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ders [1], the most prevalent group of mental disorders world-
wide with more than 300 million people affected by one of 
these conditions [2–4]. Importantly, most patients are still 
resistant or relapse to currently available pharmacological 
and/or cognitive behavioural therapies [5, 6].
Pavlovian fear conditioning is a translationally relevant 
model, key in both understanding the physiology of fear and 
the pathophysiology of anxiety disorders. Fear condition-
ing is an associative learning process in which a neutral 
stimulus, such as a tone or a specific context (conditioned 
stimulus, CS), is presented together with an aversive stimu-
lus, usually a foot shock (unconditioned stimulus, US), to 
generate a conditioned fear response [7]. The fear acquisition 
phase is followed by a consolidation phase, lasting hours to 
days, during which memories are formed [8]. Subsequent 
exposure to the CS, in absence of the US, is sufficient to 
evoke freezing behaviour, demonstrating the effective for-
mation of a conditioned fear memory. Re-exposure to the 
CS after the stabilization of fear memories causes them 
to become labile and susceptible to disruption [9]. In this 
state, depending on the characteristics of the CS exposure, 
memories can progress to extinction, or they can undergo 
reconsolidation. Reconsolidation is the process through 
which memories re-stabilize after having been destabilized 
by exposure to the CS. It is thought to allow the integration 
of new information into the memory trace, and shares most, 
but not all, molecular mechanisms with fear consolidation 
[10, 11].
Fear conditioning is mediated by the brain fear circuit, 
a complex ensemble of brain areas with highly specialized 
 * Mónica Santos 
 
mjpsantos@cnc.uc.pt; monicapsantos@hotmail.com
1 
CNC – Center for Neuroscience and Cell Biology, University 
of Coimbra, Coimbra, Portugal
2 
Institute of Interdisciplinary Research, University of Coimbra 
(iiiUC), Coimbra, Portugal
3 
Department of Life Sciences, University of Coimbra,


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Vol.:(0123456789)
1 3
https://doi.org/10.1007/s12035-023-03253-7
A Circular RNA Expressed from the FAT3 Locus Regulates Neural 
Development
Sabine Seeler1,2 · Maria Schertz Andersen1 · Tamas Sztanka‑Toth3 · Mateja Rybiczka‑Tešulov4 · 
Marleen H. van den Munkhof4 · Chi‑Chih Chang1 · Muyesier Maimaitili2,5 · Morten Trillingsgaard Venø1,6 · 
Thomas Birkballe Hansen1 · R. Jeroen Pasterkamp4 · Agnieszka Rybak‑Wolf3 · Mark Denham2,5 · Nikolaus Rajewsky3 · 
Lasse Sommer Kristensen1,2 · Jørgen Kjems1 
Received: 27 July 2022 / Accepted: 28 January 2023 
© The Author(s) 2023
Abstract
Circular RNAs (circRNAs) are key regulators of cellular processes, are abundant in the nervous system, and have putative 
regulatory roles during neural differentiation. However, the knowledge about circRNA functions in brain development is 
limited. Here, using RNA-sequencing, we show that circRNA levels increased substantially over the course of differentiation 
of human embryonic stem cells into rostral and caudal neural progenitor cells (NPCs), including three of the most abundant 
circRNAs, ciRS-7, circRMST, and circFAT3. Knockdown of circFAT3 during early neural differentiation resulted in minor 
transcriptional alterations in bulk RNA analysis. However, single-cell transcriptomics of 30 and 90 days differentiated cerebral 
organoids deficient in circFAT3 showed a loss of telencephalic radial glial cells and mature cortical neurons, respectively. 
Furthermore, non-telencephalic NPCs in cerebral organoids showed changes in the expression of genes involved in neural 
differentiation and migration, including FAT4, ERBB4, UNC5C, and DCC. In vivo depletion of circFat3 in mouse prefrontal 
cortex using in utero electroporation led to alterations in the positioning of the electroporated cells within the neocortex. 
Overall, these findings suggest a conserved role for circFAT3 in neural development involving the formation of anterior cell 
types, neuronal differentiation, or migration.


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Keywords ncRNAs · circRNAs · Cerebral organoids · scRNA-seq · Neural development · circFAT3
Introduction
The mammalian nervous system develops through a suc-
cession of tightly orchestrated processes including neural 
induction, neural tube patterning along the anterior–pos-
terior and dorsal–ventral axes, progenitor cell expansion, 
neural differentiation, and neuronal migration [1, 2]. Several 
types of non-coding RNAs (ncRNAs), including microR-
NAs (miRNAs) [3] and long ncRNAs (lncRNAs) [4, 5], 
Sabine Seeler and Maria Schertz Andersen contributed equally to 
this work.
Lasse Sommer Kristensen and Jørgen Kjems contributed equally to 
this work.
 * Lasse Sommer Kristensen 
 
lasse@biomed.au.dk
 * Jørgen Kjems 
 
jk@mbg.au.dk
1 
Interdisciplinary Nanoscience Center, Department 
of Molecular Biology and Genetics, Aarhus University, 8000 
Aarhus C, Aarhus, Denmark
2 
Department of Biomedicine, The Skou Building, Aarhus 
University, 8000 Aarhus C, Aarhus, Denmark
3 
Berlin Institute for Medical Systems Biology (BIMSB), 
MDC Berlin-Mitte, 10115 Berlin, Germany
4 
Department of Translational Neuroscience, University 
Medical Center Utrecht Brain Center, 3584 CG, Utrecht, 
Netherlands
5 
Danish Research Institute of Translational Neuroscience, 
Nordic EMBL Partnership for Molecular Medicine, Aarhus 
University, 8000 Aarhus C, Aarhus, Denmark
6 
Omiics ApS, 8200 Aarhus N, Aarhus, Denmark
/ Published online: 25 February 2023
Molecular Neurobiology (2023) 60:3239–3260


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Super-resolution in brain positron emission tomography using a 
real-time motion capture system
Yanis Chemlia,d, Marc-André Tétraultb, Thibault Marina, Marc D. Normandina, Isabelle 
Blochc,d, Georges El Fakhria, Jinsong Ouyanga,1,*, Yoann Petibona,1
aGordon Center for Medical Imaging, Department of Radiology Massachusetts General Hospital, 
Harvard Medical School, Boston, MA, United States
bDepartment of Computer Engineering, Universté de Sherbrooke, Sherbrooke, QC, Canada
cSorbonne Université, CNRS, LIP6, Paris, France
dLTCI, Télécom Paris, Institut Polytechnique de Paris, France
Abstract
Super-resolution (SR) is a methodology that seeks to improve image resolution by exploiting the 
increased spatial sampling information obtained from multiple acquisitions of the same target with 
accurately known sub-resolution shifts. This work aims to develop and evaluate an SR estimation 
framework for brain positron emission tomography (PET), taking advantage of a high-resolution 
infra-red tracking camera to measure shifts precisely and continuously. Moving phantoms and 
non-human primate (NHP) experiments were performed on a GE Discovery MI PET/CT scanner 
(GE Healthcare) using an NDI Polaris Vega (Northern Digital Inc), an external optical motion 
tracking device. To enable SR, a robust temporal and spatial calibration of the two devices was 
developed as well as a list-mode Ordered Subset Expectation Maximization PET reconstruction 
algorithm, incorporating the high-resolution tracking data from the Polaris Vega to correct motion 
for measured line of responses on an event-by-event basis. For both phantoms and NHP studies, 
the SR reconstruction method yielded PET images with visibly increased spatial resolution 
compared to standard static acquisitions, allowing improved visualization of small structures. 
Quantitative analysis in terms of SSIM, CNR and line profiles were conducted and validated our 
observations. The results demonstrate that SR can be achieved in brain PET by measuring target


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motion in real-time using a high-resolution infrared tracking camera.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
*Corresponding author at: 125 Nashua St. Suite 660, Boston, MA 02114-1101. ouyang.jinsong@mgh.harvard.edu (J. Ouyang).
1Co-senior authors
Declaration of Competing Interest
None.
Credit authorship contribution statement
Yanis Chemli: Methodology, Software, Investigation, Formal analysis, Validation, Writing – original draft, Writing – review & 
editing. Marc-André Tétrault: Investigation. Thibault Marin: Software, Writing – review & editing. Marc D. Normandin: 
Investigation, Resources. Isabelle Bloch: Supervision, Writing – review & editing. Georges El Fakhri: Supervision, Resources, 
Funding acquisition. Jinsong Ouyang: Conceptualization, Methodology, Investigation, Supervision, Writing – review & editing, 
Project administration. Yoann Petibon: Conceptualization, Methodology, Investigation, Supervision, Writing – review & editing, 
Project administration.
HHS Public Access
Author manuscript
Neuroimage. Author manuscript; available in PMC 2023 May 15.
Published in final edited form as:
Neuroimage. 2023 May 15; 272: 120056. doi:10.1016/j.neuroimage.2023.120056.
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Sequencing of Circulating Cell-free DNA during Pregnancy
Diana W. Bianchi, M.D.,
Prenatal Genomics and Fetal Therapy Section, Medical Genetics Branch, National Human 
Genome Research Institute, National Institutes of Health, Bethesda
Rossa W.K. Chiu, M.B., B.S., Ph.D.
Li Ka Shing Institute of Health Sciences and the Department of Chemical Pathology, Chinese 
University of Hong Kong, Shatin, Hong Kong
Abstract
Sequence analysis of cell-free DNA (cfDNA) fragments that circulate in the blood of pregnant 
women, along with the translation of this method into screening for fetal chromosome 
abnormalities, is a success story of modern genomic medicine. In less than a decade, prenatal 
cfDNA testing has gone from small, proof-of-principle studies to a global transformation of 
prenatal care. As of late 2017, a total of 4 million to 6 million pregnant women had had DNA 
from their plasma analyzed to screen for fetal aneuploidy.1 The exponential growth of the test has 
been a function of the role of the biotechnology industry in its development and marketing. Here 
we review what has been learned from the wide-scale implementation of this testing, how it has 
changed prenatal clinical care, and what ethical concerns have arisen, and we speculate about what 
lies ahead.
PATH TO CLINICAL IMPLEMENTATION
Twenty years ago, cfDNA was identified as a plasma analyte when Y chromosomal 
“fetal” DNA was extracted and amplified from the blood of pregnant women carrying 
male fetuses.2 During pregnancy, DNA fragments are released from the placenta into the 
maternal circulation as cytotrophoblast and syncytiotrophoblast cells undergo physiologic 
cycles of fusion and apoptosis (see video).3 In contrast to the isolation of intact fetal cells 
from maternal blood, analysis of circulating DNA fragments is performed on a plasma 
sample that contains both maternal and placental cfDNA. The ratio of placental to total 
(consisting of maternal and placental) cfDNA is known as the fetal fraction, which increases


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as pregnancy advances.4,5 Testing of cfDNA is generally performed from the 10th week of 
gestation onward, since this is when the fetal fraction in the maternal circulation reaches 
the minimum amount needed for an informative test result. The fetal fraction is only one 
of several variables that affect the sensitivity of the test. Other variables include the number 
of cfDNA molecules sequenced, the proportion of guanine and cytosine bases in a specific 
chromosome, and the presence of maternal and fetal copy-number variants.6
Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.
HHS Public Access
Author manuscript
N Engl J Med. Author manuscript; available in PMC 2023 April 24.
Published in final edited form as:
N Engl J Med. 2018 August 02; 379(5): 464–473. doi:10.1056/NEJMra1705345.
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Preventive and therapeutic benefits of nelfinavir in rhesus macaques and human beings infected with SARS-CoV-2


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ARTICLE
OPEN
Preventive and therapeutic beneﬁts of nelﬁnavir in rhesus
macaques and human beings infected with SARS-CoV-2
Zhijian Xu
1,2,3, Danrong Shi4, Jian-Bao Han5, Yun Ling6, Xiangrui Jiang1,3, Xiangyun Lu4, Chuan Li1,3, Likun Gong1,3,7, Guangbo Ge8,
Yani Zhang8, Yi Zang1,3, Tian-Zhang Song5,9, Xiao-Li Feng5, Ren-Rong Tian5,9, Jia Ji4, Miaojin Zhu4, Nanping Wu4, Chunhui Wu1,3,
Zhen Wang1,3, Yechun Xu1,2,3, Cheng Peng1,2,3, Min Zheng
4, Junling Yang1,3, Feifei Du1,3, Junliang Wu1,3,7, Peipei Wang1,3,
Jingshan Shen
1,3✉, Jianliang Zhang6✉, Yong-Tang Zheng
5,9✉, Hangping Yao
4,10✉ and Weiliang Zhu
1,2,3✉
Effective drugs with broad spectrum safety proﬁle to all people are highly expected to combat COVID-19 caused by SARS-CoV-2.
Here we report that nelﬁnavir, an FDA approved drug for the treatment of HIV infection, is effective against SARS-CoV-2 and COVID-
19. Preincubation of nelﬁnavir could inhibit the activity of the main protease of the SARS-CoV-2 (IC50 = 8.26 μM), while its antiviral
activity in Vero E6 cells against a clinical isolate of SARS-CoV-2 was determined to be 2.93 μM (EC50). In comparison with vehicle-
treated animals, rhesus macaque prophylactically treated with nelﬁnavir had signiﬁcantly lower temperature and signiﬁcantly
reduced virus loads in the nasal and anal swabs of the animals. At necropsy, nelﬁnavir-treated animals had a signiﬁcant reduction of
the viral replication in the lungs by nearly three orders of magnitude. A prospective clinic study with 37 enrolled treatment-naive
patients at Shanghai Public Health Clinical Center, which were randomized (1:1) to nelﬁnavir and control groups, showed that the
nelﬁnavir treatment could shorten the duration of viral shedding by 5.5 days (9.0 vs. 14.5 days, P = 0.055) and the duration of fever
time by 3.8 days (2.8 vs. 6.6 days, P = 0.014) in mild/moderate COVID-19 patients. The antiviral efﬁciency and clinical beneﬁts in
rhesus macaque model and in COVID-19 patients, together with its well-established good safety proﬁle in almost all ages and


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during pregnancy, indicated that nelﬁnavir is a highly promising medication with the potential of preventative effect for the
treatment of COVID-19.
Signal Transduction and Targeted Therapy  (2023) 8:169 
; https://doi.org/10.1038/s41392-023-01429-0
INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a
highly infectious positive-strand RNA virus causing COVID-19
(coronavirus disease 2019), has evolved through Alpha, Beta,
Gamma, Delta, and Omicron mutant strains, resulting in an
unabated global pandemic trend. Although quite a few COVID-19
vaccines have been used worldwide since 2021, which could
signiﬁcantly protect people from getting severely ill or dying, as
many as hundreds of thousands of infection cases are currently
reported each day.1 Therefore, effective drugs with good safety
proﬁles are highly expected to protect the SARS-CoV-2 infected
people by stopping the virus replication in the body to prevent
the people from becoming severe or dying. There are two well
known drug targets for SARS-CoV-2, one is RNA-dependent RNA
polymerase (RdRp) and the other is main protease (Mpro).
Replication of RNA from an RNA template is catalyzed by RdRp,
leading to its vital role in SARS-CoV-2 replication and transcription.
Mpro, also known as 3C-like protease (chymotrypsin-like protease),
cleaving at least 11 sites of SARS-CoV-2 polyprotein, is highly
conservable among coronaviruses. Sequence alignment revealed
that the Mpro of SARS-CoV-2 shares 96% similarity with that of
SARS-CoV-1. In addition, Mpro has no human homolog, making
Mpro the most attractive drug target to treat COVID-19.
Small
molecule
antivirals
have
the
advantages
of
easy
manufacture, low cost and good accessibility.2–4 There are several
small molecule drugs shown beneﬁcial effects in preclinical and
clinical trials. For example, in the Evaluation of Protease Inhibition
for Covid-19 in High-Risk Patients (EPIC-HR) double-blind con-
trolled trial with a total of 2246 unvaccinated patients from July 16


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to December 9, 2021 when the Delta mutant dominated,
Received: 24 September 2022 Revised: 8 March 2023 Accepted: 4 April 2023
1State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 2Drug Discovery and Design Center,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 3School of Pharmacy, University of Chinese Academy of Sciences, Beijing 100049,
China; 4State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, First Afﬁliated Hospital, Zhejiang
University School of Medicine, Hangzhou 310003, China; 5Kunming National High-level Biosafety Research Center for Non-human Primates, Center for Biosafety Mega-Science,
Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China; 6Department of Infectious Disease, Shanghai Public Health Clinical Center, Fudan
University, Shanghai 201508, China; 7Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
China; 8Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese
Medicine, Shanghai 201203, China; 9Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences, KIZ-CUHK Joint Laboratory of
Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China and 10Jinan
Microecological Biomedicine Shandong Laboratory, Jinan 250117, China
Correspondence:
Jingshan
Shen
(shenjingshan@simm.ac.cn)
or
Jianliang
Zhang
(zhangjianliang@shphc.org.cn)
or
Yong-Tang
Zheng
(zhengyt@mail.kiz.ac.cn)
or
Hangping Yao (yaohangping@zju.edu.cn) or Weiliang Zhu (wlzhu@simm.ac.cn)
These authors contributed equally: Zhijian Xu, Danrong Shi, Jian-Bao Han, Yun Ling, Xiangrui Jiang, Xiangyun Lu


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www.nature.com/sigtrans
Signal Transduction and Targeted Therapy
© The Author(s) 2023
1234567890();,:


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Nature Reviews Immunology
nature reviews immunology
https://doi.org/10.1038/s41577-023-00871-z
Review article
 Check for updates
The unique properties of IgG4  
and its roles in health and disease
Theo Rispens1 & Maartje G. Huijbers 
  2,3 
Abstract
IgG4 is the least abundant subclass of IgG in human serum and has 
unique functional features. IgG4 is largely unable to activate antibody-
dependent immune effector responses and, furthermore, undergoes 
Fab (fragment antigen binding)-arm exchange, rendering it bispecific 
for antigen binding and functionally monovalent. These properties 
of IgG4 have a blocking effect, either on the immune response or 
on the target protein of IgG4. In this Review, we discuss the unique 
structural characteristics of IgG4 and how these contribute to its 
roles in health and disease. We highlight how, depending on the 
setting, IgG4 responses can be beneficial (for example, in responses 
to allergens or parasites) or detrimental (for example, in autoimmune 
diseases, in antitumour responses and in anti-biologic responses). 
The development of novel models for studying IgG4 (patho)physiology 
and understanding how IgG4 responses are regulated could offer 
insights into novel treatment strategies for these IgG4-associated 
disease settings.
Sections
Introduction
Development of an IgG4 
response
Structure and function of IgG4
Physiological roles of IgG4
IgG4-dependent pathology
IgG4-related diseases
Future directions
1Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands. 
2Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. 3Department of 
Neurology, Leiden University Medical Center, Leiden, The Netherlands. 
 e-mail: M.G.M.Huijbers@lumc.nl


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In vitro effects of cannabidiol and its main metabolites in mouse 
and human Sertoli cells
Yuxi Li1, Qiangen Wu1, Xilin Li2, Linda S. Von Tungeln1, Frederick A. Beland1, Dayton 
Petibone2, Lei Guo1, Patrick Cournoyer3, Supratim Choudhuri3, Si Chen1,*
1Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and 
Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA
2Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. 
Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA
3Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug 
Administration, College Park, MD 20740, USA.
Abstract
Cannabidiol (CBD) is a major cannabinoid present in extracts of the plant Cannabis sativa 
(marijuana). While the therapeutic effects of CBD on epilepsy have been demonstrated, less is 
understood regarding its potential adverse effects. Recent studies revealed that CBD induced 
toxicity in the male reproductive system of animal models. In this study, we used TM4, an 
immortalized mouse Sertoli cell line, and primary human Sertoli cells to evaluate the toxicities of 
CBD and its main metabolites, 7-carboxy-CBD and 7-hydroxy-CBD. CBD induced concentration- 
and time-dependent cytotoxicity in mouse and human Sertoli cells, which mainly resulted from 
the inhibition of the G1/S-phase cell cycle transition. CBD also inhibited DNA synthesis and 
downregulated key cell cycle proteins. Moreover, CBD reduced the mRNA and protein levels 
of a functional marker, Wilms’ tumor 1. Similar to CBD, 7-carboxy-CBD and 7-hydroxy-CBD 
inhibited cellular proliferation and decreased DNA synthesis. 7-Carboxy-CBD was less cytotoxic 
than CBD, while 7-hydroxy-CBD showed comparable cytotoxicity to CBD in both mouse and 
human Sertoli cells. Compared to mouse Sertoli cells, CBD, 7-hydroxy-CBD, and 7-carboxy-CBD 
were more cytotoxic in human Sertoli cells. Our results indicate that CBD and its main metabolites


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can inhibit cell proliferation in mouse and human Sertoli cells.
Keywords
Cannabidiol; 7-Carboxy-CBD; 7-Hydroxy-CBD; Male reproductive toxicity; Sertoli cells; Cell 
cycle arrest; DNA synthesis; Wilms’ tumor 1
*Corresponding author. Division of Biochemical Toxicology, National Center for Toxicological Research, 3900 NCTR Road, 
Jefferson, AR 72079, USA. Si.Chen@fda.hhs.gov. 
Conflict of interest: The authors declare no conflict of interest.
Compliance with ethical standards
Disclaimer: This article reflects the views of the authors and does not necessarily reflect those of the U.S. Food and Drug 
Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or 
recommendation.
HHS Public Access
Author manuscript
Food Chem Toxicol. Author manuscript; available in PMC 2023 April 24.
Published in final edited form as:
Food Chem Toxicol. 2022 January ; 159: 112722. doi:10.1016/j.fct.2021.112722.
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Inhibiting degradation of 2-arachidonoylglycerol as a therapeutic strategy for neurodegenerative diseases


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Inhibiting degradation of 2-arachidonoylglycerol as a therapeutic 
strategy for neurodegenerative diseases
Chu Chen
Department of Cellular and Integrative Physiology, Center for Biomedical Neuroscience, Joe 
R. and Teresa Lozano Long School of Medicine, University of Texas Health San Antonio, San 
Antonio, Texas, 78229, USA
Abstract
Endocannabinoids are endogenous lipid signaling mediators that participate in a variety of 
physiological and pathological processes. 2-Arachidonoylglycerol (2-AG) is the most abundant 
endocannabinoid and is a full agonist of G-protein-coupled cannabinoid receptors (CB1R 
and CB2R), which are targets of Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive 
ingredient in cannabis. While 2-AG has been well recognized as a retrograde messenger 
modulating synaptic transmission and plasticity at both inhibitory GABAergic and excitatory 
glutamatergic synapses in the brain, growing evidence suggests that 2-AG also functions as an 
endogenous terminator of neuroinflammation in response to harmful insults, thus maintaining 
brain homeostasis. Monoacylglycerol lipase (MAGL) is the key enzyme that degrades 2-AG 
in the brain. The immediate metabolite of 2-AG is arachidonic acid (AA), a precursor of 
prostaglandins (PGs) and leukotrienes. Several lines of evidence indicate that pharmacological 
or genetic inactivation of MAGL, which boosts 2-AG levels and reduces its hydrolytic metabolites, 
resolves neuroinflammation, mitigates neuropathology, and improves synaptic and cognitive 
functions in animal models of neurodegenerative diseases, including Alzheimer’s disease (AD), 
multiple sclerosis (MS), Parkinson’s disease (PD), and traumatic brain injury (TBI)-induced 
neurodegenerative disease. Thus, it has been proposed that MAGL is a potential therapeutic 
target for treatment of neurodegenerative diseases. As the main enzyme hydrolyzing 2-AG, 
several MAGL inhibitors have been identified and developed. However, our understanding


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of the mechanisms by which inactivation of MAGL produces neuroprotective effects in 
neurodegenerative diseases remains limited. A recent finding that inhibition of 2-AG metabolism 
in astrocytes, but not in neurons, protects the brain from TBI-induced neuropathology might shed 
some light on this unsolved issue. This review provides an overview of MAGL as a potential 
therapeutic target for neurodegenerative diseases and discusses possible mechanisms underlying 
the neuroprotective effects of restraining degradation of 2-AG in the brain.
Correspondence should be addressed to: Chu Chen, PhD, Department of Cellular and Integrative Physiology, School of Medicine, 
University of Texas Health San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA, Tel: (210) 450-5376, Fax: (210) 
567-4410, chenc7@uthscsa.edu or chen502@gmail.com. 
Conflict of interest statement
The authors declare that there are no conflicts of interest.
HHS Public Access
Author manuscript
Pharmacol Ther. Author manuscript; available in PMC 2023 April 24.
Published in final edited form as:
Pharmacol Ther. 2023 April ; 244: 108394. doi:10.1016/j.pharmthera.2023.108394.
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Bridging to Allotransplantation—Is Pig Liver 
Xenotransplantation the Best Option?
Vladimir Lamm, MD1, Burcin Ekser, MD, PhD2, Parsia A. Vagefi, MD3, David K.C. Cooper, 
MD, PhD, FRCS4
1Division of Gastroenterology, Department of Medicine, Washington University School of 
Medicine, St. Louis, MO.
2Division of Transplant Surgery, Department of Surgery, Indiana University School of Medicine, 
Indianapolis, IN.
3Division of Surgical Transplantation, Department of Surgery, University of Texas Southwestern 
Medical Center, Dallas, TX.
4Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, 
Birmingham, AL.
Abstract
In the past 20 y, the number of patients in the United States who died while waiting for a 
human donor liver totaled >52 000. The median national wait time for patients with acute liver 
failure and the most urgent liver transplant listing was 7 d in 2018. The need for a clinical 
“bridge” to allotransplantation is clear. Current options for supporting patients with acute liver 
failure include artificial liver support devices, extracorporeal liver perfusion, and hepatocyte 
transplantation, all of which have shown mixed results with regard to survival benefit and are 
largely experimental. Progress in the transplantation of genetically engineered pig liver grafts in 
nonhuman primates has grown steadily, with survival of the pig graft extended to almost 1 mo 
in 2017. Further advances may justify consideration of a pig liver transplant as a clinical bridge 
to allotransplantation. We provide a brief history of pig liver xenotransplantation, summarize 
the most recent progress in pig-to-nonhuman primate liver transplantation models, and suggest 
criteria that may be considered for patient selection for a clinical trial of bridging by genetically 
engineered pig liver xenotransplantation to liver allotransplantation.
Correspondence: Vladimir Lamm, MD, Division of Gastroenterology Department of Medicine, Washington University School of


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Medicine, 660 S. Euclid Ave, Campus Box 8124, St. Louis, MO 63110. (lamm@wustl.edu). Correspondence: David K.C. Cooper, 
MD, PhD, FRCS, Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, LHRB 752, 701 19th 
St S, Birmingham, AL 35294. (dkcooper@uabmc.edu).
All listed authors participated in the writing of this review article. V.L. and D.K.C.C. drafted the article, revised it critically for 
important intellectual content, approved the final version of the work to be published, and agreed to be accountable for all aspects of 
the work. B.E. and P.A.V. revised the article critically for important intellectual content; approved the final version of the work to be 
published; and agreed to be accountable for all aspects of the work.
The authors declare no conflicts of interest.
HHS Public Access
Author manuscript
Transplantation. Author manuscript; available in PMC 2023 April 24.
Published in final edited form as:
Transplantation. 2022 January 01; 106(1): 26–36. doi:10.1097/TP.0000000000003722.
Author Manuscript
Author Manuscript
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Suggested Patient Selection Criteria for Initial Clinical Trials of Pig Kidney Xenotransplantation in the USA


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Suggested Patient Selection Criteria for Initial Clinical Trials of 
Pig Kidney Xenotransplantation in the USA
Abhijit Jagdale, MD1, Vineeta Kumar, MD2, Douglas J. Anderson, MD1,3, Jayme E. Locke, 
MD, MPH1,3, Michael J. Hanaway, MD3, Devin E. Eckhoff, MD1,3, Hayato Iwase, MD, PhD1, 
David K.C. Cooper, MD, PhD, FRCS1
1.Xenotransplantation Program, Division of Transplantation, Department of Surgery, University of 
Alabama at Birmingham, Birmingham, AL, USA
2.Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, 
Birmingham, AL, USA
3.Division of Transplantation, Department of Surgery, University of Alabama at Birmingham, 
Birmingham, AL, USA
Lay summary
There is a critical shortage of kidneys for transplantation into patients with kidney failure. 
Genetically-engineered pigs could provide an additional source. Increasing success is being 
reported of the transplantation of pig kidneys in nonhuman primates. Consideration is now being 
given to the selection of patients for the first clinical trial of pig kidney transplantation. In some 
US states, patients aged 55–65, particularly if of blood group O, may wait >5 years for a donor 
organ, by which time >50% are likely to have died or removed from the wait-list because they 
are no longer acceptable for transplantation. We suggest these patients, if otherwise healthy, might 
accept the opportunity of early pig kidney transplantation.
Introduction
More than 80 000 patients with end-stage renal disease (ESRD) are on the wait-list for 
kidney transplantation in the USA,1 most of whom are receiving chronic hemodialysis. 
The 5-year mortality of wait-listed patients is approximately 40%,2–5 whereas the median 
waiting time to receive a deceased human donor kidney is 4 years.6 Many patients will not 
live long enough to undergo kidney allotransplantation.
Chronic dialysis is associated with a relatively high mortality and increased comorbidities 
that may render the patient unsuitable for renal transplantation. Furthermore, the quality of


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Correspondence: David K.C. Cooper, MD, PhD, FRCS, Xenotransplantation Program, Department of Surgery, University of Alabama 
at Birmingham (UAB), Birmingham, AL 35294-0007, USA., Tel: 412-443-4294, dkcooper@uabmc.edu.
Author contribution:
DKCC, AJ: Conceived and/or designed the study. AJ: acquired data, played an important role in interpreting the results, drafted the 
manuscript. DKCC, VK, DA, JL, MH, DE, HI: revised the manuscript and approved the final version.
DKCC confirm that he has had full access to the data in the study and final responsibility.
Conflict of interest
No author has a conflict of interest
HHS Public Access
Author manuscript
Transplantation. Author manuscript; available in PMC 2023 April 24.
Published in final edited form as:
Transplantation. 2021 September 01; 105(9): 1904–1908. doi:10.1097/TP.0000000000003632.
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Sleeping Beauty transposon system for GDNF overexpression of entrapped stem cells in fibrin hydrogel in a rat model of Parkinsons disease


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Vol.:(0123456789)
1 3
https://doi.org/10.1007/s13346-023-01289-9
Sleeping Beauty transposon system for GDNF overexpression 
of entrapped stem cells in fibrin hydrogel in a rat model of Parkinson’s 
disease
Laura Stahn1  · Justyna Rasińska1 · Tilo Dehne2 · Stefanie Schreyer1 · Aileen Hakus1 · Manfred Gossen3,4 · 
Barbara Steiner1 · Shabnam Hemmati‑Sadeghi1,2
Accepted: 30 December 2022 
© The Author(s) 2023
Abstract
There is currently no causal treatment available for Parkinson’s disease (PD). However, the use of glial cell line–derived neurotrophic 
factor (GDNF) to provide regenerative effects for neurons is promising. Such approaches require translational delivery systems that 
are functional in diseased tissue. To do so, we used a non-viral Sleeping Beauty (SB) transposon system to overexpress GDNF in 
adipose tissue–derived mesenchymal stromal cells (adMSCs). Entrapment of cells in fibrin hydrogel was used to boost potential 
neurorestorative effects. Functional GDNF-adMSCs were able to secrete 1066.8 ± 169.4 ng GDNF/120,000 cells in vitro. The 
GDNF-adMSCs were detectable for up to 1 month after transplantation in a mild 6-hydroxydopamine (6-OHDA) hemiparkinson 
male rat model. Entrapment of GDNF-adMSCs enabled GDNF secretion in surrounding tissue in a more concentrated manner, also 
tending to prolong GDNF secretion relatively. GDNF-adMSCs entrapped in hydrogel also led to positive immunomodulatory effects 
via an 83% reduction of regional IL-1β levels compared to the non-entrapped GDNF-adMSC group after 1 month. Furthermore, 
GDNF-adMSC-treated groups showed higher recovery of tyrosine hydroxylase (TH)-expressing cells, indicating a neuroprotec-
tive function, although this was not strong enough to show significant improvement in motor performance. Our findings establish a 
promising GDNF treatment system in a PD model. Entrapment of GDNF-adMSCs mediated positive immunomodulatory effects.


