; yoda et al . , 2001 ) , with a step size of 0.28 mev at bl09xu and 0.27 mev at bl19lxu .
the difference is due to minor difference in energy scales for the different beamlines ; the raw step sizes before the energy calibrations were both 0.295 mev .
the 14.4 kev beam with an energy resolution of 0.8 mev was scanned through the defined energy region ; the delayed nuclear fluorescence and fe k
fluorescence from fe were detected with the 2 2 apd array , processed by the electronics , and stored / displayed via the control computer .
the scanning region covered from 30 mev ( 240 cm ) to 70125 mev ( 5601000 cm ) depending on the different samples used and the different search cases .
, 1995 ) , where the observed raw nrvs spectra ( cts ) were calibrated to the nuclear resonance peak position , normalized to i
0 , summed and converted to the single - phonon fe partial vibrational density of states ( pvdos for abbreviation ) .
the spectral conversion was optimized when the observed stokes / anti - stokes imbalance matched the imbalance calculated using the entered temperature as a parameter .
the real sample temperatures were thus obtainable with the measured nrvs s stokes / anti - stokes imbalance ( wang et al . , 2012 ) .
the energy position for each scan was calibrated by aligning the elastic peak to zero during the nrvs spectral analysis with phoenix .
the energy scales were usually calibrated with a standard sample of [ fecl4][net4 ] with a prominent peak at 380 cm .
alternatively , the energy scales can also be calibrated with fe metal powder at the quick - switch calibration stage at the back of the main measurement stage ( wang et al .
it has a clear peak at 287 cm at room temperature . the background noise level ( dark current cts / s )
was estimated prior to ( and sometimes during ) each beam time by tuning the hrm s energy position to about 100 mev or about 800 cm ( in reference to the resonance elastic energy ) , where no vibrational peak is present .
signal was accumulated for either 500 s in total or five consecutive 100 s to obtain an average cts / s . for almost all the beam times , the dark current was 0.03 cts / s for our nrvs measurement system at bl09xu and at bl19lxu . since the relative strength of nrvs transitions varies dramatically and since it is necessary to emphasize one region of interest ( e.g. for searching for ni
fe ) , the scans were divided into segments with different data collection times ( seconds per point , or s / p ) . in general , 13
s / p was used for the range from 240 to 400 cm ( covering the fe s region ) , then 510 s / p for the fe
cn and fe co region from 400 to 620 cm . a longer scanning time ( 1030 s ) was used for the candidate ni h fe searching region ( e.g. at 620770 cm ) .
this practice is used for the nrvs measurements at both bl09xu and bl19lxu , but the exact timing for different beam times varied a little .
the scan details will be discussed again in the results and discussions section . to compare the nrvs data from bl09xu and bl19lxu
, we re - scale the bl19lxu counting time based on its maximum cts / s versus the standard maximum cts / s at bl09xu and create bl09xu equivalent seconds , e.g 10 s / p at bl19lxu corresponds to 26 or 30 equivalent s / p at bl09xu [ corresponding to a cts / s ratio of 2.6 for measuring nir - h and 3 for measuring nir - d ] .
nir - h was measured once at bl09xu and twice at bl19lxu with an equivalent bl09xu time of 10 680 = 680 s / p , 20 24 2.6 = 1248 s / p and 15 12 2.6 = 468 s / p , respectively , in total 2396 s / p ( with a percentile of 28% , 52% and 20% ) .
nir - d was measured three times at bl09xu and once at bl19lxu , equivalent to 3980 bl09xu s / p [ = ( 40 20 ) + ( 30 30 ) + ( 36 30 ) + 20 20 3 = 2780 + 400 3 = 3980 ] in the same region . more s / p was used for nir - d because more time should be used to conclude that there is no signal than to find a weak signal .
fermentation was carried out in a 10 l glass fermenter under anaerobic conditions , and the [ nife ] h2ase expressed was isolated and purified as described earlier ( ogata et al .
for preparing nir - h ( nir - d ) , the as - isolated [ nife ] h2ase was transferred from 25 mm tris - hcl ( ph = 7.4 ) buffer to 100 mm mes ( ph = 5.0 or pd = 5.0 ) and was then purged with 1.2 bar h2 ( or 1.3 bar d2 ) for more than 8 h. the solutions were then loaded into nrvs cells under anaerobic conditions .
fourier transform ir ( ftir ) spectra were recorded on a bruker ifs66v / s ftir spectrometer to ensure the samples were in their supposed states ( ni r1 : one of the subforms of ni
it was measured in transmission mode in a sealed ir cell at room temperature and with a 2 cm energy resolution .
nrvs or the fe h / d - related bending positions for several fe
, 2011 ; crossland et al . , 2009 ; schilter et al . , 2012
) : ( i ) [ feh(d)6][mgbr(thf)2]4 ( or fefeh6/d6 for short ) ; ( ii ) fe(h / d)(co)(dppe)2 , dppe = 1,2-bis(diphenylphosphino)ethane ( or h / dfeco ) ; ( iii ) trans-[fe(dmeoprpe)2(n2)h ] { dmeoprpe = 1,2-bis[bis(methoxypropyl)phosphino]ethane } ( or hfen2 ) ; ( iv ) [ ( ,k2-bdt - h)(-pph2)(-h)fe2(co)5][otf ] ( or fehfe ) ; ( v ) [ ( dppe)ni(-pdt)(-h)fe(co)2(ph3p ) ] ( * = nature abundant , dppe = 1,2-ph2pch2ch2pph2 , pdt = sch2ch2ch2s ) ( or nihfex ) ; ( vi ) [ ( dppe)ni(-pdt)(-h / d)fe(co)3 ] ( or nih / dfe ) .
in addition , [ fecl4][net4 ] and metal fe were used as calibration samples for energy scales ( smith et al . , 2005 ;
the overall nrvs spectra for dvmf nir - h ( blue ) and nir - d ( red ) are presented in fig .
the nrvs spectra include the very low energy intensities from the backbone motion , the 100400 cm peaks for fe s cluster vibrations , the 420530 cm peaks for fe
cn modes , the 530630 cm peaks for fe co modes ( lauterbach et al . , 2015 ; kamali et al . ,
2013 ) and a weak but clear peak at 675 cm , which are assigned to the ni h
3 , the top bars indicate different vibrational regions while the bottom bars illustrate the total time ( s ) used to measure the nir - h ( blue text ) and nir - d ( red text ) in each region . the blue text in the middle shows the signal level ( cts / s ) for each of the various signature peaks . while these middle blue numbers illustrate the signal level ( cts / s ) for nir - h , both nir - h and nir - d have about the same signal level .
co / cn features have a much lower cts / s than the fe s features while the ni h fe has an even lower cts / s in comparison with the weak fe
this provides readers with a basic idea of how weak the ni h fe peak in ni
1(b
1)1(b
6 ) ] ( ogata , kramer et al . ,
2015 ) , the existence of ni h fe at 675 cm for nir - h ( blue ) and its absence for nir - d ( red ) provided a critical observation reference for the dft calculations , not the other way around . without this successful observation ,
r is observable or not . as per the nrvs statistics at bl09xu between 2006 and 2010 , when the hrm s energy resolution is at 0.9 mev , 1 mm of fe sample produced a 30 cts / s signal in the elastic peak ( wang et al .
4 , using 30 cts / s per 1 mm
fe .
the first two columns describe vibrational feature names and their vibrational energy positions , and the third column presents the nrvs signals for a hypothetical 1 mm h2ase ( or 12 mm
fe ) sample , which is similar to the h2ase measured in the past ( kamali et al .
it has 360 cts / s in the resonance peak , 0.71.2 cts / s for the fe s peaks ( at 150380 cm ) , and 0.06 cts / s signal for fe co ( at 600 cm ) .
co features were successfully observed with a 1 mm h2ase sample in the past ( kamali et al .
fe was too weak to be observed under previous conditions ( 1 mm h2ase concentration ) , so we have to estimate its potential cts / s ( the numbers below the
fe and fe co in available complexes , e.g. 1/5 for nihfe ( fig .
5b ) . a value of 0.012 cts / s is estimated for ni h
fe in 1 mm nir - h at bl09xu ; it is under the dark cts / s level ( 0.03 cts / s ) and is not observable under the given circumstances . since the completion of the nrvs measurements for kamali et al.s publication ( kamali et al .
, 2013 ) , further progress has been made to increase the potential nrvs signal level ( cts / s ) .
the major advance is the samples concentration increasing from 1 mm to 4 mm , which puts the ni
fe feature at a hypothetical level of 0.012 4 = 0.048 cts / s .
some other minor improvements also contribute to the increase of cts / s : ( i ) bl09xu s hrm advanced from 0.9 mev to 0.8 mev energy resolution with almost the same level of beam intensity , which led to an increase in peaks cts / s [ maximum = ( 0.9/0.8 ) = 112.5% ] ; ( ii ) due to better control of the sample temperature during the nrvs measurement ( wang et al .
, 2012 ) , we are able to move the sample even closer to the apd .
it is difficult to estimate the real movement of the sample position but we obtained about 10% signal level increase in cts / s due to sample position alternation while still maintaining the sample temperature below 70 k. these two points thus mean all the nrvs peaks have 1.2 height .
fe is then about 0.048 1.2 = 0.058 cts / s , which is comparable with the fe co feature for 1 mm h2ase in the previous measurement ( kamali et al .
4 ( not counting the narrow columns showing the arrow symbols ) shows the estimated cts / s for a hypothetical 4 mm h2ase , which will be measured at the improved bl09xu ( 1.2 ) .
then , how much time is needed to reveal such a weak peak ( 0.058 cts / s ) with a reasonable signal - to - noise ratio ( s / n ) ? in a previous publication ( wang et al .
, 2014 ) we have concluded that a 10 mm
fe sample has 1 cts / s in the fe s region and 100 s / p is thus enough for a s / n = 100 = 10 . using the same principle
fe can lead to 0.058 500 = 29 pure cts and s / n = 29 = 5.4 .
although dark - current cts / s limits the best possible s / n , as will be discussed in detail later , an as - high - as - possible statistical s / n is still required and thus 500 s / p is a kind of realistic minimum time required to achieve a reasonable s / n for a weak feature like ni h fe . as a reference , the very weak fe co features for the oxidized and the reduced 1 mm h2ase were observed with 580 and 2180 s / p total scanning time at bl09xu in the previous publication ( kamali et al . , 2013 ) . the 500 s / p scanning time means , for example , 25 20 s / p , leading to about 24 h or more beam time for one measurement with one ni
fe signal is as important as ( if not more important than ) knowing it is observable .
r , which requires long ( e.g. 24 h or more ) beam times for each search .
since no nrvs features for iron hydrides or iron deuterides in a real iron enzyme have been published before this work , we used nrvs for a series of iron hydride / deuteride complexes and their fe h - related vibrational positions ( fig . 5 ) as a starting point to discuss the possible location of the ni
the nrvs feh6 ( blue)/fed6 ( red ) spectra were among the first nrvs spectra obtained for chemical complexes ( bergmann et al .
their features include : the x fe h / x fe d bending modes at 790/572 cm and the fe
later on , many other iron - hydride - containing complexes were published or evaluated , e.g. the ( h / d)feco model complexes have the x fe h / x fe d at 740/620 cm , respectively ( pelmenschikov et al . , 2011 ) .
we noticed that the x fe d bending peak for this complex is close to the fe
the nihfe complex is used to model [ nife ] h2ases active site and it mimics the structure for the [ nife ] center with very minor structural differences ( barton et al . , 2009 ; shafaat et al .
its ni h fe wag mode is at 758 cm ( fig .
d stretching ( mixed with fe ) per dft calculation ( ogata , kramer et al .
it is not fe d - related bending , which is mixed into the fe co features .
the nrvs feature most relevant to ni r s ni h fe is the x fe h or ni h fe feature in various complexes .
the energy positions of these fe h - related bending modes for feh6 , hfeco , hfen2 , fehfe , nihfex and nihfe ( refer to 2 for the full names of these complexes ) are illustrated in fig .
5(b ) . the blue shaded area highlights the possible fe h - related bending energies while the orange ( purple ) area indicates the fe
d bending ( stretching ) mode regions . from these fe h / d complexes
we have learned the following : ( i ) although fe d - related bending has more intensity than fe h - related bending , it may mix with fe co and become unresolvable .
therefore , for fe co - containing complexes , the fe h - related bending mode has the highest observable nrvs peak among all of the fe h / d - related stretching and bending modes .
( ii ) the fe h - related bending mode positions span from 740 to 790 cm for the examined iron hydride complexes , including those whose spectra are not shown here .
dft was also used to make a prediction about the possible position of this critical vibrational peak .
however , all of the earlier dft calculations as well as normal mode analyses ( before the ni
fe was finally observed experimentally ) pointed to a region with energies higher than 760 cm ; some of them suggested a region higher ( or much higher ) than 800 cm and we experimentally searched up to 125 mev ( or 1000 cm ) , but did not find anything .
fe regions was searched one to two times but led to no success at all .
although 620720 cm became the only region not searched with a great investment of beam time , the idea to search in this region was not at all popular at the time , because all the model complexes and theoretical calculations pointed to a much higher energy position .
r model complex nihfe has its ni h fe at 758 cm , 140 cm higher than its highest - energy fe co peak at 617 cm , while ni
then the question arose : should we perform a thorough search in this unlikely but unsearched region ?
fe at 675 cm . as searching in the 620720 cm region was still not a mainstream opinion at that time , we first performed a balance measurement between acquiring a perfect fe co spectrum and searching for ni
fe with a 4.5 mm nir - h sample at ( improved ) bl09xu .
therefore an even scanning time of 10 s / p was used between 400 and 740 cm instead of spending more time ( e.g. 2030 s ) in the ni
( with a total scanning time of 680 s / p ) a weak but positive peak at 675 cm was observed for the first time as shown in fig .
6(a ) , with the raw nrvs spectrum at the top and the converted pvdos at the center ( 10 ) and the bottom ( 1 ) .
we noticed that the 675 cm energy position is much lower than any of the previous dft or normal mode analyses predicted theoretical energy positions .
it is actually still at least 32 cm lower than the lowest current dft value , which is at 707 cm [ model vi in fig .
1(b
6 ) ] ( ogata , kramer et al . , 2015 ) .
the observed cts / s for various fe s , fe co and ni h fe in ni
r are documented in fig . 4 [ column 6 ( not counting the columns showing
the first argument was that the observed peak at 675 cm was still too weak .
the second argument attributed this peak to a fe co - related feature , rather than to a fe h - related peak .
however , there is no such fe co sub - feature in the mimic complex nihfe ( ogata , kramer et al .
these nrvs measurements on nir - d took 106 scans in total (= 40 + 30 + 36 ) and 2780 total s / p [ = ( 40 20 ) + ( 30 30 ) + ( 36 30 ) ] , but concluded no peak in the same region .
this suggests that the peak at 675 cm is related to a fe h vibrational mode . in order to resolve the first issue ( to repeat the observation and to improve the spectral s / n ) and to further clarify the second issue , two samples of nir - h and one sample of nir - d were re - measured at bl19lxu , which has a higher beam intensity and more nrvs cts / s than bl09xu .
a similar pair of nrvs and pvdos for nir - h ( blue , 24 scans , 480 s / p ) and for nir - d ( red , 20 scans , 400 s / p ) are compared in fig .
6(b ) , illustrating much clearer evidence for the existence and the fe h - related nature for the 675 cm peak . from fig . 4 ( column 6 for one measurement at bl19 and column 7 for one measuremnt at bl19 , not counting the columns with the arrow symbols ) , the observed weak ni
fe wagging mode is 2729 parts per million in intensity in comparison with the nuclear resonance peak , or 0.8% in comparison with the fe
co ( s ) , this ratio is about 14.816.6% , or , equivalently , ni h fe : fe co 1:61:7 .
for such a weak signal , the apd s dark - current noise ( instrument error ) defines the best possible s / n because the instrumental error bar can not be averaged lower with more signal statistics .
therefore a low ( 0.03 cts / s ) dark current is the most important foundation for a successful observation of weak ni
fe for a hypothetical 4 mm nir - h but measured at bl19lxu , which has 2.6 times more cts / s , is listed in column 5 of fig .
the real signal level for a 4.1 mm nir - h at bl19 is listed in column 7 : ni h fe = 0.12 cts / s for measurement 1 [ and 0.13 cts / s for measurement 2 ( not listed ) ] . in comparison with the hypothetical case ( column 5 ) , the real measurement ( column 7 ) of ni h
fe has a slightly lower cts / s level although the corresponding fe co has a higher cts / s level .
this is because ni h fe : fe co is about 1:6 for nir - h ( fig .
nir - h was measured once at bl09xu ( 680 s ) and twice at bl19lxu ( 480 + 180 s ) .
the signal level at bl09xu is estimated at ( 94 60)/680 = 0.05 cts / s , leading to a s / n = 0.05/0.03 1.7 and a not - so - clear nrvs ( fig .
6b
) is ( 139 81)/480 = 0.12 cts / s , corresponding to a s / n = 0.12/0.03 = 4 , a much clearer observation for the ni
the signal level for measurement 2 at bl19lxu is about 0.13 and s / n = 4.3 .
the total equivalent bl09xu s / p ( as defined in the experimental section ) for nir - h in the nifeh region is 2396 s and the averaged signal level is 0.1 cts / s . assuming as - high - as - possible statistics , this leads to s / n = 3.3 . as the real statistics are not infinite , the real s / n is slightly lower than 3.3 .
nir - d was measured three times at bl09xu and once at bl19lxu , equivalent to 3980 bl09xu s / p in the same region ( versus 2396 s for nir - h ) . as there is no peak
, there is no meaning to estimating the signal level or s / n for nir - d .
however , a more equivalent s / p was used for nir - d because more time should be used to conclude that there is no signal than to find a weak signal .
7 summarizes three pvdos for nir - h and four pvdos for nir - d in the region around 675 cm .
again , all nir - h pvdos show a clear peak around 675 cm while none of those for nir - d show a peak in the same region , providing solid evidence that the observed 675 cm peak is real and is related to fe
there are several experimental conditions which make this work successful and a super - concentrated ( 4 mm ) and almost pure ni
r ) is one of the most important conditions but is also difficult to realise .
we proceed with exchange ph of the buffer ( 7.4 5.0 ) and with a longer period ( more than 8 h ) of h2/d2 reduction . monitoring samples integrity can never be more important than under these circumstances .
the following three steps were used to monitor the samples integrity : ( i ) nir - h / d were first checked with ftir at the sample preparation laboratory before sending to spring-8 for nrvs measurements , as shown in fig .
these subforms are distinguishable by ftir and differ in their protonation states but they are all ni r .
r1 ( co = 1946 cm ) , 16% nic ( at 1962 cm ) and a trace amount of ni r2 ( co = 1933 cm ) .
( ii ) nir - h / d after nrvs measurements were also collected and checked with ftir and both ir spectra [ blue for nir - h and red for nir - d in fig .
8(b ) ] show little change in comparison with those measured before sending for nrvs experiments ( fig .
8a
) , confirming the integrity of the samples for the whole nrvs measurement . (
iii ) immediately prior to a nrvs measurement , in situ nrvs measurement in a sample s fe
s region was another way of carrying out a last - minute check , as shown in fig .
9 . for example , the light blue curve shows one example of many good ni
7 ) , while the black curve shows one bad ( unexpectedly oxidized ) ni
the green and red spectra are the established nrvs for the oxidized and reduced dvmf h2ase ( kamali et al . , 2013 ) .
r , the real control over the samples integrity is via the ftir measurements prior to and following the nrvs experiments , as shown in fig .
8 . this in situ examination was used only as a handy check that the sample was not oxidized ( e.g. fig .
9 , black curve ) before investing a long beam time on the sample .
there are a lot of features in the fe s region which can be used to characterize the oxidation states of nife h2ase , including the peaks between 340 and 460 cm ( kamali et al .
. 4 , column 4 ) , a hypothetical 4 mm dvmf h2ase sample will have 36 cts / s in its fe s region , and three regular 5 s / p scans will produce 4590 total cts or a statistical s / n of 6.79.5 .
as the dark current is much smaller in comparison with the fe s signal cts / s , the statistical s / n is close to the real s / n .
then a s / n of 79 should be sufficient to resolve the samples oxidation states . at bl19lxu , in principle , one scan is enough , but we usually took two 3 s / p scans in order to check the repeatability .
we noticed that the low dark cts / s of the apd array ( i.e. 0.03 cts / s ) stays the same at bl19lxu , which has a higher beam intensity than bl09xu .
if this dark - current level increases at bl19lxu , the increased cts / s will become less meaningful .
we also noticed that the higher beam intensity at bl19lxu does not lead to higher sample temperatures ( wang et al .
this again illustrates the conclusion in our previous publication , which suggested that the hike in the samples temperatures in comparison with the sample base is mainly due to thermal radiation from the room - temperature window near the samples rather than from the x - ray irradiation ( wang et al .
, 2012 ) . as the hrm is inside the same hutch as the nrvs measurement chamber ,
entering the hutch can disturb the hutch temperature and the performance of the hrm . a sample change or other regular operation , which lasts 30 min , will lead to a system recovery time of about 2 h. in the case of an emergency entry for 1 min , the system can be recovered in about 2030 min . as mentioned earlier , the energy scale can be calibrated either normally or with a quick - switch calibration procedure ( wang et al . , 2013 ) .
for concentrated nir - h / d samples , the fe s peak at 365 cm ( fig .
9 ) can also be used as a calibration reference because of its high signal level .
the fe h or fe d stretching modes will have no overlaps with any possible nrvs peaks in the same regions and will allow a straightforward experimental identification of the hydride .
however , in reference to the model complexes and dft calculations ( ogata , kramer et al .
, 2015 ) , fe d has 1/5 of the intensity of ni h fe ( see fig .
5 ) while fe h has 1/15 of that of ni h fe ( not shown in fig . 5 ) , presenting further challenges . under the current experimental conditions ,
all these peaks are still not observable unless significant improvements in beam intensity , sample concentration and/or measurement solid angles are made .
different improvements and tests are underway . from the nidfe complex , the 711 cm ni
d stretching bending peak ( mixed with fe ) has about the same intensity as ni h fe .
however , it could be mixed with fe co and is not observable in ni
this publication has presented the strenuous experimental journey to discover the first spectroscopic evidence for the existence of a bridging hydride in a [ nife ] h2ase s ni
fe wagging mode has 2729 parts per million in intensity in comparison with the nuclear resonance peak , or 0.8% in comparison with a fe s feature .
improved sample concentration , increased photon flux and ultra - low noise level in the apd detectors all set the foundation for a possible nrvs measurement of the weak ni
the decision and insistence on searching in the then unlikely energy region of 620720 cm was the key step towards successful observation of the ni
the existence / non - existence of the 675 cm peak for nir - h / nir - d identified the peak as a fe h - related feature experimentally .
fe feature at 675 cm provided a critical observed reference for dft calculations and thus served as a key in the dft determination of the ni
it will be of interest to other scientists who use synchrotron radiation for measuring dilute samples and/or weak spectroscopic features in general .
|
direct spectroscopic evidence for a hydride bridge in the ni
r form of [ nife ] hydrogenase has been obtained using iron - specific nuclear resonance vibrational spectroscopy .
this article focuses on the long and strenuous experimental journey to search for and identify this first spectroscopic evidence for a hydride in ni
r .
|
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this is a retrospective cross - sectional study . there were 4,377 participants , all of whom underwent cardiac ct in a health promotion center at gangnam severance hospital ( seoul , korea ) between january 2008 and february 2013 . from 4,377 participants
, we excluded men ( n = 2,584 ) and premenopausal women ( n = 748 ) .
postmenopausal women 50 years or older with no menstrual periods for more than 12 consecutive months and women with elevated follicle - stimulating hormone levels ( > 30
iu / l ) were included in this study ( n = 1,045 ) .
women with diabetes mellitus ( n = 59 ) , excessive alcohol consumption ( n = 7 ) , viral hepatitis ( positive results for hepatitis b surface antigen or anti hepatitis c virus ; n = 19 ) , liver cirrhosis or malignancy on ultrasonography ( n = 6 ) , and self - reported or medically verified history of cvd ( n = 35 ) were excluded from this study .
height and weight were measured , and body mass index ( bmi ) was calculated by dividing weight ( kg ) by the square of height ( m ) . lifestyle , personal medical history of acute and chronic diseases , and medication history were assessed using a standard questionnaire .
systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were measured after a 5-minute rest .
fasting plasma glucose ( fpg ) , aspartate aminotransferase , alanine aminotransferase , total cholesterol ( tc ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides ( tg ) , and c - reactive protein ( crp ) levels were determined by enzymatic methods using a hitachi 7600 - 120 automated chemistry analyzer ( hitachi , tokyo , japan ) .
low - density lipoprotein cholesterol ( ldl - c ) was calculated according to the friedewald equation .
hepatitis b surface antigen levels and antibodies to hepatitis c virus were measured with a roche e-170 device ( roche diagnostics , mannheim , germany ) .
fasting serum insulin was determined by chemiluminescence ( radioimmunoassay kit ; daiichi , kyoto , japan ) , and insulin resistance was estimated using the homeostasis model assessment of insulin resistance ( homa - ir ) index calculated from the following formula : homa - ir = fasting insulin ( u / ml ) fpg ( mmol / l ) / 22.5 .
diagnosis of fatty liver disease was based on an abdominal ultrasonography scan obtained with a 3.5-mhz transducer ( hdi 5000 ; philips , bothell , wa ) .
abdominal ultrasonographic examination was performed by one of three experienced radiologists , all of whom were blinded to the laboratory and clinical details of participants at the time of the procedure . in the present study , liver with any degree of fat accumulation
the number of participants with severe fatty liver disease was too small ( n = 29 ) ; thus , the presence and severity of nafld were classified into three groups according to hyperechogenicity of liver tissue , degree of discrepancy between the liver and the right kidney , and visibility of vascular structures .
the three groups were as follows : group i , no fatty liver disease ; group ii , mild fatty liver disease ; group iii , moderate to severe fatty liver disease .
cac was determined with a multidetector ct scanner ( philips brilliance 64 ; philips medical system , best , the netherlands ) .
we used a standard prospective electrocardiogram gating protocol with a step - and - shoot technique .
the scanning protocol included a slice section collimation of 64 0.625 mm , rotation time of 420 milliseconds , tube voltage of 120 kv , and tube current of 210 mas .
quantitative cac score ( cacs ) was calculated with dedicated software and expressed as agatston score .
continuous variables with normal distribution were expressed as mean ( sd ) , whereas continuous variables with skewed distribution were presented as median ( interquartile range ) and log - transformed for analysis .
logistic regression analysis was used to analyze the association between nafld and presence of cac while controlling for potential confounders .
covariates in the multivariable model , such as age , bmi , sbp , dbp , fpg , tc , ldl - c , tg , hdl - c , crp , and homa - ir , were chosen for clinical importance and statistical significance .
spss statistical package version 20.0 ( spss inc , chicago , il ) was used for all statistical analyses .
this is a retrospective cross - sectional study . there were 4,377 participants , all of whom underwent cardiac ct in a health promotion center at gangnam severance hospital ( seoul , korea ) between january 2008 and february 2013 . from 4,377 participants
, we excluded men ( n = 2,584 ) and premenopausal women ( n = 748 ) .
postmenopausal women 50 years or older with no menstrual periods for more than 12 consecutive months and women with elevated follicle - stimulating hormone levels ( > 30
iu / l ) were included in this study ( n = 1,045 ) .
women with diabetes mellitus ( n = 59 ) , excessive alcohol consumption ( n = 7 ) , viral hepatitis ( positive results for hepatitis b surface antigen or anti hepatitis c virus ; n = 19 ) , liver cirrhosis or malignancy on ultrasonography ( n = 6 ) , and self - reported or medically verified history of cvd ( n = 35 ) were excluded from this study .
height and weight were measured , and body mass index ( bmi ) was calculated by dividing weight ( kg ) by the square of height ( m ) .
lifestyle , personal medical history of acute and chronic diseases , and medication history were assessed using a standard questionnaire .
systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were measured after a 5-minute rest .
fasting plasma glucose ( fpg ) , aspartate aminotransferase , alanine aminotransferase , total cholesterol ( tc ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides ( tg ) , and c - reactive protein ( crp ) levels were determined by enzymatic methods using a hitachi 7600 - 120 automated chemistry analyzer ( hitachi , tokyo , japan ) . low - density lipoprotein cholesterol ( ldl - c )
hepatitis b surface antigen levels and antibodies to hepatitis c virus were measured with a roche e-170 device ( roche diagnostics , mannheim , germany ) .
fasting serum insulin was determined by chemiluminescence ( radioimmunoassay kit ; daiichi , kyoto , japan ) , and insulin resistance was estimated using the homeostasis model assessment of insulin resistance ( homa - ir ) index calculated from the following formula : homa - ir = fasting insulin ( u / ml ) fpg ( mmol / l ) / 22.5 .
diagnosis of fatty liver disease was based on an abdominal ultrasonography scan obtained with a 3.5-mhz transducer ( hdi 5000 ; philips , bothell , wa ) .
abdominal ultrasonographic examination was performed by one of three experienced radiologists , all of whom were blinded to the laboratory and clinical details of participants at the time of the procedure . in the present study , liver with any degree of fat accumulation was considered nafld .
the number of participants with severe fatty liver disease was too small ( n = 29 ) ; thus , the presence and severity of nafld were classified into three groups according to hyperechogenicity of liver tissue , degree of discrepancy between the liver and the right kidney , and visibility of vascular structures .
the three groups were as follows : group i , no fatty liver disease ; group ii , mild fatty liver disease ; group iii , moderate to severe fatty liver disease .
cac was determined with a multidetector ct scanner ( philips brilliance 64 ; philips medical system , best , the netherlands ) .
we used a standard prospective electrocardiogram gating protocol with a step - and - shoot technique .
the scanning protocol included a slice section collimation of 64 0.625 mm , rotation time of 420 milliseconds , tube voltage of 120 kv , and tube current of 210 mas .
quantitative cac score ( cacs ) was calculated with dedicated software and expressed as agatston score .
continuous variables with normal distribution were expressed as mean ( sd ) , whereas continuous variables with skewed distribution were presented as median ( interquartile range ) and log - transformed for analysis .
logistic regression analysis was used to analyze the association between nafld and presence of cac while controlling for potential confounders .
covariates in the multivariable model , such as age , bmi , sbp , dbp , fpg , tc , ldl - c , tg , hdl - c , crp , and homa - ir , were chosen for clinical importance and statistical significance .
spss statistical package version 20.0 ( spss inc , chicago , il ) was used for all statistical analyses .
a total of 919 postmenopausal women were included in this study . of these , 738 women had no cac ( cacs = 0 ) , whereas the remaining 181 women exhibited some evidence of cac ( cacs > 0 ; 19.7% ) . among women in this study , 294 women ( 31.9% )
women were stratified into three groups by the presence and severity of fatty liver disease .
age , bmi , sbp , dbp , fpg , tg , aspartate aminotransferase , alanine aminotransferase , crp , and homa - ir increased according to the severity of fatty liver disease , whereas hdl - c decreased .
the prevalence of cac and the mean cac score significantly increased with the severity of fatty liver disease ( table 2 ) .
the association between fatty liver disease and presence of cac ( cacs > 0 ) was further explored by categorizing the severity of nafld into three groups and by using the first group as reference ( table 3 ) .
these relationships remained significant even after adjusting for well - established cardiovascular risk factors , including age , bmi , sbp , dbp , fpg , tc , ldl - c , tg , hdl - c , and crp ( model 2 ) .
however , this association was attenuated after additional adjustment for insulin resistance ( model 3 ) .
in this study , we investigated the relationship between nafld and prevalence of cac in postmenopausal women .
we found that the prevalence of cac was significantly associated with nafld after adjustment for conventional cardiovascular risk factors , but this association was attenuated after adjustment for insulin resistance . to the best of our knowledge ,
this is the first study to investigate the correlation between nafld and presence of early coronary artery atherosclerosis in postmenopausal women . in this study ,
this prevalence was higher than that in premenopausal women ( 19.7% ) , which was determined by analyzing the subgroup of premenopausal women among 4,377 participants .
hamaguchi et al reported that the prevalence of nafld in japanese postmenopausal women was higher than that in premenopausal women and concluded that postmenopause was a risk factor for nafld .
although it remains unclear how estrogen deficiency increases the prevalence of nafld after menopause , several reports have suggested that estrogen deficiency affects body fat distribution , which increases accumulation of gluteofemoral fat and central fat .
hepatic estrogen receptors mediate estrogen action in the liver , and estradiol has been shown to play a favorable role in chronic liver disease .
thus , estrogen seems to exert a protective effect against nafld in women . in this study
, we showed that most metabolic risk factors increased or decreased with the severity of nafld .
cac has been shown to be an independent predictor of future cardiovascular events . moreover ,
several recent studies have reported the association between nafld and cac , but results have been inconsistent . as part of the diabetes heart study ,
mckimmie et al reported that coronary arterial calcium did not correlate with hepatic steatosis in women with type 2 diabetes .
another study of 398 white and black participants by ding et al did not find associations of liver fat attenuation with calcified coronary plaque .
recently , vanwagner et al showed that obesity attenuates the relationship between nafld and cac .
in contrast , some studies have suggested that nafld might be an independent risk factor for cac , after accounting for conventional risk factors .
only a few studies have investigated the association between nafld and carotid intima - media thickness , which is another marker for subclinical atherosclerosis , after adjusting for insulin resistance and other traditional risk factors . however , whether nafld is an independent risk factor for increased intima - media thickness remains controversial . in the present study , similar to a previous study of healthy women , we found that the presence of cac increased with the severity of nafld in postmenopausal women .
however , this association was attenuated after adjusting for insulin resistance , and insulin resistance was independently associated with cac in these adjusted models .
this suggests that insulin resistance may act as a mediator of the relationship between nafld and cac .
although the pathogenesis of nafld and cac has not been fully elucidated , there are several explanations for the relationship between nafld and cac .
one of the accepted hypotheses implicates insulin resistance as a major factor leading to nafld and cac .
menopause affects body fat distribution , and the resultant increases in body weight and body fat lead to insulin resistance , which might play an important role in the pathogenesis of clinical nafld and cac .
recently , rodrigues et al reported that insulin resistance , metabolic syndrome , and abdominal obesity were risk markers for the development of nafld in postmenopausal women . therefore , although the mechanism underlying the relationship between nafld and cac in postmenopausal women is not completely understood , insulin resistance might provide a link between them .
first , this is a cross - sectional study that can not definitively establish causality .
second , this study lacked direct measures of body fat , which affects insulin resistance .
fatty liver disease was assessed by liver ultrasonography a technique that can not detect fatty infiltration below 30% .
however , ultrasonography is nonetheless a very useful noninvasive technique that is often used as the first - line imaging technique in clinical practice and epidemiological studies . finally ,
because participants in the current study were enrolled in the same health promotion center , generalizability may be limited .
increased severity of nafld is significantly associated with cac , a marker of subclinical atherosclerosis .
however , this association is attenuated after additional adjustment for insulin resistance ; thus , nafld is not an independent factor for coronary atherosclerosis in postmenopausal women .
further prospective large - scale studies are required to explore these findings and to elucidate the mechanism for this association .
|
abstractobjective : cardiovascular disease is a leading cause of death in postmenopausal women , and nonalcoholic fatty liver disease ( nafld ) has been known to be associated with cardiovascular disease .
however , little information regarding the relationship between nafld and coronary artery calcification ( cac ) in postmenopausal women is available .
the aim of this study was to investigate the association between nafld and cac in postmenopausal women.methods:among 4,377 participants who underwent cardiac computed tomography in a health promotion center , 919 postmenopausal women were enrolled .
anthropometric profiles and multiple cardiovascular risk factors were measured .
nafld was measured by ultrasonography , and cac was evaluated by cardiac computed tomography .
odds ratios and 95% ci for the presence of cac , by severity of fatty liver disease , were estimated using logistic regression.results:women were stratified into three groups by severity of nafld .
there were significant differences in cardiovascular parameters among the groups , and prevalence of cac significantly increased with severity of nafld . on logistic regression analysis
after adjustment for multiple risk factors , the odds ratios for the prevalence of cac were as follows ( p < 0.05 ) : no nafld , 1.0 ; mild nafld , 1.34 ( 95% ci , 0.92 - 2.16 ) ; moderate to severe nafld , 1.83 ( 95% ci , 1.06 - 3.16 ) .
however , this association was attenuated after adjustment for insulin resistance ( p = 0.16).conclusions : there is a significant correlation between nafld and prevalence of cac , but nafld is not an independent factor for coronary atherosclerosis in postmenopausal women .
|
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the design and baseline characteristics of the whi have been described in detail elsewhere ( 8) . in brief , the whi enrolled postmenopausal women 5079 years of age who provided written informed consent and had an expected survival and local residency of 3 years .
exclusion criteria included current alcoholism , drug dependency , dementia , or other conditions that would limit full participation in the study .
a total of 161,808 women ( 68,132 in clinical trials and 93,676 in an observational study ) were enrolled in the whi between 1993 and 1998 .
data used in this report were obtained from women at baseline and then at periodic follow - up points over an average of 10.4 years . the protocol and consent forms were approved by the institutional review boards of all participating institutions .
prevalent diabetes was defined as a self - report at baseline of ever having received a physician diagnosis of diabetes when not pregnant .
incident diabetes was based on data collected at annual follow - up visits at which participants were asked , since the date given on the front of this form , has a doctor prescribed any of the following pills or treatments ? choices included
cases of incident treated diabetes reported as of 31 august 2009 were included in the analysis .
a study of the accuracy of self - reported diabetes conducted in a subset of whi participants indicated that self - reported disease status was a reasonably valid indicator of diagnosed diabetes when compared with medication and laboratory criteria ( 9 ) .
however , self - report fails to identify undiagnosed diabetes and tends to underestimate diabetes prevalence and incidence in our study group ( 9 ) . at baseline , whi participants
self - reported their race / ethnicity , choosing from non - hispanic white ( referred to hereafter as white ) , non - hispanic black ( referred to hereafter as black ) , hispanic , american indian / alaska native , asian ( ancestry was chinese , indo - chinese , korean , japanese , pacific islander , or vietnamese ) , and other .
we excluded 1,849 women who reported race / ethnicity as other and 413 women without race / ethnicity information .
the sample size was limited among american indians ( n = 714 ) ; therefore , analyses were restricted to whites , blacks , hispanics , and asians .
body weight , height , and waist circumference were measured at baseline and year 3 .
demographic and health history data were self - reported at baseline and included age , years of education , cigarette smoking status , family history of diabetes , and hormone therapy use .
the baseline physical activity questionnaire asked about usual frequency and duration of several types of recreational and household activities using a standardized classification of physical activity intensity ( 10 ) .
total energy expenditure was summarized in metabolic equivalent ( met ) hours per week ( met - h / week ) , computed as the summed product of frequency , duration , and intensity for reported activities .
participants also completed a standardized food frequency questionnaire ( ffq ) developed for the whi to estimate average daily nutrient intake over the 3-month period prior to baseline visit ( 11 ) .
dietary quality , assessed by the alternate healthy eating index ( ahei ) ( 12,13 ) , was computed based on food items and nutrients derived from the ffq , including 1 ) fruit , 2 ) vegetables , 3 ) nuts and legumes , 4 ) ratio of white to red meat , 5 ) total dietary fiber , 6 ) trans - fat , 7 ) ratio of polyunsaturated fat to saturated fat , 8) alcohol , and 9 ) multivitamin use .
the reliability and validity of physical activity measurements were assessed among a random sample of 536 participants by a second measure of physical activity 10 weeks after the first measure . the test - retest reliability ( weighted ) for the physical activity variables ranged from 0.53 to 0.72 , and the intraclass correlation for the total physical activity variable was 0.77 ( 8) . for the ffq ,
we assessed bias and precision of the ffq by comparing the intake of 30 nutrients estimated from the ffq with means from four 24-h dietary recalls and a 4-day food record from 113 women who participated in the whi ( 11 ) . for most nutrients , means estimated by the ffq were within 10% of the records or recalls .
energy - adjusted correlation coefficients ranged from 0.2 ( vitamin b12 ) to 0.7 ( magnesium ) , with a mean of 0.5 .
we concluded that the ffq produced nutrient estimates , which were similar to those obtained in other studies comparing short - term dietary recall and recording methods .
we acknowledge that the reliability and validity are not perfect ; however , they provided reasonable measures in a large clinical trial , as published by several of our authors in high - impact articles ( 1416 ) .
racial / ethnic differences in the prevalence of diabetes were assessed using logistic regression and are expressed as odds ratios ( ors ) and associated 95% cis , with whites used as the reference group .
we present four logistic regression models : 1 ) unadjusted , including only race / ethnicity as a covariate ; 2 ) age - adjusted ; 3 ) adjusted for multiple potential confounding factors , including study arm , baseline age , bmi , waist circumference , physical activity , dietary quality , and smoking status ; and 4 ) adjusted for the same covariates as in model 3 plus educational attainment . among women who were free of diabetes at baseline ,
incidence rate was calculated as the number of newly reported postbaseline diabetes cases divided by total follow - up time in person - years .
the time to diabetes ( i.e. , time to event ) was calculated as the interval between enrollment date and the earliest of the following : 1 ) date of annual medical history update when new diagnosis of diabetes and initiation of treatment for diabetes were ascertained ( observed event ) ; 2 ) date of last annual medical update during which participants were identified to be without diabetes ( censored event ) ; or 3 ) date of death from any cause ( censored event ) .
cox proportional hazards models were used to estimate the hazard ratios ( hrs ) and associated 95% ci of incident diabetes , with whites used as the reference group .
four cox regression models that parallel those described for the analysis of diabetes prevalence are presented . to identify determinants of diabetes incidence that might vary by racial / ethnic group ,
the impact of several covariates was evaluated by assessing the extent to which the hr estimate for a specific race / ethnicity group changed when each covariate was added individually to the unadjusted model . the percentage change in hr between the model with and without the covariate was used to describe the contribution of each covariate , considered singly , to diabetes risk estimated by race / ethnic group . to identify the most parsimonious prediction model ,
a stepwise cox proportional hazards regression analysis was conducted ( p values for entry = 0.25 and for retaining in the model = 0.05 ) .
subgroup analyses also were conducted by race / ethnicity . weight change from baseline to 3 years was evaluated in relation to risk of incident disease by race / ethnicity among women who reported being free of diabetes at the 3-year measurement point .
a similar analysis was conducted for waist circumference . to assess how the combined role of healthy lifestyle habits in diabetes prevention may vary by race / ethnicity ( 6,7 ) ,
categories of four modifiable lifestyle factors were defined as follows : physical activity ( upper vs. lower two tertiles ) , dietary quality score ( upper vs. lower two tertiles ) , smoking ( nonsmokers vs. past and former smokers combined ) , and bmi ( < 25 vs. 25
kg / m ) . in subgroup analyses by race / ethnicity , hrs and 95% cis were calculated for diabetes incidence for each lifestyle risk factor individually and several lifestyle factors in aggregate , adjusting for age , family history of diabetes , hormone therapy use , study arm , and lifestyle risk factors not already included in the model .
the design and baseline characteristics of the whi have been described in detail elsewhere ( 8) . in brief , the whi enrolled postmenopausal women 5079 years of age who provided written informed consent and had an expected survival and local residency of 3 years .
exclusion criteria included current alcoholism , drug dependency , dementia , or other conditions that would limit full participation in the study .
a total of 161,808 women ( 68,132 in clinical trials and 93,676 in an observational study ) were enrolled in the whi between 1993 and 1998 .
data used in this report were obtained from women at baseline and then at periodic follow - up points over an average of 10.4 years . the protocol and consent forms were approved by the institutional review boards of all participating institutions .
prevalent diabetes was defined as a self - report at baseline of ever having received a physician diagnosis of diabetes when not pregnant .
incident diabetes was based on data collected at annual follow - up visits at which participants were asked , since the date given on the front of this form , has a doctor prescribed any of the following pills or treatments ? choices included
cases of incident treated diabetes reported as of 31 august 2009 were included in the analysis .
a study of the accuracy of self - reported diabetes conducted in a subset of whi participants indicated that self - reported disease status was a reasonably valid indicator of diagnosed diabetes when compared with medication and laboratory criteria ( 9 ) .
however , self - report fails to identify undiagnosed diabetes and tends to underestimate diabetes prevalence and incidence in our study group ( 9 ) .
at baseline , whi participants self - reported their race / ethnicity , choosing from non - hispanic white ( referred to hereafter as white ) , non - hispanic black ( referred to hereafter as black ) , hispanic , american indian / alaska native , asian ( ancestry was chinese , indo - chinese , korean , japanese , pacific islander , or vietnamese ) , and other .
we excluded 1,849 women who reported race / ethnicity as other and 413 women without race / ethnicity information .
the sample size was limited among american indians ( n = 714 ) ; therefore , analyses were restricted to whites , blacks , hispanics , and asians .
body weight , height , and waist circumference were measured at baseline and year 3 .
demographic and health history data were self - reported at baseline and included age , years of education , cigarette smoking status , family history of diabetes , and hormone therapy use .
the baseline physical activity questionnaire asked about usual frequency and duration of several types of recreational and household activities using a standardized classification of physical activity intensity ( 10 ) .
total energy expenditure was summarized in metabolic equivalent ( met ) hours per week ( met - h / week ) , computed as the summed product of frequency , duration , and intensity for reported activities .
participants also completed a standardized food frequency questionnaire ( ffq ) developed for the whi to estimate average daily nutrient intake over the 3-month period prior to baseline visit ( 11 ) .
dietary quality , assessed by the alternate healthy eating index ( ahei ) ( 12,13 ) , was computed based on food items and nutrients derived from the ffq , including 1 ) fruit , 2 ) vegetables , 3 ) nuts and legumes , 4 ) ratio of white to red meat , 5 ) total dietary fiber , 6 ) trans - fat , 7 ) ratio of polyunsaturated fat to saturated fat , 8) alcohol , and 9 ) multivitamin use .
the reliability and validity of physical activity measurements were assessed among a random sample of 536 participants by a second measure of physical activity 10 weeks after the first measure . the test - retest reliability ( weighted ) for the physical activity variables ranged from 0.53 to 0.72 , and the intraclass correlation for the total physical activity variable was 0.77 ( 8) . for the ffq ,
we assessed bias and precision of the ffq by comparing the intake of 30 nutrients estimated from the ffq with means from four 24-h dietary recalls and a 4-day food record from 113 women who participated in the whi ( 11 ) . for most nutrients , means estimated by the ffq were within 10% of the records or recalls .
energy - adjusted correlation coefficients ranged from 0.2 ( vitamin b12 ) to 0.7 ( magnesium ) , with a mean of 0.5 .
we concluded that the ffq produced nutrient estimates , which were similar to those obtained in other studies comparing short - term dietary recall and recording methods .
we acknowledge that the reliability and validity are not perfect ; however , they provided reasonable measures in a large clinical trial , as published by several of our authors in high - impact articles ( 1416 ) .
racial / ethnic differences in the prevalence of diabetes were assessed using logistic regression and are expressed as odds ratios ( ors ) and associated 95% cis , with whites used as the reference group .
we present four logistic regression models : 1 ) unadjusted , including only race / ethnicity as a covariate ; 2 ) age - adjusted ; 3 ) adjusted for multiple potential confounding factors , including study arm , baseline age , bmi , waist circumference , physical activity , dietary quality , and smoking status ; and 4 ) adjusted for the same covariates as in model 3 plus educational attainment . among women who were free of diabetes at baseline ,
incidence rate was calculated as the number of newly reported postbaseline diabetes cases divided by total follow - up time in person - years .
the time to diabetes ( i.e. , time to event ) was calculated as the interval between enrollment date and the earliest of the following : 1 ) date of annual medical history update when new diagnosis of diabetes and initiation of treatment for diabetes were ascertained ( observed event ) ; 2 ) date of last annual medical update during which participants were identified to be without diabetes ( censored event ) ; or 3 ) date of death from any cause ( censored event ) .
cox proportional hazards models were used to estimate the hazard ratios ( hrs ) and associated 95% ci of incident diabetes , with whites used as the reference group .
four cox regression models that parallel those described for the analysis of diabetes prevalence are presented . to identify determinants of diabetes incidence that might vary by racial / ethnic group ,
the impact of several covariates was evaluated by assessing the extent to which the hr estimate for a specific race / ethnicity group changed when each covariate was added individually to the unadjusted model . the percentage change in hr between the model with and without the covariate was used to describe the contribution of each covariate , considered singly , to diabetes risk estimated by race / ethnic group . to identify the most parsimonious prediction model ,
a stepwise cox proportional hazards regression analysis was conducted ( p values for entry = 0.25 and for retaining in the model = 0.05 ) .
subgroup analyses also were conducted by race / ethnicity . weight change from baseline to 3 years was evaluated in relation to risk of incident disease by race / ethnicity among women who reported being free of diabetes at the 3-year measurement point .
a similar analysis was conducted for waist circumference . to assess how the combined role of healthy lifestyle habits in diabetes prevention may vary by race / ethnicity ( 6,7 )
, categories of four modifiable lifestyle factors were defined as follows : physical activity ( upper vs. lower two tertiles ) , dietary quality score ( upper vs. lower two tertiles ) , smoking ( nonsmokers vs. past and former smokers combined ) , and bmi ( < 25 vs. 25
kg / m ) . in subgroup analyses by race / ethnicity , hrs and 95% cis
were calculated for diabetes incidence for each lifestyle risk factor individually and several lifestyle factors in aggregate , adjusting for age , family history of diabetes , hormone therapy use , study arm , and lifestyle risk factors not already included in the model .
at baseline , the average age of the 158,833 women with evaluable data was 63 years .
the racial / ethnic distribution was 84.1% white , 9.2% black , 4.1% hispanic , and 2.6% asian .
blacks and hispanics tended to have more risk factors , whereas asians tended to have fewer ( table 1 ) .
baseline characteristics of participants in the whi 19932009 ( n = 158,833 ) at enrollment , diabetes prevalence was highest among blacks ( 12.2% ) , followed by hispanics ( 7.2% ) , asians ( 5.9% ) , and whites ( 3.3% ) ( table 2 ) .
compared with the unadjusted ors , which were significantly higher in all three race / ethnic groups when compared with whites , the age - adjusted and multivariable - adjusted ors were attenuated for blacks and hispanics but strengthened for asians . additional adjustment for educational attainment further attenuated the ors for blacks and hispanics ; however , ors for all three racial / ethnic minority groups remained significantly higher than that of whites .
prevalence and incidence of diabetes in the whi 19932009 during an average of 10.4 years ( sd = 3.2 ) of follow - up , 14,604 new cases of diabetes were reported ( 11,127 whites , 2,181 blacks , 879 hispanics , and 417 asians ) , with incidence being highest in blacks and lowest in whites . compared with whites ,
unadjusted analyses showed a significantly higher diabetes incidence of 136% in blacks , 114% in hispanics , and 43% in asians . after adjusting for age , study arm , bmi , physical activity , smoking status , and educational attainment ,
1 ) , the highest cumulative incidence of diabetes was seen among women who had both high bmi and low levels of physical activity .
the cumulative incidence of diabetes was 23.4% among black women who were both obese and in the lowest tertile of physical activity , but decreased to 8.8% in those who had a healthy weight and exercised .
white women who were overweight ( bmi = 2529.9 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 8.7% , whereas white women who were obese ( bmi 30 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 18.6% . across all racial / ethnic groups , women who were normal weight ( bmi < 25 kg / m ) and in the highest tertile of physical activity had less than one - third to one - sixth the incidence of diabetes compared with women with bmi 30 kg / m and in the lowest tertile of physical activity .
cumulative incidence of diabetes by race / ethnicity , physical activity level , and bmi category .
analyses conducted to estimate the influence of individual factors that may account for the observed racial / ethnic variation in diabetes incidence by comparing hrs obtained from unadjusted versus fully adjusted models ( results not shown ) revealed that if asian women had the same waist circumference , bmi , and dietary quality intake as whites , their hr for diabetes would be increased by 44 , 29 , and 3.5% , respectively . among blacks ,
if they had the same bmi , waist circumference , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 25 , 19 , 11 , 7 , and 6% , respectively . among hispanics ,
if they had the same educational attainment , bmi , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 14 , 10 , 9 , 7 , and 6% , respectively .
results from the stepwise cox proportional hazards regression analysis conducted to identify the most parsimonious predictive model for diabetes incidence , fitting candidate predictors plus race / ethnicity , showed that race / ethnicity , age , bmi , waist circumference , education , smoking status , physical activity , family history of diabetes , and dietary quality score entered the model .
hrs of incident diabetes corresponding to single and specific combinations of lifestyle risk factors in race / ethnicity subgroups in the whi 19932009 subgroup analyses by race / ethnicity ( results not shown ) conducted to assess the effect of interval ( i.e. , baseline to 3 years ) changes in factors found to be significant in the cox proportional hazards models revealed a significant effect of 5% increased risk of diabetes for each 5-cm increase in waist circumference ( hr 1.05 [ 95% ci 1.041.07 ] ) . the observed effect was consistent across all race / ethnicity groups .
an alternative model fit to assess if weight gain could explain some of the observed racial / ethnic variation in diabetes incidence revealed an 3% increase in subsequent risk for each 1-kg / m increment in bmi ( 1.03 [ 1.021.04 ] ) .
higher levels of physical activity , better diet , and having a healthy weight tended to be associated with a significantly lower risk of diabetes in each racial / ethnic group , with some exceptions .
whites , blacks , and hispanics with all factors in the low - risk category ( 4.0 , 1.1 , and 2.2% ) had 60 , 69 , and 63% lower risk for incident diabetes ( table 3 ) .
healthy weight ( bmi < 25 kg / m ) demonstrated the greatest role in reducing risk of diabetes in each racial / ethnic group : 66% in whites , 55% in blacks , 64% in hispanics , and 66% in asians .
at baseline , the average age of the 158,833 women with evaluable data was 63 years .
the racial / ethnic distribution was 84.1% white , 9.2% black , 4.1% hispanic , and 2.6% asian .
blacks and hispanics tended to have more risk factors , whereas asians tended to have fewer ( table 1 ) .
at enrollment , diabetes prevalence was highest among blacks ( 12.2% ) , followed by hispanics ( 7.2% ) , asians ( 5.9% ) , and whites ( 3.3% ) ( table 2 ) . compared with the unadjusted ors , which were significantly higher in all three race / ethnic groups when compared with whites ,
the age - adjusted and multivariable - adjusted ors were attenuated for blacks and hispanics but strengthened for asians .