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Although the durability of the hydrogel needs to be extended to unlock its full potential for motor improvements, the neuroprotec-
tive effects of GDNF were evident and safe. Further motor behavioral tests and other disease models are necessary to evaluate this 
treatment option adequately.
Keywords Neurodegeneration · Adipose tissue–derived mesenchymal stromal cells (adMSCs) · Neurotrophic factors · 
Fibrin hydrogel · Transplantation · Tissue engineering
Abbreviations
6-OHDA 
 6-Hydroxydopamine
adMSCs 
 Adipose tissue–derived mesenchymal stromal 
cells
AAV 
 Adeno-associated virus
BSA 
 Bovine serum albumin
CPu 
 Caudoputamen
DAPI 
 4′,6-Diamidino-2-phenylindole
eGFP 
 Enhanced green fluorescent protein
ELISA 
 Enzyme-linked immunosorbent assay
FDA 
  Fluorescein diacetate
GDNF 
 Glial cell-derived neurotrophic factor
IFN-γ 
 Interferon-γ
IL 
 Interleukin
IQR 
 Interquartile range
KC/GRO  Keratinocyte chemoattractant/growth-regu-
lated oncogene
min 
 minutes
MRI 
 Magnetic resonance imaging
NuMa 
 Nuclear mitotic apparatus protein 1
PBS 
 Phosphate buffered saline
PD 
 Parkinson’s disease
PI 
 Propidium iodide
SB 
 Sleeping Beauty
SN 
 Substantia nigra
SNpc 
 Substantia nigra pars compacta
TH 
 Tyrosine hydroxylase
Barbara Steiner and Shabnam Hemmati-Sadeghi contributed equally.
 * Laura Stahn 
 
laura.stahn@charite.de
Extended author information available on the last page of the article
/ Published online: 28 February 2023
Drug Delivery and Translational Research (2023) 13:1745–1765


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Moderate-Intensity Rosuvastatin Plus Ezetimibe Versus High-Intensity Rosuvastatin for Target Low-Density Lipoprotein Cholesterol Goal Achievement in Patients With Recent Ischemic Stroke: A Randomized Controlled Trial


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Copyright © 2023  Korean Stroke Society
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which 
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
pISSN: 2287-6391 • eISSN: 2287-6405
242 
https://j-stroke.org
Original Article
Journal of Stroke 2023;25(2):242-250
https://doi.org/10.5853/jos.2022.02957
Moderate-Intensity Rosuvastatin Plus Ezetimibe Versus 
High-Intensity Rosuvastatin for Target Low-Density 
Lipoprotein Cholesterol Goal Achievement in Patients 
With Recent Ischemic Stroke: A Randomized 
Controlled Trial
Keun-Sik Hong,
1 Oh Young Bang,
2 Jong-Ho Park,
3 Jin-Man Jung,
4 Sang-Hun Lee,
4 Tae-Jin Song,
5  
Hyo Suk Nam,
6 Hee-Kwon Park,
7 Keun-Hwa Jung,
8 Sung Hyuk Heo,
9 Jaseong Koo,
10 Kyung-Ho Yu,
11  
Kwang-Yeol Park,
12 Chi Kyung Kim,
13 Hong-Kyun Park,
1 Jiyoon Lee,
14 Juneyoung Lee,
14 Woo-Keun Seo
2
1Department of Neurology, Ilsan Paik Hospital, Inje University, Goyang, Korea
2Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
3Department of Neurology, Myongji Hospital, Hanyang University College of Medicine, Goyang, Korea 
4Department of Neurology, Korea University Ansan Hospital, Korea University College of Medicine, Ansan, Korea
5Department of Neurology, Seoul Hospital, Ewha Women’s University College of Medicine, Seoul, Korea
6Department of Neurology, Yonsei University College of Medicine, Seoul, Korea
7Department of Neurology, Inha University Hospital, Incheon, Korea
8Department of Neurology, Seoul National University College of Medicine, Seoul, Korea
9Department of Neurology, Kyung Hee University Medical Center, Seoul, Korea
10Department of Neurology, Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Korea
11Department of Neurology, Hallym University Sacred Heart Hospital, Anyang, Korea


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12Department of Neurology, Chung-Ang University Hospital, Seoul, Korea
13Department of Neurology, Korea University Guro Hospital, Seoul, Korea
14Department of Biostatistics, Korea University College of Medicine, Seoul, Korea
Background and Purpose Moderate-intensity statin plus ezetimibe versus high-intensity statin 
alone may provide a greater low-density lipoprotein cholesterol (LDL-C) reduction in patients with 
recent ischemic stroke. 
Methods This randomized, open-label, controlled trial assigned patients with recent ischemic 
stroke <90 days to rosuvastatin/ezetimibe 10/10 mg once daily (ROS10/EZT10) or to rosuvastatin 
20 mg once daily (ROS20). The primary endpoint was LDL-C reduction ≥50% from baseline at 90 
days. Key secondary endpoints were LDL-C <70 mg/dL and multiple lipid goal achievement, and 
composite of major vascular events. 
Results Of 584 randomized, 530 were included in the modified intention-to-treat analysis. The 
baseline LDL-C level was 130.2±34.7 mg/dL in the ROS10/EZT10 group and 131.0±33.9 mg/dL in the 
ROS20 group. The primary endpoint was achieved in 198 patients (72.5%) in the ROS10/EZT10 group 
and 148 (57.6%) in the ROS20 group (odds ratio [95% confidence interval], 1.944 [1.352–2.795]; P= 
0.0003). LDL-C level <70 mg/dL was achieved in 80.2% and 65.4% in the ROS10/EZT10 and ROS20 
groups (P=0.0001). Multiple lipid goal achievement rate was 71.1% and 53.7% in the ROS10/EZT10 
and ROS20 groups (P<0.0001). Major vascular events occurred in 1 patient in the ROS10/EZT10 group 
and 9 in the ROS20 group (P=0.0091). The adverse event rates did not differ between the two groups. 
Correspondence: Keun-Sik Hong
Department of Neurology, Inje University 
Ilsan Paik Hospital, 170 Juhwa-ro, 
Ilsanseo-gu, Goyang 10380, Korea
Tel: +82-31-910-7680
Email: nrhks455@gmail.com
https://orcid.org/0000-0002-4684-6111
Received: September 15, 2022
Revised: January 17, 2023
Accepted: February 14, 2023


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The Journal of Clinical Investigation   
R E S E A R C H  A R T I C L E
1
Introduction
Efforts to clear HIV infection require a careful assessment of all 
the tissue reservoirs in which the virus persists despite durable 
antiretroviral therapy (ART). The CNS is a site with unique biolog-
ical, immunological, and pharmacological features (1). However, 
rigorous evidence of viral persistence in the CNS cells of humans 
on durable suppressive ART is incomplete (2–5). Circulating T cells 
have been well characterized as the major HIV reservoir in the 
peripheral blood and may circulate into the CNS, contributing to 
HIV persistence in the brain (6). Myeloid cells are another major 
cellular component of the immune system infected by SIV and HIV, 
and infection may persist in brain myeloid cells (BrMCs) (7–13). 
Viral rebound in the CNS was reported upon interruption of therapy 
in the humanized myeloid-only mouse model of HIV latency (11). 
Replication-competent SIV has also been reported in brain macro-
phages of ART-suppressed, SIV-infected rhesus macaques (14–16).
Given the obvious challenges in accessing human brain tis-
sue, the HIV reservoir in BrMCs, including long-lived and self- 
renewing microglia (MG), has not been rigorously studied in 
people with HIV (PWH) on long-term ART. The lack of physio-
logically relevant cellular model systems that faithfully replicate 
persistent HIV infection in BrMCs or MG poses another challenge 
to the study of these CNS cell types. Immortalized MG cell lines, 
such as HMC3 and C20, have been used as HIV infection models 
but have transcriptional profiles distinct from that of primary MG 
(17), making them less relevant to the study of the biology of HIV 
persistence in the CNS. Human studies of HIV persistence in the 
CNS have been limited to the examination of archived postmor-
tem brain tissues. Most studies of HIV reservoirs in brain tissue 
have lacked confirmation of ART adherence and viral suppres-
sion prior to death and may have utilized brain tissues that had


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begun to degrade prior to collection. Thus, it remains unclear 
whether human BrMCs, and especially long-lived MG, are latent-
ly infected, and if they encode replication-competent HIV despite 
durable, successful ART.
To address these challenges, we first developed protocols to 
isolate highly pure populations of BrMCs and MG from the tissues 
of nonhuman primates (NHPs). We then adapted these protocols 
to the study of human brain tissues containing large numbers of 
Brain microglia (MG) may serve as a human immunodeficiency virus 1 (HIV) reservoir and ignite rebound viremia following 
cessation of antiretroviral therapy (ART), but they have yet to be proven to harbor replication-competent HIV. Here, we 
isolated brain myeloid cells (BrMCs) from nonhuman primates and rapid autopsy of people with HIV (PWH) on ART and 
sought evidence of persistent viral infection. BrMCs predominantly displayed microglial markers, in which up to 99.9% 
of the BrMCs were TMEM119+ MG. Total and integrated SIV or HIV DNA was detectable in the MG, with low levels of cell-
associated viral RNA. Provirus in MG was highly sensitive to epigenetic inhibition. Outgrowth virus from parietal cortex 
MG in an individual with HIV productively infected both MG and PBMCs. This inducible, replication-competent virus and 
virus from basal ganglia proviral DNA were closely related but highly divergent from variants in peripheral compartments. 
Phenotyping studies characterized brain-derived virus as macrophage tropic based on the ability of the virus to infect cells 
expressing low levels of CD4. The lack of genetic diversity in virus from the brain suggests that this macrophage-tropic 
lineage quickly colonized brain regions. These data demonstrate that MG harbor replication-competent HIV and serve as  
a persistent reservoir in the brain.
Brain microglia serve as a persistent HIV reservoir 
despite durable antiretroviral therapy


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Yuyang Tang,1,2 Antoine Chaillon,3 Sara Gianella,3 Lilly M. Wong,1 Dajiang Li,1 Theresa L. Simermeyer,1 Magali Porrachia,3  
Caroline Ignacio,3 Brendon Woodworth,3 Daniel Zhong,1 Jiayi Du,1 Eduardo de la Parra Polina,1 Jennifer Kirchherr,1 Brigitte Allard,1 
Matthew L. Clohosey,1 Matt Moeser,4 Amy L. Sondgeroth,4 Gregory D. Whitehill,5 Vidisha Singh,6 Amir Dashti,6 Davey M. Smith,3 
Joseph J. Eron,2,7 Katherine J. Bar,5 Ann Chahroudi,6,8 Sarah B. Joseph,1,4,9 Nancie M. Archin,1,2 David M. Margolis,1,2,7,9 and Guochun Jiang1,2,10
1University of North Carolina (UNC) HIV Cure Center, and 2Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 3Department 
of Medicine, UCSD, La Jolla, California, USA. 4UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, Chapel Hill, North Carolina, USA. 5Department of Medicine, 
University of Pennsylvania, Philadelphia, Pennsylvania, USA. 6Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA. 7Department of Epidemiology, Gillings School of Global 
Public Health, University of North Carolina at Chapel Hill, North Carolina, USA. 8Center for Childhood Infections and Vaccines of Children’s Healthcare of Atlanta and Emory University, Atlanta, Georgia, USA. 
9Department of Microbiology and Immunology, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 10Department of Epidemiology, Gillings School of Global 
Public Health, University of North Carolina at Chapel Hill, North Carolina, USA.
Conflict of interest: The authors have declared that no conflict of interest exists.
Copyright: © 2023, Tang et al. This is an open access article published under the 
terms of the Creative Commons Attribution 4.0 International License.
Submitted: November 23, 2022; Accepted: April 25, 2023; Published: June 15, 2023.
Reference information: J Clin Invest. 2023;133(12):e167417.


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Journal of Neurology, Neurosurgery, and Psychiatry 1983;46:533-539
Crossed optic ataxia: possible role of the dorsal
splenium
JOSE M FERRO,* JM BRAVO-MARQUES,* A CASTRO-CALDAS,* LOBO ANTUNESt
From the Language Research Laboratory* and the Neuropathology Laboratory, t Centro de Estudos Egas
Moniz (INIC), Department ofNeurology, Hospital de Santa Maria, Lisbon, Portugal
SUMMARY An unusual combination of disconnective syndromes is reported: transcortical motor
aphasia,
left arm apraxia and optic ataxia. Neuropathological examination showed
a
left
parieto-occipital and a subcortical frontal infarct and a lesion of the dorsal part of the posterior
two-fifths of the callosum. The frontal lesion caused the transcortical motor aphasia and pro-
duced the left arm apraxia. Visuomotor incoordination in the right hemispace was due to the left
parieto-occipital infarct, while the crossed optic ataxia in the left hemispace was attributed to the
callosal lesion. It is proposed that the pathway that serves crossed visual reaching passes through
the dorsal part of the posterior callosum. This case reinforces the growing evidence that fibres in
the corpus callosum are arranged in ventro-dorsal functional lamination.
Optic ataxia or visuomotor ataxia' consists of a dis-
turbance in reaching under visual control that may
affect one or both hands, and be present either in
the whole or in part of the visual fields. A probable
explanation for this defect is a disconnection be-
tween the visual and motor areas, in such a way that
visual perception, naming, and reading are possible,
although visual information cannot gain access to
the hand motor areas in order to steer manual reach-
ing. This explanation was supported by the experi-
mental study of Haaxma and Kuypers2 who could
disturb visual guided hand movements by means of a
parietal leucotomy in the split-brain monkey. The
reports by Castaigne and associates3 4 of visual
ataxia following surgical section of the parieto-
occipital junction, which severed association fibres


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between the occipital and frontal lobes, are also in
keeping with this view. It is suggested from these
studies that the pathway subserving visuomotor
behaviour connects the occipital with the frontal
lobe and crosses through the posterior parietal lobe.
The pathway connecting the visual areas of one
hemisphere with the contralateral motor and pre-
motor areas (in which a lesion, according to Rondot,
Address for reprint requests: Jos6 M Ferro, MD, Neurobehavioral
Unit, Centro de Estudos Egas Moniz, Hospital Santa Maria, 1600
Lisboa, Portugal.
Received 14 May 1982 and in revised form 23 January 1983.
Accepted 7 February 1983
Recondo and Dumas',
induces
a crossed
optic
ataxia, that is, inability to reach in one hemifield
with the hand of the opposite side) is, according to
most animal studies, via the callosum.5-9 Further, it
appears that adequate interhemispheric transmis-
sion for the visual control of the ipsilateral hand and
fingers is effected by the splenium.'0 Evidence from
clinical studies is scanty because the majority of
cases of optic ataxia which underwent anatomical
study
had
bilateral
optic
ataxia
and
bilateral
parieto-occipital lesions.' Stenvers" stated that for
crossed visuomotor reaching,
visual information
could travel from the occipital to the ipsilateral fron-
tal lobe and then to the contralateral one, crossing
the anterior callosum (case 34) or be transferred
from one occipital lobe to the opposite, via the post-
erior callosum, and then, ipsilaterally, to the frontal
lobe. Indeed, crossed visuomotor ataxia in Boller S12
case is explained by lesions severing both pathways.
In the present clinico-pathological study we propose
that the pathway that serves crossed visuomotor
control travels through the dorsal part of the post-
erior callosum.
Case report
A 73-year-old right-handed woman had speech and visual
disturbances and slight right-sided weakness of sudden
onset. Blood pressure was 100/70 mmHg, the pulse was
irregular with premature beats, and a systolic blowing


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Reduced cerebello-cerebral functional connectivity correlates with disease severity and impaired white matter integrity in Friedreich ataxia


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Vol:.(1234567890)
Journal of Neurology (2023) 270:2360–2369
https://doi.org/10.1007/s00415-023-11637-x
1 3
ORIGINAL COMMUNICATION
Reduced cerebello‑cerebral functional connectivity correlates 
with disease severity and impaired white matter integrity in Friedreich 
ataxia
Rebecca Kerestes1 · Hannah Cummins1 · Nellie Georgiou‑Karistianis2 · Louisa P. Selvadurai1 · Louise A. Corben3 · 
Martin B. Delatycki3 · Gary F. Egan2,4 · Ian H. Harding1,4 
Received: 17 November 2022 / Revised: 7 February 2023 / Accepted: 19 February 2023 / Published online: 1 March 2023 
© The Author(s) 2023
Abstract
Friedreich ataxia (FRDA) is a rare, inherited neurodegenerative disease characterised in most cases by progressive and 
debilitating motor dysfunction. Degeneration of cerebellar white matter pathways have been previously reported, alongside 
indications of cerebello-cerebral functional alterations. In this work, we examine resting-state functional connectivity changes 
within cerebello-cerebral circuits, and their associations with disease severity (Scale for the Assessment and Rating of Ataxia 
[SARA]), psychomotor function (speeded and paced finger tapping), and white matter integrity (diffusion tensor imaging) 
in 35 adults with FRDA and 45 age and sex-matched controls. Voxel-wise seed-based functional connectivity was assessed 
for three cerebellar cortical regions (anterior lobe, lobules I-V; superior posterior lobe, lobules VI-VIIB; inferior posterior 
lobe, lobules VIIIA-IX) and two dentate nucleus seeds (dorsal and ventral). Compared to controls, people with FRDA 
showed significantly reduced connectivity between the anterior cerebellum and bilateral pre/postcentral gyri, and between 
the superior posterior cerebellum and left dorsolateral PFC. Greater disease severity correlated with lower connectivity in 
these circuits. Lower anterior cerebellum-motor cortex functional connectivity also correlated with slower speeded finger


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tapping and less fractional anisotropy in the superior cerebellar peduncles, internal capsule, and precentral white matter in 
the FRDA cohort. There were no significant between-group differences in inferior posterior cerebellar or dentate nucleus 
connectivity. This study indicates that altered cerebello-cerebral functional connectivity is associated with functional status 
and white matter damage in cerebellar efferent pathways in people with FRDA, particularly in motor circuits.
Keywords Friedreich ataxia · Cerebellum · Resting state · Anterior · Superior · Dentate
Introduction
Friedreich ataxia (FRDA) is a recessively inherited neuro-
degenerative disorder principally characterised by progres-
sive motor incoordination [1] that manifests secondary to 
degeneration in the spinal cord, cerebellum, and cerebello-
cerebral connections [2]. Quantitative structural magnetic 
resonance imaging (MRI) studies have been key to defining 
the pattern and evolution of anatomical brain changes in 
people with FRDA. MRI studies indicate early, robust, and 
progressive volume loss in the dentate nuclei [3, 4], which 
are the primary sites of pathology in the brain [5]. Atro-
phy of the cerebellar cortex is more subtle and considered a 
late feature of the disease. This volume loss is particularly 
weighted to the anterior lobe and adjacent areas of the pos-
terior lobe (i.e., lobules I-VI) [2, 6]], which connect to motor 
and premotor areas of the cerebral cortex. Loss of volume 
Rebecca Kerestes and Hannah Cummins equally contributed.
 * Ian H. Harding 
 
ian.harding@monash.edu
1 
Department of Neuroscience, Central Clinical School, 
Monash University, Melbourne, Australia
2 
Turner Institute for Brain and Mental Health, School 
of Psychological Sciences, Monash University, Melbourne, 
Australia
3 
Bruce Lefroy Centre for Genetic Health Research, Murdoch 
Children’s Research Institute, Melbourne, Australia
4 
Monash Biomedical Imaging, Monash University, 
Melbourne, VIC 3800, Australia


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Quantitative immunopeptidomics reveals a tumor stroma–
specific target for T cell therapy
Gloria B. Kim1,†, Jens Fritsche2,†, Sebastian Bunk2,†, Andrea Mahr2, Felix Unverdorben2, 
Kevin Tosh1, Hong Kong1, Colby R. Maldini1, Chui Lau1, Sriram Srivatsa1, Shuguang 
Jiang1, Joshua Glover1, Derek Dopkin1, Carolyn X. Zhang1, Heiko Schuster2, Daniel 
J. Kowalewski2, Valentina Goldfinger2, Martina Ott2, David Fuhrmann2, Maike Baues2, 
Hans Boesmueller3, Christoph Schraeder2, Gisela Schimmack2, Colette Song2, Franziska 
Hoffgaard2, Michael Roemer2, Chih-Chiang Tsou4, Martin Hofmann2, Thomas Treiber2, 
Meike Hutt2, Leonie Alten2, Maike Jaworski2, Amir Alpert4, Sarah Missel2, Carsten 
Reinhardt2, Harpreet Singh2,4, Oliver Schoor2, Steffen Walter4, Claudia Wagner2, Dominik 
Maurer2,‡, Toni Weinschenk2,4,*,‡, James L. Riley1,*,‡
exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works
*Corresponding author. toni.weinschenk@immatics.com (T.W.); rileyj@upenn. edu (J.L.R.).
†These authors contributed equally to this work.
‡These authors contributed equally to this work.
Author contributions: G.B.K., K.T., H. Singh, C.R., S.W., T.W., and J.L.R. designed the study. G.B.K., H. Singh, S.W., T.W., and 
J.L.R. provided funding. D.M., T.W., S.M., O.S., and J.L.R. supervised the project. The original draft was written by G.B.K., S.B., 
J.F., D.M., T.W., and J.L.R. Editing and review were supported by all authors. Sample procurement and analysis was performed and 
supervised by M.O. MS was performed and supervised by D.J.K. and T.W. RNA sequencing was analyzed by F.H. Data processing, 
database development, bioinformatics, and statistical analyses were performed by F.H., M.R., C.-C.T., and J.F. Target discovery was 
performed by A.M., C. Song, and J.F. Absolute peptide copy number determination was performed by V.G., C. Schraeder, and H. 
Schuster. ISH was performed by D.F., M.B., and M.O. Pathology review was performed by H.B. Selection of similar peptides and cell


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lines for off-target toxicity studies was performed by G.S., S.B., and H. Schuster. TCR discovery and characterization of wild-type 
TCR variants were performed by G.B.K., K.T., C.L., L.A., A.A., and S.B. Affinity enhancement of F9 TCR and selection of maturated 
variants were performed by F.U., M. Hutt, M. Hofmann, T.T., and S.B. In vitro characterization of antitumor efficacy and safety 
profiling of affinity-enhanced F9 TCR variants were performed by G.B.K., C.X.Z., S.S., M.J., A.A., and C.W. In vivo studies were 
performed by G.B.K., K.T., H.K., C.R.M., S.J., J.G., D.D., and C.X.Z.
SUPPLEMENTARY MATERIALS
www.science.org/doi/10.1126/scitranslmed.abo6135 
Figs. S1 to S8
Tables S1 to S4
MDAR Reproducibility Checklist 
View/request a protocol for this paper from Bio-protocol.
Competing interests: J.F., S.B., A.M., F.U., H. Schuster, D.J.K., V.G., M.O., D.F., M.B., C. Song, G.S., C. Schraeder, F.H., M.R., 
C.-C.T., M. Hofmann, T.T., M. Hutt, L.A., M.J., A.A., S.M., O.S., H. Singh, C.R., S.W., C.W., D.M., and T.W. were employees 
of Immatics in the course of this work and may have securities from Immatics. J.L.R. is a cofounder and holds an equity interest 
in Tmunity Therapeutics. C.R., H. Singh, S.W., and T.W. are management board members of Immatics. All other authors have no 
competing interests. The following patent applications relate to subject matter in this paper: PCT/EP2011/053863 (entitled “Novel 
Immunotherapy against several tumors including gastrointestinal and gastric cancer”), disclosing subject matter coinvented by J.F., 
H. Singh, S.W., and T.W.; PCT/EP2017/066630 (entitled “T cell receptors and immune therapy using the same”), disclosing subject 
matter coinvented by S.B., S.W., D.M., and L.A.; PCT/EP2018/080176 (entitled “Novel engineered T cell receptors and immune 
therapy using the same”), disclosing subject matter coinvented by S.B., F.U., M. Hofmann, M. Hutt, C.W., D.M., and L.A.; PCT/


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EP2014/066755 (entitled “Novel immunotherapy against several tumors, such as lung cancer, including NSCLC”), disclosing subject 
matter coinvented by J.F., C. Song, H. Singh, S.W., and T.W.; PCT/EP2016/055817 (entitled “Novel peptides and combination 
of peptides for use in immunotherapy against pancreatic cancer and other cancers”), disclosing subject matter coinvented by J.F., 
A.M., M.O., H. Singh, O.S., C.W., and T.W.; PCT/EP2016/059053 (entitled “Novel peptides and combination of peptides for use 
in immunotherapy against lung cancer, including NSCLC and other cancers”), disclosing subject matter coinvented by J.F., A.M., 
C. Song, H. Singh, O.S., C.W., and T.W.; PCT/EP2016/060007 [entitled “Novel peptides and combination of peptides and scaffolds 
thereof for use in immunotherapy against colorectal carcinoma (CRC) and other cancers”], disclosing subject matter coinvented 
by J.F., A.M., H. Singh, O.S., and T.W.; and PCT/EP2016/079059 (entitled “Peptides and combination of peptides for use in 
immunotherapy against breast cancer and other cancers”), disclosing subject matter coinvented by J.F., A.M., H. Singh, O.S., and T.W.
HHS Public Access
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Published in final edited form as:
Sci Transl Med. 2022 August 31; 14(660): eabo6135. doi:10.1126/scitranslmed.abo6135.
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Journal of Neurology, Neurosurgery, and Psychiatry 1983 ;46: 266-273
Biopsy and post-mortem findings in a patient
receiving cerebellar stimulation for epilepsy
GDS WRIGHT, RO WELLER
From the Wessex Neurological Centre and Department ofNeuropathology, Southampton General Hospital,
Southampton, UK
SUMMARY
Cerebellar stimulating equipment was implanted in a 24-year-old male with severe
epilepsy. He received continuous alternating stimulation for at least two months, intermittent
bilateral stimulation on demand for at least two months and numerous short bursts of stimulation
during preliminary testing and calibration of the apparatus. The patient died during a prolonged
grand mal seizure 16 months after implantation and this paper reports the findings at necropsy. A
special study was made of the degree of tissue damage in the cerebellar hemispheres resulting
from implantation of the electrodes and subsequent cerebellar stimulation. There was deep
grooving of the upper aspect of the cerebellum under the electrodes but only a minor degree of
neuronal loss and gliosis was observed in the underlying cortex. Purkinje cell populations in the
cerebellum at necropsy were similar to those in a biopsy specimen taken 16 months previously
during implantation. The electrodes were covered with a thick layer of fibrous tissue but they
were not adherent to the cerebellar surface. Thus, the main significant findings in the present case
were the very slight structural damage to the cerebellum in contact with the electrodes and the
intense fibrosis around the electrodes themselves. These features are discussed in relation to the
efficacy of long-term cerebellar stimulation.
Cerebellar stimulation has been recommended as a
useful treatment in severe epilepsy and in a variety
of motor disorders.' 2 There is, however, still consid-
erable uncertainty about the degree of tissue dam-
age caused by implantation of the electrodes and by
chronic electrical stimulation of the cerebellum.3
Although the study of a number of human and ani-


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mal brains has shown that pathological changes
can occur with cerebellar stimulation, firm criteria
have not yet been determined to enable optimal
parameters for cerebellar stimulation to be defined.3
There are only a few reports of post-mortem
studies in patients who have received cerebellar
stimulation.249 The present paper is a post-mortem
study of a 24-year-old male with severe epilepsy
who died 16 months after the implantation of cere-
bellar electrodes. Electrode access resistance was
measured
at
implantation
and
the
stimulating
Address for reprint requests: Dr GDS Wright, Department of
Neurology, St James's University Hospital, Beckett Street, Leeds
LS9 7TF, UK.
Received 16 July 1982
Accepted 4 October 1982
266
equipment was accurately calibrated. The function-
ing of the stimulating equipment was regularly
reviewed and reasonable estimates of the charge
density at the cerebellar surface and the total charge
delivered to the cerebellum are therefore available.
Case history
The patient, (SB, born 8/1/55), was 24 years old at the time
of his admission to the Southampton cerebellar stimulation
trial. His epilepsy began at the age of seven years and there
were no predisposing factors. He suffered from grand mal
epilepsy, complex partial epilepsy and petit mal epilepsy.
The grand mal attacks occurred rarely, but could be severe
and prolonged. Complex partial attacks occuffed up to five
times a day and the petit mal attacks up to forty or more in
a day. Medication consisted of phenytoin 200 mg twice
daily and carbamazepine 200 mg four times a day and he
had been on this combination for over six months before
admission. It was considered the best combination and the
optimal
dosage
and
gave serum
levels
of phenytoin
1-3 mg/100 ml and carbamazepine 0-7 mg/100 ml. Previ-
ously he had, at different times, been treated with these
drugs in different dosages and with primidone.
At operation on 20 March 1979 under general anaes-
thesia, two eight-button electrode pads with an electrode


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Safety and preservation of cardiac function following therapeutic vaccination against Trypanosoma cruzi in rhesus macaques


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Safety and preservation of cardiac function following therapeutic 
vaccination against Trypanosoma cruzi in rhesus macaques
Eric Dumonteila,*, Claudia Herreraa, Preston A. Marxa,b
aDepartment of Tropical Medicine, School of Public Health and Tropical Medicine, Vector-Borne 
and Infectious Disease Research Center, Tulane University, New Orleans, LA, USA
bDivision of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, 
LA, USA
Abstract
Background: Chronic Chagasic cardiomyopathy is responsible for a large disease burden in 
the Americas, and a therapeutic vaccine would be highly desirable. We tested the safety and 
efficacy of a therapeutic DNA vaccine encoding antigens TSA-1 and Tc24 for preventing cardiac 
alterations in experimentally infected macaques. A secondary objective was to evaluate the 
feasibility of detecting changes in cardiac alterations in these animals.
Methods: Naïve rhesus macaques were infected with Trypanosoma cruzi and treated with three 
doses of DNA vaccines.
Results: Blood cell counts and chemistry indicated that therapeutic vaccination was safe, as 
hepatic and renal function appeared unaffected by the vaccination and/or infection with T. cruzi. 
Electrocardiographic (ECG) recordings indicated that no marked arrhythmias developed up to 7 
months post-infection. Univariate analysis of ECG parameters found no significant differences in 
any of these parameters between vaccinated and control macaques. However, linear discriminant 
analysis revealed that control macaques presented clear alterations in their ECG patterns at 7 
months post-infection, indicative of the onset of conduction defects and cardiac alterations, and 
these changes were prevented in vaccine treated macaques.
Conclusions: This is the first evidence that therapeutic vaccination against T. cruzi can prevent 
cardiac alterations in non-human primates, strengthening the rationale for developing a human 
vaccine against Chagas disease.
Keywords


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Keywords
Chagas disease; DNA vaccine; Cardiomyopathy; Non-human primates; Electrocardiograph
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
*Corresponding author. Department of Tropical Medicine, Vector-Borne and Infectious Disease Research Center, School of Public 
Health and Tropical Medicine, Tulane University, 1440 Canal St., New Orleans, LA, 70112, USA. edumonte@tulane.edu (E. 
Dumonteil). 
Declaration of competing interest
The authors have no conflict of interest.
HHS Public Access
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J Microbiol Immunol Infect. Author manuscript; available in PMC 2023 April 26.
Published in final edited form as:
J Microbiol Immunol Infect. 2023 April ; 56(2): 400–407. doi:10.1016/j.jmii.2022.09.003.
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The Journal of Clinical Investigation   
R E S E A R C H  A R T I C L E
1
Introduction
Atherosclerosis, the buildup of plaque and hardening of the arter-
ies over time, is a widespread and debilitating disease in older 
populations. Many risk factors contribute to the development of 
atherosclerosis, including unhealthy diets, smoking, genetics, 
diabetes, aging, and obesity. Among them, aging is one of the 
most important factors, increasing the prevalence, incidence, and 
mortality associated with atherosclerosis (1, 2). The most serious 
complication and leading cause of death associated with athero-
sclerosis is the thrombotic occlusion of the arteries following ero-
sion or rupture of an atherosclerotic plaque. The cap of the plaque 
consists of a layer of vascular smooth muscle cells (VSMCs) that 
have dedifferentiated into synthetic, noncontractile cells that 
typically proliferate and migrate, but also a small population of 
senescent, inflammatory VSMCs (3).
Previous research has shown that cellular senescence is 
associated with the development and progression of atheroscle-
rosis (4). Cellular senescence, a hallmark phenotype of cells in 
aging tissues, is characterized by increased production of cyclin- 
dependent kinase inhibitors and tumor suppressors such as p16 
(CDKN2A), p21 (CDKN1A), and p53 (TP53) as well as many fac-
tors of the senescence-associated secretory phenotype (SASP), 
including cytokines (e.g., IL-6, IL-8), chemokines (e.g., CCL2), 
adhesion molecules (e.g., ICAM1), matrix remodeling enzymes 
(e.g., MMPs), and angiogenic factors (e.g., VEGF). Senescent cells 
also display a senescence-associated β-galactosidase (SA–β-gal) 
activity (5, 6). Although senescent cells possess the ability to be 
both beneficial and harmful depending on the biological situa-
tion, there is little doubt that the accrual of senescent cells in older 
human tissues is detrimental and contributes to pathologies asso-
ciated with aging (7–9). As a result of their widespread detrimen-


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tal effects in many diseases, interest is escalating in developing 
therapies directed against senescent cells (senotherapies) and in 
discovering senescence-associated cell-surface proteins. Impor-
tantly, VSMCs cultured from atherosclerotic plaques display a 
myriad of these senescence traits; thus, further research on senes-
cent VSMCs in atherosclerosis is crucial to our understanding of 
the progression and treatment of this age-associated disease (10).
The development of age-related multifactorial diseases such 
as atherosclerosis is associated with persistent systemic inflam-
mation. However, the dynamic interplay among aging, inflamma-
tion, and VSMC senescence is not well understood. A recent study 
found that dipeptidyl peptidase 4 (DPP4) is highly expressed on 
the surface of senescent cells (11). While the effect of DPP4 on 
VSMCs is not characterized, DPP4 inhibitors, known as gliptins, 
are clinically used to treat diabetes (12). In animal models, gliptins 
have the ability to reduce atherosclerosis and inflammation inde-
pendently of the canonical role of DPP4 in glucose metabolism 
(13–17). Thus, we set out to investigate the ability of DPP4 inhib-
Senescent vascular smooth muscle cells (VSMCs) accumulate in the vasculature with age and tissue damage and secrete 
factors that promote atherosclerotic plaque vulnerability and disease. Here, we report increased levels and activity of 
dipeptidyl peptidase 4 (DPP4), a serine protease, in senescent VSMCs. Analysis of the conditioned media from senescent 
VSMCs revealed a unique senescence-associated secretory phenotype (SASP) signature comprising many complement and 
coagulation factors; silencing or inhibiting DPP4 reduced these factors and increased cell death. Serum samples from persons 
with high risk for cardiovascular disease contained high levels of DPP4-regulated complement and coagulation factors. 
Importantly, DPP4 inhibition reduced senescent cell burden and coagulation and improved plaque stability, while single-cell


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resolution of senescent VSMCs reflected the senomorphic and senolytic effects of DPP4 inhibition in murine atherosclerosis. 
We propose that DPP4-regulated factors could be exploited therapeutically to reduce senescent cell function, reverse 
senohemostasis, and improve vascular disease.
DPP4 inhibition impairs senohemostasis to improve 
plaque stability in atherosclerotic mice
Allison B. Herman,1 Dimitrios Tsitsipatis,1 Carlos Anerillas,1 Krystyna Mazan-Mamczarz,1 Angelica E. Carr,1 Jordan M. Gregg,1 
Mingyi Wang,2 Jing Zhang,2 Marc Michel,1 Charnae’ A. Henry-Smith,1 Sophia C. Harris,2 Rachel Munk,1 Jennifer L. Martindale,1 
Yulan Piao,1 Jinshui Fan,1 Julie A. Mattison,3 Supriyo De,1 Kotb Abdelmohsen,1 Robert W. Maul,4 Toshiko Tanaka,3  
Ann Zenobia Moore,3 Megan E. DeMouth,5 Simone Sidoli,6 Luigi Ferrucci,3 Yie Liu,1 Rafael de Cabo,3  
Edward G. Lakatta,2 and Myriam Gorospe1
1Laboratory of Genetics and Genomics, 2Laboratory of Cardiovascular Sciences, 3Translational Gerontology Branch, and 4Laboratory of Molecular Biology and Immunology, National Institute on Aging (NIA) 
Intramural Research Program (IRP), NIH, Baltimore, Maryland, USA. 5Department of Microbiology and Immunology and 6Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.
Conflict of interest: The authors have declared that no conflict of interest exists.
Copyright: © 2023, Herman et al. This is an open access article published under the 
terms of the Creative Commons Attribution 4.0 International License.
Submitted: October 3, 2022; Accepted: April 19, 2023; Published: June 15, 2023.
Reference information: J Clin Invest. 2023;133(12):e165933. 
https://doi.org/10.1172/JCI165933.


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Antiviral polysaccharide and antiviral peptide delivering nanomaterials for prevention and treatment of SARS-CoV-2 caused COVID-19 and other viral diseases


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Development of \215hunger neurons\216 and the unanticipated relationship between energy metabolism and mother-infant interactions


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Development of “hunger neurons” and the unanticipated 
relationship between energy metabolism and mother-infant 
interactions
Onur Iyilikci1,#, Marcelo R. Zimmer1,#, Marcelo O. Dietrich1,2,3,*
1Laboratory of Physiology of Behavior, Department of Comparative Medicine, Yale School of 
Medicine, New Haven, CT, USA.
2Department of Neuroscience, Yale School of Medicine, New Haven, CT, USA.
3Yale Center for Molecular and Systems Metabolism, Yale School of Medicine, New Haven, CT, 
USA.
Abstract
Over the course of a lifetime, the perinatal period plays an outsized role in the function of 
physiological systems. Here, we discuss how neurons that regulate energy metabolism contribute 
to the infant’s relationship with the mother. We focus our discussion on Agrp neurons, which 
are located in the arcuate nucleus of the hypothalamus. These neurons heavily regulate energy 
metabolism. Because offspring transition from a period of dependence on the caregiver to 
independence, we discuss the importance of the caregiver-offspring relationship for the function 
of Agrp neurons. We present evidence that, in the adult, Agrp neurons motivate the animal to 
eat, while, in the neonate, they motivate the offspring to seek the proximity of the caregiver. 
We specifically highlight the peculiarities in the development of Agrp neurons and how they 
relate to the regulation of metabolism and behavior over the course of a lifetime. In sum, this 
review considers the unique insights that ontogenetic studies can offer toward our understanding 
of complex biological systems, such as the regulation of energy metabolism and mother-infant 
attachment.
Keywords
Hypothalamus; obesity; diabetes; development; melanocortin system; mother-infant relationship; 
energy balance; metabolism
Life as an infant
Compared to adults, infants deal with distinct physiological disturbances, and, thus, they 
need regulatory processes that are specific to each stage of development. For example, due


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to their small body size, limited fat resources, and greater surface area to volume ratio—
which increases heat loss—neonates are particularly vulnerable to cold exposure. As an 
*Correspondence to: marcelo.dietrich@yale.edu.
#These authors contributed equally.
HHS Public Access
Author manuscript
Biol Psychiatry. Author manuscript; available in PMC 2023 May 15.
Published in final edited form as:
Biol Psychiatry. 2022 May 15; 91(10): 907–914. doi:10.1016/j.biopsych.2022.02.962.
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Bone Marrow Mesenchymal Stem Cells Reversed Ovarian Aging-related m6A RNA Methylation Modification Profile in Aged Granulosa Cells


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Vol.:(0123456789)
1 3
https://doi.org/10.1007/s12015-022-10485-y
Bone Marrow Mesenchymal Stem Cells Reversed Ovarian 
Aging‑related m6A RNA Methylation Modification Profile in Aged 
Granulosa Cells
Chuan Tian1 · Yuanyuan An2 · Jing Zhao1 · Xiangqing Zhu1 · Wei Wei1 · Guangping Ruan1 · Ye Li1 · Xinghua Pan1 
Accepted: 1 December 2022 
© The Author(s) 2022
Abstract
Background Ovarian ageing causes endocrine disturbances and the degeneration of systemic tissue and organ functions 
to seriously affect women's physical and mental health, and effective treatment methods are urgently needed. Based on our 
previous studies using juvenile rhesus monkey bone marrow mesenchymal stem cells (BMMSCs) to treat ovarian ageing 
in rhesus monkey, we found that BMMSCs improved ovarian structure and function. This study continues to explore the 
mechanism by which BMMSCs reversed granulosa cell (GC) ageing.
Methods A GC ageing model and coculture system of BMMSCs were established, changes in the level of the N6-methyl-
adenosine (m6A) methylation modification were detected, m6A-modified RNA immunoprecipitation sequencing (MeRIP-
seq) were performed, correlations between m6A peaks and mRNA expression were determined, and the expression of hub 
genes was identified using Q-PCR, immunofluorescence staining, and western blot.
Results Our results showed that  H2O2 successfully induced GC ageing and that BMMSCs reversed measures of GC ageing. 
BMMSCs increased the expression of the FTO protein and reduced the overall level of m6A. We identified 797 m6A peaks 
(348 hypomethylated and 449 hypermethylated peaks) and 817 differentially expressed genes (DEGs) (412 upregulated and 
405 downregulated) after aged GCs were cocultured with BMMSCs, which significantly associated with ovarian function 
and epigenetic modification. The epigenetic repressive mark and important cell cycle regulator lysine demethylase 8 (KDM8) 
was downregulated at both the mRNA and protein levels, histone H3 was upregulated in aged GCs after BMMSC coculture,


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and KDM8 was upregulated after FTO was inhibited through FB23.
Conclusions Our study revealed an essential role for m6A in BMMSCs in reversing GC ageing, and FTO regulated KDM8 
mediates histone H3 changes may as a novel regulatory mechanism in BMMSCs to reverse GC ageing.
Keywords BMMSCs · Ovarian ageing · m6A · Lysine demethylase 8 (KDM8)
Abbreviations
BMMSCs 
 Bone marrow mesenchymal stem cells
hGC 
 Human granulosa cell
MeRIP-seq  M6A-modified RNA immunoprecipitation 
sequencing
KDM8 
 Lysine demethylase 8
m6A 
 N6-methyladenosine
DEGs 
 Differentially expressed genes
Introduction
The world's population is ageing rapidly, and the number 
of elderly individuals has increased significantly, which 
has placed great pressure on society and become one of 
the major economic challenges facing contemporary soci-
ety [1–3]. The ovary is a dynamic reproductive endocrine 
organ that enacts female reproductive function through 
ovulation and the secretion of sex hormones and affects 
tissues and organs throughout the body [4, 5], it is also 
one of the most sensitive organs to ageing. A variety of 
 * Xinghua Pan 
 
xinghuapankm@163.com
1 
The Basic Medical Laboratory of the 920Th Hospital 
of Joint Logistics Support Force of PLA, The Transfer 
Medicine Key Laboratory of Cell Therapy Technology 
of Yunan Province, The Integrated Engineering Laboratory 
of Cell Biological Medicine of State and Regions, 
Kunming 650032, Yunnan Province, China
2 
The Affiliated Stomatology of Kunming Medical University, 
Kunming 650106, Yunnan Province, China
/ Published online: 7 January 2023
Stem Cell Reviews and Reports (2023) 19:953–967


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Dichotomous Regulation of Striatal Plasticity by Dynorphin
Renzhi Yang1,*, Rupa R. Lalchandani Tuan2,3,*, Fuu-Jiun Hwang2, Daniel W. Bloodgood2, 
Dong Kong4, Jun B. Ding2,5,6,†
1Biology Graduate Program, Stanford University, Stanford, CA 94305, USA
2Department of Neurosurgery, Stanford University, Stanford, CA 94305, USA
3Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94158, USA
4Division of Endocrinology, Department of Pediatrics, F.M. Kirby Neurobiology Center, Boston 
Children’s Hospital and Harvard Medical School, Boston MA 02115, USA
5Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA 94305, 
USA
6Stanford Bio-X, Stanford University, Stanford, CA 94305, USA
Abstract
Modulation of corticostriatal plasticity alters the information flow throughout basal ganglia 
circuits and represents a fundamental mechanism for motor learning, action selection, and reward. 
Synaptic plasticity in the striatal direct- and indirect-pathway spiny projection neurons (dSPNs and 
iSPNs) is regulated by two distinct networks of GPCR signaling cascades. While it is well-known 
that dopamine D2 and adenosine A2a receptors bi-directionally regulate iSPN plasticity, it remains 
unclear how D1 signaling modulation of synaptic plasticity is counteracted by dSPN-specific Gi 
signaling. Here, we show that striatal dynorphin selectively suppresses long-term potentiation 
(LTP) through Kappa Opioid Receptor (KOR) signaling in dSPNs. Both KOR antagonism and 
conditional deletion of dynorphin in dSPNs enhance LTP counterbalancing with different levels 
of D1 receptor activation. Behaviorally, mice lacking dynorphin in D1 neurons show comparable 
motor behavior and reward-based learning, but enhanced flexibility during reversal learning. These 
findings support a model in which D1R and KOR signaling bidirectionally modulate synaptic 
plasticity and behavior in the direct pathway.
† Correspondence: J.B.D. (dingjun@stanford.edu).
*Equal contribution
AUTHOR CONTRIBUTIONS


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R.Y., R.R.L., and J.B.D. conceived this project. R.Y. performed electrophysiology recording and operant chamber behavior 
experiments. R.R.L. characterized lox-pDyn mice, and performed immunostaining and behavioral experiments. F.J.H. performed 
the spine imaging. D.K. and J.B.D. designed and generated lox-pDyn mice. R.Y. and R.R.L. analyzed the data. R.Y., R.R.L., D.W.B. 
and J.B.D. wrote the manuscript with input from all authors.
CONFLICT OF INTEREST
The authors declare no competing interests.
HHS Public Access
Author manuscript
Mol Psychiatry. Author manuscript; available in PMC 2023 May 17.
Published in final edited form as:
Mol Psychiatry. 2023 January ; 28(1): 434–447. doi:10.1038/s41380-022-01885-0.
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New genetic and epigenetic insights into the chemokine system: the latest discoveries aiding progression toward precision medicine


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REVIEW ARTICLE
OPEN
New genetic and epigenetic insights into the chemokine
system: the latest discoveries aiding progression toward
precision medicine
Hanli Xu
1,4, Shuye Lin2,4, Ziyun Zhou1, Duoduo Li1, Xiting Zhang1, Muhan Yu1, Ruoyi Zhao1, Yiheng Wang1, Junru Qian1, Xinyi Li1,
Bohan Li1, Chuhan Wei1, Keqiang Chen3, Teizo Yoshimura3, Ji Ming Wang3 and Jiaqiang Huang1,2,3✉
© The Author(s) 2023
Over the past thirty years, the importance of chemokines and their seven-transmembrane G protein-coupled receptors (GPCRs) has been
increasingly recognized. Chemokine interactions with receptors trigger signaling pathway activity to form a network fundamental to
diverse immune processes, including host homeostasis and responses to disease. Genetic and nongenetic regulation of both the
expression and structure of chemokines and receptors conveys chemokine functional heterogeneity. Imbalances and defects in the system
contribute to the pathogenesis of a variety of diseases, including cancer, immune and inﬂammatory diseases, and metabolic and
neurological disorders, which render the system a focus of studies aiming to discover therapies and important biomarkers. The integrated
view of chemokine biology underpinning divergence and plasticity has provided insights into immune dysfunction in disease states,
including, among others, coronavirus disease 2019 (COVID-19). In this review, by reporting the latest advances in chemokine biology and
results from analyses of a plethora of sequencing-based datasets, we outline recent advances in the understanding of the genetic variations
and nongenetic heterogeneity of chemokines and receptors and provide an updated view of their contribution to the pathophysiological
network, focusing on chemokine-mediated inﬂammation and cancer. Clariﬁcation of the molecular basis of dynamic chemokine-receptor
interactions will help advance the understanding of chemokine biology to achieve precision medicine application in the clinic.


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Keywords: Chemokine; Chemokine receptor; Migration; Homeostasis; Genetics; Epigenetics
Cellular & Molecular Immunology (2023) 20:739–776; https://doi.org/10.1038/s41423-023-01032-x
Leukocyte migration is a central component of physiological and
pathological responses [1–9]. Chemokines are the largest family of
cytokines and have chemotactic activity that is essential for host
responses in homeostasis and diseases. Chemokines activate cell-
surface G-protein-coupled
receptors
(GPCRs) to generate
a
regulatory
network
and
play
indispensable
roles
in
many
processes in immunobiology [10–17]. Imbalances and defects in
this system alter host susceptibility to diseases, including diverse
inﬂammatory disorders, infections and malignancies [17–20]. In
this article, we highlight the most recent ﬁndings related to
chemokines and receptors regarding their genetic variations and
nongenetic heterogeneity. Our review provides molecular insights
for chemokine biology to realize precision medicine.
BACKGROUND
History
Since the discovery of the human chemokine CXCL8 or IL-8
(CXCL8/IL-8) in the last century [21–23], chemokines have been
recognized to exist in a complicated mega system [10–12, 14–17].
The rather short but rich history in the ﬁeld includes two waves of
chemokine identiﬁcation [11, 23–26]: the ﬁrst discovery of
inﬂammatory chemokines and receptors that mainly attract
neutrophils and macrophages (Mφs) in the early 1990s and the
second round of chemokines and receptor discovery after the
mid-1990s, which identiﬁed those chemokines and receptors
related to homeostasis and the trafﬁcking of lymphocytes and
dendritic cells (DCs). However, chemokine research was really
initiated in 1977 after the discovery of platelet factor 4 (PF4), also
called CXCL4, which was the ﬁrst identiﬁed peptide containing a
prototypical chemokine structure with uncharacterized chemoat-
tractant activity [24, 26–28]. The discovery that CXCL8 and CCL2
(originally called MCP-1) [21, 22] have chemotactic activity was


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nevertheless a landmark ﬁnding in immunology [23, 24].
Recently,
rapid advances in technologies,
such as next-
generation sequencing (NGS), mass spectrometry and nuclear
magnetic resonance (NMR), have created abundant datasets
allowing integrative multiomics analysis of chemokines even at
Received: 11 October 2022 Accepted: 14 April 2023
Published online: 17 May 2023
1College of Life Sciences and Bioengineering, School of Physical Science and Engineering, Beijing Jiaotong University, 3 ShangyuanCun, Haidian District, 100044 Beijing, P.R.
China. 2Cancer Research Center, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, 101149 Beijing, China. 3Laboratory of Cancer
Innovation, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA. 4These authors contributed equally: Hanli Xu, Shuye Lin.
✉email: jqhuang@bjtu.edu.cn
www.nature.com/cmi
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Frequency Variation and Dose Modification of Benznidazole Administration for the Treatment of Trypanosoma cruzi Infection in Mice, Dogs, and Nonhuman Primates


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Frequency Variation and Dose Modiﬁcation of Benznidazole
Administration for the Treatment of Trypanosoma cruzi
Infection in Mice, Dogs, and Nonhuman Primates
Juan M. Bustamante,a Brooke E. White,a Gregory K. Wilkerson,b* Carolyn L. Hodo,c§ Lisa D. Auckland,c Wei Wang,a^
Stephanie McCain,d
Sarah A. Hamer,c Ashley B. Saunders,e
Rick L. Tarletona,f
aCenter for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA
bMD Anderson Cancer Center, Michale E. Keeling Center for Comparative Medicine and Research, Bastrop, Texas, USA
cDepartment of Veterinary Integrative Biosciences, School of Veterinary Medicine and Biomedical Research, Texas A&M University, College Station, Texas, USA
dBirmingham Zoo, Birmingham, Alabama, USA
eDepartment of Small Animal Clinical Sciences, School of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA
fDepartment of Cellular Biology, University of Georgia, Athens, Georgia, USA
ABSTRACT
Trypanosoma cruzi naturally infects a broad range of mammalian species
and frequently results in the pathology that has been most extensively characterized
in human Chagas disease. Currently employed treatment regimens fail to achieve parasi-
tological cure of T. cruzi infection in the majority of cases. In this study, we have extended
our previous investigations of more effective, higher dose, intermittent administration pro-
tocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice
and in naturally infected dogs and nonhuman primates (NHP). Collectively, these studies
demonstrate that twice-weekly administration of BNZ for more than 4 months at doses
that are ;2.5-fold that of previously used daily dosing protocols, provided the best
chance to obtain parasitological cure. Dosing less frequently or for shorter time periods
was less dependable in all species. Prior treatment using an ineffective dosing regimen
in NHPs did not prevent the attainment of parasitological cure with an intensiﬁed BNZ


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dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed
nearly identical susceptibility to BNZ as those obtained prior to treatment, conﬁrming the
low risk of induction of drug resistance with BNZ and the ability to adjust the treatment
protocol when an initial regimen fails. These results provide guidance for the use of BNZ
as an effective treatment for T. cruzi infection and encourage its wider use, minimally
in high value dogs and at-risk NHP, but also potentially in humans, until better options are
available.
KEYWORDS Trypanosoma cruzi, Chagas disease, benznidazole, cure, dogs, nonhuman
primates
C
hagas disease is a vector-borne parasitic disease caused by the hemoﬂagellate proto-
zoan Trypanosoma cruzi and is one of the major worldwide causes of infection-induced
myocardial disease. T. cruzi is widely distributed and transmitted to a broad range of mam-
mals, from the southern United States to the far south of Chile and Argentina. Managing
Chagas disease is particularly challenging; human infections are not routinely diagnosed
before health-impacting tissue damage is substantial and without treatment, the infection
generally persists for life. Systematic vector control can limit new human infections but can
be expensive and labor-intensive and may have less impact on the rate of new infections in
animals in some settings (1–3). The current treatment for human T. cruzi infection depends
on two nitroheterocyclic compounds, benznidazole (BNZ) and nifurtimox (NFX), that are
given in an intensive treatment regimen (usually 30 to 60 twice-daily doses for BNZ and
Copyright © 2023 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Rick L. Tarleton,
tarleton@uga.edu.
*Present address: Gregory K. Wilkerson,
Department of Clinical Sciences, North Carolina
State University College of Veterinary Medicine,
Raleigh, North Carolina, USA.
§Present address: Carolyn L. Hodo, MD
Anderson Cancer Center, Michale E. Keeling
Center for Comparative Medicine and


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Research, Bastrop, Texas, USA.
^Present address: Wei Wang, General
Dynamics Information Technology, Inc., Falls
Church, Virginia, USA.
The authors declare no conﬂict of interest.
Received 30 January 2023
Returned for modiﬁcation 22 February 2023
Accepted 22 March 2023
Published 11 April 2023
May 2023
Volume 67 Issue 5
10.1128/aac.00132-23
1
EXPERIMENTAL THERAPEUTICS


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The G118R plus R263K Combination of Integrase Mutations Associated with Dolutegravir-Based Treatment Failure Reduces HIV-1 Replicative Capacity and Integration


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The G118R plus R263K Combination of Integrase Mutations
Associated with Dolutegravir-Based Treatment Failure Reduces
HIV-1 Replicative Capacity and Integration
Meng A. Xiao,a,b Jenna Cleyle,a,b Sunbin Yoo,a,b Mekayla Forrest,a,b
Zoë Krullaars,c Hanh Thi Pham,a,b
Thibault Mesplèdea,b,*
aMcGill AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Quebec, Canada
bDepartment of Microbiology and Immunology, Faculty of Medicine, McGill University, Montréal, Quebec, Canada
cViroscience Department, Erasmus Medical Center, Rotterdam, Zuid-Holland, The Netherlands
ABSTRACT
Human immunodeﬁciency virus (HIV) treatment with antiretroviral regi-
mens containing integrase strand transfer inhibitors such as dolutegravir (DTG) and
bictegravir (BIC) offers high levels of protection against the development of drug re-
sistance mutations. Despite this, resistance to DTG and BIC can occur through the
development of the R263K integrase substitution. Failure with DTG has also been
associated with the emergence of the G118R substitution. G118R and R263K are usu-
ally found separately but have been reported together in highly treatment-experi-
enced persons who experienced treatment failure with DTG. We used cell-free strand
transfer and DNA binding assays and cell-based infectivity, replicative capacity, and
resistance assays to characterize the G118R plus R263K combination of integrase
mutations. R263K reduced DTG and BIC susceptibility ;2-fold, in agreement with
our previous work. Single-cycle infectivity assays showed that G118R and G118R plus
R263K conferred ;10-fold resistance to DTG. G118R alone conferred low levels of re-
sistance to BIC (3.9-fold). However, the G118R plus R263K combination conferred
high levels of resistance to BIC (33.7-fold), likely precluding the use of BIC after DTG
failure with the G118R plus R263K combination. DNA binding, viral infectivity, and
replicative capacity of the double mutant were further impaired, compared to single


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mutants. We propose that impaired ﬁtness helps to explain the scarcity of the
G118R plus R263K combination of integrase substitutions in clinical settings and that
immunodeﬁciency likely contributes to its development.
KEYWORDS HIV drug resistance, dolutegravir, bictegravir, integrase inhibitors,
integrase, R263K, G118R
C
urrent treatment for human immunodeﬁciency virus (HIV) infection consists of combi-
nations of two or three antiretroviral drugs that can be taken orally daily or intramuscu-
larly every 2 months (1). In addition, every-6-month subcutaneous injections of lenacapavir
were approved recently in combination with other antiretroviral drugs for the treatment of
highly treatment-experienced individuals with limited therapeutic options (2). Regardless of
their form of administration, for all regimens, treatment failure occurs in some cases when vi-
ral suppression cannot be achieved or maintained, and drug resistance mutations can
emerge (3). The ability of an antiretroviral treatment regimen, or of a particular drug, to pro-
tect against the development of HIV resistance mutations upon treatment failure is called its
barrier to resistance. Although antiretroviral drugs with a low barrier to resistance can be
combined to form regimens sufﬁcient to maintain virological suppression in the context
of optimal treatment adherence, drugs with high barriers to resistance may improve clin-
ical outcomes on population and individual levels. Advantages associated with a high
barrier to resistance include cost-effectiveness associated with reduced genotyping and
Copyright © 2023 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Thibault Mesplède,
t.mesplede@erasmusmc.nl.
*Present address: Thibault Mesplède,
Viroscience Department, Erasmus Medical
Center, Rotterdam, Zuid-Holland,
The Netherlands.
The authors declare no conﬂict of interest.
Received 13 October 2022
Returned for modiﬁcation 2 November 2022
Accepted 16 March 2023
Published 18 April 2023
May 2023
Volume 67 Issue 5


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REVIEW ARTICLE
OPEN
Targeting strategies for bone diseases: signaling pathways and
clinical studies
Hao Xu1, Wentao Wang1, Xin Liu1, Wei Huang2, Chen Zhu2, Yaozeng Xu1, Huilin Yang1,3✉, Jiaxiang Bai1,3✉ and Dechun Geng
1,3✉
Since the proposal of Paul Ehrlich’s magic bullet concept over 100 years ago, tremendous advances have occurred in targeted
therapy. From the initial selective antibody, antitoxin to targeted drug delivery that emerged in the past decades, more precise
therapeutic efﬁcacy is realized in speciﬁc pathological sites of clinical diseases. As a highly pyknotic mineralized tissue with lessened
blood ﬂow, bone is characterized by a complex remodeling and homeostatic regulation mechanism, which makes drug therapy for
skeletal diseases more challenging than other tissues. Bone-targeted therapy has been considered a promising therapeutic
approach for handling such drawbacks. With the deepening understanding of bone biology, improvements in some established
bone-targeted drugs and novel therapeutic targets for drugs and deliveries have emerged on the horizon. In this review, we
provide a panoramic summary of recent advances in therapeutic strategies based on bone targeting. We highlight targeting
strategies based on bone structure and remodeling biology. For bone-targeted therapeutic agents, in addition to improvements of
the classic denosumab, romosozumab, and PTH1R ligands, potential regulation of the remodeling process targeting other key
membrane expressions, cellular crosstalk, and gene expression, of all bone cells has been exploited. For bone-targeted drug
delivery, different delivery strategies targeting bone matrix, bone marrow, and speciﬁc bone cells are summarized with a
comparison between different targeting ligands. Ultimately, this review will summarize recent advances in the clinical translation of
bone-targeted therapies and provide a perspective on the challenges for the application of bone-targeted therapy in the clinic and
future trends in this area.


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Signal Transduction and Targeted Therapy  (2023) 8:202 
; https://doi.org/10.1038/s41392-023-01467-8
INTRODUCTION
Bone is a solid structure undergoing perpetual renewal with
crucial functions such as kinematic support, visceral protection,
and regulation of hematopoiesis and mineral balance.1 Main-
tenance of these functions depends on normal bone mass and
strength, which are achieved through bone remodeling. Skeletal
diseases, such as osteoporosis, are usually accompanied by
abnormal bone remodeling, in which osteoclast-mediated bone
resorption preponderates over osteoblast-mediated bone forma-
tion, leading to decreased bone mass, deteriorated microstructure,
and increased fragile fracture risk.2 In light of the perplexing
pathological
mechanism
underlying
abnormal
remodeling,
symptom-relief therapy by anti-resorption and pro-formation is
the main choice, in addition to the elemental calcium and vitamin
D supplements.
Since the proposal of Paul Ehrlich’s “magic bullet” concept over
100 years ago, great advances have occurred in drugs that target
intended cellular structures.3 In bone-targeted pharmacological
therapy, anti-resorption agents, such as bisphosphonates, selec-
tive estrogen receptor modulators (SERMs), receptor activator of
nuclear factor-kB (RANK) ligand (RANKL) inhibitors, and anabolic
medications, such as type 1 parathyroid hormone receptor
(PTH1R)
ligands,
sclerostin
inhibitors,
have
emerged
with
demonstrated efﬁcacy in treating diseases characterized by
abnormal bone remodeling.4,5 Nevertheless, the improvement in
bone parameters by these agents does not simply imply a regain
of normal bone remodeling, as is observed by the attendant
suppression of anti-resorption agents to the bone formation or a
slight increase in bone resorption by anabolic agents.6,7 Further-
more, side effects such as osteonecrosis,8 rebound fractures,9
cardiovascular events,10 and osteosarcoma genesis,11 impede
effective long-term management of bone diseases, which under-


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scores a need to improve these established targets for more
precise therapy.
Fortunately, with the deepening knowledge of bone biology,
the mechanism underlying some side effects has been recognized
with a prominent decrease by preclinical improvements of these
drugs. In addition, recent insights have revealed that bone
remodeling is a coordinated process spatiotemporally mediated
by all bone cells instead of single activities by the basic
multicellular units consisting of osteoblasts (OBs) and osteoclasts
(OCs).12 Osteocytes, immune cells, vessel endothelial cells, and
bone marrow cells were shown to possess multiple inﬂuences
during the remodeling process,13–15 and some emerging ther-
apeutic strategies targeting these bone cells, especially the
crosstalk between them, have shown promise in promoting bone
Received: 1 January 2023 Revised: 2 April 2023 Accepted: 19 April 2023
1Department of Orthopedics, The First Afﬁliated Hospital of Soochow University, 188 Shizi Street, Suzhou, Jiangsu 215006, P. R. China; 2Department of Orthopedics, The First
Afﬁliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230031 Anhui, China and 3Orthopaedic Institute, Medical
College, Soochow University, Suzhou 215006 Jiangsu, China
Correspondence: Huilin Yang (suzhouspine@163.com) or Jiaxiang Bai (jxbai1995@163.com) or Dechun Geng (szgengdc@suda.edu.cn)
These authors contributed equally: Hao Xu, Wentao Wang, Xin Liu
www.nature.com/sigtrans
Signal Transduction and Targeted Therapy
© The Author(s) 2023
1234567890();,:


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Abatacept GVHD prophylaxis in unrelated hematopoietic cell transplantation for pediatric bone marrow failure


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Abatacept GVHD prophylaxis in unrelated hematopoietic cell
transplantation for pediatric bone marrow failure
Elizabeth O. Stenger,1 Benjamin Watkins,1 Kelsey Rogowski,1 Kuang-Yueh Chiang,2 Ann Haight,1 Kathryn Leung,1 Muna Qayed,1
Sharmila Raghunandan,1 Yvonne Suessmuth,1 Leslie Kean,3 and John Horan1,3
1Division of Pediatric Hematology/Oncology, Aﬂac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Emory University, Atlanta, GA; 2Division of Pediatric
Hematology/Oncology, University of Toronto/The Hospital for Sick Children, Toronto, Canada; and 3Division of Pediatric Hematology/Oncology, Dana-Farber/Boston
Children’s Hospital, Boston, MA
Key Points
• Four doses of Aba can
be safely added to
standard 2-drug GVHD
prophylaxis in URD
HCT for pediatric bone
marrow failure.
• No severe acute or
chronic GVHD was
seen in pediatric
patients with bone
marrow failure with
Aba-containing
prophylaxis after URD
HCT.
Hematopoietic cell transplantation (HCT) is the only readily available cure for many life-
threatening pediatric nonmalignant diseases (NMD), but most patients lack a matched
related donor and are at higher risk for graft-versus-host disease (GVHD). Use of abatacept
(Aba) to target donor T-cell activation has been safe and effective in preventing GVHD after
unrelated donor (URD) HCT for malignant diseases (Aba2 trial). Our primary objective
was to evaluate the tolerability of Aba added to standard GVHD prophylaxis (cyclosporine
and mycophenolate mofetil) in pediatric patients with NMD undergoing URD HCT. In this
single-arm, single-center phase 1 trial, 10 patients receiving reduced intensity or
nonmyeloablative conditioning underwent URD HCT. Immune reconstitution was assessed
longitudinally via ﬂow cytometry and compared to pediatric patients on Aba2. Nine
patients successfully engrafted, with 1 primary graft rejection in the setting of inadequate
cell dose; secondary graft rejection occurred in 1 patient with concurrent cytomegalovirus


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viremia. Two deaths occurred, both unrelated to Aba. One patient developed probable
posttransplant lymphoproliferative disease, responsive to rituximab and immune
suppression withdrawal. No patients developed severe acute or chronic GVHD, and 8
patients were off systemic immunosuppression at 1 year. Immune reconstitution did not
appear to be impacted by Aba, and preservation of naïve relative to effector memory CD4+ T
cells was seen akin to Aba2. Thus, 4 doses of Aba were deemed tolerable in pediatric
patients with NMD following URD HCT, with encouraging preliminary efﬁcacy and
supportive immune correlatives in this NMD cohort.
Introduction
Allogeneic hematopoietic cell transplantation (HCT) is the only standard-of-care curative therapy for
many severe nonmalignant diseases (NMDs) which affect children.1-4 While a diverse group of dis-
eases, including highly prevalent hemoglobinopathies and less prevalent diseases such as severe
aplastic anemia (SAA) and Fanconi anemia (FA), they universally lead to signiﬁcant morbidity and early
mortality. These diseases can be cured with a HCT, without which patients face a myriad of
Submitted 11 July 2022; accepted 28 November 2022; prepublished online on Blood
Advances
First
Edition
1
February
2023.
https://doi.org/10.1182/
bloodadvances.2022008545.
Deidentiﬁed individual participant data from this clinical trial will be made available,
upon request, 3 months after publication and for a period of up to 5 years.
Data are available on request from the corresponding author, Elizabeth O. Stenger
(estenge@emory.edu).
The study protocol is included as a data supplement available with the online version
of this article.
The full-text version of this article contains a data supplement.
© 2023 by The American Society of Hematology. Licensed under Creative Commons
Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0),
permitting only noncommercial, nonderivative use with attribution. All other rights
reserved.
REGULAR ARTICLE
2196


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Lateral cerebellothalamic tract activation underlies DBS therapy 
for Essential Tremor
AnneMarie Brindaa,1, Julia P. Slopsemaa,1, Rebecca D. Butlera, Salman Ikramuddinc, 
Thomas Bealla, William Guoa, Cong Chua, Remi Patriatb, Henry Braunb, Mojgan Goftaria, 
Tara Palnitkarb, Joshua Amanc, Lauren Schrockc, Scott E. Cooperc, Joseph Matsumotoc, 
Jerrold L. Vitekc, Noam Harelb, Matthew D. Johnsona,d,*
aDepartment of Biomedical Engineering, University of Minnesota, Minneapolis, MN, 55455, USA
bDepartment of Radiology, CMRR, University of Minnesota, Minneapolis, MN, 55455, USA
cDepartment of Neurology, University of Minnesota, Minneapolis, MN, 55455, USA
dInstitute for Translational Neuroscience, University of Minnesota, Minneapolis, MN, 55455, USA
Abstract
Background: While deep brain stimulation (DBS) therapy can be effective at suppressing tremor 
in individuals with medication-refractory Essential Tremor, patient outcome variability remains a 
significant challenge across centers. Proximity of active electrodes to the cerebellothalamic tract 
(CTT) is likely important in suppressing tremor, but how tremor control and side effects relate to 
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
*Corresponding author. Department of Biomedical Engineering, University of Minnesota, 7-105 Nils Hasselmo Hall, 312 Church 
Street SE, Minneapolis, MN, 55455, USA. john5101@umn.edu (M.D. Johnson).
1These authors contributed equally to this work.
Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: 
Tara Palnitkar: consultant for Surgical Information Sciences; Remi Patriat: consultant for Surgical Information Sciences; Jerrold Vitek: 
consultant for Medtronic, Boston Scientific, Abbott, and Surgical Information Sciences; Noam Harel: consultant and a shareholder for 
Surgical Information Sciences; all other authors declare no conflict of interest.