additional adjustment for educational attainment further attenuated the ors for blacks and hispanics ; however , ors for all three racial / ethnic minority groups remained significantly higher than that of whites .
prevalence and incidence of diabetes in the whi 19932009 during an average of 10.4 years ( sd = 3.2 ) of follow - up , 14,604 new cases of diabetes were reported ( 11,127 whites , 2,181 blacks , 879 hispanics , and 417 asians ) , with incidence being highest in blacks and lowest in whites . compared with whites ,
unadjusted analyses showed a significantly higher diabetes incidence of 136% in blacks , 114% in hispanics , and 43% in asians . after adjusting for age , study arm , bmi , physical activity , smoking status , and educational attainment ,
1 ) , the highest cumulative incidence of diabetes was seen among women who had both high bmi and low levels of physical activity .
the cumulative incidence of diabetes was 23.4% among black women who were both obese and in the lowest tertile of physical activity , but decreased to 8.8% in those who had a healthy weight and exercised .
white women who were overweight ( bmi = 2529.9 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 8.7% , whereas white women who were obese ( bmi 30 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 18.6% . across all racial / ethnic groups ,
women who were normal weight ( bmi < 25 kg / m ) and in the highest tertile of physical activity had less than one - third to one - sixth the incidence of diabetes compared with women with bmi 30 kg / m and in the lowest tertile of physical activity .
cumulative incidence of diabetes by race / ethnicity , physical activity level , and bmi category .
analyses conducted to estimate the influence of individual factors that may account for the observed racial / ethnic variation in diabetes incidence by comparing hrs obtained from unadjusted versus fully adjusted models ( results not shown ) revealed that if asian women had the same waist circumference , bmi , and dietary quality intake as whites , their hr for diabetes would be increased by 44 , 29 , and 3.5% , respectively . among blacks , if they had the same bmi , waist circumference , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 25 , 19 , 11 , 7 , and 6% , respectively . among hispanics ,
if they had the same educational attainment , bmi , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 14 , 10 , 9 , 7 , and 6% , respectively .
results from the stepwise cox proportional hazards regression analysis conducted to identify the most parsimonious predictive model for diabetes incidence , fitting candidate predictors plus race / ethnicity , showed that race / ethnicity , age , bmi , waist circumference , education , smoking status , physical activity , family history of diabetes , and dietary quality score entered the model .
hrs of incident diabetes corresponding to single and specific combinations of lifestyle risk factors in race / ethnicity subgroups in the whi 19932009 subgroup analyses by race / ethnicity ( results not shown ) conducted to assess the effect of interval ( i.e. , baseline to 3 years ) changes in factors found to be significant in the cox proportional hazards models revealed a significant effect of 5% increased risk of diabetes for each 5-cm increase in waist circumference ( hr 1.05 [ 95% ci 1.041.07 ] ) . the observed effect was consistent across all race / ethnicity groups .
an alternative model fit to assess if weight gain could explain some of the observed racial / ethnic variation in diabetes incidence revealed an 3% increase in subsequent risk for each 1-kg / m increment in bmi ( 1.03 [ 1.021.04 ] ) .
higher levels of physical activity , better diet , and having a healthy weight tended to be associated with a significantly lower risk of diabetes in each racial / ethnic group , with some exceptions .
whites , blacks , and hispanics with all factors in the low - risk category ( 4.0 , 1.1 , and 2.2% ) had 60 , 69 , and 63% lower risk for incident diabetes ( table 3 ) .
kg / m ) demonstrated the greatest role in reducing risk of diabetes in each racial / ethnic group : 66% in whites , 55% in blacks , 64% in hispanics , and 66% in asians .
previous reports , mainly focused on younger men and women , have indicated significant racial / ethnic disparities in diabetes in the u.s . ( 1,4,5 ) .
the 20072009 national health interview survey found that 7.1% of whites , 8.4% of asians , 11.8% of hispanics , and 12.6% of blacks reported having a diagnosis of diabetes ( 1 ) .
the whi allows a unique opportunity to deepen understanding of the patterns and determinants of diabetes in older women , including racial / ethnic minorities that represent growing segments of the u.s .
we found that the prevalence and incidence of diabetes ranged from approximately two to three times higher in blacks and approximately two times higher in hispanics and in asians , compared with whites . observed racial / ethnic differences in diabetes incidence
were explained , in large part , by modifiable lifestyle factors that included diet quality , physical activity , and smoking status or factors resulting from lifestyle behaviors including bmi and waist circumference .
adjustment for differences in these variables indicates that asians have the highest inherent risk , though women of all four racial / ethnic groups would experience a large reduction in diabetes risk by maintaining a healthy body weight , healthy diet , and a physically active lifestyle .
maintaining a bmi < 25 kg / m appears to be particularly important , and interval changes in both bmi and waist circumference predicted newly incident disease . in this study ,
both bmi and waist circumference were found to be related to prevalent diabetes , and interval changes were associated with risk of incident disease . because waist circumference reflects centralized obesity , and the propensity toward large waist circumference varies according to race / ethnicity , its effect may differ from that of bmi ( 17,18 ) . although bmi and waist circumference among asian , postmenopausal women in the whi were relatively lower than whites , asians were at higher risk of diabetes at lower levels of bmi and with smaller waist circumferences ( i.e. , by 44 and 29% , respectively ) compared with whites , a result consistent with that seen in the multi - ethnic study of atherosclerosis ( 19 ) .
our results on asians suggest that additional risk factors , which might be biological , social , or a combination , drive the higher prevalence and incidence of diabetes in this population .
diabetes risk in blacks with the same bmi and waist circumference as whites was lower by 25 and 17% , respectively .
similarly , diabetes risk in hispanics with the same bmi and waist circumference as whites was lower by 10 and 2% , respectively .
we noted that within each group , bmi was the most important determinant for diabetes incidence .
physical inactivity also was found to be associated with increased risk of diabetes in all groups . in a previous publication using whi data , we reported the association between physical inactivity and development of diabetes ( 20 ) .
our present study showed that , compared with whites , given the same level of physical activity , the risk for diabetes for blacks and hispanics is 6% lower .
several studies have found differences in dietary intake by race / ethnicity ( 26,27 ) .
although their diet quality was better than that of whites , we found , for example , if their overall dietary quality were to decrease to that of whites , their risk of diabetes would increase by 4% relative to whites .
in fact , if blacks and hispanics improved their diet to a quality similar to that of whites , their risk of diabetes would be 7% lower .
although previous studies demonstrated that diabetes is preventable by relatively simple lifestyle modifications ( 6,7 ) , our data suggest that some minority groups might obtain greater benefit from improving lifestyle factors ( i.e. , blacks and hispanics ) than others ( i.e. , asians ) due to differences in the amount of change possible or the vulnerability to these factors .
the divergent findings in asian women are of interest , suggesting that additional approaches to prevention deserve attention .
for example , asians may need to achieve even greater weight loss to have the same low risk of diabetes as nonoverweight whites , and the world health organization already uses a lower bmi cut point for asians .
the fact that asians in the whi had much healthier body weights at baseline set the stage for much lower overall diabetes risk .
however , further research with this population is warranted because our statistical models , with additional covariates , may be unstable and less reliable due to small numbers .
we found that blacks and hispanics are more sensitive to lifestyle modifications and weight loss than whites , and this is corroborated by our previous lifestyle intervention study results from a hispanic population ( 28 ) .
hispanic sensitivity to the development of an insulin - resistant state or diabetes with lower weight gain is well described ( 29 ) .
if confirmed in weight - loss studies , the sensitivity to modest weight loss that we observed in high - risk groups in the whi will have important clinical and public health implications .
it will also be important to explore possible social and genetic underpinnings for such population sensitivity .
two recent studies indicate that social economic status ( ses ) may account for some of the observed race / ethnicity disparities in diabetes prevalence ( 30,31 ) .
similarly , in our study , adjustment for educational attainment resulted in the largest decrease in diabetes incidence in hispanics ; if hispanics had achieved the same education levels as whites , their risk of diabetes would be 14% lower . however , education is only one component of ses , and thus consideration of an adjustment for other ses parameters ( e.g. , income and occupation ) would likely account for a greater proportion of the disparities observed in black and hispanic women .
first , observed prevalence of diabetes in the whi was lower than expected , as relatively healthy postmenopausal women were enrolled in the study .
second , reported education level among black women in the whi was considerably higher than education level among black women in the u.s .
the education differential for blacks and hispanics is much more extreme than that observed in whites , and this may underlie some of the other observations in the data ( 32,33 ) .
however , observed patterns are consistent with the population - based literature in terms of race / ethnic patterns of diabetes prevalence and incidence of diabetes .
third , asian women appear to have the greatest inherent risk in that their risk factor profile was in fact better than whites , and large increases in diabetes risk would occur if the risk factors deteriorate .
( 19 ) , which showed that asians had a higher diabetes risk per unit increase in bmi and waist circumference . recognizing that each of the racial / ethnic groups is heterogeneous due to differences by national origin and other relevant parameters
fourth , it is encouraging to note the effect of interval changes in weight amounting to a 3% reduction in risk of diabetes for each unit decrease in bmi .
individual efforts to reduce weight and increase physical activity are difficult to sustain ; however , current efforts are underway to tackle weight regulation through a variety of approaches that go beyond the individual .
lessons from the tobacco literature indicate that behavior change is influenced by our social and political structure ; thus , multilevel approaches are likely needed . in the u.s . , the race / ethnicity categories used most often in medical and public health research are from self - report , the same as the u.s . census categories . genetic race / ethnicity information known as admixture data ( i.e. , ancestry - informative markers ) from another study indicates great diversity within all four of the groups that we examined ( 34 ) .
yet , few studies obtain such data due to issues concerning cost , feasibility , practicality , and comparability . thus , the differences observed and reported here reflect any inherent biological differences across the groups studied as well as differences in life experiences ( e.g. , exposure to specific environments and stressors ) , which may also contribute to acquired physiological differences in reactivity , and in turn diabetes risk ( 35 ) .
the challenges for interpreting the results associated with studying self - reported racial / ethnic groups will likely increase in future studies , as more people identify themselves as belonging to multiple racial / ethnic groups .
although geographically diverse , racial / ethnic groups vary in their representation of the general population .
data for whites show that many characteristics of the whi participants are similar to white women participating in the national health and nutrition examination survey ( 11 ) ; however , ethnic groups are underrepresented in the whi .
women from parts of the country where we see large disparities in certain minorities ( e.g. , rural southern blacks ) were not represented .
thus , both prevalence and racial differentials are smaller in the whi than what we might expect to see in the u.s . as a whole .
second , only self - reported prevalence of diabetes and treated incident diabetes were ascertained ; thus , prevalence and incidence of diabetes may be underestimated .
we acknowledge that this is a limitation , and we did not account for nontreated diabetes .
however , self - reported diabetes in the whi was found to be reliable and sufficiently accurate to allow its use in epidemiologic studies ( 9 ) .
third , there could be other factors for which we did not control that further contribute to racial / ethnic disparities , such as health care access ( 36 ) . however , >
90% of the whi participants had insurance coverage , and diabetes prevalence and incidence were assessed at regular study visits .
fourth , although incident diabetes in older women is likely to be type 2 diabetes , the whi question did not specify type of diabetes .
other limitations include missing data ; however , the rates of retention in the whi were > 95% during an average of 7 years of follow - up ( 37 ) . balancing the limitations , there are several major strengths .
in addition , the whi collected detailed information on a comprehensive range of diabetes risk factors relevant to this investigation , with a 10-year follow - up for diabetes outcome . in conclusion ,
significant disparities exist between the major ethnic groups in diabetes prevalence and incidence in postmenopausal women ; these differences withstand adjustment for a very comprehensive group of physiological and behavioral risk factors .
although these results highlight the potential benefits of tailored diabetes prevention strategies directed at those specific factors that are most likely to increase the risk of diabetes among each racial / ethnic group , it is prudent to recommend avoidance of weight gain , weight loss , a healthy diet , and adequate levels of physical activity to all postmenopausal women for the purpose of diabetes risk reduction .
|
objectiveto examine determinants of racial / ethnic differences in diabetes incidence among postmenopausal women participating in the women s health initiative.research design and methodsdata on race / ethnicity , baseline diabetes prevalence , and incident diabetes were obtained from 158,833 women recruited from 19931998 and followed through august 2009 .
the relationship between race / ethnicity , other potential risk factors , and the risk of incident diabetes was estimated using cox proportional hazards models from which hazard ratios ( hrs ) and 95% cis were computed.resultsparticipants were aged 63 years on average at baseline .
the racial / ethnic distribution was 84.1% non - hispanic white , 9.2% non - hispanic black , 4.1% hispanic , and 2.6% asian .
after an average of 10.4 years of follow - up , compared with whites and adjusting for potential confounders , the hrs for incident diabetes were 1.55 for blacks ( 95% ci 1.471.63 ) , 1.67 for hispanics ( 1.541.81 ) , and 1.86 for asians ( 1.682.06 ) .
whites , blacks , and hispanics with all factors ( i.e. , weight , physical activity , dietary quality , and smoking ) in the low - risk category had 60 , 69 , and 63% lower risk for incident diabetes .
although contributions of different risk factors varied slightly by race / ethnicity , most findings were similar across groups , and women who had both a healthy weight and were in the highest tertile of physical activity had less than one - third the risk of diabetes compared with obese and inactive women.conclusionsdespite large racial / ethnic differences in diabetes incidence , most variability could be attributed to lifestyle factors .
our findings show that the majority of diabetes cases are preventable , and risk reduction strategies can be effectively applied to all racial / ethnic groups .
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several types of studies have suggested that infectious agents may play a role in atherosclerosis and coronary heart disease ( chd ) .
atherosclerosis in chickens is exacerbated by infection with marek 's disease virus , a member of the herpes virus family 12 , and numerous studies have suggested an association of viral pathogens such as cmv and bacterial pathogens such as helicobacter and chlamydia with human atherosclerosis 345 .
most 678 though not all 910 studies have found that patients with chd are more likely to carry anti - chlamydia antibodies than healthy subjects , and several studies have demonstrated that chlamydia can be detected immunohistologically in > 70% of atherosclerotic lesions , whereas it is virtually undetectable in undiseased arteries 5678 . c. pneuomonia is a gram - negative bacteria that may produce persistent infection through intracellular growth in macrophages 11 , a cell type that plays a dominant and necessary role in atherosclerotic lesions 1213 .
these observations have led to the hypothesis that chlamydia may play a causal role in atherosclerosis and chd 1415161718 .
this hypothesis is made plausible by studies on the association of helicobacter pylori and duodenal ulcer 1920 .
this condition was originally thought to be caused by excess gastric acid and was treated by reducing or neutralizing acid .
it is now clear that ulcers are most commonly caused by infection with h. pylori , and the most effective treatment involves antibiotics . in the same way
, atherosclerosis is currently thought to be caused by excess plasma cholesterol and is treated by agents that reduce cholesterol levels .
if chlamydia or any other pathogen causes atherosclerosis , then a more effective treatment may be an antibiotic or vaccine . we have sought evidence for a causal role of infectious agents in atherosclerosis using the murine apolipoprotein ( apo ) e ( deficient ) model .
because of the deficiency of apo e , these animals have high levels of plasma cholesterol and develop atherosclerosis with a reproducible time course 212223 . in a first experiment
, we bred the apo e with a strain of mouse that is unable to respond to bacterial lps , but found no alteration in the rate or extent of atherogenesis . in a second study
we conclude that neither the response to infectious agents nor the presence of infectious agents themselves is absolutely necessary for atherogenesis .
mice with the lps gene crossed onto a c57bl background ( c57bl/10scn ) were the gift of dr .
these mice exhibited minimal increases in serum levels of tnf- and il-6 in response to intraperitoneal injection of 50 g of lps , whereas c57bl and apo e animals exhibited > 100-fold elevations in both ( not shown ) .
c57bl/10scn mice were crossed with apo e mice on a c57bl/6 background ( jackson laboratories ) .
apo e/lpsanimals were identified in the f2 progeny by the presence of plasma cholesterol levels > 500 mg / dl and the absence of plasma elevations of tnf- 90 min after injection of 50 g lps .
germ - free and control apo e mice were generated and maintained at taconic farms .
offspring were weaned at 4 wk of age onto a high - fat western - type diet containing 21.22% ( g/100 g ) fat , 17.01% protein , 48.48% carbohydrate , and 0.15% cholesterol ( td88137 ; harlan teklad ) and maintained on the diet for the remainder of the study . autoclaved food and water were provided ad libitum .
the institutional animal care and use committee of merck research laboratories approved the animal use for experimentation , and all animals were cared for in accordance with the guide for the care and use of laboratory animals ( 1996 , national research council ) . to measure aortic cholesterol and cholesteryl ester ,
mice were killed and gently perfused through the left ventricle with cold pbs with 5 mm edta .
all branches and any adipose tissue connected to the aorta were removed , and each aorta was carefully excised from the aortic root to the right renal artery .
the aortas were blotted dry , minced , and extracted with chloroform / methanol ( 2:1 ) according to the method used by folch et al . 24 .
total and free cholesterol in the aortic extracts were determined using an enzymatic fluorometric assay modified from previously described methods 2526 , and data are expressed on a per aorta basis .
plasma cholesterol and triglyceride levels were determined using standard enzymatic kits ( sigma chemical co. ) .
for histology , mouse hearts were perfused in situ with pbs and removed with 1 mm of proximal aorta attached .
the top half of the heart was embedded in o.c.t . embedding medium ( fisher scientific ) and prepared for cryosectioning .
6-m sections were collected of the aortic root area , defined as having aortic valve leaflets present , and mounted on 10-well masked slides .
additional sections were immunolabeled with mab l3t4 for cd4 or m1/70 for cd11b ( pharmingen ) using the horseradish peroxidase method with 3 - 3-diamino benzadine as substrate .
the time course of atherosclerogenesis in apo e mice was followed by measuring aortic cholesteryl ester ( fig .
1 ) , a parameter that tracks with histological progression of atherosclerosis ( 23 ; data not shown ) . to determine a role for gram - negative bacteria such as chlamydia , we first used lps mice 27 , animals with a congenital deficiency in responses to the principal inflammatory component of these bacteria , lps ( endotoxin )
. lps is known to stimulate expression of cytokines ( tnf , monocyte chemotactic protein 1 [ mcp-1 ] , and others ) , adhesion molecules ( vascular cell adhesion molecule 1 [ vcam-1 ] , intercellular adhesion molecule 1 [ icam-1 ] , 2 integrins ) , and enzymes ( inducible nitric oxide synthetase [ inos ] , matrix metalloproteinase 9 [ mmp9 ] ) observed in atherosclerotic lesions 28 and is a prime candidate to mediate possible proatherogenic effects of chlamydia .
lps mice fail to respond to lps because of a deficiency in toll - like receptor 4 ( tlr-4 [ 29 , 30 ] ) , a transmembrane protein with homology to the il-1 receptor .
the inability of these animals to respond to lps was verified by measurements of plasma tnf in response to lps challenge , and their deficiency in apo e was verified by measurements of plasma cholesterol ( described in materials and methods ) .
we observed that development of atherosclerotic lesions in mice fed a high - fat western - type diet was not altered by the deletion of tlr-4 ( fig .
further studies showed that plasma cholesterol was also not affected by the absence of tlr-4 ( data not shown ) .
this result suggests that responses to gram - negative bacterial products are not critical for murine atherogenesis .
to more thoroughly ascertain the role of infectious agents in atherogenesis , we generated a germ - free colony of apo e mice .
founder germ - free apo e pups were delivered by caesarian section and reared by germ - free foster mothers .
the colony resulting from these founders was maintained germ free : weekly tests showed that no bacteria could be grown from feces , bedding , or swabs of the isolator .
in addition , animals chosen at random showed no antibodies against any of several viral pathogens .
the absence of intestinal bacteria is known to cause enlargement of the caecum 31 , and dramatically enlarged caeca were observed in all of the animals from the colony .
the progress of hyperlipidemia and atherogenesis in germ - free animals was compared with that of a second set of animals taken from the germ - free colony and reared with ambient pathogens .
as expected , control apo e animals exhibited extremely high levels of plasma cholesterol and moderately high triglyceride at both 22 and 32 wk of age ( fig .
germ - free animals showed an essentially identical plasma lipid profile , and lipid changes are thus unlikely to obscure effects of gnotobiosis on atherogenesis . in both male and female animals
, we observed that the exclusion of infectious agents caused no consistent differences in the rate or extent of free cholesterol or cholesteryl ester accumulation in the aorta ( fig . 2c and fig .
d ) . we did observe slightly reduced aortic cholesterol ester in the germ - free animals that attained statistical significance at 22 wk of age . however , these small differences disappeared upon correction for reduced body weight observed in the germ - free animals at the 22-wk time point ( 28 0.6 g for germ - free males vs. 34 0.8 g for control males , and 22 .02 g for germ - free females vs. 24 0.6 g for controls ; p = 0.001 ) .
the values for aortic cholesteryl ester in germ - free mice were also similar to those seen in a control colony that had never been made germ free ( values in fig .
the similarity of atherogenesis in control and germ - free animals was confirmed by histologic examination of the aortic root in animals taken at the 22-wk time point .
well - developed lesions with necrotic cores , large foam cells , and fibrous caps were observed in both control and germ - free specimens ( fig .
b ) , and neither quantitative nor qualitative differences could be detected in observations of either male or female lesions . a difference in aortic size reflecting the smaller body size of the germ - free animals 31 was readily detected histologically .
t cells are found in human and murine atherosclerotic lesions 1223 , suggesting activity of the adaptive immune system .
we identified t cells by immunohistochemistry in both control and germ - free animals ( fig . 3c and fig .
d ) , indicating that pathogens are not required to drive the influx of t cells into atherosclerotic lesions .
the observations presented here indicate that infectious agents , whether bacterial , viral , or fungal , are not necessary for murine atherogenesis .
infectious agents do not appear necessary either for initiation or progression of atherosclerosis , nor does the presence of infectious agents alter the morphology or cellular composition of the atherosclerotic lesions .
finally , the presence of infectious agents does not appear to alter the pattern of distribution of lesions along the aorta ( not shown ) .
rather , it appears that the high - circulating cholesterol levels in the apo e animals are sufficient to initiate and drive atherogenesis .
we conclude that koch 's postulates can not be fulfilled for any infectious agent in murine atherosclerosis .
atherosclerosis in apo e mice closely resembles that in humans with respect to histology , progression , and dependence on circulating cholesterol 23 , and we may thus speculate that infectious agents may not be necessary for the development of human atherosclerosis .
we wish to emphasize that the observations reported here relate to atherogenesis , not to plaque rupture , thrombus formation , or myocardial infarction .
it is possible that infectious agents play an important role in one or more of these acute processes .
several current studies are seeking to define a role for bacterial infection in human chd by studying endpoints such as myocardial infarction in patients randomized to placebo or antibiotic therapy of varying duration .
the largest of these studies to report results thus far has failed to demonstrate a decline in cardiovascular events in the 6 mo after treatment with antibiotic 32 .
although our results suggest that pathogens do not serve as etiologic agents for atherosclerosis , they may still have a role in exacerbating the disease .
recent reports suggest that inoculation of atherosclerosis - prone mice with high doses of chlamydia may cause an approximately twofold increase in the size of atherosclerotic lesions 3334 .
it is important to note that stresses such as infection provoke a set of physiological responses that are uniformly proatherogenic 353637 .
these include insulin resistance and hyperglycemia , elevation of plasma triglycerides , elevated white blood cell counts , elevated acute phase reactants ( e.g. , c - reactive protein and fibrinogen ) , and depression of high density lipoprotein ( hdl ) cholesterol levels .
it is thus possible that infection may exacerbate atherosclerogenesis , and antibiotics may ameliorate atherogenesis not through the action of an etiologic agent , but through indirect effects on metabolism and known risk factors .
|
recent work has revealed correlations between bacterial or viral infections and atherosclerotic disease .
one particular bacterium , chlamydia pneumoniae , has been observed at high frequency in human atherosclerotic lesions , prompting the hypothesis that infectious agents may be necessary for the initiation or progression of atherosclerosis . to determine if responses to gram - negative bacteria are necessary for atherogenesis , we first bred atherosclerosis - prone apolipoprotein ( apo ) e/ ( deficient ) mice with animals incapable of responding to bacterial lipopolysaccharide .
atherogenesis was unaffected in doubly deficient animals .
we further tested the role of infectious agents by creating a colony of germ - free apo e/ mice .
these animals are free of all microbial agents ( bacterial , viral , and fungal ) .
atherosclerosis in germ - free animals was not measurably different from that in animals raised with ambient levels of microbial challenge .
these studies show that infection is not necessary for murine atherosclerosis and that , unlike peptic ulcer , koch 's postulates can not be fulfilled for any infectious agent in atherosclerosis .
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according to the world health organisation ( who ) , the current estimates are that more than 62 million individuals are affected by diabetes mellitus ( dm ) , with 79.4 million cases expected by 2030 [ 1 , 2 ] .
the success of the treatments such as the one started in south india is closely related to early detection and explicit risk factors of diabetes .
this is a public health challenge and a number of diabetic patients ( estimated one in two ) will remain undiagnosed .
there are a number of people who are hesitant in taking the blood test till the disease has advanced and for such cases the treatment options for retinopathy or neuropathy become very limited .
there have been successes with population screening for diabetic retinopathy ( dr ) using digital photography of the retina [ 4 , 5 ] .
studies have shown that changes to both retinal nonvascular and vascular parameters are associated with different stages of dr .
there is also evidence of foveal and macular thickening to be an indicator of minimal nonproliferative dr [ 7 , 8 ] .
vascular parameters such as venule dilation and larger retinal arteriolar calibre [ 9 , 10 ] , changes to the vasculature shapes and arteriolar branching angle , increased tortuosity [ 1113 ] , and fractal dimension ( fd ) of the retinal vascular network [ 1416 ] are associated with dr .
these methods , however , require significant manual supervision and are not suitable for automatic analysis .
this study has identified the association between retinal vascular parameters that can be automatically obtained without manual supervision with type ii diabetes and no diagnosed dr .
this work provides an explanatory model of diabetes from retinal vascular parameter and knowledge about candidate clinical information , using retinal images with no sign of dr .
rivas , a retinal imaging software that automatically measures a range of parameters , was used .
the parameters that have already been reported in the literature were measured along with introduction of two new parameters : total number and average variability of the acute branching angles .
the study has developed statistical models using retinal vascular parameters , patients ' demographic information , and clinical parameters as the explanatory variables .
this is a follow - up to a recent similar work where only fd of optic disk centred ( odc ) retinal images was analysed .
a potential impact of this study is to allow for development of a predictive mode in the future to identify diabetic patients with no dr during a routine visit to an ophthalmologist or an optometrist .
experiments were conducted using database collected in department of the retina , save sight centre hospital located in delhi . approval for this study
was granted by the human research ethics committee ( hrec ) of the royal melbourne institute of technology ( rmit university ) , melbourne , australia , and also by the institutional review board at save sight centre hospital in accordance with the declaration of helsinki ( 1975 , as revised in 2004 ) .
all participants were respondents to a request advertised in the save sight centre in delhi . the purpose and experimental procedure in plain language
all the volunteers self - evaluated themselves to be reasonably active and none of them were pregnant .
the participants were classified into two groups of type ii diabetes ( case ) with no observable retinopathy and nondiabetic ( control ) patients .
the diabetic cases were confirmed by the patients ' physician based on either ( i ) their fasting or ( ii ) postprandial glucose plasma levels being greater than 126 mg and 200 mg / decilitre , respectively .
none of the diabetic patients had any observable intraretinal haemorrhages or venous beading , hard exudates , and neovascularisation according to the classification levels by international clinical disease severity scale for dr .
this was confirmed by an ophthalmologist after examination of both eyes . the participants ' demographic information , including age , gender , weight ( kg ) , height ( m ) , systolic and diastolic blood pressure ( mmhg ) , skin fold ( mm ) , and cholesterol level ( ldl and hdl ) , was recorded .
the participants ' age was limited to a narrow range of 40 to 73 years to remove confounding effect of the age factor on the analysis outcome and provide a better balance between the number of diabetic cases and the control groups .
all the participants were nonsmoker , did not consume alcohol , had no history of any cardiovascular disease , and did not have any history of antihypertensive and lipid - lowering medications .
one odc and one macula centred ( mc ) fundus images were taken from both eyes of each subject making total of four images for each participant .
the photographs were taken in mydriatic mode in a dimmed light room using a mydriatic kowa vx alpha camera ( kowa , japan ) .
the original image resolution was 300 dpi ( 4288 2848 pixels ) and the camera was set to an angle of 30. all the images were examined in pairs for quality assurance ( i.e. , vessel to background contrast and illumination artefacts ) . after checking the quality discarding ungradable images and age adjustment , 180 retinal images ( two ( left and right ) two ( odc and mc ) 45 subjects ( 13 diabetics and 32 nondiabetics ) ) were obtained .
the demographic information of the patients at the baseline has been provided in table 1 . in this study ,
odc and mc images were analysed separately , but for each image category ( i.e. , odc / mc ) the retinal vascular parameters of the left and right eyes of each subject were averaged .
all the original images were first cropped and resampled to identical sizes of 729 485 pixels .
image enhancement and segmentation ( binarization ) was performed to reduce degrading image background artefacts and improve vessel to background contrast .
this process was performed using mlvessel v1.3 , software provided online by soares et al . .
an example of the enhanced retinal vessels has been shown in figure 1 . for more information on the exact technique
retinal vascular geometry features were measured quantitatively using unsupervised retinal image and vasculature assessment software ( rivas ) v1.0 which has been specifically developed by the authors and described in . in brief
some of these are ( i ) vessel calibre of a specified segment , ( ii ) simple tortuosity , ( iii ) number of different fractal dimensions ( fd ) , ( iv ) vessel - to - background ratio / percentage ( v / b ( % ) ) , ( v ) average of acute branching angles ( aba ) defined as the smallest angle between two daughter vessels , and ( vi ) the total number of branching angles ( tba ) .
the fd measures include binary and grayscale box - counting ( also known as differential box - counting ( dbc ) ) and fourier fractal dimension ( ffd ) . in this study ,
simple tortuosity was measured as the ratio between the actual length of a vessel segment and the shortest ( euclidean ) distance between the two endpoints within the same segment providing a reflection of the shape / curvature of the vessel .
binary box - counting fd was calculated on skeletonized image as indicator of vascular network complexity without comprising any vessel calibre information .
figure 2(a ) shows the thinned vascular network ( skeletonized ) and the candidate points for location of the branches ( red dots ) . the blue line in this figure is the shortest path connecting the two end points of the vessel segments identified by two consecutive true candidate branch points .
prior to segment identification , the candidate points were automatically labelled in rivas , as true ( green circle ) and false ( red circle ) branch points as in figure 2(b ) .
true candidates were defined as the points which correspond to the bifurcations ( junction between two daughter vessels and a mother vessel ) and false candidates were defined as the points corresponding to other possibilities : crossovers or noise .
vessel diameter summary was also measured using ivan software ( university of wisconsin , madison , wi , usa ) based on the calibre summary of the biggest 6 arterioles and venules separately and vessel summary represented by central retinal arteriolar equivalent ( crae ) and central retinal venular equivalent ( crve ) as well as the ratio of the calibre of arterioles to venules ( avr ) .
the measurements were performed within a fixed region and in between the margins of 0.5 to 1.0 disc diameters from the disc margin .
crae and crve were obtained based on the revised knudtson - parr - hubbard formula .
statistical analyses were performed using minitab v.16.1.0 and r studio ( r statistical software v.3.3.0 ) . as the number of observations was relatively small , the data was statistically upsampled to the new size of 200 samples ( i.e. , 58 diabetic cases and 142 nondiabetic ) using the bootstrapping technique with sample replacement . the upsampled data was then standardized and centred to decrease the multicolinearity between an interaction term and its corresponding main effects as well as making categorical parameters such as gender , comparable with continuous parameters .
sixteen predictors were used in this analysis : vessel tortuosity ( both mean and standard deviation ( sd ) ) , aba , sd of angle , tba , crae , crve , avr , fd ( binary box - counting dimension of skeletonized images ) , and vb plus the patient 's demographic information ( i.e. , gender , age , systolic and diastolic blood pressure , body mass index ( bmi ) , and skin fold ) .
multiple linear regression model was built using all the 16 parameters as dependent variable and diabetes status with two possible categories ( i.e. , case and control ) as independent variable .
analysis of variance ( anova ) test was performed and f statistic was calculated to check the model fit .
r was also calculated to examine if the model is close to the regression line and obtain the percentage from the dependent variable 's variation explained by the model . for each predictor in the model
the coefficients and their significance level were calculated to identify potential nonsignificant variables and remove them from the model .
the test for multicolinearity was performed by looking into the variance inflation factor ( vif ) for standard error of the regression coefficients .
this paper also reports a stepwise regression approach , where the aim was to improve the exploratory stages of model building , but without compromising the physiological understanding of the predictors by using methods such as principal component analysis ( pca ) for dimensionality reduction .
this is essential for medical applications because the clinicians are keen to identify the relevant health parameters along with improved labelling of the data .
for this purpose , stepwise regression analysis was performed to select the variables that are significantly important . in this process
the most important variables are first selected with a forward searching algorithm followed by a backward elimination process to provide a reduced model with most suitable variables .
this method adds and removes the predictors in each step until all the variables used in the model have p value and the ones which are not used in the model have p value > with = 0.05 . for each predictor in the reduced model , vif
was calculated to test for the multicolinearity followed by testing the model for fitting performance using anova and r statistics .
the demographic information ( mean sd ) of the patients at the baseline prior to bootstrapping the data has been summarized in table 1 .
the bmi was calculated as weight ( kg ) divided by squared height ( m ) .
data analysis was performed separately on both odc and mc images to identify the relationship between potential predictors and diabetes factor .
the result from linear regression analysis and anova test for the first full model has been shown in table 2 . in this model ,
the r of 96.6% indicates 96.6% from the diagnosis variation is due to the model ( or due to change in predictors ) and only 3.4% is due to error or some unexplained factors .
p values of < 0.05 represent the fact that predictors play a significant role in the regression model .
the anova test also shows that the multiple linear regression model fits well to the data ( f = 3094.49 , p < 0.001 ) .
however , in this model , vif is greater than 5 for almost all the predictors ( except diastolic blood pressure ) showing that there is problem concerning the predictors ' colinearity .
a second model was built from the full model using stepwise procedure to reduce the number of parameters and the multicolinearity of the predictors .
the result has been provided in table 3 . for the second model , anova test shows that the multiple linear regression model fits significantly with the data ( f = 50.11 , p < 0.001 ) .
the vif factor in all cases is smaller than 5 showing negligible colinearity between predictors . in this model
the coefficients are weaker than the first model with reduced r. this result is in line with general expectations that there is reduction in model goodness of fit with reduction in number of features ; however , this does not represent decline in the explanatory power of the reduced model compared to the full model .
also interpretation of the coefficients in the second model with reduced number of variables and negligible degree of colinearity is more valid and accurate compared to the full model .
comparison between the two models shows that some coefficients have lower magnitude in the second model .
also some predictors ( i.e. , crae , mean tortuosity , vb , and age ) change their sign from the full model to the reduced model , resulting in some degree of uncertainty for the interpretation of the full model with highly colinear predictors .
the results also show that the retinal vasculature parameters with level of 0.05 that play a significant role in the reduced explanatory model of diabetes are the crae , mean tortuosity , aba , sd of branch angles , vb , and tba . from the clinical and demographical information , only systolic blood pressure and age were found to be significant predictor .
the same analysis as explained above was performed on odc images ; however , no model was found to provide adequate fit to the mc data for this database .
this research has studied retinal vasculature parameters to detect dm in the indian population with no dr .
the analyses were performed on both mc and odc images using a 30 nonmydriatic eye - fundus camera .
the significance of this work is that it reports automatic analysis of the eye - fundus images and provides an explanatory model for early changes in some retinal vascular parameters as a result of dm .
this study has also introduced a set of new retinal vascular parameters , tba and aba , and employed them together with other features and patients demographic information to create an explanatory model for prediction of diabetes in the absence of evident features of diabetic retinopathy . in this work
, linear regression analysis was performed to model association between a large number of explanatory variables as the predictors ( i.e. , retinal vascular parameters and clinical information ) and dm as the response variable .
the application of stepwise regression allowed for dimensionality reduction as well as solving the multicolinearity problem making the send model clinically interpretable .
it was important that predictors should be clinically relevant and without compromising the physiological understanding of the predictors .
the result showed that six retinal vascular parameters of ( 1 ) crae , ( 2 ) mean tortuosity , ( 3 ) aba , ( 4 ) sd of angle , ( 5 ) vb , and ( 6 ) tba are associated with diabetes in indian population when there is no observable dr . the anova test and resulting
the significant outcome of this study is that it provides the basis for an alternate technique to detect diabetes among people with minimal or no dr .
another major outcome of this study is that it has introduced two retinal vascular parameters that may find application in a predictive model to predict the risk of developing dr in diabetic patients with no dr .
therefore , conducting longitudinal study is proposed to monitor the progress of the patients and identify changes among those who may later develop dr .
the measurements are suitable for automation and for being used with simple eye - fundus imaging , which could be put in form of a modified smart - phone in the future .
the limitation of this study is that it was limited in the population type , having been conducted only in delhi in india , and represents narrow demographics with limited number of samples and matching cases .
it is essential to include subjects with similar cultural , ethnic , and socioeconomic conditions .
there is also the need for conducting similar tests on a larger dataset and for other demographics to observe potential differences and better evaluate the performance of the model .
|
this study has investigated the association between retinal vascular parameters with type ii diabetes in indian population with no observable diabetic retinopathy .
it has introduced two new retinal vascular parameters : total number of branching angles ( tba ) and average acute branching angles ( aba ) as potential biomarkers of diabetes in an explanatory model .
a total number of 180 retinal images ( two ( left and right ) two ( odc and mc ) 45 subjects ( 13 diabetics and 32 nondiabetics ) ) were analysed .
stepwise linear regression analysis was performed to model the association between type ii diabetes with the best subset of explanatory variables ( predictors ) , consisting of retinal vascular parameters and patients ' demographic information .
p value of the estimated coefficients ( p < 0.001 ) indicated that , at level of 0.05 , the newly introduced retinal vascular parameters , that is , tba and aba together with crae , mean tortuosity , sd of branching angle , and vb , are related to type ii diabetes when there is no observable sign of retinopathy .
|
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thymoglobulin , rabbit anti - human thymocyte immunoglobulin ( atg ) , is an immunosuppressive drug used as an anti - rejection therapy in solid organ transplantation and in hematological diseases .
an association between atg and acute lung injury was first described in an experimental model in 1975 . since then
, few cases have been reported from which it is believed that in rare cases , atg is responsible for a spectrum of lung injuries varying from transient infiltrate to full - blown acute respiratory distress syndrome ( ards ) .
we report a case of atg - induced non - cardiogenic pulmonary edema ( ncpe ) , its diagnosis and management under general anesthesia .
a 28-year - old , 50 kg man with asa risk iii was posted for laparoscopic renal transplantation .
he was diagnosed to have hypertension since 2 years and chronic interstitial nephritis leading to chronic renal failure ( crf ) since 6 months and was on maintenance hemodialysis twice a week .
he had undergone laparoscopic cholecystectomy and bilateral nephrectomy 2 months back without any complications . in preoperative examination
, his blood pressure was controlled with nifedipine 20 mg , metoprolol 50 mg , and clonidine 0.1 mg .
preoperative electrocardiogram ( ecg ) was normal nd 2-dimensionl echocardiography showed 60% ejection fraction with no wall motion abnormality .
induction was done with thiopentone sodium 350 mg , and suxamethonium 75 mg was given to facilitate endotracheal intubation .
right - sided internal jugular vein was cannulated by seldinger 's technique for central venous pressure ( cvp ) monitoring .
continuous monitoring of ecg , pulse oxymetry , capnography , invasive blood pressure , cvp , airway pressure and temperature was done . for surgical procedure ,
airway pressure was 21 cm h2o after induction of anesthesia , which increased to 25 cm h2o after pneumoperitonium and stabilized at 28 cm h2o in head down position .
after confirming negative reaction to test dose of atg and prophylactic administration of 100 mg iv hydrocortisone and 45.5 mg of iv chlorpheniramine maleate , 75 mg of atg diluted in 100 ml normal saline was started in central line which was to be given over a period of 4 hours .
methyl prednisolone 500 mg in 500 ml of normal saline was also started as an anti - rejection therapy .
intraoperatively , 3 l of normal saline and 100 ml albumin 20% was given to keep the cvp between 15 and 20 mm hg .
ureteric reimplant was done . at the end of surgery , which lasted for 4.5 hours
, the urine output was around 2 l. throughout the procedure , all parameters remained normal , but at the time of port closure , suddenly airway pressures were elevated up to 3540 cm of h2o . on manual ventilation , resistance was felt .
gradually , oxygen saturation started falling to less than 90% even with 8 l / min of oxygen , with persistent high airway pressures .
cvp line was changed to pa catheter and pulmonary capillary wedge pressure ( pcwp ) of 15 mm hg was noted .
diagnosis of ncpe was confirmed . when surgery was completed , the patient was given inj .
diazepam 5 mg , furosemide 100 mg iv and shifted to icu . in icu , the patient was kept on ventilator on synchronized intermittent mandatory ventilation ( pressure control)+ pressure support mode with 10 cm of positive end - expiratory pressure and 100% fio2 ) chest x - ray was done which showed diffuse infiltrates consistent with pulmonary edema . gradually , as the patient 's oxygenation and x - ray chest improved , he was weaned off the ventilator and extubated by next day morning .
pulmonary edema can be cardiogenic due to increase in the net hydrostatic pressure across the pulmonary capillaries or fluid overload or non - cardiogenic due to increase in the permeability of alveolar capillary membrane .
ncpe is characterized by simultaneous presence of severe hypoxemia , bilateral alveolar infiltrates on chest radiograph and no evidence of left atrial hypertension / congestive heart failure / fluid overload .
the common causes are gastric aspiration , sepsis , trauma , and respiratory obstruction leading to negative pressure pulmonary edema .
less appreciated is the fact that various drugs , either taken as standard therapy or as an overdose , may precipitate ncpe .
the first extensive review on agents causing pulmonary insult was published in 1972 which included 20 medications . since that time
bauman et al . published a paper on drug - induced lung diseases in which atg is mentioned as a causal agent for drug - induced ncpe .
dean et al . and murdock reported atg - induced ards . like the other case reports
, it was impossible for us to definitely prove that pulmonary edema was secondary to atg .
cardiogenic pulmonary edema and fluid overload were ruled out by the absence of preexisting heart disease , good left ventricular systolic function and normal pcwp .
the other causes of ncpe are less likely as it was an elective surgery , there was no respiratory obstruction and pulmonary edema developed at the end of surgery with endotracheal tube in situ .
pulmonary edema developed at the time of completion of atg infusion probably due to administration of antihistaminics and steroids during surgery . although rechallenging can definitely establish the causal link , we did not believe it safe for purely diagnostic purposes .
diagnosing drug - induced ncpe is actually an exercise of exclusion as there is no diagnostic test available .
it is related to the time proximity of administration of drugs , and pathogenesis involves direct cytotoxic insults to the lung epithelial cells and induction of cytokine triggered inflammatory responses .
various postulates are as follows : ( 1 ) atg contains antibodies of animal origin which are active against human t lymphocyte antigen , with cellular activation , degranulation and respiratory burst response damaging pulmonary endothelium .
pulmonary capillary endothelial permeability increases in response to tumor necrosis factor and alpha interleukin 1 and 8 released from damaged or activated lymphocytes similar to the pathogenesis of ards in sepsis .
our purpose of reporting this case is to alert anesthesiologists involved in transplantation program of this acute complication related to atg infusion so that they are prepared to treat it promptly .
|
non - cardiogenic pulmonary edema ( ncpe ) is a clinical syndrome characterized by simultaneous presence of severe hypoxemia , bilateral alveolar infiltrates on chest radiograph , without evidence of left atrial hypertension / congestive heart failure / fluid overload .
the diagnosis of drugrelated ncpe relies upon documented exclusion of other causes of ncpe like gastric aspiration , sepsis , trauma , negative pressure pulmonary edema .
we describe a 28year - old , 50 kg male with asa risk iii posted for laparoscopic renal transplantation , who developed ncpe after 4 hours of administration of rabbit anti - human thymocyte immunoglobulin ( atg ) .
he was successfully treated with mechanical ventilatory support and adjuvant therapy .
this report emphasizes that this fatal complication may occur with use of atg .
|
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arthritis affects around 0.51% of the world population with more women being affected than men .
the deregulation of the immune system may lead to the development of autoimmune diseases such as rheumatoid arthritis ( ra ) which is a prototype of the groups of illnesses with chronic systemic disorders with destructive inflammatory polyarticular joint potentially resulting in progressive destruction of articular and periarticular structure .
persistent inflammation produces swollen joints with severe synovitis , decreased nociceptive threshold , and massive subsynovial infiltration of mononuclear cells , which along with angiogenesis leads to pannus formation .
expansion of the pannus induces bone erosion and cartilage thinning , leading to the loss of joint function in due course .
this results in a high degree of morbidity and disturbed daily life of the patient .
corticosteroids have not been able to fully control the incidence because of its limitations and risk of side effects .
many patients and practitioners are seeking alternative approach to provide an effective cure in the treatment of arthritis and to overcome the serious drawbacks such as gastrointestinal bleeding on treatment with corticosteroids .
hence , there is an urgent need to find safer drugs for the management of rheumatoid arthritis which is linked to inflammation of joints .
many herbal formulations in the form of a single drug or compound drugs have been used for the treatment of joint pain , fever , and inflammation since ancient times as per the indian system of ayurvedic medicine .
saraca asoca has been traditionally used in the indian system from time immemorial for the treatment of uterine , genital , and other reproductive disorders in women , ailments of urogenital tract , fever , pain , and so forth .
its properties have been mentioned in the ancient ayurvedic text charak samhita under the vedanasthapan ( analgesic , antipyretic , and anti - inflammatory ) category [ 25 ] .
the legumes of saraca asoca are 610 inches long containing 48 grey dicotyledonous seeds like a chest nut .
the seeds are 35 cm long with average diameter of 8 - 9 cm , smooth surface , ellipsoid - oblong , and compressed .
the seed coating is brown or slightly black in colour while sun - dried seeds are dark brown coloured having a smooth surface with hard texture .
the stem bark part of this plant contains tannin , catechol , sterol , organic calcium compounds , essential oil , haematoxylin , a ketosterol , a crystalline glycosidal constituent , saponin , organic iron compound , leucocyanidin , and quercetin .
the pharmacological activities of stem bark are uterogenic , antibacterial , oxytocic , antitumour , anticancer , and antiprogestational .
saracin , a seed integument lectin from saraca indica , is highly specific for binding n - acetylneuraminyl - n - acetyllactosamine [ neu5ac--(26)/(2 - 3)-d - gal--(14)-d - glcnac ] .
this lectin has been found to be mitogenic for human lymphocytes , and this mitogenic activity could be inhibited in presence of fetuin .
further , treatment with saracin could induce secretion of il-2 in a culture of resting human peripheral blood mononuclear cells ( pbmc ) after 48 h. saracin has a higher affinity for the cd8 + than cd4 + t cells as revealed by facs analysis .
the medicinal plant extracts exhibiting inhibitory activity on cell proliferation should undergo analysis for possible antitumor activity , while extracts displaying inhibition of tfs / dna interactions without effects on cell growth kinetics might be employed to control tfs - dependent gene expression without cytotoxic effects , including the case of inflammatory processes involved in relevant human pathologies , such as rheumatoid arthritis and cystic fibrosis .
the scientific pharmacological evaluation of the analgesic , antipyretic , and acute anti - inflammatory activities of the acetone extract of seeds of saraca asoca has given significant and positive results during animal experimentation . therefore , its antiarthritic pharmacological action was evaluated on animals following the adjuvant test to find out its chronic anti - inflammatory effect which could validate the possible usage of these seeds as an effective nonsteroidal anti - inflammatory antiarthritic drug having the property of antioxidant , immune modulator , analgesic , and so forth .
the pharmacognostical , chemical , and experimental studies were carried out in the laboratory of the department of dravyaguna ( medicinal plant pharmacology ) at the institute of post graduate ayurvedic education & research , kolkata .
the acute and subacute toxicity and adjuvant antiarthritis studies of acetone extract of the seeds of saraca asoca were done on rodent animals after getting approval from the institutional animal ethical committee ( iaec ) in the animal house of ipgae&r kolkata ( registration number 1180/ac/08/cpsea dated 27.03.2008 of cpcsea ) according to the guidelines of cpcsea .
the seeds of saraca asoca were collected from the medicinal plant garden of narendrapur ramakrishna mission , kolkata , and the state government herbal garden at kalyani , west bengal , india , in the month of july .
the identification of seeds was done by the botanist at the botanical survey of india , howrah , india , vide ref .
an authentic herbarium specimen was deposited in the herbarium museum of the department of dravyaguna at ipgae&r , kolkata , for future reference .
the cfa reagent was purchased from m / s sigma aldrich , usa , while hydroxyproline and quercetin were purchased from m / s srl , india , and indomethacin was purchased from m / s jagsonpal pharmaceuticals ltd . ,
the macroscopic and microscopic examination of the seeds were done for the purpose of standardization .
coarse powder ( number 40 mesh ) was used for extraction and fine powder was used to ascertain the macroscopic and microscopic pharmacognostical characteristics under high resolution microscope ( dewinter , italy ) at the department of dravyaguna of ipgae&r according to established procedures [ 9 , 10 ] .
the seeds were washed and cleaned thoroughly to remove any extraneous matter and kept initially under sunlight covered with fine net for 5 hours .
they were dried under shade for 10 days taking all precautions to prevent them from any contamination and other foreign matters .
the completely dried whole seeds were powdered with a grinding machine ( hammer mill ) , passed through a number 40 mesh sieve , and stored in airtight containers for experimental purposes .
the powder of research drug was subsequently extracted sequentially in petroleum ether ( 6080 ) , chloroform , acetone , methanol , and water in a soxhlet 's extractor and then filtered .
the acetone extract was concentrated under vacuum in a rotary evaporator to yield semisolid mass .
this was further dried under a vacuum oven drier to give solid residue and preserved in refrigerator below 10c for experimental work .
the various important physiochemical parameters such as ph value , extractive values , moisture content , ash values , total flavonoid and phenol content , seed oil properties , and fluorescence of the powdered seeds were estimated using standard methods .
the presence of important phytochemical constituents such as phenols , flavonoids , tannins , and saponins was assessed using standard techniques [ 11 , 12 ] .
swiss albino mice of either sex , weighing about 2030 gm , and albino ( wistar ) rats of either sex , weighing about 120130 gm , were used for different in vivo evaluation .
all animals were procured from the government of west bengal approved breeder , m / s satyacharan ghosh , kolkata , and housed under standard environmental conditions with fixed 12 h light / dark cycles and a temperature of approximately 25c in animal house of ipgae&r .
the animals were kept in standard polypropylene cages and provided with food ( standard pellet diet ) and water ad libitum .
these animals were acclimatized for a period of 14 days prior to performing any experiments .
acute toxicity study was carried out on healthy swiss albino mice following oecd guideline 423 .
the animals of both sexes were selected by random sampling technique and divided into 5 groups of 3 animals each .
a single oral dose of the extract was administered orally at the level of 100 mg , 300 mg , 500 mg , 700 mg , and 1000 mg / kg body weight , respectively .
all the animals were observed for appearance of toxic symptoms including muscle spasm , loss of righting reflex , tremors , behavioural changes , locomotion , convulsions , and mortality for 1 , 2 , 4 , 8 , and 24 h. long - term supervision was continued for a period of 14 days for observing any occurrence of toxic symptoms and mortality .
the six wistarrats were dosed daily , starting at 300 mg / kg body weight ( the expected therapeutic level ) and increased stepwise every two to three days on to 400 , 500 , 600 , 700 , 800 , 900 , and 1000 mg / kg dosage , respectively , until toxic signs were observed .
hematological and biochemical monitoring were carried out and blood level of the compound was checked to ensure its absorption .
the animals were maintained at the maximum tolerated dose for a period of two to three weeks to allow development of any pathological changes , killed thereafter , and subjected to full pathological and histological examination .
the purpose of this test was to determine the maximum tolerated dose and to ascertain the nature of toxic reactions , so that suitable chronic toxicity studies can be designed to fully evaluate the toxic potential of the compound .
the clinical symptoms of each animal in terms of behavioral patterns detailed in the acute toxicity section mentioned above were also observed in all groups [ 15 , 16 ] .
fca - induced arthritis model in rats is suggested as the most suitable model of chronic and subchronic inflammation .
rat adjuvant arthritis is an experimental model of polyarthritis which has been widely used for preclinical testing of numerous antiarthritic agents which are either under preclinical or clinical investigation or are currently used as therapeutics in this disease [ 1719 ] .
animals like wistar rats were randomly divided into four groups of six animals each ( n = 6 ) .
the control group received 10 ml / kg of vehicle in which the extract was going to be suspended while the standard group received indomethacin ( 2.5 mg / kg p.o ) as the reference standard .
the third and fourth groups of animals were administered acetone extract of saraca asoca ( ae ) at a dose of 300 mg / kg and 500 mg / kg , respectively .
arthritis was induced by a single subplanter injection of 0.1 ml of complete freund 's adjuvant ( cfa ) containing 1.0 mg dry heat - killed mycobacterium tuberculoi per ml sterile paraffin oil into foot pad of left hind paw of male wistar rats .
drug treatment was started in all the groups from the day of adjuvant injection ( 0 day ) 30 min before adjuvant injection and continued till 21st day .
the swelling in hind paw ( oedema ) and paw ankle joint of left foot was periodically examined using screw gauge . the arthritic effect and other clinical symptoms
it was observed during the experiment that rats of standard group expired within a week when administered indomethacin at the dose of 10 ml / kg daily due to gastric ulceration and other toxicities in the body .
therefore , the dose of indomethacin was lowered and after several rounds of trial , the dose of 2.5 mg / kg body weight was selected which provided highly significant results and no mortality of animals .
the diameter of left paw and left ankle joint was measured in mm on 0 , 2nd , 5th , 7th , 9th , 11th , 14th , 18th , and 21st days by using screw gauge and the body weight of animals was measured by digital balance .
the mean changes in weight and diameter of paw oedema and ankle joint as well as their percentage inhibition with respect to control group were calculated on the 7th day , 14th day , and 21st day , respectively .