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CRediT authorship contribution statement
AnneMarie Brinda: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Writing – original draft, 
Writing – review & editing, Visualization. Julia P. Slopsema: Conceptualization, Methodology, Software, Validation, Formal 
analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Rebecca D. Butler: Conceptualization, 
Methodology, Software, Validation, Formal analysis, Investigation, Writing – review & editing, Visualization. Salman Ikramuddin: 
Investigation, Formal analysis, Writing – review & editing. Thomas Beall: Methodology, Software, Writing – review & editing. 
William Guo: Methodology, Software, Writing – review & editing. Cong Chu: Methodology, Software, Writing – review & 
editing. Remi Patriat: Methodology, Software, Validation, Investigation, Writing – review & editing, Visualization. Henry Braun: 
Methodology, Software, Validation, Investigation, Writing – review & editing. Mojgan Goftari: Methodology, Software, Validation, 
Writing – review & editing. Tara Palnitkar: Methodology, Software, Validation, Investigation, Writing – review & editing, 
Visualization. Joshua Aman: Investigation, Writing – review & editing. Lauren Schrock: Investigation, Writing – review & editing. 
Scott E. Cooper: Conceptualization, Investigation, Writing – review & editing. Joseph Matsumoto: Conceptualization, Investigation, 
Writing – review & editing. Jerrold L. Vitek: Conceptualization, Investigation, Writing – review & editing. Noam Harel: 
Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Writing – review & editing, Supervision, 
Project administration, Funding acquisition. Matthew D. Johnson: Conceptualization, Methodology, Software, Validation, Formal 
analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration, 
Funding acquisition.
Appendix A. Supplementary data


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Supplementary data to this article can be found online at https://doi.org/10.1016/j.brs.2023.02.002.
HHS Public Access
Author manuscript
Brain Stimul. Author manuscript; available in PMC 2023 May 21.
Published in final edited form as:
Brain Stimul. 2023 ; 16(2): 445–455. doi:10.1016/j.brs.2023.02.002.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript


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A Holistic Review of the Preclinical Landscape for Long-Acting Anti-infective Drugs Using HIV as a Paradigm


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Clinical Infectious Diseases                                          
S U P P L E M E N T  A R T I C L E
A Holistic Review of the Preclinical Landscape for 
Long-Acting Anti-infective Drugs Using HIV as a Paradigm
Megan Neary, Andrew Owen, and Adeniyi Olagunju
Department of Pharmacology and Therapeutics, Centre of Excellence for Long-acting Therapeutics, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, 
Merseyside, United Kingdom
Lack of predictive preclinical models is a key contributor to the steep attrition rate in drug development. Successful clinical 
translation may be higher for new chemical entities or existing approved drugs reformulated for long-acting (LA) 
administration if preclinical studies designed to identify any new uncertainties are predictive of human exposure and response. 
In this review, we present an overview of standard preclinical assessments deployed for LA formulations and delivery systems, 
using human immunodeficiency virus LA therapeutics preclinical development as a paradigm. Key progress in the preclinical 
development of novel LA antiretrovirals formulations and delivery systems are summarized, including bispecific broadly 
neutralizing monoclonal antibody and small molecule technologies for codelivery of multiple drugs with disparate solubility 
properties. There are new opportunities to take advantage of recent developments in tissue engineering and 3-dimensional in 
vitro modeling to advance preclinical modeling of anti-infective activity, developmental and reproductive toxicity assessment, 
and to apply quantitative modeling and simulation strategies. These developments are likely to drive the progression of more 
LA anti-infective drugs and multipurpose technologies into clinical development in the coming years.
Keywords. long-acting; antiretroviral; preclinical; in vitro models.
Correspondence: A. Olagunju, Centre of Excellence for Long-acting Therapeutics, Institute of


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Systems, Molecular and Integrative Biology, Biosciences Building, Crown Street, Liverpool L69 
7BE, UK (olagunju@liverpool.ac.uk).
Clinical Infectious Diseases®
2022;75(S4):S490–7 
© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases 
Society of America. All rights reserved. For permissions, please e-mail: journals.permissions 
@oup.com
https://doi.org/10.1093/cid/ciac685
Decisions about progressing new drug candidates to clinical de-
velopment are guided by evidence from preclinical studies. 
These are the cornerstone of new drug development and clin-
ical translation, providing the critical data that inform 
first-in-human trials (see Figure 1). The landscape for long- 
acting (LA) anti-infective development over the past 12 months 
was marked by some significant developments, particularly for 
human immunodeficiency virus (HIV), which will be used as a 
paradigm to discuss the preclinical development of LA anti- 
infective drugs. LA therapeutics offer a transformational ap-
proach to HIV treatment and preexposure prophylaxis, largely 
circumventing established issues around treatment fatigue and 
associated nonadherence by offering regimens with extended 
dosing intervals.
Multiple LA candidates for treatment of HIV are in develop-
ment and are at different points in the drug development pipe-
line. Multicountry approval of LA-injectable cabotegravir plus 
rilpivirine administered intramuscularly every two months 
was a major milestone for LA HIV treatment, despite virological 
failure in patients with baseline nonnucleoside reverse 
transcriptase inhibitor or integrase strand transfer inhibitor re-
sistance [1–3]. The US Food and Drug Administration (FDA) 
issued a Complete Response Letter with easily resolvable con-
cerns about incompatibility with borosilicate glass vials in re-
sponse to a New Drug Application for the LA lenacapavir 
(capsid inhibitor) administered every 6 months for treatment 
in heavily treatment-experienced patients with multidrug resis-


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tant HIV-1 infection [4]. The clinical hold has since been lifted 
by the FDA for injectable lenacapavir [5]. Investigational New 
Drug Applications for LA islatravir (nucleoside reverse tran-
scriptase translocation inhibitor) monthly oral formulation 
and annual implant formulation have been placed on clinical 
hold due to observations of reductions in total lymphocyte 
and CD4+ T-cell count.
From the 12% of novel drug candidates that pass preclinical 
testing, only 11% successfully make it to the market, mostly 
because of failures resulting from unanticipated toxicity 
(about 50%) or suboptimal efficacy. About 50% of postap-
proval withdrawals are also from toxicity. Lack of predictive 
preclinical models is a key driver of this steep attrition rate, 
and it has been suggested that clinical trial success rate can 
be theoretically increased to > 55% with better preclinical 
models [6]. If preclinical studies designed to identify any 
new uncertainties are predictive of human exposure and re-
sponse, translation of LA medicines should have a much high-
er success rate. However, additional considerations are 
evident for LA medicines and key knowledge gaps relating 
to drug absorption remain.
S490 • CID 2022:75 (Suppl 4) • Neary et al


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REVIEW ARTICLE
OPEN
Macrophages in immunoregulation and therapeutics
Shanze Chen1, Abdullah F.U.H. Saeed2, Quan Liu3, Qiong Jiang1, Haizhao Xu1,4, Gary Guishan Xiao5✉, Lang Rao
6✉ and
Yanhong Duo7✉
Macrophages exist in various tissues, several body cavities, and around mucosal surfaces and are a vital part of the innate immune
system for host defense against many pathogens and cancers. Macrophages possess binary M1/M2 macrophage polarization
settings, which perform a central role in an array of immune tasks via intrinsic signal cascades and, therefore, must be precisely
regulated. Many crucial questions about macrophage signaling and immune modulation are yet to be uncovered. In addition, the
clinical importance of tumor-associated macrophages is becoming more widely recognized as signiﬁcant progress has been made
in understanding their biology. Moreover, they are an integral part of the tumor microenvironment, playing a part in the regulation
of a wide variety of processes including angiogenesis, extracellular matrix transformation, cancer cell proliferation, metastasis,
immunosuppression, and resistance to chemotherapeutic and checkpoint blockade immunotherapies. Herein, we discuss immune
regulation in macrophage polarization and signaling, mechanical stresses and modulation, metabolic signaling pathways,
mitochondrial and transcriptional, and epigenetic regulation. Furthermore, we have broadly extended the understanding of
macrophages in extracellular traps and the essential roles of autophagy and aging in regulating macrophage functions. Moreover,
we discussed recent advances in macrophages-mediated immune regulation of autoimmune diseases and tumorigenesis. Lastly,
we discussed targeted macrophage therapy to portray prospective targets for therapeutic strategies in health and diseases.
Signal Transduction and Targeted Therapy  (2023) 8:207 
; https://doi.org/10.1038/s41392-023-01452-1
INTRODUCTION
The primary formation of host resistance, contrary to infectious


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pathogens in disease, is the innate immune framework mainly
made of innate immune cellular entities and cells originating from
myeloid, comprising macrophages, monocytes, granulocytes, and
dendritic cells (DCs).1,2 When disease ensues, pathogen-associated
molecular patterns (PAMPs) and damage-associated molecular
patterns (DAMPs) are recognized via the cell surface or intracellular
pattern recognition receptors (PPRs) from innate immune cells, for
example,
Toll-like
receptors
(TLRs),
advanced
glycation
end
products (RAGE) and Nod-like receptors (NLRs).2 Successively, the
interaction of PAMPs or DAMPs through particular receptors
triggers
signaling
cascades
following
association
at
reactive
oxygenic and nitrogen-related species, chemokines, proinﬂamma-
tory cytokines, and antimicrobial-associated peptides, including
augmented phagocytosis and the efﬁcient elimination of microbial
infection.2,3 In addition, instigating the innate immune system leads
to the follow-up with the trigger of more speciﬁc adaptive
immunity. However, given the advantages of the innate immune
system, this tends to remain a double-edged sword employing
inﬂammation that will damage the host.2 Hence, it is signiﬁcant to
comprehend the immune regulatory strategies that govern the
inﬂammatory progression’s commencement, extent, and goals.
Macrophages are innate immune cells ﬁrst identiﬁed by Elia
Metchnikoff in starﬁsh hatchlings in 1882 when tangerine tree
thistles were used, then in Daphnia magna or essential water ﬂea
infested with fungal spores as the cells responsible for the cycle of
phagocytosis of foreign materials. For this accomplishment, the
Nobel Prize (Physiology and Medicine) was presented to Elia
Metchnikoff in 1908. In this way, macrophages are the primary
innate immune cells identiﬁed 130 years ago.4
Macrophages are crucial in innate immunity by regulating
several homeostatic and evolutionary host defense immune
responses. Besides, macrophages partake in many other biological


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events, including modulating reactive oxygen species (ROS)
endogenous intensities, iron homeostasis, tissue injury repair,
and numerous other metabolic functions.5,6 In addition, macro-
phages have three essential functions, i.e., immunomodulation,
phagocytosis, and antigen presentation. They are vital for
performing normal immune reactions under various pathophy-
siological conditions.7
As the relationship between TAMs and malignant tumors
expands into more apparent, TAMs are suggested as possible
biomarkers for diagnosing and prognosis tumors and therapeutic
targets in various cancers. Inhibiting monocyte recruitment,
targeting
TAM
activation,
converting
TAMs
to
anticancer
Received: 5 September 2022 Revised: 6 March 2023 Accepted: 26 April 2023
1Department of Respiratory Diseases and Critic Care Unit, Shenzhen Institute of Respiratory Disease, Shenzhen Key Laboratory of Respiratory Disease, Shenzhen People’s Hospital
(The Second Clinical Medical College, Jinan University; The First Afﬁliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; 2Department of
Cancer Biology, Beckman Research Institute of City of Hope National Medical Center, Los Angeles, CA 91010, USA; 3Department of Laboratory Medicine, Huazhong University of
Science and Technology Union Shenzhen Hospital (Nanshan Hospital), Shenzhen University, Shenzhen 518052, China; 4Department of Respiratory, The First Afﬁliated Hospital,
School of Medicine, Southern University of Science and Technology, Shenzhen 518055, China; 5State Key Laboratory of Fine Chemicals, Department of Pharmaceutical Sciences,
School of Chemical Engineering, Dalian University of Technology, Dalian, China; 6Institute of Biomedical Health Technology and Engineering, Shenzhen Bay Laboratory, Shenzhen
518132, China and 7Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden
Correspondence: Gary Guishan Xiao (gxiao@dlut.edu.cn) or Lang Rao (lrao@szbl.ac.cn) or Yanhong Duo (duoduo7753@126.com)


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These authors contributed equally: Shanze Chen, Abdullah F.U.H. Saeed, Quan Liu
www.nature.com/sigtrans
Signal Transduction and Targeted Therapy
© The Author(s) 2023
1234567890();,:


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Maternal outcomes of placental abruption with intrauterine fetal death and delivery routes: A nationwide observational study


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708 |   
 
Acta Obstet Gynecol Scand. 2023;102:708–715.
wileyonlinelibrary.com/journal/aogs
Received: 20 October 2022 | Revised: 14 February 2023 | Accepted: 21 March 2023
DOI: 10.1111/aogs.14569  
O R I G I N A L  R E S E A R C H  A R T I C L E
Maternal outcomes of placental abruption with intrauterine 
fetal death and delivery routes: A nationwide observational 
study
Yoshimitsu Wada1
 |   Hironori Takahashi1 |   Yusuke Sasabuchi2 |   Rie Usui1
 |   
Manabu Ogoyama1
 |   Hirotada Suzuki1 |   Akihide Ohkuchi1 |   Hiroyuki Fujiwara1
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction 
in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2023 The Authors. Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of 
Obstetrics and Gynecology (NFOG).
Abbreviations: CD, cesarean delivery; CI, confidence interval; DIC, disseminated intravascular coagulation; IPTW, inversed probability weighting; IQR, interquartile range; IUFD, 
intrauterine fetal death; OR, odds ratio; PA, placental abruption; VD, vaginal delivery.
1Department of Obstetrics and 
Gynecology, Jichi Medical University, 
Tochigi, Japan
2Data Science Center, Jichi Medical 
University, Tochigi, Japan
Correspondence
Hironori Takahashi, Jichi Medical 
University, 3311- 1 Yakushiji, Shimotsuke, 
Tochigi 329- 0498, Japan.
Email: hironori@jichi.ac.jp
Abstract
Introduction: Placental abruption is a serious complication, especially when accom-
panied by intrauterine fetal death. The optimal delivery route for placental abruption 
with intrauterine fetal death for reducing maternal complications is still unclear. In 
this study we aimed to compare the maternal outcomes between cesarean delivery 
and vaginal delivery in women with placental abruption with intrauterine fetal death.


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Material and methods: Using the Japan Society of Obstetrics and Gynecology na-
tionwide perinatal registry database, we identified pregnant women with placental 
 abruption with intrauterine fetal death between 2013 and 2019. The following women 
were excluded: those with multiple pregnancies, placenta previa, placenta accreta 
spectrum, amniotic fluid embolism, or whose delivery route was missing data. The 
association between delivery routes (cesarean delivery and vaginal delivery) and the 
maternal outcome was examined using a linear regression model with inverse prob-
ability weighting. The primary outcome was the amount of bleeding during delivery. 
Missing data were imputed using multiple imputation.
Results: The number of women with placental abruption with intrauterine fetal death 
was 1218/1601932 (0.076%). Of 1134 women analyzed, 608 (53.6%) underwent ce-
sarean delivery. Bleeding during delivery (median [interquartile range]) was 1650.00 
(950.00– 2450.00) (mL) and 1171.00 (500.00– 2196.50) (mL) in cesarean and vaginal 
delivery, respectively. Bleeding during delivery (mL) was significantly greater in cesar-
ean delivery than in vaginal delivery (regression coefficient, 1086.39; 95% confidence 
interval, 130.96– 2041.81; p = 0.026). Maternal death and uterine rupture occurred in 
four (0.4%) and five (0.4%) women, respectively. The four maternal deaths were noted 
in the vaginal delivery group.
Conclusions: Bleeding during delivery was significantly greater in cesarean delivery than 
that in vaginal delivery in women with placental abruption with intrauterine fetal death.


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R E S E A R C H A R T I C L E
Similar somatotopy for active and passive digit representation
in primary somatosensory cortex
Zeena-Britt Sanders1
|
Harriet Dempsey-Jones2,3
|
Daan B. Wesselink1,2
|
Laura R. Edmondson4
|
Alexander M. Puckett3,5
|
Hannes P. Saal5
|
Tamar R. Makin1,2,6
1Wellcome Centre of Integrative
Neuroimaging, FMRIB, John Radcliffe Hospital,
Oxford, UK
2Institute of Cognitive Neuroscience,
University College London, London, UK
3School of Psychology, The University of
Queensland, Brisbane, Australia
4Department of Psychology, University of
Sheffield, Sheffield, UK
5Queensland Brain Institute, The University of
Queensland, Brisbane, Australia
6MRC Cognition and Brain Sciences Unit,
University of Cambridge, Cambridge, UK
Correspondence
Harriet Dempsey-Jones, School of Psychology,
The University of Queensland, St. Lucia,
Brisbane 4067, Australia.
Email: h.dempseyjones@uq.edu.au
Funding information
H2020 European Research Council,
Grant/Award Number: 715022; Leverhulme
Trust Research Project Grant, Grant/Award
Number: RPG-2022-031; Wellcome Trust,
Grant/Award Number: 215575/Z/19/Z;
Medical Research Council, Grant/Award
Number: MC_UU_00030/10
Abstract
Scientists traditionally use passive stimulation to examine the organisation of primary
somatosensory cortex (SI). However, given the close, bidirectional relationship
between the somatosensory and motor systems, active paradigms involving free
movement may uncover alternative SI representational motifs. Here, we used 7 Tesla
functional magnetic resonance imaging to compare hallmark features of SI digit rep-
resentation between active and passive tasks which were unmatched on task or stim-
ulus properties. The spatial location of digit maps, somatotopic organisation, and
inter-digit representational structure were largely consistent between tasks, indicat-
ing representational consistency. We also observed some task differences. The active
task produced higher univariate activity and multivariate representational information


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content (inter-digit distances). The passive task showed a trend towards greater
selectivity for digits versus their neighbours. Our findings highlight that, while the
gross features of SI functional organisation are task invariant, it is important to also
consider motor contributions to digit representation.
K E Y W O R D S
finger representations, fMRI, hand map, motor, representational similarity analysis,
somatosensation, topography
1
|
INTRODUCTION
Traditionally, the organisation of the somatosensory system has been
studied using passive tactile stimulation protocols, that is, contacting
stationary digit(s) with tactile stimuli (examples in humans: (Besle
et al., 2014; Sanchez-Panchuelo et al., 2010) and in primates: (Michael
M. Merzenich et al., 1978; Sur et al., 1980)). While providing a highly
controlled means to study the primary somatosensory cortex (SI),
active touch tasks (Kikkert et al., 2016; Schellekens et al., 2018),
involving movement of the digits, may provide somatosensory inputs
more like those that occur habitually. In daily life, the majority of hand
inputs to SI occur from deliberate action (i.e., driven by motor control),
or are directly relevant for supporting motor control (Scott, 2004).
Accordingly, somatosensory hand representation reflects habitual pat-
terns of coordinated action between digits (Ejaz et al., 2015; Ingram
et al., 2008). These usage effects manifest in greater overlap between
Zeena-Britt Sanders and Harriet Dempsey-Jones contributed equally to this work.
Lead author: Tamar R. Makin, MRC Cognition and Brain Sciences Unit, University of
Cambridge, Cambridge CB2 7EF, UK.
Received: 20 July 2022
Revised: 11 December 2022
Accepted: 13 March 2023
DOI: 10.1002/hbm.26298
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2023 The Authors. Human Brain Mapping published by Wiley Periodicals LLC.
3568


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Long-Lasting effects of Subthalamic Nucleus Coordinated Reset Deep Brain Stimulation in the Non-Human Primate Model of Parkinson\
ism: A case report


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Long-Lasting effects of Subthalamic Nucleus Coordinated Reset 
Deep Brain Stimulation in the Non-Human Primate Model of 
Parkinsonism: A case report
Joyce Chelangat Borea, Brett A. Campbella,b, Hanbin Choa, Francesco Puccic,d, Raghavan 
Gopalakrishnanc, Andre G. Machadoa,c,d, Kenneth B. Baker, PhDa,c
aDepartment of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, 
44106, United States.
bDepartment of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, 
44106
cCenter for Neurological Restoration, Neurological Institute, Cleveland Clinic, Cleveland, OH, 
44106, United States.
dDepartment of Neurosurgery, Neurological Institute, Cleveland Clinic, Cleveland, OH, 44106, 
United States.
Dear Editor,
Continuous, high-frequency isochronal (i.e., “traditional”) deep brain stimulation (tDBS), 
is a standard treatment option for advanced-stage Parkinson’s disease (PD). However, 
tDBS is limited by side effects [1] and its chronic nature can necessitate frequent 
battery replacement. To address these issues, a novel stimulation method, coordinated reset 
(CR)-DBS, was developed through computational modeling [2–3]. To date, however, data 
addressing its underlying electrophysiological effects are limited [3]. The goal of this study 
was to characterize the sub-acute and carryover motor and electrophysiological effects of 
CR-DBS in terms of both local field potential (LFP) power changes and brain connectivity 
in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) non-human primate (NHP) 
model of parkinsonism.
Animal care complied with the National Institutes of Health Guide for the Care and Use 
of Laboratory Animals and a protocol approved by the Cleveland Clinic Institutional 
Animal Care and Use Committee (IACUC). A 14-year-old adult female NHP (Macaca 
mulatta) was instrumented with a DBS lead in the subthalamic nucleus (STN) and a pair of 
twelve-contact electrocorticography (ECoG) arrays spanning from medial somatosensory to


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prefrontal cortex (Figure 1A) as described previously [4–5]. The M1 region was identified 
as the contacts showing the lowest threshold for corticospinal activation, with localization 
confirmed by 3D reconstruction of the co-registered preoperative MRI and postoperative CT 
(Figure 1B) [6].
Corresponding author: Kenneth B. Baker, PhD, Department of Neurosciences, Lerner Research Institute (NC-30), Cleveland Clinic, 
Cleveland, OH, USA. Tel: (216) 445-2244 Fax: (216) 444-7927, bakerk6@ccf.org. 
HHS Public Access
Author manuscript
Brain Stimul. Author manuscript; available in PMC 2023 May 23.
Published in final edited form as:
Brain Stimul. 2022 ; 15(3): 598–600. doi:10.1016/j.brs.2022.04.005.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript


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Vol.:(0123456789)
1 3
Drug Delivery and Translational Research 
https://doi.org/10.1007/s13346-023-01361-4
REVIEW ARTICLE
Innate immune regulations and various siRNA modalities
Anju Kaushal1 
Accepted: 29 April 2023 
© Controlled Release Society 2023
Abstract
RNAi therapeutics are designed to produce the precise silencing effects against the gene-linked diseases which were known 
to be untreatable in the past. The highly immunostimulatory nature of siRNA enhances the off-target effects and easily get 
attacked by nucleases; hence, their modulation is essentially required for accurate alterations to be made in the structures to 
intensify the pharmacological attributes. The phosphonate modifications act as shield against undue phosphorylation effects, 
and the molecular changes in ribose sugar lowers the level of immunogenicity and increases the binding efficacy. When 
bases are substituted with virtual/or pseudo bases, they eventually reduce the off-target effects. These changes modulate the 
nucleic acid sensors and control the hyper-activation of innate immune response. Various modification designs based on 
STC (universal pattern), ESC, ESC + (advanced patterns) and disubstrate have been explored to silence the gene expression 
of various diseases e.g., hepatitis, HIV, influenza, RSV, CNV and acute kidney injury. This review describes the various 
innovative siRNA therapeutics and their implications on the developed immune regulations to silence the disease effects.
Keywords RNAi · siRNA modification · Innate immune immune response · Gene silencing
Introduction
Interfering RNA (RNAi) technology is being employed to 
target the disease-associated gene to knockdown/or silence 
its debilitating effect by halting the activity of associated 
mRNA, therefore, repressing the translation process. Fire 
and Mello, in 1998, have demonstrated the RNAi concept 
for the first time, using dsRNA in Caenorhabditis elegans 
to observe the silencing effects. These discoveries on the


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RNA-mediated process of gene regulations have advanced 
our knowledge in this field [1]. Later on, the work has been 
extended to watch the effects of siRNA molecular machines 
in plants. The final effects demonstrated the guide sequence-
dependent endo-nucleolytic cleavage was linked to inhibit the 
translation by mRNA [2]. By 2001, microRNA (miRNA) has 
also been explored and classified in small RNA regulators 
[3]. In the past, siRNA therapeutics development has faced 
many challenges regarding stability and specificity along 
with accurate drug delivery to the target tissues. But, the 
most recent advancements in the arena of chemistry, genetics 
and biotechnology have revolutionized the field of nucleic 
acid developments to the next level.
In the past decade, incredible efforts have been made 
to study many chemical modification geometries and 
to analyse and evaluate their efficacy and biosafety in 
modified-siRNA drugs. siRNA/or mRNA used in higher 
concentration, without modification-treatments, can 
enhance the immunostimulatory effects of nucleic acid 
sensors initiating the strong innate immune response, by 
secreting excessive IFN-α. The nucleoside modifications 
are an effective tool to increase stability and reduce the 
immunogenicity of nucleic-acid therapeutics [6]. Various 
siRNA modification chemistries are being used to control  
the innate immune stimulations in context with increasing  
potency and decreasing the involved toxicities. Off-target 
effects mediated by siRNAs, however, is one more param-
eter which considerably affect the precision and accu-
racy in the drug effectiveness. Chemical modifications 
of nucleotides can be placed at the phosphate backbone, 
ribose moiety and base. Phosphothioate (PS)-modified oli-
gonucleotides are hydrophobic and more stable with an 
increased half-life of oligonucleotides and enhance their 
affinity to specific proteins. The protein binding capacity 
of oligonucleotides is advantageous to penetrate into the


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cells, but excessive bindings could lead the in-vivo toxic-
ity with a consequence of eliminating of drug metabo-
lites from the body. siRNA joined with PS will not make 
any changes in-vivo biodistribution and can accumulate 
 * Anju Kaushal 
 
anjukaushal186@hotmail.com; anjukaushal186@gmail.com
1 
New Zealand Organization for Quality-Member, Auckland, 
New Zealand


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Safety and efficacy of a biodegradable implant releasing tenofovir alafenamide for vaginal protection in a macaque model


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J Antimicrob Chemother 2022; 77: 2964–2971 
https://doi.org/10.1093/jac/dkac252 Advance Access publication 1 August 2022                 
Safety and efficacy of a biodegradable implant releasing 
tenofovir alafenamide for vaginal protection in a macaque model
I. Massud1, A. Krovi2, K. Nishiura1, S. Ruone1, L. Li2, A. Holder1, J. Gary3†, P. Mills4‡, J. Mitchell1, G. Khalil1, Y. Pan1, 
E. Luecke2, G. Gatto2, W. Heneine1, J. G. Garcίa-Lerma1, L. Johnson2, A. van der Straten5 and C. Dobard
1*
1Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and 
Prevention, Atlanta, GA, USA; 2RTI International, Research Triangle Park, NC, USA; 3Infectious Diseases Pathology Branch, Division of High- 
Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infection Diseases, Centers for Disease Control and 
Prevention, Atlanta, GA, USA; 4Comparative Medicine Branch, Division of Scientific Resources, National Center for Emerging and Zoonotic 
Infection Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA; 5Center for AIDS Prevention Studies (CAPS), Department 
of Medicine, University of California San Francisco, San Francisco, CA and ASTRA Consulting, Kensington, CA, USA
*Corresponding author. E-mail: gok5@cdc.gov
†Present address: Neuropathology, StageBio, Frederick, MD, USA.
‡Present address: Department of Comparative Medicine, Tulane University, New Orleans, LA, USA.
Received 9 March 2022; accepted 24 June 2022
Objectives: To advance the initiative of ending the global epidemic, long-lasting HIV protection is needed 
through sustained release of antiretroviral drugs for months to years. We investigated in macaques the safety 
and efficacy of biodegradable polycaprolactone implants releasing tenofovir alafenamide for HIV pre-exposure 
prophylaxis (PrEP). 
Methods: Implants were administered subcutaneously in the arm using a contraceptive trocar. Efficacy against


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vaginal simian-HIV (SHIV) infection was investigated in six pigtailed macaques that received two tenofovir ala-
fenamide implants (0.35 mg/day), one in each arm, for a total release rate of tenofovir alafenamide at 0.7 mg/ 
day. Macaques were exposed to SHIV twice weekly for 6 weeks. Statistical analyses were used to compare out-
come with eight untreated controls. Histological assessments were performed on skin biopsies collected near 
implantation sites. 
Results: Median (range) tenofovir diphosphate level in PBMCs was 1519 (1068–1898) fmol/106 cells. All maca-
ques with tenofovir alafenamide implants were protected against vaginal SHIV infection. In contrast, 7/8 con-
trols were infected after a median of 4 SHIV exposures (P = 0.0047). Histological assessment of tissues near 
tenofovir alafenamide implant sites showed inflammation and necrosis in 5/6 animals, which were not evident 
by visual inspection. 
Conclusions: We demonstrated complete protection against vaginal SHIV infection with two implants releasing 
a total of 0.7 mg of tenofovir alafenamide per day. We also identified tenofovir diphosphate concentrations in 
PBMCs associated with complete vaginal protection. Consistent with previous findings, we observed adverse lo-
cal toxicity and necrosis near the tenofovir alafenamide implant site. Improved tenofovir alafenamide implants 
that are safe and maintain high efficacy have the potential to provide long-lasting protection against vaginal 
HIV infection.
Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy 2022. 
This work is written by (a) US Government employee(s) and is in the public domain in the US.
Introduction
The HIV epidemic remains one of the highest global public health 
priorities.1 Although new HIV infections have steadily declined 
since the peak in 1997, an estimated 1.5 million became newly 
infected in 2020.2 Daily oral pre-exposure prophylaxis (PrEP) 
with the combination of emtricitabine and tenofovir disoproxil


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fumarate or tenofovir alafenamide has been highly effective in 
preventing HIV acquisition when taken as prescribed.3,4
However, inadequate adherence to the daily oral regimen re-
duces effectiveness and public health impact. As efforts to scale 
up PrEP worldwide accelerate, it is a high priority to identify novel 
PrEP modalities that accommodate the different needs among 
users. To this end, the pipeline for HIV prevention options is 
2964


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Second-Generation Human Immunodeficiency Virus Integrase Inhibitors Induce Differentiation Dysregulation and Exert Toxic Effects in Human Embryonic Stem Cell and Mouse Models


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The Journal of Infectious Diseases                                
M A J O R  A R T I C L E
Second-Generation Human Immunodeficiency Virus 
Integrase Inhibitors Induce Differentiation Dysregulation 
and Exert Toxic Effects in Human Embryonic Stem Cell 
and Mouse Models
Marie-Soleil R. Smith,1,2 Haneesha Mohan,3 Abhinav Ajaykumar,1,2,a Anthony Y. Y. Hsieh,1,2 Lou Martineau,1 Ronil Patel,1 Izabella Gadawska,1,2
Christopher Sherwood,4 Lena Serghides,3,5,6, James M. Piret,4,7,8 and Hélène C. F. Côté1,2,9
1Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada; 2Centre for Blood Research, University of British Columbia, Vancouver, Canada; 3Toronto 
General Hospital Research Institute, University Health Network, Toronto, Canada; 4Michael Smith Laboratories, University of British Columbia, Vancouver, Canada; 5Department of Immunology and 
Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada; 6Women’s College Research Institute, Toronto, Canada; 7Department of Chemical and Biological Engineering, 
University of British Columbia, Vancouver, Canada; 8School of Biomedical Engineering, The University of British Columbia, Vancouver, Canada; and 9Women’s Health Research Institute, Vancouver, 
Canada
Background. Each year, approximately 1.1 million children are exposed in utero to human immunodeficiency virus 
antiretrovirals, yet their safety is often not well characterized during pregnancy. The Tsepamo study reported a neural tube 
defect signal in infants exposed to the integrase strand transfer inhibitor (InSTI) dolutegravir from conception, suggesting that 
exposure during early fetal development may be detrimental
Methods. The effects of InSTIs on 2 human embryonic stem cell (hESC) lines were characterized with respect to markers of 
pluripotency, early differentiation, and cellular health. In addition, fetal resorptions after exposure to InSTIs from conception 
were analyzed in pregnant mice.


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Results. At subtherapeutic concentrations, second-generation InSTIs bictegravir, cabotegravir, and dolutegravir decreased 
hESC counts and pluripotency and induced dysregulation of genes involved in early differentiation. At therapeutic 
concentrations, bictegravir induced substantial hESC death and fetal resorptions. It is notable that first-generation InSTI 
raltegravir did not induce any hESC toxicity or differentiation, at any concentration tested.
Conclusions. Exposure to some InSTIs, even at subtherapeutic concentrations, can induce adverse effects in hESCs and pregnant 
mice. Given the increasingly prevalent use of second-generation InSTIs, including in women of reproductive age, it is imperative to 
further elucidate the effect of InSTIs on embryonic development, as well as their long-term safety after in utero exposure.
Keywords. antiretroviral; hESC; HIV; integrase inhibitor; pregnancy.
Received 19 July 2022; editorial decision 14 September 2022; accepted 16 September 2022; 
published online 19 September 2022
aPresent affiliation: Scientist at AbCellera Biologics, Vancouver, BC, Canada.
Presented in part: 17th European AIDS Conference (EACS), November 2019. Basel, 
Switzerland; 27th Conference on Retroviruses and Opportunistic Infections (CROI), March 
2020, Boston (Virtual), MA, USA; 29th Annual Canadian Conference on HIV/AIDS Research 
(CAHR), May 2020, Quebec (Virutal), Canada; 28th CROI, February 2022, Denver (Virtual), CO, 
USA; 30th CAHR, April 2022, London (Virtual), Canada.
Correspondence: Hélène C. F. Côté, PhD, Department of Pathology and Laboratory Medicine, 
University of British Columbia, G227-2211 Wesbrook Mall, Vancouver, B.C. Canada, V6T 2B5 
(helene.cote@ubc.ca).
The Journal of Infectious Diseases®
2022;226:1992–2001 
© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases 
Society of America. All rights reserved. For permissions, please e-mail: journals.permissions 
@oup.com
https://doi.org/10.1093/infdis/jiac386


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More than half of the estimated 36 million adults with human 
immunodeficiency virus (HIV) are women [1]. With increasing 
access to combination antiretroviral therapy (cART), 85% of 
pregnant women with HIV worldwide now receive cART dur-
ing pregnancy. This has successfully reduced vertical transmis-
sion rates to <2% globally [1]. Although most antiretrovirals 
(ARVs) can cross the placenta [2], the short- and long-term 
safety of exposure in the context of conception and pregnancy 
have not been fully characterized. Given how common in utero 
ARV exposure is, this represents an important knowledge gap 
identified by many researchers and care providers [2–4].
Recommended first-line cART for pregnant women typically 
include a nucleoside reverse-transcriptase inhibitor backbone 
with a base drug consisting of either the nonnucleoside reverse- 
transcriptase inhibitor efavirenz (EFV), the protease inhibitors 
ritonavir-boosted darunavir (DRVr), or ritonavir-boosted ataza-
navir (ATVr), or one of the integrase strand transfer inhibitors 
(InSTIs) dolutegravir (DTG), or raltegravir (RAL) [5, 6]. Owing 
to their efficacy and generally favorable tolerability, InSTIs have 
gained popularity and mass use [5]. Both DTG and RAL effective-
ly suppress HIV viremia during pregnancy [7, 8], but the use of 
first-generation InSTI RAL is much less common than DTG, ow-
ing in part to its requirement for twice-daily administration and 
lack of a fixed-dose combination. Regarding other InSTIs, stan-
dard dosing of cobicistat-boosted elvitegravir (EVGc) during 
pregnancy results in lessened drug exposure due to reduced 
1992 • JID 2022:226 (1 December) • Smith et al


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A post-mortem analysis of tenofovir, lamivudine, efavirenz and fluconazole penetration in female genital tissues


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J Antimicrob Chemother 2022; 77: 3180–3186 
https://doi.org/10.1093/jac/dkac300 Advance Access publication 14 September 2022         
A post-mortem analysis of tenofovir, lamivudine, efavirenz and 
fluconazole penetration in female genital tissues
Fan Wang1, Olivie C. Namuju2, Katelyn A. Pastick
3,4, Kizito Abdusalaamu2, Usha Mishra1, Lindsey Collins1, 
David R. Boulware
3, Robert Lukande5, David B. Meya3,5 and Melanie R. Nicol
1*
1Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, MN, USA; 2Infectious Disease Institute, 
Kampala, Uganda; 3Department of Medicine, University of Minnesota, Minneapolis, MN, USA; 4Massachusetts General Hospital, Boston, 
MA, USA; 5Makerere University, Kampala, Uganda
*Corresponding author. E-mail: mrnicol@umn.edu
Received 11 April 2022; accepted 11 August 2022
Background: Optimal penetration of anti-infectives in the female genital tract (FGT) is paramount in the treat-
ment and prevention of infectious diseases. While exposure of anti-infectives in lower FGT tissues (e.g. cervix, 
vagina) has been described, little data exist on upper genital tissues (e.g. ovary, uterus). 
Methods: Autopsies were performed and post-mortem tissues were collected within 24 h of death for female 
participants with advanced HIV in Uganda (n = 27). Tenofovir, lamivudine, efavirenz and fluconazole concentra-
tions were measured using LC-MS/MS in plasma, ovarian, uterine, cervical and vaginal tissues. Tissue penetration 
was calculated as tissue-to-plasma concentration ratios (TPRs). 
Results: TPRs of tenofovir, lamivudine and fluconazole were highest in vaginal tissue (medians 1.86, 1.83 and 
0.94, respectively), while the TPR of efavirenz was highest in ovarian tissue (median 0.65). With cervix as a ref-
erence compartment, vaginal TPRs were significantly higher than cervical for all four drugs; TPRs of efavirenz in 
uterine and ovarian compartments were also significantly higher than cervical. Most of the post-mortem FGT


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samples had a TPR of greater than 1 for tenofovir and lamivudine, while less than 50% had a TPR of greater 
than 1 for both efavirenz and fluconazole. 
Conclusions: Penetration of anti-infectives was not homogeneous among the FGT compartments. 
Approximately 70% of FGT tissues had a TPR of greater than 1 for tenofovir and lamivudine, favouring the pre-
vention of local HIV replication and transmission in the FGT.
© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. 
For permissions, please e-mail: journals.permissions@oup.com
Introduction
The female genital tract (FGT) is a significant anatomical site of 
pathogen persistence and sexual transmission of infectious dis-
eases. Optimal drug exposure in the FGT is paramount for preven-
tion and treatment of FGT-involved infectious diseases, such as 
HIV and vaginal candidiasis. Although much of the focus on mu-
cosal HIV transmission in women has been the lower FGT, HIV in-
fection can occur throughout the entire FGT, as demonstrated in 
the rhesus macaque vaginal transmission model of simian im-
munodeficiency virus.1 Candidiasis, while more often defined as 
vulvovaginal compared with other FGT sites, can, in rare cases, 
occur in the upper FGT as well.2 Despite this, our knowledge of 
FGT tissue exposure to antiretrovirals and antifungals has pre-
dominantly been obtained through sampling of the lower FGT, 
mostly via cervicovaginal tissue and cervicovaginal fluid.3–9
Studies related to FGT drug exposure in upper tissue tracts (i.e. 
ovarian and uterine tissue) in non-pregnant women are scarce 
due to lack of easiness of sampling.
In order to fill the knowledge gap surrounding exposure of 
anti-infectives in solid tissues of the upper and lower FGT, we ex-
plored tissue penetration of two commonly prescribed NRTIs, te-
nofovir and lamivudine, and an NNRTI, efavirenz, in addition to an 
antifungal, fluconazole, in post-mortem FGT tissues (i.e. ovarian,


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uterine, cervical and vaginal) from a population of Ugandan wo-
men living with HIV/AIDs prior to the time of death.
Methods
Study participants and sample collection
All study participants were hospitalized patients living with HIV/AIDs who 
passed away at Mulago National Referral Hospital in Kampala, Uganda 
from 2017 to 2020. Written informed consent was obtained from the 
next of kin. ART history, including recent medication adherence, was ex-
tracted from medical charts and from interviews with caretakers. 
3180


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ARTICLE
OPEN
Inside-out assembly of viral antigens for the enhanced
vaccination
Fengqiang Cao1,2,3, Sha Peng1,2,3, Yaling An4,5, Kun Xu4,6, Tianyi Zheng4,7, Lianpan Dai
4, Kenji Ogino2,3✉, To Ngai
8,
Yufei Xia1,9,10✉ and Guanghui Ma
1,3,9,10✉
Current attempts in vaccine delivery systems concentrate on replicating the natural dissemination of live pathogens, but neglect
that pathogens evolve to evade the immune system rather than to provoke it. In the case of enveloped RNA viruses, it is the natural
dissemination of nucleocapsid protein (NP, core antigen) and surface antigen that delays NP exposure to immune surveillance.
Here, we report a multi-layered aluminum hydroxide-stabilized emulsion (MASE) to dictate the delivery sequence of the antigens. In
this manner, the receptor-binding domain (RBD, surface antigen) of the spike protein was trapped inside the nanocavity, while NP
was absorbed on the outside of the droplets, enabling the burst release of NP before RBD. Compared with the natural packaging
strategy, the inside-out strategy induced potent type I interferon-mediated innate immune responses and triggered an immune-
potentiated environment in advance, which subsequently boosted CD40+ DC activations and the engagement of the lymph nodes.
In both H1N1 inﬂuenza and SARS-CoV-2 vaccines, rMASE signiﬁcantly increased antigen-speciﬁc antibody secretion, memory T cell
engagement, and Th1-biased immune response, which diminished viral loads after lethal challenge. By simply reversing the
delivery sequence of the surface antigen and core antigen, the inside-out strategy may offer major implications for enhanced
vaccinations against the enveloped RNA virus.
Signal Transduction and Targeted Therapy  (2023) 8:189 
; https://doi.org/10.1038/s41392-023-01414-7
INTRODUCTION
A major challenge in vaccine design is stimulating the potency
and duration of the immune responses.1,2 The immune responses
to infection or vaccination are temporal sequences of events,
which depend on the ordered exposure of antigenic components


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to the immune system,3,4 as well as the coordinated actions of the
lymph
nodes,
immunocytes,
cytokines,
etc.5,6
Thus,
potent
vaccines are expected to harness spatial and temporal control
over sequential immune activation.7
To address
this,
nano- and micro-delivery
systems
with
controllable physicochemical properties and multi-level nanos-
tructures are engineered to deliver multiple vaccine compo-
nents.8,9 Additionally, since pathogens are the perfect vehicles of
natural selection, there is a trend to mimic their structures or
physiochemical properties.10,11 Increased lymph node accumula-
tion of antigen, antigen uptake, and antigen cross-presentation
have been witnessed in previous attempts to replicate live
pathogens’ sizes, shapes, charges, and softness.12,13 As for the
delivery kinetics, it is thought to replicate the natural dissemina-
tion of multiple antigenic components, which may dictate the
exposure sequence for subsequent immune activation in a
biomimetic manner.14
Nonetheless, pathogens usually evolve to escape the immune
system rather than to provoke it.15–17 In the case of enveloped
RNA viruses, genome replication results in the accumulation of
pathogen-associated molecular patterns, which can lead to a
strong host anti-viral response.18,19 To circumvent this, immuno-
genic components, such as viral genes and proteins (e.g.,
nucleocapsid
protein,
NP),
are
tightly
bound
and
hidden
inside.20,21 Subsequently, the embedded NP is delayed in its
exposure to immune surveillance, leading to suppressed type I
interferon (IFN-I) expression, as well as impeded anti-viral effects.22
Accordingly, the exact replicas of natural dissemination may not
be an optimal solution. As a preliminary test, we treated bone
marrow-derived dendritic cells (BMDCs) with the surface antigen
and NP of H1N1 inﬂuenza virus (A/Puerto Rico/8/1934)23 and
SARS-CoV-2 (hCoV-19/China/CAS-B001/2020),24 respectively. In
the presence of the surface antigen, higher doses of NP resulted


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in the up-regulated expression of IFN-α, suggesting a robust anti-
viral effect (Supplementary Fig. 1).
Received: 12 July 2022 Revised: 13 January 2023 Accepted: 8 March 2023
1State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; 2Graduate School of Bio-Applications
and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan; 3Institute of Global Innovation Research, Tokyo
University of Agriculture and Technology, 2-24-16 Nakacho, Koganei-shi, Tokyo 184-8588, Japan; 4CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of
Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China; 5Savaid Medical School, University of Chinese Academy of Sciences, Beijing, PR China; 6Key Laboratory of
Tropical Translational Medicine of Ministry of Education, School of Tropical Medicine and Laboratory Medicine, The First Afﬁliated Hospital, Hainan Medical University, Hainan
571199, PR China; 7Zhejiang University School of Medicine, Hangzhou 310058, PR China; 8Department of Chemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong
Kong 999077, PR China; 9University of Chinese Academy of Sciences, Beijing 100049, PR China and 10Innovation Academy for Green Manufacture Chinese Academy of Sciences,
Beijing 100190, PR China
Correspondence: Kenji Ogino (kogino@cc.tuat.ac.jp) or Yufei Xia (yfxia@ipe.ac.cn) or Guanghui Ma (ghma@ipe.ac.cn)
These authors contributed equally: Fengqiang Cao, Sha Peng, Yaling An
www.nature.com/sigtrans
Signal Transduction and Targeted Therapy
© The Author(s) 2023
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Current Neuropharmacology
	

	

	





Send Orders for Reprints to reprints@benthamscience.net 
560
 
Current Neuropharmacology, 2023, 21, 560-577 
 
REVIEW ARTICLE 
Therapeutic Approaches to Non-Motor Symptoms of Parkinson's Disease: 
A Current Update on Preclinical Evidence 
 
Poornima D.E. Weerasinghe-Mudiyanselage1,#, Sohi Kang1,#, Joong-Sun Kim1 and Changjong Moon1,* 
1Department of Veterinary Anatomy and Animal Behavior, College of Veterinary Medicine and BK21 FOUR Program, 
Chonnam National University, Gwangju 61186, South Korea 
 
Abstract: Despite being classified as a movement disorder, Parkinson’s disease (PD) is characterized 
by a wide range of non-motor symptoms that significantly affect the patients' quality of life. However, 
clear evidence-based therapy recommendations for non-motor symptoms of PD are uncommon. Ani-
mal models of PD have previously been shown to be useful for advancing the knowledge and treat-
ment of motor symptoms. However, these models may provide insight into and assess therapies for 
non-motor symptoms in PD. This paper highlights non-motor symptoms in preclinical models of PD 
and the current position regarding preclinical therapeutic approaches for these non-motor symptoms. 
This information may be relevant for designing future preclinical investigations of therapies for non-
motor symptoms in PD. 
 
A R T I C L E  H I S T O R Y 
 
 
Received: June 19, 2022 
Revised: August 30, 2022 
Accepted: September 02, 2022 
 
DOI:  
10.2174/1570159X20666221005090126 
 
 
Keywords: Non-motor symptoms, Parkinson’s disease, patients' quality of life, preclinical models, therapeutics, rapid eye move-
ment (REM). 
1. INTRODUCTION 
 
Parkinson’s disease (PD) is one of the most prevalent 
neurodegenerative disorders owing to the main impairment 
of the nigrostriatal system with dopamine (DA) deficiency, 
which is characterized by a combination of motor and non-
motor symptoms [1, 2]. Furthermore, PD is a chronic, pro-


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gressive disease that requires continued treatment to prevent 
the deterioration of quality of life [3]. Although various clin-
ical trials have been conducted for the most current sympto-
matic therapies [4, 5], therapies for PD are still not complete-
ly satisfactory. Moreover, continuous preclinical investiga-
tions are essential to clarify the efficacy of existing therapies 
in different animal models that mimic specific pathological 
aspects of PD.  
 
In patients, PD is defined not only by motor symptoms 
but also by a complex range of non-motor symptoms present 
throughout disease progression [6]. The prodromal period of 
PD is characterized by a variety of non-motor symptoms, 
such as hyposmia, rapid eye movement (REM), constipation, 
fatigue, erectile dysfunction, pain, depression, and cognitive 
impairment [6, 7]. Alternatively, these and other non-motor 
symptoms may arise later in the disease course [6, 8]. For 
example, cognitive deficits may become more severe as the 
disease progresses [8]. Mild cognitive impairment is an 
 
*Address correspondence to this author at the Department of Veterinary 
Anatomy and Animal Behavior, College of Veterinary Medicine and BK21 
FOUR Program, Chonnam National University, 77 Yongbong-ro, Buk-gu, 
Gwangju 61186, South Korea, Tel: +82-62-530-2838;  
E-mail: moonc@chonnam.ac.kr  
#These first two authors contributed equally to this work. 
intermediate stage on the gradient between normal cognitive 
function and dementia and is linked with an increased risk of 
dementia [9]. Thus, mild cognitive impairment has been 
suggested as an important predictor of the progression to PD 
dementia [10, 11]. Some non-motor symptoms remain undis-
closed because patients are embarrassed or unaware that the 
symptoms are linked to PD [12]. Consequently, the non-
motor symptoms of PD are poorly recognized and treated 
[6]. However, non-motor symptoms can be equally debilitat-
ing in patients [13, 14], and the majority of non-motor symp-


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toms do not respond to treatments targeting motor symptoms 
[15, 16].  
 
Non-motor symptoms of PD most likely involve different 
mechanisms, such as DAergic, non-DAergic, genetic, or 
drug-induced pathways [6]. For instance, DAergic and nora-
drenergic denervation of the limbic system are noted in pa-
tients with PD and depression [17, 18]. The appearance of 
non-motor symptoms in PD patients with Parkin [19], SNCA 
[20], LRRK2 [21] or GBA [22] mutations implies a genetic 
basis for non-motor symptoms. Long-term use of DA re-
placement therapies can lead to non-motor side effects, such 
as diarrhea, nausea, hallucination, blurred vision, and hypo-
tension [2, 23-26]. The heterogeneous nature and differential 
responses to DAergic and non-DAergic treatments make the 
management of non-motor symptoms challenging in clinical 
practice. Some non-motor symptoms, such as apathy, anxie-
ty, and primary pain, are responsive to DA replacement ther-
apy [2]. However, PD-associated dementia is responsive to 
non-DAergic therapies such as cholinesterase inhibitors and 
N-methyl-d-aspartate receptor (NMDAR) antagonists [27].  
1875-6190/23 $65.00+.00
© 2023 Bentham Science Publishers


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Oligodendrocyte death initiates synchronous remyelination to restore cortical myelin patterns in mice


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Oligodendrocyte death initiates synchronous remyelination to 
restore cortical myelin patterns in mice
Timothy W. Chapman1, Genaro E. Olveda1, Xhoela Bame1, Elizabeth Pereira1, Robert A. 
Hill1,#
1Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA
Abstract
Myelin degeneration occurs in neurodegenerative diseases and aging. In these conditions, 
resident oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes that carry 
out myelin repair. To investigate the cellular dynamics underlying these events, we developed a 
noninflammatory demyelination model that combines intravital two-photon imaging with a single-
cell ablation technique called two-photon apoptotic targeted ablation (2Phatal). Oligodendrocyte 
2Phatal in both sexes results in a myelin degeneration cascade that triggers rapid forms of 
synchronous remyelination on defined axons. This remyelination is driven by oligodendrocytes 
differentiated from a subset of morphologically distinct, highly branched OPCs. Moreover, 
remyelination efficiency depends on the initial myelin patterns, as well as the age of the organism. 
In summary, using 2Phatal, we show a form of rapid synchronous remyelination, mediated by a 
distinct subset of OPCs, capable of restoring the original myelin patterning in adulthood but not 
aging.
INTRODUCTION
Myelination increases neural circuit processing speed and efficiency1,2. Many 
neurodegenerative conditions result from myelin dysfunction and myelin is one of the 
earliest structures to degenerate in aging3-5. The cellular mechanisms that allow for the 
continued refinement and myelin plasticity observed in adulthood6-8 are thought to be 
co-opted for myelin repair. Yet, it is not fully known how these cycles of degeneration and 
repair occur at the cellular level, in vivo.
Remyelination is primarily carried out by resident, self-renewing oligodendrocyte precursor 
cells (OPCs), which can differentiate into oligodendrocytes throughout life9,10. OPCs are


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dynamic, engaging in tissue surveillance, response to damage, and remyelination11-14. 
There is some evidence that functional heterogeneity within the OPC population exists15-20. 
#corresponding author: robert.hill@dartmouth.edu.
AUTHOR CONTRIBUTIONS
T.W.C and R.A.H conceived of, designed, and performed all experiments and most of the data analysis and quantification. 
G.E.O. performed OPC morphological and migration analyses and contributed to quantification associated with sheath and node 
remyelination. Xh.B. contributed to data analysis and quantification. E.P. contributed to OPC fate analysis and imaging data collation. 
T.W.C and R.A.H. wrote the paper and R.A.H. directed the study.
COMPETING INTERESTS
The authors declare no competing interests.
HHS Public Access
Author manuscript
Nat Neurosci. Author manuscript; available in PMC 2023 October 01.
Published in final edited form as:
Nat Neurosci. 2023 April ; 26(4): 555–569. doi:10.1038/s41593-023-01271-1.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript


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Frequency dependent emotion differentiation and directional coupling in amygdala, orbitofrontal and medial prefrontal cortex network with intracranial recordings


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ARTICLE
OPEN
Frequency dependent emotion differentiation and directional
coupling in amygdala, orbitofrontal and medial prefrontal
cortex network with intracranial recordings
Saurabh Sonkusare
1,2,3, Ding Qiong1,4, Yijie Zhao
3,4, Wei Liu1, Ruoqi Yang1, Alekhya Mandali
2,5,6, Luis Manssuer1,2,3,
Chencheng Zhang
1, Chunyan Cao1, Bomin Sun
1, Shikun Zhan
1✉ and Valerie Voon
2,3✉
© The Author(s) 2022
The amygdala, orbitofrontal cortex (OFC) and medial prefrontal cortex (mPFC) form a crucial part of the emotion circuit, yet their
emotion induced responses and interactions have been poorly investigated with direct intracranial recordings. Such high-ﬁdelity
signals can uncover precise spectral dynamics and frequency differences in valence processing allowing novel insights on
neuromodulation. Here, leveraging the unique spatio-temporal advantages of intracranial electroencephalography (iEEG) from a
cohort of 35 patients with intractable epilepsy (with 71 contacts in amygdala, 31 in OFC and 43 in mPFC), we assessed the spectral
dynamics and interactions between the amygdala, OFC and mPFC during an emotional picture viewing task. Task induced activity
showed greater broadband gamma activity in the negative condition compared to positive condition in all the three regions.
Similarly, beta activity was increased in the negative condition in the amygdala and OFC while decreased in mPFC. Furthermore,
beta activity of amygdala showed signiﬁcant negative association with valence ratings. Critically, model-based computational
analyses revealed unidirectional connectivity from mPFC to the amygdala and bidirectional communication between OFC-
amygdala and OFC-mPFC. Our ﬁndings provide direct neurophysiological evidence for a much-posited model of top-down
inﬂuence of mPFC over amygdala and a bidirectional inﬂuence between OFC and the amygdala. Altogether, in a relatively large
sample size with human intracranial neuronal recordings, we highlight valence-dependent spectral dynamics and dyadic coupling


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within the amygdala-mPFC-OFC network with implications for potential targeted neuromodulation in emotion processing.
Molecular Psychiatry (2023) 28:1636–1646; https://doi.org/10.1038/s41380-022-01883-2
INTRODUCTION
The amygdala, orbitofrontal cortex (OFC) and medial prefrontal
cortex (mPFC) are critical to emotional processing and regulation
[1]. Human neuroimaging studies with functional magnetic
resonance imaging (fMRI) provide substantial evidence for their
involvement in emotion perception and evaluation [2, 3]. How-
ever, fMRI is dependent on the sluggish haemodynamic response
(>3 s) and which remains an indirect measure of neuronal activity
[4]. In contrast, intracranial electroencephalography (iEEG) records
direct neuronal
population
level
activity
at
high
temporal
resolution enabling insights into precise spectral dynamics. Thus,
iEEG can potentially uncover speciﬁc frequency and timing
differences underlying various emotions which may inform
stimulation and targets for neuromodulation. Although a few
studies have investigated emotional processing in the amygdala
using iEEG, they have focussed predominantly on event related
potentials (ERPs) [5, 6] with limited reporting of precise time-
resolved spectral dynamics [7, 8]. Furthermore, only one iEEG
study has examined OFC and mPFC activity in emotion processing
[9] to the best of our knowledge and crucially none have
investigated the interactions amongst this key emotion circuit
with direct recordings. Consequently, direct evidence of valence-
dependent coupling between the amygdala, the OFC, and mPFC
remains poorly investigated in humans despite it being strongly
implicated in affect and psychopathology [10].
The prefrontal cortex is believed to play a role in top-down
modulation of amygdala activity providing contextualisation and
evaluation of stimuli while the amygdala drives bottom-up
salience processing [11]. However, the prefrontal cortex is a
relatively large heterogenous structure with various distinct


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anatomical and functional divisions including the OFC and mPFC
[12, 13]. Structurally, the amygdala is highly interconnected with
both the OFC [14] and mPFC [15]. Tract tracing studies from non-
human primate brains reveal afferent ﬁbres from the OFC and
mPFC terminating in the amygdala [15]. In turn, efferent ﬁbres
from the amygdala project to the OFC and mPFC [16]. Such
structural connectivity constrains functional interactions between
brain regions for fast and efﬁcient transfer of information [17]
highlighting the integrated coupling within this amygdala-OFC-
mPFC network.
Received: 8 March 2022 Revised: 4 November 2022 Accepted: 10 November 2022
Published online: 2 December 2022
1Department of Neurosurgery, Centre for Functional Neurosurgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 2Department of
Psychiatry, University of Cambridge, Cambridge, UK. 3Institute of Science and Technology for Brain-Inspired Intelligence, Fudan University, Shanghai, China. 4Key Laboratory of
Computational Neuroscience and Brain-Inspired Intelligence, Fudan University, Ministry of Education, Shanghai, China. 5Nufﬁeld Department of Clinical Neurosciences, University
of Oxford, Oxford, UK. 6MRC Brain Network Dynamics Unit, University of Oxford, Oxford, UK. ✉email: shikun_zhan@hotmail.com; voonval@gmail.com
www.nature.com/mp
Molecular Psychiatry
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ARTICLE
OPEN
Acute psilocybin enhances cognitive ﬂexibility in rats
Alejandro Torrado Pacheco
1✉, Randall J. Olson1, Gabriela Garza
1,2 and Bita Moghaddam1✉
© The Author(s) 2023
Psilocybin has been shown to improve symptoms of depression and anxiety when combined with psychotherapy or other clinician-
guided interventions. To understand the neural basis for this pattern of clinical efﬁcacy, experimental and conceptual approaches
that are different than traditional laboratory models of anxiety and depression are needed. A potential novel mechanism is that
acute psilocybin improves cognitive ﬂexibility, which then enhances the impact of clinician-assisted interventions. Consistent with
this idea, we ﬁnd that acute psilocybin robustly improves cognitive ﬂexibility in male and female rats using a task where animals
switched between previously learned strategies in response to uncued changes in the environment. Psilocybin did not inﬂuence
Pavlovian reversal learning, suggesting that its cognitive effects are selective to enhanced switching between previously learned
behavioral strategies. The serotonin (5HT) 2 A receptor antagonist ketanserin blocked psilocybin’s effect on set-shifting, while a
5HT2C-selective antagonist did not. Ketanserin alone also improved set-shifting performance, suggesting a complex relationship
between psilocybin’s pharmacology and its impact on ﬂexibility. Further, the psychedelic drug 2,5-Dimethoxy-4-iodoamphetamine
(DOI) impaired cognitive ﬂexibility in the same task, suggesting that this effect of psilocybin does not generalize to all other
serotonergic psychedelics. We conclude that the acute impact of psilocybin on cognitive ﬂexibility provides a useful behavioral
model to investigate its neuronal effects relevant to its positive clinical outcome.
Neuropsychopharmacology (2023) 48:1011–1020; https://doi.org/10.1038/s41386-023-01545-z
INTRODUCTION
Recent clinical trials indicate that psilocybin is an effective
treatment for symptoms of several psychiatric disorders, including


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anxiety and depression [1–5]. A unique aspect of these studies is
that sustained therapeutic efﬁcacy is observed when one or a few
doses of psilocybin are administered during clinician-guided
facilitation or intervention [6–11]. To date, clinical data showing
that psilocybin has therapeutic efﬁcacy as a stand-alone treatment
is lacking, suggesting that its effects may be, at least in part, due
to the combination of therapeutic intervention and exposure to
acute psilocybin. Accordingly, multiple ongoing clinical trials are
assessing the efﬁcacy of joining psychotherapy or cognitive
behavioral therapy with psilocybin treatment (e.g., NCT05227612).
While the pharmacology of psilocybin is relatively well character-
ized, how this translates to its clinical effects remains unclear.
What are the mechanisms that underlie psilocybin’s clinical
efﬁcacy when combined with psychotherapy or other clinician-
guided interventions? Though several ideas have been proposed
[12], we focused on the hypothesis that acutely psilocybin creates
a cognitive state that improves therapeutic intervention by
relaxing prior beliefs, promoting ﬂexible thinking, and increasing
cognitive ﬂexibility [1, 13, 14].
Cognitive ﬂexibility is the ability to switch between mental
processes in order to appropriately adapt behavior to changes in
the environment. Impairments in cognitive ﬂexibility are a
prominent feature of many psychiatric disorders, including major
depression (MDD) and substance use disorders [15–19], which
have shown a positive response to psilocybin-assisted treatment.
Thus, the capacity to improve cognitive ﬂexibility in order to
facilitate breaking out of rigid behavioral patterns and beliefs is a
plausible mechanism by which psilocybin may exert its therapeu-
tic beneﬁts [1, 14, 20].
Preclinical studies are critical for advancing our mechanistic
understanding of the therapeutic efﬁcacy of psilocybin. There is a
growing appreciation that traditional behavioral tests used to


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study antidepressant effects do not reliably translate to effects in
human populations [21]. While several traditional preclinical
approaches to study depression-like behaviors in rodents have
been used in conjunction with psilocybin [22, 23], little is known
about the acute effect of this drug on cognitive ﬂexibility and
related constructs. Other work has examined the acute effects of
psychedelics on cognitive ﬂexibility, but none of these studies
used psilocybin [24, 25]. We hypothesize that the acute effects of
psilocybin may be inducing a state of increased cognitive
ﬂexibility
that
enhances
the
therapeutic
response
during
clinician-assisted sessions.
To assess cognitive ﬂexibility, we used a set-shifting task, which
tests an animal’s ability to modify behavior, among competing
and previously learned strategies, in response to changes in
environmental contingencies. We ﬁnd that acute psilocybin
treatment improves set-shifting behavior without inﬂuencing
appetitive or aversive learning. Psilocybin’s effect on cognitive
ﬂexibility was diminished by a 5HT2A but not by a 5HT2C
serotonin receptor antagonist. Consistent with other studies
showing impaired cognitive ﬂexibility with psychedelics other
than
psilocybin
[24,
25],
we
ﬁnd
that
2,5-Dimethoxy-4-
Received: 20 October 2022 Revised: 27 January 2023 Accepted: 30 January 2023
Published online: 20 February 2023
1Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, OR 97239, USA. 2Present address: Department of Biomedical Engineering, University of
Michigan, Ann Arbor, MI 48109, USA. ✉email: pachecoa@ohsu.edu; bita@ohsu.edu
www.nature.com/npp
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Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict the Liver-Tropism of Adeno-Associated Virus Vectors