the rats were anaesthetized under light ether anaesthesia and blood was collected by retroorbital puncture for estimation of serum parameters such as hb , rbc , wbc , esr , and prostaglandin ( e2 eia kit monoclonal cayman chemical item number 514010 ) by using various diagnostic kits on the 14th day and 21st day which were compared with the data obtained from untreated rats before injection of adjuvant to the rats .
this procedure is used to assess the urinary excretion of connective tissue metabolites such as hydroxyproline and glucosamine during the antiarthritic analysis of the test drug .
hydroxyproline is produced by hydroxylation of the amino acid proline by the enzyme prolyl hydroxylase following protein synthesis ( as a posttranslational modification ) .
although it is not directly incorporated into proteins , hydroxyproline comprises roughly 4% of all amino acids found in animal tissue , an amount greater than seven other amino acids that are translationally incorporated .
glucosamine ( c6h13no5 ) is an amino sugar and a prominent precursor in the biochemical synthesis of glycosylated proteins and lipids .
glucosamine is part of the structure of the polysaccharides chitosan and chitin , which compose the exoskeletons of crustaceans and other arthropods , cell walls in fungi , and many higher organisms .
glucosamine is one of the most abundant naturally occurring amino monosaccharides that has been used to treat or prevent osteoarthritis in humans .
the effect of adjuvant - induced arthritis on the urinary excretion of hydroxyproline and glucosamine was investigated in rats on the 2nd , 14th , and 21st days of the treatment after the injection of cfa .
the rats were kept overnight in metabolic cages on the 14th and 21st days to collect urine samples for the estimation of hydroxyproline and glucosamine .
the hydroxyproline and glucosamine content in urine was also calculated in mg / ml from the absorbance at 540 nm calibration curve which was generated with different concentrations of these two compounds , respectively .
the radiographic examination of animals of all groups was done using dental x - ray plate in the probe diagnostic laboratory , kolkata , by performing x - ray images of left and right paws on 28th day to assess swelling or other changes in the cartilages or destruction and irregular margin of the bone and cartilage in the paws .
this test is done to find out the degree of chronic inflammation which has occurred in the affected parts .
all the animals were sacrificed at the end of the experiment on the 28th day .
the left hind paws of all the animals were removed , fixed in formal saline for 7 days , and then decalcified in 5% formic acid .
joints were then trimmed and embedded and sections were cut just above the ankle joint having a thickness of 6 m by microtome .
these sections were stained with haematoxylin and eosin and mounted on the slide permanently for histopathological observation to ascertain the presence of dense inflammatory infiltrate in the joints , arthritis with destruction of cartilage , pannus formation , and so forth under microscope ( dewinter , italy ) . since use of nsaids on long - term basis has been known to be associated with toxicity , this experiment was done to assess the pharmacological effect or any toxicity of the prescribed drugs upon the stomach , liver , and kidney of the rats in all groups after the end of the experiment . fresh portions of lateral lobes of the liver , stomach , and kidney from each sacrificed rat were cut rapidly , fixed in neutral buffered formalin ( 10% ) , and then dehydrated with varying grades of ethanol ( 70 , 80 , 90 , 95 , and 100% ) followed by clearing of the samples in xylene .
these samples were then impregnated with 2 changes of molten paraffin wax , embedded , and blocked out .
paraffin sections ( 4 - 5 m ) were stained with hematoxylin and eosin , the conventional histological stainer according to pearse .
stained sections of control and treated rats were examined for alterations in the architecture , portal triads , hepatocytes , and sinusoids and for the presence of degeneration , necrosis , fatty change , and portal fibrosis .
the data were statistically analyzed using one - way anova followed by dunnett 's t - test for individual comparison of groups with control .
the powder of the seeds is light brown in colour with an aromatic odour and has a slightly sweet taste .
the microscopic structure of the powder showed the presence of cells containing tannins , stone cells , crystals , endospermic cells , starch grains , and vessels .
the values of various important physiochemical parameters for the purpose of quality control and standardization such asmoisture content , ash value , and extractive value were evaluated following methods prescribed in the ayurvedic pharmacopoeia as detailed in table 1 .
the animals tested in acute toxicity tests up to the dose of 1000 mg / kg showed no significant toxic symptoms like sedation , convulsion , diarrhoea , irritation , and so forth and no signs of behavioural changes . no mortality was reported up to 24 hrs and even later during the subsequent 14 days at 1000 mg / kg dose .
it is observed that one rat at the dose of 900 mg and 1000 mg / kg out of 6 rats died due to some other factors during daily dose pattern toxicity .
this was confirmed since no pathological changes occurred in the histopathological slides of the liver , stomach , and kidney shown below .
the assessment of physical changes and behaviour activities was also recorded in the subacute toxicity test like acute toxicity analysis of this research drug ( table 2 ) .
no noticeable changes were noticed in the values obtained during the haematological test of the blood sample of treated groups up to the dose of 1000 mg / kg as compared to the normal values .
the histopathological images of the stomach , liver , and kidney of the rats administered the highest dose of 1000 mg / kg showed normal internal structure of mucosal lining , hepatic cells , kupffer cells , and cell content as shown in figures 1(a)1(c ) .
the values of mean body weight and left paw oedema and diameter of left paw ankle joint after daily administration of the prescribed dose were calculated over time following the adjuvant arthritis model in case of all groups .
the size of right paw oedema was measured and the clinical features of inflammation were observed after 14th day and 21st day of this study because these changes may occur in this test as per protocol of the cfa reagent .
the changes in body weight of rats were monitored up to 21 days and the results are shown in table 3 .
the significant decrease in mean body weight of control group rats from 119.8 gm on day 0 to 110.8 gm on the 21st day may be due to decrease in their immune response and increased symptoms of inflammation , pain , fever , and swelling due to subplantar administration of cfa .
on the other hand , the other groups showed appreciable increase in body weight during this period , ranging from 5.57% in case of the standard group to 3.68% in case of the 300 mg / kg group and 4.53% in case of the 500 mg / kg drug group ( figure 2 ) , which may be attributed to the response of the treatment drug against the induced inflammation .
the paw volume was measured in terms of the diameter in mm for all the groups and compared with the control group on the 7th , 14th , and 21st days . in case of the control group ,
the paw oedema increased consistently from 4.37 mm to 6.77 mm over this period .
the group which was administered 500 mg / kg drug exhibited inhibition of 48.85% , 67.88% , and 93.75% as compared to the control group on the 7th , 14th , and 21st days ( table 4 ) .
these results are very close to the figures of 55.72% , 88.21% , and 99.58% obtained over the same period in case of the standard indomethacin group , signifying very strong and significant anti - inflammatory activity .
strong and sustained anti - inflammatory action was also noticed in case of the lower dose of acetone extract ( 300 mg / kg ) where the observed inhibition values were 38.93% , 45.52% , and 53.33% after 7 , 14 , and 21 days ( figure 3 ) .
the left ankle joint oedema in the control group increased significantly over the 21-day period , while substantial inhibition was observed in case of the other research groups . in case of the drug extract group at higher dose , 50.75% , 91.96% , and 98.63% inhibition
was noticed after 7 , 14 , and 21 days , respectively , as compared to the control group , indicating significantly high anti - inflammatory activity which is quite close to the corresponding values of 51.49% , 92.77% , and 98.97% in case of the standard drug group ( table 5 ) .
the drug group extract at the lower dose ( 300 mg / kg ) also exhibited significantly high activity especially on the 14th and 21st days ( figure 4 ) when 67.06% and 89.11% inhibition was noticed .
the hydroxyproline and glucosamine content in urine was calculated in mg / ml from the calibration curve which was generated with different concentrations of these two compounds on the 2nd , 11th , and 21st days .
these parameters were found to exhibit an increasing pattern in the control group due to excessive passing of connective tissues in the form of amino acid proline and amino monosaccharide in the urine which indicated that there is no anti - inflammatory effect on the induced inflammation of the body parts . however , these two metabolites showed a decreasing pattern in the other groups ; the standard group exhibiting the highest decrease followed by the higher drug dose group and the lower drug dose group , respectively ( table 6 ) .
there was a significant decrease in the values of rbcs along with hb% and significant increase in wbcs and esr in the control group rats as compared to normal rats after 21 days of treatment which indicated that the second stage of inflammation had occurred in the body .
however , the haematological parameters of the blood of rats treated with acetone extract ( 300 mg / kg and 500 mg / kg ) and the standard drug indomethacin showed a lower decrease in the red cells counts and hb% and also a lower increase in the wbc count and esr values ( table 7 ) . in comparison , it can be seen that the highest impact was seen in case of the standard group , while the lowest impact was seen in the lower dose of the acetone extract , even though significant and sustained impact was noticed in all the three groups as compared to the control group .
the anti - inflammatory effect of these three groups on the basis of the above haematological results was confirmed by the changes observed in the various groups after 21 days in the level of the prostaglandin which was found to be lower in the standard and higher drug dose treated groups as compared to the control group . the x - ray image of the tibia - tarsal joints of left and right paw of adjuvant - induced control group showed marginal bone swelling and a slight destruction of the cartilage bone in the left and right paw after 28 days of treatment .
the radiographic images ( figures 5(b ) and 5(c ) ) of the adjuvant - induced standard indomethacin drug group and higher dose ( 500 mg / kg ) of acetone extract after 28 days ( figures 5(a)5(c ) ) showed no callus formation , deformity , or irregular margins of cartilage and bone , which indicated the significant pharmacological anti - inflammatory action as compared to the control group .
the results of the histopathological analysis of the left ankle joints after 28 days as seen under the microscope are shown in figures 6(a)6(c ) which reveal signs of mild bone destruction by synovial proliferation , cellular infiltration , and cartilage erosion in the control group , while no such changes are seen in the other two groups .
the histopathological analysis of the three important internal organs of the body , namely , the kidney , liver , and stomach , was done in order to evaluate the changes that may have occurred in their internal structure after 28 days of this study in respect of the adjuvant - induced treated rats administered with standard drug indomethacin and acetone extract at 300 mg / kg and 500 mg / kg dosage .
it was observed from the obtained results shown in figures 7(a)7(i ) that no changes or abnormal features were noticed in the slides of kidney and liver in respect of all the three groups .
however , the slide of stomach of the standard drug indomethacin showed the irregular and broken margins of the endothelium of stomach , possibly indicating the initial stage of the production of ulcer because usage of nsaid drugs has been shown to be associated with such side effects in some cases . on the other hand , the slides of both doses of the acetone extract of the research drug showed the features of normal structure of the stomach of rat which indicated its safety and nontoxic effect .
the saraca asoca bark has a stimulating effect on the endometrium and ovarian tissue and is useful in menorrhagia during uterine fibroids .
flowers of this tree are used to treat cervical adenitis , biliousness , syphilis , hyperdipsia , burning sensation , hemorrhagic dysentery , piles , scabies in children , and inflammation in a traditional system of medicine .
fca - induced arthritis has been used as a model for assessing subchronic or chronic inflammation in rats and is of considerable relevance for the study of pathophysiology and pharmacological control of inflammatory processes . during the study ,
the initial inflammatory response was developed within few hours , but more critical clinical signs were seen during the 1st week of postinoculation and thereafter for several weeks [ 19 , 23 ] .
the chemical analysis of acetone extract of the seed exhibited the presence of high concentrations of phenolic compounds such as flavonoids , tannins , carbohydrates , and acetyl salicylic acid after following the standard pharmacognostical methods . during the acute and subacute toxicity assessment of the acetone
extract , there was no mortality and no other severe symptoms were observed up to the dose of 1000 mg / kg body weight of mice . during the antiarthritis studies , the body weight of the rats after 21 days of treatment increased appreciably ranging from 5.57% in case of the standard group to 3.68% in case of the 300 mg / kg group and 4.53% in case of the 500 mg / kg drug group . at the same time , there was a decrease of 7.51% in body weight in case of the control group which may be due to the nonsubsidence of the clinical symptoms and pathological changes in the induced paw edema .
the changes in the left paw oedema volume as compared with the control as measured on the 7th , 14th , and 21st days showed that the inhibition of inflammation in case of the drug - treated group at higher dose ( 500 mg / kg ) was comparable to that in case of the standard group and a little lower in case of the lower drug dose ( 300 mg / kg ) group .
measurements of the ankle joint size over the same time period in case of the different groups were also compared with the control group and similar results were obtained , with the lower drug group also exhibiting very high inhibition of inflammation after 21 days . the similarity in the pattern of inhibition of the oedema in the paw and the ankle joints of the acetone extract (
500 mg / kg ) as compared to the standard group could be attributed to the presence of high concentrations of flavonoidic phenolic compounds in the extract which are known to exhibit antimicrobial , antiviral , antiulcerogenic , cytotoxic , antioxidant , antihepatotoxic , antipyretic , and anti - inflammatory activities .
mild oedema in the right paw and the other clinical features were noticed in the control group after 21 days , but no similar changes were noticed in the drug and standard groups which also indicated the positive effect of drug .
the amount of hydroxyproline and glucosamine in urine as measured on the 2nd , 11th , and 21st days was found to have an increasing pattern in the control group indicating absence of any anti - inflammatory effect upon the induced inflammation . however , their concentrations showed a decreasing pattern in the other groups ; the standard group exhibiting the highest decrease followed by the higher drug dose group and the lower drug dose group , respectively .
the results of the changes in the haematological parameters such as hb% , rbc count , wbc count , and esr levels as well as the level of prostaglandin also indicated that the maximum anti - inflammatory effect and mechanism of the action of the drug were observed in case of the standard group , but a comparable significant impact was seen in case of the 500 mg / kg drug group followed by the lower drug group .
indomethacin inhibits the catalytic activity of the cox enzymes , the enzymes responsible for catalyzing the rate - limiting step in prostaglandin synthesis via the arachidonic acid pathway .
the radiographic images obtained during the x - ray of experimental animals showed that none of the changes , namely , bony swelling , destruction of the cartilage , articular meniscus , and so forth , were exhibited in the acetone extract 500 mg / kg treated and the standard groups , while the erosion in synovial membrane and cartilage was found in the control group .
the histopathological images of the stomach , liver , and kidney showed no adverse impact upon the liver and kidney in any of the groups , while some ulcerogenic effect was noticed in the stomach in case of the standard group .
the test drug showed no toxic effect upon the three main organs of the body , stomach , liver , and kidney , when administered orally for a long time ( up to 21 days ) .
this is particularly important because it is well known that nsaids can produce some toxic effect on these organs when used for a long time or in high doses .
inflammation defined as a biological process characterized by redness , oedema , fever , and pain can result in locally increased production of free radicals by inflammatory enzymes , as well as the release of inflammatory mediators that promote cell proliferation and angiogenesis and inhibit apoptosis .
as per the modern pathophysiology concept , fever and inflammation are produced due to exogenous pyrogens which are generally a form of microorganism such as bacteria and virus .
these exogenous pyrogens act on the host cells and produce endogenous pyrogens in the form of cytokines , which are regulatory polypeptides .
the endothelial cells of anterior hypothalamus release arachidonic acid metabolites when exposed to these endogenous pyrogenic cytokines .
one of the arachidonic acid metabolite prostaglandins e2 ( pge2 ) is a very potent fever producing autacoid and also inflammation in the body .
in fact , interleukin-6 is a cytokine not only involved in inflammation and infection responses but also in the regulation of metabolic , regenerative , and neural processes .
the medicinal plant extracts exhibiting inhibitory activity on cell proliferation should undergo analysis for possible antitumor activity , while extracts displaying inhibition of tfs / dna interactions without effects on cell growth kinetics might be employed to control tfs - dependent gene expression without cytotoxic effects , including the case of inflammatory processes involved in relevant human pathologies , such as rheumatoid arthritis and cystic fibrosis .
several transcription factors ( tfs ) play crucial roles in governing the expression of different genes involved in the immune response , embryo or cell lineage development , cell apoptosis , cell cycle progression , oncogenesis , repair and fibrosis processes , and inflammation . as far as inflammation , tfs playing pivotal roles are nuclear factor kappa b ( nf-b ) , activator protein ( ap-1 ) , signal transducer and activator of transcription ( stats ) , camp response element binding protein ( creb ) , and gata-1 factors .
all these tfs regulate the expression of proinflammatory cytokines and are involved in the pathogenesis of a number of human disorders , particularly those with an inflammatory component .
the findings indicate that the acetone extract of saraca asoca possesses antiarthritic pharmacological action similar to the nonsteroidal anti - inflammatory drug indomethcin due to the presence of flavonoidic compounds quercetin and gallic acid in the test drug which may be responsible for decreasing the level of cytokine and interleukin ( responsible for inflammation in the body ) by measuring the responsible prostaglandin in serum .
indomethacin is a nonsteroidal anti - inflammatory drug ( nsaid ) that reduces fever , pain , and inflammation by reducing the production of prostaglandins .
prostaglandins are autacoids that the body produces to cause fever and pain that are associated with inflammation .
the decreased level of prostaglandin in the blood and other changes in the hematological parameters have also confirmed the pharmacokinetic action of the acetone extract during this study .
indomethacin blocks the enzymes that make prostaglandins ( cyclooxygenases 1 and 2 ) and thereby reduces the levels of prostaglandins leading to reduction in fever , pain , and inflammation .
a similar mechanism of action probably exists in the research drug at the dose of 500 mg / kg acetone extract due to the presence of flavonoidic compounds that are well known to possess antipyretic , analgesic , and anti - inflammatory properties .
the research effort makes a detailed and comprehensive assessment of the antiarthritic efficacy of the seeds of saraca asoca by evaluating the impact on various diverse parameters employing standard techniques of scientific analysis associated with freund 's adjuvant - induced arthritic rat model .
the findings confirmed the significant nontoxic , antiarthritic , and anti - inflammatory pharmacological effect of saraca asoca 's acetone extract which is comparable to the standard drugs in a dose - dependant manner , possibly due to the presence of flavonoidic chemical compounds and the resultant lowering in the level of prostaglandin in the blood .
however , further research is required for isolation and identification of the specific chemical compounds responsible for the antiarthritic effect .
|
saraca asoca has been traditionally used in indian system for treatment of uterine , genital , and other reproductive disorders in women , fever , pain , and inflammation .
the hypothesis of this study is that acetone extract of saraca asoca seeds is an effective anti - inflammatory treatment for arthritis in animal experiments .
the antiarthritic effect of its oral administration on freund 's adjuvant - induced arthritis has been studied in wistar albino rats after acute and subacute toxicities .
phytochemical analysis revealed presence of high concentrations of phenolic compounds such as flavonoids and tannins , while no mortality or morbidity was observed up to 1000 mg / kg dose during acute and subacute toxicity assessments .
regular treatment up to 21 days of adjuvant - induced arthritic rats with saraca asoca acetone extract ( at 300 and 500 mg / kg doses ) increases rbc and hb , decreases wbc , esr , and prostaglandin levels in blood , and restores body weight when compared with control ( normal saline ) and standard ( indomethacin ) groups . significant (
p < 0.05 ) inhibitory effect was observed especially at higher dose on paw edema , ankle joint inflammation , and hydroxyproline and glucosamine concentrations in urine .
normal radiological images of joint and histopathological analysis of joint , liver , stomach , and kidney also confirmed its significant nontoxic , antiarthritic , and anti - inflammatory effect .
|
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medulloblastoma is the most common pediatric brain tumor , with an incidence of ~0.5/100,000 children < 15 years old ( 1 ) .
it is classified as grade iv according to the world health organization ( who ) ( 2 ) .
current treatment consisting of surgery , radiotherapy and adjuvant chemotherapy has a limited role in improving the prognosis of patients ( 3,4 ) . novel therapeutic methods based on the specific mechanism of medulloblastoma carcinogenesis
an increasing number of studies have revealed that micrornas ( mirnas / mirs ) serve a role in tumorigenesis ( 59 ) .
gene expression is regulated by thousands of known mirnas , which bind to imperfect complementary sites within the 3 untranslated regions of their target protein - coding mrnas , and repressing the expression of these genes at the level of mrna stability and translation ( 10 ) .
previous research has revealed that mirnas are involved in the regulation of the majority of physiological and pathological process , including development , life span , and metabolism , and their dysregulation contributes to a number of types of disease , notably cancer ( 11 ) .
these small rnas may function as oncogenes or tumor suppressors by modulating the levels of critical proteins , and their relevance in human disease and therapy is currently under investigation ( 12 ) .
an increasing number of studies have demonstrated abnormal mirna expression levels in the central nervous system ( cns ) of cancer patients , suggesting that mirnas may serve as key regulatory components in cancer of the cns ( 5,10,13 ) .
it was observed that 22 mirnas were upregulated and 26 mirnas were downregulated in the tumor tissue .
ingenuity pathway analysis ( ipa ) was used to analyze the regulatory network of the differentially expressed mirna in medulloblastoma .
it was proposed that the pathways exhibited in the network may have an important role in the tumorigenesis of medulloblastoma .
the data were downloaded from the ncbi gene expression omnibus ( geo ) database ( gse42657 , www.ncbi.nlm.nih.gov/geo ) .
the data include 7 control samples ( consisting of 3 cerebellum and 4 frontal lobe controls ) and 9 medulloblastoma samples .
the data from all samples was normalized using the limma package in r software version 3.3.0 ( https://www.r-project.org/ ) .
the selected samples of this profile are exhibited in table i. the differentially expressed genes ( degs ) were obtained with the thresholds of |logfc|>1.0 and p<0.01 , using linear models and empirical bayes methods for assessing differential expression in microarray experiments in the limma package ( table ii ) .
a heat map of the different groups was produced to visualize the degs and cluster the corresponding groups with the differentially expressed mirnas from the tumor tissue , using the r package gplots ( fig .
the dataset ( 48 differentially expressed mirnas ) was uploaded to the ingenuity pathway analysis software version 2016 ( qiagen , inc . ,
valencia , ca , usa ) , using the microrna target filter to find the targeting information .
a total of 40 micrornas targeting 11,757 mrnas were identified by the filter . the core analysis function ( rapid assessment of the signaling and metabolic pathways , molecular networks , and biological processes that are most markedly perturbed in the dataset of interest ) to analyze the associated network functions of the mirnas .
using mirna microarray data from the geo database , 48 mirnas which are differentially expressed in medulloblastoma were identified , compared with the control group ( table ii ) .
the core analysis demonstrated the molecular network interactions and signaling pathways associated with 28 differentially expressed mirnas of medulloblastoma , and their predicted molecular targets were rebuilt using ipa . the network
organismal injury and abnormalities , reproductive system disease , cancer had an ipa score of 41 , focusing on 17 mirna molecules , and the network cancer , organismal injury and abnormalities , cell death and survival had an ipa score of 23 , focusing on 11 mirna molecules ( fig .
the most impacted biological processes and diseases regulated by the analyzed mirnas included organismal injury and abnormalities , reproductive system disease and cancer .
the molecular network maps demonstrated that three primary components were identified to be at the central hub of the most significant network with a score of 41 ; these were tumor protein p53 ( tp53 ) , argonaute 2 ( ago2 ) and mitogen activated kinases 3 and 1 ( erk1/2 ) ( fig .
tp53 , sirtuin 1 ( sirt1 ) and y box protein 1 ( ybx1 ) were located in the center of the second network with a core analysis score of 23 ( fig .
the first network included mir-17 - 5p , mir-130a-3p , mir-182 - 5p , mir-18a-5p , mir-19b-3p , mir-4288 , mir-96 - 5p , which were upregulated in the medulloblastoma samples , and mir-128 - 3p , mir-132 - 3p , mir-133a-3p , mir-299a-5p , mir-409 , mir-668 - 3p , which were downregulated . the second notable network ( fig .
2b ) involved upregulated mir-217 - 5p and mir-216a-5p , and downregulated mir-329 , mir-330 and mir-584 , which are associated with the regulation of key regulatory genes in medulloblastoma development , including tp53 , sirt1 and ybx1 .
however , when the cerebellum samples were used as the control , it was observed that 4 mirnas were overexpressed and 5 mirnas were underexpressed in the medulloblastoma ( table iii ) .
the network cell death and survival , gastrointestinal disease , hepatic system disease had an ipa score of 9 , focusing on 4 mirna molecules ( fig .
mirnas function as master regulators and signal modulators by fine - tuning gene expression in multiple complex pathways .
a number of the mirnas which exhibit dysregulated expression in medulloepithelioma have been reported to serve various roles in carcinogenesis ( 6,7,9 ) . through analysis of a geo mirna expression profiling dataset , mirnas which are over- and underexpressed in medulloblastoma
were identified , including mir-217 , mir-216 , mir-183 , mir-182 and mir-96 , which are upregulated in tumor tissue .
previous research demonstrates that mir-183 - 96 - 182 regulates cell survival , proliferation and migration in medulloblastoma , and increased mir-183 - 96 - 182 expression is associated with aggressive clinical course , high rates of metastasis and poor overall survival ( 8,9,14 ) .
mir-217 and mir-216 were overexpressed in medulloblastoma . however , the role of mir-217 was controversial .
mir-217 is an oncogene that is overexpressed in aggressive human b cell lymphomas ( 15 ) .
however , it may be a tumor suppressor in hepatocellular carcinoma ( 16 ) . in pancreatic intraepithelial neoplasm , pancreatic ductal adenocarcinomas and
further studies are required to determine whether increased expression of mir-217 in medulloblastoma represents an oncogenic effect , or if the dysregulation functions as a potential tumor suppressor . while mir-216 was underexpressed in various types of cancer , including breast cancer , pancreatic cancer and nasopharyngeal carcinoma ( 1921 )
, it may serve as a tumor suppressor to inhibit cell proliferation , invasion and tumor growth .
further studies are required to determine whether increased expression of mir-216 in medulloblastoma represents an oncogenic effect or is a potential tumor suppressor . in the present study ,
marked downregulation of mir-383 , mir-206 , mir-138 , mir-128a / b and mir-133b was identified .
mir-383 , repressing peroxiredoxin 3 at transcriptional and translational levels , suppressed the cell growth of medulloblastoma ( 6 ) .
additionally , mir-206 was downregulated in all the four molecular subgroups of medulloblastoma as well as in cell lines .
orthodenticle homeobox 2 ( otx2 ) , a target gene of mir-206 which is overexpressed in all non - sonic - hedgehog - driven medulloblastomas , is an oncogene in medulloblastoma .
therefore , underexpression of mir-206 contributed to the upregulation of otx2 expression and enhanced growth of medulloblastoma cell lines ( 7 ) .
studies have demonstrated a reduction in the mirnas mir-383 , mir-138 , mir-128a / b , mir-133 , mir-124 and let-7 g in medulloblastoma .
nevertheless , mir-328 , mir-133b , mir-149 , mir-181b and mir-154 were downregulated in the study of ferretti et al ( 5 ) and the present study .
in addition , the present study revealed that mir-433 , mir-488 , mir-584 , mir-329 , mir-299 - 5p , mir-330 , mir-770 - 5p , mir-656 and mir-642 were underexpressed in medulloblastoma , and further research is required to identify whether these mirnas have a specific role in the tumorigenesis of medulloblastoma .
based on microarray analysis , mirnas that distinguish normal brain from medulloblastoma tissue were identified . as presented in the heat map ,
the dysregulated mirnas are more closely associated with the medulloblastoma samples compared with the normal control samples .
additionally , by uploading the mirnas from the first network to the ipa , it was identified that the dysregulated mirnas were associated with key regulatory genes in tumorigenesis , including tp53 , insulin - like growth factor 1 receptor , ago2 and erk1/2 .
the overexpressed mirnas mir-17 - 5p and mir-182 ( log fold change > 2 ) have interactions with tp53 ; studies have demonstrated an association between these mirnas and tp53 in tumorigenesis ( 22,23 ) .
agos are associated with mirnas , due to their function in the rna induced silencing complex ( risc ) , wherein they use small rna guides to recognize targets .
ago2 had an interaction with mirnas that are dysregulated in medulloblastoma , including mir-17 - 5p , mir-128 , mir-96 , mir-433 , mir-383 and mir-138 .
the second network , including mir-217 and mir-216 , which are upregulated in medulloblastoma , exhibits associations with key regulatory genes in medulloblastoma development , including tp53 , sirt1 and ybx1 .
sirt1 is a well understood member of the sirtuin protein class , preventing cell aging and apoptosis of normal cells ( 24,25 ) .
the effects of sirt1 are controversial ; the protein was considered to be a tumor promoter in neuroblastoma , prostate and skin cancer ( 2628 ) , whereas it was considered to be a tumor suppressor in breast and colon cancer ( 29,30 ) .
therefore , sirt1 may serve a role in medulloblastoma and correlate with the formation and prognosis of human medulloblastomas ( 31 ) .
recently , dey et al ( 32 ) proved that ybx1 ( yb1 ) is upregulated across human medulloblastoma subclasses and is required for medulloblastoma cell proliferation .
furthermore , the study identified that the shh : yap : yb1:igf2 axis may be a powerful target for therapeutic intervention in medulloblastoma . when the medulloblastoma samples were compared with the normal cerebellum , 9 dysregulated mirnas were identified .
the 9 mirnas were uploaded to the ipa in order to perform the core analysis .
the network involved 4 downregulated mirnas and was associated with important regulators of tumorigenesis , including met proto - oncogene ( met ) , akt serine - threonine kinase 1 and sterol regulatory element - binding factor 1 ( srebf1 ) .
previous evidence has demonstrated the association between aberrant met signaling and medulloblastoma development ( 33,34 ) .
previous studies have revealed that overexpressed mir-206 may inhibit met in lung cancer ( 35,36 ) , however the function of downregulated mir-206 in medulloblastoma is unknown .
srebf1 is a transcription factor , regulating lipogenesis , which is also involved in tumorigenesis ( 37 ) .
these network components alone are not sufficient to initiate medulloblastoma formation ; the interactions between these dysregulated factors and additional genetic insults promote tumorigenesis . in conclusion , based on the mirna array data in the geo database , mirnas which are differentially expressed in medulloblastoma samples were identified .
certain dysregulated mirnas have been confirmed ; however , further research is required to verify the mirnas which have not been previously identified .
additionally , the ipa core analysis , presenting the interaction of mirnas and counterpart targets , provides a method to understand the tumorigenesis of medulloblastoma .
the results of the present study may provide a potent target for therapeutic intervention or diagnosis in medulloblastomas .
|
medulloblastoma is the most common malignant brain tumor of the central nervous system among children .
medulloblastoma is an embryonal tumor , of which little is known about the pathogenesis .
several efforts have been made to understand the molecular aspects of its tumorigenic pathways ; however , these are poorly understood .
microrna ( mirna ) , a type of non - coding short rna , has been proven to be associated with a number of physiological processes and pathological processes of serious diseases , including brain tumors .
differentially expressed mirnas serve an important role in numerous types of malignancy .
the present study aims to define a differentially expressed set of mirnas in medulloblastoma tumor tissue , compared with normal samples , to improve the understanding of the tumorigenesis .
it was identified that 22 mirnas were upregulated and 26 mirnas were downregulated in the tumor tissue compared with the normal group . however , when the medulloblastoma tissue was compared with normal cerebellum tissue , 9 mirnas were identified to be up or downregulated in the tumor samples .
the differentially expressed mirnas in the tumor tissue were identified in order to clarify the networks and pathways of tumorigenesis using ingenuity pathway analysis .
subsequently , key regulatory genes associated with the development of medulloblastoma were identified , including tumor protein p53 , insulin like growth factor 1 receptor , argonaute 2 , mitogen - activated protein kinases 1 and 3 , sirtuin 1 and y box binding protein 1 .
|
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kindler syndrome ( ks ) is a rare autosomal recessive genodermatosis , which was first described in a 14-year - old girl in 1954 by kindler and later by forman et al . in 1989 .
more than 120 cases have been reported since the original report by kindler ; the largest series being a cluster of 26 patients identified within a tribe in the bocas del toro province on the northwestern caribbean coast of panama .
the syndrome is a combination of features of inherited blistering skin disorders ( e.g. , dystrophic epidermolysis bullosa ) and congenital poikilodermas ( e.g. , rothmund - thompson syndrome ) and should be differentiated due to clinical overlap with hereditary acrokeratotic poikiloderma ( hap ) and dystrophic epidermolysis bullosa .
apart from the skin changes , changes in the oral and conjunctival mucosa , phimosis and radiological changes , namely a dome - shaped skull ( turricephaly ) , rib and mandibular abnormalities have been reported .
other features that vary between cases include acral hyperkeratosis , nail dystrophy , webbing and contractures of the fingers and toes , alopecia , actinic changes , pigmentation of lips and onchodystrophy .
the association of aggressive periodontitis with ks was based on a single case in 1996 and later confirmed with a larger population of patients .
a number of oral features have been described , such as gingival swelling , advanced periodontal bone loss , mild - to - severe gingivitis , dental caries , and leukokeratosis of buccal mucosa .
dental findings had been briefly reported for kindler patients in dermatologic and pediatric publications including oral lesions , atrophy of buccal mucosa , limited oral opening , malocclusion , dystrophic teeth , ankyloglossia , bleeding gums , lip erosions and geographic tongue , atrophy of gingiva , erosion of the hard palate , gingival swelling and desquamative gingivitis .
a 16-year - old female patient presented with the chief complaint of bleeding gums , ulcerations of buccal mucosa , missing teeth and difficulty in swallowing .
patient 's mother reported that she had two children ; one was healthy physically and mentally . both of her pregnancies and deliveries were normal , but history of the affected child revealed skin blisters beginning at the age of 3 months .
these blisters were filled with clear fluid and left scars after their rupture , and occurred until the age of 1 year .
cutaneous examination revealed multiple hypopigmented and a few hyperpigmented macules of variable sizes , distributed over his face , neck , trunk , and limbs [ figure 1 ] .
skin over the hands and neck was dry , atrophic and photosensitive to the sunlight .
areas of hyper- and hypo - pigmentation on face absence of palmar creases hyperkeratotic plaques on the flexures scarring was evident on the soft tissues of the buccal mucosa and tongue .
tongue showed reduced mobility and was quite hard due to fibrosis [ figure 4 ] .
in certain areas , erythema extended to mucogingival junction ; resulting in appearance of desquamative gingivitis [ figure 5 ] .
desquamative gingivitis restricted mouth opening due to fibrosed commissures orthopantomograph showed areas of moderate bone loss around all the teeth present .
multiple congenitally missing teeth , and retained deciduous teeth were reported [ figure 7 ] .
orthopantomograph showing retained deciduous and congenitally missing permanent teeth histopathologic examination of atrophic skin lesions in patients with ks reveals nonspecific features of poikiloderma .
the epidermis is flattened and atrophic ; edema is present at the dermoepidermal junction , and the basal layer shows focal vacuolization with basal cell degeneration .
other histologic features include a prominence of dermal capillaries , pigmentary incontinence , and possibly , perivascular lymphocytic infiltrate .
a 16-year - old female patient presented with the chief complaint of bleeding gums , ulcerations of buccal mucosa , missing teeth and difficulty in swallowing .
patient 's mother reported that she had two children ; one was healthy physically and mentally . both of her pregnancies and deliveries were normal , but history of the affected child revealed skin blisters beginning at the age of 3 months .
these blisters were filled with clear fluid and left scars after their rupture , and occurred until the age of 1 year .
cutaneous examination revealed multiple hypopigmented and a few hyperpigmented macules of variable sizes , distributed over his face , neck , trunk , and limbs [ figure 1 ] .
skin over the hands and neck was dry , atrophic and photosensitive to the sunlight .
areas of hyper- and hypo - pigmentation on face absence of palmar creases hyperkeratotic plaques on the flexures
tongue showed reduced mobility and was quite hard due to fibrosis [ figure 4 ] .
deep pockets with marked bleeding on minimal probing were present around the teeth . in certain areas , erythema extended to mucogingival junction ; resulting in appearance of desquamative gingivitis [ figure 5 ] .
multiple congenitally missing teeth , and retained deciduous teeth were reported [ figure 7 ] .
histopathologic examination of atrophic skin lesions in patients with ks reveals nonspecific features of poikiloderma .
the epidermis is flattened and atrophic ; edema is present at the dermoepidermal junction , and the basal layer shows focal vacuolization with basal cell degeneration .
other histologic features include a prominence of dermal capillaries , pigmentary incontinence , and possibly , perivascular lymphocytic infiltrate .
in 2003 , siegel et al . mapped the disease locus to band 20p12.3 by using linkage and homozygosity analysis in an isolated cohort of patients with ks .
loss - of - function mutations were identified in the candidate gene flj20116 , which was renamed kindlin-1 ( kind1 ) .
this gene encodes a 677-amino acid protein , kind1 , which is thought to play a regulatory role in inhibiting oversecretion of basement membrane components by basal keratinocytes at the dermoepidermal junction .
an autosomal recessive pattern of transmission is usual , but sporadic cases are not uncommon . in ks , there is an unusual interruption and reduplication of the basement membrane and a broad reticular pattern of type vii collagen staining deep into the connective tissue beneath the basement membrane . it has also been suggested that ks and weary 's hap are variants of the same disease . however , the mode of inheritance , onset of blistering , photosensitivity and presence of eczema is different in these two syndromes .
the level of ultrastructural cleavage for blistering appears to be junctional in ks and intraepidermal in hap .
the dominant cutaneous findings in ks are increased skin fragility , acral blistering , photosensitivity , atrophy , and poikiloderma .
although increased skin fragility may be explained by the weakening of basal keratinocyte - extracellular matrix adhesion , the pathomechanisms of other features such as photosensitivity and skin atrophy remain unclear .
mucosal involvement is very common and may lead to urethral , anal and esophageal stenosis . our patient presented with acral blisters in the neonatal period and childhood , diffuse poikiloderma , skin fragility and atrophic changes , which were more prominent on skin exposed areas .
a set of clinical diagnostic criteria has recently been proposed for this condition to facilitate clinical diagnosis .
the major criteria are acral blistering in infancy and childhood , progressive poikiloderma , skin atrophy , photosensitivity , and gingival fragility , and/or swelling .
the additional features of these criteria are nail dystrophy , ectropion , palmoplantar keratoderma , leukokeratosis of lips , squamous cell carcinoma , anhidrosis , skeletal abnormalities , and dental problems . according to the proposed criteria ,
the presence of four major criteria makes the diagnosis certain , the presence of three major and two minor criteria makes the diagnosis probable , and diagnosis is considered to be likely if two major and two minor / additional features are present . according to these criteria a definitive diagnosis of ks was made .
several conditions that can cause blistering , cutaneous atrophy , and/or poikilodermatous skin changes must be differentiated from ks . ks might be difficult to differentiate from variants of epidermolysis bullosa in newborns .
progressive improvement of blistering , photosensitivity , poikilodermatous changes , and cutaneous atrophy with age help to differentiate ks from epidermolysis bullosa . in dystrophic epidermolysis bullosa
, there is a mutation in the gene encoding type vii0 collagen ( cola7a0 ) distinguishing it from ks .
rothmund - thomson syndrome shows poikiloderma and photosensitivity like ks , but additional features like sparse hair , hypogonadism , and cataracts in the former condition distinguish it from ks .
bloom 's syndrome is characterized by telangiectasia and photosensitivity with the presence of erythema on the face and other sun - exposed areas without showing true poikiloderma .
short stature , recurrent infections , and increased frequency of hematological malignancies are also features of this disease .
patients with cockayne 's syndrome develop erythema in photo - distributed areas , atrophy , and hyperpigmentation .
the associated features of dwarfism , cachexia , progressive pigmentary retinopathy , deafness , and birdlike faces differentiate cockayne 's syndrome from ks .
reticulated hyperpigmentation , nail dystrophy , and leukoplakia are characteristic features of dyskeratosis congenita . unlike ks ,
the pigmentary changes are not truly poikilodermatous and bullae are not an important feature of this rare genodermatosis .
good wound care including the use of topical and systemic antibiotics for infected bullous lesions and ulcerations might reduce the morbidity .
patients usually have a normal life span , but significant morbidity may be caused by secondary infections of congenital blisters ; mucosal involvement leading to urethral , anal and esophageal stenosis ; aggressive periodontitis , and ocular complications .
|
kindler syndrome is a rare hereditary disorder , associated with skin fragility .
the syndrome involves the skin and mucous membrane with radiological changes .
the genetic defect has been identified on the short arm of chromosome 20 .
this report describes a 16-year - old patient with classical features like blistering and photosensitivity in childhood and the subsequent development of poikiloderma .
|
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myelodysplastic syndromes ( mdss ) represent a group of clonal hematological disorders characterized by progressive cytopenia reflecting defects in erythroid , myeloid and megakaryocytic maturation .
anemia , neutropenia and thrombocytopenia , separated or in combination , despite a hyper- or normocellular marrow define mds .
hypoplastic mds ( h - mds ) accounts for 1217% of all patients with mds .
clonality studies of mature blood cells and immature progenitors suggest that a myeloid lymphoid stem cell might be the principal target for clonal transformation at least in a major part of mds .
an association between mds and autoimmune disorders and with chronic t - cell disorders like large granular lymphocytic leukemia ( lgl ) has been recognized and is suggestive of an abnormal functioning immune system in mds .
lymphoproliferative disorders , especially t - prolymphocytic leukemia ( pll ) developing in an h - mds patient , is extremely rare and has an aggressive course .
we are reporting a unique case of t - pll developing in h - mds , and this is probably the first case report in the literature .
a 45-year - old male was admitted with generalized weakness , palpitation , gradual weight loss and frequent syncopal attacks for more than 2 years . on examination ,
the iron profile was : serum iron 115.50 g / dl , total iron binding capacity
177.10 ng / ml and markedly elevated b12 levels ( > 2000 pg / ml ) .
hemoglobin was 3.9 gm% , total leukocyte count was 2100 cells / cumm , platelet count was 70,000 cells / cumm and reticulocyte count was 0.8% at the time of admission .
pancytopenia [ figure 1a ] was persistent on every visit and the peripheral smear showed normocytic and macrocytic rbcs , leukopenia , hypogranular and pseudo pelger - huet neutrophils [ figure 1b and 1c ] , which were characteristic with relative lymphocytosis and no blasts in the peripheral smear ; the platelet count was reduced .
the bone marrow aspiration smears and cell block preparation showed severe hypocellularity ( < 20% for his age ) [ figure 1d ] .
sparse erythroid cells with dyserythropoiesis [ figure 2a and 2b ] , minimal dysmyelopoietic ( hypogranular and hyposegmented myeloid cells - 10% ) and dysmegakaryopoietic ( hypolobated and micromegakaryocytes [ figure 2c and 2d ] features were evident with blasts < 5% and absence of fibrosis in biopsy sections .
the possibility of aplastic anemia and hypoplastic acute myeloid leukaemia ( h - aml ) were ruled out .
a diagnosis of primary h - mds was made in the absence of chemotherapy and radiotherapy .
the patient was transfusion dependent and later started on thalidomide . on follow - up within few months , because of persistent pancytopenia , the patient was subjected for repeat bone marrow study , where the smear and section showed cellular marrow with diffuse infiltrate of mature lymphocytes ( 60% ) [ figure 3a and b ] with no evidence of improved hematopoiesis .
the neoplastic cells showed a mature t - cell immunophenotype , cd3 + , cd4 + , cd5 + , cd7 + and cd8 + , and tdt- , cd20- , cd23- , cd79a- , cd34- , bcl2- and cyclind1- , and was diagnosed as t - pll .
( d ) hypocellular marrow - cell block preparation h and e , 10 ( a ) dyserythropoiesis with multinucleation in bma .
( c ) dysmegakaryopoiesis - cell block h and e , 40. ( d ) dysmegakaryopoiesis bma ( a ) lymphoid infiltrate in bma .
the diagnosis may be difficult in h - mds if the aspirate is so sparsely cellular as to preclude cytogenetic studies .
significant clonal cytogenetic abnormalities are present in only half of all mds cases , and full karyotype analysis , which requires cells in metaphase , may not be possible because cells may be scarce or senescent .
we encountered similar problems when there was hypocellular marrow , but later , when diagnosed as t - pll , the marrow culture showed a normal karyotype .
there is growing scientific evidence supporting the hypothesis that cytogenetic abnormalities are not the initiating clonal event but are acquired during disease progression .
h - mds has yet to be shown to alter the disease course or prognosis , but some studies suggest benefit from immunosuppressive therapeutics .
it is presently unknown whether h - mds patients have a preceding , but unrecognised , phase of excessive apoptosis insufficiently compensated by hyperproliferation , or whether their disease is more related to aplastic anemia .
several years ago , in vitro studies have proven that autologous cytotoxic t - lymphocytes can exert an inhibitory effect on mds myelopoiesis .
an association between mds and autoimmune disorders and with chronic t - cell disorders like lgl has been recognized and is suggestive of an abnormal functioning immune system in mds .
but , occurrence of t - pll in h - mds is extremely rare and , possibly , this is the first case in the literature .
the immunophenotype is that of mature t - cell , which is usually cd4 + but , occasionally , can be cd8 + , which was evident in our case .
expression of cd7 helps to distinguish it from other clpds , like sezary syndrome and adult t - cell leukemia / lymphoma .
to conclude , the finding of activated and clonal t - cell population in mds is further argument for the use of immunosuppression to treat the cytopenias of mds .
|
myelodysplastic syndrome ( mds ) represents one of the most challenging health - related problems in the elderly , characterized by dysplastic morphology in the bone marrow in association with ineffective hematopoiesis .
hypoplastic mds ( h - mds ) accounts for 1217% of all patients with mds and has yet to be shown to alter the disease course or prognosis .
the concept that t - cell - mediated autoimmunity contributes to bone marrow failure in mds has been widely accepted due to hematologic improvement after immunosuppressive therapy .
t - cell expansion is known to occur in these patients , but development of chronic t - cell disorders , especially t - prolymphocytic leukemia ( pll ) in a hypocellular mds is extremely rare , which has an aggressive course .
the possible explanation for the association between the two disorders is that t - pll might arise from a clonally arranged mds stem cell .
we report a unique case of h - mds with non - progressive pancytopenia and severe hypocellular marrow for 2 years , followed by t - pll within few months .
|
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bacteria of the genus actinomyces are gram - positive bacilli which generally colonize mouth , colon and vagina .
, if actinomycosis occurs , it often presents as an indolent , slowly progressive infection characterized by abscess formation , which can progress across tissue boundaries , can present with mass like features which simulate malignancy , can develop into a sinus tract and/or develop clinically into a refractory or relapsing infection after a short course of therapy .
a 43-year old woman with no significant medical history presented with complaints of an unilateral slowly growing painful mass in the left breast .
mammography ( fig . 1 ) and ultrasonography were conducted and showed a small abscess - like density retroareolarly.fig .
. 1 mammography of the left breast showing a confined density in the retroareolar area .
species identification was performed using matrix - assisted laser desorption ionization - time - of - flight mass spectrometry ( maldi - tof ms ; bruker ) .
susceptibility testing for a. neuii was performed using e - tests ( biomerieux ) . at the time that culture findings became available ,
therefore , the cultured actinomyces was considered as skin commensals and a wait and see policy was installed .
after several months , the patient returned to the outward clinic with complaints of pain in the breast .
psychical examination of the breast showed mild erythema and ultrasonography showed a minimal increase of the lesion .
the abscess was surgically drained and new purulent fluid from the abscess was sent to the laboratory . the material was processed as described above .
in contrast to initial culture findings , determination by the maldi - tof ms showed peptostreptococcus magnus ( score value 2.056 ) . after four days there was also growth observed in the brewer thioglycollate medium .
the isolate was shown to be susceptible to penicillin , amoxicillin , clindamycin , meropenem and vancomycin .
histopathological examination of the obtained tissue showed abscess formation in an infection with a sinus tract .
subsequently , the patient was treated for six weeks of intravenous penicillin followed by six months of oral amoxicillin successfully .
secondly , a 73-year old woman with a history of bilateral breast cancer was seen at with severe pain and swelling of the left breast ten days after her ablation which was complicated by a large hamatoma treated conservatively .
gram strain showed many leukocytes , some gram - positive cocci and many gram - positive rods .
determination using maldi - tof ms identified a staphylococcus aureus after 2 days there were colonies visible on the anaerobically incubated schaedler agar .
determination by the maldi - tof ms revealed an a. neuii susceptibility testing was performed using e - tests .
unfortunately , patient returned four days after discharge with a relapse of the hematoma which was surgically evacuated . a month later at the outward clinic she showed full recovery.fig .
other isolates that have been identified as pathogens causing primary breast actinomycosis include actinomyces viscosus , actinomyces turincensis and actinomyces radingae
.
it differs from the other actinomyces species because of its aerobic growth and its microscopic morphology without branching .
we were able to retrieve only three former cases of a. neuii causing primary actinomycosis .
in the first case , treatment with antibacterials was unsuccessful and the lesion had to be surgically removed . in the second case ,
patient was successfully treated with amoxicillin for 21 days . in the third case ,
actinomycosis , regardless of species , should be treated with high doses of antibacterials for a prolonged period of time although therapy needs to be individualized .
current guidelines recommend high dose of penicillin intravenously for two to six weeks , followed by oral therapy with penicillin or amoxicillin for 6 to 12 months .
alternative antibacterial treatments include doxycycline , erythromycin or clindamycin . if actinomycosis presents with well - defined abscess then surgical drainage followed by long - term antibacterial therapy is indicated .
we describe this combination of treatment in our cases . in the first case initial cultures showed islolated a. neuii . because signs of infection had decreased this finding was interpreted as skin flora and not as a pathogen . therefore medical treatment was considered unnecessary .
in retrospect , if antibacterial therapy had been given at first presentation recurrence of the infection might have been prevented .
actinomyces species morphologically resemble diphtheroids and could be dismissed as skin commensals , even when isolated from an abscess sample . in both of our presented cases we found different organisms in the culture additional to a. neuii . in
the first case we found p. magnus and in the second case s. aureus was present .
although a. neuii infection is known for its chronicity , the influence of these mixed organisms on the severity of infections remains unclear .
further research on the effect of mixed organism on the severity of an a. neuii infection is required .
|
actinomycosis is a slowly progressive infection caused by anaerobic bacteria , primarily from the genus actinomyces .
primary actinomycosis of the breast is rare and presents as a mass like density which can mimic malignancy .
mammography , ultrasonography and histopathologic examination is required for diagnosis .
treatment should consist of high doses of antibacterials for a prolonged period of time and possibly surgical drainage .
primary actinomycosis infections are commonly caused by a. israelii .
actinomyces neuii is a less common cause of classical actinomycosis .
we present two cases of primary actinomycosis of the breast in two female patients caused by a. neuii .
|
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indeed , while their short - term effects for acute exposure are well known [ 1 , 2 ] and some fatal cases [ 25 ] have been reported , chronicle exposures to these xenobiotics are not studied yet , even if toxic effects ( abortion and liver damage ) are suspected in people involved in these jobs .
in addition , scientific research is leading to the development of protocols in which the use of anesthetic mixtures reduces their concentration ; on the other hand , these require more sensitivity and selective analytical methods for their determination .
inhalation anesthesia is indeed a widely used method for inducing complete anesthesia in surgeries . several collateral or secondary effects due to acute exposure to inhalatory anesthetics
are reported in the literature [ 1 , 2 , 6 ] , while little information has been collected on chronicle exposure to small amounts of these drugs .
nowadays , the most representative class of anesthetics used is perfluorinated molecules such as desflurane and sevoflurane .
these drugs are very often employed in synergy with nitrogen dioxide and sometimes with other anesthetics in order to reduce doses and anesthetic induction times .
the surgical operators exposure to the drugs is today drastically reduced due to the use of efficient air recirculation and refreshing systems in the surgical theatres and the development of anesthesia protocols .
nevertheless , symptoms such as drowsiness , reduction of concentration , and lack of clarity are sometimes still reported from operators , requiring a more effective control on working areas in order to reduce risks directly or indirectly connected to the inhalatory anesthetics use . in order to establish
if an effective amount of anesthetic is inhaled from operators , a very sensitive technique is required to determine the concentration of anesthetic in surgical theatres since a relatively small xenobiotics concentration is required to improve the working conditions .
while first approaches to determine anesthetics were based on hexane extraction [ 7 , 8 ] , more recently , gas chromatography ( gc ) equipped with different kinds of detectors has been used to identify these drugs [ 914 ] .
the most recent method relies on rapid determination of sevoflurane and hexafluoroisopropanol ( free , unconjugated form ) based on direct injection of human plasma into gc / ms apparatus without any sample clean - up procedures .
indeed , gc / ms technique is nowadays a very common analysis tool and its field of application can be extended to moderately polar to very polar molecules by appropriate derivatization / modification procedures [ 16 , 17 ] . in our work ,
a very sensitive method based on solid phase microextraction gas chromatography mass spectrometry for determination of anesthetics in urine has been developed .
the analysis results were also evaluated in the light of independent environmental anesthetics determinations obtained through a complementary technique based on the photo - acoustic detection principle . in particular , sevoflurane , its metabolite hexafluoroisopropyl alcohol , and desflurane
were taken into consideration ( figure 1 ) , using halothane as internal standard and matrix matched calibration curves .
this approach took into consideration the matrix effect , which also , in our case , proved to be relevant especially when spme was used .
due to its high sensitivity , rapidity , and the simultaneous determination of different anesthetics in urine samples , the present method represents a further development in comparison with the literature [ 1921 ] .
water ( hplc grade ) , methanol ( hplc grade ) , -glucuronidase from bovine liver ( 1,000,000 units / g solid ) , and sodium chloride were purchased from sigma - aldrich ( supelco , bellafonte ) .
spme experiments were carried out using three kinds of coated fibers : 100 m pdms , 65 m pdms / car , and 50/30 m dvb / car / pdms .
sevoflurane ( 99,9% ) and desflurane ( 99,8% ) were purchased from baxter ( deerfield , il ) ; halothane ( 99,5% ) and hexafluoroisopropyl alcohol ( 99% ) were purchased from sigma - aldrich ( supelco , bellafonte ) .
standard solutions preparation was made according to this procedure : 20 l aliquots of sevoflurane , desflurane , and hexafluoroisopropanol ( hfip ) were weighed and dissolved in methanol in 100 ml flasks . due to their high volatility , for all analytes ( sevoflurane , boiling point : 331.75 k at 101.3 kpa and density 1.5 10 kg / m at 293 k ; desflurane , boiling point : 296.65 k at 101.3 kpa and density 1.5 10 kg / m at 293 k ; hfip , boiling point : 332 k at 101.3 kpa and density 1.6 10 kg / m at 293 k ) , pure standards volumes were picked through cold ( 255 k ) tips and were rapidly weighed into volumetric flasks containing 10 ml of cold methanol .
these solutions were then diluted with a further amount of cold methanol ( 255 k ) up to the mark ( 100 ml ) at ambient temperature ( 298 k ) .
the solutions obtained had a final concentration of 302 g ml for desflurane and sevoflurane and 320 g ml for hfip . these stock solutions , stored in refrigerator at 277 k 2 k , were checked weekly and were found to be stable in these conditions up to three months .
an aqueous mother solution containing all the analytes at a concentration of 250 g l has been prepared using these standard solutions and it has been found stable for no more than a month .
further diluted solutions , used to make the matrix matched calibration curves , were prepared weekly by simple dilution of the mother solution in hplc grade water . internal standard solution was prepared picking 20 l of halothane standard ( density 1,86 10
kg / m at 293 k ) through cold ( 255 k ) tips and rapidly weighing into volumetric flasks containing a small amount of cold methanol ( about 10 ml at 293 k ) .
the obtained solution was then further diluted up to the mark ( 100 ml ) with methanol at ambient temperature ( 298 k ) .
the solution obtained has a final concentration of 380 g ml and was stored in refrigerator at 277 k 2 k. this solution was checked weekly and it was found to be stable in these conditions up to three months .
analyses were carried out using focus gc thermo equipped with a capillary column supel q - plot ( 30 m 0.32 mm i.d .