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RESEARCH ARTICLES
Open camera or QR reader and
scan code to access this article
and other resources online.
Assessment of Pre-Clinical Liver Models Based on Their Ability
to Predict the Liver-Tropism of Adeno-Associated Virus Vectors
Adrian Westhaus,1,2,{ Marti Cabanes-Creus,1,{ Kimberley L. Dilworth,1 Erhua Zhu,3 David Salas Go´ mez,4
Renina G. Navarro,1 Anais K. Amaya,3 Suzanne Scott,1,3,5 Magdalena Kwiatek,6 Alexandra L. McCorkindale,7
Tara E. Hayman,7 Silke Frahm,8 Dany P. Perocheau,9–11 Bang Manh Tran,12 Elizabeth Vincan,12–14
Sharon L. Wong,15,16 Shafagh A. Waters,15–17 Georgina E. Riddiough,12,18 Marcos V. Perini,18
Laurence O.W. Wilson,5,19 Julien Baruteau,9–11 Sebastian Diecke,8 Gloria Gonza´lez-Aseguinolaza,4
Giorgia Santilli,2 Adrian J. Thrasher,2 Ian E. Alexander,3,20 and Leszek Lisowski1,21,22,*
1Translational Vectorology Research Unit, Children’s Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, Australia; 2Great
Ormond Street Institute of Child Health, University College London, London, United Kingdom; 3Gene Therapy Research Unit, Children’s Medical Research Institute and
Sydney Children’s Hospitals Network, Faculty of Medicine and Health, The University of Sydney, Westmead, Australia; 4Gene Therapy and Regulation of Gene Expression
Department, IdiSNA,InstitutodeInvestigacio´n SanitariadeNavarra,Universidad deNavarra, CIMA, Pamplona,Spain; 5Australian e-HealthResearch Centre,Commonwealth
Scientiﬁc and Industrial Research Organisation, Sydney, Australia; 6Military Institute of Hygiene and Epidemiology, The Biological Threats Identiﬁcation and Countermeasure
Centre,Puławy,Poland; 7Inventia Life SciencePty Ltd.,Sydney,Australia; 8StemCellTechnology Platform,Max Delbru¨ckCentrumforMolecular Medicine, Berlin,Germany;
9Genetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College London, London, United Kingdom; 10Metabolic Medicine


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Department, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom; 11National Institute of Health Research Great Ormond Street
Hospital Biomedical Research Centre, London, United Kingdom; 12Department of Infectious Diseases, Melbourne Medical School, The University of Melbourne at the Peter
Doherty Institute for Infection and Immunity, Melbourne, Australia; 13Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital at the Peter Doherty
Institute for Infection and Immunity, Melbourne, Australia; 14Curtin Medical School, Curtin University, Perth, Australia; 15Molecular and Integrative Cystic Fibrosis (miCF)
Research Centre, University of New South Wales and Sydney Children’s Hospital, Sydney, Australia; 16School of Biomedical Sciences, Faculty of Medicine, University of
New South Wales, Sydney, Australia; 17Department of Respiratory Medicine, Sydney Children’s Hospital, Faculty of Medicine, University of New South Wales, Sydney,
Australia; 18Department of Surgery, Austin Health Precinct, The University of Melbourne, Austin Health, Heidelberg, Australia; 19Applied BioSciences, Faculty of Science and
Engineering, Macquarie University, Sydney, Australia; 20Discipline of Child and Adolescent Health, Faculty of Medicine and Health, The University of Sydney, Sydney,
Australia; 21Vector and Genome Engineering Facility, Children’s Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, Australia;
22Laboratory of Molecular Oncology and Innovative Therapies, Military Institute of Medicine, Warszawa, Poland.
{These authors’ contributed equally to this work.
An earlier draft of this article was posted as a preprint at bioRxiv (doi: content/10.1101/2022.09.28.510021v1).
The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors. Multiple clinical trials
have been undertaken for this target in the past 15 years; however, we are still to see market approval of the ﬁrst liver-targeted


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adeno-associated virus (AAV)–based gene therapy. Inefﬁcient expression of the therapeutic transgene, vector-induced liver
toxicity andcapsid, and/or transgene-mediated immune responsesreportedathigh vectordosesare the main challengestodate.
One of the contributing factors to the insufﬁcient clinical outcomes, despite highly encouraging preclinical data, is the lack of
robust, biologically and clinically predictive preclinical models. To this end, this study reports ﬁndings of a functional
evaluation of6 AAV vectors in 12 preclinical models of the human liver, withthe aim to uncover which combinationof models
is the most relevant for the identiﬁcation of AAV capsid variant for safe and efﬁcient transgene delivery to primary human
hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepa-
tocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses
of each system. This should allow the development of novel gene therapies targeting the human liver.
*Correspondence: Dr. Leszek Lisowski, Vector and Genome Engineering Facility, Children’s Medical Research Institute, Faculty of Medicine and Health, The University of
Sydney, 214 Hawkesbury Road, Westmead, NSW 2145, Australia. E-mail: llisowski@cmri.org.au
ª Adrian Westhaus et al. 2023; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License [CC-BY]
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In the April 2023 issue of Human Gene Therapy (vol. 34, no. 7–8; 273–288), the article entitled ‘‘Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict
the Liver-Tropism of Adeno-Associated Virus Vectors’’ has been updated on May 2, 2023 after ﬁrst online publication of April 17,2023 to reﬂect Open Access, with copyright


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transferring to the author(s), and a Creative Commons License (CC-BY) added (http://creativecommons.org/licenses/by/4.0).
HUMAN GENE THERAPY, VOLUME 34, NUMBERS 7 and 8
DOI: 10.1089/hum.2022.188 j 273
Mary Ann Liebert, Inc.


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Mapping the primate thalamus: systematic approach to analyze the distribution of subcortical neuromodulatory afferents


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Brain Structure and Function (2023) 228:1153–1176 
https://doi.org/10.1007/s00429-023-02619-w
METHODOLOGY
Mapping the primate thalamus: systematic approach to analyze 
the distribution of subcortical neuromodulatory afferents
Isabel Pérez‑Santos1,2  · Miguel Ángel García‑Cabezas1,2,3  · Carmen Cavada1,2 
Received: 12 September 2022 / Accepted: 9 February 2023 / Published online: 8 March 2023 
© The Author(s) 2023
Abstract
Neuromodulatory afferents to thalamic nuclei are key for information transmission and thus play critical roles in sensory, 
motor, and limbic processes. Over the course of the last decades, diverse attempts have been made to map and describe 
subcortical neuromodulatory afferents to the primate thalamus, including axons using acetylcholine, serotonin, dopamine, 
noradrenaline, adrenaline, and histamine. Our group has been actively involved in this endeavor. The published descriptions 
on neuromodulatory afferents to the primate thalamus have been made in different laboratories and are not fully comparable 
due to methodological divergences (for example, fixation procedures, planes of cutting, techniques used to detect the afferents, 
different criteria for identification of thalamic nuclei…). Such variation affects the results obtained. Therefore, systematic 
methodological and analytical approaches are much needed. The present article proposes reproducible methodological and 
terminological frameworks for primate thalamic mapping. We suggest the use of standard stereotaxic planes to produce 
and present maps of the primate thalamus, as well as the use of the Anglo-American school terminology (vs. the German 
school terminology) for identification of thalamic nuclei. Finally, a public repository of the data collected under agreed-on 
frameworks would be a useful tool for looking up and comparing data on the structure and connections of primate thalamic 
nuclei. Important and agreed-on efforts are required to create, manage, and fund a unified and homogeneous resource of


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data on the primate thalamus. Likewise, a firm commitment of the institutions to preserve experimental brain material is 
much needed because neuroscience work with non-human primates is becoming increasingly rare, making earlier material 
still more valuable.
Keywords Thalamus · Thalamic nuclei · Acetylcholine · Dopamine · Adrenaline · Noradrenaline · Serotonin · Histamine
Introduction
The thalamus is a nuclear complex in the diencephalon, 
whose neurons participate in sensory, motor, and limbic 
functions. These functions are supported by bidirectional 
connections with the cerebral cortex and a variety of subcor-
tical afferents from sensory pathways, globus pallidus, cer-
ebellum, amygdala, and the hypothalamus. The information 
carried by the subcortical afferents, as well as from cortical 
layers V and VI, is processed, modulated, integrated, and 
transmitted to the cerebral cortex by thalamocortical projec-
tion neurons (Jones 2007).
The thalamus receives cortical afferents, which are 
glutamatergic, and subcortical afferents, which are either 
glutamatergic (for example, from the cerebellum or the 
somatosensory gracile and cuneate nuclei), GABAergic 
(for example, from the internal globus pallidus or the sub-
stantia nigra reticulata), as well as cholinergic, adrenergic, 
noradrenergic, dopaminergic, serotoninergic, histaminergic, 
and peptidergic. This review focuses on the cholinergic and 
aminergic afferents, which are functionally modulatory; 
they shall be referred to as “neuromodulatory afferents” 
throughout the text, to distinguish them from glutamatergic 
Isabel Pérez-Santos and Miguel Ángel García-Cabezas have equally 
contributed.
 * Carmen Cavada 
 
carmen.cavada@uam.es
1 
Department of Anatomy, Histology and Neuroscience, 
School of Medicine, Facultad de Medicina, Universidad 
Autónoma de Madrid, Calle Arzobispo Morcillo 4, 
28029 Madrid, Spain
2 
PhD Program in Neuroscience, Universidad Autónoma de 
Madrid-Cajal, Madrid, Spain
3 
Neural Systems Laboratory, Department of Health Sciences,


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Emotion, motivation, decision-making, the orbitofrontal cortex, anterior cingulate cortex, and the amygdala


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Brain Structure and Function (2023) 228:1201–1257 
https://doi.org/10.1007/s00429-023-02644-9
REVIEW
Emotion, motivation, decision‑making, the orbitofrontal cortex, 
anterior cingulate cortex, and the amygdala
Edmund T. Rolls1,2 
Received: 27 January 2023 / Accepted: 12 April 2023 / Published online: 13 May 2023 
© Springer-Verlag GmbH Germany, part of Springer Nature 2023
Abstract
The orbitofrontal cortex and amygdala are involved in emotion and in motivation, but the relationship between these functions 
performed by these brain structures is not clear. To address this, a unified theory of emotion and motivation is described in 
which motivational states are states in which instrumental goal-directed actions are performed to obtain rewards or avoid 
punishers, and emotional states are states that are elicited when the reward or punisher is or is not received. This greatly 
simplifies our understanding of emotion and motivation, for the same set of genes and associated brain systems can define 
the primary or unlearned rewards and punishers such as sweet taste or pain. Recent evidence on the connectivity of human 
brain systems involved in emotion and motivation indicates that the orbitofrontal cortex is involved in reward value and 
experienced emotion with outputs to cortical regions including those involved in language, and is a key brain region involved 
in depression and the associated changes in motivation. The amygdala has weak effective connectivity back to the cortex in 
humans, and is implicated in brainstem-mediated responses to stimuli such as freezing and autonomic activity, rather than in 
declarative emotion. The anterior cingulate cortex is involved in learning actions to obtain rewards, and with the orbitofrontal 
cortex and ventromedial prefrontal cortex in providing the goals for navigation and in reward-related effects on memory 
consolidation mediated partly via the cholinergic system.


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Keywords Emotion · Motivation · Reward · Human orbitofrontal cortex · Cingulate cortex · Amygdala · Ventromedial 
prefrontal cortex · Memory · Depression · Consciousness · Mind-brain problem · Welfare
Introduction and aims
There have been considerable advances recently in under-
standing the connectivity and connections of the human 
orbitofrontal cortex and amygdala, and how they relate 
to emotion (Rolls et al. 2023a, d), but how these systems 
and processes are related to motivation has been much less 
explored. This paper shows how the brain systems involved 
in motivation are similar to those involved in emotion, and 
provides a framework for understanding how emotion and 
motivation are related to each other, and how similar brain 
systems are involved in both. This paper aims to make key 
advances in our understanding of how the orbitofrontal 
cortex and amygdala structure (anatomy and connectivity) 
is related to the two key functions performed by these brain 
regions, emotion and motivation.
To understand the neuroscience of both emotion and 
motivation, it is important to have a framework for under-
standing the relation between emotion and motivation. This 
paper first sets out a theory of emotion, and a framework for 
understanding the relation between emotion and motivation, 
and then considers how brain regions involved in emotion 
and motivation, the orbitofrontal cortex, anterior cingulate 
cortex, and amygdala, are involved in emotion and moti-
vation. Special reference is made to these brain regions in 
primates including humans, to ensure that what is described 
in relevant to understanding brain systems involved in emo-
tion and motivation in humans, and their disorders. Recent 
evidence about the connectivity of these systems in humans 
makes this paper very timely (Rolls et al. 2023a, d). A sec-
ond aim is to show how emotion and its brain systems are 
highly adaptive from an evolutionary and gene specification 
perspective. The third aim is to consider where and how 
 * Edmund T. Rolls


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Edmund.Rolls@oxcns.org
 
https://www.oxcns.org
1 
Oxford Centre for Computational Neuroscience, Oxford, UK
2 
Department of Computer Science, University of Warwick, 
Coventry, UK


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Brain Structure and Function (2023) 228:1259–1281 
https://doi.org/10.1007/s00429-023-02646-7
ORIGINAL ARTICLE
Changes in brain rhythms and connectivity tracking fear acquisition 
and reversal
Gabriele Pirazzini1 · Francesca Starita2 · Giulia Ricci1 · Sara Garofalo2 · Giuseppe di Pellegrino2 · Elisa Magosso1 · 
Mauro Ursino1
Received: 21 November 2022 / Accepted: 17 April 2023 / Published online: 2 May 2023 
© The Author(s) 2023
Abstract
Fear conditioning is used to investigate the neural bases of threat and anxiety, and to understand their flexible modifications 
when the environment changes. This study aims to examine the temporal evolution of brain rhythms using electroencephalo-
graphic signals recorded in healthy volunteers during a protocol of Pavlovian fear conditioning and reversal. Power changes 
and Granger connectivity in theta, alpha, and gamma bands are investigated from neuroelectrical activity reconstructed 
on the cortex. Results show a significant increase in theta power in the left (contralateral to electrical shock) portion of 
the midcingulate cortex during fear acquisition, and a significant decrease in alpha power in a broad network over the left 
posterior-frontal and parietal cortex. These changes occur since the initial trials for theta power, but require more trials (3/4) 
to develop for alpha, and are also present during reversal, despite being less pronounced. In both bands, relevant changes in 
connectivity are mainly evident in the last block of reversal, just when power differences attenuate. No significant changes in 
the gamma band were detected. We conclude that the increased theta rhythm in the cingulate cortex subserves fear acquisi-
tion and is transmitted to other cortical regions via increased functional connectivity allowing a fast theta synchronization, 
whereas the decrease in alpha power can represent a partial activation of motor and somatosensory areas contralateral to


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the shock side in the presence of a dangerous stimulus. In addition, connectivity changes at the end of reversal may reflect 
long-term alterations in synapses necessary to reverse the previously acquired contingencies.
Keywords Fear conditioning · Reversal · Granger connectivity · Theta rhythm · Alpha rhythm
Introduction
Fear conditioning is a paradigm used in neuroscience to 
study the neurobiological bases of threat and anxiety (Duits 
et al. 2015; Starita et al. 2016, 2019a). In this experimental 
protocol, a neutral stimulus (conditioned stimulus, CS +) is 
paired with an aversive stimulus (unconditioned stimulus, 
US) to trigger neural adjustments at the basis of fear acqui-
sition, finally resulting in the expression of a fear response 
in the presence of the CS + alone. In some experiments, a 
second conditioned stimulus (CS-) is unpaired with the US 
to serve as a control condition.
Results in rodents, primates, and humans provide a 
precise scenario showing the amygdala, hippocampus, 
and medial prefrontal cortex (mPFC) involvement in fear 
acquisition (Bechara et al. 1995, 1999; LaBar et al. 1995, 
1998; Knight et al. 2005; Cheng et al. 2006; Fullana et al. 
2016; Bertini et al. 2020; Battaglia et al. 2020). Further-
more, brain oscillations in the theta range (4–8 Hz) play a 
pivotal role in this process (Sperl et al. 2019). In rodents 
enhanced theta synchrony between the amygdala and 
mPFC has been observed, which differentiated between 
threat and safety (Likhtik et al. 2014). In rats, behavioral 
fear expression, like freezing, coincides with internally 
generated theta oscillations in prefrontal-amygdala cir-
cuits (Karalis et al. 2016). In primates, theta power and 
coherence in the amygdala and anterior cingulate cortex 
increase during fear learning, but progressively decline 
 * Gabriele Pirazzini 
 
gabriele.pirazzini3@unibo.it
1 
Department of Electrical, Electronic, and Information 
Engineering “Guglielmo Marconi”, University of Bologna, 
Area di Campus Cesena, Via Dell’Università 50,


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47521 Cesena, Italy
2 
Center for Studies and Research in Cognitive Neuroscience, 
Department of Psychology “Renzo Canestrari”, University 
of Bologna, 40126 Bologna, Italy


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Brain Structure and Function (2023) 228:1125–1151 
https://doi.org/10.1007/s00429-022-02598-4
REVIEW
Mapping the primate thalamus: historical perspective and modern 
approaches for defining nuclei
Miguel Ángel García‑Cabezas1,2,3  · Isabel Pérez‑Santos1,2  · Carmen Cavada1,2 
Received: 9 May 2022 / Accepted: 14 November 2022 / Published online: 9 January 2023 
© The Author(s) 2023
Abstract
The primate thalamus has been subdivided into multiple nuclei and nuclear groups based on cytoarchitectonic, myeloarchi-
tectonic, connectional, histochemical, and genoarchitectonic differences. Regarding parcellation and terminology, two main 
schools prevailed in the twentieth century: the German and the Anglo-American Schools, which proposed rather different 
schemes. The German parcellation and terminology has been mostly used for the human thalamus in neurosurgery atlases; 
the Anglo-American parcellation and terminology is the most used in experimental research on the primate thalamus. In 
this article, we review the historical development of terminological and parcellation schemes for the primate thalamus over 
the last 200 years. We trace the technological innovations and conceptual advances in thalamic research that underlie each 
parcellation, from the use of magnifying lenses to contemporary genoarchitectonic stains during ontogeny. We also discuss 
the advantages, disadvantages, and practical use of each parcellation.
Keywords Thalamic nucleus · Nuclear parcellation · Cytoarchitecture · Myeloarchitecture · Genoarchitecture · New 
Neuromorphology
Introduction
The thalamus is a mass of subcortical gray matter on the 
lateral walls of the third ventricle. Some thalamic neurons 
relay sensory information from the periphery to the cerebral 
cortex and others participate in cognitive and emotional pro-
cessing through strong reciprocal connections with the cer-
ebral cortex and connections with the amygdala, striatum, 
and hypothalamus (Jones 2007).


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The structure of the thalamus is not homogeneous. Tha-
lamic neurons are diverse and, together with glial cells, are 
distributed unevenly across multiple nuclei that can be iden-
tified and delineated by microscopic examination of brain 
sections stained with different techniques. Neuroanatomists 
of the last 200 years identified multiple nuclei and nuclear 
groups in the primate thalamus based on cytoarchitectonic, 
myeloarchitectonic, connectional, and histochemical differ-
ences that prompted different theoretical paradigms. Regard-
ing terminology, two main schools, the German and the 
Anglo-American Schools, prevailed in the twentieth century 
and proposed rather different schemes for primate thalamic 
nuclei. The German terminology is mostly used in atlases of 
human neurosurgery (e.g., Schaltenbrand and Bailey 1959; 
Andrew and Watkins 1968; Van Buren and Borke 1972; 
Tasker 1990). In contrast, the Anglo-American terminol-
ogy is mostly used in experimental research on primates, 
including humans (Hirai and Jones 1989) and non-human 
primates (e.g., Olszewski 1952; Goldman-Rakic and Porrino 
1985; Sánchez-González et al. 2005). More recently, geno-
architectonic studies using in situ hybridization in vertebrate 
embryos to detect the spatial and temporal expression of 
morphogenetic genes across the neural plate and neural tube 
have opened the path for a New Neuromorphology (Nieu-
wenhuys and Puelles 2016), which is based on the origin of 
brain structures in ontogeny and phylogeny, and will likely 
impact future parcellations and terminologies for thalamic 
nuclei in human and non-human primates.
 * Carmen Cavada 
 
carmen.cavada@uam.es
1 
Department of Anatomy, Histology and Neuroscience, 
School of Medicine, Universidad Autónoma de Madrid, 
Calle Arzobispo Morcillo 4, 28029 Madrid, Spain
2 
PhD Program in Neuroscience, Universidad Autónoma de 
Madrid-Cajal, Madrid, Spain
3 
Neural Systems Laboratory, Department of Health Sciences, 
Boston University, Boston, MA, USA


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Phenotypic and functional analysis of CD1a+ dendritic cells from cats chronically infected with feline immunodeficiency virus


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Phenotypic and functional analysis of CD1a+ dendritic cells from 
cats chronically infected with feline immunodeficiency virus
Lin Zhang1, Stacie Reckling2, Gregg A. Dean1
Gregg A. Dean: gregg.dean@colostate.edu
1Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine, 
Colorado State University, Fort Collins, CO 80523, USA, Telephone: 970-491-6144, FAX: 
970-491-0603
2Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina 
State University, Raleigh, NC 27606, USA
Abstract
Numerous studies suggest dendritic cell (DC) dysfunction is central to the dysregulated immune 
response during HIV infection; however, in vivo studies are lacking. In the present study we 
used feline immunodeficiency virus (FIV) infection of cats as a model for HIV-1 infection to 
assess the maturation and function of dendritic cells, in vivo and in vitro. We compared CD1a+ 
DC migration, surface phenotype, endocytosis, mixed leukocyte reaction (MLR) and regulatory T 
cell (Treg) phenotype induction by CD1a+ cells isolated from lymph nodes of FIV-infected and 
control cats. Results showed that resident CD1a+ DC in lymph nodes of chronically FIV-infected 
cats are phenotypically mature, can stimulate normal primary T cell proliferation, override Treg 
suppression and do not skew toward Treg induction. In contrast, FIV infection had deleterious 
effects on antigen presentation and migratory capacity of CD1a+ cells in tissues.
Keywords
Feline immunodeficiency virus; dendritic cells; CD1a; regulatory T cells
1. Introduction
Feline immunodeficiency virus (FIV) is a naturally occurring pathogenic lentivirus that 
infects domestic and non-domestic cats causing an immunodeficiency syndrome similar 
to HIV infection of people [1]. In addition to well-studied deficiencies in the adaptive 
immune response, innate immune defects caused by FIV have also been described [1, 2]. 
We have previously shown that FIV-infected cats have a delayed and impaired response


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Correspondence to: Gregg A. Dean, gregg.dean@colostate.edu.
Conflict of interest statement: The authors declare no conflicts of interest.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our 
customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review 
of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be 
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
HHS Public Access
Author manuscript
Comp Immunol Microbiol Infect Dis. Author manuscript; available in PMC 2023 June 09.
Published in final edited form as:
Comp Immunol Microbiol Infect Dis. 2015 October ; 42: 53–59. doi:10.1016/j.cimid.2015.07.003.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript


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Antibody-dependent cellular cytotoxicity, infected cell binding and neutralization by antibodies to the SIV envelope glycoprotein


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RESEARCH ARTICLE
Antibody-dependent cellular cytotoxicity,
infected cell binding and neutralization by
antibodies to the SIV envelope glycoprotein
Michael W. Grunst1¤, Ruby A. Ladd2, Natasha M. Clark1, Hwi Min Gil2, Vadim A. Klenchin1,
Rosemarie Mason3, Genoveffa Franchini4, Mario Roederer3, David T. EvansID1,2*
1 Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin,
United States of America, 2 Wisconsin National Primate Research Center, University of Wisconsin-Madison,
Madison, Wisconsin, United States of America, 3 Vaccine Research Center, National Institute of Allergy and
Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of
America, 4 Animal Models and Retroviral Vaccines Section, National Cancer Institute, Bethesda, Maryland,
United States of America
¤ Current address: Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut, United
States of America
* dtevans2@wisc.edu
Abstract
Antibodies specific for diverse epitopes of the simian immunodeficiency virus envelope gly-
coprotein (SIV Env) have been isolated from rhesus macaques to provide physiologically
relevant reagents for investigating antibody-mediated protection in this species as a nonhu-
man primate model for HIV/AIDS. With increasing interest in the contribution of Fc-mediated
effector functions to protective immunity, we selected thirty antibodies representing different
classes of SIV Env epitopes for a comparison of antibody-dependent cellular cytotoxicity
(ADCC), binding to Env on the surface of infected cells and neutralization of viral infectivity.
These activities were measured against cells infected with neutralization-sensitive (SIV-
mac316 and SIVsmE660-FL14) and neutralization-resistant (SIVmac239 and SIVsmE543-3)
viruses representing genetically distinct isolates. Antibodies to the CD4-binding site and
CD4-inducible epitopes were identified with especially potent ADCC against all four viruses.


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ADCC correlated well with antibody binding to virus-infected cells. ADCC also correlated
with neutralization. However, several instances of ADCC without detectable neutralization
or neutralization without detectable ADCC were observed. The incomplete correspondence
between ADCC and neutralization shows that some antibody-Env interactions can uncouple
these antiviral activities. Nevertheless, the overall correlation between neutralization and
ADCC implies that most antibodies that are capable of binding to Env on the surface of viri-
ons to block infectivity are also capable of binding to Env on the surface of virus-infected
cells to direct their elimination by ADCC.
Author summary
Antibodies to the envelope glycoprotein (Env) of simian immunodeficiency virus (SIV)
have been isolated to investigate mechanisms of antibody-mediated protection in rhesus
PLOS PATHOGENS
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011407
May 30, 2023
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OPEN ACCESS
Citation: Grunst MW, Ladd RA, Clark NM, Gil HM,
Klenchin VA, Mason R, et al. (2023) Antibody-
dependent cellular cytotoxicity, infected cell binding
and neutralization by antibodies to the SIV
envelope glycoprotein. PLoS Pathog 19(5):
e1011407. https://doi.org/10.1371/journal.
ppat.1011407
Editor: Guido Silvestri, Emory University, UNITED
STATES
Received: November 17, 2022
Accepted: May 8, 2023
Published: May 30, 2023
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced,
distributed, transmitted, modified, built upon, or
otherwise used by anyone for any lawful purpose.
The work is made available under the Creative
Commons CC0 public domain dedication.
Data Availability Statement: All relevant data are
available within the manuscript and Supporting
information files.
Funding: Research reported in this publication was
supported by National Institutes of Heath grants
AI121135, AI095098, AI148379, AI161816,
AI167732 and AI098485 to DTE. This work was


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also supported in part by the Office of the Director,
National Institutes of Health award number
P51OD011106 to the Wisconsin National Primate


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HIV, asymptomatic STI, and the rectal mucosal immune environment among young men who have sex with men


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RESEARCH ARTICLE
HIV, asymptomatic STI, and the rectal mucosal
immune environment among young men who
have sex with men
Vanessa E. Van DorenID1*, S. Abigail Smith1, Yi-Juan Hu2, Gregory Tharp3,
Steven Bosinger3,4, Cassie G. Ackerley1, Phillip M. Murray1, Rama R. Amara3,4, Praveen
K. Amancha1, Robert A. Arthur5, H. Richard Johnston5, Colleen F. Kelley1,6
1 The Hope Clinic of the Emory Vaccine Center, Division of Infectious Diseases, Department of Medicine,
Emory University School of Medicine, Atlanta, Georgia, United States of America, 2 Department of
Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, Georgia, United
States of America, 3 Emory National Primate Research Center, Emory University, Atlanta, Georgia, United
States of America, 4 Department of Microbiology and Immunology, Emory University, Atlanta, Georgia,
United States of America, 5 Emory Integrated Computational Core, Emory University, Atlanta, Georgia,
United States of America, 6 Grady Health System, Atlanta, Georgia, United States of America
* vvandor@emory.edu
Abstract
Young men who have sex with men (YMSM) are disproportionately affected by HIV and bac-
terial sexually transmitted infections (STI) including gonorrhea, chlamydia, and syphilis; yet
research into the immunologic effects of these infections is typically pursued in siloes. Here,
we employed a syndemic approach to understand potential interactions of these infections on
the rectal mucosal immune environment among YMSM. We enrolled YMSM aged 18–29
years with and without HIV and/or asymptomatic bacterial STI and collected blood, rectal
secretions, and rectal tissue biopsies. YMSM with HIV were on suppressive antiretroviral ther-
apy (ART) with preserved blood CD4 cell counts. We defined 7 innate and 19 adaptive
immune cell subsets by flow cytometry, the rectal mucosal transcriptome by RNAseq, and the
rectal mucosal microbiome by 16S rRNA sequencing and examined the effects of HIV and


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STI and their interactions. We measured tissue HIV RNA viral loads among YMSM with HIV
and HIV replication in rectal explant challenge experiments among YMSM without HIV. HIV,
but not asymptomatic STI, was associated with profound alterations in the cellular composi-
tion of the rectal mucosa. We did not detect a difference in the microbiome composition asso-
ciated with HIV, but asymptomatic bacterial STI was associated with a higher probability of
presence of potentially pathogenic taxa. When examining the rectal mucosal transcriptome,
there was evidence of statistical interaction; asymptomatic bacterial STI was associated with
upregulation of numerous inflammatory genes and enrichment for immune response path-
ways among YMSM with HIV, but not YMSM without HIV. Asymptomatic bacterial STI was
not associated with differences in tissue HIV RNA viral loads or in HIV replication in explant
challenge experiments. Our results suggest that asymptomatic bacterial STI may contribute
to inflammation particularly among YMSM with HIV, and that future research should examine
potential harms and interventions to reduce the health impact of these syndemic infections.
PLOS PATHOGENS
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011219
May 30, 2023
1 / 23
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Van Doren VE, Smith SA, Hu Y-J, Tharp
G, Bosinger S, Ackerley CG, et al. (2023) HIV,
asymptomatic STI, and the rectal mucosal immune
environment among young men who have sex with
men. PLoS Pathog 19(5): e1011219. https://doi.
org/10.1371/journal.ppat.1011219
Editor: Daniel C. Douek, Vaccine Research Center,
UNITED STATES
Received: February 17, 2023
Accepted: May 10, 2023
Published: May 30, 2023
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:


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https://doi.org/10.1371/journal.ppat.1011219
Copyright: © 2023 Van Doren et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting information
files.


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Hydroxychloroquine Causes Early Inner Retinal Toxicity and Affects Autophagosome\205Lysosomal Pathway and Sphingolipid Metabolism i\
n the Retina


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Hydroxychloroquine Causes Early Inner Retinal Toxicity and 
Affects Autophagosome–Lysosomal Pathway and Sphingolipid 
Metabolism in the Retina
Koushik Mondal1, Hunter Porter2, Jerome Cole II1, Hemang K. Pandya2, Sandip K. 
Basu1, Sufiya Khanam1, Chi-Yang Chiu3, Vinay Shah2, Daniel J. Stephenson4, Charles E. 
Chalfant4,5,6, Nawajes Mandal1,2,7,8,9
1Department of Ophthalmology, Hamilton Eye Institute, The University of Tennessee Health 
Science Center, Memphis, TN 38163, USA
2Department of Ophthalmology, Dean McGee Eye Institute, The University of Oklahoma Health 
Sciences Center, Oklahoma City OK-73104, USA
3Division of Biostatistics, Department of Preventive Medicine, University of Tennessee Health 
Science Center, Memphis, TN 38163, USA
4Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 
Tampa, FL 33620, USA
5The Moffitt Cancer Center, Tampa, FL 33620, USA
6Research Service, James A. Haley Veterans Hospital, Tampa, FL 33612, USA
7Department of Anatomy and Neurobiology, The University of Tennessee Health Science Center, 
Memphis, TN 38163, USA
✉Nawajes Mandal, nmandal@uthsc.edu.
Koushik Mondal, Hunter Porter, Jerome Cole II, Hemang K. Pandya and Sandip K. Basu are equally contributing authors.
Author Contribution Koushik Mondal performed experiment, analyzed data, prepared illustrations and wrote the manuscript; 
Hunter Porter performed experiment, analyzed data, prepared illustrations, and wrote and edited the manuscript; Jerome Cole II 
performed experiment; Hemang K. Pandya recruited patients, retrieved patient and clinical data, and analyzed data; Sandip K. Basu 
analyzed data, and wrote and edited the manuscript; Vinay Shah recruited patients and retrieved patient and clinical data; Daniel 
J Stephenson performed experiment and analyzed data; Charles E Chalfant provided facility and analyzed data; Nawajes Mandal 
designed experiment, obtained IRB and IACUC approvals, provided facility, analyzed data, and wrote and edited manuscript.
Conflict of Interests None.


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Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/
s12035-022-02825-3.
Ethics Approval and Consent to Participate Human study was approved by University of Oklahoma Institutional Review Board for 
the Protection of Human Subjects; IRB#: 5218; Approval Date: 02/27/2015
Study Title: Sphingolipids, Autoimmune Diseases and Retinopathies.
Consent for Publication Human study and consent to publish was approved by University of Oklahoma; Institutional Review Board 
for the Protection of Human Subjects; IRB#: 5218; Approval Date: 02/27/2015.
Author Consent All the authors have seen the manuscript and approved for publication.
Research involving Human Participants Human study was approved by University of Oklahoma; Institutional Review Board for 
the Protection of Human Subjects; IRB#: 5218; Approval Date: 02/27/2015; Study Title: Sphingolipids, Autoimmune Diseases and 
Retinopathies.
Animals: Animal studies approved by UTHSC IACUC committee. Approval # 19-0104.
Informed Consent Obtained for all participants according to the approved IRB#: 5218; protocol at the University of Oklahoma. 
Approval Date: 02/27/2015.
Study Title: Sphingolipids, Autoimmune Diseases and Retinopathies.
HHS Public Access
Author manuscript
Mol Neurobiol. Author manuscript; available in PMC 2023 June 12.
Published in final edited form as:
Mol Neurobiol. 2022 June ; 59(6): 3873–3887. doi:10.1007/s12035-022-02825-3.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript


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Food insecurity is associated with higher respiratory quotient 
and lower glucagon-like peptide-1
Jetaun M. Booker, BS,
Douglas C. Chang, MD,
Emma J. Stinson, MPH,
Cassie M. Mitchell, PhD, RD,
Susanne B. Votruba, PhD, RD,
Jonathan Krakoff, MD,
Marci E. Gluck, PhD,
Tomás Cabeza de Baca, PhD**
Obesity and Diabetes Clinical Research Section, Phoenix Epidemiology and Clinical Research 
Branch National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of 
Health, Department of Health and Human Services, Phoenix, AZ, USA
Abstract
Objective: Food insecurity is known to be associated with obesity but its association with 
physiological measures is unclear. Therefore, we hypothesized that compared to food secure, 
individuals with food insecurity would have higher 24-h energy expenditure (EE[kcal/d]) and a 
24-h respiratory quotient (RQ[ratio]). Subsequently, we explored hormones involved in appetite 
regulation, substrate oxidation, and EE.
Methods: One hundred-thirteen non-diabetic healthy participants (75M; Age 40±12 [years]; BMI 
30±8 [kg/m2]) were included in this analysis. Participants completed the Food Security Short 
Form, a DXA scan, and spent 24 hours in a human respiratory chamber following a weight 
maintaining diet.
Results: Compared to individuals with food security, participants with food insecurity had no 
difference in 24-h EE. However, they had higher carbohydrate oxidation rates (p = 0.03), and 
lower lipid oxidation rates (p = 0.02), resulting in higher 24-h RQ (p<0.01). They also had lower 
fasting glucagon-like peptide 1 (GLP-1 [p = 0.03]) concentrations.
Conclusions: Food insecurity is associated with higher 24-h RQ and lower fasting GLP-1 
concentrations, metabolic and hormonal differences previously shown to drive greater calorie 
**Contact Info: Tomás Cabeza de Baca, PhD, Obesity and Diabetes Clinical Research Section, Phoenix Epidemiology and Clinical


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Research Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 4212 North 16th 
Street, Phoenix, Arizona, 85016, USA, tommy.cabezadebaca@nih.gov, 602-200-5300. 
Disclosure: The authors declare no conflict of interest.
Competing Interests Statement
The authors declare no competing financial interests.
Clinical Trial Registration: clinicaltrials.gov: NCT00342732
HHS Public Access
Author manuscript
Obesity (Silver Spring). Author manuscript; available in PMC 2023 June 13.
Published in final edited form as:
Obesity (Silver Spring). 2022 June ; 30(6): 1248–1256. doi:10.1002/oby.23437.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript


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Identification of antigenic epitopes recognized by tumor infiltrating lymphocytes in high grade serous ovarian cancer by multi-omics profiling of the auto-antigen repertoire


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Vol.:(0123456789)
1 3
Cancer Immunology, Immunotherapy (2023) 72:2375–2392 
https://doi.org/10.1007/s00262-023-03413-7
RESEARCH
Identification of antigenic epitopes recognized by tumor infiltrating 
lymphocytes in high grade serous ovarian cancer by multi‑omics 
profiling of the auto‑antigen repertoire
Douglas G. Millar1 · S. Y. Cindy Yang1 · Azin Sayad1 · Qingchuan Zhao2 · Linh T. Nguyen1 · Kathrin Warner1 · 
Ami G. Sangster1 · Munehide Nakatsugawa1 · Kenji Murata1 · Ben X. Wang1 · Patricia Shaw1 · Blaise Clarke1 · 
Marcus Q. Bernardini3 · Trevor Pugh1 · Pierre Thibault2 · Naoto Hirano1,4 · Claude Perreault2 · Pamela S. Ohashi1,4
Received: 2 November 2022 / Accepted: 16 February 2023 / Published online: 21 March 2023 
© The Author(s) 2023
Abstract
Immunotherapeutic strategies aimed at enhancing tumor cell killing by tumor-specific T cells hold great potential for reduc-
ing tumor burden and prolonging survival of cancer patients. Although many potential tumor antigens have been described, 
identifying relevant targets when designing anti-cancer vaccines or targeted cell therapies remains a challenge. To identify 
novel, potentially immunogenic candidate tumor antigens, we performed integrated tumor transcriptomic, seromic, and 
proteomic analyses of high grade serous ovarian cancer (HGSC) patient tumor samples. We identified tumor neo-antigens 
and over-expressed antigens using whole exome and RNA sequencing and examined these in relation to patient-matched 
auto-antibody repertoires. Focusing on MHC class I epitopes recognized by  CD8+ T cells, HLA-binding epitopes were 
identified or predicted from the highly expressed, mutated, or auto-antibody target antigen, or MHC-associated peptides 
(MAPs). Recognition of candidate antigenic peptides was assessed within the tumor-infiltrating T lymphocyte (TIL) popula-
tion expanded from each patient. Known tumor-associated antigens (TAA) and cancer/testis antigens (CTA) were commonly


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found in the auto-antibody and MAP repertoires and  CD8+ TILs recognizing epitopes from these antigens were detected, 
although neither expression level nor the presence of auto-antibodies correlated with TIL recognition. Auto-antibodies 
against tumor-mutated antigens were found in most patients, however, no TIL recognition of the highest predicted affinity 
neo-epitopes was detected. Using high expression level, auto-antibody recognition, and epitope prediction algorithms, we 
identified epitopes in 5 novel antigens (MOB1A, SOCS3, TUBB, PRKAR1A, CCDC6) recognized by HGSC patient TILs. 
Furthermore, selection of epitopes from the MAP repertoire identified 5 additional targets commonly recognized by multi-
ple patient TILs. We find that the repertoire of TIL specificities includes recognition of highly expressed and immunogenic 
self-antigens that are processed and presented by tumors. These results indicate an ongoing autoimmune response against a 
range of self-antigens targeted by HGSC TILs.
Keywords Auto-antibodies · Seromics · Transcriptomics · Epitope prediction · Tumor-associated antigens · Cancer vaccine
Introduction
Therapeutic vaccination against tumor-expressed antigens 
can promote tumor-specific T cell-mediated killing of can-
cer cells and represents a promising form of immunother-
apy for cancer. Tumor target antigens fall broadly into two 
classes: tumor-associated antigens (TAAs), including tis-
sue differentiation antigens, tumor over-expressed antigens, 
and cancer/testis antigens (CTA); and tumor-specific anti-
gens (TSA) including mutated neo-antigens and aberrantly 
expressed translation products from non-canonical reading 
frames [1–3]. Neo-antigens arise from somatic mutations 
 * Pamela S. Ohashi 
 
pohashi@uhnresearch.ca
1 
Tumor Immunotherapy Program, Princess Margaret Cancer 
Centre, 610 University Avenue, Toronto, ON M5G 2M9, 
Canada
2 
Institute for Research in Immunology and Cancer, Université 
de Montréal, Montréal, Québec, Canada
3


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3 
Division of Gynecologic Oncology, Cancer Clinical Research 
Unit (CCRU), Princess Margaret Cancer Centre, Toronto, 
ON, Canada
4 
Department of Immunology, University of Toronto, Toronto, 
ON, Canada


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An atlas of white matter anatomy, its variability, and reproducibility based on constrained spherical deconvolution of diffusion\
 MRI


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An atlas of white matter anatomy, its variability, and 
reproducibility based on constrained spherical deconvolution of 
diffusion MRI
Ahmed M. Radwana,b,∗, Stefan Sunaerta,b,c, Kurt Schillingd, Maxime Descoteauxe, Bennett 
A. Landmanf, Mathieu Vandenbulckeb,g,h, Tom Theysb,i,j, Patrick Dupontb,k, Louise 
Emsella,b,g,h
aKU Leuven, Department of Imaging and pathology, Translational MRI, Leuven, Belgium
bKU Leuven, Leuven Brain Institute (LBI), Department of Neurosciences, Leuven, Belgium
cUZ Leuven, Department of Radiology, Leuven, Belgium
dVanderbilt University Medical Center, Department of Radiology and Radiological Sciences, 
Nashville, TN, USA
eSCIL, Université de Sherbrooke, Quebec, Canada
fVanderbilt University, Department of Electrical Engineering and Computer Engineering, Nashville, 
TN, USA
gKU Leuven, Department of Neurosciences, Neuropsychiatry, Leuven, Belgium
hKU Leuven, Department of Geriatric Psychiatry, University Psychiatric Center (UPC), Leuven, 
Belgium
iKU Leuven, Department of Neurosciences, Research Group Experimental Neurosurgery and 
Neuroanatomy, Leuven, Belgium
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
∗Corresponding author at: Department of Imaging and pathology, Translational MRI, KU Leuven, Leuven, Belgium. 
ahmed.radwan@kuleuven.be, radwanphd@gmail.com (A.M. Radwan). 
Data and code sharing statement
All data used in this work can be recovered from their respective online sources mentioned above. The accompanying supplementary 
information document contains more detail on the datasets used in this work, further explanation of the FWT method, and additional 
similarity analysis results. The FWT workflows can be downloaded from (https://github.com/KUL-Radneuron/KUL_FWT.git). 
Finally, the Open Science Framework repository (https://osf.io/snq2d/) contains additional screenshots of single subject bundles, 
bundle heatmaps, original and symmetric versions of the HCP-template atlas bundles, and additional tractograms.


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Declaration of Competing Interest
Maxime Descoteaux is a founder and co-owner of Imeka Solutions Inc. No conflicts related to the current work.
Credit authorship contribution statement
Ahmed M. Radwan: Conceptualization, Methodology, Data curation, Software, Writing – original draft, Visualization, Investigation, 
Formal analysis. Stefan Sunaert: Software, Resources, Supervision, Visualization, Investigation, Formal analysis, Writing – 
original draft, Writing – review & editing. Kurt Schilling: Conceptualization, Methodology, Writing – review & editing. Maxime 
Descoteaux: Supervision, Writing – review & editing. Bennett A. Landman: Supervision, Writing – review & editing. Mathieu 
Vandenbulcke: Supervision, Writing – review & editing. Tom Theys: Supervision, Writing – review & editing. Patrick Dupont: 
Supervision, Investigation, Writing – review & editing. Louise Emsell: Conceptualization, Supervision, Methodology, Visualization, 
Investigation, Formal analysis, Writing – original draft, Writing – review & editing.
Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.neuroimage.2022.119029.
HHS Public Access
Author manuscript
Neuroimage. Author manuscript; available in PMC 2023 June 14.
Published in final edited form as:
Neuroimage. 2022 July 01; 254: 119029. doi:10.1016/j.neuroimage.2022.119029.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript


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Neutralizing antibodies targeting a novel epitope on envelope protein exhibited broad protection against flavivirus without risk of disease enhancement


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Yen et al. 
Journal of Biomedical Science           (2023) 30:41  
https://doi.org/10.1186/s12929-023-00938-y
RESEARCH
Open Access
© The Author(s) 2023. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which 
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the 
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or 
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line 
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory 
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this 
licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco 
mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Neutralizing antibodies targeting a novel 
epitope on envelope protein exhibited broad 
protection against flavivirus without risk 
of disease enhancement
Li‑Chen Yen1, Hsin‑Wei Chen2, Chia‑Lo Ho3, Chang‑Chi Lin1,4, Yi‑Ling Lin1,5, Qiao‑Wen Yang1, Kuo‑Chou Chiu6,7, 
Shu‑Pei Lien2, Ren‑Jye Lin8 and Ching‑Len Liao1,2,8*   
Abstract 
Background Flavivirus causes many serious public health problems worldwide. However, licensed DENV vaccine has 
restrictions on its use, and there is currently no approved ZIKV vaccine. Development of a potent and safe flavivirus 
vaccine is urgently needed. As a previous study revealed the epitope, RCPTQGE, located on the bc loop in the E 
protein domain II of DENV, in this study, we rationally designed and synthesized a series of peptides based on the


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sequence of JEV epitope RCPTTGE and DENV/ZIKV epitope RCPTQGE.
Methods Immune sera were generated by immunization with the peptides which were synthesized by using five 
copies of RCPTTGE or RCPTQGE and named as JEV‑NTE and DV/ZV‑NTE. Immunogenicity and neutralizing abilities of 
JEV‑NTE or DV/ZV‑NTE‑immune sera against flavivirus were evaluated by ELISA and neutralization tests, respectively. 
Protective efficacy in vivo were determined by passive transfer the immune sera into JEV‑infected ICR or DENV‑ and 
ZIKV‑challenged AG129 mice. In vitro and in vivo ADE assays were used to examine whether JEV‑NTE or DV/ZV‑NTE‑
immune sera would induce ADE.
Results Passive immunization with JEV‑NTE‑immunized sera or DV/ZV‑NTE‑immunized sera could increase the 
survival rate or prolong the survival time in JEV‑challenged ICR mice and reduce the viremia levels significantly in 
DENV‑ or ZIKV‑infected AG129 mice. Furthermore, neither JEV ‑NTE‑ nor DV/ZV‑NTE‑immune sera induced antibody‑
dependent enhancement (ADE) as compared with the control mAb 4G2 both in vitro and in vivo.
Conclusions We showed for the first time that novel bc loop epitope RCPTQGE located on the amino acids 73 to 79 
of DENV/ZIKV E protein could elicit cross‑neutralizing antibodies and reduced the viremia level in DENV‑ and ZIKV‑
challenged AG129 mice. Our results highlighted that the bc loop epitope could be a promising target for flavivirus 
vaccine development.
Keywords Japanese encephalitis virus, Dengue virus, Zika virus, Flavivirus vaccine, Neutralizing epitope, Envelop 
protein domain II
*Correspondence:
Ching‑Len Liao
chinglen@gmail.com
Full list of author information is available at the end of the article


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Gut 1997; 40: 689-690
Familial hyperamylasaemia
P M Cuckow, A Y Foo, A Jamal, M D Stringer
Abstract
A six year old boy underwent extensive
investigation for recurrent abdominal pain
and was found to have a persistently raised
serum amylase. Endoscopic retrograde
cholangiopancreatography
was
normal
and macroamylasaemia
was
excluded.
Serum amylase concentrations were found
to be raised in other family members
spanning three generations, all of whom
were
asymptomatic.
Clearance
studies
suggested no evidence of a renal tubular
defect and serum lipase concentrations
were normal. This is the first report of
apparently familial hyperamylasaemia and
the mode of inheritance is consistent with
an autosomal dominant pattern.
(Gut 1997; 40: 689-690)
Keywords: amylase, pancreatitis.
Although a greatly raised serum amylase in a
patient with abdominal pain is most often due
to pancreatic disease, there are many recog-
nised causes of a mildly raised concentration.'
We report a hitherto undescribed cause of
hyperamylasaemia.
Methods
INDEX CASE
In 1993, a four year old white boy presented
acutely with colicky periumbilical pain, leth-
argy, anorexia, and vomiting. He had experi-
enced
several
similar
episodes
during
the
2.5 r
2
0)
0
35.
E
E
1.5
0
0
0
0.5
o
I
r'V
r'rmc
r'm
C ')C r)
14
14
I-
le
d.
it le
l
0
e
LO
LO
LO
LO
LO
ULO
LO
LO
LO
LO
LO
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a,
a,
a, Oa
w
a,
a,
a,
o
) aOa,
a,
a
a,
a,
a,
a,
a,
a,
a,
ac
a
a,
a,o
a, Oa
< E * n < cn ° Z a
X) u E < E n
<
0° z a n u E < E:
< w0
z° a
-t
Date
Figure 1: Serial serum amylase concentrations for the index case. *Serum amylase is
expressed as the ratio of the measured value to the upper limit of the normal range.
previous year, each resolving after a few hours
with simple analgesics. Between episodes he
was well. As an infant he had been investigated
for poor weight gain, vomiting with occasional
loose stools, and a persistent cough. Routine
haematology and biochemistry, liver and renal
function
tests,
serum
ferritin
and
vitamin
B12, thyroid function tests, urine and stool


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string_value:
microbiology, a sweat test, xylose absorption
test, chest radiography, and barium meal had
been
normal.
At two
years
his
persistent
symptoms prompted an upper gastrointestinal
endoscopy and jejunal biopsy, which were
normal. Subsequently, his problems resolved
and his growth became normal with height and
weight increasing between the 10th and 25th
centiles.
On admission, he had a mild pyrexia with no
signs
of an
acute
abdomen.
Routine
in-
vestigations were normal except for a slightly
raised serum amylase of 168 Somogyi U/dl
(normal range 40-160 U/dl). A plain abdomi-
nal
radiograph
and
ultrasound
scan
were
normal. A laparoscopy disclosed no pathology
and a non-inflamed appendix was removed.
Six
weeks
later
he
was
readmitted
with
recurrent abdominal pain and a repeat serum
amylase was raised at 336 Somogyi U/dl but a
repeat
abdominal
ultrasound
scan
again
showed no abnormality. His pain settled and
outpatient
investigations
including
a
small
bowel
meal,
abdominal
computed
tomog-
raphy,
sweat
test,
and DNA
analysis
for
common cystic fibrosis gene mutations were all
normal. His serum amylase remained raised at
226 Somogyi U/dl and he was referred to us for
further investigation.
During the next year he remained well but
his serum amylase was raised at 160 inter-
national units/l (normal range 19-95 IU/1). He
had a normal lipid profile, liver function tests,
urine metabolic screen, faecal chymotrypsin,
and faecal fat content. Endoscopic retrograde
cholangiopancreatography
showed
normal
ductal anatomy with a normal pancreatico-
biliary junction. Macroamylasaemia was con-
sidered likely after finding a normal urinary
amylase excretion (38 IU/4 h) in the presence
of a persistently raised serum amylase (149 IU/
1).2 Macroamylasaemia was
definitively
ex-
cluded by the absence of diffuse streaking in
the y-globulin region after electrophoretic sep-
aration on cellulose acetate.3 Autoantibodies,
rheumatoid factor, and Ro and La antigens were
negative. Currently the child is well with normal


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growth. He has had no further attacks of non-
specific abdominal pain. During a three year
period,
the
patient's
serum
amylase
was
estimated on multiple
occasions by various
standard laboratory methods4 and was found to
be consistently raised (Fig 1).
Department of
Paediatric Surgery,
Leeds General
Infirmary,
Leeds
P M Cuckow
M D Stringer
Department of
Chemical Pathology,
St James' University
Hospital
A Jamal
Department of
Chemical Pathology,
Royal Free Hospital,
London, UK
A Y Foo
Correspondence to:
Mr M D Stringer,
Department of Paediatric
Surgery, Clarendon Wmg,
Leeds General Infirmary,
Leeds LS2 9NS.
Accepted for publication
23 January 1997
689
1


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Original Article
Postnatal intestinal mucosa and gut microbial
composition develop hand in hand: A mouse study
Uday Pandey a,b, Palok Aich a,b,*
a School of Biological Sciences, National Institute of Science Education and Research (NISER), P.O. Jatni, Khurda,
Odisha, India
b Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai, India
a r t i c l e i n f o
Article history:
Received 14 October 2021
Accepted 10 March 2022
Available online 16 March 2022
Keywords:
Gut barrier integrity
Intestinal epithelial cells (IECs)
Gut microbiota
Postnatal period
Akkermansia muciniphila (A. mucini-
phila)
Goblet cells
a b s t r a c t
Background: During the early postnatal life, gut microbiota development experiences
dynamic changes in their structural and functional composition. The postnatal period is
the critical window to develop a host defense mechanism. The maturation of intestinal
mucosal barrier integrity is one of the essential defense mechanisms to prevent the entry
of pathogens. However, the co-development of intestinal microbial colonization, formation
of barrier integrity, and intestinal epithelial cell layer is not entirely understood.
Methods: We studied the gut microbial composition and diversity using 16S rRNA marker
gene-based sequencing in mice to understand postnatal age-dependent association ki-
netics between gut microbial and intestinal development. Next, we assessed the intestinal
development by in vivo gut permeability assay, mRNA gene expression of different tight
junction proteins and intestinal epithelial cell markers, goblet cells population, villus
length, and cecal IgA quantiﬁcation.
Results: Our results showed a signiﬁcant shift in gut microbial structural and functional
composition from postnatal day 14 onwards with early life Proteobacteria abundance.
Relative abundance of Verrucomicrobia was maximum at postnatal day 14 and showed a
gradual decrease over time. We also observed an age-dependent biphasic pattern in barrier


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integrity improvement and differentiation of intestinal epithelial cells (IECs). A signiﬁcant
improvement in barrier integrity between days 1 and 7 showed the host factor contribu-
tion, while that beyond day 14 revealed an association with changes in microbiota
composition. Our temporal correlation analysis associated Bacteroidetes phylum with the
mucosal barrier formation during postnatal development.
Conclusions: The present study revealed the importance and interplay of host factors and
the microbiome in gut development and intestinal mucosal homeostasis.
* Corresponding author. School of Biological Sciences, National Institute of Science Education and Research (NISER), P.O. Jatni, Khurda
752050, Odisha, India
E-mail address: palok.aich@niser.ac.in (P. Aich).
Peer review under responsibility of Chang Gung University.
Available online at www.sciencedirect.com
ScienceDirect
Biomedical Journal
journal homepage: www.elsevier.com/locate/bj
b i o m e d i c a l j o u r n a l 4 6 ( 2 0 2 3 ) 1 0 0 5 1 9
https://doi.org/10.1016/j.bj.2022.03.004
2319-4170/© 2022 The Authors. Published by Elsevier B.V. on behalf of Chang Gung University. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).


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RESEARCH ARTICLE
Ecological drivers of sustained enzootic yellow
fever virus transmission in Brazil, 2017–2021
Nata´lia Ingrid Oliveira Silva1¤‡, Gregory F. Albery2‡, Matheus Soares Arruda1☯, Gabriela
Fernanda Garcia Oliveira1☯, Thaı´s Alkifeles Costa1, E´ rica Munhoz de Mello1,3, Gabriel
Dias Moreira1, Erik Vinı´cius Reis1, Simone Agostinho da Silva3, Marlise Costa Silva3,
Munique Guimarães de Almeida3, Daniel J. Becker4, Colin J. Carlson2,5,
Nikos Vasilakis6,7,8, Kathryn A. Hanley9, Betaˆnia Paiva DrumondID1*
1 Department of Microbiology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 2 Department
of Biology, Georgetown University, Washington, DC, United States of America, 3 Laborato´rio de Zoonoses—
Centro de Controle de Zoonoses, Prefeitura de Belo Horizonte, Belo Horizonte, Minas Gerais, Brazil,
4 Department of Biology, University of Oklahoma, Norman, Oklahoma, United States of America, 5 Center
for Global Health Science and Security, Georgetown University, Washington, D.C., United States of America,
6 Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of
America, 7 Center for Vector-Borne and Zoonotic Diseases, The University of Texas Medical Branch,
Galveston, Texas, United States of America, 8 Institute for Human Infection and Immunity, University of
Texas Medical Branch, Galveston, Texas, United States of America, 9 Department of Biology, New Mexico
State University, Las Cruces, New Mexico, United States of America
☯ These authors contributed equally to this work.
¤ Current address: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA
‡ These authors share first authorship on this work.
* betaniadrumond@gmail.com, betaniadrumond@ufmg.br
Abstract
Beginning December 2016, sylvatic yellow fever (YF) outbreaks spread into southeastern
Brazil, and Minas Gerais state experienced two sylvatic YF waves (2017 and 2018). Follow-
ing these massive YF waves, we screened 187 free-living non-human primate (NHPs) car-


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casses collected throughout the state between January 2019 and June 2021 for YF virus
(YFV) using RTqPCR. One sample belonging to a Callithrix, collected in June 2020, was
positive for YFV. The viral strain belonged to the same lineage associated with 2017–2018
outbreaks, showing the continued enzootic circulation of YFV in the state. Next, using data
from 781 NHPs carcasses collected in 2017–18, we used generalized additive mixed mod-
els (GAMMs) to identify the spatiotemporal and host-level drivers of YFV infection and inten-
sity (an estimation of genomic viral load in the liver of infected NHP). Our GAMMs explained
65% and 68% of variation in virus infection and intensity, respectively, and uncovered strong
temporal and spatial patterns for YFV infection and intensity. NHP infection was higher in
the eastern part of Minas Gerais state, where 2017–2018 outbreaks affecting humans and
NHPs were concentrated. The odds of YFV infection were significantly lower in NHPs from
urban areas than from urban-rural or rural areas, while infection intensity was significantly
lower in NHPs from urban areas or the urban-rural interface relative to rural areas. Both YFV
infection and intensity were higher during the warm/rainy season compared to the cold/dry
season. The higher YFV intensity in NHPs in warm/rainy periods could be a result of higher
exposure to vectors and/or higher virus titers in vectors during this time resulting in the deliv-
ery of a higher virus dose and higher viral replication levels within NHPs. Further studies are
PLOS NEGLECTED TROPICAL DISEASES
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June 5, 2023
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OPEN ACCESS
Citation: Silva NIO, Albery GF, Arruda MS, Oliveira
GFG, Costa TA, de Mello E´M, et al. (2023)
Ecological drivers of sustained enzootic yellow
fever virus transmission in Brazil, 2017–2021.
PLoS Negl Trop Dis 17(6): e0011407. https://doi.
org/10.1371/journal.pntd.0011407


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Editor: Eric H. Y. Lau, The University of Hong
Kong, CHINA
Received: October 20, 2022
Accepted: May 22, 2023
Published: June 5, 2023
Copyright: © 2023 Silva et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: The authors confirm
that all data underlying the findings are fully
available without restriction. All relevant data are
within the paper and its Supporting Information
files.
Funding: This study was funded by National
Institute of Allergy and Infectious Diseases (NIAID)
(https://www.niaid.nih.gov/) through the CREID
Pilot Research Program 2021 (https://creid-
network.org/pilot-program) awarded to BPD by the
Centers for Research in Emerging Infectious


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Journal of Neurology, Neurosurgery, and Psychiatry 1983;46:576-578
Short report
Psychophysical assessment of a patient with
Tolosa-Hunt syndrome
LESLEY J FALLOWFIELD, JOHN E REES
From the Laboratory of Experimental Psychology Sussex University, Falmer, and Hurstwood Park
Neurological Centre, Haywards Heath, Sussex, UK
SUMMARY
Patients may complain of impaired vision and yet exhibit no abnormalities when tested
with conventional tests of visual acuity. Some sensitive psychophysical tests used by psychologists
and neurophysiologists to examine the function of the normal visual system are beginning to gain
recognition as useful tools in clinical situations. They are particularly sensitive indicators of
damage to the visual pathways. The results are presented of one such application which character-
ises the visual defects of a patient with a suspected diagnosis of Tolosa-Hunt Syndrome.
Clinical History
In 1979 the patient, aged 51 years, presented with a history
of 2 episodes of pain in and around the left orbit and upper
gum. Diagnosis was thought to be an unusual presentation
of migrainous neuralgia. By January 1980 there had been
two further episodes of painful ophthalmoplegia with dip-
lopia. This pain was predominantly peri-orbital and there
was 3rd and 6th cranial nerve paresis and ptosis on the left
side. His visual fields were full and Snellen acuity was nor-
mal. A carotid angiogram showed narrowing of the entire
intracavernous section of the left carotid artery, and eleva-
tion of the syphon. The CT scan (fig 1) shows a mass lesion
in the left orbital apex surrounding the optic nerve, and
involvement of the superior orbital fissure. Full blood
count, ESR, urea, electrolytes, protein electrophoresis
were
all
normal.
Predisone
treatment
resolved
the
headache and improved the ocular pareses.
A diagnosis of Tolosa-Hunt syndrome' was confirmed at
the Moorfields Eye Hospital. By May 1981 there was no
diplopia, although the patient still complained of transient


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lack of clarity in the left eye. Central field analysis now
showed a left para-central scotoma despite a visual acuity
of 6/5. Steroid treatment had been stopped and the CT
scan was now normal. Visual evoked responses to checker-
board pattern reversal showed a delay across the left visual
pathway with a major P2 potential at 124 ms. The right
side had a normal latency at 109ms. The patient continued
Address for reprint requests: Lesley J Fallowfield, Laboratory of
Experimental Psychology, Sussex University, Brighton BN1 2QG,
UK.
Received 6 October 1982 and in revised form 13 January 1983
Accepted 26 January 1983
Fig 1
CT scan showing high attenuation area in orbital
apex taken 18 February 1980.
to complain of poor vision in the left eye when psychophys-
ical measurements were made in June 1981.
Measurement of contrast sensitivity.
The limits of normal spatial vision can be character-
ised by measuring the contrast sensitivity to sinusoi-
dal gratings of different spatial frequency. Periodic
exchange of the dark and light bars produces a flick-
ering grating. Different flicker-rates or temporal
frequencies produce specific changes in the shape of
the spatial contrast sensitivity curve as can be seen in
576


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Development/Plasticity/Repair
Loss of Motor Cortical Inputs to the Red Nucleus after CNS
Disorders in Nonhuman Primates
Simon Borgognon1,2,3 and Eric M. Rouiller2
1Center for the Neural Basis of Cognition, Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, 2Department of
Neurosciences and Movement Sciences, Section of Medicine, Faculty of Science and Medicine, University of Fribourg, CH-1700 Fribourg, Switzerland, and
3Center for Neuroprosthetics and Brain Mind Institute, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
The premotor (PM) and primary motor (M1) cortical areas broadcast voluntary motor commands through multiple neuronal
pathways, including the corticorubral projection that reaches the red nucleus (RN). However, the respective contribution of
M1 and PM to corticorubral projections as well as changes induced by motor disorders or injuries are not known in nonhu-
man primates. Here, we quantified the density and topography of axonal endings of the corticorubral pathway in RN in
intact monkeys, as well as in monkeys subjected to either cervical spinal cord injury (SCI), Parkinson’s disease (PD)-like
symptoms or primary motor cortex injury (MCI). Twenty adult macaque monkeys of either sex were injected with the biotin-
ylated dextran amine anterograde tracer either in PM or in M1. We developed a semiautomated algorithm to reliably detect
and count axonal boutons within the magnocellular and parvocellular (pRN) subdivisions of RN. In intact monkeys, PM and
M1 preferentially target the medial part of the ipsilateral pRN, reflecting its somatotopic organization. Projection of PM to
the ipsilateral pRN is denser than that of M1, matching previous observations for the corticotectal, corticoreticular, and corti-
cosubthalamic projections (Fregosi et al., 2018, 2019; Borgognon et al., 2020). In all three types of motor disorders, there


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was a uniform and strong decrease (near loss) of the corticorubral projections from PM and M1. The RN may contribute to
functional recovery after SCI, PD, and MCI, by reducing direct cortical influence. This reduction possibly privileges direct
access to the final output motor system, via emphasis on the direct corticospinal projection.
Key words: motor disorders; plasticity; premotor cortex; primary motor cortex; primate; red nucleus
Significance Statement
We measured the corticorubral projection density arising from the PM or the M1 cortices in adult macaques. The premotor
cortex sent denser corticorubral projections than the primary motor cortex, as previously observed for the corticotectal, corti-
coreticular, and corticosubthalamic projections. The premotor cortex may thus exert more influence than primary motor cor-
tex onto subcortical structures. We next asked whether the corticorubral motor projections undergo lesion-dependent
plasticity after either cervical spinal cord injury, Parkinson’s disease-like symptoms, or primary motor cortex lesion. In all
three types of pathology, there was a strong decrease of the corticorubral motor projection density, suggesting that the red nu-
cleus may contribute to functional recovery after such motor system disorders based on a reduced direct cortical influence.
Introduction
The multiple motor cortical areas in primates broadcast com-
mands through parallel neuronal pathways to generate and coor-
dinate voluntary movements (Luppino and Rizzolatti, 2000; Dum
and Strick, 2002; Rouiller, 2012). These routes reach multiple
regions of the CNS including subcortical relays, the brainstem,
and the spinal cord. These projections are specifically organized
depending on their origin and their targets (Lemon, 2008). For
instance, in intact macaques, the premotor cortex (PM) sends
denser projections to the subthalamic nucleus (Borgognon et
al., 2020), the superior colliculus (Fregosi and Rouiller, 2017),
and the reticular formation (Fregosi et al., 2017) than the pri-