15 m average thickness ) , coupled with a dsq ii mass spectrometer , working in selected ions monitoring ( sim ) mode .
the analysis method was optimized in order to reach the maximum efficacy in terms of rapidity , selectivity , and sensitivity .
the best trade - off obtained leads to a total analysis time of 13 minutes ( see figure 2 ) .
operating conditions were the following : injector temperature , 473 k ; splitless ( gas flow 1 ml min , splitless flow for 1 min with a gas flow 10 ml min ) ; constant flow 1 ml min ; and carrier gas , helium .
the thermal program starts at 309 k and holds for 3 min and then the temperature increases at 40 k min up to 403 k and holds for 7 minutes at this temperature . according to the literature and the experimental data obtained from pure standards full scan ms spectra , the most representative mass peaks have been chosen ( see table 1 ) .
these ions were used in sim mode in order to increase sensitivity , with respect to full scan ms .
the rationale fragmentation processes that could explain these peaks are briefly reported in figure 2 .
the following procedure was applied for the real samples preparation : 18 ml sealed vials were prepared with 1 ml of enzyme solution ( -glucuronidase 999 ui / ml ) , 10 l of acetic acid , 40 l of internal standard solution , and 2.0 10 kg of nacl .
3950 l of surgery 's operator urine was added by means of gastight syringe into the previously prepared vials . in order to avoid loss in
is concentration , the urine volumes were placed into the vials , without unscrewing the cap and piercing the septum with the syringe 's needle .
after having placed the vials at 310 k for 16 h to obtain the enzymatic hydrolysis of the glucuronate - hfip , the samples were analyzed .
matrix matched calibration samples were prepared weekly according to the following procedure : 1 ml of enzyme solution ( -glucuronidase 999 ui / ml ) , 10 l of acetic acid in order to generate the optimal conditions ( ph 4 - 5 ) for the enzyme activation , 40 l of internal standard solution , 1 ml of standard solution containing the mix of anesthetics at increasing concentration , and an aliquot of urine ( pooled 50% from female and 50% from male voluntary healthy subjects ) up to final volume of 5 ml were added in a 18 ml vial , sealed with ptfe / silicon septum ; finally , in order to get a salting - out effect , 2.0 10 kg of nacl was added . for the sake of strict repeatability
, standards were subjected to 16 hours of incubation at 310 k ( normally necessary for the enzymatic hydrolysis of the glucuronate - hfip ) before analysis .
the final optimal conditions were the following : fiber dvb / car / pdms ; equilibration time ( t1 ) : 30 minutes ( 298 k 1 k ) ; extraction time ( t2 ) : 10 minutes ( 298 k 1 k ) .
the best fiber desorption conditions were the following : injector at 473 k , desorption time ( t3 ) of 1 minute ( splitless mode ) .
anesthetic concentrations in different selected operating rooms were measured by multi - gas monitor type 1302 ( brel & kjr , denmark ) , whose design was based on the photo - acoustic detection principle .
this instrument is capable of continuously recording the gaseous concentrations of fluorinated anesthetics within the surgery room .
more than 25 real samples were collected from operators working in three different surgical rooms .
analyses were carried out in duplicate on pre- and post shift urine samples of both the operators coming in contact with the anesthetics during their shift ( i.e. , surgeons or anesthetists ) and the ones that are not usually in contact with xenobiotics during shift ( i.e. , medical attendants ) .
solid phase microextraction is a sample preparation method , invented by pawliszyn in 1989 , which does not require solvents . since its first applications to environmental and food analysis
, its use is becoming wider and frequent , due to its environmentally friendly features , effectiveness , and reduced costs .
the principle of spme is based on a fused - silica fiber that is coated with an appropriate stationary phase .
all analytes in the sample are directly extracted to the fibre coating , in a single step that involve both extraction and concentration from the headspace of the vial . because of the high volatility of the compounds , optimizing the efficiency and sensitivity of the analysis method is necessary to identify the most suitable spme sampling conditions .
therefore , three different spme fibers ( pdms ; pdms / car ; dvb / car / pdms ) have been tested .
the analyte peak areas , obtained from analyses of standard solutions at the same concentration of anesthetics , were compared and the higher efficiency of extraction of all the analytes was reached using the dvb / car / pdms fiber ( according also to poli et al . , 1999 ) .
the second step was the determination of the optimal saturation time , needed for the analytes to reach equilibrium between the matrix , the headspace , and the fiber .
the headspace sampling was performed at the same temperature ( 298 k 1 k ) for all the analytes and optimal saturation time was determined ; the analyte peak areas after 15 , 30 , and 60 minutes of equilibration were compared , showing that 30 minutes were enough to reach equilibrium in headspace . finally , in order to improve the extraction efficiency , various extraction times were evaluated .
the comparison between the responses obtained from extraction times of 5 , 10 , 20 , and 30 minutes , at the same temperature ( 298 k 1 k ) , indicates that after 10 minutes the extraction efficiency reached a plateau .
no further raise in analyte peak areas resulted from increase of the fiber exposition time in hs .
a preliminary calibration curve was made in water in order to achieve the order of magnitude of both limit of detection ( lod ) and limit of quantification ( loq ) and to verify the linearity range of the whole method without any interference due to matrix .
these provided a very strong linear correlation ( r > 0.99 ) in a range from 0.32 g
l to 40 g l. gc / ms chromatogram of 5 g l fortified ( a ) and blank ( b ) urine are reported in figure 5 to show analytes separation and method sensitivity in matrix .
the effect of the matrix has been evaluated in order to verify its eventual impact on linearity , sensitivity , and slope of the calibration curves using matrix matched calibration curves to determine effects of the matrix at different levels of concentration .
in addition , in our experience , the intrinsic low reproducibility of precise experimental conditions ( i.e. , intrinsic sample differences , fiber wearing , etc . ) often requires an internal standard to effectively reduce errors . when a matrix effect is observed , as in this case , both a matrix matched calibration curve and an internal standard should be used in order to reduce errors and improve the accuracy of the measures . as shown in figure 6 ,
a marked matrix effect is observed when determining the same anesthetic concentration in water and urine .
indeed , very different ( sample / internal standard ) area ratios are observed looking at calibration curves obtained in distilled water ( inserts ) and calibration curves made using pooled urine as matrix .
this matrix effect markedly lowers the calibration curve slopes ( and consequently the sensitivity ) with respect to calibration curves obtained in pure water .
this defeats any attempt to use an external calibration and requires a matrix effect correction . however , matrix matched calibration curves repeated using different urine pools at distance of one to several weeks demonstrate good reproducibility , with similar slopes and intercepts .
this demonstrates that matrix effect is not peculiar for each sample and does not require more complicated and time consuming multiple standard addition procedures in order to get reliable measures .
the linearity of the method using matrix matched standards remains fair , even if it has been ascertained in a range of concentration that is smaller ( from 0.30 g
l to 22.5 g l for sevoflurane and desflurane and in the range 0.32 g
l to 24 g l for hfip ) than the corresponding linearity obtained in water solution ( from 0.06 g
l to 37.5 g l for sevoflurane and desflurane and in the range 0.6 g l to 40 g l for hfip ; see figure 6 ) standards .
the three curves obtained were weighed on 1/x in order to enhance the curve fitting at a lower concentration and show a very good coefficient of correlation r ( see table 2 ) . also lod and loq have been calculated according to this procedure : average and standard deviation values of at least 20 blanks have been reckoned and lod and loq values are defined as blanks signal average plus 3 and 10 times the standard deviation of the blanks , respectively .
the following performances studies were , moreover , carried out : within - laboratory repeatability ( wlr ) and within - laboratory reproducibility ( wlr ) expressed as % rsd on sample at 5.0 g l ( see table 2 ) .
the extended performances reached through this method were useful to evaluate the anesthetic exposure effects on a small number of operators working in surgical theaters . in the selected theaters , a parallel evaluation of the environmental instantaneous levels of anesthetic
was also conducted in order to evaluate eventual relationships between the environmental pollution and the anesthetics biological levels .
the observed correlation was indeed scarce and no correlation was found between the exposition time and the anesthetics biological levels .
actually , it emerged that , during the surgical operations , the staff exposition time to anesthetics is largely variable and not linked to the working hours .
another exposition factor such as the distance between the vaporizer and each individual is variable as well .
the samples collected from operators working in three different surgical theaters were used to determine desflurane levels , while those collected from the other two surgical theaters were employed to determine the sevoflurane levels and its metabolite hfip in urine .
it is worth noting that , in all cases , the anesthetics levels determined after shift were higher than the corresponding levels in pre - shift samples . in particular , high levels of desflurane anesthetic were determined in people working in emergency b surgical theaters . from anamnesis , it was ascertained that several people mostly working in this theatre reported symptoms such as headache , drowsiness , asthenia , and concentration diseases , while the same diseases were rarely found in people working in the other theaters .
urine levels can be taken as a clue of the air condition system efficiency being mean levels of anesthetics well grouped for each surgical theatre ; see figure 7 . it is also interesting to note that levels before and after shift were in the g l order , mostly corresponding to the lods of the previously reported methods [ 19 , 20 ] .
this implies that , in most cases , up to now , only few positive cases could be detected , of subject being exposed to anesthetics . from our determinations , instead
, it seems that , even if in different extents , exposition to anesthetics involves all the personnel working in the theatres .
indeed , post - shift urine mean levels were always significantly higher than pre - shift ones .
the developed analytical method proved to be very quick , sensitive , and robust ( precision , calculated as rsd , was always below 13% for all intra- and interday determinations ) .
it represents a further development in comparison with the literature being the first method for the quantification of desflurane in biological fluids .
indeed , this method exhibits lod and loq values at least 2 magnitude orders lower than those previously reported [ 1921 ] for similar volatile anaesthetics .
this is probably due to the synergism of a well - optimized spme procedure and gc / sim - ms analysis .
the preliminary data obtained by this pilot study on surgical theatres stressed the effectiveness of this method as a useful tool to monitor the exposition to anaesthetics in occupationally exposed people .
indeed , with this method , it has been possible to ascertain significant difference between the anesthetics levels in urine before and after the work shifts in 25 individuals .
further , significant differences in post - shift urine levels have been found in personnel working in different surgical theaters .
this means that different working conditions and probably differences in efficiency of the environmental treatment effectively reflect on personnel exposition to these xenobiotics .
|
in this work , a new sensitive analytical method has been developed and evaluated for the determination of the most commonly used gaseous anesthetics , desflurane , sevoflurane , and this latter 's hepatic metabolite hexafluoroisopropanol ( hfip ) in the urine .
in addition , an evaluation of anesthetics exposition on the urine levels of a small population of surgical operators has been performed and results are briefly discussed .
|
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in addition to being a crucial underlying circumstance in the primary cardiovascular risk factors ( cvrfs ) , obesity is an independent risk factor for cardiovascular illness and mortality [ 1 , 2 ] . increased body mass index ( bmi ) alters the behavior of adipose tissue , which provides insulin resistance as well as resistance to type-2 diabetes , arterial hypertension , dyslipidemia , and proinflammatory and prothrombotic states , thereby favoring the onset of ischemic cardiopathy .
an estimated 42.3% of coronary episodes in the spanish population may be attributable to excess weight after adjusting for age , sex , and other risk factors
. it would therefore be logical to expect obesity to have a lethal effect on patients who have suffered a coronary event .
however , some studies have reported better short- and medium - term prognoses in overweight coronary patients [ 5 , 6 ] .
this situation , in which obesity seems to protect patients with acute coronary syndrome ( acs ) , has been called the obesity paradox and has been described in other facilities .
other authors have obtained conflicting results and question the existence of this protective effect [ 7 , 8 ] . in light of this controversy
, the goal of this study is to determine the relationship between bmi and intrahospital mortality in patients admitted consecutively for acs .
all patients admitted consecutively between 2009 and 2010 for acs were included in the renaci database of the working group on ischemic heart disease and coronary care units of the spanish cardiology society .
a total of 853 patients were initially admitted during the period studied with a discharge diagnosis of unstable angina or myocardial infarction .
therefore , the final sample consisted of 824 patients . the demographic characteristics and base anthropometry were recorded for all the patients studied .
the patients were divided into three groups : normal ( bmi < 25 kg / m ) , overweight ( bmi = 2530 kg / m ) , and obese ( bmi > 30
we analyzed various clinical variables including the classic cvrfs ( smoking , diabetes , hypertension , and dyslipidemia ) , cardiovascular background and treatment prior to the acute event , hemodynamic variables at admission , risk scores according to the timi and grace scales , electrocardiographic data , analytical parameters and noninvasive cardiologic examination parameters , treatment administered at admission , coronarography examination , and results , as well as percutaneous and surgical revascularization .
all data were analyzed with the one - sample kolmogorov - smirnov test to evaluate the normality of their distribution .
continuous variables were expressed as the average and standard deviation and were compared using an unpaired student 's t - test . the one - way anova test
was used to compare more than two groups , and the bonferroni test was used to detect differences between groups .
percentages were compared using chi - squared statistics and fisher 's exact test when any of the expected values in any of the cells were below 10 .
univariate and multivariate logistic regression analyses were used to determine the variables associated with hospital mortality .
the average age of the sample was 65.84 + 0.4 years ( ranging from 2595 ) , and the sample was predominantly male ( 73.5% ) .
bmi had an inverse relation to mortality , that is , to say , higher bmis were associated with a reduced mortality rate . in the multivariate logistic regression analysis ( table 1 ) ,
bmi along with other clinical variables ( age , diabetes mellitus , grace score , and cardiogenic shock ) were the independent predictors of death in our sample .
table 2 shows the hospital admission data for the sample , and table 3 shows not only the distribution of subjects in the normal weight , overweight , and obese categories but also the main clinical variables ( risk factors and relevant background factors ) .
the incidence of risk factors is high in this population ( 95.8% ) . in the obese group ,
the prevalence of diabetes mellitus and chronic obstructive pulmonary disease is significantly higher than in the other two groups .
a larger proportion of the obese group had previously undergone treatment with angiotensin ii receptor antagonists and diuretics although there were no other differences in prior treatments . at admission ,
overweight patients presented higher blood pressure levels , and lower killip classes and timi scores , as well as higher initial glycemia and cholesterol levels .
a coronariography was conducted in 76% of the patients in our sample , and no differences were found between obese , overweight , or normal weight subjects .
no differences were found among the three groups in the percentage of angioplasties conducted or in the medical treatment received during admission .
the prevalence of complications such as angina pectoris , reinfarction , hemorrhage , and mechanical complications was similar across groups with the exception of stroke , which was lower in obese patients .
thirty - five patients died ( 4.2% ) with no statistically significant differences among the different bmi groups .
most deaths occurred within 48 hours of having been admitted ( 19 patients ) , followed by 7 patients between day 2 and day 7 , and a further 9 cases after day 7 .
no statistical differences were found in the combined endpoint ( mortality , reinfarction , bleeding , and cerebrovascular accident ) .
our study shows that bmi is an independent predictor of hospital mortality in that a higher bmi is associated with a lower mortality rate .
this finding is correlated with a lower mortality rate in obese and overweight patients compared to patients in the normal range although this result is not statistically significant . unlike other authors [ 5 , 9 ] ,
we did not find pronounced differences among these groups in terms of clinical data , hemodynamics , electrocardiographs , analyses , echocardiographs , angiographs , treatments administered , or resulting complications except that obese subjects have a greater incidence of diabetes , cardiovascular history , chronic obstructive pulmonary disease , and lower killip classes and timi scores .
this data suggests that evolution would be less favorable and the mortality rate higher during hospital stays .
although bmi is an inverse predictor of mortality in our series , indicating that overweight patients with acs have better survival rates than normal weight subjects , when subjects are classified into bmi subgroups ( normal weight , overweight , and obese ) , we were unable to demonstrate with statistical significance that patients classified as obese have lower hospital mortality although this tendency does exist . among other reasons , this may be due to the sample size .
our data is consistent with other data published , among which are the results of the synergy , merlin - timi 36 , and crusade studies , which also describe lower mortality rates in obese patients suffering from acs . in a meta - analysis of 40 studies with more than 250,000 patients , romero - corral et al
. observed that overweight patients with coronary disease have a lower risk of cardiovascular and total mortality than patients in the low and normal weight groups .
however , this tendency disappears in the bmi 35 kg / m patient group ( morbid obesity ) , which runs a greater risk of cardiovascular mortality .
analyzed the prognostic value of bmi in medium - term hospital mortality in a cohort of 1,063 consecutive patients with first infarction in 15 hospitals in spain and found no association between bmi and medium - term hospital mortality .
furthermore , in patients with acute st - elevated myocardial infarction , das et al . described a less - favorable prognosis for very obese patients in a sample of 501,489 patients .
several hypotheses have been proposed to explain the inverse relationship between obesity and the prognosis of patients with ischemic cardiopathy .
the higher mortality rate in the normal weight group may be due to a higher mortality rate in underweight patients .
some studies [ 1 , 2 ] show that the optimal bmi for the general population is 22.525
kg / m in smokers , and that people in the lower range of normal bmi ( 18.522.5 kg / m ) have a higher mortality rate than individuals in the upper range of overweight bmi ( 27.530 kg / m ) .
so the high - risk , underweight group is subsumed within the group of patients that is often considered normal . in contrast , most studies have found that a higher mortality rate is associated with extreme obesity [ 8 , 9 , 11 ] .
another aspect that is often not given appropriate consideration is the role of pharmacological treatment , especially the adverse effects of multiple medicines used in acs ( fibrinolytic agents , antiplatelet drugs , anticoagulants , inotropes , antiarrhythmic agents , diuretics , nitrates , beta blockers , etc . ) , which could explain a higher mortality rate .
given the difficulty of anthropometric measurement in the first hours of cardiologic emergency treatment , errors in medicinal dosage may arise , primarily in those patients with lower body weight .
nevertheless , unlike other authors , in our series we found that bmi group had no effect on either treatments administered or hemorrhages .
since bmi can not differentiate between muscle and fat mass , overweight and obese subjects with coronary disease may have more muscle mass .
when bmi is very high and better reflects body adiposity , the obesity paradox disappears .
the main approaches used to measure obesity are bmi , waist - hip ratio , and waist circumference .
there is considerable disagreement on which of them is best [ 3 , 1315 ] .
most authors recommend the simultaneous use of all of these parameters to better assess patient risk .
although the worst scenario is the obese individual with a high waist circumference , patients categorized as normal with a high waist circumference also face an elevated risk .
other authors suggest that obesity should be expressed as the percentage of body fat ( > 25 for men and > 35 for women ) , but this is difficult to quantify clinically .
correctly identifying the different compartments of body fat , and specifically visceral fat , using more sophisticated techniques [ 16 , 17 ] may help to clarify the role of obesity in acs patient mortality .
studies have also been conducted which measure the degree of activity or physical condition and classify the subgroups normal weight - sedentary , overweight active , obese active , overweight sedentary , and obese sedentary from least to greatest risk , with obese - sedentary being the group at greatest risk [ 18 , 19 ] . although the sample consisted of more than 800 patients , classifying the patients into three groups may prevent hospital mortality from being appropriately assessed because its incidence is relatively low .
the clinical appraisal of obesity has many limitations , and an alternative might be the combined appraisal of other anthropometric measurements such as waist circumference , which was an unknown variable in our sample .
better anthropometric appraisal using data on abdominal obesity and an understanding of the physical condition of the patients so that they can be more accurately classified into groups could play a critical role in clarifying this paradox .
the relationship between higher bmi and a greater incidence of coronary disease in the general population is well documented , and excess weight should clearly be avoided . however ,
once coronary heart disease arises , the association between bmi and the prognosis becomes more complex , even paradoxical according to some authors .
so the controversy over the predictive value of bmi in patients with acs remains latent .
our study confirms that bmi is an independent predictor of hospital mortality ; a higher bmi is associated with a lower mortality rate .
our overweight and obese patients had a higher incidence of diabetes , cardiovascular history , chronic obstructive pulmonary disease , and lower killip classes and timi scores .
however , they did not show an increased mortality rate , which is apparently paradoxical .
|
although obesity is a well - established cardiovascular risk factor , some controversy has arisen with regard to its effect on hospital mortality in patients admitted for acute coronary syndrome .
methods .
clinical and anthropometric variables were analyzed in patients consecutively admitted for acute coronary syndrome to a university hospital between 2009 and 2010 , and the correlation of those variables with hospital mortality was examined . results .
a total of 824 patients with a diagnosis of myocardial infarction or unstable angina were analyzed .
body mass index was an independent factor in hospital mortality ( odds ratio 0.739 ( ic 95% : 0.597 0.916 ) , p = 0.006 ) .
mortality in normal weight ( n = 218 ) , overweight ( n = 399 ) , and obese ( n = 172 ) subjects was 6.1% , 3.1% , and 4.1% , respectively , with no statistically significant differences between the groups .
conclusions .
there is something of a paradox in the relationship between body mass index and hospital mortality in patients with acute coronary syndrome in that the mortality rate decreases as body mass index increases .
however , no statistically significant differences have been found in normal weight , overweight , or obese subjects .
|
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in early november 2001 , a 57-year - old female laboratory worker ( laboratory worker 1 ) began experiencing nonspecific symptoms of malaise , vomiting , headache , lower leg cramping , anorexia , and fever .
one week after onset of symptoms , she was evaluated for severe headaches at a local emergency room , where cerebrospinal fluid ( csf ) and blood cultures were collected .
small , gram - positive bacilli were isolated and characterized as coryneform bacilli , which are usually interpreted as contaminants of unknown clinical importance . despite multiple hospital admissions ,
approximately 5 weeks after symptom onset , colleagues from the hospital microbiology laboratory where she was employed ( laboratory 1 ) drew her blood for culture again . after 5 days of incubation , gram - variable coccobacilli ,
the brucella serum agglutination test ( sat ) was reactive ( 1:640 ) at the nysdoh laboratory , wadsworth center .
laboratory worker 1 was initially treated with doxycycline and gentamicin , followed by doxycycline and rifampin , for 6 weeks of outpatient therapy .
the isolate was later identified as b. melitensis by the wadsworth center and confirmed by the centers for disease control and prevention .
the patient has not relapsed 18 months after completing treatment . in a second incident in mid - january 2002 ,
a 48-year - old woman had nocturnal temperature spikes to 40c , chills , drenching sweats , and weight loss .
symptoms persisted , and uveitis developed . in early march 2002 , a diffuse , erythematous rash appeared on the anterior aspect of both legs .
a blood culture and serologic tests for lyme disease , ehrlichiosis , and rocky mountain spotted fever ( rmsf ) were performed . from the blood culture ,
gram - positive cocci were isolated and identified as micrococcus spp . by a commercial laboratory .
rmsf titers were immunoglobulin ( ig ) m - negative with a reactive igg of 1:256 .
subsequently , she was referred to an infectious disease specialist , who found repeat rmsf titers unchanged , which made acute rmsf unlikely .
when interviewed by nysdoh staff , the patient reported that she was a laboratory worker ( laboratory worker 2 ) at laboratory 2 .
her initial blood culture specimen , which had originally been identified as micrococcus , was reassessed by the commercial laboratory .
the commercial laboratory referred the original isolate to the wadsworth center , where the isolate was identified as b. melitensis .
. they denied traveling outside of the united states , consuming imported or domestic unpasteurized dairy products , knowing ill family or friends who may have traveled , attending events with potentially contaminated foods , or handling farm or laboratory animals .
both laboratory workers denied any accidental contamination or spills in the laboratory during the 6 months before their respective illnesses .
site visits to laboratories 1 and 2 were conducted by nysdoh . on the open bench at laboratory 1
a syringe was used to directly plate contents of the blood culture media bottle onto agar .
subsequent biochemical tests , including a catalase test , which may generate aerosols by introducing hydrogen peroxide to the specimen , were performed on the open laboratory bench . at laboratory 2 , subculturing occurred in a similar manner , but it took place in a class ii biosafety cabinet .
however , biochemical tests were performed on the open laboratory bench when gram stain results indicated that spores were not present .
laboratory workers 1 and 2 wore gloves when processing specimens , and both denied having dermatitis or skin lesions on their hands .
after the diagnosis of brucellosis in these two laboratory workers , serum samples from their co - workers from laboratories 1 and 2 were tested by brucella sat ; samples from seven of eight co - workers were nonreactive ( < 1:20 ) .
a co - worker of laboratory worker 1 had an initial agglutination titer of 1:40 ( indeterminate ) and 1 month later had a repeat titer of < 1:20 ; she denied having symptoms . to determine the source of these laboratory workers ' infections , each laboratory reviewed gram stains of blood culture specimens processed within 3 to 6 months of symptom onset .
laboratory worker 2 reviewed prior gram stain slides from laboratory 2 and identified one slide containing a questionable coryneform bacillus .
this blood culture had been drawn from laboratory worker 1 during her visit to the emergency room of laboratory worker 2 's hospital , early in november 2001 .
approximately 2 months before her illness , laboratory worker 2 had personally processed laboratory worker 1 's blood culture but had characterized the isolate as coryneform bacilli .
wadsworth center staff reviewed the original gram stain from laboratory worker 1 's blood culture specimen and noted gram - variable organisms with similar size and shape to brucella organisms .
however , laboratory worker 2 did not identify any other gram stains that resembled brucella organisms during the period before her illness . the likely source of laboratory worker 1 's infection
was found after reassessing gram stains performed in laboratory 1 in august , september , and october 2001 .
a 76-year - old woman visited the emergency room served by laboratory 1 in early september 2001 .
the patient had had fever ( temperature 38.3c ) for 1 day and headache for 3 weeks .
she received one dose of ciprofloxacin for a possible urinary tract infection and was discharged .
after 4 days of incubation , a gram stain of this patient 's blood culture specimen showed tiny , gram - negative coccobacilli , reported as coryneform bacilli .
as part of the investigation for the source of infection for laboratory worker 1 , this patient 's prepared gram stain slide was referred to the wadsworth center , where small , gram - variable cocci were identified . more than 1 year after being seen in the emergency room , this patient was offered brucella sat and a repeat blood culture .
she refused brucella sat , and her physician did not identify symptoms consistent with brucellosis .
six months after this identification , further experimental analysis by the wadsworth center with polymerase chain reaction tests performed on gram stain material detected b. melitensis dna ( wadsworth center , nysdoh , unpub .
the patient was again contacted , and brucella sat ( > 1.5 years after her initial blood culture ) showed a titer of 1:80 ( indeterminate ) .
, the patient denied any visits outside the united states since 1989 , when she emigrated from peru .
although brucellosis is a rare disease in the united states , its potential use as a bioweapon highlights the need for accurate and rapid identification ( 15 ) . in this investigation
, brucellosis was diagnosed weeks to years after initial positive blood cultures were misidentified , and laboratory personnel were unknowingly exposed to the organism .
this investigation suggests that transmission occurred from the 76-year - old index patient to laboratory worker 1 , on the basis of b. melitensis dna found in the gram stain material .
processing the index patient 's blood culture specimen on an open laboratory bench was most likely the reason for laboratory worker 1 's illness approximately 5 weeks later .
the same mechanism of transmission probably occurred when laboratory worker 2 handled laboratory worker 1 's blood culture specimen on the open bench for biochemical testing . in these instances ,
blood culture bottle media were transferred to slides and agar without much risk for aerosolization , since contents were manipulated with a syringe .
biochemical tests , however , included the catalase test , which creates bubbles as a result of exposure to 3% hydrogen peroxide in positive specimens .
neither laboratory worker could identify any other possible sources of infection , and because brucellosis is a rare disease in new york , the connection between these three patients is plausible . with the initial interpretations of these gram stains as gram - variable , which resulted in misidentification of the organism by three different laboratories , nysdoh initiated an effort to educate clinical laboratories in new york state about the potential difficulties in characterizing the organism and the importance of primary gram stain interpretation .
additional investigation into the staining properties of brucella spp . under various conditions is now in progress and may help differentiate brucella spp . from other organisms .
because of immigration and foreign travel , brucellosis remains an occupational hazard for laboratory personnel , even in industrialized countries where animal control efforts have virtually eliminated the disease . because of the nonspecific symptoms and the rarity of the disease in the united states , healthcare providers may not consider brucellosis in a differential diagnosis .
. moreover , improved communication among healthcare providers and laboratory personnel should facilitate prompt and accurate identification and appropriate handling of the organism .
its potential use as a bioweapon necessitates that healthcare providers , as well as microbiologists in hospitals and commercial laboratories , be knowledgeable about the diagnosis , identification , and handling of brucella spp .
reporting brucellosis cases to public health officials is another component in protecting others from this disease .
public health officials should notify laboratory personnel who may have handled cultures taken from patients ultimately diagnosed with brucellosis .
early notification of exposed personnel could lead to their timely diagnosis and treatment , should symptoms occur , and could prevent further laboratory exposures .
|
we report two laboratory - acquired brucella melitensis infections that were shown to be epidemiologically related .
blood culture isolates were initially misidentified because of variable gram stain results , which led to misdiagnoses and subsequent laboratory exposures .
notifying laboratory personnel who unknowingly processed cultures from brucellosis patients is an important preventive measure .
|
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chikungunya virus ( chikv ) is an arthropod - borne virus belonging to the family , togaviridae , and genus , alphavirus . in this group of 29 viruses , six cause arthralgia and arthritis upon infection in humans .
these six viruses are chikv , ross river virus ( rrv ) , semliki forest virus ( sfv ) , onyong - nyong virus ( onnv ) , sindbis virus ( sinv ) and mayaro virus ( mayv ) .
the name chikungunya means that which bends up and refers to the contorted posture adopted by infected patients .
symptomatic chikv infections are characterised by fever , rashes and arthralgia leading to arthritis . in the last 50 years
there have been frequent outbreaks of chikv disease in asia and africa . in 2005 - 2006
an outbreak that occurred in la runion island resulted in approximately 260,000 cases of chikv associated fever with 237 deaths reported2 .
additionally , in 2006 - 2007 around 1.5 million cases of chikv disease were reported in india3 . through viral adaptation chikv has broadened its vector competence and therefore increased its potential to cause human disease .
a single mutation , a226e in the envelope protein 1 ( e1 ) of chikv , now allows aedes albopictus as well as ae .
currently there are no vaccines or antivirals for the prevention or treatment of chikv infections .
treatment is symptomatic with analgesics , antipyretics and non - steroidal anti - inflammatory agents .
passive immunization with human iggs derived from chikv infected patients has been shown to be protective in mice against chikv challenge5 .
this approach may prove effective for the prevention and treatment of neonates who are at the risk of infection from viraemic mothers .
however , this is not cost - effective and it is critical to develop antiviral drugs and/or vaccines for the control of chikv infections .
owing to the recent outbreaks of chikv in asia , africa and la runion island there has been a renewed interest in the development of antiviral drugs and vaccines to manage chikv infections . in this article
, we report on the advancements made towards the development of anti - inflammatory agents , antivirals and vaccines for the efficient control of chikv infections .
chloroquine phosphate was reported to be effective in the treatment of chronic chikv arthritis6 . however , in a clinical trial of chloroquine in chikv infected patients , no difference was observed between the placebo group and the treatment group7 .
maheshwari et al8 have also reported that in a mouse model , chloroquine enhances alphavirus replication and aggravates disease pathogenesis .
ribavirin , an antiviral agent was used to treat chikv induced arthritis in a small cohort of patients and was found to be beneficial in resolving joint and soft tissue swelling9 .
briolant et al10 have reported a synergistic effect of ribavirin and interferon- in the inhibition of chikv and sfv replication in cell culture .
recently rulli et al11 have reported using bindarit , a small molecule anti - inflammatory drug , in a mouse model to effectively treat chikv induced arthritis .
an additional 356 compounds ( natural and clinically approved drugs ) were initially screened for their antiviral effects using a chikv replicon cell line13 . a recombinant chikv with rluc ( renilla luciferase ) fused with nonstructural protein 3 ( chikv - rluc ) was constructed and used to validate the most active compounds from the initial screening studies . out of 12 selected compounds , coumarin 30
was found to be the most potent in inhibiting the replication of chikv - rluc virus ( ic50 value of 6.4 m ) .
arbidol , licensed for the treatment of influenza and other respiratory infections , was found to be effective in inhibiting chikv replication in vero and mrc-5 cell lines ( ic50<10g / ml)14 .
recently , screening of a highly purified natural compound library for inhibition of chikv replication identified 44 inhibitors15 . among the selected compounds , harringtone , a cephalotoxine alkaloid ,
was found to be a potent inhibitor of chikv infection with an ec50 value of 0.24 m .
was also found to inhibit sinv replication suggesting the drug may be effective against other alphaviruses . in another study , an optimal homology model of chikv nonstructural protein 2(nsp2 )
based on the active site of nsp2 model , a virtual screening was performed with commercially available compounds subsequently 26 compounds were evaluated for the anti - chikv activity in a cell culture assay16 .
levitt et al17 described the development of an attenuated strain of chikv ( 181/25 ) obtained by serial passage in mrc-5 cells .
this vaccine candidate was tested in both mice and non - human primates and was found to protect the animals against challenge with parent virus . however , in phase ii trials the vaccine caused mild transient arthralgia in some of vaccinees18 .
recent attempts to develop a chikv vaccine have used formalin inactivated vaccine19 , virus like particles ( vlps)20 and dna vaccines2122 .
immunization with vlps and dna vaccines resulted in immunogenicity and protected the mice and non - human primates against subsequent challenge with chikv .
chimaeric viruses containing venezeulean equine encephalitis virus ( veev ) or eastern equine encephalitis virus ( eeev ) or sinv non - structural proteins with chikv structural proteins elicited neutralizing antibodies and protected the mice against chikv challenge23 . however , these viruses still had the ability to infect mosquitoes and attenuation was dependent on an intact interferon response in mice .
another chimaeric vaccine using veev strain tc-83 and encepahalomyocarditis virus ( emcv ) ires ( internal ribosome entry site ) in the subgenomic promoter was developed to reduce the transmission to mosquitoes .
recently chattopadhyay et al25 developed a chimaeric vaccine using a vesiculostomatitis virus ( vsv ) backbone and chikv structural proteins ( vsvg - chikv ) .
this vsvg - chikv chimaeric virus induced a good neutralizing antibody response and protected mice against chikv infection .
plante et al24 designed a vaccine by introducing the emcv ires sequence into the chikv subgenomic promoter .
this reduced the replication of the vaccine strain in mosquitoes and was highly immunogenic in mice .
sirnas and shrnas against envelope protein e1 , nsp3 , capsid and nsp1 proteins of the chikv have been used to inhibit the replication of chikv in mammalian cells with promising results2627 .
furthermore , in vivo studies have shown that shrna e1 offers a strong and sustained protection against chikv infection in suckling mice .
studies on the role of mannose binding lectin ( mbl ) in rrv - induced arthritis in mice28 suggest that mbl may be a target for therapeutic intervention in the treatment of alphavirus induced arthritis / myositis .
inhibitors targeting the mbl pathway of complement may be useful in the treatment of alphavirus induced diseases . additionally , a peptide corresponding to the autophagy inducing peptide , beclin , has been shown to be effective against chikv infections in cell culture and in mice29 .
a recent study on gene profiling has revealed a significant overlap of differentially expressed genes involved in the inflammatory processes of both chikv- induced arthritis and rheumatoid arthritis ( ra ) .
thus , new drugs being developed for ra might also be beneficial for the treatment of alphavirus induced arthritis30 .
chikungunya fever is a global health problem and several outbreaks have occurred in the last 50 years in asia and africa .
recent outbreaks in la runion and italy have raised the awareness of the need to develop effective antiviral drugs or vaccines against chikv .
although many antiviral compounds have been shown to be effective in cell culture , very few compounds have been evaluated in animal models .
intensive studies are needed to evaluate the effect of appropriate compounds in animal models and humans .
|
chikungunya virus , a re - emerging mosquito - borne alphavirus , causes fever , rash and persistent arthralgia / arthritis in humans .
severe outbreaks have occurred resulting in infections of millions of people in southeast asia and africa .
currently there are no antiviral drugs or vaccines for prevention and treatment of chikungunya infections .
herein we report the current status of research on antiviral drugs and vaccines for chikungunya virus infections .
|
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] |
thousands of children and adolescents across the united states suffer from musculoskeletal conditions each year [ 1 , 2 ] .
common musculoskeletal conditions in children include cerebral palsy ( cp ) , congenital muscular torticollis ( cmt ) , duchenne muscular dystrophy , idiopathic clubfoot , idiopathic toe walking ( itw ) , legg - calv - perthes disease ( lcpd ) , limb length discrepancy , and neonatal brachial plexus palsy ( nbpp ) .
musculoskeletal abnormalities and deformities can deprive children of physical activities , childhood experiences , and a healthy lifestyle . besides the physical and psychosocial burden these conditions and injuries place on the child and family , these conditions also incur a financial burden for the patient and the healthcare system as multiple hospital visits are often required .
proper and timely treatment including standard approaches such as physiotherapy , casting , bracing , and surgery is essential to ensure the child optimal growth and development . besides these traditional modalities
, the use of botulinum toxins appears as a promising treatment on its own , as an adjunct to other treatment modalities and as an alternative to surgery .
several authors have suggested the use of btx - a in children with musculoskeletal conditions , yet the evidence supporting its safety and effectiveness is not well established .
the use of btx - a in children with cp has been widely documented [ 35 ] and is beyond the scope of this paper .
the objectives of this systematic review were to establish the evidence on the effectiveness , safety , and functional outcome of btx - a in children with musculoskeletal conditions .
botulinum toxin is an extremely potent , naturally occurring poison resulting from the fermentation of the anaerobic spore - forming bacterium clostridium botulinum .
these toxins cause flaccid paralysis by blocking acetylcholine release , which is required for muscle contraction at the neuromuscular junction .
thus , the toxins have the capacity to reduce muscular activity in a dose - dependent manner .
muscle weakness occurs within a few days to one week after local injection , peaks within two weeks for several weeks , and then plateaus in milder form ( the desired clinical effect ) before gradually returning to baseline .
recovery from the toxin - induced paralysis begins with resprouting of axon terminals and slow recovery of the neurons ability to release acetylcholine , which results in nerve conduction to be reestablished .
clinical observations suggest that these neurotoxins have three mechanisms of action : paralytic , antisecretory , and analgesic ( antinociceptive ) .
a number of studies suggest that several pathways play a role in the analgesic effects of botulinum toxins , such as in conditions of pathologic muscle overactivity ( dystonia and spasticity ) and in the role of the calcitonin gene - related peptide in the afferent signaling of pain .
the fda has approved a total - body dose of 400 units administered every 12 to 16 weeks or at longer intervals to avoid toxicity . identifying the appropriate muscle sites for injection
is done through palpation , electrical stimulation , electromyography , ultrasound , fluoroscopy , and computerized tomography depending on the muscle size and location . a needle between 23 and 27 gauge
is selected based on muscle depth , difficulty palpating the muscle , and use of electromyography .
there are seven different serotypes of the neurotoxin , named botulinum toxin ( btx ) types a to g. although they all inhibit acetylcholine release from nerve terminals , they differ according to their intracellular protein targets , potency , dosing , and duration of effect .
btx - a is the serotype that has been most studied in terms of therapeutic application .
btx - b and btx - f have also been used in clinical practice , but are less potent than btx - a , and have a shorter duration of action .
btx - b has also been shown to have regional and systemic anticholinergic adverse side effects , which limits its clinical use .
there are currently three types a and one type b brand of botulinum toxins available in the us market . in 2010 , the us food and drug administration ( fda ) announced generic names for all of the versions of injectable botulinum toxins .
this change in terminology is expected to differentiate between these different brands and provide each brand with its own identity thereby improving its clinical use and reducing errors and misinterpretation .
hence , onabotulinumtoxina ( botox by allergan inc in the united states ) , abobotulinumtoxina ( dysport by ipsen in france ) , incobotulinumtoxina ( xeomin by merz pharmaceuticals gmbh in germany ) , and rimabotulinumtoxinb ( myobloc / neurobloc by solstice neurosciences in the united states ) are the new four generic names used in the usa .
btx - a was first approved by the fda in 1989 for the treatment of strabismus and blepharospasm ( two eye muscle disorders ) , making it the first botulinum toxin type a product approved in the world . in the usa ,
btx - a is also approved to treat cervical dystonia and severe primary axillary hyperhidrosis in adults .
recently , the fda has also approved the use of btx - a to treat increased muscle stiffness in the elbow , wrist , and finger muscles in adults with upper limb spasticity .
in addition to its therapeutic uses , the same formulation of btx - a was approved by the fda in 2002 under the trade name botox cosmetic to improve the look of facial wrinkles in adults less than 65 years of age .
although the use of btx - a has not yet been approved for use in children , it has been used in a variety of clinical conditions both for its neuromuscular and analgesic effects due to its safe , predictable , and reversible effects on motor weakness .
these off - label indications include cmt , cp , idiopathic clubfoot , itw , lcpd , lower limb lengthening and nbpp .
btx - a is used in these children to decrease spasticity , manage postoperative pain , and improve quality of life . the most common use of btx - a in children is for the treatment of spasticity in cp .
studies have shown that btx - a decreases muscle tone in children with upper and lower limbs spasticity associated with cp and can help prevent the development of muscle contractures and bony deformities , as well as improve upper limb movement and function [ 1517 ] .
patient selection , btx - a dosing , dilution and administration , identification of muscle groups , and outcome measurement must be carefully considered [ 13 , 16 ] .
adverse events following botulinum toxin injection have been found to be mild , temporary , as well as dose and site related .
these may include local reactions , such as bruising and pain at the site of injection , excessive localized muscle weakness , and incontinence .
systemic effects are very rare and may include flu - like symptoms , headaches , light - headedness , fever , chills , hypertension , diarrhea and abdominal pain , generalized weakness , dysphagia , dry mouth , and subsequent aspiration .
these are far less common , are generally short - lived , and may result from the systemic spread of the toxin to adjacent muscles . a boxed warning label describing the spread of the toxin and
its potentially life - threatening effects appears on the labels following the death of a child following btx - a injections .
however , until today , no causal relationship confirmed the evidence relating this specific adverse event to the toxins .
caution should be exerted when injecting btx - a in children with preexisting swallowing or respiratory problems .
the incidence of antibody - mediated resistance in long - term treated patients ranges from 3 to 23% , depending on the patient sample , treatment regimen and toxin preparation .
a recent australian study prospectively documented the presence of adverse events in 334 children with cp in the month before and in the month after btx - a injection .
they found that the children had significant morbidities prior to the injection , adverse events were present in 23.2% of children , and no deaths occurred .
cmt is common and refers to unilateral contracture of the sternocleidomastoid muscle that restricts the infant 's range of motion at the neck .
infants with cmt display head tilt toward the shortened side , which is often combined with rotation of the head to the opposite side [ 20 , 21 ] .
it is reported to occur in 1 infant in every 250300 live births [ 21 , 22 ] .
manual stretching is still the most common form of treatment , and about 90% of cmt resolves with stretching exercises . when conservative treatment is ineffective , surgery is considered .
however , as an alternative to invasive surgical intervention , btx - a may be an option to increase the effectiveness of stretching on the side of the contracture and allow strengthening of overstretched and weakened muscles on the opposite side of the neck [ 12 , 21 , 22 ] .
it is a progressive x - linked recessive disorder and is caused by a defective gene for dystrophin affecting approximately 1 out of every 3,600 male infants .
the proximal muscles of the lower extremities are affected first , with decreased range of motion , flexion contracture of the hip and knee , and extension contracture of the ankle .
symptoms usually appear before age 6 and typically include fatigue , muscle weakness , and progressive difficulty in walking .
braces may improve mobility and self - care function , and a wheelchair is often used by age 12 .
there is no known cure for duchenne muscular dystrophy and by the late teens or twenties the condition is severe enough to shorten life expectancy .
treatment aims to control symptoms to maximize quality of life and maintain muscle strength and function .
btx - a may be indicated to decrease muscle contractures of the lower extremities and facilitate standing and mobility . in this congenital deformity ,
clubfoot is one of the most common birth defects , occurring in 13 per 1000 live births .
a child with an untreated clubfoot will walk on the outer edge of the foot instead of the sole , develop painful callosities , be unable to wear shoes , and have painful feet that often limit activity .
nonsurgical modalities include serial manipulation and casting , such as ponseti 's technique [ 24 , 25 ] , as well as taping , physical therapy , and continuous passive motion .
surgery is indicated if satisfactory clinical and radiographic correction by nonsurgical methods is not obtained .
btx - a injection into the child 's calf muscle is indicated to facilitate nonsurgical techniques , to supplement surgical release and to serve as an alternative to achilles tendon lengthening by reducing the tone in the most contracted muscles [ 12 , 14 ] .
this condition is present in children older than 3 years of age still walking on their toes without any neurological , orthopaedic , or psychiatric diseases .
treatment recommendations include physical therapy , casting , bracing , surgical release , and achilles tendon lengthening .
btx - a may be useful to manage the contractures in cases in which toe walking recurs despite conservative and surgical treatment .
lcpd is a degenerative disease of the hip joint affecting about 10.8 children of 100,000 children and is more common in boys .
it is characterized by an interruption of the blood supply of the head of the femur with loss of bone mass and joint deformity at the hip .
the disease is typically found in young children , and it can lead to osteoarthritis in adults .
common symptoms include hip , knee , or groin pain , exacerbated by hip / leg movement , reduced range of motion at the hip and painful and limping gait .
physical activity such as standing , walking , running , and kneeling may cause severe irritation or inflammation of the damaged area .
the goal of treatment is to prevent deformity of the femoral head and avoid severe degenerative arthritis later on .
btx - a may be indicated to weaken selected muscles to restore muscle balance at the hip joint .
children undergoing lower limb lengthening using an external fixator exhibit excellent results in most cases , yet the postoperative pain can be significant and often requires prolonged use of analgesics and even narcotics [ 28 , 29 ] .
other aspects of the limb lengthening , such as the osteotomy and incision sites , gradual distraction increasing soft tissue tension , range of motion exercises , and gait training result in ongoing pain for weeks after the application .
appropriate pain management is essential for optimal quality of life and functional outcomes in children undergoing limb lengthening .
btx - a has been shown to reduce postoperative pain in children secondary to reduced muscle spasm and may be indicated in children undergoing lower limb lengthening to alleviate spasm and pain during the lengthening process .
nbpp is defined as a flaccid paresis of the upper extremity secondary to unwanted muscular cocontraction or inappropriate activation of antagonist muscles due to increased forces of distraction to the neck during delivery .
associated injuries may include fractures to the clavicle and humerus , facial nerve palsy , and torticollis .
incidence varies between 0.38 and 3 per 1000 live births in industrialized countries and occurs more frequently in infants born over 4 kg , breech deliveries , maternal diabetes , and vacuum / forceps extraction .
while early physical therapy yields complete return to function in many infants , infants whose elbow flexion and shoulder abduction have not recovered before 6 months are indicated for surgical reconstruction of the brachial plexus .
a novel approach for children with nbpp injuries is the therapeutic use of btx - a to inhibit unwanted cocontractions and activate antagonist muscles .
early treatment of these children with btx - a injections may also result in stronger , normal muscles and may prevent the development of glenoid dysplasia [ 12 , 31 ] .
a literature search using the electronic databases medline , pubmed , cochrane , trip , and web of science for published articles in english from 1980 to september 2011 was conducted using botulinum toxin , congenital muscular torticollis , duchenne muscular dystrophy , idiopathic clubfoot , idiopathic toe walking , legg - calv - perthes disease , lower limb lengthening , neonatal brachial plexus palsy , and relevant search terms . to be included , a study had to be written in english and to include children between 0 to 21 years of age with one of the above - mentioned musculoskeletal conditions .
research studies that were included in this review were classified as levels i to iv , based on the american academy of neurology ( aan ) levels of evidence .
data was independently extracted by two reviewers ( n. dahan - oliel and b. kasaai ) .
the majority of studies were on children with nbpp ( n = 10 ) , followed by idiopathic clubfoot ( n = 5 ) , cmt ( n = 4 ) , itw ( n = 3 ) , duchenne muscular dystrophy ( n = 1 ) , and lower limb lengthening ( n = 1 ) . no studies that looked at the effect of btx - a in children with lcpd were found .
two of these studies [ 32 , 33 ] included both children and adults but were included as separate results for children were provided . in total
, outcome was provided for forty - five children aged 3 months to 18 years following btx - a injections .
the following muscles were injected in isolation or in combination : sternocleidomastoid , trapezius , splenius , and scalenus .
two studies reported mild dysphagia , neck weakness , neck bruising and soreness , and brief fever following injection in five children [ 34 , 35 ] .
there were variations in btx - a dosage , number of injections per muscle , length of follow - up , and outcomes assessed between studies as well as within the same study , which limited comparability of findings .
it is difficult to attribute clinical improvements to the btx - a treatment since the children in these studies often received a combination of both btx - a injections and physical therapy .
furthermore , none of these studies compared btx - a treatment with either traditional treatment or surgical treatment .
one study on 15 infants who had a significant risk of progressing to surgery because of severe torticollis found promising results .
indeed , only the oldest infant required surgical release following btx - a injections , while the other 14 infants showed significantly improved neck rom and did not require surgery .
these findings suggest that btx - a may be a safe and effective treatment modality when traditional treatments ( home program , physical therapy ) do not yield acceptable results .
btx - a treatment may obviate surgical interventions in certain cases , but randomized controlled trials with larger samples are needed to confirm this finding .
although it seems that the initial injections of btx - a should be administered at a young age , the exact age is not yet established .
an 11-year - old boy was injected with btx - a for tightness in the left knee flexors in order to enable standing exercises .
knee range of motion was increased by 20 degrees following injection , with no side - effects .
btx - a may be indicated in children with duchenne muscular dystrophy when temporary improvement of range of motion is needed to minimize knee contracture and encourage exercises for muscle stretching , prevention of osteoporosis , and retaining lung function .
the first study by delgado and colleagues included four patients who had severe clubfoot deformities and rapidly reached a plateau following physical therapy .
btx - a injections helped most of the patients , despite their severe deformity , allowing two of them to avoid surgery .
the remaining two patients had a demyelinating neuropathy and did not respond to btx - a treatment .
another study by mitchell and colleagues reported on three children under the age of 13 months who had a recurrence of their deformity after surgery and found a marked improvement of the deformity following btx - a injection and application of molded plaster casts .
the largest study by alvarez and colleagues [ 37 , 38 ] included 51 children .
following ponseti - type manipulations , casting , and btx - a injection , all but one child improved in terms of dorsiflexion and a decrease in the severity of the deformity was noted .
bracing was provided to maintain the correction . at the five - year follow - up visit , 48% ( 31 of 65 clubfeet ) successfully responded to a single btx - a injection and experienced no recurrence over the follow - up period .
at least one repeat btx - a injection was required in 34 clubfeet , and surgery was required in 10 clubfeet .
these four studies found a positive effect of btx - a as an adjunct to manipulation , casting , and physical therapy to correct muscle imbalance and to correct recurrent deformity in idiopathic clubfoot .
all five studies concluded that btx - a may be an effective and safe treatment alternative and can decrease the number of patients requiring surgery .
a limitation of these four studies is that they did not include a control group and that the results can not be attributed solely to btx - a as other treatment modalities were used . cummings and colleagues conducted a randomized double - blind controlled trial on 20 infants ( 32 clubfeet ) comparing a single btx - a injection to placebo following serial manipulation and casting according to the ponseti technique .
the study found no significant difference in time of correction , need for tenotomy , or relapse between both groups .
however , this trial included a small sample size and low btx - a dosage that may have compromised positive findings of btx - a .
no adverse events were reported following btx - a injections in children with clubfoot . in order to address the need for additional btx - a injections , a larger trial is required with regular follow - up as well as a good maintenance - bracing program , so as to provide each child with an individualized treatment . the exact dose and number of injections
three studies [ 4244 ] reported improvement in gait pattern , function , and decreased toe walking severity following btx - a injection to gastrocnemius and soleus muscles .
in addition to the btx - a injection , children also received a home exercise program , physical therapy , and orthotics .
further studies are needed to evaluate whether repeated injections and btx treatment in combination with other treatment interventions ( such as orthotics and physical therapy ) improve outcome .
this was a pilot randomized controlled trial comparing the effects of btx - a injection versus placebo at the time of surgery .
fifty - two children with limb length discrepancies of various etiologies , as well as children with surgical correction of clubfoot deformities , were included .
findings showed that compared to placebo , the btx - a group had a trend for lower pain at middistraction , less parenteral pain medication after - surgery , higher functional mobility scores , and better quality of life at three of five time points , although these differences were not statistically significant .
no adverse events related to the btx - a injection were reported , indicating that btx - a may be safe and effective in alleviating pain , improving functional mobility and quality of life in children undergoing lower limb lengthening and/or deformity correction .
future studies with larger sample sizes are required and with homogeneous study populations to verify whether btx - a injections are beneficial at the time of surgery in these children .
ten studies [ 4655 ] reported on the outcomes of using btx - a , in which one study by hierner and colleagues was a follow - up report of the previous study by rollnik and colleagues .
although all children were diagnosed with nbpp , there were a number of variations between studies in the specific mechanism of injury and in the underlying limitations , such as in severity of biceps - triceps cocontractions , persistence in shoulder paralysis , medial rotation deformity of the shoulder , response to serial cast treatment , and posterior shoulder subluxation or dislocation .
however , different muscle injection sites were used across the studies , indicating an individualized protocol according to each child 's condition and needs .
four studies reported administering additional btx - a injections , as needed . all ten studies reported positive outcomes following btx - a injections , including improved ability to make hand - to - mouth movements , avoidance of open surgical procedures , improved active shoulder abduction and elbow extension , better functional scores , and reduction of triceps cocontractions during elbow flexion . two studies [ 46 , 48 ] reported that those children who did not experience improved outcomes following btx - a injections were older .
desiato and risina found that the gain in shoulder abduction was directly related to younger age ( r = 0.6 ) .
transient weakness was reported in a 6-year - old female and mild to moderate discomfort at the injection site was reported in two children .
these findings indicate that btx - a may be a promising treatment modality in young children with nbpp ; however , stronger methodologies are required to confirm the effectiveness of btx - a for this condition .
the objectives of this systematic review were to establish the evidence on the effectiveness of btx - a in several musculoskeletal conditions in children and to show whether these studies reported improved functional outcome .
out of the 24 studies reviewed , only two randomized clinical trials were conducted , one in children with clubfeet and the other in children undergoing lower limb lengthening .
furthermore , treatment of children with musculoskeletal conditions is often multimodal , including bracing , casting , rehabilitation , and home programs .
therefore , attributing the improvements in outcome to a single intervention , such as btx - a , is not straightforward .
a recent systematic review on the indications for the use of btx - a treatment for children with nbpp was conducted by gobets and colleagues .
they included 10 full - text papers and six congress abstracts , involving 343 children .
four groups of indications were identified : internal rotation / adduction contracture of the shoulder , limited active elbow flexion , limited active elbow extension , and pronation contracture of the lower arm .
overall , positive results were reported for all except the indication for limited active elbow extension .
however , only one study was comparative in nature ; all others were classified as having a low level of evidence .
these authors conclude that multicentre randomized controlled trials are needed to provide better evidence of btx - a in this population .
these factors include the different dosages and commercial sources of btx - a used across the different studies .
two brands of btx - a were used across the 24 studies ( allergan and dysport ) .
btx - a brand was not always specified in the reviewed studies and this information was made available in several cases by contacting the authors .
these two preparations should not be used interchangeably , either in terms of predicting outcome or in determining doses to be used . though the units are not interchangeable , various published reports support a conversion ratio from 1 : 5 to 1 : 3 . the technique used to identify which
muscle groups will be injected also varied ( palpation technique , electrical stimulation , etc . ) .
indeed , different studies have used different techniques and thus may lead to varying outcomes following btx - a injection .
there is no consensus as to the exact age at which a child with a musculoskeletal condition should be first administered btx - a .
age of injection varied across the different studies reviewed . however , several authors [ 46 , 48 , 54 ] found that the younger children at the onset of btx - a treatment for nbpp benefited most compared to older children .
another factor to keep in mind is the need for administering repeat btx - a injections for sustained benefit .
some authors administered repeated btx - a doses , whereas other authors gave just one dose , even in cases when the children did not demonstrate improved outcome following the initial btx - a injection .
this may have been due to financial and time restrictions as repeated injections are both expensive and time - consuming .
findings indicate that repeat btx - a doses appear safe in children with musculoskeletal conditions .
longer follow - up periods are required to inform health care providers whether the benefits of btx - a are temporary and transient and , then if indicated , at which time should an additional injection be administered .
they reported that 48% of clubfeet were successfully corrected after a single dose of btx - a in 44 children .
serious adverse events related to btx - a injection were not reported in the studies included in this review .
minor adverse events such as transient muscular weakness and local discomfort at the injection site were reported in few studies . while this may be suggestive of the clinical safety of btx - a , it is important to note that several studies did not actually report on the presence or absence of adverse events .
therefore , more rigor is required in reporting these events to establish the safety of btx - a in children with musculoskeletal conditions .
btx - a is a promising treatment adjunct in improving functional outcomes in children with musculoskeletal conditions by causing a flaccid paralysis of the affected muscles .
further studies should include a prospective methodology , longer follow - up periods , and comparison group and evaluate whether repeated injections are required to improve the outcome of children , thus providing evidence on the effectiveness and safety of this drug in children with musculoskeletal conditions .
n. dahan - oliel and b. kasaai carried out the systematic review and data extraction .
|
children with musculoskeletal conditions experience muscle weakness , difficulty walking and limitations in physical activities .
standard treatment includes physiotherapy , casting , and surgery .
the use of botulinum toxins appears as a promising treatment on its own , but usually as an adjunct to other treatment modalities and as an alternative to surgery .
the objectives were to establish the evidence on the effectiveness , safety and functional outcome of btx - a in children with musculoskeletal conditions .
a literature search using five electronic databases identified 24 studies that met our inclusion criteria .
two randomized clinical trials were included ; most studies were case studies with small sample sizes and no control group .
improvements in gait pattern , function , range of motion , reduction of co - contractions , and avoidance of surgical procedures were found following btx - a injections .
adverse events were not reported in 10 studies , minor adverse events were reported in 13 children and there were no severe adverse events .
additional doses appear safe .
btx - a is a promising treatment adjunct in improving functional outcomes in children with musculoskeletal conditions . future studies including larger samples , longer follow - up periods and a comparison group are required to provide evidence on the effectiveness and safety of this drug in children with musculoskeletal conditions .
|
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the ability of mammals to respond to an inadequate o2 supply , commonly termed hypoxia , is crucial for their survival .
although a proper response to changed o2 tensions triggers adaptation , a number of pathological conditions or failures in the o2 response are associated with various diseases such as anemia , myocardial infarction , thrombosis , atherosclerosis , or cancer . when exposed to hypoxia or even anoxic conditions , mammalian organisms initiate a variety of responses in different organs , aiming to increase the delivery of oxygen to the tissues .
in addition to the switch from an aerobic to an anaerobic metabolism and the suppression of energy - using reactions , the carotid body chemoreceptor cells stimulate the brain stem center controlling the respiratory and cardiovascular systems to enhance ventilation , heart rate , and blood pressure ( reviewed by prabhakar1 ) .
in addition , neuroepithelial cells in the lung contribute to adjusting pulmonary perfusion and gas exchange .
moreover , organs and cells switch their gene expression profile : the kidneys produce erythropoietin , which increases red blood cell production in the bone marrow , and vascular cells produce vascular endothelial growth factor to promote angiogenesis and flow of enhanced blood volume ( reviewed by semenza2 ) . in addition to the expression of erythropoietin and vascular endothelial growth factor , the expression of more than 500 genes , products of which are involved in glycolysis , angiogenesis , erythropoiesis , cell death , and differentiation , is also changed on exposure to hypoxia ( reviewed by wenger and stiehl3 and semenza4 ) . in mammals ,
the hypoxia - dependent changes on the level of gene expression are mainly mediated by the -subunits of hypoxia - inducible transcription factors ( hifs ) .
hif -subunits are tightly regulated , and post - translational hydroxylations in response to hypoxia appear to be of major importance .
in addition to hypoxia , hif -subunits were also found to respond to various growth and coagulation factors , hormones , cytokines , or stress factors already under normoxia .
indeed , different kinases , among them glycogen synthase kinase 3 ( gsk-3 ) , have been identified to directly phosphorylate hif- proteins .
this review discusses the regulation of hif- by gsk-3 and compares it with hydroxylase - dependent hif- protein regulation .
in their active form , hifs are heterodimeric transcription factors consisting of an - and -subunit .
the hif -subunit represents the stable nuclear subunit primarily represented by the ubiquitously found arnt ( arylhydrocarbon receptor - nuclear translocator ) protein ; however , arnt2 or artn3 , although to a lesser extent , also appear to be able to take part in the formation of hif dimers ( reviewed by semenza5 ) .
so far , three -subunit proteins , hif-1 , hif-2 ( also known as epas,6 hlf,7 hrf,8 or mop29 ) , and hif-3 have been identified .
together , the different hif - and -subunits may give rise to the formation of several combinations of hif dimers.5,10 hif-1 and hif-2 are the best - studied hif- isoforms .
although they share structural and functional similarities , it appears that differences in the cell - type expression pattern , the target genes , the embryonic deletion phenotypes , and the effects on tumorigenesis exist between hif-1 and hif-2.1114 the function of hif-3 , from which several splice variants exist in humans,15,16 is largely unknown , although some human hif-3 variants and a mouse splice variant termed inhibitory pas protein ( ipas ) appear to act as negative regulators of the hypoxic response.1619 similar to the arnt proteins , the hif -proteins belong to the basic helix - loop - helix pas ( per - arnt - sim ) protein family . in particular , hif-1 and
hif-2 show the highest degree of sequence identity in the basic helix- loop - helix ( 85% ) , pas - a ( 68% ) , and pas - b ( 73% ) domains .
both also contain two nuclear localization sequences responsible for translocation to the nucleus under hypoxia ; they are localized in the n terminus ( amino acids 1733 in hif-1 and amino acids 150 in hif-2 ) and in the c terminus ( amino acids 718721 in hif-1 and amino acids 689870 in hif-2).20,21 with the exception of hif-3 , which does not contain a c - terminal transactivation domain ( c - tad),22,23 hif -subunits also contain n- and c - terminal transcriptional activation domains ( n - tad and c - tad ) . a unique oxygen - dependent degradation domain ( oddd , amino acids 401603 in hif-1 and amino acids 517682 in hif-2 ) overlaps n - tad .
the residues between n - tad and c - tad represent an inhibitory domain ( amino acids 604785 in hif-1 and amino acids
hif -subunit activation under hypoxia is mainly the result of an increased protein stability and coactivator recruitment , although transcriptional and translational mechanisms also were shown to be involved in hif -subunit activation.22,2630 as a result , hif- proteins accumulate , translocate to the nucleus , and dimerize with hif- to form a functional transcription factor.31 thus , in the presence of oxygen ( ie , normoxia ) , hif- proteins become degraded .
this is primarily achieved by oxygen - dependent hydroxylations at the oddd.32 under normoxia , prolyl hydroxylase domain proteins ( phds),33,34 in particular phd2 , hydroxylate two crucial residues in the oddd of hif -subunits ( p402 and p564 in hif-1 and p405 and p531 in hif-2).25,32,35 prolyl hydroxylation is required for binding the von hippel - lindau protein ( vhl),36,37 which represents the substrate recognition subunit of an e3 ubiquitin protein ligase consisting of elongin c , elongin b , ring box 1 , cullin 2 , and an e2 ubiquitin - conjugating enzyme ( figure 1 ) .
the prolyl hydroxylation and ubiquitination can be further promoted by the binding of phd2 to os938 and that of hif-1 , vhl , and elongin c to ssat2 , respectively.39,40 in addition to prolyl hydroxylation , a conserved asparagine residue ( n803 in hif-1 and n852 in hif-2 ) in the c - tad is hydroxylated by the factor - inhibiting hif in an oxygen - dependent manner .
this hydroxylation prevents interaction with the coactivator proteins cbp / p300.4144 thus , the major posttranslational modification appears to be the oxygen - dependent hydroxylation.36,37
in addition to hydroxylation , hif- transcriptional activity and protein stability appear also to be dynamically regulated by other posttranslational modifications such as acetylation , s - nitrosylation , sumoylation , and phosphorylation ( for review , see dimova and kietzmann45 ) .
phosphorylation appears to be of special importance under normoxic conditions , mediating the response of hif- to various growth and coagulation factors , hormones , cytokines , or stress factors ( reviewed by dimova et al46 ) under normoxia .
indeed , a panel of protein kinases is reported to be involved in hif-1 phosphorylation , either directly ( table 1 ) or indirectly.4752 although the individual action of certain kinases on hif-1 regulation was mainly studied in vitro ( table 1 ) , the in vivo mechanisms are likely much more complex .
at least the extent to which the kinases can be involved in hif- phosphorylation may vary according to the signal , cell type , or tissue . given the different developmental and/or differentiation status of a cell or tissue , the expression of various growth factors , their receptors , and respective signaling components and
thus , it seems not to be surprising that phosphorylation of hif- by different kinases or after modulation of signaling pathways may be a highly cell type - specific event .
although direct proof is currently lacking , it is plausible that the phosphorylation pattern of hif- in a certain cell may be explained by different layers of regulations that affect kinases depending on the cellular context .
in addition to being activated by a variety of extracellular signals , the pi3k / akt cascade appeared also to be regulated by hypoxia , thus integrating hypoxia signaling with extracellular signals affecting multiple cellular processes such as apoptosis , metabolism , cell proliferation , and cell growth ( for review , see braccini et al53 ) .
the pi3k / akt pathway is considered to control hif-1 within the cell via regulation of hif-1 protein synthesis and stability .
however , it appeared that hif- proteins are not directly phosphorylated by pkb / akt but , rather , by a pkb / akt target .
the pkb / akt targets hdm2,54,55 mammalian target of rapamycin ( mtor),56 and gsk-357 were shown to contribute to changes in hif- protein levels ; however , only gsk-3 was shown to do this directly , ie , by phosphorylating hif- proteins .
gsk-3 is a serine / threonine kinase that was first identified as a negative regulator of glycogen synthesis ; inhibition is achieved through phosphorylation of glycogen synthase.58,59 since its initial discovery , gsk-3 has been found to be involved in numerous signaling pathways initiated by diverse stimuli and to contribute to the regulation of cell proliferation , stem cell renewal , apoptosis , and development , which are processes often associated with hypoxia . because of these multiple involvements ,
dysregulation of gsk-3 has been implicated in the pathogenesis of human diseases , including type 2 diabetes , bipolar disorders , inflammation , alzheimer s disease , and cancer ( reviewed by frame and cohen,60 grimes and jope,61 and woodgett62 ) .
two isoforms , gsk-3 ( 51 kda ) and gsk-3 ( 47 kda ) , have been identified in mammals . despite their homology in the catalytic domain ( 98% ) , they significantly differ in their n- and c - terminal parts63,64 and do not have entirely overlapping roles in metabolism ( reviewed in force and woodgett65 ) .
moreover , gsk-3 ( gsk-3 ) homozygous knockout mice showed an embryonic lethal phenotype around day 16 because of hepatic apoptosis or a cardiac pattern defect,66,67 whereas homozygous gsk-3 ( gsk-3 ) knockout mice are viable and fertile.68,69 gsk-3 is a target of pkb / akt , which can phosphorylate both gsk-3 isoforms ( serine 21 of gsk-3 and serine 9 of gsk-3 ) , leading to an inhibition of gsk-3 activity.70 interestingly , these serine residues can also be phosphorylated by other kinases such as erk1/2,71 p70 ribosomal s6 kinase 1,72 camp ( cyclic adenosine monophosphate)-dependent protein kinase a ( pka),73 and protein kinase c ( pkc).74 in contrast , an autophosphorylation event leading to phosphorylation of tyrosine 279 in gsk-3 and of tyrosine 216 in gsk-3 increases gsk-3 activity.75,76 neurons seem to possess a spliced gsk-3 variant called gsk-32 that contains a 13 amino acid residue insert within the kinase domain , leading to reduced kinase activity.77,78 although gsk-3 is mostly known in the insulin field as a regulator of glycogen synthesis , it has been shown that early hypoxia enhanced pi3k / akt activity and increased hif-1 protein levels.57 similarly , hypoxia was capable of inhibiting gsk-3 by phosphorylation in different cell types , such as pc-12 ( rat pheochromocytoma cell line ) cells,79 ht1080 ( human fibrosarcoma cell line ) cells,80 and hepg2 ( human liver hepatocellular cell line ) cells,57 as well as in vivo.81 although this effect was not observed in other cell types , including some breast cancer cell lines,82 pc-3 prostate cancer cells,83 and 3t3 cells,84 it was considered to have a cell type - specific component .
however , the findings that gsk-3 inhibition57 and small interfering rna - mediated depletion induced hif-1 , whereas gsk-3 overexpression reduced hif-1 protein levels,85 suggested that hif-1 is a direct target of gsk-3. indeed , the oddd86 and three sites , s-551 , t-555 , and s-589 , located within the oddd overlapping the n - tad of hif-1 were found to be directly phosphorylated by gsk-3.85 another study reported five sites , t-498 , s-502 , s-505 , t-506 , and s-510 , within the n - tad of hif-1 as gsk-3 phosphorylation sites.87 the disparity of the different phosphorylation sites is difficult to explain , but the different oxygen concentrations ( 8% o2 compared with 2% o2 ) used in these studies may contribute to the differences .
it is possible that different oxygen levels may induce variable signaling pathways that have unequal effects on hif-1 and its ability to act as a substrate for gsk-3. another possibility could be the different cell types ( hepg2 compared with sk - ov-3 [ human ovarian cancer cells ] ) that were used in the studies . despite the differences in the phosphorylation sites , both studies demonstrated that the regulation of hif-1 by gsk-3 is independent of o2 , hydroxylation , and recruitment of the vhl - containing e3 ubiquitin ligase .
experiments with vhl - deficient cells showed that gsk-3-dependent hif-1 degradation occurred independent of vhl , indicating that the phosphorylation of hif-1 by gsk-3 target hif-1 for proteasomal degradation in an oxygen - independent manner.85 this suggested involvement and recruitment of another so - far - unknown e3 ubiquitin ligase to gsk-3-phosphorylated hif-1. indeed , two groups demonstrated that the f - box and wd protein fbw7 ( also known as hcdc4 in yeast , hsel10 in caenorhabditis elegans , or ago in drosophila ) acted as the substrate - recognition component of a multisubunit e3 ubiquitin ligase , which was crucial for the proteasomal degradation of gsk-3 phosphorylated hif-1.85,87 in this e3 ligase , fbw7 interacts with skp1 ( s - phase kinase - associated protein 1 ) , cul1 ( cullin 1 ) , and rbx1 , forming the so - called scf complex .
similar to vhl , fbw7 is considered to serve as a tumor suppressor , and three fbw7 isoforms ( fbw7 , fbw7 , and fbw7 ) are known to be produced by alternative splicing .
they are found in the nucleoplasm , cytoplasm , and nucleolus , respectively.88 in addition to hif-1 , fbw7 was shown to be involved in the degradation of various oncogenic proteins , including cyclin e,89 c - myc,90,91 c - jun,92,93 and notch.94 several studies have shown that loss of the fbw7 gene is associated with malignant transformation , especially in ovarian cells and t cells,95 in breast cancer cells,96 and later also in human colorectal cancers,97 which leads then to chromosomal instability and some types of malignancy .
furthermore , investigation of more than 1,500 human tumors revealed that approximately 6% of those tumors showed mutations in the fbw7 coding region .
specifically , cholangiocarcinomas ( 35% ) , t - cell acute lymphocytic leukemia ( 31% ) , and endometrial ( 9% ) , colon ( 9% ) , and stomach ( 6% ) cancer98 had the highest mutation rates .
strikingly , nearly half ( 43% ) of these were missense mutations that resulted in amino acid substitutions within the wd40 domain ( arg465 and arg479 ) , which are shared by all three fbw7 isoforms , suggesting that all fbw isoforms might collectively contribute to the tumor - suppressor function.98 with respect to hif-1 , all three fbw7 isoforms were able to induce hif-1 degradation , and the loss of the fbw7 wd domain abolishes gsk-3-initiated degradation , leading to higher hif-1 levels , which has been found to be associated with several tumors.99101 the finding that hif subunits can be targeted for degradation by two different e3 substrate recognition proteins indicates that the system is highly dynamic .
the human genome encodes nearly 100 deubiquitylating enzymes that are predicted to be active and that oppose the function of around 600 e3 ligases.102,103 similar to e3s , deubiquitylating enzymes have a central role in cell cycle regulation and dna damage response and , depending on the context , can act either as a tumor promoter or suppressor ( see references in love et al104 ) .
with respect to vhl , two different deubiqiutinating enzymes , vdu1 ( usp33 ) and vdu2 ( usp20 ) , were suggested to oppose the vhl - e3 ubiquitin ligase.105,106 later , it was shown that vdu2 but not vdu1 can interact with hif-1.107 experiments with cycloheximide and hypoxia showed that the half - life of hif-1 was significantly increased upon overexpression of vdu2 , whereas a catalytic inactive vdu2 c154a mutant had no effect .
in addition , it was shown that only vdu2 , not vdu1 , deubiquitinated hif-1 , resulting in the stabilization of hif-1 protein107 ( figure 1 ) .
experiments with gsk-3- and fbw7-deficient cells revealed that gsk-3- and fbw7-dependent hif-1 degradation can be antagonized by ubiquitin - specific protease 28 ( figure 1).99 these findings suggest that the gsk-3-dependent degradation of hif-1 is not limited by the presence of oxygen and is therefore independent of vhl .
together , these results demonstrate that hif-1 protein stability is regulated in a dynamic manner involving different ubiquitin ligases and deubiquitinases .
as such , the hydroxylation- and vhl - dependent ubiquitination and degradation of hif-1 under normoxia is opposed by the deubiquitinase vdu2 .
in contrast , the oxygen - independent but phosphorylation - dependent ubiquitination of hif-1 is counteracted by ubiquitin - specific protease 28-mediated deubiquitination .
the latter process allows the integration of the hif system into the cellular response to various physiologic and pathophysiologic signals independent of the oxygen tension .
the finding that gsk-3 is involved in the degradation of hif-1 indicated similarities with the destruction of -catenin in the canonical wnt signaling pathway . in this pathway ,
gsk-3 and -catenin are part of a destructive complex in which binding of gsk-3 and -catenin promotes phosphorylation of -catenin by gsk-3 , which requires priming phosphorylation by casein kinase 1 , -isoform .
the phosphorylated -catenin is recognized by the f - box / wd protein -trcp and subsequently ubiquitylated and targeted for proteasomal degradation ( for review , see cohen and frame108 and metcalfe and bienz109 ) .
when this phosphorylation event is blocked , -catenin accumulates and binds to the t - cell - specific transcription factor / lymphoid enhancer - binding factor 1 family of transcriptional activators to activate numerous target genes ( reviewed by reya and clevers110 ) contributing to embryonic development and adult tissue homeostasis ( reviewed by clevers111 ) .
similarly , gsk-3-mediated phosphorylation of hif-1 recruits fbw7 , and thus targets hif-1 for ubiquitylation and proteasomal degradation.99 those very similar scenarios imply interference or interconnection of both the wnt/-catenin and hypoxia / hif-1 signaling on the level of gsk-3 .
actually , crosstalk between the hypoxia and/or hif-1 and wnt/-catenin pathway was reported and appears to be quite complex because of controversial and likely cell / tissue / differentiation - stage specific data.112117 indeed , it was reported that hypoxia and/or hif-1 can inhibit wnt/-catenin signaling .
several mechanisms , such as binding of hif-1 to hard1 ( human arrest - defective-1 protein ) with subsequent interference with acetylation of -catenin,112 blocking processing and secretion of wnt proteins,113 down - regulating -catenin via p53-dependent activation of siah-1 ( seven in absentia homolog 1),118 or direct interaction between hif-1 and -catenin119,120 were proposed to contribute to these effects .
in contrast , hypoxia was also shown to activate wnt/-catenin signaling in undifferentiated cells and in vivo.115,117 in hypoxic embryonic stem cells , this occurred via hif-1-mediated expression of lymphoid enhancer - binding factor 1 and t - cell - specific transcription factor , followed subsequently by increased interaction of -catenin with lymphoid enhancer - binding factor 1/t - cell - specific transcription factor , and thus activating wnt/-catenin targets.115 in addition , hypoxia was able to activate -catenin via gsk-3 inactivation116,121 in different human cell lines such as ht-29 ( human colorectal adenocarcinoma cell line ) and hepg2 ; this activation contributed to an endothelial mesenchymal transition program , leading to significantly increased invasiveness,121 and in renal tubular cells , this process impaired wound healing.116 together , the reported findings indicate that complex interconnections between hypoxia and/or the hif-1 and wnt/-catenin pathway exist and that cell- , tissue- , and differentiation - specific aspects contribute to their functional consequences .
hypoxia and hifs play important roles in many critical aspects of physiological and pathological processes .
most solid cancers contain hypoxic areas , and clinical data demonstrate that overexpression of hif-1 is associated with an increased risk for patient mortality . in line ,
downregulation of hifs interferes with tumor growth , vascularization , invasion , and metastasis , as well as radiation and chemotherapy .
activation of multiple oncogenic pathways including growth factor signaling coupled with enhanced kinase signaling is a common event in tumors , thus making it likely that kinases are involved in the modulation of hif- function .
because regulation of hif- protein stability is critical for its activation , identification of kinases contributing to hif- stability may provide a link explaining normoxic hif- stabilization by extracellular stimuli . in light of this , dysregulation of gsk-3 is thought to underlie the pathogenesis of various diseases that are also associated with hypoxia and changed hif- levels , such as type 2 diabetes mellitus , alzheimer s disease , mood disorders , cardiovascular diseases , and cancer.122 thus , given that gsk-3 upstream regulation leads to inhibition of gsk-3 and hif-1 accumulation , this raises the question whether it is an option to target gsk-3 in those diseases and disregard the adverse effects . although research from the last decade has demonstrated that a number of kinase pathways contribute to hif-1 regulation , data for hif-2 or hif-3 are limited .
taking into consideration the overlapping , but different , roles of the hif- proteins , more knowledge about the phosphorylation - dependent regulation of hif-2 and hif-3 is necessary to better understand both already - observed general and different effects .
|
hypoxia - inducible factors ( hifs ) , consisting of - and -subunits , are critical regulators of the transcriptional response to hypoxia under both physiological and pathological conditions .
to a large extent , the protein stability and the recruitment of coactivators to the c - terminal transactivation domain of the hif -subunits determine overall hif activity .
the regulation of hif -subunit protein stability and coactivator recruitment is mainly achieved by oxygen - dependent posttranslational hydroxylation of conserved proline and asparagine residues , respectively . under hypoxia ,
the hydroxylation events are inhibited and hif -subunits stabilize , translocate to the nucleus , dimerize with the -subunits , and trigger a transcriptional response . however , under normal oxygen conditions , hif -subunits can be activated by various growth and coagulation factors , hormones , cytokines , or stress factors implicating the involvement of different kinase pathways in their regulation , thereby making hif--regulating kinases attractive therapeutic targets . from the kinases known to regulate hif -subunits , only a few phosphorylate hif- directly . here , we review the direct phosphorylation of hif- with an emphasis on the role of glycogen synthase kinase-3 and the consequences for hif-1 function .
|
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although considered the gold standard for evaluating treatment effectiveness , randomized clinical trials ( rcts ) have important limitations .
because randomization removes potential bias from unknown and unmeasured confounders , observed differences in measured outcomes can be reasonably attributed to the treatment alone.1 for valid experimental reasons , however , rcts frequently restrict enrollment based on existing comorbidities , treatment history , and disease severity , among other criteria . as a result
, outcomes observed in rcts can not necessarily be generalized to the real world of clinical practice , where patients present with varying degrees of disease severity and a range of comorbidity profiles . while there has been a call for increasing the number of pragmatic clinical trials , trials that examine outcomes among diverse populations of patients in typical practice settings are still rare.2 retrospective , observational studies are valuable because they contribute pragmatic knowledge about treatment risk , effectiveness , and cost in clinical practice settings knowledge that is critical to health care decision makers .
in addition , observational studies are less costly and more quickly accomplished than rcts , and can utilize large databases , permitting analysis of infrequent events . because observational studies do nt involve randomization of patients to treatment groups , however
, selection bias can occur , and unmeasured variables can confound the associations between treatments and outcomes .
multivariable regression ( mr ) methods are commonly used to control for confounding factors in observational studies .
it can be done at the individual level , as in case control matching , or at the group or frequency level , as in stratified random sampling .
the matching process involves diagnostic checks regarding the balance of covariates across groups and provides information about the quality of the inferences that can be drawn from the subsequent analysis.3 propensity score matching ( psm ) has been increasingly used in epidemiologic studies of medical treatment effectiveness.1 a propensity score represents the propensity of a particular subject to receive a particular treatment , based on the subject s pre - treatment characteristics.1,4,5 the score combines many covariates into a single variable and enables individuals from each treatment group with similar covariate values to be matched , as a quasi - randomization method.3 subjects who can not be matched are excluded from the analysis .
an advantage of psm is that matched sets with comparable covariate distributions can be created without the need for exact matches of each variable , which is problematic when there are more than a few covariates.3 propensity matching works best if there is a fairly large overlap between the groups in terms of propensity to be given a treatment .
when there is not , underlying selection bias may exist.3 despite this method s theoretical benefits , in studies where both mr and psm analysis methods have been used , only a small percentage of results ( 10% in one review and 13% in another ) have been markedly different.1,6 disease exacerbations are important events in the course of copd .
moderate and severe exacerbations adversely affect lung function , potentially accelerating disease progression.7,8 frequency of exacerbations is a significant factor in deteriorating health.9,10 exacerbations also contribute substantially to health care utilization and costs . in the united states in 2010 ,
direct medical costs were estimated to be $ 29.5 billion , including $ 13.2 billion for copd - related hospital care.11 reducing exacerbations is thus a singularly important goal of copd management , both to improve patient quality of life and to reduce the indirect and direct medical costs of the disease .
as pharmacotherapy is a primary means for reducing exacerbations , data concerning real world treatment effectiveness is of interest to health care providers , health care organizations , and health plans .
agents for the relief and prevention of symptoms in copd include short- and long - acting beta - agonists ( including albuterol and salmeterol ) , short - and long - acting anticholinergics ( including ipratropium bromide [ ipr ] , tiotropium [ tio ] ) , and inhaled corticosteroids ( ics).12 fluticasone propionate 250 g / salmeterol 50 g combination therapy ( fsc ) is an ics plus long - acting beta - agonist used for treatment of airflow obstruction and reduction of exacerbations .
previously , we reported cost and utilization outcomes following initiation of copd maintenance therapy with tio , ipr ( with or without albuterol ) , or fsc , using mr as the analysis method.13 to our knowledge , this was the first observational study to directly compare these three maintenance therapies . compared to tio and ipr , fsc was associated with lower copd - related costs and utilization ( hospitalizations , emergency department [ ed ] visits , and outpatient visits associated with an antibiotic or oral corticosteroid prescription ) .
the objective of the present study was to perform a parallel analysis employing psm to investigate the equivalency of results with those obtained in the prior mr analysis .
using psm methods , we conducted a parallel analysis of copd - related health care utilization and costs in patients with copd receiving initial maintenance therapy ( imt ) with fsc , tio , or ipr , and we compared the results to those of a previous mr analysis .
the term imt refers to the patient s first instance of a pharmacy claim for a copd maintenance medication ; prior to this point , the patient s records indicated that he / she had not received maintenance therapy , only reliever medications or no medication .
we assessed exacerbations using claims data to measure health care utilization events related to exacerbations .
there is no universally accepted definition of exacerbation . in clinical research , exacerbations generally are defined in terms of worsening symptoms , unscheduled medical attention , and courses of antibiotics and/or oral corticosteroids . 14 in observational studies such as ours ,
in which clinical and laboratory data are absent , exacerbations typically have been defined in terms of copd - related health care utilization events , including hospitalizations , ed visits , physician visits , and outpatient pharmacy fills for oral corticosteroids / antibiotics .
administrative data were obtained from the ims lifelink health plan claims database ( ims health , watertown , wa ) , which contains enrollment and demographic data , and health care and outpatient pharmacy claims from more than 40 million members of more than 70 us health plans .
calculated costs were based on allowed amounts , which most closely resemble the direct health care cost burden of illness .
this is typically the amount the health plan pays , plus any member liability ( eg , co - payment , deductible , or coinsurance amount ) . for claims with missing charges due to capitation arrangements ,
the dataset included patient demographic and enrollment data , outpatient pharmacy claims , and medical services claims ( outpatient , ed , and inpatient claims , including both facility claims and professional services claims ) for january , 2004 to june , 2009 .
the specific content of the dataset has been described previously.13 in the prior retrospective , observational cohort study , copd - related clinical and economic outcomes were evaluated in patients who received one of three imt medications for copd ( fsc , tio , or ipr).13 the study perspective was that of the health plan provider organization , and only direct costs were considered .
the study population included health plan members with diagnosed copd who were new to maintenance therapy with fsc 250 g/50 g , tio , or ipr ( alone or in fixed dose combination with albuterol ) .
the members were age 40 years and older , had a primary or secondary diagnosis of copd ( at least one ed visit , one hospitalization , or two outpatient visits with a primary or secondary international classification of disease , 9th edition , clinical modification [ icd-9-cm ] diagnosis code of 491.xx , 492.xx , or 496.xx ) , had an imt pharmacy claim between july 1 , 2004 and june 30 , 2008 ( the date of the first identified prescription claim was the index date ) , had at least 6 months of continuous health plan enrollment prior to the index date ( the baseline period ) , and at least 12 months of continuous enrollment following the index date ( the follow - up period ) .
patients were excluded if they had a prescription drug fill for a copd maintenance medication ( fsc , ipr , tio , budesonide / formoterol , inhaled corticosteroid alone , or long - acting beta - agonist alone ) during the baseline period , or a pharmacy fill for an alternate study imt medication ( fsc , tio , or ipr ) within 60 days of the index date .
patients were excluded if they had a primary or secondary diagnosis of respiratory tract cancer ( larynx , trachea , or pleura [ icd-9-cm codes of 161 , 161.x , 162 , 163 , 163.x , 231 , 231.x ] ) during the baseline period .
the patient eligibility criteria and selection process have been described in detail previously.13 the primary utilization outcomes were incidence and mean number of copd - related outpatient visits , outpatient visits associated with an antibiotic prescription fill , outpatient visits associated with an oral corticosteroid fill , hospitalizations , ed visits , and hospitalization and/or ed visits ( combined endpoint ) .
encounters with a primary diagnosis code of 491.xx , 492.xx , or 496 were defined as copd - related .
the primary cost outcomes were mean copd - related medical services costs , outpatient pharmacy costs ( copd controller and relief medications , oral corticosteroids , and antibiotics ) , and total costs ( the sum of the two ) .
medical services costs comprised inpatient , outpatient , and ed care ( including facility charges and professional service fees ) .
costs were inflated to 2009 dollars on a monthly basis using the medical care portion of the us consumer price index.15 as outcomes were evaluated over a 12-month follow - up period , no discounting was applied to events or costs .
bivariate analyses were used to compare differences between treatment cohorts in health care utilization and cost outcomes for the 12-month follow - up period .
multivariable logistic regression was used to model the risk for any health care utilization event as an odds ratio ( or ) .
negative binomial and poisson regression were used to calculate incidence rate ratios ( irrs ) . because of the right - skewed nature of the cost distribution , a generalized linear model using a gamma distribution was used to estimate differences in treatment costs .
estimates of mean differences and 95% confidence intervals ( cis ) were calculated from the predicted cost values .
the multivariable models controlled for age , sex , treatment , comorbidities ( including asthma and heart disease ) , and copd - related health care utilization at baseline . starting with the original patient sample identified for the mr analysis , we created matched cohorts for the psm analysis .
the tio patients and ipr patients were separately matched to fsc patients based on propensity score ; that is , patients initiating therapy with tio were matched to patients initiating with fsc , and patients initiating therapy with ipr were matched to patients initiating with fsc .
the matched samples were created based on each patient s predicted probability ( propensity ) of assignment to the case treatment ( tio or ipr ) .
the propensity to be a patient whose initial maintenance therapy was tio ( or alternatively , ipr ) incorporated the following baseline factors in the logistic regression equation : sex , age category , geographic region , comorbidities , copd - related health care utilization , non - copd - related health care utilization , copd medication use , and copd - related medical services costs .
the utilization factors were hospitalization count and binary variables for outpatient visit , outpatient visit associated with an oral corticosteroid fill , outpatient visit associated with an antibiotic fill , ed visit , and hospitalization and/or ed visit ( combined endpoint ) .
medication use was included using binary variables for short - acting beta - agonist ( sabas ) , oral corticosteroid , oral antibiotic , leukotriene modifier , and methylxanthine .
the greedy match algorithm was used , which performs matching using as much information as possible through the
nearest available pair ( or nearest - neighbor ) matching method with a caliper component.1618 once a match is made , the greedy algorithm does not reconsider the match . because no available matches could be identified for some patients in the original cohorts , the patient sample for the psm analysis was a smaller subset of the sample used in the mr analysis .
bivariate analyses were used to compare differences in outcomes in the 12-month period following initiation of maintenance therapy for the fsc - tio and fsc - ipr matched cohorts .
logistic regression was used to model the risk for any health care utilization event ( or ) , and negative binomial and poisson regression were used to calculate irrs .
mean cost differences and 95% cis were assessed using least squares estimates from generalized linear models using a gamma distribution . since the psm treatment groups were already matched for baseline characteristics , and our interest was only in the treatment effect , the psm regression models contained only a factor for case imt ( tio or ipr ) , with fsc used as the reference medication .
for both the mr and psm analyses , statistical tests were two - sided , with an -level of 0.05 for statistical significance .
demographic , clinical , and health care utilization characteristics were assessed as counts and percentages for categorical variables , and as standard measures ( mean and sd ) for continuous variables . both unpaired and paired t - tests
the chi - square test and mcnemar s test were used to test paired proportion differences because significance tests that do not consider the non - independence of matched data have been found to be more conservative than tests for paired comparisons.19,20 adequacy of matching was assessed using p values for comparison tests and standardized percentage differences.4,21 all analyses were conducted using sas software ( v 9.2 for windows ; sas institute , cary , nc ) .
administrative data were obtained from the ims lifelink health plan claims database ( ims health , watertown , wa ) , which contains enrollment and demographic data , and health care and outpatient pharmacy claims from more than 40 million members of more than 70 us health plans .
calculated costs were based on allowed amounts , which most closely resemble the direct health care cost burden of illness .
this is typically the amount the health plan pays , plus any member liability ( eg , co - payment , deductible , or coinsurance amount ) . for claims with missing charges due to capitation arrangements ,
the dataset included patient demographic and enrollment data , outpatient pharmacy claims , and medical services claims ( outpatient , ed , and inpatient claims , including both facility claims and professional services claims ) for january , 2004 to june , 2009 .
in the prior retrospective , observational cohort study , copd - related clinical and economic outcomes were evaluated in patients who received one of three imt medications for copd ( fsc , tio , or ipr).13 the study perspective was that of the health plan provider organization , and only direct costs were considered .
the study population included health plan members with diagnosed copd who were new to maintenance therapy with fsc 250 g/50 g , tio , or ipr ( alone or in fixed dose combination with albuterol ) .
the members were age 40 years and older , had a primary or secondary diagnosis of copd ( at least one ed visit , one hospitalization , or two outpatient visits with a primary or secondary international classification of disease , 9th edition , clinical modification [ icd-9-cm ] diagnosis code of 491.xx , 492.xx , or 496.xx ) , had an imt pharmacy claim between july 1 , 2004 and june 30 , 2008 ( the date of the first identified prescription claim was the index date ) , had at least 6 months of continuous health plan enrollment prior to the index date ( the baseline period ) , and at least 12 months of continuous enrollment following the index date ( the follow - up period ) .
patients were excluded if they had a prescription drug fill for a copd maintenance medication ( fsc , ipr , tio , budesonide / formoterol , inhaled corticosteroid alone , or long - acting beta - agonist alone ) during the baseline period , or a pharmacy fill for an alternate study imt medication ( fsc , tio , or ipr ) within 60 days of the index date .
patients were excluded if they had a primary or secondary diagnosis of respiratory tract cancer ( larynx , trachea , or pleura [ icd-9-cm codes of 161 , 161.x , 162 , 163 , 163.x , 231 , 231.x ] ) during the baseline period .
the patient eligibility criteria and selection process have been described in detail previously.13 the primary utilization outcomes were incidence and mean number of copd - related outpatient visits , outpatient visits associated with an antibiotic prescription fill , outpatient visits associated with an oral corticosteroid fill , hospitalizations , ed visits , and hospitalization and/or ed visits ( combined endpoint ) .
encounters with a primary diagnosis code of 491.xx , 492.xx , or 496 were defined as copd - related .
the primary cost outcomes were mean copd - related medical services costs , outpatient pharmacy costs ( copd controller and relief medications , oral corticosteroids , and antibiotics ) , and total costs ( the sum of the two ) .
medical services costs comprised inpatient , outpatient , and ed care ( including facility charges and professional service fees ) .
costs were inflated to 2009 dollars on a monthly basis using the medical care portion of the us consumer price index.15 as outcomes were evaluated over a 12-month follow - up period , no discounting was applied to events or costs .
bivariate analyses were used to compare differences between treatment cohorts in health care utilization and cost outcomes for the 12-month follow - up period .
multivariable logistic regression was used to model the risk for any health care utilization event as an odds ratio ( or ) .
negative binomial and poisson regression were used to calculate incidence rate ratios ( irrs ) .
because of the right - skewed nature of the cost distribution , a generalized linear model using a gamma distribution was used to estimate differences in treatment costs .
estimates of mean differences and 95% confidence intervals ( cis ) were calculated from the predicted cost values .
the multivariable models controlled for age , sex , treatment , comorbidities ( including asthma and heart disease ) , and copd - related health care utilization at baseline .
starting with the original patient sample identified for the mr analysis , we created matched cohorts for the psm analysis .
the tio patients and ipr patients were separately matched to fsc patients based on propensity score ; that is , patients initiating therapy with tio were matched to patients initiating with fsc , and patients initiating therapy with ipr were matched to patients initiating with fsc .
the matched samples were created based on each patient s predicted probability ( propensity ) of assignment to the case treatment ( tio or ipr ) .
the propensity to be a patient whose initial maintenance therapy was tio ( or alternatively , ipr ) incorporated the following baseline factors in the logistic regression equation : sex , age category , geographic region , comorbidities , copd - related health care utilization , non - copd - related health care utilization , copd medication use , and copd - related medical services costs .
the utilization factors were hospitalization count and binary variables for outpatient visit , outpatient visit associated with an oral corticosteroid fill , outpatient visit associated with an antibiotic fill , ed visit , and hospitalization and/or ed visit ( combined endpoint ) .
medication use was included using binary variables for short - acting beta - agonist ( sabas ) , oral corticosteroid , oral antibiotic , leukotriene modifier , and methylxanthine .
the greedy match algorithm was used , which performs matching using as much information as possible through the
nearest available pair ( or nearest - neighbor ) matching method with a caliper component.1618 once a match is made , the greedy algorithm does not reconsider the match . because no available matches could be identified for some patients in the original cohorts , the patient sample for the psm analysis was a smaller subset of the sample used in the mr analysis .
bivariate analyses were used to compare differences in outcomes in the 12-month period following initiation of maintenance therapy for the fsc - tio and fsc - ipr matched cohorts .
logistic regression was used to model the risk for any health care utilization event ( or ) , and negative binomial and poisson regression were used to calculate irrs .
mean cost differences and 95% cis were assessed using least squares estimates from generalized linear models using a gamma distribution .
since the psm treatment groups were already matched for baseline characteristics , and our interest was only in the treatment effect , the psm regression models contained only a factor for case imt ( tio or ipr ) , with fsc used as the reference medication .
for both the mr and psm analyses , statistical tests were two - sided , with an -level of 0.05 for statistical significance .
demographic , clinical , and health care utilization characteristics were assessed as counts and percentages for categorical variables , and as standard measures ( mean and sd ) for continuous variables .
both unpaired and paired t - tests were used for determining significant differences in mean measures .
the chi - square test and mcnemar s test were used to test paired proportion differences because significance tests that do not consider the non - independence of matched data have been found to be more conservative than tests for paired comparisons.19,20 adequacy of matching was assessed using p values for comparison tests and standardized percentage differences.4,21 all analyses were conducted using sas software ( v 9.2 for windows ; sas institute , cary , nc ) .
a total of 32,338 patients met patient selection criteria in the mr analysis : 12,595 fsc patients , 9126 tio patients , and 10,617 ipr patients . for the psm analysis , 89.1% ( 8135 ) of the tio patients
were matched to fsc patients ( 64.6% of the original fsc cohort ) and 80.2% ( 8514 ) of the ipr patients were matched to similar fsc patients ( 67.6% of the original fsc cohort ) .
there was a large degree of overlap in the populations and good balance was achieved between the matched groups .
baseline demographic , clinical , and utilization characteristics of the cohorts after matching on propensity score are shown in table 1 ( tio - fsc ) and table 2 ( ipr - fsc ) , along with p values for unpaired significance tests .
paired significance tests showed similar results although the p values were almost always lower ( data not shown ) . after matching ,
the tio and fsc groups were well balanced with respect to baseline characteristics ; the groups were different only in mean copd - related outpatient visits ( p < 0.001 ) . matching between the ipr and fsc patients
involved more factors . after matching , differences were present for some baseline characteristics : mean copd - related outpatient visits ( p = 0.02 ) , mean all - cause outpatient visits ( p < 0.001 ) , and mean days supply of sabas ( p = 0.007 ) .
figures 2 and 3 show absolute standardized difference percentages for baseline characteristics prior to and after matching .
these graphs further illustrate that , while some significant differences remained after matching , they were small from a clinical standpoint .
absolute standardized percentage differences were less than 10% for all assessed baseline characteristics in both groups , supporting an assessment of balance between groups.22 we compared differences in baseline characteristics between excluded patients and matched patients ( figures 2 and 3 ) .
characteristics with large differences between excluded and matched patients tended to be the same characteristics as those associated with large standardized differences prior to matching .
the excluded tio and ipr patients were older , and had more comorbidities and higher health care utilization .
the excluded tio patients , when compared to fsc patients who were not matched to tio patients , were older ( mean , 66.9 vs 60.1 years , p < 0.001 ) and more likely to be male ( 68.7% vs 39.8% , p < 0.001 ) , not to have asthma ( 4.4% vs 46.8% , p < 0.001 ) , to have lower use of leukotriene modifiers ( 2.4% vs 12.6% , p < 0.001 ) , and sabas ( 15.4% vs 32.9% , p < 0.001 ) , and to have significantly higher copd - related medical service costs ( us$3734 vs $ 365 , p < 0.001 ) . similarly , excluded ipr patients , when compared to fsc patients not matched to ipr patients , were older ( 68.6 vs 60.4 years , p < 0.001 ) and more likely to be male ( 58.4% vs 37.0% , p < 0.001 ) , not to have asthma ( 6.6% vs 45.3% , p < 0.001 ) , to have lower use of leukotriene modifiers ( 1.5% vs 14.6% , p < 0.001 ) and sabas ( 6.2% vs 45.1% , p < 0.001 ) , and to have significantly higher copd - related medical service costs ( $ 5437 vs $ 220 , p < 0.001 ) .
utilization and cost outcomes in the 12 months following initiation of maintenance therapy are shown in table 3 and figure 4 , respectively . for the utilization comparisons ,
as described above , some subjects in the original mr cohorts were excluded from the psm cohorts during the matching process .
those excluded were predominantly older ( and costlier ) tio and ipr patients , and younger fsc patients .
this resulted in changes in the frequencies and means for outcomes in all treatment groups in the psm analysis .
for example , in the mr analysis , 3.6% of fsc patients , 4.7% of tio patients , and 7.3% of ipr patients had one or more ed visit ( p < 0.001 for all differences between tio and fsc and between ipr and fsc).19 in the tio - fsc psm analysis ,
3.4% of fsc patients and 4.5% of tio patients had one or more ed visit . in the ipr - fsc psm analysis , 3.8% of fsc patients and
6.6% of ipr patients had one or more ed visit ( p < 0.001 for both comparisons ) .
thus , in both analysis methods , the incidence of ed visits was lower in the fsc group , and differences between treatment groups were similar in magnitude .
the excluded fsc patients had almost no impact on mean cost estimates for patients treated with fsc .
however , for patients treated with tio and ipr , the exclusion of older and sicker patients resulted in lower cost estimates for copd - related medical services and total copd - related costs ( figure 4 ) .
several significant differences between the tio and fsc groups seen in the mr analysis were also seen in the psm analysis .
the fsc group had a lower percentage of patients with an outpatient visit , outpatient visit associated with an oral corticosteroid , ed visit , or hospitalization / ed visit .
in contrast to the mr analysis , the psm analysis found no difference in the percentage of patients with a hospitalization ( p = 0.25 ) or outpatient visit associated with an oral corticosteroid ( p = 0.08 ) .
for each outcome measure , the percentage of patients with an encounter was lower in the fsc cohort than in the tio and ipr cohorts , although , in the psm analysis , because of the excluded younger fsc and older tio patients , the fsc percentages increased slightly and the tio percentages decreased slightly , diminishing the absolute differences between the two groups . with the exception of pharmacy costs , differences in costs that were significant in the mr analysis were also significant in the psm analyses .
fsc was associated with lower medical services costs ( fsc , us$1085 [ 95% ci : $ 10611108 ] ; tio , us$1316 [ 95% ci : $ 12881345 ] ) , and total health care costs compared to tio ( fsc , $ 2037 [ 95% ci : $ 19932081 ] ; tio , us$2267 [ 95% ci : $ 22182316 ] ) .
the original mr analysis found that , in each of the five categories of utilization events , a lower percentage of fsc patients compared to ipr patients experienced events .
these findings were essentially duplicated in the psm analysis , despite the exclusion of 20% of the ipr patients .
( p values for all differences were < 0.001 in the mr analysis , and ranged from < 0.001 to 0.03 in the psm analysis ) .
differences in copd - related costs that were significant in the mr analysis were also significant in the psm analysis .
fsc was associated with higher pharmacy costs ( fsc , $ 917 [ 95% ci : $ 897936 ] ; ipr , us$614 [ 95% ci : $ 601627 ] ) , but lower medical service costs ( fsc , $ 1122 [ 95% ci : $ 10991146 ] ; ipr , us$1746 [ 95% ci : $ 17091784 ] ) , and total costs compared to ipr ( fsc , us$2039 [ 95% ci : $ 19962083 ] ; ipr , us$2360 [ 95% ci : $ 23112411 ] ) .
the mr and psm analyses produced fairly similar ors for various categories of health care utilization , with ors produced by the psm analysis being slightly lower .
for example , in the mr analysis , the statistically significant hospitalization / ed visit ors for tio and ipr ( with respect to fsc ) are 1.28 and 1.72 , respectively ; these values are 1.21 and 1.67 in the psm analysis , respectively .
nonetheless , both analyses show that tio and ipr patients have higher ors , compared to fsc patients , for outpatient visit , outpatient visit with oral corticosteroid , ed visit , and hospitalization / ed visit .
the ipr and fsc comparison also showed higher ors for hospitalization and for an outpatient visit with an antibiotic .
however , while the mr analysis calculated slightly higher odds for hospitalization for tio ( or : 1.19 [ 95% ci : 1.041.37 ] ) compared to fsc , the psm analysis found no difference ( or : 1.10 [ 95% ci : 0.941.28 ] ) , nor was any difference in risk found between tio and fsc for an outpatient visit with an antibiotic ( or : 1.14 [ 95% ci : 0.981.32 ] ) .
the irrs for health care utilization events in the tio and ipr groups with reference to the fsc group are shown in figure 5 .
again , both analytic methods yielded fairly similar irrs , with the psm analysis producing slightly lower irrs for all categories of utilization . in all comparisons in the psm analysis , as in the mr analysis , ipr patients were found to be at significantly higher risk for events , compared to fsc patients .
for the tio group compared to the fsc group , all irrs in the mr analysis were significantly higher .
however , in the psm analysis , irrs for outpatient visits with oral corticosteroid and for hospitalizations were no longer significant .
baseline demographic , clinical , and utilization characteristics of the cohorts after matching on propensity score are shown in table 1 ( tio - fsc ) and table 2 ( ipr - fsc ) , along with p values for unpaired significance tests . paired
significance tests showed similar results although the p values were almost always lower ( data not shown ) . after matching ,
the tio and fsc groups were well balanced with respect to baseline characteristics ; the groups were different only in mean copd - related outpatient visits ( p < 0.001 ) .
matching between the ipr and fsc patients involved more factors . after matching , differences were present for some baseline characteristics : mean copd - related outpatient visits ( p = 0.02 ) , mean all - cause outpatient visits ( p < 0.001 ) , and mean days supply of sabas ( p = 0.007 ) .
figures 2 and 3 show absolute standardized difference percentages for baseline characteristics prior to and after matching .
these graphs further illustrate that , while some significant differences remained after matching , they were small from a clinical standpoint .
absolute standardized percentage differences were less than 10% for all assessed baseline characteristics in both groups , supporting an assessment of balance between groups.22 we compared differences in baseline characteristics between excluded patients and matched patients ( figures 2 and 3 ) . characteristics with large differences between excluded and matched patients tended to be the same characteristics as those associated with large standardized differences prior to matching .
the excluded tio and ipr patients were older , and had more comorbidities and higher health care utilization .
the excluded tio patients , when compared to fsc patients who were not matched to tio patients , were older ( mean , 66.9 vs 60.1 years , p < 0.001 ) and more likely to be male ( 68.7% vs 39.8% , p
< 0.001 ) , not to have asthma ( 4.4% vs 46.8% , p < 0.001 ) , to have lower use of leukotriene modifiers ( 2.4% vs 12.6% , p < 0.001 ) , and sabas ( 15.4% vs 32.9% , p < 0.001 ) , and to have significantly higher copd - related medical service costs ( us$3734 vs $ 365 , p < 0.001 ) .
similarly , excluded ipr patients , when compared to fsc patients not matched to ipr patients , were older ( 68.6 vs 60.4 years , p < 0.001 ) and more likely to be male ( 58.4% vs 37.0% , p < 0.001 ) , not to have asthma ( 6.6% vs 45.3% , p < 0.001 ) , to have lower use of leukotriene modifiers ( 1.5% vs 14.6% , p < 0.001 ) and sabas ( 6.2% vs 45.1% , p < 0.001 ) , and to have significantly higher copd - related medical service costs ( $ 5437 vs $ 220 , p < 0.001 ) .
utilization and cost outcomes in the 12 months following initiation of maintenance therapy are shown in table 3 and figure 4 , respectively . for the utilization comparisons ,
as described above , some subjects in the original mr cohorts were excluded from the psm cohorts during the matching process .
those excluded were predominantly older ( and costlier ) tio and ipr patients , and younger fsc patients .
this resulted in changes in the frequencies and means for outcomes in all treatment groups in the psm analysis .
for example , in the mr analysis , 3.6% of fsc patients , 4.7% of tio patients , and 7.3% of ipr patients had one or more ed visit ( p < 0.001 for all differences between tio and fsc and between ipr and fsc).19 in the tio - fsc psm analysis ,
3.4% of fsc patients and 4.5% of tio patients had one or more ed visit . in the ipr - fsc psm analysis ,
3.8% of fsc patients and 6.6% of ipr patients had one or more ed visit ( p < 0.001 for both comparisons ) .
thus , in both analysis methods , the incidence of ed visits was lower in the fsc group , and differences between treatment groups were similar in magnitude .
the excluded fsc patients had almost no impact on mean cost estimates for patients treated with fsc .
however , for patients treated with tio and ipr , the exclusion of older and sicker patients resulted in lower cost estimates for copd - related medical services and total copd - related costs ( figure 4 ) .
several significant differences between the tio and fsc groups seen in the mr analysis were also seen in the psm analysis .
the fsc group had a lower percentage of patients with an outpatient visit , outpatient visit associated with an oral corticosteroid , ed visit , or hospitalization / ed visit .
in contrast to the mr analysis , the psm analysis found no difference in the percentage of patients with a hospitalization ( p = 0.25 ) or outpatient visit associated with an oral corticosteroid ( p = 0.08 ) .
for each outcome measure , the percentage of patients with an encounter was lower in the fsc cohort than in the tio and ipr cohorts , although , in the psm analysis , because of the excluded younger fsc and older tio patients , the fsc percentages increased slightly and the tio percentages decreased slightly , diminishing the absolute differences between the two groups . with the exception of pharmacy costs , differences in costs that were significant in the mr analysis were also significant in the psm analyses .
fsc was associated with lower medical services costs ( fsc , us$1085 [ 95% ci : $ 10611108 ] ; tio , us$1316 [ 95% ci : $ 12881345 ] ) , and total health care costs compared to tio ( fsc , $ 2037 [ 95% ci : $ 19932081 ] ; tio , us$2267 [ 95% ci : $ 22182316 ] ) .
the original mr analysis found that , in each of the five categories of utilization events , a lower percentage of fsc patients compared to ipr patients experienced events .
these findings were essentially duplicated in the psm analysis , despite the exclusion of 20% of the ipr patients .
( p values for all differences were < 0.001 in the mr analysis , and ranged from < 0.001 to 0.03 in the psm analysis ) .
differences in copd - related costs that were significant in the mr analysis were also significant in the psm analysis .
fsc was associated with higher pharmacy costs ( fsc , $ 917 [ 95% ci : $ 897936 ] ; ipr , us$614 [ 95% ci : $ 601627 ] ) , but lower medical service costs ( fsc , $ 1122 [ 95% ci : $ 10991146 ] ; ipr , us$1746 [ 95% ci : $ 17091784 ] ) , and total costs compared to ipr ( fsc , us$2039 [ 95% ci : $ 19962083 ] ; ipr , us$2360 [ 95% ci : $ 23112411 ] ) .
several significant differences between the tio and fsc groups seen in the mr analysis were also seen in the psm analysis .
the fsc group had a lower percentage of patients with an outpatient visit , outpatient visit associated with an oral corticosteroid , ed visit , or hospitalization / ed visit .
in contrast to the mr analysis , the psm analysis found no difference in the percentage of patients with a hospitalization ( p = 0.25 ) or outpatient visit associated with an oral corticosteroid ( p = 0.08 ) .
for each outcome measure , the percentage of patients with an encounter was lower in the fsc cohort than in the tio and ipr cohorts , although , in the psm analysis , because of the excluded younger fsc and older tio patients , the fsc percentages increased slightly and the tio percentages decreased slightly , diminishing the absolute differences between the two groups . with the exception of pharmacy costs , differences in costs that were significant in the mr analysis were also significant in the psm analyses .
fsc was associated with lower medical services costs ( fsc , us$1085 [ 95% ci : $ 10611108 ] ; tio , us$1316 [ 95% ci : $ 12881345 ] ) , and total health care costs compared to tio ( fsc , $ 2037 [ 95% ci : $ 19932081 ] ; tio , us$2267 [ 95% ci : $ 22182316 ] ) .
the original mr analysis found that , in each of the five categories of utilization events , a lower percentage of fsc patients compared to ipr patients experienced events .
these findings were essentially duplicated in the psm analysis , despite the exclusion of 20% of the ipr patients .
( p values for all differences were < 0.001 in the mr analysis , and ranged from < 0.001 to 0.03 in the psm analysis ) .
differences in copd - related costs that were significant in the mr analysis were also significant in the psm analysis . fsc was associated with higher pharmacy costs ( fsc , $ 917 [ 95% ci : $ 897936 ] ; ipr , us$614 [ 95% ci : $ 601627 ] ) , but lower medical service costs ( fsc , $ 1122 [ 95% ci : $ 10991146 ] ; ipr , us$1746 [ 95% ci : $ 17091784 ] ) , and total costs compared to ipr ( fsc , us$2039 [ 95% ci : $ 19962083 ] ; ipr , us$2360 [ 95% ci : $ 23112411 ] ) .
the mr and psm analyses produced fairly similar ors for various categories of health care utilization , with ors produced by the psm analysis being slightly lower .
for example , in the mr analysis , the statistically significant hospitalization / ed visit ors for tio and ipr ( with respect to fsc ) are 1.28 and 1.72 , respectively ; these values are 1.21 and 1.67 in the psm analysis , respectively .
nonetheless , both analyses show that tio and ipr patients have higher ors , compared to fsc patients , for outpatient visit , outpatient visit with oral corticosteroid , ed visit , and hospitalization / ed visit .
the ipr and fsc comparison also showed higher ors for hospitalization and for an outpatient visit with an antibiotic .
however , while the mr analysis calculated slightly higher odds for hospitalization for tio ( or : 1.19 [ 95% ci : 1.041.37 ] ) compared to fsc , the psm analysis found no difference ( or : 1.10 [ 95% ci : 0.941.28 ] ) , nor was any difference in risk found between tio and fsc for an outpatient visit with an antibiotic ( or : 1.14 [ 95% ci : 0.981.32 ] ) .
the irrs for health care utilization events in the tio and ipr groups with reference to the fsc group are shown in figure 5 .
again , both analytic methods yielded fairly similar irrs , with the psm analysis producing slightly lower irrs for all categories of utilization . in all comparisons in the psm analysis , as in the mr analysis , ipr patients were found to be at significantly higher risk for events , compared to fsc patients . for the tio group compared to the fsc group ,
however , in the psm analysis , irrs for outpatient visits with oral corticosteroid and for hospitalizations were no longer significant .
in this analysis of data from an observational , retrospective cohort study of initial maintenance therapies for copd , we demonstrated the similarity of results using two analytic approaches to observational research .
specifically , we compared results from a psm analysis with those from a previously published , parallel mr analysis.13 we found that both methods yielded similar health care utilization and cost outcomes .
general agreement between mr and psm methods has been found in other studies . in a review of 177 comparative method studies
, sturmer concluded that substantial changes in treatment effects were seen when point estimates were calculated with and without adjustment for covariates , but that the method of adjustment itself mr or psm made little difference.1 in psm , a high degree of propensity score overlap after matching is desirable in terms of internal validity . when overlap is minimal ,
unmeasured confounding bias in treatment groups probably can not be resolved using either mr or psm techniques.1 in the present psm analysis , large proportions of patients in both the tio and ipr cohorts ( 89.1% and 80.2% , respectively ) were matched to fsc patients , and there was substantial overlap in propensity scores between groups . in other words , matching produced cohorts with similar baseline characteristics . in general , the few statistically significant differences remaining after matching were minor in terms of effect size and practical significance , and had small absolute standardized differences .
characteristics that would be expected to considerably skew utilization outcomes , such as comorbid cardiovascular disease , were not different between matched groups .
some differences may be due to the smaller psm sample size , since the excluded patients were a contributing explanatory factor for the lower psm utilization and cost estimates . while the mr analysis was a population - based study , the psm analysis , as a result of the matching process , excluded some younger individuals , with minimal comorbidities , who were treated with fsc , and some older , sicker individuals who were treated with ipr or tio .
exclusion of the older and sicker individuals resulted in lower mean costs for ipr and tio patients , while costs for fsc patients were quite similar in both analyses .
event frequency also may be a factor in the differences in findings between analysis methods .
multivariable logistic regression and propensity matching have been found to produce similar results when events are not infrequent.2325 through simulations , cepeda and colleagues found that the use of propensity scores yielded less biased estimates than multivariable logistic regression only when there were eight or fewer modeled events per covariate.26 when the ratio of modeled events was higher , multivariable logistic regression was the better method .
other studies have determined that ten events per covariate is a desired ratio when using maximum likelihood methods.27 the main outcomes in our analyses , ( copd - related outpatient visits associated with an antibiotic or oral corticosteroid , ed visits , and hospitalizations ) , although of great concern clinically , occur relatively infrequently , from a statistical standpoint , when averaged across a large population of copd patients that is unrestricted in terms of disease severity .
the outcome of outpatient visit with an oral corticosteroid had the smallest number of events per covariate modeled , with 877 of 32,338 patients having at least one event , which translates to 20 events per covariate in the multivariate logistic regression analysis .
this compares to 65 events per covariate for the combined endpoint of hospitalization / ed visit
. the lower ratios of events per covariate for some outcomes may have been a factor in the different findings of the two analyses for ors and irrs . on the other hand , costs ( copd - related medical service costs and pharmacy costs )
were universally incurred , and both analyses found that , compared to fsc , tio and ipr were associated with higher costs for copd - related medical services , and higher total costs , even though costs associated with tio and ipr were reduced in the psm analyses .
both mr and psm methods adjust associations between treatment effects and outcomes to reduce potential bias from observed covariates .
other researchers have reported that results from the two methods appear to be consistent when there is large overlap between groups in propensity for a given treatment , which ensures minimal loss of observations , and when outcomes can be modeled with a relatively large number of events per covariate.1,4,24 our findings support this view and suggest that , with regard to less frequent events , in particular when effect sizes may be small , consideration should be given to analyzing outcomes using both methods , assuming a large proportion of subjects can be matched . while psm is a more transparent method , in the sense that it allows one to see the degree of equality between groups after matching , in this study , psm provided little advantage over mr in terms of the validity of the results . because of the inevitable reduction in sample size and change in overall composition of treatment groups being compared , the choice of whether to use psm or mr will depend on the question being investigated , whether a population effect is being measured , and whether review of a non - representative population of patients receiving treatment is acceptable ( or even preferred ) .
this point is addressed by dagostino , who recommended that when patients are excluded in matched analyses , researchers need to be particularly clear in their descriptions of the included and excluded patients , and of the populations to which study results are applicable.28 strengths of this study include the large sample sizes of both analyses and the high degree of propensity matching , with approximately 80% and 89% of the original ipr and tio cohorts matched , respectively , to fsc patients .
the exclusion of some original subjects from the psm cohorts due to lack of a match does mean , however , that any additional information these subjects might have provided was lost , and statistical power affected .
we measured exacerbations using claims data , defining exacerbations as copd - related health care events . using an alternative definition of exacerbation based on symptoms , lung function , or other clinical parameters could influence observed effect sizes.14 however , we would not expect a different definition of exacerbations to influence effect sizes differently for mr than for psm , or for it to change the overall findings of this study .
since both mr and propensity matched analyses attempt to reduce bias through adjustment using covariates , the ability to do this is dependent on the capture of all relevant factors . in this analysis
was an observational study utilizing administrative claims data , information about patients clinical status was not available .
we could not ascertain lung function status , disease severity , or other clinical characteristics .
however , we did control for two key characteristics of interest disease severity and exacerbation frequency by using prior copd - related health care and pharmacy utilization ( particularly oral corticosteroids / antibiotics ) as proxy measures .
results obtained in our analysis suggest that both mr and psm methods are appropriate analytic techniques for addressing and mitigating bias in observational research . in this example of an observational study of maintenance therapy for copd ,
more than 80% of the original treatment groups used in the mr analysis were matched to a comparison group for the psm analysis .
while some sample size was lost in the psm analysis , results from both methods were similar in direction and statistical significance .
further , this analysis underscores the need for researchers to have a good understanding of the populations undergoing treatment and the factors associated with both receipt of treatment and occurrence of the measured outcomes .
|
purposeto investigate equivalency of results from multivariable regression ( mr ) and propensity score matching ( psm ) models , observational research methods used to mitigate bias stemming from non - randomization ( and consequently unbalanced groups at baseline ) , using , as an example , a large study of chronic obstructive pulmonary disease ( copd ) initial maintenance therapy.methodspatients were 32,338 health plan members , age 40 years , with copd initially treated with fluticasone propionate / salmeterol combination ( fsc ) , tiotropium ( tio ) , or ipratropium ( ipr ) alone or in combination with albuterol . using mr and psm methods , the proportion of patients with copd - related health care utilization , mean costs , odds ratios ( ors ) , and incidence rate ratios ( irrs ) for utilization events were calculated for the 12 months following therapy initiation.resultsof 12,595 fsc , 9126 tio , and 10,617 ipr patients meeting mr inclusion criteria , 89.1% ( 8135 ) of tio and 80.2% ( 8514 ) of ipr patients were matched to fsc patients for the psm analysis .
methods produced substantially similar findings for mean cost comparisons , ors , and irrs for most utilization events .
in contrast to mr , for tio compared to fsc , psm did not produce statistically significant ors for hospitalization or outpatient visit with antibiotic or significant irrs for hospitalization or outpatient visit with oral corticosteroid .
as in the mr analysis , compared to fsc , ors and irrs for all other utilization events , as well as mean costs , were less favorable for ipr and tio.conclusionin this example of an observational study of maintenance therapy for copd , more than 80% of the original treatment groups used in the mr analysis were matched to comparison treatment groups for the psm analysis .
while some sample size was lost in the psm analysis , results from both methods were similar in direction and statistical significance , suggesting that mr and psm were equivalent methods for mitigating bias .
|
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] |
adult soft - shelled turtles ( 2 males and 4 females ) were purchased from a local supplier
tobun ( kamikita , japan ) .
all procedures were done in accordance with the guideline for care
and use of animal experiments and approved by the animal care and use committee at iwate
university .
the animals were anesthetized by intraperitoneal injection of sodium
pentobarbital ( 64.8 mg / kg ) .
two animals ( 1 male and 1 female ) were perfused transcardially
with ringer s solution followed by bouin s solution without acetic acid .
the upper jaw was
dissected , immersed in the same fixative solution overnight at 4c and decalcified in 10%
ethylenediamine tetra - acetic acid ( edta ) in 0.1 m phosphate buffer at 4c for two weeks .
the
specimens were routinely embedded in paraffin and cut at 57 m thick .
sections were stained with pas or ab ( ph 1.0 or ph 2.5 ) , or processed for lectin
histochemistry as described previously . for transmission electron microscopy , samples were collected from four animals ( 1 male and
3 females ) and processed as described previously .
the nasal cavity of soft - shelled turtles was divided into the upper and lower chambers
( fig . 1a and 1bfig . 1.the olfactory organs in soft - shelled turtle .
uc , upper chamber ; lc , lower chamber ; on , olfactory nerve ; ob , olfactory
bulb . coronary section at line b is shown in b. ( b ) the right nasal cavity .
( c ) he stained sections showing upper
chamber epithelium ( upper left ) , the bowman s glands ( lower left , bg ) and the lower
chamber epithelium ( right ) in the nasal cavity of soft - shelled turtles .
the nuclei of
supporting cells ( sp ) , receptor cells ( rc ) and basal cells ( bc ) were situated in the
apical , intermediate and basal layers of the upper and lower chamber epithelia .
( d )
pas , alcian blue ( ab ) ph 1.0 and ab ph 2.5 stainings in the upper chamber epithelium
( top panels ) , lower chamber epithelium ( middle panels ) and the bowman s glands ( bottom
panels ) .
the apical portion of the upper chamber epithelium was stained with pas , ab
ph 1.0 and ab ph 2.5 ( bidirectional arrows ) . in the lower chamber epithelium ,
cytoplasm near the free border ( arrowheads ) was more intensely stained than the
supranuclear region of the supporting cells ( bidirectional arrows ) with pas and ab ph
2.5 .
the bowman s glands were
positive for pas , but negative for ab ph 1.0 and ab ph 2.5 .
the nuclei were
counterstained with nuclear fast red in the sections stained with ab ph 1.0 .
scale
bars : 50 m . ) . both of the upper and lower chamber epithelia consist of three types of
cells , the supporting cells , receptor cells and basal cells ( fig .
nuclei of the supporting , receptor and basal
cells were situated in the apical , middle and basal layers of the epithelium , respectively .
the associated glands ( the bowman s glands ) were observed in the upper chamber epithelium ,
but not in the lower chamber epithelium ( fig .
in addition , staining patterns for pas and ab ph 2.5 differed between the two
epithelia ( fig .
pas and ab ph 2.5 intensely
stained the apical one - third of the upper chamber epithelium .
in contrast , they stained the
apical portion near the free border intensely and the supranuclear region of supporting
cells less intensely in the lower chamber epithelium .
moreover , the upper chamber epithelium
and the lower chamber epithelium were differently stained by ab ph 1.0 : the upper chamber
epithelium was positive for ab ph 1.0 , but the lower chamber epithelium was negative .
the
bowman s glands were positive for pas , but negative for ab ph 1.0 and ab ph 2.5 .
uc , upper chamber ; lc , lower chamber ; on , olfactory nerve ; ob , olfactory
bulb . coronary section at line b is shown in b. ( b ) the right nasal cavity .
( c ) he stained sections showing upper
chamber epithelium ( upper left ) , the bowman s glands ( lower left , bg ) and the lower
chamber epithelium ( right ) in the nasal cavity of soft - shelled turtles .
the nuclei of
supporting cells ( sp ) , receptor cells ( rc ) and basal cells ( bc ) were situated in the
apical , intermediate and basal layers of the upper and lower chamber epithelia .
( d )
pas , alcian blue ( ab ) ph 1.0 and ab ph 2.5 stainings in the upper chamber epithelium
( top panels ) , lower chamber epithelium ( middle panels ) and the bowman s glands ( bottom
panels ) .
the apical portion of the upper chamber epithelium was stained with pas , ab
ph 1.0 and ab ph 2.5 ( bidirectional arrows ) .
in the lower chamber epithelium ,
cytoplasm near the free border ( arrowheads ) was more intensely stained than the
supranuclear region of the supporting cells ( bidirectional arrows ) with pas and ab ph
2.5 .
the bowman s glands were
positive for pas , but negative for ab ph 1.0 and ab ph 2.5 .
the nuclei were
counterstained with nuclear fast red in the sections stained with ab ph 1.0 .
scale
bars : 50 m . in the apical part of the upper chamber epithelium , the dendrites of the receptor cells
bearing cilia on the tip ( fig .
( a ) secretory granules filled the supranuclear region of the supporting cells ( sp )
in the upper chamber epithelium .
( b ) most secretory granules were distributed near
the free border of supporting cells in the lower chamber epithelium .
arrows in a and
b indicate receptor cells bearing cilia on the tip of the dendrites .
( c ) secretory
granules of the supporting cells in the upper chamber epithelium , 12
m in diameter , consisted of the marginal part of moderate
density and central part of low density .
some of the secretory granules contained a
core of high density ( arrowheads ) .
( d ) secretory granules of the supporting cells in
the lower chamber epithelium , 0.51 m in diameter , consisted of
two parts of moderate and high density .
( e ) the glandular cells in the bowman s
glands contained secretory granules of high density .
scale bars : 2
m in a and b , 0.5 m in c e .
, arrows ) alternated with the cytoplasm of supporting cells bearing
microvilli on the free border ( fig .
the secretory
granules of supporting cells in the upper chamber epithelium , measuring 12
m in diameter , showed a bipartite structure with the marginal part
displaying moderate density and central part displaying low density ( fig . 2c ) .
in addition , a core of high density was observed in some
secretory granules ( fig . 2c , arrowheads ) . in the
apical part of lower chamber epithelium , the dendrites of receptor cells bearing cilia
alternated with the cytoplasm of supporting cells bearing microvilli ( fig .
most secretory granules were distributed near the
free border of supporting cells in the lower chamber epithelium ( fig .
the secretory granules of supporting cells , 0.51
m in diameter , showed a bipartite structure of moderate and high
densities ( fig .
secretory granules of high
density , approximately 1 m in diameter , were abundant in the glandular
cells of the bowman s gland ( fig .
( a ) secretory granules filled the supranuclear region of the supporting cells ( sp )
in the upper chamber epithelium .
( b ) most secretory granules were distributed near
the free border of supporting cells in the lower chamber epithelium .
arrows in a and
b indicate receptor cells bearing cilia on the tip of the dendrites .
( c ) secretory
granules of the supporting cells in the upper chamber epithelium , 12
m in diameter , consisted of the marginal part of moderate
density and central part of low density .
some of the secretory granules contained a
core of high density ( arrowheads ) .
( d ) secretory granules of the supporting cells in
the lower chamber epithelium , 0.51 m in diameter , consisted of
two parts of moderate and high density .
( e ) the glandular cells in the bowman s
glands contained secretory granules of high density .
scale bars : 2
m in a and b , 0.5 m in c e . cellular expression of carbohydrate chains
staining patterns for lectins were evaluated in each region where the secretory
granules were observed by transmission electron microscopy : the apical one - third of the
upper chamber epithelium ( fig . 3afig .
( a ) the apical
portions of the upper chamber epithelium ( top panels ) and lower chamber epithelium
( bottom panels ) .
bidirectional arrows indicate the supranuclear region , and arrowheads
indicate the cytoplasmic region near the free border of the supporting cells .
( left
panels ) uea - i stained the supporting cells in the lower chamber epithelium more
intensely than those in the upper chamber epithelium .
( middle panels ) rca120 equally
stained the supporting cells in the upper chamber epithelium and those in the lower
chamber epithelium .
( right panels ) lel stained the supporting cells in the upper
chamber epithelium more intensely than those in the lower chamber epithelium .
( b )
bowman s glands were stained intensely by swga , moderately by vva and weakly by pna .
scale bars : 25 m . , top panels ) , the superficial portion ( fig .
3a , bottom panels , bidirectional arrows ) of the lower chamber
epithelium , and the glandular cells of the bowman s gland ( fig .
eighteen lectins stained the secretory granules of
supporting cells in the upper and/or lower chamber epithelium ( table 1table 1.lectin binding patterns in the supporting cells of the upper and lower
chamber epithelia and the bowman s glandslectin ( abbreviation)upper chamber epitheliumlower chamber epitheliumbowman s glandswheat germ agglutinin ( wga)+++++succinylated wheat germ agglutinin ( s - wga)+/+/++lycopersicon esculentum lectin ( lel)++++solanum tuberosum lectin ( stl)++/+datura stramonium lectin ( dsl)++++bandeiraea simplicifolia lectin - ii ( bsl - ii)++dolichos biflous agglutinin ( dba)+++soybean agglutinin ( sba)+/++bandeiraea simplicifolia lectin - i ( bsl - i)+/+/vicia villosa agglutinin ( vva)++++sophora japonica agglutinin ( sja)ricinus communis agglutinin - i ( rca120)+++++jacalin++/peanut agglutinin ( pna)+++/erythrina cristagalli lectin ( ecl)+++ulex europaeus agglutinin - i ( uea - i)+/+++concanavalin a ( con a)++++++pisum satibum agglutinin ( psa)++lens culinaris agglutinin ( lca)++phaseolus vulgaris agglutinin - e ( pha - e)+++phaseolus vulgaris agglutinin - l ( pha - l)+/ , negative staining ; + / , faint staining ; + , moderate staining ; + + , intense
staining . ) . among them , 14 lectins stained the supporting cells in the upper chamber
epithelium and those in the lower chamber epithelium differently .
eleven lectins , including
dsl , dba , sba , bsl - i , vva , jacalin , pna , uea - i , psa , lca and pha - e , stained the supporting
cells in the lower chamber epithelium more intensely than those in the upper chamber
epithelium ( fig .
four lectins ,
including swga , rca120 , ecl and cona , stained the supporting cells in the upper chamber
epithelium and those in the lower chamber epithelium equally ( fig .
three lectins , including wga , lel and stl , stained
the supporting cells in the upper chamber epithelium more intensely than those in the lower
chamber epithelium ( fig .
twenty
lectins , excluding sja , stained the secretory granules in the glandular cells of the
bowman s glands ( table 1 ) .
five lectins ,
including wga , swga , bsl - ii , dba and cona , stained the bowman s glands intensely .
eleven
lectins , including lel , stl , dsl , sba , vva , rca120 , ecl , uea - i , psa , lca and pha - e , stained
moderately , and 4 lectins , including bsl - i , jacalin , pna and pha - l , stained weakly ( fig .
fourteen lectins stained
the supporting cells of upper chamber epithelium and the bowman s glands differently ( table 1 ) .
( a ) the apical
portions of the upper chamber epithelium ( top panels ) and lower chamber epithelium
( bottom panels ) .
bidirectional arrows indicate the supranuclear region , and arrowheads
indicate the cytoplasmic region near the free border of the supporting cells .
( left
panels ) uea - i stained the supporting cells in the lower chamber epithelium more
intensely than those in the upper chamber epithelium .
( middle panels ) rca120 equally
stained the supporting cells in the upper chamber epithelium and those in the lower
chamber epithelium .
( right panels ) lel stained the supporting cells in the upper
chamber epithelium more intensely than those in the lower chamber epithelium .
( b )
bowman s glands were stained intensely by swga , moderately by vva and weakly by pna .
, negative staining ; + / , faint staining ; + , moderate staining ; + + , intense
staining .
as we demonstrated in this study , the supporting cells contained secretory granules both in
the upper chamber epithelium and lower chamber epithelium in the nasal cavity of
soft - shelled turtles .
supporting cells of the upper chamber epithelium contained secretory
granules more abundantly than those of the lower chamber epithelium : the secretory granules
filled the supranuclear region of the supporting cells in the upper chamber epithelium :
whereas , most of the secretory granules were localized near the free border and were
scarcely distributed in the supranuclear region of the supporting cells in the lower chamber
epithelium . in addition , the upper chamber epithelium possessed the associated glands , but
the lower chamber epithelium did not . these evidences
suggest variable lubrication ability
between the upper and lower chamber epithelia : secretory activity is higher in the upper
chamber epithelium than in the lower chamber epithelium . in squamates ,
the presence or
absence of associated glands and the amount of the secretory granules are different between
the oe and the vne [ 21 , 27 ] .
for example , the oes of snakes , gekkos and scincomorpha associate
with glands and contain abundant secretory granules in the supporting cells : whereas , their
vnes lack the associated glands and contain few secretory granules in the supporting cells
[ 25 , 26 ] .
the lumen of squamate vno is always filled with fluids , which have both an intrinsic source
and , at least partially , an extrinsic source ( most likely the harderian glands ) . in the case
of soft - shelled turtles ,
the surface of the lower chamber epithelium is always in contact
with extrinsic water , possibly leading to the lower secretory activity in the lower chamber
epithelium as compared with the upper chamber epithelium . in general
, the secretory products of exocrine cells are classified into 3 types : serous
type containing neutral mucopolysaccharides , mucous type containing acid mucopolysaccharides
and seromucous type containing both neutral and acid mucopolysaccarides .
the secretory granules in the bowman s glands of
soft - shelled turtles have been suggested to be serous , because they were pas - positive and
ab - negative and showed high electron - density .
the bowman s glands , which present in all
vertebrate species except fish and some amphibians , are generally serous in amphibians ,
reptiles and birds . also , in the soft - shelled
turtles , the bowman s glands contain serous secretory granules , suggesting that their
secretory products play a significant role shared among non - mammalian bowman s glands .
on
the other hand , the supporting cells of both upper chamber epithelium and lower chamber
epithelium were positive for pas and ab ph 2.5 , suggesting that they contain mucous or
seromucous secretory granules .
by the electron microscopic observation , the supporting cells
of both chamber epithelia have been demonstrated to contain secretory granules of
heterogeneous electron - density , suggesting that they are seromucous .
furthermore , supporting
cells of upper chamber epithelium and lower chamber epithelium were suggested to contain
secretory granules of different compositions , because they exhibited inconsistent staining
for ab ph 1.0 . in order to make further confirmation for the differences in the properties of secretory
granules
, we analyzed the expression of carbohydrate chains by lectin histochemistry in the
area where secretory granules were distributed , and compared them between supporting cells
of upper chamber epithelium and those of the lower chamber epithelium .
although 4 lectins
stained the supporting cells of both chamber epithelia equally , 14 lectins stained the
supporting cells of either upper or lower chamber epithelium more intensely . among them , 3
lectins stained the supporting cells of upper chamber epithelium more intensely than those
of the lower chamber epithelium , and 11 lectins stained the supporting cells of lower
chamber epithelium more intensely than those of the upper chamber epithelium .
although
electron - microscopic data indicated that the amount of the secretory granules in the
supporting cells of lower chamber epithelium is apparently smaller than those of the upper
chamber epithelium , most lectins stained the supporting cells of lower chamber epithelium
more intensely than those of the upper chamber epithelium .
thus , it seemed that the
differences in the lectin staining between them are not due to the differences in the amount
of secretory granules but instead due to the differences in their carbohydrate expressions ,
i.e. the differences in the properties of secretory products .
in addition to the differences in staining patterns for pas and ab or in the
electron - density , the secretory granules in the bowman s glands showed different binding
patterns to lectins from the patterns of the supporting cells in the upper and lower chamber
epithelia .
for example , supporting cells of both epithelia were negative for bsl - ii and
pha - l , whereas the bowman s glands were positive .
the supporting cells of both epithelia
were weakly positive for s - wga , whereas the bowman s glands were intensely positive .
these
evidences suggest that the properties of secretory granules are different among them . in this study
, we revealed that the associated glands are present only in the upper chamber
epithelium , that the supporting cells of upper chamber epithelium contain secretory granules
more abundantly than those of the lower chamber epithelium and that the properties of
secretory granules in the supporting cells are different between the upper and lower chamber
epithelia in the nasal cavity of soft - shelled turtles .
the fluid layer covering the surface
of olfactory sensory epithelium plays important roles in olfaction as mentioned above , and
the properties of fluid layer differ among functionally distinct epithelia .
considering the
lubricating ability as one of the indices for olfactory functions , differences in the
olfactory function are suggested by the differences in the amount and properties of
secretory granules between the upper and lower chamber epithelia in the olfactory organ of
soft - shelled turtles . in general ,
semi - aquatic turtles show sniffing behavior under water as
well as on land .
even when turtles are under water and the lower chamber is filled with
water , the upper chamber could be filled with air owing to the structure of nasal cavity .
thus , in the nasal cavity of semi - aquatic turtles , the upper and lower chamber epithelia
could be considered as the olfactory organs that function in the air and under water ,
respectively [ 5 , 28 ] .
also , in some amphibians , the olfactory organ that functions in the air is
different from the olfactory organ that functions under water in the amount and properties
of secretory granules .
two distinct sensory epithelia present in the main olfactory system
of adult xenopus laevis , the oe ( functioning in the air ) and the middle
chamber epithelium ( functioning under water ) , are such examples [ 9 , 10 ] : the bowman s glands are
present , and the supporting cells contain abundant secretory granules in the oe ; whereas ,
associated glands are not present , and the supporting cells contain few secretory granules
in the middle chamber epithelium [ 14 , 24 ] .
the xenopus oe functions under
water in the larval stage and functions in the air during adulthood . during metamorphosis ,
the bowman s glands are formed , the amount and size of the secretory granules increase , and
their electron - density and lectin - binding patterns change in the supporting cells of the oe
[ 6 , 14 , 33 ] .
also , in the olfactory organs of soft - shelled
turtles , the upper and lower chamber epithelia seem to be the olfactory organs that function
in the air and under water , respectively .
|
in general , the nasal cavity of turtles is divided into two chambers : the upper chamber ,
lined with the olfactory epithelium containing ciliated olfactory receptor cells , and the
lower chamber , lined with the vomeronasal epithelium containing microvillous receptor
cells . in the nasal cavity of soft - shelled turtles , however , differences between the upper
and lower chamber epithelia are unclear due to the presence of ciliated receptor cells in
both epithelia . in the olfactory organ of vertebrates ,
the surface of sensory epithelium
is covered with secretory products of associated glands and supporting cells , playing
important roles in the olfaction by dissolving odorants and transporting them to the
olfactory receptors . here ,
the associated glands and supporting cells in the olfactory
organ of soft - shelled turtles were analyzed histochemically and ultrastructurally .
the
upper chamber epithelium possessed associated glands , constituted by cells containing
serous secretory granules ; whereas , the lower chamber epithelium did not . in the upper
chamber epithelium
, secretory granules filled the supranuclear region of supporting cells ,
while most of the granules were distributed near the free border of supporting cells in
the lower chamber epithelium .
the secretory granules in the supporting cells of both
epithelia were seromucous , but alcian blue stained them differently from each other . in
addition , distinct expression of carbohydrates was suggested by the differences in lectin
binding .
these data indicate the quantitative and qualitative differences in the secretory
properties between the upper and lower chamber epithelia , suggesting their distinct roles
in the olfaction .
|
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] |
they are congenital benign tumor accounting for 15 - 20 % of intraventricular mass but only about 1% of intracranial ones ( 1 - 3 ) .
they can be diagnosed at any age but usually become symptomatic in the third to sixth decades and more common in men than women .
they usually found incidentally and are asymptomatic ; but in some instances may associate with rapid neurologic deterioration , herniation , and sudden death .
so , recognition of this rare but important diagnosis may result in decreasing mortality ( 4 ) . in this report
, we presented a 13-year - old boy with complaint of two times drop attack and final diagnosis of colloid cyst in the third brain ventricle .
a 13-year - old boy was brought to the emergency department ( ed ) with complaint of two times drop attack .
the patient mentioned that he felt sudden weakness in both lower limbs , which led to drop .
these attacks were happened about 4 hours before admission and he did not have any same experience previously .
he did not have complaint of nausea , vomiting , headache , vertigo , blurred vision , or palpitation .
the subject did not have any known structural or congenital heart disease , but suffered from asthma and used salbutamol spray irregularly .
the patient did not have trauma history and there was no positive history of any known medical illness in his parents or closed relatives . on arrival , he had 36.9c axillary temperature , 16/minute respiratory rate , 90/minute pulse rate , 120/80 mmhg blood pressure , and 96% oxygen saturation at room air . on physical examination , he did not have focal neurologic findings or even paresthesia or paraplegia .
general examination of head and neck , chest , abdomen , and limbs did not reveal any positive findings .
following the evaluation process , a brain computed tomography ( ct ) was performed ( figure-1 ) .
a hyperdense round lesion was seen in the third ventricle consequently caused that the corresponding physician requested a neurologic consultation in the ed . finally , the brain magnetic resonance imaging ( mri ) confirmed the diagnosis of third ventricle colloid cyst and the patient was underwent surgery and discharged without any problem ( figure 2 ) .
the colloid cysts commonly settle near the foramen of monro in the anterior third ventricle and so may encounter with drainage of the cerebrospinal fluid ( csf ) ( 3 ) .
since even a small lesion can block the mentioned foramen , these cysts may result in hydrocephalus and increase the intra cranial pressure ( 5 ) .
increased intracranial pressure can be manifested with headache described as severe and intermittent , with short duration , usually located frontally .
in contrast with usual headaches , secondary to intracranial tumors , the colloid cyst induced headache can be relieved by lying down ( 4 ) .
other symptoms include drop attacks , gait abnormalities , progressive dementia , and transient loss of consciousness . in children ,
the most common symptoms are nausea , vomiting , headache , diplopia , and papilledema ( 6 ) .
the classic clinical description of intermittent headaches and drop attacks occurs in only one - third of patients .
sudden obstruction of the ventricular system and following rapid rising of intracranial pressure can lead to herniation and rarely sudden death ( 7 , 8) .
colloid cysts size varies from 3 - 40 millimeters in diameter , but the size do not related to their symptoms or outcome , as even small ones may lead to sudden death ( 9 ) .
colloid cyst is usually diagnosed by non - contrast computed tomography ( ct ) as an oval or rounded hyperdense mass on the anterior aspect of the third ventricle .
they may occasionally be hypodense or isodense to the brain , or found in other areas of the brain .
colloid cysts have different manifestation on mri . despite their variable signal characteristics , their location and shape help to the correct preoperative diagnosis in most patients ( 6 ) .
half of the cases are hyperintense on t1-weighted mri images and hypointense on t2-weighted mri images respected to brain .
isointense cysts are not easily identified on mri , and in such instances ct scan is more useful ( 9 , 10 ) .
small asymptomatic colloid cyst can be considered for close follow up by serial examinations and neuroimaging ( 11 ) .
there is also the rare report of spontaneous resolving of the third ventricle colloid cyst ( 12 , 13 ) .
all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors .
|
colloid cysts are mucous - filled masses with an outer fibrous layer .
these cysts are rare developmental malformation and not a true neoplasm .
they usually found incidentally and are asymptomatic ; but in some cases may associate with rapid neurologic deterioration , herniation , and sudden death .
recognition of this rare but important diagnosis may result in decreasing mortality . in this report
, we presented a 13-year - old boy with complaint of two times drop attack and final diagnosis of colloid cyst in the third brain ventricle .
|
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microbial contamination and side effects of synthetic antioxidants are two important major concerns of food and pharmaceutical industries .
increasing propensity for replacing synthetic antioxidant by natural one on one side and development of microbial resistance to existing antibiotics from the other has encouraged researchers toward appraising medicinal plants for dual antioxidant and antimicrobial properties .
though , since immemorial time , medicinal plants have been used to treat and prevent various human ailments and they are considered as reservoir of bioactive compounds.1 , 2 till date , biological properties and bioactive compounds of many medicinal plants are not studied .
extensive investigation of medicinal plants for biological activities and bioactive compounds is the crucial and the foremost step in development of effective alternative medications . in view of this , in the present study , bergenia ciliata sternb .
( family saxifragaceae ) , a high value plant of the sikkim himalaya , has been investigated for antioxidant , antimicrobial activity and bioactive compounds .
it is an important perennial medicinal herb that grows widely in the temperate himalaya between 1500 and 3000 m asl .
the plant has been in use as folklore medicine since ancient times for dissolution of kidney and gall bladder stones.4 , 5 in sikkim himalaya , bergenia rhizome is used to treat fractured bones , fresh cuts , wounds , diarrhea , pulmonary infections , vomiting , fever , cough and boils by locales.6 , 7 , 8
bergenia is also used for the treatment of heart disease , haemorrhoids , stomach disorders and ophthalmia . in addition
, it is accredited with analgesic , antiviral , anti - inflammatory and antimalarial properties.9 , 10 the plant 's virtues , to a large extent , are attributed to its secondary metabolites such as bergenin , gallic acid and catechin which are therapeutic and account for its use in traditional medicine.11 , 12 despite the widespread use of this plant in traditional medicine , the scientific literature with respect to its biological properties is scanty . to the best of our knowledge , so far , antioxidant , antimicrobial properties and the bioactive compounds associated with the leaf of bergenia plant , growing in the sikkim himalaya , have not been investigated . therefore , the present study was conducted with the objectives to ( 1 ) obtain the most effective solvent for extracting the potent bioactive compounds , especially phenolics and flavonoids , ( 2 ) investigate different extracts for antioxidant and antimicrobial activities and , ( 3 ) quantify the amount of bergenin , catechin and gallic acid present in the leaf extracts of b. ciliata .
leaves of b. ciliata were collected during flowering season ( march 2014 ) from the plants growing in the arboretum of g.b .
pant institute of himalayan environment and development ( gbpihed ) , sikkim unit , gangtok , india ( latitude 27 21 35.7n ; longitude 88 37 24.4e ) , situated at the elevation of 2047 m asl .
the leaves were washed thoroughly under running tap water and finally with distilled water and dried on blotting paper at room temperature to get consistent weight .
2 g powder was soaked separately in 10 ml of different solvents ( ethyl acetate , methanol and hexane ) for 24 h. the supernatant was transferred into a new tube and the residue was re - extracted twice with 10 ml solvent .
each extract was resuspended in the methanol to yield a 50 mg / ml stock solution .
the yield of the extraction was calculated from { ( w1/w2 ) 100 } , where w1 is the weight of extract after evaporation of solvent and w2 is the dry weight of the plant sample .
solutions of each extract ( 100 l ; 1 mg / ml ) were taken individually in test tubes . to this solution , 2.5 ml of 10-fold diluted folin
after 3 min , 2.0 ml of 7.5 % na2co3 solution was added and the mixtures were incubated for 30 min .
the absorbance of the reaction mixtures was measured at 760 nm by using a spectrophotometer ( uv-1800 , shimadzu , kyoto , japan ) .
gallic acid was used as a standard and tpc of bergenia extracts was expressed in milligram gallic acid equivalents ( mg gae / g extract ) .
total flavonoid content was determined by the aluminum chloride calorimetric method , with some modifications .
then , the sample solution ( 2 ml ) was mixed with 2 ml of 2% alcl3 .
after 10 min of incubation at ambient temperature , the absorbance of the solution was measured at 435 nm by using a spectrophotometer ( uv-1800 , shimadzu , kyoto , japan ) .
the flavonoid content was expressed as milligram quercetin equivalent ( mg qe / g extract ) .
the effect of extracts on 2,2-diphenyl-1-picrylhydrazyl ( dpph ) radical was determined using the method of liyana - pathiranan and shahidi .
a solution of 0.135 mm dpph in methanol was prepared and 1.5 ml of this solution was mixed with 1.5 ml of extract in methanol .
the reaction mixture was mixed thoroughly and left in the dark at room temperature for 30 min .
the ability of sample to scavenge dpph radical was calculated by the following equation : dpph radical scavenging activity ( % ) = [ ( abs control abs sample)]/(abs control ) 100where abs control is the absorbance of dpph radical + methanol ; abs sample is the absorbance of dpph radical + extract / standard .
ic50 value is the concentration of extracts at which dpph radicals are scavenged by 50% .
the lower ic50 value indicates higher radical scavenging capacity and vice versa . the 2,2-azino - bis(3-ethylbenzothiazoline-6-sulfonic acid )
the abts radical cation solution was prepared by mixing equal quantities of abts ( 7 mm ) and ammonium persulfate ( 2.45 mm ) and allowing them to react for 1620 h at room temperature , in dark .
the working solution was then prepared by diluting the previous solution with methanol until the absorbance at 734 nm was 0.706 0.02 .
plant extracts ( 1.5 ml ) were allowed to react with equal volume of the abts working solution and the absorbance was taken at 734 nm after 7 min using the spectrophotometer .
the abts scavenging capacity of the extracts was compared with that of bht and the percentage inhibition was calculated as : abts radical scavenging activity ( % ) = [ ( abs control abs sample)]/(abs control ) 100where abs control is the absorbance of abts radical + methanol ; abs sample is the absorbance of abts radical + extract / standard .
abts radical scavenging activity of extracts was determined by ic50 value as mentioned above in dpph assay .
total eight strains ( 4 bacteria , 2 actinomycetes and 2 fungi ) were taken from microbial culture collection established in the microbiology laboratory of gbpihed institute , almora , india .
initially test organisms were grown on the respective media i.e. bacteria and actinomycetes in tryptone yeast extract ( ty ) and fungus on potato dextrose broth ( pd ) in conical flask , at 25c for 24 h. antimicrobial activity was performed on ty and pd agar plates following disc diffusion assay at 25c , 2 days for bacteria and 6 days for actinomycetes and fungus . minimum inhibitory concentration ( mic ) of extracts was determined following the protocol of clinical and laboratory standards institute ( clsi).17 , 18 all of the experiments were performed in triplicate , and results were expressed as diameters ( mm ) of inhibition .
detection and quantification of catechin , gallic acid and bergenin were carried out using shimadzu 20 ad , hplc system ( shimadzu , japan ) consisted of uv detector , a binary pump , a 10 l injection loop , and reverse phase c-18 column of dimensions 4.6 250 mm .
the mobile phase used for catechin was 75% a ( water + 0.1% trifluoroacetic acid ) and 25 % b ( methanol ) with a flow rate of 0.8 ml / min .
the eluted samples were detected by the uv detector at 280 nm . for gallic acid ,
mobile phase used was 90 % a ( water + 1% acetic acid ) and 10% b ( acetonitrile ) with a flow rate of 1.0 ml / min .
for analysis of bergenin , elution was carried out with solvent 75% a ( water + 0.1% trifluoroacetic acid ) and 25% b ( acetonitrile ) as mobile phase having flow rate of 1.0 ml / min .
calibration curve was constructed by plotting the peak area ( y ) against concentration in g / ml of standard solutions ( x ) . the standard equation obtained from the curve
all the experiments were performed in triplicate and the experimental data were expressed as mean standard deviation ( sd ) .
one - way analysis of variance ( anova ) and duncan 's multiple range tests were carried out to determine significant differences ( p < 0.05 ) between the means by spss ( version 16.0 ) .
results showed that leaf extraction yield of b. ciliata varied considerably as a function of solvent nature and ranged from 8.3 to 38.2 % with a descending order of methanol > ethyl acetate > hexane ( table 1 ) .
extraction with methanol resulted in the highest amount of total extractable compounds whereas the extraction yield with hexane was small in comparison to other solvents .
higher extraction yield in methanol might be due to the fact that it easily penetrates the cellular membrane and extracts the intracellular ingredients from the plant material .
moreover , results indicated that the plant contains more of polar substances than the others .
earlier reports have also suggested that methanol give higher extraction yield than the other solvents such as acetone , diethyl ether , ethyl acetate and water.19 , 20 the antioxidant activity of medicinal plants , fruits and vegetables has been reported to be positively correlated to their total phenolic contents due to their ability to scavenge free radicals.21 , 22 it becomes , therefore , mandatory to estimate total phenolic compounds present in the bergenia extracts . in the present study , the content of extractable phenolic compounds was determined through a linear gallic acid standard curve ( y = 2.271x + 0.18 ; r = 0.995 ) .
the highest content of total phenolic compounds was detected in the bergenia methanolic extract ( 473.4 mg
gae / g extract ) followed by ethyl acetate extract ( 249.7 mg gae / g extract ) ( p < 0.05 ) . in the hexane
these results demonstrate clearly that the content of phenolic compounds is dependent on the polarity of the solvent used ; higher the polarity of the solvent , higher the content of phenolic compounds .
these results are in agreement with the previous study of singh et al . that reported methanol as an effective solvent for antioxidant extraction , phenolic compounds in particular .
flavonoids are common secondary metabolites present in plants which are responsible for many plant biological activities .
the solvent efficiency on tfc , in ascending order was : hexane < methanol < ethyl acetate .
the highest flavonoid content of 208.4 mg qe / g extract was observed in the extract of ethyl acetate followed by methanol extract ( 89.9 qe / g extract ) .
these results are in agreement with the report of hajji et al . which reported that flavonoid content in extract depend on solvent polarity .
the dpph free radical scavenging activity in leaf extracts of b. ciliata is presented in fig . 1 .
the methanolic leaf extract showed excellent free radical scavenging activity ( ic50 = 53.5 g / ml ) .
in other two leaf solvent extracts , the free radical scavenging activity in ethyl acetate extract ( ic50 = 2593.3 g / ml ) was superior to that of the hexane extract ( ic50 = 3026.7 g / ml ) .
nevertheless , when compared to standard , the bht , all the tested bergenia leaf extracts showed significantly ( p < 0.05 ) lower dpph radical scavenging activity .
these results are indicative of the influence of the solvent on the antioxidant activity of the biological extracts . it has been reported that in plant compounds with different polarity and structure are present that dissolve in specific solvents having similar polarity.24 , 25 the difference in the dpph radical scavenging activity in different solvent extracts implies towards the preference of the solvents for extraction of different types and concentrations of bioactive compounds.24 , 25 strong dpph radical scavenging activity of b. ciliata methanolic extract can be due to higher content of total phenolic compounds . the free radical scavenging activity of bergenia extracts was also determined using abts radical .
significant difference ( p < 0.05 ) was revealed between abts scavenging capacities of extracts measured as ic50 value ( fig . 2 ) .
the highest abts radical scavenging activity was found in methanol extract with an ic50 value of 5.4 g / ml , whereas hexane extract exerted the lowest ability to scavenge abts radical with an ic50 value of 51 g / ml .
strong abts radical scavenging ability of b. ciliata methanol and ethyl acetate extracts can be attributed to the presence of flavonoids .
earlier , it has been reported that abts radical scavenging ability of bioactive compounds depends on its molecular weight , structure and presence of number of aromatic rings .
the correlation coefficients determined between the antioxidant activities and the total phenolic and flavonoid contents in all the b. ciliata leaf extracts revealed the presence of significant high correlation ( p < 0.1 ) .
the correlation for dpph ic50 value vs tpc and dpph ic50 value vs tfc were 0.913 and 0.056 , respectively .
results indicate clearly that the dpph activities of extracts are mainly due to the total phenolic compounds .
negative correlation indicated that with the increase in phenolic and flavonoid content , concentration of extract required to inhibit free radical decreases .
the correlation for abts ic50 value vs tpc and abts ic50 value vs tfc were 0.911 and 0.782 , respectively , imply that both the total phenol and flavonoids of b. ciliata are responsible for abts activities .
these differences in correlation between phytochemicals and antioxidant assays could be attributed to the different mechanism of the radical antioxidant reaction . in accordance with this study , li et al .
have also reported high negative linear correlation between the phenolic contents and ic50 values in angelicae sinensis confirming that phenolics are likely to contribute to the antioxidant activity of the extracts .
however , presence of other metabolites which have significantly contributed in antioxidant activity of b. ciliata extracts can not be ruled out .
the results of the disc diffusion assay ( table 2 ) revealed that all the extracts tested were effective against the bacteria and actinomycetes studied , but showed no activity against fungi . among the tested extracts ,
methanol extract was found to be the most effective which showed the highest effects against bacillus megaterium strain with inhibition zone of 9.8 mm , followed by nocardia tenerifensis and bacillus subtilis with inhibition zones of 9.7 and 8.8 mm , respectively .
the ethyl acetate extract showed the similar pattern of microbial inhibition with highest inhibition zone of 7.5 mm for b. megaterium , followed by 6.2 and 5.5 mm inhibition zone for n. tenerifensis and b. subtilis , respectively .
in contrary to methanol and ethyl acetate extract , the hexane extract had the greatest inhibitory effects against serratia marcescense ( 5.0 mm ) and b. subtilis ( 4.7 mm ) .
the minimum inhibitory concentration ( mic ) was determined in the methanolic extracts of b. ciliata due to its higher antimicrobial activity in disc diffusion assay against the tested organisms .
the methanol extract gave the lowest minimal inhibitory concentration against b. subtilis with mic of 1250 g / ml ( table 3 ) .
the highest antimicrobial activity of methanolic extract could be due to the high contents of phenolic compounds and flavonoids present in the extract .
these results are in accordance with a previous study where similar observations were recorded in case of bergenia ligulata leaf extracts .
b. ciliata is reported to contain a wide array of pharmaceutically important bioactive compounds.9 , 29 , 30 among all , three main compounds viz .
, bergenin catechin and gallic acid are the most sought after compounds of the plant species . during the past years , efforts have been made for identification and quantification of the major bioactive compounds from bergenia spp.5 , 28 , 30 , 31 , 32 however , till date , b. ciliata plant of the sikkim himalaya has never been analyzed for secondary metabolites . to the best of our knowledge ,
this is the first report on this aspect . in the present study , different extracts of b.
ciliata were analyzed by hplc for the identification and quantification of bergenin , catechin and gallic acid .
standards showed high linearity at tested concentrations with correlation coefficients ( r ) of 0.99 , 0.96 and 0.99 for catechin , gallic acid and bergenin , respectively .
the chromatographic peaks of the analytes were confirmed by comparing their retention time with those of the standards . from the standard equation obtained ( table 4 ) , the amount of bergenin , catechin and gallic acid calculated in different extracts is presented in table 5 .
bergenin was detected in all the extracts and amount of bergenin was found in the order : methanol > ethyl acetate > hexane .
the methanol extract contained bergenin ( 2.43 0.08% ) in amounts that were orders of magnitude higher than those in the ethyl acetate ( 1.63 0.17% ) and hexane ( 0.0024 0.00% ) extracts .
hplc analysis of catechin and gallic acid revealed that these compounds were present in methanol extracts only ( table 5 ) .
in this study , the antioxidant and antimicrobial activities of b. ciliata plant growing in the sikkim himalaya are reported for the first time .
the methanolic extracts of b. ciliata leaves , that were shown to contain the highest total phenolic and flavonoid compounds , exhibited high free radical scavenging activity against dpph and abts radicals .
further , hplc analysis of different extracts for catechin , gallic acid , and bergenin showed that methanolic extract contained significantly higher amount of these bioactive compounds .
|
bergenia ciliata sternb . , commonly known as paashaanbhed , is a well known herb of sikkim himalaya with various pharmaceutical properties .
however , scientific exploration of b. ciliata , growing in the sikkim himalaya , for phytochemicals and pharmacological properties is in infancy . with this view
, the present study was undertaken to investigate b. ciliata leaf extracts for antioxidant , antimicrobial activity and bioactive compounds .
three solvents viz . ,
methanol , ethyl acetate and hexane were used for extraction and the respective leaf extracts were analyzed for total phenolic and flavonoid contents along with the antioxidant and antimicrobial activities . amongst the tested solvents ,
methanol was found to be the best solvent for extraction with highest total phenolic contents and the lowest ic50 values for the 2,2-diphenyl-1-picrylhydrazyl ( dpph ) and 2,2-azino - bis(3-ethylbenzothiazoline-6-sulfonic acid ) ( abts ) assays .
methanol extract also exhibited effective antimicrobial activity , particularly against bacteria and actinomycetes .
further , high performance liquid chromatography ( hplc ) analysis revealed that methanolic extract contains the highest amount of all the three analyzed bioactive compounds viz .
bergenin , catechin and gallic acid .
the current study suggests that the methanol extract of b. ciliata is a potential source of natural antioxidant and antimicrobial compounds that can be used in food and drug industries .
|
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an 18-year - old male transferred to the emergency department presented loss of consciousness , cyanosis , and deteriorated vital signs . according to the rescue team transfer records , he called the control center himself because he was home alone .
he complained of aggravating dyspnea after eating dinner and of sudden onset vomiting . during ambulance transfer
the patient could not remember what happened after the arrival of the rescue team at his house and had no memory of his stay at the emergency room and intensive care unit ( icu ) .
upon arrival to the emergency room , the patient presented with a cyanotic lip , stupor or semi - comatose mental state , and severe tachyarrhythmia .
endotracheal intubation was performed immediately . after drawing blood for an emergency laboratory study and portable chest x - ray ,
1 ) , a large bore needle ( 16 g ) was inserted into the second intercostal space of the mid - clavicular line to decompress intrathoracic pressure .
immediately after insertion of the needle , percutaneously checked sao2 increased dramatically to higher than 95% . closed bilateral thoracostomy followed , using a 24 fr chest tube .
the patient was transferred to the icu for close monitoring of the patient 's mental state and other parameters related to tension pneumothorax .
the abga results upon arrival and after insertion of the chest tube were a ph of 7.272 , pco2 of 56.1 mmhg , po2 of 78.6 mmhg , and sao2 78.6% , and a ph of 7.373 , pco2 of 42.7 mmhg , po2 of 171.3 mmhg , and a sao2 of 99.3% , respectively .
within several hours after admission into the icu , the patient 's mental state and vital signs were restored to within normal limits .
two hours after admission into the icu , endotrachial intubation was removed and spontaneous breathing was maintained with an adequate sao2 level .
the possibility of hypoxic brain damage was evaluated by a neurosurgeon , who confirmed that there were no significant sequela . on the second day at the hospital ,
bilateral pneumothorax recurred during the application of continuous negative wall suction ( -18 cmh2o ) .
the operation was performed under general anesthesia with double - lumen endotracheal intubation . to avoid hemodynamic instability during the operation ,
the patient was positioned with the head elevated 30 degrees ( semifowler 's position ) and with the legs slightly elevated , with both upper arms extended . during the operation , to improve the operation field view , the operation table was tilted to the contralateral side ( fig .
a large bullae on the left lung and single giant bulla on right upper lung were resected with an endo - gia linear stapling device .
one long thoracic drain ( 28 fr ) was inserted bilaterally into the thoracic cavity after mechanical abrasion of upper portion of the parietal pleura .
the incidence of simultaneous bilateral primary spontaneous pneumothorax has been reported to be as low as 1.4% to 7.6% .
simultaneous bilateral primary spontaneous pneumothorax can result in a severely deteriorated condition , usually requiring intubation or resuscitation .
the reported causes of bilateral pneumothorax include trauma , tumor , and iatrogenic causes [ 1 - 3 ] .
other more rare causes have been reported , including catamenial pneumothorax , sarcoidosis , pregnancy , and radiation .
simultaneous spontaneous bilateral tension pneumothorax is defined as when no tracheal shift occurs and when the degree of bilaterally lung collapse is similar in a chest x - ray .
patients with simultaneously developed bilateral tension pneumothorax may deteriorate rapidly , and immediate decompression is recommended .
a vicious cycle may occur , leading to a deteriorated condition , with a chain reaction consisting of lowered venous return , lowered preload , lowered cardiac output and so on .
the patient under study experienced a critically life threatened state , loss of consciousness , cyanosis , and tachyarrhythmia .
the treatment methods include needle aspiration , percutaneous catheter drainage , and tube thoracostomy [ 1 - 4 ] .
indication for surgical management of pneumothorax include continuous air leakage after insertion of chest tube , recurring pneumothorax on the same side , simultaneous bilateral pneumothorax , pneumothorax developed after pneumonectomy , and having an occupational cause . at present , video - assisted thoracic surgery ( vats ) is the most frequently used form of surgery for treating pneumothorax .
kim et al . reported a series of 66 patients that were randomized to treatment with either transaxillary minithoracotomy ( tamt ) or vats .
first , a smaller incision was required when compared with tamt , which generally resulted in less pain .
second , the cosmetic effect is superior to that of tamt . finally , most procedures are possible ( mechanical pleurodesis , chemical pleurodesis , etc . ) . prospective studies of simultaneous bilateral spontaneous pneumothorax management are rare .
generally , cases of bilateral pneumothorax require definitive surgical therapy to reduce the risk of recurrence .
this can be done either through vats or open thoracotomy , depending on the surgeon 's preference .
our case presented the rare clinical situation of simultaneous bilateral tension pneumothorax , and a decision was made to treat with immediate large size needle decompression at the emergency room by a properly trained doctor .
the procedure was a critical step which restored this patient from life - threatening tension pneumothorax .
|
spontaneous pneumothorax is a common clinical problem in emergency care .
however , the overall incidences of primary spontaneous pneumothorax has been reported from as low as 1.4% to 7.6% .
the clinical findings of simultaneous bilateral spontaneous pneumothorax can be variable .
clinical presentation is variable , ranging from mild dyspnea to tension pneumothorax .
bilateral tension pneumothorax can defined as cases where no tracheal deviation is detected in chest x - ray , and symptoms may be equal bilaterally . herein , we present a case with simultaneous bilateral tension pneumothorax , severely deteriorated ( i.e. with loss of consciousness , cyanosis , and hemodynamically unstable ) , that was successfully treated with immediate large - size needle decompression .
|
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lung cancer is the main cause of cancer deaths both in poland and in the world .
non - small cell lung cancer accounts for about 85% of all lung cancers , and the progress in its treatment is still not very satisfactory . the therapeutic procedure of choice is surgery , but only every fifth patient qualifies for it .
this is due to the fact that most patients at the time of diagnosis are in an advanced stage of the disease , usually accompanied with tumour metastases . in about 25% of such patients extension of life by a few month
such disappointing results force us to search for new therapeutic options [ 2 , 3 ] .
one of the new molecular targets for non - small cell lung cancer ( nsclc ) therapy is the epidermal growth factor receptor ( egfr ) .
egfr ( her1 ) belongs to the erbb ( her ) receptor family . for its activation , not only
a connection with a suitable ligand is necessary , but also homo- or heterodimerisation on the cell surface with other receptors of the her family ( her 2 - 4 ) .
the intracellular domain of egfr has activity of tyrosine kinase and it is responsible for the phosphorylation of cellular proteins in the pi3k / akt signalling pathway .
furthermore , this domain has regulatory functions . in many cancers , including nsclc , activating mutations or overexpression of the egfr gene are found . in some cases progression of the disease
uncontrolled cell proliferation , inhibition of apoptosis , and the ability to metastasise is the effect of excessive activation of intracellular pathways constantly stimulated by egfr .
therefore , there is more and more interest in treatment associated with inhibition of egfr [ 46 ] .
one of them is blocking of the extracellular domain of the receptor through monoclonal antibodies ( cetuximab , panitumumab ) , which prevents connection of the egfr ligand or receptor dimerisation . blocking of signal transduction from the cell membrane to the nucleus by small molecule inhibitors of tyrosine and serine - threonine kinase is still in the experimental phase . the biggest hopes for improving the prognosis in nsclc
are associated with the introduction of small molecule , reversible tyrosine kinase inhibitors of egfr ( egfr tki ) gefitinib and erlotinib .
the mechanism of their action is based on reversible binding to the intracellular tyrosine kinase domain of egfr and blocking of atp binding .
selective connectivity of the inhibitor prevents phosphorylation of the tyrosine kinase domain , and in consequence activates the pathway of cellular signal transduction , and leads to cell cycle arrest in g1 phase and increase in apoptosis of tumour cells .
the therapeutic effect of egfr tki depends on the amino acid structure of the tyrosine kinase domain conditioned upon the state of the egfr gene .
appearance of the most frequent activating mutation deletion of 15 nucleotides in codons 746 - 750 in exon 19 and the l858r substitution in exon 21 of the egfr gene is associated with permanent stimulation of the receptor , but they also lead to an increase in both efficiency of reversible egfr tki and effectiveness of radiotherapy . activating mutations of the egfr gene have been reported in only about 10% of caucasians patients with nsclc , more often in non - smokers , women , and patients with adenocarcinoma .
therefore , the first studies on the efficacy of erlotinib and gefitinib in second - line treatment ( br.21 , isel , interest ) have shown that an objective response to egfr tki treatment occurs in less than 10% of patients in the general population [ 1014 ] . in recent clinical trials ( ipass , optimal , eurtac ) conducted on carriers of activating mutations in the egfr gene , over 70% response and almost one year progression - free survival ( pfs )
for this reason , both inhibitors were granted registration in the treatment of locally advanced and advanced nsclc in first - line of treatment , and are an alternative to standard chemotherapy in patients with activating mutations of the egfr gene .
the method of qualification for egfr tki monotherapy in second- or third - line treatment in nsclc is still debatable and unclear [ 1016 ] . in the case of the wild type form of the egfr gene ,
even if the receptor is overexpressed , egfr tki have not shown strong antitumor activity ( none or a few percent of objective response ) mainly for two reasons : lack of changes in the structure of the atp - binding pocket of tyrosine kinase domain and the small role of the normal egfr form in carcinogenesis of lung cancer .
therefore , investigation of the expression of egfr on the cell surface ( by immunohistochemistry ) or amplification of the egfr gene detected by molecular probes and fluorescence in situ hybridization ( fish ) did not find a wider application in qualification for egfr tki therapy . in some cases ,
it is possible to consider the usefulness of the determination of egfr gene amplification by the fish method , if examination of the mutation is unfeasible or the outcome is uncertain , bearing in mind the frequent co - existence of egfr gene mutations ( 10% of patients ) with an increase in copy number of the gene ( 40% of patients ) in tumour cells [ 7 , 8 ] .
unfortunately , regardless of the applied line of egfr tki treatment , at some point of therapy , even in patients who carry an activating mutation in the egfr gene and show a response , there is a recurrence of the disease and the patient 's life is not prolonged [ 17 , 18 ] .
some mechanisms associated with resistance to reversible egfr tki in nsclc are already known . the t790 m mutation in exon 20 of the egfr gene
was detected for the first time in tumour cells of a patient with progression after a few months of efficacious therapy with gefitinib .
that point mutation is caused by a single nucleotide substitution ( c- > t ) at position 2369 ( codon 790 ) in exon 20 of the egfr gene .
the t790 m mutation leads to a substitution of threonine to methionine in the catalytic centre of egfr tyrosine kinase , which is located in the binding pocket of tki and atp .
substitution of threonine at codon 790 into larger methionine probably results in blocking of the binding sites of erlotinib and gefitinib aromatic residues with their point of action . in vitro studies demonstrated that the effective concentration of erlotinib in tumour cells harbouring both the l858r and t790 m mutations has to be 300 times higher to induce apoptosis than in cells only with an activating mutation in codon 858 [ 19 , 20 ] .
presence of a mutation in exon 20 of the egfr gene is a potential prognostic and predictive marker of egfr tki therapy and may play an important role in the planning of therapy .
activating mutations in egfr were detected in cells obtained from primary tumour , circulating tumour cells in peripheral blood ( consistency of results 92% ) and in free circulating dna ( consistency of results
the authors also investigated presence of the t790 m mutation in similar samples , and they detected cells harbouring that mutation in 10 patients ( n = 26 ; 38.5% ) .
median survival in patients with the t790 m mutation treated with reversible egfr tki was 7.7 months against 16.5 months in patients without that mutation ( p < 0.001 ) .
mutations were detected using sarms real - time pcr technique . that investigation has also shown that mutation in exon 20 of the egfr gene leads to acquired resistance to treatment in patients not only with the wild type egfr gene , but also in patients with confirmed clinical sensitivity to egfr tyrosine kinase inhibitors .
presence of t790 m substitution may be an indication for treatment with irreversible egfr tki , such as neratinib , keratinib and especially afatinib , which is currently in the third phase of clinical trials .
the lux - lung 1 study has shown that afatinib induces an objective response and prolongation of progression - free survival ( pfs ) in nsclc patients who have been previously treated with egfr tki for at least 12 weeks .
the rationale for this type of study is the fact that afatinib has a different molecular structure when compared to gefitinib and erlotinib .
this structure causes that afatinib forms a covalent bond with cysteine at codon 733 of the egfr tyrosine kinase domain .
therefore , inhibition of afatinib is only possible due to synthesis of new receptor proteins .
inhibition of phosphorylation by egfr tyrosine kinase in tumour cells after afatinib exposure remains active for 48 hours ( after gefitinib only 8 hours ) , and phosphorylation activity of the kinase after that time ranges from 50 to 70% of the primary activity . in vitro studies and some case reports
have shown that afatinib is effective ( cell apoptosis and disease stabilisation ) in cells harbouring the t790 m mutation in nsclc patients [ 19 , 2226 ] .
the mechanism of mutation appearance in nsclc recurrence in egfr tki treated patients should be thoroughly examined .
a resistant cell subpopulation harbouring t790 m can emerge in the tumour probably as a result of massive apoptosis of tumour cells harbouring an activating mutation associated with response to therapy .
the authors examined presence of mutations in exons 18 - 24 of the egfr gene in patients treated with egfr tki in order to determine if additional mutations in the egfr gene are associated with progression of the disease . in patients with mutations identified in exon 19 or 21 additional mutations
samples obtained from patients tumour cells before treatment were re - examined to exclude the possibility of missing changes in exon 20 of egfr . however , no evidence for the presence of the sought mutations was found .
mutations associated with resistance to egfr tki were observed in three tumor recurrences that emerged after treatment .
it was the t790 m mutation coexisting with deletion in exon 19 or l858r substitution in the egfr gene .
results of the investigations suggest that the resistance to treatment is acquired during therapy with egfr tki , and mutations may form de novo .
it is worth mentioning that tumour cell clones harbouring t790 m substitution may be rare , and the mutation may not be detected ( due to the small number of cells with the t790 m mutation in the whole sample ) .
it is observed especially when the dna sequencing technique is employed , which is characterised by relatively low sensitivity and requires the presence of at least 50% of mutated tumour cells in the examined sample .
currently it is believed that t790 m substitution occurs in over 50% of cases of acquired resistance to reversible egfr tki in nsclc patients and in approximately 5% of patients before egfr tki treatment .
before the therapy with egfr tki , the t790 m mutation emerges along with activating mutations of the egfr gene , and secondarily it coexists with the primary activating mutation in egfr , especially l858r [ 17 , 2831 ] . with time
, it was found that other mutations in exon 20 and 21 of egfr ( insertion - deletion , substitutions : d761y , t854a , etc . )
these mutations account for less than 5% of all known mutations in the egfr gene .
they are detected very rarely , so reports about their presence should be made carefully , and egfr tki treatment outcomes in patients harbouring these mutations should be confirmed on a larger group of patients .
furthermore , it is necessary to understand the mechanism of resistance caused by these mutations .
d761y and t854a mutations in the egfr gene were detected for the first time in patients with activating l858r mutation .
probably substitution of aspartic acid with tyrosine at position 761 is responsible for acquisition of resistance to tki treatment of tumour cells with an activating mutation which are sensitive to the treatment ( this theory was confirmed in tumour cell lines in vitro ) .
however , this resistance to egfr tki has been many times lower than the resistance in cells harbouring both the l858r and t790 m mutations . in case of occurrence of both l858r and t790 m mutations ,
the concentration of erlotinib should be three times higher in order to achieve a therapeutic effect .
analysis of the egfr structure in patients harbouring the t854a mutation suggests the presence of an additional side chain , localised near the egfr tki binding site .
the role of rare mutations in exon 20 such as s768i and v769l in development of acquired resistance to egfr tki is still unknown .
these substitutions were reported for the first time in nsclc patients with progression , bearing activating mutations , after gefitinib therapy . in vitro cultures of cell lines harbouring
the s768i substitution showed slightly more resistance to egfr tki than cells with wild type egfr . in these cells permanent hyperphosphorylation of tyrosine at codon 1045 of the tyrosine kinase domain has been observed [ 17 , 3234 ] .
insertion - deletion mutations in exon 20 of egfr are also associated with acquired resistance to egfr tki .
these mutations , similarly to activating mutations in the egfr gene , are more often detectable in asians , non - smokers , women and in patients with adenocarcinoma . most of the pre - clinical investigations allowed observation of their impact on resistance to therapeutic doses of gefitinib or erlotinib , and some of the irreversible egfr tki , e.g. neratinib . however , in the most common mutations such as n771gy and a767-v769 duplication , even though lack of sensitivity to erlotinib has been reported , low sensitivity to irreversible egfr tki such as afatinib and pf-00299804 was observed .
crystallographic analysis of epidermal growth factor receptor structure has shown that both mutations have an influence on the spatial structure of tyrosine kinase c - helix , which plays an important role in phosphorylation activity of that enzyme .
other insertions in exon 20 of the egfr gene such as v738inskipvai , m766insasv , d770insnpg and d770insnph seem to be associated with that process , but their role in development of resistance to reversible egfr tki is still unknown [ 35 , 36 ] .
many studies have demonstrated that the presence of mutations at codons 12 , 13 or 61 in the k - ras gene is not a predictive factor in treatment with egfr tki .
these observations stem from the fact that mutations in the k - ras gene almost never coexist with mutations in the egfr gene .
lack of mutations in egfr determines the inefficiency of egfr tki used in therapeutic doses , which means that the occurrence of mutations in the k - ras gene is no longer relevant . therefore there were no objective responses to treatment with egfr tki among carriers of k - ras mutations , which was initially considered as an independent , adverse predictive factor in egfr tki treatment .
in fact , in vitro studies have shown that the destruction of tumour cells with the wild - type egfr and mutant k - ras gene is only possible with very high , non - therapeutic doses of egfr tki [ 3739 ] . however , in some individual case reports in which coexistence of both egfr and k - ras gene mutations have been observed , the efficacy of egfr tki was not entirely confirmed .
the situation is presented in a different manner when monoclonal anti - egfr antibodies , such as cetuximab , are used in the treatment of colorectal cancer , head and neck cancer , and perhaps soon in nsclc with strong overexpression of egfr .
mutations in the k - ras gene are a negative predictor here , and are routinely determined in qualification of patients for therapy . however , in the case of therapy with egfr tki the only test eligible for treatment is investigation of egfr gene mutations [ 37 , 41 , 42 ] .
activation of an alternative signalling pathway from stimulated egfr protein leads to activation of ras protein and subsequently kinases associated with raf / mapk / erg proteins and transcription factors such as myc , fos and jun .
mutations which are present in the k - ras gene lead to constant activation of ras protein , so the tumour cells become independent of the signals from the egfr , which explains the mechanism of resistance conferred by the egfr tki and monoclonal anti - egfr antibodies .
mutations in the k - ras gene occur with a frequency of 1520% in caucasian nsclc patients .
substitutions in codons 12 ( g12v , g12c , g12a , g12r , g12d , or g12s ) and 13 ( g13d ) in the k - ras gene are much more common than in codon 61 and they are mostly observed in smokers with adenocarcinoma ( about 30% of patients with this type of cancer ) [ 37 , 38 ] .
egfr is not the only receptor on the cell surface responsible for initiation of signal transduction for cell activation and proliferation through pi3k / akt and ras / raf / mapk / erg pathways .
insulin - like growth factor 1 receptor ( igf-1r ) and the c - met receptor for hepatocyte growth factor ( hgf ) also play an important role in this signal transduction .
overexpression of these receptors and amplification of the genes encoding them often occur in nsclc cells .
overexpression of c - met is associated with generation of an additional excitation signal ( without egfr ) in protein kinase of akt , which allows tumour cells to survive and proliferate and induces the formation of metalloproteinases responsible for invasion and metastasis .
this mechanism is observed in 37% of patients who were not treated with egfr tki and may explain the initial resistance to this kind of treatment .
however , approximately 20% of patients with egfr activating mutations develop met gene amplification as a result of the creation of acquired resistance to egfr tki . moreover
blocking of c - met can restore the sensitivity of cancer cells to the egfr tki .
unfortunately , more than half of patients with met gene amplification also presented the t790 m mutation in the egfr gene . despite the lack of unequivocal efficacy , simultaneous inhibition of c - met and egfr function seems to be an attractive alternative therapeutic option in resistance to reversible egfr tki .
there are ongoing second and third phase trials of the c - met inhibitors tivantinib and pf-2341066 , used together with erlotinib in patients previously untreated with egfr tki or in case of progression after successful monotherapy with erlotinib [ 17 , 4348 ] . similarly to the c - met receptor , the igf-1r also leads to activation of the akt pathway without egfr in nsclc cells .
development of effective small - molecule inhibitors of igf-1r encountered difficulties due to the similarity in the tyrosine kinase domain of insulin and igf-1 receptors .
attempts to use such drugs ( e.g. linsitinib ) had ended with serious side effects in the form of disorders in carbohydrate and lipid metabolism .
monoclonal anti - igf-1r antibodies , such as figitumumab , proved to be more selective for the igf-1r , but were also not devoid of side effects . as a result
, attempts of the combined administration of figitumumab and erlotinib were discontinued in 2010 [ 4951 ] .
mutations or amplifications of the her2 gene , whose product as well as egfr ( her1 ) belongs to the family of her receptors , plays an important role in the development of human tumours , such as breast , ovarian and stomach cancers .
reactions between these receptors may play an important role in pathogenesis of nsclc . in the case of increased expression of her2 protein and amplification of its gene ( about 2030% of patients with nsclc , mainly patients with adenocarcinoma subtype )
the dimerisation between her2 and egfr occurs more easily and the ability to increase proliferation of tumour cells is higher .
mutations in the tyrosine kinase domain of her2 are reported in lung cancer as very rare ( about 24% , more often in adenocarcinoma , non - smokers and women ) .
they occur as insertion - deletion changes , which change the reading frame in eight codons in exon 20 of the her2 gene .
the most common disorders are insertion - deletion of 12 base pairs : a775yvma ( 66% of all discovered mutations in the her2 gene ) and m774ayvm .
these mutations are analogous to a mutation in exon 20 of the egfr gene and cause changes in the tyrosine kinase domain of the her2 receptor . on the basis of this similarity , it is assumed that a mutation in the her2 gene is caused by similar factors as in the egfr gene and causes similar effects .
narrowing of the atp binding gap leads to an increase in tyrosine kinase activity and excessive phosphorylation of signalling proteins and subsequent secondary resistance to the reversible egfr tki [ 53 , 54 ] .
although the main mechanism of the decreased response to reversible egfr tki is caused by mutations in exon 20 ( t790 m ) of the egfr gene , equally strong resistance to reversible egfr tki may be caused by insertion - deletion changes in exon 20 of the her2 gene .
however , studies of this phenomenon have been carried out only in cell cultures [ 5355 ] .
egfr tki , which are effective for the t790 m mutation of the egfr gene , may also prove to be effective in patients with mutations in exon 20 of the her2 gene , but only if they have the ability to block the function of the tyrosine kinase associated with both egfr and her2 .
a double reversible inhibitor of egfr and her2 receptors called lapatinib has not shown efficacy in this case .
however , irreversible inhibitors , such as afatinib and neratinib , which additionally have a likely inhibition effect on the her4 tyrosine kinase , may be effective in the case of mutations in both genes .
mutations in exon 20 of the her2 gene do not eliminate the possibility of afatinib binding to the tyrosine kinase domain , as the drug is covalently associated with cysteine at codon 805 of this domain . in in vitro cultures afatinib is characterised by about 10-fold greater ability to inhibit phosphorylation than gefitinib in case of mutations in exon 20 of egfr or her2 genes .
also several cases have been reported where afatinib was effective in nsclc patients with mutations in exon 20 of the her2 gene .
there are also premises about the effectiveness of combined use of afatinib and mtor inhibitors ( mammalian target of rapamycin ) if her2 mutation is present .
signalling mtor protein is activated by the pi3k / akt cascade and provides the signal which regulates both mrna translation and cell growth .
its inhibition by such agents as temsirolimus , everolimus or deforolimus can increase the potency of dual inhibitors of egfr and her2 and overcome the resistance to their use [ 25 , 5355 ] .
the greatest hope for an extension of progression - free survival in patients treated with reversible egfr tki , in whom resistance has developed , is provided by test results on the effectiveness of irreversible egfr tki . in vitro studies
have shown that these drugs exhibit activity against cells with the wild - type or mutated ( with activating mutation ) form of the egfr gene , as well as against cells which are resistant to them because of the presence of the t790 m mutation in the egfr gene and insertion- deletion changes in the her2 gene . however , in the case of wild - type egfr and mutations which cause resistance to drugs , the concentration of agents required to effectively induce apoptosis in tumour cells may exceed the tolerated dose level in therapy .
currently , it is known that afatinib , which is in the most advanced trials , is effective in some patients who have benefited from pre - treatment with reversible egfr tki .
therefore , the expansion of molecular tests with studies concerning mechanisms of resistance to reversible egfr tki is necessary .
determination of presence of resistance mutations that cause insensitivity of cancer cells to reversible egfr tki , but which does not rule out the effectiveness of irreversible egfr tki , may be considered as a qualifying factor for changing the type of therapy [ 5557 ] .
there are also investigations on the effectiveness of other irreversible egfr tki such as pf-00299802 , ekb-569 , canertinib ( ci-1033 ) and neratinib ( hki-272 ) .
all of these agents are required in relatively high doses in order to overcome the resistance to egfr tki in cancer cells .
however , an additional advantage of the irreversible egfr tki is their ability to suppress other receptors of the her group ( pan - erbb inhibitors ) , which are necessary for heterodimerisation with egfr .
use of irreversible egfr tki and monoclonal anti - egfr ( cetuximab ) antibodies or concomitant administration of egfr and c - met inhibitors may present prospects for the future .
the results of clinical trials of new generation drugs are promising and their usefulness in molecularly targeted therapies will probably be proven in the near future [ 5557 ] .
|
abnormalities of epidermal growth factor receptor ( egfr ) in non - small - cell lung cancer ( nsclc ) patients consist of egfr overexpression and egfr ( her1 ) gene mutations .
structural dysfunction of the tyrosine kinase domain of egfr is associated with the clinical response to tyrosine kinase inhibitors ( tki ) in patients with nsclc .
the most common egfr gene mutations occur as either deletions in exon 19 or as substitution l858r in exon 21 and cause a clinically beneficial response to gefinitib or erlotinib treatment .
unfortunately , the majority of patients finally develop resistance to these drugs .
acquired resistance is linked to secondary mutations localised in the egfr gene , mainly substitution t790 m in exon 20 . through intense research
a few different mechanisms of resistance to reversible tyrosine kinase inhibitors have been identified : amplification of met or igf-1r genes , abnormalities of pten and mtor proteins as well as rare mutations in egfr and her2 genes .
extensively investigated new drugs could be of significant efficiency in nsclc patients with secondary resistance to reversible egfr tki .
|
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a 50 year male presented on july 2011 to the emergency department with right heel pain and inability to bear weight following falling from height . clinical examination revealed mild uniform swelling of the right heel and moderate tenderness with full ankle range of motion .
1 ) revealed undisplaced calcaneal fracture which was treated conservatively and the lesion was not noticed initially .
he was followed up in outpatient clinic and the new x - rays ( fig .
2 ) revealed the presence of pathological fracture with a well - defined osteolytic lesion .
mri showed a well demarcated expansile lesion ( 34 mm 17.3 mm 27.8 mm ) .
it was hypointense on t1 and hyperintense on t2 and the findings were suggestive of giant cell tumor ( fig .
3 ) . lab results including complete blood count , sedimentation rate and c - reactive protein were within normal ranges .
after 4 months he underwent curettage of lesion using burrs through posterolateral approach ( fig .
histopathological examination revealed multiple bone fragments and the soft tissue infiltrated by multinucleated giant cells , hemosiderin laden macrophages , cholesterol cleft and hemorrhages ( fig .
postoperative lipid profile was normal and the diagnosis of primary xanthoma of calcaneus bone was made .
postoperatively the patient was well with complete relief of pain and the patient started full weight bearing ambulation after 6 weeks .
the patient is still under follow up with no signs of recurrence after 2 years .
xanthomatous changes have been reported in various lesions of bone , e.g. fibrous dysplasia , giant cell tumor , aneurysmal bone cysts , non - ossified fibroma , chondroblastoma , fibrous histiocytoma , and xanthogranulomatous osteomyelitis .
defined primary xanthoma as a condition in which the xanthomatous pattern is extensive and when it is not confirmed as secondary phenomenon in pre - existing lesion .
some authors define this lesion as a variant , because xanthoma and giant cells may be seen in many non - neoplastic and neoplastic lesions of the bone .
classification of xanthoma may be helpful to allow diagnosis and treatment:(1)xanthomatous variant : xanthomatous changes in advanced stage of skeletal benign or malignant pre - existing lesions.(2)secondary xanthoma : forms in the skeletal system of type-2 and 3 hyperlipidemic patients.(3)primary xanthoma with normal lipid metabolism .
xanthomatous variant : xanthomatous changes in advanced stage of skeletal benign or malignant pre - existing lesions .
all material should be examined microscopically , the radiological features of the lesion should be studied properly and lipid profile should be investigated to differentiate between primary and secondary xanthoma .
primary xanthoma may be treated with curettage and bone graft while secondary xanthoma is treated nonsurgically and the skeletal manifestations would disappear with systemic treatment of hyperlipidemia .
skeletal primary xanthoma is a rare benign lesion and it is difficult to diagnose using only clinical and imaging techniques .
detailed histopathological examination throughout the specimens is essential to establish the diagnosis of primary xanthoma and to exclude the underlying lesion .
it is important to histologically and radiologically differentiate a primary xanthoma from secondary changes in other benign or malignant tumors .
as the prognosis of primary xanthoma is excellent , curettage and bone grafting is an effective way of treatment .
written informed consent was obtained from the patient for publication of this case report and accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request .
dr . ghalib ahmed : operating surgeon 1 , writing the manuscript and review of literature .
moutasim el mahi : obtaining all the patient 's data ( imaging and histopathology ) .
sameh abolfotouh : writing the manuscript , reviewing the patient 's file & author of correspondence .
|
introductionxanthoma ( or xanthofibroma ) is a benign proliferative lesion , mostly seen in soft tissue .
xanthoma of bone is very rare benign primary bone tumor , more frequently seen in men and in patients over 20 years of age .
histologically , it is characterized by mononuclear macrophage - like cells , abundant foam cells , and multinucleated giant cells .
it is sometimes discovered coincidentally and the most frequent symptom is pain.presentation of casewe present a 50-year - old healthy male patient with primary xanthoma of the calcaneus , who was treated by curettage and bone cement .
he presented with a pathological fracture in a calcaneus bone lesion .
giant cell tumor was suspected on x - ray and mri .
curettage and bone cementing was done through the posterolateral approach .
lipid profile was normal and histological examination revealed findings consistent with primary xanthoma of calcaneus bone.discussionto avoid an erroneous diagnosis , all material should be examined microscopically , the radiological features of the lesion should be studied properly and lipid profile should be investigated to differentiate between primary and secondary xanthoma .
primary xanthoma may be treated with curettage and bone graft while secondary xanthoma is treated nonsurgically and the skeletal manifestations will disappear with systemic treatment of hyperlipidemia.conclusionwe present this case to raise the suspicion of this lesion that is rarely described in the literatures .
this is the first case of primary xanthoma of calcaneus bone that has been reported in qatar .
|
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] |
computational probe mapping is frequently
applied in structure - based
drug design ( sbdd ) to identify potential binding pockets along a protein
surface ( i.e. , hot spots ) . however , mapping is usually limited by the implementation
of gas - phase minimizations , wherein protein flexibility and solvent
competition are ignored .
this leads to a rugged potential surface
and many local energy minima , where druggable sites are indistinguishable
from irrelevant minima .
furthermore , predictions based on a static
receptor structure often neglect important binding - site features ,
such as the presence of transient cavities or bridging water molecules . as a result ,
appropriately modeling the binding potential continues to be a challenge
for sbdd .
several computational and experimental models have
emerged that
address the limitations of traditional solvent mapping .
the multiple
protein structure ( mps ) method addressed the issue of protein flexibility
by creating pharmacophore models from consensus mapping of conformational
ensembles with solvent probes .
while this technique has demonstrated success in
mapping the binding sites of pharmaceutically relevant targets , it can not account for desolvation
penalties or water - bridging contacts , which may be essential to accurate
prediction .
the multiple solvent crystal structure ( mscs ) technique uses x - ray crystallography to determine receptor structures
in the presence of competing water and organic solvent .
this paved
the way for fragment - based methods by providing the first experimental
identification of preferred sites for probe binding .
mscs results
have demonstrated high potential for use in drug development and have
inspired several new computational approaches .
simultaneous
protein flexibility and solvent mapping was first
implemented in 2009 when molecular dynamics ( md ) simulations were
used as a tool to map hot spots .
seco
et al . performed md simulations with seven proteins that were solvated
by a 20% volume / volume ( v / v ) solution of water and isopropanol ( ipa ) .
although their method sought to detect binding sites and predict druggability ,
it did not address aromatic hotspots and was unable to reproduce some
of the experimental binding sites .
yang and wang have conducted similar
studies that included phenol with the alcohol / water mix to address
aromatic interactions .
another md - based approach , site
identification by ligand competitive saturation ( silcs ) , was implemented
with a ternary - solvent box composed of 1 m benzene ( bnz ) , 1 m propane ,
and water to locate hot spots and to reproduce experimental binding
sites .
the authors introduced a dummy atom with
a virtual repulsion term for both bnz and propane in order to solubilize
the hydrophobic solvents and prevent probe
their fragment
maps for bnz along the trypsin surface found abundant local minima
that were mapped equally or better than the valid binding site .
in addition , guvench and mackerell noted that
the repulsive term may also prevent the mapping of secondary binding
sites due to the unnatural physical interactions that are enforced
between the hydrophobic probes .
the most
recent technique was developed by bakan et al . , which uses mixtures of water with multiple probes simultaneously
( ipa , acetamide , acetic acid , isopropylamine ) .
although each of these
md - based techniques have shown some success in identifying binding
sites , they were limited in their ability to selectively map hot spots
without the use of either a weighting or artificial repulsion term .
many spurious minima were also identified . over the past few
years , we have worked to develop mixed - solvent
molecular dynamics ( mixmd ) using amber , with an aim of solvent mapping
across a wide range of targets . instead of simply showing
that probes could occupy regions of known binding sites
, we sought
to establish a stronger foundation that would allow for application
of our method to target systems without requiring a priori knowledge of the binding sites .
our initial study with mixmd used
a 50% w / w solvent box composed of acetonitrile ( acn ) and water , and
the results showed that mixmd reproduced binding sites from mscs studies
with excellent convergence to the true hot spots and no spurious minima .
we were interested in extending the probe set for mixmd to
fully
enable identification of hydrogen - bonding sites , hydrophobic contacts ,
protein protein interactions , and aromatic pockets .
here , we
show what happens when poor parameters are used in mapping a protein .
we then use md simulations of water - probe boxes to examine which remain
evenly mixed .
it is important to identify additional functional sites ,
particularly aromatic sites , for mixmd to be most useful for sbdd .
we also
identify additional probe solvents that expand the ability of mixmd
to locate important interaction profiles in protein
ligand
binding : acetone ( ace ) , n - methylacetamide ( nma ) ,
imidazole ( imi ) , and pyrimidine ( 1p3 ) .
we highlight the importance
of developing a solid foundation for mapping with md , particularly
as it relates to the validation of parameters through detailed evaluation
of metrics for probe probe dispersion .
solvent probes were selected to represent
specific interaction types for use in mixmd ( table 1 ) .
parameters for ace , acn , and nma were obtained
from studies of liquid solvent with amber .
therefore ,
we explored the suitability of opls parameters , which were developed
to work with tip3p water and to reproduce
the empirical data for pure liquids .
the
adaptation of nonbonded parameters from opls for use in amber simply
required the conversion of to rmin , where 2 /2 = rmin .
this is necessary because boss and amber use different combining rules
for van der waals ( vdw ) parameters .
a comprehensive list of the parameters
used in our mixmd simulations is given in table s1 and figure s1 of
the supporting information .
pure solvent boxes of no fewer than
200 probe molecules were built using the tleap module
in ambertools .
md simulations were performed in amber10 through the sander module with
shake and a 1 fs time step .
each solvent box was subjected to 1000 steps
of steepest descent followed by 49,000 cycles of conjugate gradient
minimization and then heated over 20 ps from 10 to 300 k at constant
volume .
two nanoseconds of equilibration were followed by a 5 ns simulation
at constant pressure with the temperature held at 300 k by a weak - coupling
algorithm .
the berendsen coupling method
was chosen because it was used in the original publication of amber
parameters for the solvent molecules used in this study .
the behavior of the pure - probe boxes was used
to confirm proper behavior in our setup .
the final boxes of organic solvent were also used in the setup
of the mixed boxes .
mixed - solvent boxes were created in tleap by solvating a single probe molecule with a layer
of tip3p water and then a layer of the
equilibrated box of pure , organic solvent .
the size of the outer box
of probes is adjusted to achieve the desired solvent ratio .
at least
2500 water molecules and the equivalent mass of probe molecules necessary
to achieve a 50% w / w solution were used in order to ensure that realistic
solvent behavior would be observed ( table s3 , supporting information ) .
each layered mixed - solvent box underwent
the same procedure for minimization , heating , equilibration , and production
simulation as described for the pure - solvent boxes above .
it
should be noted that berendsen s coupling method can sometimes be problematic for simulations
of mixtures because differential heating can occur for different components
of the system .
our use of the method
with the small boxes could be considered a worst - case scenario , where
probes may ( or may not ) have a small bias to aggregate .
any mixture
of water and probe solvent that displays homogeneous behavior under
these circumstances is likely to be robust to a wide range of applications .
in our examination of thermolysin with ipa+water
, we chose the andersen
coupling method to be consistent with our other published applications
of mixmd .
there is no bias from the temperature coupling
that can cause the differences seen in the two parameter choices for
ipa when used in mixmd of thermolysin .
we characterized the
distribution of probe density by computing
atom atom radial distribution functions ( rdfs ) with the radial
command in ptraj .
the final 1 ns of production time
was used to calculate the rdf with a bin size of 0.1 .
when
the solvent probes are fully solubilized into water , they are well
dispersed throughout the solvent box , and the rdf converges toward
unity at the vdw cutoff .
when phase separation occurs in simulations
of mixed solvent , too many probes and too little water occupy the
local microenvironment around each probe .
this makes the local environment
too dense with organic probes , and the rdf will not converge to unity
within the vdw cutoff .
accordingly , we used these rdf descriptions
of mixed vs aggregated solvent systems to evaluate the structure of
each mixed - solvent box . the carbon
carbon ( rcc ) , carbon oxygen
( rco ) , carbon nitrogen ( rcn ) , nitrogen nitrogen ( rnn ) , nitrogen oxygen ( rno ) , and oxygen oxygen ( roo ) distributions
were assessed between probe probe , water water , and
probe water as was relevant for each probe molecule . for brevity ,
cumulative
density distributions of roo for water water
distances in mixed solvent were also examined to determine the number
density of water within the volume sphere . for the case of a 50% w / w
mixed - solvent box , equitable distribution of solvent probes and water
within the system will give a cumulative plot for roo of
water
the distribution of water molecules
relative to one another is less than in pure solvent because obviously
the presence of a miscible probe reduces the number of water molecules
that can occupy the local microenvironment around each water .
earlier papers ours included have
used the behavior
at the edges of the simulation box to prove even mixing of probes
and water .
the reasoning was that the box edges are far from the protein ,
outside the vdw cutoff , and should have minimal bias in the ratio
of probe to water .
if the edges of the box had the same ratio as the
setup solvent box , then there was even mixing .
however , taking a step
back to these basic simulations of solvent boxes provided a better
means of measuring mixing : the use of a rdf .
rdfs of the solvents
relative to one another can be calculated for simulations of proteins ,
and we recommend they be used in all mixed - solvent simulations to
judge whether the behavior of the probes and waters are reasonable .
the rdf , or pair correlation function g(r ) , can be used to describe the structure of the solvent
based on atom / molecule pairs in a system .
the function g(r ) enables a precise description of how likely
a probe molecule will have another probe present at a separation distance
of r. this function may be simplified for a liquid
system in a solvent box , where g(r ) is proportional to the observed number density ( o ) divided by the expected number density ( e ) .
the
value o is the total number of atoms at a given
distance , and e is the number expected at that same
distance if the solvent were uniformly distributed .
if the probe solvent
and water are evenly mixed , the rdf will converge to 1.0 at long ranges .
if it converges to a larger number , this indicates phase separation
( further discussion below ) .
previously , mixmd was validated
with acn as a probe , which specifies
hot spots for amphipathic nitrogen - containing ligands .
we applied amber parameters for acn from grabuleda
et al . , and they accurately reproduce
experimental data from mscs . for completeness , we ran simulations
of acn+h2o boxes to confirm that the rdf demonstrated appropriate
convergence to unity ( figure s2 , supporting information ) . in trying to reproduce simulations of thermolysin in ipa+h2o published by seco et al . , we
found that the ipa phase - separated from water ( figure 1a ) .
it is unclear whether the authors observed this same behavior ,
and they do not note which temperature - coupling method was used .
they
noted partial phase separation in the paper , and they
rescaled the reference values for probe density to account for the
solvent behavior .
however , the bulk phase separation we observed signifies
the implementation of inadequate parameters for liquid ipa , which
were derived from the amber parameter files for threonine with charges
assigned after resp calculations ( ipaseco ) .
snapshots of mixmd simulations
of thermolysin in ipa+h2o executed using the protocol outlined
in seco et al .
( a ) fully flexible simulations
of thermolysin
were performed with a mixed box of 50% w / w ipaseco+h2o .
five independent runs were calculated , and every simulation
resulted in the solvent layers separating between 4 and 5 ns and remaining
separated when simulations were extended to 10 ns .
( b )
the same simulation using ipaopls resulted in solvent remaining well distributed for the entire equilibration
and 10 ns of production in all five independent simulations .
we turned to the work of jorgensen
et al . for alcohol parameters
because they were developed to specifically reproduce a variety of
pure - solvent behaviors and interactions with tip3p water . for small boxes without protein , we compared
our simulations of ipaopls+h2o to experiments
by langdon and keyes that determined the density for ipa+h2o systems at different temperatures .
we found that our mixed box of ipaopls+h2o
reproduced the experimental density at 308 k ( 0.830g / ml ) to within
0.06% .
our ability to replicate this fundamental data confirmed the
use of good parameters for our ipa+h2o systems .
indeed ,
mixmd simulations of thermolysin resulted in proper mixing using the
ipa parameters from jorgensen et al .
the rdfs for the ipa probes ( roo and rcc ) and water ( roo ) in mixed - solvent boxes further justified
the use of opls parameters .
figure 2 compares the o o rdf for both
mixed - solvent and pure - solvent boxes of ipa .
the roo for ( ipa - ipa)opls converged to unity with
an appropriately shorter peak than that of pure liquid alcohol .
however ,
the roo for ( ipa - ipa)seco remained
well above 1.0 at 8 and featured a large peak for the first
solvent shell , the same as seen in the simulation of a pure ipa box .
all simulations with ipaseco feature rdfs that indicate
aggregation of the solvent molecules . for this first example , the
count data behind the rdfs will be discussed in detail to further
explain our interpretation , which is supported by viewing the boxes .
water
and ( b , c ) probe probe in a mixed - solvent environment with ipa .
the impact of different parameters for ipa is demonstrated by the
poor convergence to unity for o o in simulations of 50% w / w
ipaseco ( b ) when compared to the appropriate convergence
obtained for the same mixed solvent with ipaopls parameters
( c ) .
ipa and the great majority of
organic solvents chosen for this
study are miscible with water ( table 3 ) .
the
definition of miscibility is that the two solutions are homogeneously
distributed at all ratios of the two solvents .
it might be reasonable
to find some bias or local structure in the first and second solvent
shells , but bulk separation should not be seen at long ranges .
this
has critical correspondence with one of the basic underlying assumptions
of rdfs .
rdfs are founded on a uniform distribution of solvent to
describe e ( solvent count / box volume ) ; any deviations
from uniformity ( 1.0 ) reflects the local structure of the solvent .
in all the rdfs in
figure 2 , water has its
maximum g(r ) at 2.75 , and
the ipa maxima are at 2.85 . for pure water at 2.75 ,
the maximum g(r )
is 2.6 , and the
cumulative count of water is 1.4 molecules ( table 4 ) .
if we define the first solvent shell between 2.45 and 3.15
, it contains 3.9 neighboring water molecules .
when simulated
in a mixture with ipaopls , there are 1.1 waters within
2.75 and 2.9 waters within 3.15 ( table 4 ) . of course , both counts are reduced because some of the
interactions are occasionally fulfilled by ipa .
this is also incorporated
into e , where the larger box volume reduces the
expected density .
it is that reduced expectation ( actually 0.244 at
2.75 ) that results in a g(r)of 4.6 .
this is higher than the g(r ) maximum from the pure water simulation , but it does not reflect
more interactions with water molecules . within a 7.95 radius
sphere ,
the observed and expected counts are nearly equal and g(r ) approaches 1.0 , showing that the molecules
have a uniform distribution over larger scales .
both o and e depend
on the volume of the simulation box ; this drops out
and makes each g(r ) = no(r)/ne(r ) .
the values in this table have been rounded ; the g(r ) in the figures are the exact values .
values for ne(r ) are based on the volume
of the count
sphere , the number of solvent in the whole box , and size of the whole
box during the simulation . for the mixture of water and ipaseco ,
the
expected count of water was 34.0 , but the observed count was 51.6
water molecules ( table 4 ) . clearly , there are
too many waters close to one another ! furthermore ,
phase separation ( as in figure 1a ) is best
quantified by high g(r ) at long r , but even the local changes reflected the phenomenon .
within 2.75 and 3.15
, there are 1.3 and 3.4 waters ( table 4 ) , respectively , which is much closer to the values
shown in the pure - water boxes .
the mixed box equilibrated to a slightly
larger volume ( 1% change ) , which is reflected in the slight differences
in the expected counts between ipaseco+water and ipaopls+water .
however , the 1% change does not explain the maximum g(r ) of 5.1 , which is based on the increase
in number of water molecules .
the same patterns are seen in
the rdfs of the ipa oxygens ( table 4 ) .
pure
ipa simulations show appropriate behavior
for both sets of parameters ( black lines in figure 2bc , table s2 and text in supporting information ) .
though the rdfs may imply ipaopls has slightly tighter
solvent shells , they both sum to the same number of ipa molecules
in the first solvent shell ( 2.1 for ipaopls and 2.0 for
ipaseco ) . for mixed simulations ,
both ipaseco and ipaopls show lower counts than pure - solvent boxes
at their g(r ) maxima ( 2.85 )
and within their solvent shells ( 3.95 ) .
influenced by the discrepancies in solvent behavior
observed for ipaseco and ipaopls , we carefully
examined the behavior of existing parameters for additional solvent
probes to determine their applicability in mixmd .
the inclusion of
various probe types , including complicated molecular fragments , would
facilitate the development of accurate pharmacophores for druggable
hot spots , ligand binding sites , and protein protein interactions .
the selection of additional probes for expanding the functional groups
represented in mixmd was based on existing mscs data and common interaction
types found in protein ligand systems .
ipa maps both hydrogen
bond - accepting and -donating locations , but additional hydrogen - bonding
probes are needed to develop robust pharmacophore maps of binding
sites .
ace and nma were selected because each molecule represents
a different hydrogen - bonding profile .
ace locates where the protein
donates hydrogen bonds , and nma represents protein protein
or peptide binding .
specific
parameters for ace and nma had been developed for use with amber and opls .
simulations of ace+h2o and nma+h2o were performed using each parameter set
to assess their suitability .
the g(r ) was calculated for probe probe , probe water , and
water
analysis
of the resulting rdf plots indicated that in each case all g(r ) converged to unity near 8 .
we found that both the amber and opls parameters for liquid ace and
nma demonstrated correct behavior according to rdf plots and visual
inspection ( figure 3 ) .
the o o rdf for
water from the mixed - solvent simulations also illustrate that solvent
mixing has occurred .
analysis of the cumulative plots for the roo of water in solution with ace or nma showed
that the number density of water within the local volume sphere was
half the number density observed in pure water simulations , which
is the expected result for a well - distributed system of 50% w / w probe
and water .
visual analysis of the trajectory snapshots served to corroborate
the occurrence of proper solvent mixing .
however , the rdf s
deviation from unity at 8 was slightly higher for the opls
descriptions of ace and nma ( table 2 ) . for
this reason
, we would recommend that the amber parameters be used
for ace and nma in our mixmd method , especially when the amber parameters
are also used to describe the protein .
( a , d ) o o and
( b , e ) n n radial distribution functions
for probe probe and o o rdfs for water water
( c , f ) in a mixed - solvent environment with ace and nma .
the impact
of optimized parameters from amber versus general solvent parameters
from opls is shown in the slightly better convergence to unity for
simulations using amber parameters ( d f ) compared to opls ( a c )
parameters .
in particular , we wanted
to find parameters for soluble heterocycles that could map aromatic
hot spots without requiring an artificial repulsive term .
no other
md approach for probe mapping has successfully integrated aromatic
probes without an artificial interaction term .
futhermore , we were
particularly interested in developing probes that matched common heterocycles
used in modern pharmaceuticals . as a result ,
a highly polar
diazole , imi , is miscible with water and is present as a functional
group in a wide range of biologically active molecules , including
mercaptopurine ( anticancer ) , ketoconazole ( antifungal ) , and moxonidine
( antihypertensive ) .
imi parameters were derived from jorgensen and
mcdonald , and then simulations of pure
imi and imi+h2o were conducted to assess the potential
of imi as a probe for mixmd .
the proper convergence of the rdf plots
to 1.0 showed that imi was properly solubilized and well distributed
within the binary - solvent system ( figure 4 ) .
our pure - solvent rdfs were consistent with the results of jorgensen
and co - workers and further validated our parameter
choice and the accuracy of our protocol .
also , visualization of simulation
snapshots confirmed that the imi probe was well dispersed in the aqueous
solution .
( c ) water water in a mixed - solvent environment with ( a )
imi and ( b ) 1p3 .
opls
parameters for pyr , 1p2 , 1p3 , and 1p4 were each simulated in a binary
water probe solution .
visualization of the simulations of pyr+h2o , 1p2+h2o , and 1p4+h2o clearly showed
that they underwent phase separation .
this was proved by rdfs that
converged to values well above 1.0 at 8 ( figure 5 ) .
however , 1p3+h2o yielded a well - dispersed system
with well - behaved rdf plots ( figure 4 ) .
probe probe
radial distribution functions for mixed solvent
environments composed of ( a ) bnz+h2o , ( b ) pyr+h2o , ( c ) iph+h2o , ( d ) 1p2+h2o , and ( e ) 1p4+h2o clearly showed phase separation , with g(r ) values well above 1.0 at 8 .
we also examined two nonpolar aromatic probes ,
bnz and iph , simply
to confirm that they are not appropriate probes when an artificial
repulsive term is not used .
comparison of the resultant rdf plots
to the reference rdf plot of pure water showed that the g(r ) values for bnz+h2o and iph+h2o do not converge to unity within 8 ( figure 5 ) .
in fact , the g(r ) for bnz+h2o indicated convergence to a value of 2 . the
simulations with iph as a probe molecule showed a converged g(r ) of approximately 1.45 at 8 .
visualization of snapshots from these simulations showed significant
aggregation of the probe molecules , verifying that these probes are
not be soluble enough for use with mixmd without inclusion of a repulsive
term . because bnz is one of the solvent probes used in silcs
,
we were
able to compare the rdf results for our mixmd simulations of bnz+h2o to the published results from silcs mapping studies .
the bnz probe used in silcs was parametrized
based on the charmm force field and contained a repulsive term to
correct for probe probe aggregation .
although a nonbonded cutoff
of 8 with a 58 switching term was applied in
silcs , the probe probe distance for bnz converged to 1.0 at
13 ( solvent box size was 72 58
43 ) . in comparison , the soluble aromatic probes applied
in mixmd converged at approximately 67 .
the first solvent
shell of bnz in silcs was observed at 9 , while we observed
the first peak at 45 .
the presence of the second hydrophobic
probe ( propane ) in silcs simulations may have influenced these differences
in rdf .
however , the use of a repulsive term to prevent probe probe
aggregation does not correspond with the experimental properties of
bnz , suggesting that silcs simulations with high concentrations of
bnz may yield incorrect results . to determine whether bnz and
iph would disperse more readily in
the presence of an intermediary probe ,
ternary mixed - solvent
boxes were constructed to contain 1 m aromatic probe and 1 m ipa in
water .
we hypothesized that the introduction of the ipa molecule would
enhance available interactions and aid in the dispersion of the aromatic
probes into water .
however , this was not observed in simulations of
the ternary - solvent boxes . at 5
ns of production time , the bnz molecules
were predominantly clustered along one side of the solvent box , while
the ipa molecules were dispersed throughout the water .
an additional
20 ns of production time did not change the observed aggregation ( figure 6 ) .
ternary - solvent simulations with iph indicated
a similar result ; after 5 ns of production time , iph was separated
with some dispersion toward the interior . elongating the run time
out to 25 ns
representative snapshots of md trajectory for solvent boxes of
1 m aromatic probe ( yellow ) and 1 m ipa ( purple ) in water ( cyan ) for
( a ) bnz+ipa+h2o at 25 ns and ( b ) iph+ipa+h2o
at 25 ns .
although
all of the probes selected for
use in our validation study had experimental data establishing their
solubility in water , not all of these probes were soluble in simulation
( table 3 ) .
the solvation conditions applied
in a mixmd simulation included high probe concentrations , which may
have affected probe solubility . to determine whether the outcomes
we observed were justified based on experimental data , we considered
their miscibility .
the terms soluble and miscible are frequently used
interchangeably ; however , they are two distinct properties .
solubility
is dependent upon temperature and pressure and refers to the ability
of a solute ( solid , liquid , or gas ) to dissolve into a solvent ( solid ,
liquid , or gas ) , thereby forming a homogeneous mixture .
miscibility
refers to the ability of two liquids to form a homogeneous solution ,
independent of proportion .
all probes that have been experimentally
categorized as miscible were well dispersed in our computational simulations ,
with two exceptions ( table 3 ) .
pyridine is
a weak base with a nontrivial concentration of protonated pyridinium
ions ( pka of 5.3 ) following solubilization
into water .
this effect was not simulated in our studies , and we suggest
that the charged species may be responsible for solubilizing the uncharged
pyridine molecules into water . a similar explanation may also be extended
to pyridazine .
one advantage of using silcs as a tool for solvent
mapping has been that the ternary - solvent box can enable the development
of a complete pharmacophore model from a single md run because the
three probe types identify aromatic , hydrophobic , and hydrogen - bonding
sites .
using three of the probes with parameter
sets we have validated for mixmd , we constructed a ternary system
with imi ( aromatic probe ) , ipa ( hydrophobic and hydrogen - bonding probe ) ,
and water at a concentration of 33% w / w by probe .
visualization of
the trajectory data indicated appropriate solvent mixing , and our
rdf results showed that both organic probes were fully dispersed into
solution ( figure 7 ) .
total run time required
a comparable amount of simulation time to a binary mixmd simulation ;
the total cost was an additional 4 or 8 h of production time on an
8-core cpu as compared to a binary ipa+h2o or imi+h2o simulation , respectively .
( a ) final snapshot from the production
simulation of a mixed - solvent
box containing imi+ipa+h2o illustrates that the probes
are well mixed within the system .
( b d ) probe probe
and water water radial distribution functions for imi and ipa
establish convergence to unity within the trisolvent environment .
we suggest that the use of this
ternary mixmd model may offer several
advantages over the silcs approach .
we have shown that mixmd can be
used to identify maximally occupied sites without recovering additional
spurious minima , which is a feature that was not consistently seen
in the silcs studies .
in addition , instead
of requiring water to act as a hydrogen - bonding probe , in mixmd , the
druggable hydrogen - bonding sites can be identified in competition
with water using ipa as the probe .
this is important for ascertaining
whether a drug - like molecule that contains a similar function group
could displace water molecules at the proposed hydrogen - bonding site .
a further advantage of using mixmd is the array of available probes ,
which can be tailored to an investigator s mapping needs . for
example
, either imi or 1p3 could be used in the ternary box to locate
aromatic hotspots that are specific to the size of the ring .
the ternary
mixture of 33% w / w ace+acn+h2o was also simulated , and
the results depicted a well - distributed solvent system for use in
hot spot mapping with mixmd ( figure s3 , supporting
information ) .
we have examined solvent
parameters for neat liquid from the literature
for use in hot spot mapping through mixmd .
our work highlights the
importance of parameter validation and analysis of simulation data
when performing computational solvent mapping in order to obtain appropriate
behavior .
influenced by the poor results obtained using a standard
set of parameters used in the field for liquid isopropanol in water ,
we identified a set of functional group probes with specific parameters
available for liquid simulation .
we pursued validation of these parameters
for neat and mixed - solvent simulations and identified six probes for
use with tip3p in mixmd : acetonitrile , acetone , imidazole , isopropanol , n - methylacetamide , and pyrimidine .
of course , these probes
should be used with more proteins beyond thermolysin ( figure 1 ) to prove their applicability .
we have previously
applied opls ipa parameters to also examine elastase , hewl , hewl ,
p53 core , and rnase a. furthermore , we
are currently using all of these probes to map 10 additional protein
systems , but these are separate studies in their own right .
the extent of ideal dispersion is most easily quantified by using
the cumulative rdf of roo for water .
although
published results for pyridine and pyridazine indicated that they
are miscible with water , these two probes were observed to separate
from the aqueous phase during our simulations , rendering them unsuitable
for solvent mapping in our context .
we hypothesized that this disparity
between experiment and computation was caused by the presence of the
corresponding ionized species , which were not simulated . in order
to avoid unphysical mapping or reduced accuracy ,
our simulations did
not employ the use of an artificial repulsion or weighted density
term .
we have successfully expanded the range of probes that can be
incorporated into mixmd studies to allow for the mapping of druggable
sites , including hydrogen - bonding regions , aromatic pockets , and protein
, a full pharmacophore
model could be created from the simulation of a single protein+probes+water
system , which would greatly reduce the computational costs of mixmd
studies .
this is a clear advantage of the silcs method that we would
like to incorporate .
ultimately , investigators could mix and
match the probes used in mixmd to identify hotspots with a
greater degree of specificity .
|
probe mapping is a common approach
for identifying potential binding
sites in structure - based drug design ; however , it typically relies
on energy minimizations of probes in the gas phase and a static protein
structure .
the mixed - solvent molecular dynamics ( mixmd ) approach was
recently developed to account for full protein flexibility and solvation
effects in hot - spot mapping .
our first study used only acetonitrile
as a probe , and here , we have augmented the set of functional group
probes through careful testing and parameter validation .
a diverse
range of probes are needed in order to map complex binding interactions .
a small variation in probe parameters can adversely effect mixed - solvent
behavior , which we highlight with isopropanol .
we tested 11 solvents
to identify six with appropriate behavior in tip3p water to use as
organic probes in the mixmd method .
in addition to acetonitrile and
isopropanol , we have identified acetone , n - methylacetamide ,
imidazole , and pyrimidine .
these probe solvents will enable mixmd
studies to recover hydrogen - bonding sites , hydrophobic pockets , protein
protein
interactions , and aromatic hotspots . also , we show that ternary - solvent
systems can be incorporated within a single simulation .
importantly ,
these binary and ternary solvents do not require artificial repulsion
terms like other methods . within merely 5 ns , layered solvent boxes
become evenly mixed for soluble probes .
we used radial distribution
functions to evaluate solvent behavior , determine adequate mixing ,
and confirm the absence of phase separation .
we recommend that radial
distribution functions should be used to assess adequate sampling
in all mixed - solvent techniques rather than the current practice of
examining the solvent ratios at the edges of the solvent box .
|
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] |
illness is a challenge to our physical , psychological and spiritual wellbeing that has repercussions on our identity and our social context . in a pathogenetic approach ,
diagnostic tests are used to look for the underlying disease and treatments are aimed at removing it .
a person is considered cured when the disease is no longer detectable and agreed parameters have normalised
. however daily clinical practice is characterised by people who suffer from chronic illness where there is often a discrepancy between their biochemical results and the way they feel .
good or even normal parameters do not necessarily correlate with a good perceived state of health , and vice versa .
health is therefore a concept that goes beyond the absence of disease to include physical , mental and social wellbeing .
salutogenesis , developed by antonovsky in the 1970s , looks at what generates health by exploring the reasons why some people stay healthy in the face of hazardous influences whilst others , faced with the same hardship fall ill .
antonovsky s research shows how adverse events and stress can become the opportunity to generate health if certain personal characteristics are present .
resilience to difficult situations depends on a person s sense of coherence ( soc ) , a global orientation towards life that is based on self - reliance in the face of challenges , self - confidence in one s ability to deal with demanding events and the trust that difficult events hold meaning for one s life [ 2 , 3 ] . there is a growing body of research on all age groups [ 46 ] , different socioeconomic backgrounds and across cultures that shows how a strong soc is related to better health , healthier ageing [ 812 ] and is a protective factor against alcohol addiction despite similar rates of recreational consumption in teenagers .
conversely , a weak soc is related to poorer health and lower mood [ 4 , 5 ] .
although soc develops naturally in the first 30 years of life it is not a static orientation .
it can be strengthened through personal activity and care [ 4 , 14 ] . a dynamic , personally driven element that can change through activity and care
it is not the absence of hardship or disease that determines health but our ability to deal with them positively and assign meaning to them .
health is not a steady state , effortlessly maintained once it has been achieved , but an active process of continuous adjustment , a subtle equilibrium between our physiological , psychological and spiritual integrity and the outer or inner influences that can strengthen or undermine this .
the concept of health as a state of complete well - being advocated by the declaration of alma ata , becomes health as the ability to adjust and self - manage in the face of physical , emotional or personal challenges .
a salutogenetic healthcare system needs to be oriented in such a way that illness is considered an adverse event that can become the foundation for better future health .
methods need to be adopted that allow the ill person to be treated in all their characteristics and in context .
each medical intervention should aim to strengthen soc and a person s physiological , psychological and spiritual resilience .
the increasing prevalence of chronic diseases , the very different but constant environmental challenges faced by people in the developing and developed world alike , provide us with constant salutogenetic opportunities .
person - centred medicine ( pcm ) takes into account the physical , psychological and spiritual aspects of a person in health and illness in order to individualise health promotion practices , diagnosis and treatment .
it broadens the technological advances of biomedicine with the epistemological approach , the relationship - based care and the salutogenetic treatments of non - conventional medicine .
complementary and alternative medicine ( cam ) and traditional medicine ( tm ) are grouped together as cam / tm or non - conventional medicine ( ncm ) .
the term includes a variety of different medical systems and healthcare methods whose roots lie in different philosophical backgrounds and cultural origins .
although very different from one another , they all share a holistic view of the human being as a physical , psychological and spiritual entity .
they value the complexity of natural phenomena and study the relationship between the human being and nature in health and illness .
their treatment systems are based on knowledge , skills and practices that restore health and encourage the self - healing abilities of the human being .
they advocate respect for the dignity of every person and promote responsibility for keeping healthy at individual and community levels .
this holistic , person - centred , salutogenetic approach is the reason for the growing interest in all forms of ncm by patients and healthcare workers alike .
salutogenetic healthcare practices are personalised using the principles and methods of person - centred medicine . in salutogenetic healthcare the caregiver - person relationship
each professional role needs to be developed to provide salutogenetic care from as early as undergraduate training . in order to have a person - centred approach , technological proficiency , solid grounding in evidence - based medicine ( ebm ) and knowledge of international protocols need to be complemented by knowledge of the epistemological systems and practical tools of ncm .
doctors and other healthcare workers are then encouraged to the use their clinical judgment as the final decision - making tool .
clinical judgment is central to the role of the doctor and their professionalism and can be strengthened and developed to become an accurate decision - making tool .
this complements the application of ebm and protocols , which are based on large numbers and therefore not always applicable to particular cases .
following protocols ensures a basic standard of care that has lead to improvements in safety on a large scale . however following them
automatically can lead to repetitive , monotonous practice which in turn can lead to errors as well as frustration and dissatisfaction both on the part of the healthcare worker , who feels like a generic cog in a smooth machine , and the patient who does not feel personally addressed . on the contrary ,
assessing and treating each person in their physical , psychological and spiritual uniqueness , encourages active personal involvement on the part of the both healthcare worker and the patient .
the use of sound clinical judgment allows personalisation of guidelines and protocols to each case .
moreover , learning and teaching about salutogenetic practices may mean that healthcare workers adopt healthier lifestyles for themselves . greater work satisfaction and active involvement
strengthen a healthcare worker s resilience and soc , ultimately improving their health as well as that of their patients . with this person - centred method ,
any treatment is chosen as the result of an informed choice that takes all aspects of the person into consideration .
for any therapeutic process to become a salutogenetic experience , physical as well as psychological , social and personal factors need to be addressed at the appropriate time with attentive care to the physical emotional and spiritual needs of the person .
the patient needs to be able to rely on the doctor or the team who take responsibility for their treatment and recovery . during rehabilitation or at other times during the process there may be a chance to bring the experience to greater consciousness , to explore the reasons that led to the event and the things that could be changed in order to prevent similar episodes from happening again .
all this works towards developing an awareness that overcoming this difficult experience was meaningful because it allowed the development of new life skills , new coping strategies , and new confidence in one s own healing abilities .
this change needs to be sustained over time with appropriate follow - up as habits are difficult to change and may slip if they are not followed closely and encouraged over time . in order to evaluate the success of medical interventions broadened with a salutogenetic approach , parameters need to be developed and integrated into the current evaluation systems .
they need to have a suitable epistemological basis , a methodological approach that considers the value of small numbers , individual cases and clinical judgement .
salutogenetic parameters such as the quality of life of patients and caregivers , sustainable change , treatment satisfaction , job satisfaction and prevention of burn - out need to be assessed as well as cost - effectiveness and technological excellence .
health education is both a therapeutic intervention and the key form of disease prevention in salutogenetic healthcare .
it should take place at population levels , in primary care , in hospitals , as part of any rehabilitation process , in schools and families .
environmental obstacles that prevent people from adopting healthy lifestyles should be removed as far as possible .
the ottawa charter advocates the implementation of eight conditions that should underlie any change in health .
these are peace , shelter , education , food , income , a stable eco - system , sustainable resources , social justice , and equity . at the same time , individuals need to be encouraged to actively increase control over their health in order to improve it . to reach a state of complete physical , mental and social well - being , an individual or group must be able to identify and to realize aspirations , to satisfy needs , and to change or cope with the environment .
encouraging locally sourced , high quality foods , encouraging biodiversity and fair - trade , has positive effects on the sustainability of primary resources and the stability of the eco - system as well as people s health . in order to be adopted by individuals in their lifestyles ,
there should be variations between the diet of a child and that of an elderly person , of an office worker compared to that of a builder , the diet of someone who suffers from inflammatory illnesses compared to that of someone who suffers from cancer , the diet of the same person when they are well or ill .
if diet were a better used resource in healthcare and people were taught its principles and practical applications , this may decrease the need to use medication in some cases .
altered sleeping patterns , eating rhythms and patterns of physical activity are both a measure and a cause of pathology and health .
variable shift workers have a higher degree of stress , of alcohol misuse and increased use of neuroleptic drugs compared to their colleagues with regular working patterns .
many others have altered eating rhythms as they skip meals , or constantly pick at food during the day .
it is important for people to become aware of their own biorhythms , which alter with age , gender , state of health , personal and constitutional characteristics . with the help of education ,
improved environmental conditions and appropriate care during illness , people can adjust or at least compensate for altered biorhythms and therefore actively improve their health .
chronic stress can strengthen or weaken a person depending on how strong or weak their resilience and soc are .
chronic stress has been shown to be greater risk factor for developing ischaemic heart disease than hypertension or hypercholesterolaemia .
emotions can have adverse effects on the incidence of complication and prognosis during an acute event .
apart from reducing known risk factors and encouraging positive emotions , we can strengthen emotional resilience and soc through artistic activity in the form of painting , music , dance , drama , creative writing . a study carried out in sweden showed how music therapy improved quality of life and soc in a group of elderly people . through artistic activities
people learn new skills and their achievements have a positive effect on their self - esteem .
this can be applied to overcome future challenges or unknown life - situations with strengthened soc .
the initial steps towards a salutogenetic healthcare reform , are to broaden undergraduate training and to develop pilot projects in the fields of health promotion and salutogenetic rehabilitation . the person and the caregiver - person relationship needs to be placed at the centre of the therapeutic process through the principles of person - centred medicine both in education and in practice .
caregivers need to be given the tools to consider the person in all their aspects and characteristics through the adoption of the epistemological basis of ncm that broadens the technological advances of biomedicine .
medical interventions should not only be aimed at removing the disease but also at improving health by strengthening a person s soc and their resilience at physical , psychological and spiritual levels . reasons for illness can be explored during rehabilitation . through health promotion programmes , during rehabilitation , in education , people are taught how to take better care of themselves by adopting individually tailored , healthier lifestyles better suited to their own physiological , emotional and personal needs .
active involvement needs to be encouraged both on the part of the to patient and healthcare giver , the use of clinical judgment needs to be taught .
methods for the scientific investigation of effects on small numbers or single cases need to be included in the evaluation of a person - centred , salutogenetic healthcare system alongside ebm .
|
the purpose of this review is to discuss how a salutogenetic approach that takes into consideration the human being as physical , psychological and spiritual entity may provide some answers to the difficulties faced by healthcare systems .
the choice of medical intervention needs to take into account the technological advances of biomedicine but tailor them to the physical , psychological and spiritual needs of the patient in the context of their biography .
such person - centred medicine aims to strengthen antonovsky s concepts of resilience and sense of coherence with each therapeutic intervention so that overcoming illness becomes the foundation for better future health .
appropriate evaluation parameters need to be developed and included in order to evaluate the success of interventions in a person - centred , salutogenetic system .
|
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oral lichen planus ( olp ) , a common mucocutaneous disease , was first described by wilson in 1869 .
poor described the formation of cavities in lichen planus of the mucosa , corresponding in character to subepithelial bullae and gougerot described the reticular , annular , plaque , ulcerated , dotted and sclerotic forms of lichen planus .
olp is considered to be a t - cell mediated autoimmune disease in which cytotoxic cd8 + t - cells trigger the apoptosis in oral epithelial cells .
the lichen planus antigen has not been definitely identified and it may be a self - peptide . in most of the studies , olp is considered to be a t - cell - mediated chronic inflammatory oral mucosal disease .
it is mainly observed among women and symmetrically affects the buccal mucosa , tongue , gingiva , floor of the mouth , lips , and palate .
the role of angiogenesis in the pathogenesis of chronic inflammatory diseases is of considerable interest . a positive feedback loop , in which an inflammatory state promotes angiogenesis and the angiogenesis in turn facilitates chronic inflammation , has been found in some inflammatory diseases .
there is increasing evidence that chronic inflammation is tightly linked to diseases associated with endothelial dysfunction , and plays a role in the induction of aberrant angiogenesis .
microvessel density ( mvd ) evaluation is commonly applied for the estimation of tumor angiogenesis and is widely accepted to play a role in the pathogenesis of some inflammatory conditions also .
cd34 monoclonal antibody can be used to highlight the microvessels in inflammatory or neoplastic disorders because of its capability of staining the vascular endothelial cells ( membranous staining pattern ) .
the purpose of this study was to evaluate the role of angiogenesis in the pathogenesis of olp , using cd34 stain to highlight the blood vessels for measuring the mvd .
better understanding of the etiopathological mechanism underlying olp will help in the development of new treatment strategies , as well as to manage persistent inflammation in patients showing poor response to conventional immunosuppressive drug regimes .
this study included 40 cases , categorized into two groups : group i and group ii .
group i , the control group , consisted of 15 specimens of normal oral mucosa ( buccal mucosa / gingival tissue ) from individuals who underwent extractions as part of orthodontic treatment and from the paraffin blocks available with the archives of the department of oral and maxillofacial pathology and microbiology , m. m. college of dental sciences and research , mullana .
group ii consisted of 25 diagnosed cases of olp ; these cases were subdivided into two groups : group iia and group iib .
group iia consisted of 18 diagnosed cases of reticular olp and group iib consisted of 7 diagnosed cases of erosive olp .
these cases were obtained either from the blocks of previously diagnosed cases from the archives of the department of oral and maxillofacial pathology and microbiology , m. m. college of dental sciences and research , mullana , or from fresh cases identified at the department of oral medicine and radiology . case history pertaining to the lesion was obtained and recorded .
sections of 4-m thickness were cut from the paraffin blocks and were used for immunohistochemical staining .
briefly , endogenous peroxidase activity was blocked with 0.6% h2o2 and sections were incubated at room temperature with 100 l of prediluted primary cd34 antibody ( dako ) .
after washing in phosphate - buffered saline ( pbs ) , secondary antibody was applied on the section and then washed with pbs .
after counterstaining with hematoxylin , the sections were mounted with dibutyl phthalate xylol ( dpx ) and the slides were observed under a nikon research microscope .
negative and positive controls were used simultaneously to assess the specificity and reliability of the staining process .
pbs was substituted for the primary antibodies of cd34 as a negative control , and placental tissue sections with known cd34 positivity were used as the positive control .
microvessels were highlighted on the sections of olp and normal oral mucosal tissue by immunostaining with anti - cd34 monoclonal antibody [ figures 13 ] .
any single brown - stained cell or cluster of endothelial cells that were clearly separated from adjacent microvessels , histiocytes , and other connective tissue elements were considered as a single vessel .
cd34-stained brown - colored blood vessels in normal oral mucosal tissue cd34-stained brown - colored blood vessels in reticular oral lichen planus cd34-stained brown - colored blood vessels in erosive oral lichen planus cd34-stained sections were scanned at low magnification to identify the most vascular areas ( hot spot areas ) , which were almost exclusively localized within the inflammatory infiltrate .
a maximum of five fields were selected and the images were captured on the computer under the 20 objective of a nikon research microscope ( y - thr - l ; japan ; 0132508 ) for counting of the number of blood vessels in hot spot areas .
briefly , endogenous peroxidase activity was blocked with 0.6% h2o2 and sections were incubated at room temperature with 100 l of prediluted primary cd34 antibody ( dako ) . after washing in phosphate - buffered saline ( pbs ) ,
after counterstaining with hematoxylin , the sections were mounted with dibutyl phthalate xylol ( dpx ) and the slides were observed under a nikon research microscope .
negative and positive controls were used simultaneously to assess the specificity and reliability of the staining process .
pbs was substituted for the primary antibodies of cd34 as a negative control , and placental tissue sections with known cd34 positivity were used as the positive control .
microvessels were highlighted on the sections of olp and normal oral mucosal tissue by immunostaining with anti - cd34 monoclonal antibody [ figures 13 ] .
any single brown - stained cell or cluster of endothelial cells that were clearly separated from adjacent microvessels , histiocytes , and other connective tissue elements were considered as a single vessel .
cd34-stained brown - colored blood vessels in normal oral mucosal tissue cd34-stained brown - colored blood vessels in reticular oral lichen planus cd34-stained brown - colored blood vessels in erosive oral lichen planus cd34-stained sections were scanned at low magnification to identify the most vascular areas ( hot spot areas ) , which were almost exclusively localized within the inflammatory infiltrate .
a maximum of five fields were selected and the images were captured on the computer under the 20 objective of a nikon research microscope ( y - thr - l ; japan ; 0132508 ) for counting of the number of blood vessels in hot spot areas .
in the present study , of the difference between mean mvd in group i ( 44.47 ) and that in group ii ( 97.24 ) was statistically highly significant ( p=.001 ) [ table 1 ] .
a comparison of the mean mvd in group iia ( 84.61 ) with that in group iib ( 129.71 ) also showed a statistically significant difference ( p=.015 ) [ table 2 ] .
comparison of microvessel density between group i and group ii comparison of mvd between group iia and group iib
various concepts have been quoted by different authors but the differences have not yet been sorted . as an autoimmune disease with inflammatory origin and chronic progression ,
macrophages and the other cells of the immune system produce vascular endothelial growth factor ( vegf ) , which stimulates the degradation , proliferation , and migration of endothelial cells .
tnf - alpha , il-1 alpha , interleukin-6 , and interleukin-8 also upregulate the expression of vegf , and these factors have been found in the oral fluids of patients with olp . also , olp shows significantly increased vegf as compared to normal oral mucosa .
proangiogenic and angiogenic factors such as histamine , heparin , chymase , bfgf , vegf , and tgf - beta are produced by mast cells , and an increase in the number of mast cells has been observed in olp .
a significant correlation between mast cell density and mvd has been found during the evolution of oral squamous cell carcinoma from normal oral tissue through premalignant lesion with various degrees of dysplasia to carcinoma .
the malignant potential of olp remains controversial , and different research groups have proposed different approaches and interpretations .
it appears advisable to carry out a meticulous follow - up of patients with olp for early detection of the malignant transformation of suspected lesions .
malignant transformation of olp may be related to , or dependent on , a series of molecular stimuli originating in the inflammatory infiltrate .
some molecules and radicals generated by inflammatory cells can act as mutagenic agents for epithelial cells or influence important cell cycle regulation mechanisms , e.g. , apoptosis , cell cycle arrest , and cell proliferation , among others .
a source of possible mutation in olp derives from the action of cyclooxygenase-2 ( cox-2 ) that is produced by inflammatory infiltrating cells .
apart from its other actions , cox-2 also intervenes in the metabolism of arachidonic acid , generating the carcinogenic metabolite malondialdehyde , which can damage dna .
attraction of lymphocytes to a particular site would require cytokine - mediated upregulation of adhesion molecules on endothelial cells and concomitant expression of receptor molecules by circulating lymphocytes .
in olp , there is in fact increased expression of several vascular adhesion molecules ( known by the acronyms elam-1 , icam-1 , and vcam-1 ) and infiltrating lymphocytes that express reciprocal receptors ( known as l - selectin , lfa-1 , and vla4 ) , supporting the hypothesis that there is activation of a lymphocyte homing mechanism in lichen planus .
thus , if angiogenesis is increased , it will lead to more recruitment and retention of lymphocytes or inflammatory infiltrate , or progression of disease or recurrence of the lesions ; the inflammatory infiltrate can further lead to carcinogenesis .
cd34 , a myeloid progenitor cell antigen present in endothelial cells , is detectable in all types of endothelium .
the monoclonal antibody against cd34 reacts with the endothelium of arteries and venules and has been found to stain capillary endothelium most intensely , because it is highly sensitive for endothelium and produces lowest background staining .
therefore , in the present study , mvd was estimated by cd34 immunostaining . in the present study , comparison of mean mvd between group
i ( 44.47 ) and group ii ( 97.24 ) showed a statistically highly significant result .
our study suggests that angiogenesis is significantly increased in olp as compared to normal oral mucosa [ figure 1 ] , also in erosive olp [ figure 3 ] as compared to reticular olp [ figure 2 ] ; this suggests that angiogenesis is one of the main contributing factors in the progression of olp .
malignant transformation in olp is said to occur in 0.3%3% of cases and is more common in the erosive form .
thus , the increased mvd in erosive olp as compared to reticular olp in our study could be suggested as one of the factors contributing to this higher malignant potential .
angiogenesis has long been known to be closely linked to chronic inflammation , and it is a component of various chronic inflammatory diseases . however , the exact etiopathological mechanism of olp is still not clear .
most of the studies have not been able to demonstrate a direct relation between angiogenesis and olp .
if antiangiogenic drugs can be demonstrated to be beneficial in olp patients , it would reduce the dependency on corticosteroid drugs . considering the role of angiogenesis in the inflammatory lesions of olp , and the fact that some olp patients respond poorly to conventional immunosuppressive drugs
, angiogenesis could be a possible therapeutic target to reverse the persistent and stubborn inflammation .
from this study , we conclude that angiogenesis can be considered to play a role in both the etiopathogenesis and progression of olp .
our findings could be helpful for formulating new treatment strategies for olp utilizing antiangiogenic medications .
|
background : the etiology of oral lichen planus ( olp ) is not fully understood .
it is generally considered to be a t - cell mediated chronic inflammatory oral mucosal disease .
there is increasing evidence that chronic inflammation is linked to the diseases associated with endothelial dysfunction and is involved in the induction of aberrant angiogenesis.aim:our aim was to evaluate the role of angiogenesis in the pathogenesis of olp by immunohistochemistry , using the cd34 antibody.materials and methods : forty tissue sections ( 7 of erosive lichen planus , 18 of reticular oral lichen planus , and 15 of normal oral mucosa ) , were assessed for microvessel density ( mvd ) in five selected areas of high inflammatory infiltrate by immunohistochemistry for the expression of cd34 antibody.results and conclusion : the mean mvd was 44.47 in the control group ( normal oral mucosa ) and 97.24 in the olp group , showing that there is increased angiogenesis in the latter .
reticular olp had mean mvd of 84.61 and erosive olp had mean mvd of 129.71 , showing relatively greater angiogenesis in erosive olp as compared to reticular olp .
thus , angiogenesis can be considered to play a role in both the etiopathogenesis and the progression of olp .
|
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] |
chf is not only the failure of the heart to generate sufficient cardiac output , but is a multisystemic disorder with immune activation leading to increased concentrations of several cytokines . in chf
several studies showed increased concentrations of proinflammatory cytokines such as tnf , interleukin ( il)-1 , il-6 , il-18 , and cardiotrophin-1 ( ct-1 ) [ 25 ] .
one of the most examined proinflammatory molecules in chf is tnf. tnf is a trimeric 17-kda polypeptide mainly produced by monocytes and macrophages .
short - term tnf expression is thought to be an adaptive mechanism ; whereas prolonged expression causes left ventricular dysfunction and cardiomyopathy leading to chf propagation . however , tnf influences not only the heart itself but causes endothelial dysfunction and peripheral muscle wasting .
cardiotrophin-1 ( ct-1 ) is a member of the il-6 cytokine family that consists of il-6 , il-11 , ciliary neurotrophic factor ( cntf ) , cardiotrophin-1 ( ct-1 ) , cardiotrophin - like cytokine ( clc ) , leukemia inhibitory factor ( lif ) , neuropoietin ( npn ) , and oncostatin m ( osm ) and has recently been supplemented by the addition of two newly characterized cytokines : il-27 and il-31 .
all these cytokines bind to a specific receptor chain ( il-6r , il-11r , or lifr for ct-1 , lif , osm ) .
following cytokine binding the cytokine / receptor complex associates with glycoprotein 130 ( gp130 ) causing tyrosine phosphorylation of gp130 and the signal is transduced via the janus kinase ( jak)/signal transducer and activation of transcription 3 ( stat3 ) pathway [ 810 ] .
ct-1 is expressed in a time - dependent manner during embryogenesis of organs , is expressed in the heart during life , induces cardiac myocyte hypertrophy , and is able to prevent myocyte apoptosis via a mitogen dependent kinase pathway [ 8 , 11 ] .
increased ct-1 concentrations were detected in patients with acute myocardial infarction and chronic heart failure ( chf ) .
furthermore , ct-1 plasma concentrations correlate with the severity of left ventricular dysfunction [ 1114 ] .
however , ct-1 has not only effects on myocytes but also on vasculature by decreasing systemic vascular resistance in an animal model , by induction of acute phase proteins in rat hepatocytes , and by attenuation of endotoxin - induced acute lung injury .
there are several studies showing that in chf pbmcs produce tnf [ 18 , 19 ] .
but so far the mechanisms responsible for tnf production in these cells under these circumstances are not determined . in this study we investigated whether ct-1 induces tnf expression in human pbmc of healthy volunteers .
recombinant human ct-1 was purchased from r&d systems ( wiesbaden , germany ) and dissolved according to the manufacturer 's instruction .
actinomycin d , brefeldin a , and parthenolide were purchased from sigma chemicals ( deisenhofen , germany ) .
the blocking antibody against ct-1 was purchased from r&d systems ( wiesbaden , germany ) .
human peripheral blood mononuclear cells were obtained from healthy volunteers by ficoll - paque ( amersham bioscience , uppsala , sweden ) centrifugation .
the cells were washed three times with pbs , resuspended in rpmi 1640 supplemented with 10% fetal calf serum , 1% penicillin , streptomycin ( all from biochrom ag , berlin , germany ) , and cultured in plastic dishes at 37c in a humified 5% co2 atmosphere .
cells were cultivated for 24 hour with rpmi 1640 supplemented with 10% fetal calf serum , 1% penicillin , streptomycin .
afterwards , cells were subconfluent and medium was replaced by fresh medium . after 24 hours , over 90% of pbmc were alive tested by trypan blue exclusion .
primary cultures from human vein endothelial cells were purchased from promocell ( heidelberg , germany ) .
cell culture was done according to the manufacturer 's manual in endothelial growth medium with 2% fetal calf serum ( egm , promocell , heidelberg , germany ) .
cells were grown to confluence in collagen i coated tissue culture plastic ( becton dickinson , franklin lakes , usa ) .
all stimulants , inhibitors and media were without significant endotoxin levels according to the manufacturers ' instructions .
pharmacological agents , dissolved in fresh medium , were added to the cells for defined time intervals and concentrations . as a control ,
approval for this study was given by the ethics committee of the friedrich schiller university of jena , and subjects gave their written informed consent according to the university guidelines .
total rna from cultivated pbmc was extracted according to the rneasy protocol ( qiagen , hilden , germany ) .
one g of total rna was reversely transcribed into cdna in a volume of 20 l with avian myeloma leukaemia virus ( amv ) reverse transcriptase and oligo dt primers ( promega , madison , usa ) according to the manufacturers manual .
real - time pcr measurement of tnf cdna was performed with the light cycler instrument using the fast start dna master sybr green i kit ( roche diagnostics , mannheim , germany ) . for verification of the correct amplification product ,
the specific primer for human tnf was purchased from r&d systems . the amplification program for tnf consisted of 1 cycle of 94c with a 4-minute hold followed by 40 cycles of 95c with a 45-second hold ,
59c annealing temperature with a 45-second hold , and 72c with a 45-second hold . the specific primer pair for gapdh was : sense primer 5 ggg aag gtg aag gtc gg 3 , antisense primer 5 tgg act cca cga cgt act cag 3. the amplification program for gapdh consisted of 1 cycle of 95c with a 30-second hold followed by 30 cycles of 95c with a 5-second hold , 59c annealing temperature with a 10-second hold , and 72c with a 20-second hold . each reaction ( 20 l ) contained 2 l cdna , 2.5 mm mgcl2 , 1 pmol of each primer , and 2 l of fast starter mix ( containing buffer , dntps , sybr green dye and taq polymerase ) .
a negative control without cdna was run with every pcr to assess the specificity of the reaction .
pcr efficiency was determined by analysing a dilution series of cdna ( external standard curve ) .
the identity of the pcr product was confirmed by comparing its melting temperature ( tm ) with the tm of amplicons from standards or positive controls .
gapdh was analyzed in parallel to each pcr and the resulting gapdh values were used as standards for presentation of tnf transcripts .
tnf concentrations in the culture supernatants were determined by elisa ( quantiglo , r&d systems , wiesbaden , germany ) according to the manufacturer 's instructions .
nuclear extracts were achieved by the epiquik nuclear extraction kit i ( epigentek , ny , usa ) according to the manufacturer 's manual .
afterwards , protein concentrations of nuclear extracts were determined according to the bradford methode . for determination of nfb 2 g of nuclear proteins
were used and further analyzed by gel electrophoretic mobility shift assay ( emsa ) according to the suppliers manual .
emsa kits and probes were purchased from panomics , redwood city , usa . for intracellular staining peripheral blood was collected in lithium - heparin tubes .
100 l of blood was added to rpmi-1640 medium including brefeldin a ( final concentration : 1 g / ml ) ( sigma , taufkirchen , germany ) , and incubated for 6 hours time at 37c .
were stained with monoclonal antibodies against the surface antigens cd3 ( coulter - immunotech , krefeld , germany ) , cd4 ( caltag , hamburg , germany ) cd8 or cd14 ( bd - pharmingen , heidelberg , germany ) ( 15 minute , rt ) , followed , after a washing step , by fixation with 100 l 2% paraformaldehyde for 10 min at rt . after a wash
the cells were incubated in 100 l permeabilisation solution ( 0,1% saponin and 0,1% nan3 in pbs ) together with 1 l directly conjugated anti - tnf antibody ( bd - pharmingen , heidelberg , germany ) for 15 minute at rt .
followed by a wash with permeabilisation solution the cells were resuspended in pbs/2% fcs and fluorescence intensity was analyzed by flow cytometry ( facscalibur , becton - dickinson , heidelberg , germany ) . for analysis regions were defined by forward scatter and side scatter as well as cd3/cd4- or cd3/cd8-lymphocyte populations and cd14 monocyte population .
data were analyzed with cellquest software . because the amount of the cytokines produced was different in each experiment , the effects on tnf production were normalized to unstimulated cells , which were set as one .
data were analysed by nonparametric methods to avoid assumptions about the distribution of the measured variables .
in the first sets of experiments we analysed whether ct-1 is able to induce tnf in pbmc . as shown in figure 1(a ) , ct-1 induced in a concentration - dependent manner tnf in the supernatant determined by a commercial available elisa .
maximal tnf concentration was found after 3 to 6 hours and declined afterwards reaching nearly control values after 24 hours , indicating that ct-1 causes only a transient tnf release in pbmc . in the next experiments we determined intracellular tnf protein in monocytes , cd4 and cd8 lymphocytes after stimulation with various concentrations of ct-1 in the presence of brefeldin a using immunofluorescent flow cytometry .
intracellular tnf determination in cd4 and cd8 lymphocytes did not show an effect of ct-1 on tnf expression ( data not shown ) .
in monocytes we found a concentration - dependent increase of intracellular tnf after ct-1 application ( figure 1(b ) ) .
these results showed that ct-1 induced tnf in pbcm independent of culture conditions and independent of determination methodes . on tnf mrna level we found maximal mrna after 1 hour .
blocking ct-1 by an antibody against ct-1 inhibited ct-1 induced tnf mrna ( data not shown ) indicating that tnf induction is specifically caused by ct-1 . with the next experiments we addressed the question whether tnf protein expression is dependent on mrna synthesis and intracellular protein transport . in figure 3
it is shown that both inhibition of mrna synthesis by actinomycin d and intracellular protein transport by brefeldin a were able to abolish ct-1 induced tnf protein induction in the supernatant .
as shown in figure 4 ct-1 caused a concentration - dependent nfb translocation to the nucleus determined by emsa reaching maximal translocation with 100 ng / ml ct-1 .
in the next sets of experiments we used emsa to verify that nfb activation was responsible for ct-1 induced tnf expression in pbmc . human umbilical vein endothelial cells ( huvecs ) stimulated with tnf were used as a control .
unstimulated cells did not show significant nfb protein in the nucleus ; whereas ct-1 caused translocation of nfb into the nucleus .
parthenolide , an inhibitor of nfb activation , was able to inhibit nfb translocation to the nucleus ( figure 5 ) .
nfb translocation is essential for tnf expression as shown in figure 6(a ) and 6(b ) . because parthenolide was able to inhibit tnf expression both on protein and mrna level
we conclude that ct-1 not only was responsible for nfb translocation to the nucleus but this translocation was responsible for tnf expression . using flow cytometry we found in monocytes an increase of intracellular tnf after ct-1 application which could be inhibited by parthenolide ( figure 6(c ) ) .
these results show that ct-1 induced tnf in pbcm independent of culture conditions and independent of determination methodes and nfb seems to be essential for the effect of ct-1 on tnf induction in pbmc .
the first result of our study is that ct-1 is able to induce tnf mrna and protein in pbmc of healthy volunteers .
tnf is increased in serum of patients with chf and correlates with the severity of heart failure , cachexia , and clinical outcome .
tnf may be involved in progression of chf because high levels of tnf can induce left ventricular dysfunction , ventricular remodelling , cardiomyopathy , and pulmonary edema [ 22 , 23 ] .
cultured human pbmc can synthesize and secrete tnf. in heart failure , both the heart itself and activated monocytes are able to secrete tnf [ 18 , 24 ] .
furthermore , the capacity of pbmc of chf patients to secrete tnf is increased compared to control .
our data are in good agreement with these former studies and in opposite to shimokawa et al . who found decreased cytokine generation capacity of monocytes in severe heart failure after stimulation with lipopolysaccharide .
besides the hypothesis that in chf the failing heart itself is the main source of tnf it is speculated by other groups that activated monocytes are responsible for increased tnf serum concentrations .
monocytes may be activated by lps from the gut because the barrier function of the gut by cardial edema is disturbed and bacteria can easily translocate from the gut lumen to the blood stream . as a third possibility our data suggest at least in theory a new mechanism for tnf production of pbmc in heart failure .
ct-1 produced by the failing ventricle is able to induce tnf in pbmc without lps .
the here presented mechanism might also explain why tnf may be still elevated in chf even after edema were treated successfully with diuretics and the integrity of gut mucosa was restored .
furthermore , our data support the study of petretta et al . who found that tnf is not produced by the failing heart or the gut in patients with mild to severe heart failure . the second result of our study is the fact that ct-1 activates the nfb system in a concentration - dependent manner in pbmc of healthy volunteers
our in vitro data are in line with studies that found an activation of the nfb system in peripheral blood cells in chf .
jankowska et al . reported an activation of the nfb system in peripheral blood leukocytes in chf patients measured by immuncytochemistry .
found an augmented activation of nfb activation in blood mononuclear cells using electromobility shift assay in patients with chf compared to healthy controls .
the exact pathway responsible for nfb activation in chf is still unknown and remains to be determined .
but within 3 hours , there is no cytotoxic effect as shown by o'neill et al . in .
we also used high ct-1 concentrations compared to concentrations reported in patients with chf by ng et al . .
on the other hand a paper published in 2008 reported serum ct-1 concentration in healthy controls and patients with metabolic syndrome of about 100 ng / ml . so far serum concentration of ct-1 in healthy controls and patients is a matter of discussion . but
independent of reported ct-1 serum concentration the concentration of ct-1 should be much higher in the myocardium which is the source of ct-1 in chf .
exact intramyocardial ct-1 concentrations are not determined so far , only mrna and immunohistochemical studies showed increased ct-1 in hearts of patients with chf . in our experiments
both tnf mrna expression and tnf protein production of pbmc showed a large standard variation .
first one explanation for the large standard deviation may be a different genetic susceptibility of pbmc from different persons to stimuli .
second , we used the low basal mrna concentration as the basis of normalization explaining the large standard variation .
third , the fact that the increase of tnf mrna expression after ct-1 application is much higher compared to the increase of protein in the supernatant may be explained methodically .
because in chf many proinflammatory cytokines are elevated and pbmc are activated , it is not easy to study the effect of a single cytokine in pbmc of patients with chf .
for this reason we used pbmc from healthy volunteers in culture and stimulated them with recombinant ct-1 . in conclusion ,
interestingly , in our study lps is not needed for elevated tnf expression in pbmc .
elevated tnf concentrations may be important in the pathogenesis and perpetuation of heart failure by modulating systemic metabolism , causing apoptosis and having a negative inotropic effect . in the light of our results modulating ct-1
|
chronic heart failure ( chf ) is associated with elevated concentrations of tumor necrosis factor ( tnf ) and cardiotrophin-1 ( ct-1 ) and altered peripheral blood mononuclear cell ( pbmc ) function .
therefore , we tested whether ct-1 induces tnf in pbmc of healthy volunteers .
ct-1 induced in pbmc tnf protein in the supernatant and tnf mrna in a concentration- and time - dependent manner determined by elisa and real - time pcr , respectively .
maximal tnf protein was achieved with 100 ng / ml ct-1 after 36 hours and maximal tnf mrna induction after 1 hour .
elisa data were confirmed using immunofluorescent flow cytometry .
inhibitor studies with actinomycin d and brefeldin a showed that both protein synthesis and intracellular transport are essential for ct-1 induced tnf expression .
ct-1 caused a dose dependent nuclear factor ( nf ) b translocation .
parthenolide inhibited both nfb translocation and tnf protein expression indicating that nfb seems to be necessary .
we revealed a new mechanism for elevated serum tnf concentrations and pbmc activation in chf besides the hypothesis of pbmc activation by bacterial translocation from the gut .
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a 48-year - old female patient 158 cm in weight weighing 53 kg was admitted to receive functional endoscopic sinus surgery and septoplasty for a diagnosis of chronic sinusitis and nasal septal deviation .
the patient was a non - smoker and her medical history stated that she had received an appendectomy one year prior to her visit and had a benign tumor removed from her bladder six months before her visit .
there were no abnormalities other than light anemia with an hb of 9.7 g / dl and hct at 30.7% from the blood test .
glycopyrrolate 0.2 mg was i m injected as premedication , and preoxygenation was sufficiently achieved with 100% oxygen before induction of anesthesia .
propofol 100 mg and rocuronium 50 mg was slowly iv injected and after fully confirming loss of consciousness and muscle relaxation , oral endotracheal intubation was attempted with a curved laryngoscope ( macintosh size3 blade ) .
the patient was class 1 based on the cormack and lehane laryngeal endoscopic classification , and endotracheal intubation was performed smoothly in the first attempt with a preformed ring , adair , elwyn tube ( rae , mallinckrodt ) 7.0 mm in diameter .
proper placement of the tube was confirmed by chest auscultation and it was fixed at a tube length of 20 cm .
a tidal volume of 450 ml and respiration rate of 12 times / min were maintained with mechanical ventilation ( aestiva/5 , datex - ohmeda , madison , usa ) ; airway pressure was 14 - 16 cmh2o and positive end expiratory pressure was 1 cmh2o .
anesthesia was maintained with o2 2 l / min , n2o 2 l / min , and sevoflurane 2.0 - 2.5 vol% . during surgery , the blood pressure was 105 - 150/60 - 95 mmhg , heart rate 75 - 100 beats / min , and oxygen saturation 99 - 100% , and end - tidal carbon dioxide tension was maintained at 33 - 37 mmhg .
the duration of the surgery was 2 hours and 30 minutes , and there were no abnormalities during surgery .
after surgery , pyridostigmine 15.0 mg and glycopyrrolate 0.4 mg were iv injected to reverse the muscle relaxation . when spontaneous respiration was sufficiently recovered and extubation of the endotracheal tube was about to be performed , suddenly , the patient severely shook her head and started to struggle so the bandage fixing the tube was immediately removed ; air was removed from the balloon , and extubation was performed . during extubation , the patient was severely bucking while raising her neck and upper body so surrounding staff were called for help in safely holding down the patient .
after extubation , the patient was soon stabilized after a few coughs and was moved to the recovery room .
a blood pressure of 120 - 140/70 - 80 mmhg , heart rate of 65 - 76 beats / min , and oxygen saturation of 99 - 100% were maintained in the recovery room , and other than weak pain in the operated area , there were no specific complaints of symptoms so she was moved to general ward without abnormalities .
three hours after surgery , the patient started to feel nauseous and five hours after surgery , she vomited severely several times thus metoclopramide 10 mg was iv injected .
seven hours after surgery , the patient 's neck and face swelled up with complaints of pain , and physical examination showed crepitation .
vital signs at the time was blood pressure 100/60 mmhg , heart rate 80 beats / min , respiration rate 20 times / min , temperature 36.3 , and oxygen saturation 98% .
arterial blood gas analysis was immediately performed as well as chest and neck x - ray , and neck ct .
the patient was put in semi - fowler 's position to provide oxygen 2 l / min through a nasal cannula , and was put on a fast .
results of arterial blood gas analysis showed ph 7.42 , paco2 31.7 mmhg , pao2 80.2 mmhg , hco3 20.7 meq / l , and sao2 96.8% . in the chest x - ray pneumothorax , subcutaneous emphysema in the chest wall , pneumomediastinum , and pneumoperitoneum were observed ( fig .
the neck ct showed extensive subcutaneous emphysema in the neck and face area , and emphysema in the retropharyngeal space ( fig .
the patient was immediately transferred to icu and oxygen 5 l / min was provided through a mask in fowler 's position .
no abnormalities were discovered in the fiberoptic laryngoscope examination performed by the ent department when investigating the cause . 4 ml of air were extracted from the retropharyngeal space with an 18 g needle using the intraoral approach , and the thoracic surgery performed insertion of chest tube on the right chest and incision of the subcutaneous emphysema on the chest wall .
the patient received esophago gastro duodenoscopy ( egd ) and bronchoscopy for differential diagnosis , and there were no abnormalities found in the egd . in the bronchoscopy , there were no abnormalities in the laryngopharynx , vocal cords , and carina , but mucosal damage and bloody secretion were observed in the right second carina ( fig .
the patient was positive for mycobacterium tuberculosis polymerase chain reaction , and was diagnosed with tuberculosis .
the patient received conservative treatment and anti - tubercular treatment , and was discharged from the hospital 2 weeks later with improved symptoms ( fig .
clinical symptoms of pneumomediastinum are usually chest pain with accompanying radiating pain from the neck or back , breathing difficulties , tachypnea , and throat discomfort .
physical findings include subcutaneous emphysema , the loss of cardiac dullness to percussion , and mediastinal crepitation ( hamman 's sign ) .
diagnosis can be achieved by the above mentioned clinical symptoms and radiological observations . in a simple chest x - ray , usually a line due to an air shadow
can appear along the left heart disinsertion and between the heart and diaphragm ( continuous diaphragm sign ) , while in the lateral view of chest x - ray , there can be subcutaneous emphysema of the chest and neck , air shadows that appear along the back of the sternum or aortic arches , and air shadow around the outer pericardium of the right pulmonary artery ( ring around the artery sign ) .
fifty percent of pneumomediastinum can not be found just with chest pa x - rays ; therefore , a lateral chest x - ray must be taken together .
chest ct can clarify the diagnosis or help confirm lesions that did not appear in simple chest x - rays .
explain the occurrence of pneumomediastinum by the elements inside the thorax and by the elements outside the thorax , depending on the place where the air originated .
elements inside the thorax are rupturing the pulmonary alveoli from asthma , vomiting , giving birth , lifting heavy objects , and applying force with the glottis closed , as well as secondary rupture of pulmonary alveoli or damage to the trachea and bronchus from external physical impact .
elements outside the thorax are cases where anatomical boundaries surrounding the mediastinum is pierced and air is injected , such as in surgery or injury to the head and neck area , tooth extraction , infection or penetration in the abdominal or peritoneal cavity . from these various causes ,
those that are associated with surgery and anesthesia can be complications from the surgery itself , pressure damage from positive ventilation during anesthesia , airway damage from endotracheal intubation , and airway damage from movement of an endotracheal tube .
one case of pneumomediastinum that occurred after functional endoscopic sinus surgery was reported by sohail et al . , where a few hours after receiving functional endoscopic sinus surgery under general anesthesia , pneumomediastinum occurred together with subcutaneous emphysema in the face , neck , and chest wall , and the patient recovered after conservative treatment .
the authors inferred that the surgery itself could not be seen as the direct cause of pneumomediastinum , and that pulmonary alveoli rupture or airway damage from endotracheal intubation were possible causes .
also in our case , the fiberoptic laryngoscope examination performed by the ent department showed no abnormalities from the operated area to the glottis , so there is low possibility that the surgery itself was the direct cause for pneumomediastinum .
the possibility of pressure damage from positive ventilation during anesthesia is also low since various indicators such as ecg , oxygen saturation , airway pressure , positive end expiratory pressure , and end - tidal carbon dioxide tension were all in the normal range .
the possibility of mucosal damage due to endotracheal intubation while inducing anesthesia is also low since there were no difficulties in the intubation process and the 7.0 mm diameter rae tube forms a curve at a 20 cm length so it is difficult to have deeper intubation .
, when mean height of korean females is 157.8 3.7 cm ; the mean length from the front teeth to carina was 25.3 0.9 cm , and the height of the patient in our case was 158 cm so the possibility of deep intubation is low .
additionally , if the tube had been inserted into right second carina , there should have been a clear reduction in breath sound in the non - ventilated lung , increased airway pressure , and a decrease in oxygen saturation , but these situations did not occur up to the end of surgery .
therefore , the authors believe that there is low possibility of mucosal damage due to endotracheal intubation . in addition , since a stilet was not used , damage from a stilet can also be ruled out .
next , there is the possibility of mucosal damage due to the movement of the endotracheal tube . in a study regarding the movement of the endotracheal tube , conrardy et al .
reported that the tube moves 1.5 cm towards the carina when bending the neck , and moves 2.4 cm towards the vocal cords when the neck is extended . in our case , there were no excessive position changes to the neck area during surgery , but in the process of regaining consciousness and spontaneous respiration , there were several bouts of severe struggling and coughing before extubation .
the tube could have moved during the neck bending and the position could have changed during this incident . especially immediately before extubation , the air was removed from the balloon of the tube so the resistance between the tube and airway mucous membrane was largely reduced , and as the bandage was removed and the rae tube was about to be extracted , the patient suddenly lifted her upper body and coughed while bending her neck .
hence , it is conjectured that the tube was inserted much deeper than in the experiment where the neck was simply bent , and the end jabbed the mucous membrane of the airway .
macklin has shown the course of air which causes pneumomediastinum in an animal experiment , and also in our case , the air inflow through the damaged mucous membrane of the airway slowly expanded along the bronchovascular sheath to develop pneumomediastinum , and then expanded along the aorta and membrane surrounding the esophagus to develop pneumoperitoneum .
in addition , it is considered that it detached the carotid perivascular sheath to develop subcutaneous emphysema in the head and neck , and ruptured into the pleural space causing the pneumothorax . according to
the report by hood and sloan , in patients who suffered injury to the trachea or bronchial tubes , the time taken from damage to diagnosis was diverse , and a delay of 24 hours or more appeared in 66% . in our case , it took about 7 hours from the symptoms to appear from bronchial damage to be diagnosed , and such delay in time is because when there is partial damage to the tracheal wall , the air enters by detaching the adventitia and expanding to form an air sack .
this expansion of air can take many hours , and there could be no symptoms until it ruptures into the mediastinum or pleura space .
mccoy et al . reported that causes for endobronchial intubation were head and neck surgery and laparoscopy which can affect the location of the tube during surgery , incapacity of the anesthesiologist , the movements of the surgeons , and usage of the rae tube .
the rae tube has a relatively large elasticity and structurally forms a sharp angle ; thus , it is easy to damage the bronchial tubes when deep intubation occurs , hence there is need for more caution .
it is conjectured that also in our case , these structural characteristics of the rae tube caused more damage to the bronchial tubes than the generally used single lumen endotracheal tube . in our case ,
the patient severely vomited several times 5 hours after surgery and the symptoms occurred 2 hours later , and for pneumomediastinum caused by vomiting , there are 2 reasons for esophagus rupture and pulmonary alveoli rupture .
differential diagnosis is performed with fiberoptic esophagoscopy or esophagography , and in our case , an egd was performed which confirmed that there were no abnormalities in the esophagus .
hence , it is considered that the pulmonary alveoli ruptured from the valsalva maneuver during vomiting , and the inflow of air expanded through the bronchovascular sheath to cause the pneumomediastinum .
treatment for pneumomediastinum caused by tracheal damage is conservative treatment consisting of bed rest , a supply of oxygen , and use of antibiotics when the symptoms are not severe and the vital signs are satisfactory .
when there is severe damage , the vital signs are unstable , and there is a possibility of dyspnea or septicemia , it is better to perform surgical suturing in the early stages .
in addition , to reduce pressure , extraction of air through subcutaneous dilacerations and insertion of a subcutaneous catheter are performed ; chest tube insertion is performed for a pneumothorax larger than 15 - 20% of the thoracic volume , and endotracheal intubation or tracheostomy can be performed when there is severe circulatory disturbance or respiration difficulty .
the patient in our case received conservative treatment consisting of bed rest , oxygen therapy , use of antibiotics , and administration of analgesics together with prompt investigation of the cause after presenting symptoms .
the vital signs were not bad so the patient could be observed and monitored , but under the decision that the accompanying pneumothorax and subcutaneous emphysema were severe , chest tube insertion , dilacerations of the subcutaneous emphysema , and aspiration in the retropharyngeal space were performed .
although invasive , it is considered that these prompt decompression procedures prevent cardiac tamponade and an increase in pulmonary vascular pressure caused by air in the vascular sheath and hence , contribute to the faster recovery of the patient . as a method to prevent excessive excitement of the patient while waking up
, continuous iv injection of a low concentration of remifentanil has the effect of hemodynamic stability , cough reflex suppression , and pain relief , which can lead to smooth awakening and reduce serious complications as in our case .
the patient subsequently received conservative treatment and antitubercular treatment , and recovered enough to be discharged 2 weeks later . before discharge , we wanted to check what happened to the lesions in the bronchial mucous membrane , but the patient refused so it is a pity that we were unable to follow - up . from the various cases and literature reviewed above
, it is believed that in our case , the struggling and coughing of the patient during regaining consciousness pushed the endotracheal tube into the bronchial tubes , and the end of the tube damaged the bronchial mucous membrane to gradually develop emphysema . the valsalva maneuver during severe
vomiting additionally increased the airway pressure , leaking a large amount of air through the damaged area in a short period of time , which accelerated the emphysema .
pneumomediastinum can be completely treated with conservative treatment , but according to the circumstances , it can proceed to mediastinitis or septicemia and lead to the death for the patient , so diagnosis , treatment , and methods of prevention must be well informed .
in addition , the possibility of pneumomediastinum from anesthesia should also be considered and caution should be taken for positive ventilation and endotracheal intubation , and there should be close observation of the patient 's state before and after surgery . especially , there is need for meticulous care after regaining consciousness since the patient 's struggling or coughing during extubation can push the endotracheal tube inside to damage the trachea or bronchial tubes , or cause unintended extubation and damage the glottis causing difficulties in maintaining respiration .
|
the occurrences of pneumothorax and pneumomediastinum are rare , but considered to be potentially life - threatening conditions in patients undergoing functional endoscopic sinus surgery under general anesthesia .
tracheobronchial rupture may results in serious complications , such as pneumothorax and pneumomediastinum .
it may occur accidentally by endotracheal tube when the patient 's neck is flexed or extended .
we report the case of a 48-year - old female patient who developed massive subcutaneous emphysema , pneumothorax , pneumomediastinum and pneumoperitoneum seven hours after functional endoscopic sinus surgery under general anesthesia .
|
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