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mary motor cortex (M1). After motor disorders, these projec-
tions undergo unique regional plasticity that depends on the
Received Oct. 17, 2022; revised Dec. 14, 2022; accepted Jan. 13, 2023.
Author contributions: S.B. and E.M.R. designed research; S.B. and E.M.R. performed research; S.B. analyzed
data; S.B. and E.M.R. wrote the paper.
This work was supported by Swiss National Science Foundation Grants 110005, 132465, 144990, 149643,
Sinergia CRSII3_160696, Sinergia Prometheus CRSI33_125408 (E.M.R.), and 210986 (S.B.) and the Swiss
Primate Competence Center for Research. We thank Prof. Joceylne Bloch, Prof. Patrick Freund, Dr. Jean-
François Brunet, Dr. Simon Badoud, and Dr. Eric Schmidlin for surgical assistance; Prof. Martin Schwab, Prof.
Joceylne Bloch, Prof. Patrick Freund, Dr. Anis Mir, Dr. Thierry Wannier, Dr. Eric Schmidlin, Dr. Marie-Laure
Beaud, Dr. Alexander Wyss, Dr. Shahid Bashir, Dr. Adjia Hamadjida, Dr. Aderraouf Belhaj-Saïf, Dr. Mélanie
Kaeser, Dr. Julie Savidan, Dr. Anne-Dominique Gindrat, Dr. Yu Liu, Dr. Michela Fregosi, Dr. Simon Badoud,
Alessandro Contestabile, and Jérôme Cottet for contributions to experimental sessions and elaborations of
experimental protocols; Laurent Bossy, Jacques Maillard, Bernard Bapst, Bernard Morandi and Joseph
Corpataux for animal care assistance; Christine Roulin, Véronique Moret, and Christiane Marti for tissue processing;
Felix Meyenhofer for technical microscopical assistance; and Dr. Erinn M. Grigsby for proofreading.
The authors declare no competing financial interests.
Correspondence should be addressed to Simon Borgognon at simon.borgo@pitt.edu.
https://doi.org/10.1523/JNEUROSCI.1942-22.2023
Copyright © 2023 the authors
1682 • The Journal of Neuroscience, March 8, 2023 • 43(10):1682–1691


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Spatio-Temporal Characterization of Brain Inflammation in a Non-human Primate Stroke Model Mimicking Endovascular Thrombectomy


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Vol.:(0123456789)
1 3
Neurotherapeutics (2023) 20:789–802 
https://doi.org/10.1007/s13311-023-01368-2
ORIGINAL ARTICLE
Spatio‑Temporal Characterization of Brain Inflammation 
in a Non‑human Primate Stroke Model Mimicking Endovascular 
Thrombectomy
Guillaume Becker1  · Justine Debatisse1 · Margaux Rivière1 · Claire Crola Da Silva1 · Maude Beaudoin‑Gobert2 · 
Omer Eker3,4 · Océane Wateau5 · Tae Hee Cho1,4 · Marlène Wiart1 · Léon Tremblay6 · Nicolas Costes7 · 
Inès Mérida7 · Jérôme Redouté7 · Christelle Léon1 · Jean‑Baptiste Langlois7 · Didier Le Bars4,7 · Sophie Lancelot4,7 · 
Norbert Nighoghossian1,4 · Laura Mechtouff1,4 · Emmanuelle Canet‑Soulas1
Accepted: 13 March 2023 / Published online: 28 March 2023 
© The Author(s) 2023
Abstract
Reperfusion therapies in acute ischemic stroke have demonstrated their efficacy in promoting clinical recovery. However, 
ischemia/reperfusion injury and related inflammation remain a major challenge in patient clinical management. We evaluated 
the spatio-temporal evolution of inflammation using sequential clinical  [11C]PK11195 PET-MRI in a non-human primate 
(NHP) stroke model mimicking endovascular thrombectomy (EVT) with a neuroprotective cyclosporine A (CsA) treat-
ment. The NHP underwent a 110-min transient endovascular middle cerebral artery occlusion. We acquired  [11C]PK11195 
dynamic PET-MR imaging at baseline, 7 and 30 days after intervention. Individual voxel-wise analysis was performed 
thanks to a baseline scan database. We quantified  [11C]PK11195 in anatomical regions and in lesioned areas defined on per-
occlusion MR diffusion-weighted imaging and perfusion  [15O2]H2OPET imaging.  [11C]PK11195 parametric maps showed 
a clear uptake overlapping the lesion core at D7, which further increased at D30. Voxel-wise analysis identified individuals 
with significant inflammation at D30, with voxels located within the most severe diffusion reduction area during occlusion,


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mainly in the putamen. The quantitative analysis revealed that thalamic inflammation lasted until D30 and was significantly 
reduced in the CsA-treated group compared to the placebo. In conclusion, we showed that chronic inflammation matched 
ADC decrease at occlusion time, a region exposed to an initial burst of damage-associated molecular patterns, in an NHP 
stroke model mimicking EVT. We described secondary thalamic inflammation and the protective effect of CsA in this region. 
We propose that major ADC drop in the putamen during occlusion may identify individuals who could benefit from early 
personalized treatment targeting inflammation.
Keywords Stroke · Inflammation · Endovascular thrombectomy · PET-MRI · TSPO
Abbreviations
ADC 
 Apparent diffusion coefficient
AIS 
 Acute ischemic stroke
BBB 
 Blood-brain barrier
CBF 
 Cerebral blood flow
CIR 
 Cerebral ischemia reperfusion
CsA 
 Cyclosporine A
DAMPs 
 Damage-associated molecular patterns
DCE 
 Dynamic contrast enhancement
DSC-MRI  Dynamic susceptibility contrast MRI
DVR 
 Distribution volume ratio
DWI 
 Diffusion-weighted imaging
 * Guillaume Becker 
 
guillaume.becker@univ-lyon1.fr
1 
CarMeN Laboratory, INRAE U1397, INSERM U1060, 
Groupement Hospitalier Est, University Claude Bernard 
Lyon 1, 59 Boulevard Pinel, 69500 Lyon, Bron, France
2 
Lyon Neuroscience Research Center, UMR5295, INSERM 
U1028, CNRS, Université Claude Bernard Lyon 1, Lyon, 
France
3 
UMR-5220, CREATIS, CNRS, INSERM U1206, Université 
Lyon 1, INSA Lyon, Villeurbanne, France
4 
Hospices Civils de Lyon, Lyon, France
5 
Cynbiose SAS, Lyon, France
6 
Cognitive Neuroscience Center, CNRS UMR5229, 
Université Claude Bernard Lyon 1, Lyon, France
7 
CERMEP, Lyon, France


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Opioid Use, Gut Dysbiosis, Inflammation, and the Nervous 
System
Richa Jalodia1, Yaa Fosuah Abu1,2, Mark Ryan Oppenheimer1, Bridget Herlihy1, Jingjing 
Meng1, Irina Chupikova1, Junyi Tao1, Nillu Ghosh1, Rajib Kumar Dutta1, Udhghatri 
Kolli1, Yan Yan1, Eridania Valdes1, Madhulika Sharma1, Umakant Sharma1, Shamsudheen 
Moidunny1, Sabita Roy1
1 Department of Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA
2 Department of Microbiology and Immunology, University of Miami Miller School of Medicine, 
Miami, FL 33136, USA
Abstract
Opioid use disorder (OUD) is defined as the chronic use or misuse of prescribed or illicitly 
obtained opioids and is characterized by clinically significant impairment. The etiology of OUD is 
multifactorial as it is influenced by genetics, environmental factors, stress response and behavior. 
Given the profound role of the gut microbiome in health and disease states, in recent years 
there has been a growing interest to explore interactions between the gut microbiome and the 
central nervous system as a causal link and potential therapeutic source for OUD. This review 
describes the role of the gut microbiome and opioid-induced immunopathological disturbances at 
the gut epithelial surface, which collectively contribute to OUD and perpetuate the vicious cycle of 
addiction and relapse.
Keywords
Opioids; OUD; Microbiome; Gut-brain axis; Enteric nervous system
Introduction
According to the 2019 National Survey on Drug Use and Health, non-medical opioid use in 
the Unites States is estimated at 2.3% among adolescents (age 12–17), 5.3% among young 
adults (age 18–25), and 3.6% among adults (age ≥ 26) with the rates of opioid use being 
significantly higher than that of any other illicit drug (SAMSHA 2020). Consequently, this 
has resulted in the rise of OUD and a concomitant increase in drug-related overdoses, with 
increased morbidity and mortality among patients. Additionally, the opioid crisis poses a


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huge economic burden, partly attributable to higher direct medical costs for patients (Ghate 
et al. 2010) and indirect costs due to loss of productivity (Reinhart et al. 2018).
✉ Umakant Sharma usharma@med.miami.edu, Shamsudheen Moidunny sxm1464@med.miami.edu, Sabita Roy 
sabita.roy@miami.edu. 
Conflict of Interest All authors declare that they have no conflict of interest in this manuscript.
HHS Public Access
Author manuscript
J Neuroimmune Pharmacol. Author manuscript; available in PMC 2023 June 17.
Published in final edited form as:
J Neuroimmune Pharmacol. 2022 June ; 17(1-2): 76–93. doi:10.1007/s11481-021-10046-z.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript


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The spontaneous immortalization probability of mammalian cell culture strains, as their proliferative capacity, correlates with species body mass, not longevity


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Original Article
The spontaneous immortalization probability of
mammalian cell culture strains, as their
proliferative capacity, correlates with species body
mass, not longevity
Matteo Perillo a,*, Angela Punzo a, Cristiana Caliceti a, Christian Sell b,
Antonello Lorenzini a
a Department of Biomedical and Neuromotor Sciences, University of Bologna, Italy
b Department of Biochemistry & Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, USA
a r t i c l e i n f o
Article history:
Received 17 March 2023
Accepted 29 April 2023
Available online 5 May 2023
Keywords:
Cell culture immortalization
Replicative senescence
Longevity
Body mass
Partial correlation
Phylogenetic regression
a b s t r a c t
Background: The Peto's paradox consists in the observation that individuals from long-lived
and large animal species do not experience a higher cancer incidence, despite being
exposed for longer time to the possibility of accumulating mutations and having more
target cells exposed to the phenomenon. The existence of this paradox has been recently
conﬁrmed (Vincze et al., 2022). Concurrently, robust evidence has been published that
longevity involves a convergent evolution of cellular mechanisms that prevent the accu-
mulation of mutations (Cagan et al., 2022). It remains unclear which cellular mechanisms
are critical to allow the evolution of a large body mass while keeping cancer at bay.
Methods: Adding to existing data linking cellular replicative potential and species body
mass (Lorenzini et al., 2005), we have grown a total of 84 skin ﬁbroblast cell strains from 40
donors of 17 mammalian species and analyzed their Hayﬂick's limit, i.e., their senescent
plateau, and eventual spontaneous immortalization escape. The correlation of immortal-
ization and replicative capacity of the species with their longevity, body mass and meta-
bolism has been assessed through phylogenetic multiple linear regression (MLR).
Results: The immortalization probability is negatively related to species body mass. The


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new evaluation and additional data about replicative potential strengthen our previous
observation, conﬁrming that stable and extended proliferation is strongly correlated with
the evolution of a large body mass rather than lifespan.
Conclusion: The relation between immortalization and body mass suggests a need to evolve
stringent mechanisms that control genetic stability during the evolution of a large body
mass.
* Corresponding author. Department of Biomedical and Neuromotor Sciences, University of Bologna, Via Zamboni, 33, Bologna 40126,
Italy.
E-mail address: matteo.perillo2@unibo.it (M. Perillo).
Peer review under responsibility of Chang Gung University.
Available online at www.sciencedirect.com
ScienceDirect
Biomedical Journal
journal homepage: www.elsevier.com/locate/bj
b i o m e d i c a l j o u r n a l 4 6 ( 2 0 2 3 ) 1 0 0 5 9 6
https://doi.org/10.1016/j.bj.2023.100596
2319-4170/© 2023 The Authors. Published by Elsevier B.V. on behalf of Chang Gung University. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).


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Our sense of direction: progress, controversies and challenges
Kathleen E Cullen1, Jeffrey S Taube2
1Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, Maryland, 
USA.
2Department of Psychological & Brain Sciences, Dartmouth College, Hanover, New Hampshire, 
USA.
Abstract
In this Perspective, we evaluate current progress in understanding how the brain encodes our sense 
of direction, within the context of parallel work focused on how early vestibular pathways encode 
self-motion. In particular, we discuss how these systems work together and provide evidence that 
they involve common mechanisms. We first consider the classic view of the head direction cell 
and results of recent experiments in rodents and primates indicating that inputs to these neurons 
encode multimodal information during self-motion, such as proprioceptive and motor efference 
copy signals, including gaze-related information. We also consider the paradox that, while the 
head-direction network is generally assumed to generate a fixed representation of perceived 
directional heading, this computation would need to be dynamically updated when the relationship 
between voluntary motor command and its sensory consequences changes. Such situations include 
navigation in virtual reality and head-restricted conditions, since the natural relationship between 
visual and extravisual cues is altered.
Sense of direction versus a self-motion-detection system
Our ability to keep track of where we are going relative to where we have been requires 
knowledge of our ongoing location and orientation. In everyday life, this ability, or sense 
of direction, depends on the integration of both visual and nonvisual cues, including 
vestibular and proprioceptive information. The visual system provides retinal image-motion 
(optic flow) cues, which have long been known to provide a powerful sense of motion 
through an otherwise stationary environment1. In addition, extravisual cues make important


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contributions to navigation and are thought to contribute to path integration or dead 
reckoning (Box 1) in the absence of visual cues (for review see refs. 2,3). Notably, 
information sent to the brain by the vestibular nerve allows the estimation of head 
displacement (both linear and angular) in the absence of visual cues4–8. Further, during 
locomotion, proprioceptive and/or motor-related signals also contribute significantly to the 
estimation of self-motion. For example, estimates of distance traveled and self-velocity are 
Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.
Correspondence should be addressed to K.E.C. (kathleen.cullen@jhu.edu) or J.S.T. (jeffrey.taube@dartmouth.edu). 
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
HHS Public Access
Author manuscript
Nat Neurosci. Author manuscript; available in PMC 2023 June 19.
Published in final edited form as:
Nat Neurosci. 2017 October 26; 20(11): 1465–1473. doi:10.1038/nn.4658.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript


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m Effect of Selective Lesions of Nucleus Accumbens -Opioid Receptor-Expressing Cells on Heroin Self-Administration in Oprm1-Cre Male and Female Rats: A Study with Novel Knock-in Rats


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Systems/Circuits
Effect of Selective Lesions of Nucleus Accumbens m-Opioid
Receptor-Expressing Cells on Heroin Self-Administration in
Male and Female Rats: A Study with Novel Oprm1-Cre
Knock-in Rats
Jennifer M. Bossert,1 Carlos A. Mejias-Aponte,1
Thomas Saunders,2 Lindsay Altidor,1 Michael Emery,2
Ida Fredriksson,1 Ashley Batista,1 Sarah M. Claypool,1 Kiera E. Caldwell,1 David J. Reiner,1 Jonathan J. Chow,1
Matthew Foltz,2
Vivek Kumar,2 Audrey Seasholtz,2 Elizabeth Hughes,2 Wanda Filipiak,2
Brandon K. Harvey,1
Christopher T. Richie,1 Francois Vautier,1 Juan L. Gomez,1
Michael Michaelides,1 Brigitte L. Kieffer,3
Stanley J. Watson,2 Huda Akil,2 and
Yavin Shaham1
1Intramural Research Program, National Institute on Drug Abuse–National Institutes of Health, Baltimore, Maryland, 21224, 2University of
Michigan, Ann Arbor, Michigan, 48104, and 3University of Strasbourg–Institut National de la Santé et de la Recherche Médicale U1114, Strasbourg,
France, 67084
The brain m-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs.
However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known
because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in
transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic
and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-
expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH
chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hip-
pocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and
wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no


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genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-
related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing
cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had
a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an
Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of
opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indi-
cates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and
female rats.
Key words: mu opioid receptor; knockin; CRISPR; heroin self-administration; pain; caspase 3 lesion
Received Nov. 2, 2022; revised Dec. 29, 2022; accepted Jan. 18, 2023.
Author contributions: J.M.B., C.A.M.-A., T.S., V.K., A.S., B.K.H., C.T.R., J.L.G., M.M., B.L.K., S.J.W., H.A., and Y.S.
designed research; J.M.B., C.A.M.-A., T.S., L.A., M.E., I.F., A.B., S.M.C., K.E.C., D.J.R., M.F., V.K., E.H., W.F., C.T.R., J.L.G.,
and M.M. performed research; J.M.B., C.A.M.-A., L.A., M.E., A.B., K.E.C., J.J.C., M.F., V.K., E.H., W.F., J.L.G., M.M., and Y.S.
analyzed data; J.M.B. and Y.S. wrote the first draft of the paper; J.M.B., C.A.M.-A., T.S., M.E., I.F., A.B., S.M.C., K.E.C.,
D.J.R., J.J.C., M.F., V.K., A.S., E.H., W.F., B.K.H., C.T.R., F.V., J.L.G., M.M., B.L.K., S.J.W., H.A., and Y.S. edited the paper;
T.S. and F.V. contributed unpublished reagents/analytic tools.
The work was supported by Intramural Research Program of National Institute on Drug Abuse ZIA-DA000434-
22 and ZIA-DA000069; and National Institute on Drug Abuse U01DA043098, ONR 00014-19-1-2149, and HDRF to
H.A. and S.J.W. The rats bred at National Institute on Drug Abuse were registered with the Rat Genome


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Database (RGD #155641245) and deposited at the Rat Resource and Research Center (RRRC #975).
C.A. Mejias-Aponte’s present address: Intramural Research Program, National Eye Institute-National
Institutes of Health, Bethesda, Maryland.
The authors declare no competing financial interests.
Correspondence should be addressed to Jennifer M. Bossert at jennifer.bossert@nih.gov or Yavin Shaham
at yavin.shaham@nih.gov.
https://doi.org/10.1523/JNEUROSCI.2049-22.2023
Copyright © 2023 the authors
1692 • The Journal of Neuroscience, March 8, 2023 • 43(10):1692–1713


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The use of new CRISPR tools in cardiovascular research and 
medicine
Masataka Nishiga1, Chun Liu1, Lei S. Qi2, Joseph C. Wu1,3
1Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA.
2Departments of Bioengineering and Chemical & Systems Biology and ChEM-H, Stanford 
University School of Medicine, Stanford, CA, USA.
3Division of Cardiovascular Medicine, Department of Medicine and Department of Radiology, 
Stanford University School of Medicine, Stanford, CA, USA.
Abstract
Many novel CRISPR-based genome editing tools, with a wide variety of applications, have been 
developed in the past few years. The original CRISPR/Cas9 (clustered regularly interspaced 
short palindromic repeats/CRISPR-associated protein 9) system was developed as a tool to alter 
genomic sequences within living organisms in a simple way. However, the functions of new 
CRISPR tools are not limited to conventional genome editing mediated by nonhomologous end-
joining or homology-directed repair, but expand into gene-expression control, epigenome editing, 
single nucleotide editing, RNA editing and live-cell imaging. Furthermore, genetic perturbation 
screening by multiplexing guide RNAs is gaining popularity as a method of identifying causative 
genes and pathways in an unbiased manner. New CRISPR tools can also be applied to ex vivo 
or in vivo therapeutic genome editing for the treatment of conditions such as hyperlipidaemia. 
In this Review, we first provide an overview of the diverse new CRISPR tools that have been 
developed to date. Second, we summarize how these new CRISPR tools are being used to study 
biological processes and disease mechanisms in cardiovascular research and medicine. Finally, we 
discuss the prospect of therapeutic genome editing by CRISPR tools to cure genetic cardiovascular 
diseases.
Introduction
The development of genome editing technology has revolutionized biomedical research, 
particularly since the introduction of the CRISPR/Cas9 (clustered regularly interspaced


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short palindromic repeats/CRISPR-associated protein 9) method. This system was initially 
discovered as part of the immune response in bacteria1, and was then applied to eukaryotic 
genome editing2–4. The simplicity of the CRISPR/Cas9 system has made genome editing 
more accessible and easier than with traditional DNA-editing techniques. As a genome 
editing tool, the system consists of two basic components — an endonuclease Cas9, which 
joewu@stanford.edu (JCW), mnishiga@stanford.edu (MN). 
Competing interest statement
The authors declare no competing interests.
HHS Public Access
Author manuscript
Nat Rev Cardiol. Author manuscript; available in PMC 2023 June 21.
Published in final edited form as:
Nat Rev Cardiol. 2022 August ; 19(8): 505–521. doi:10.1038/s41569-021-00669-3.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript


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Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response


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RESEARCH ARTICLE
Viral vector delivered immunogen focuses
HIV-1 antibody specificity and increases
durability of the circulating antibody recall
response
LaTonya D. WilliamsID1,2,3☯*, Xiaoying Shen2,3☯, Sheetal S. Sawant1,2,3, Siriwat Akapirat4,
Lindsay C. Dahora1,2,3,5, Matthew Zirui Tay1,2,3,6, Sherry Stanfield-Oakley1,2,3,
Saintedym Wills1,2,3,5, Derrick Goodman1,2,3, DeAnna Tenney1,2,3, Rachel L. Spreng1,2,3,
Lu Zhang1,2,3, Nicole L. Yates1,2,3, David C. Montefiori2,3, Michael A. Eller7,8¤a,
David Easterhoff3,9¤b, Thomas J. Hope10, Supachai Rerks-Ngarm11,
Punnee Pittisuttithum12, Sorachai Nitayaphan12, Jean-Louis Excler7¤c, Jerome H. Kim7¤c,
Nelson L. Michael7, Merlin L. Robb7,8, Robert J. O’Connell4, Nicos Karasavvas4¤d¤e,
Sandhya Vasan7,8, Guido Ferrari1,2,3,6‡, Georgia D. TomarasID1,2,3,5,6‡*, RV305 study team¶
1 Center for Human Systems Immunology, Duke University School of Medicine, Durham, North Carolina,
United States of America, 2 Department of Surgery, Duke University School of Medicine, Durham, North
Carolina, United States of America, 3 Duke Human Vaccine Institute, Duke University School of Medicine,
Durham, North Carolina, United States of America, 4 Department of Retrovirology, US Army Medical
Directorate, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand, 5 Department of
Immunology, Duke University School of Medicine, Durham, North Carolina, United States of America,
6 Department of Molecular Genetics Microbiology, Duke University School of Medicine, Durham, North
Carolina, United States of America, 7 US Military HIV Research Program, Walter Reed Army Institute of
Research, Silver Spring, Maryland, United States of America, 8 Henry M. Jackson Foundation for the
Advancement of Military Medicine, Bethesda, Maryland, United States of America, 9 Department of
Medicine, Duke University School of Medicine, Durham, North Carolina, United States of America,
10 Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University,


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Chicago, Illinois, United States of America, 11 Thai Ministry of Public Health, Nonthaburi, Thailand, 12 Royal
Thai Army Component, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand
☯ These authors contributed equally to this work.
¤a Current address: Vaccine Translational Research Branch, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Rockville, Maryland, United States of America
¤b Current address: Moderna Therapeutics, Cambridge, Massachusetts, United States of America
¤c Current address: International Vaccine Institute, Seoul, Republic of Korea
¤d Current address: Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda,
Maryland, United States of America
¤e Current address: Diagnostics and Countermeasures Branch, Walter Reed Army Institute of Research,
Silver Spring, Maryland, United States of America
‡ These authors are joint senior authors on this work.
¶ RV305 study team members are listed in the Acknowledgements
* latonya.williams@duke.edu (LDW); gdt@duke.edu (GDT)
Abstract
The modestly efficacious HIV-1 vaccine regimen (RV144) conferred 31% vaccine efficacy
at 3 years following the four-shot immunization series, coupled with rapid waning of putative
immune correlates of decreased infection risk. New strategies to increase magnitude and
durability of protective immunity are critically needed. The RV305 HIV-1 clinical trial evalu-
ated the immunological impact of a follow-up boost of HIV-1-uninfected RV144 recipients
after 6–8 years with RV144 immunogens (ALVAC-HIV alone, AIDSVAX B/E gp120 alone,
PLOS PATHOGENS
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011359
May 31, 2023
1 / 46
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OPEN ACCESS
Citation: Williams LD, Shen X, Sawant SS, Akapirat
S, Dahora LC, Tay MZ, et al. (2023) Viral vector
delivered immunogen focuses HIV-1 antibody
specificity and increases durability of the circulating
antibody recall response. PLoS Pathog 19(5):


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e1011359. https://doi.org/10.1371/journal.
ppat.1011359
Editor: Daniel C. Douek, Vaccine Research Center,
UNITED STATES
Received: November 30, 2022
Accepted: April 14, 2023
Published: May 31, 2023
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced,
distributed, transmitted, modified, built upon, or
otherwise used by anyone for any lawful purpose.
The work is made available under the Creative
Commons CC0 public domain dedication.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Funding: This work was supported by the Bill and
Melinda Gates Foundation (https://www.
gatesfoundation.org/) Collaboration for AIDS
Vaccine Discovery grants (OPP1033098 [Dr.
Barton F. Haynes (P.I).], OPP1032144 [D.C.M.]),
Henry M. Jackson Foundation (https://www.hjf.org/
) for the Advancement of Military Science


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Lack of Toxicity in Nonhuman Primates Receiving Clinically Relevant Doses of an AAV9U7snRNA Vector Designed to Induce DMD Exon 2 Skipping


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RESEARCH ARTICLES
Lack of Toxicity in Nonhuman Primates Receiving Clinically
Relevant Doses of an AAV9.U7snRNA Vector Designed
to Induce DMD Exon 2 Skipping
Liubov V. Gushchina,1 Emma C. Frair,1 Natalie Rohan,1 Adrienne J. Bradley,1 Tabatha R. Simmons,1
Hemantkumar D. Chavan,2 Hsin-Jung Chou,3 Michelle Eggers,3 Megan A. Waldrop,1,4
Nicolas Wein,1,4 and Kevin M. Flanigan1,4,5,*
1The Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children’s Hospital, and the Departments of 4Pediatrics and 5Neurology, The Ohio State
University, Columbus, Ohio, USA.
2Charles River Laboratories, Wilmington, Massachusetts, USA.
3Audentes Therapeutics, San Francisco, California, USA.
Therapeutic exon skipping as a treatment for Duchenne muscular dystrophy (DMD) has largely concentrated on the
delivery of antisense oligomers to treat out-of-frame exon deletions. Here we report on the preclinical development of an
adeno-associated virus (AAV)-encapsidated viral vector containing four copies of the noncoding U7 small nuclear RNA
(U7snRNA), each targeted to either the splice donor or the splice acceptor sites of DMD exon 2. We have previously
shown that delivery of this vector (scAAV9.U7.ACCA) to the Dup2 mouse model results in expression of full-length
dystrophin from wild-type DMD mRNA, as well as an internal ribosome entry site (IRES)-driven isoform translated only
in the absence of exon 2 (deletion exon 2 [Del2] mRNA). Here we present the data from a rigorous dose escalation
toxicity study in nonhuman primates, encompassing two doses (3 · 1013 and 8 · 1013 vg/kg) and two time points (3 and
6 months postinjection). No evidence for signiﬁcant toxicity was seen by biochemical, histopathologic, or clinical
measures, providing evidence for safety that led to initiation of a ﬁrst-in-human clinical trial.
Keywords: Duchenne muscular dystrophy, adeno-associated virus, noncoding U7snRNA
INTRODUCTION
THERAPEUTIC RESTORATION OF an open reading frame by
selectively excluding exons from a mature mRNA tran-


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script, or exon skipping, is an approach that is already in
clinical use for a subset of patients with Duchenne mus-
cular dystrophy (DMD).1 Currently approved therapies
use repeated intravenous infusions of antisense oligomers
complementary to target exons of interest adjacent to out-
of-frame exon deletions, restoring an open mRNA reading
frame and allowing translation of internally deleted but
partially functional proteins. An alternative viral vector-
based approach uses a noncoding U7snRNA modiﬁed with
a terminal sequence targeting exon. As a result, the single-
stranded U7snRNA, stabilized by interaction with the
SmOpt protein, binds to the pre-mRNA, resulting in
the exclusion of this exon.2–7 This approach is likely to
be advantageous in that expression of U7snRNA from the
adeno-associated virus (AAV) genome and subsequent
exon skipping is long-standing,8 which may allow a single
treatment rather than weekly infusions.
We have developed such a viral vector carrying se-
quences targeting exon 2 within a self-complementary (sc)
AAV genome. Our vector, scAAV9.U7.ACCA, carries
four copies of U7snRNA, with two directed toward the
splice acceptor sequence (sequence A) and two targeting
the splice donor sequence (sequence C) encapsidated
within AAV9. In silico analysis has shown these sequen-
ces to be speciﬁc to the intron/exon junctions for human
exon 2, and the targeted sequences are 100% identical
between human and nonhuman primates (NHPs). We have
developed this vector intending to initially target not out-
of-frame deletions, but instead single-exon duplications
in the DMD gene. In contrast to the case with deletions,
skipping of one of the two duplicated exons would restore
*Correspondence: Dr. Kevin M. Flanigan, The Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children’s Hospital, 700 Children’s Drive, Columbus,
OH 43205, USA. E-mail: kevin.ﬂanigan@nationwidechildrens.org


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ª Liubov V. Gushchina et al., 2021; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License [CC-BY]
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
882 j
HUMAN GENE THERAPY, VOLUME 32, NUMBERS 17–18
DOI: 10.1089/hum.2020.286
Mary Ann Liebert, Inc.


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Concentration and reactivity of the sulphydryl population the membrane of intact erythrocytes in patients with rheumatoid arthritis


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Annals ofthe Rheumatic Diseases 1990; 49: 668-671
Concentration and reactivity of the sulphydryl
group population on the membrane of intact
erythrocytes in patients with rheumatoid arthritis
C Chilles, M Mulheron, F M McCrae, J Reglinksi, W E Smith, M Brzeski, R D Sturrock
Abstract
The
sulphydryl
population
on
erythrocyte
membrane is shown to vary as a function of the
nutritional status of the cell. When an assay
based on reaction with Eliman's reagent and
controlled conditions were used the mean values
(SEM) for the sulphydryl population on the
membranes of normal erythrocytes incubated
overnight in the presence and absence ofglucose
were found to be 3-29 (0.27) and 2 56 (0.25)
million sulphydryl functions per cell respect-
ively. Under identical conditions rheumatoid
erythrocytes incubated
in the presence and
absence
of glucose were found
to have
a
significantly lower sulphydryl population-1-54
(0.08) and 1-15 (0.08) million respectively.
The predominant concentrations ofsulphydryl
groups on the membrane are found at sites on
the transmembrane proteins and, in particular,
on the hexose transport protein. By influencing
the nutritional status of the cells significant
differences in activity between the normal and
diseased state have been identified and these
may have a role in the aetiology of rheumatoid
arthritis by altering the response of cells to
oxidative stress.
Department of Pure and
Applied Chemistry,
295 Cathedral St,
Strathelyde University,
Glasgow GI 1XL, UK
C Chilles
M Mulberon
F McCrae
J Reglinski
W E Smith
Centre for Rheumatic
Diseases and University
Department of Medicine,
Glasgow Royal Infirmary,
Glasgow G4 OSF, UK
M Brzeski
R D Sturrock
Correspondence to:
Dr Reglinski
Accepted for publication
23 October 1989
*-As a direct result of the ionic
strength of the medium used
in this study the molar
extinction coefficient of
Ellman's anion was found to
be lower than the published
value of 14 150 cm2/mol at
412 nm.9
Plasma
sulphydryl
group
concentrations
are


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are
known to be low in rheumatoid arthritis and to
be
raised
to
some
extent
during
treatment
with penicillamine' and gold compounds.2 The
sulphydryl
group
concentration
can
be
sub-
divided into fast or reactive thiol and slow or
comparatively unreactive thiol. It is mainly the fast
thiol which is affected during treatment.3 Both
gold compounds
and
penicillamine would
be
expected to react with the available thiols in vivo.
During treatment with these compounds, however,
alteration in the plasma thiol concentration tends
to be slow and follows signs ofclinical improvement
rather than rises in plasma gold or penicillamine
concentrations. Thus the change is likely to follow
amelioration of disease activity rather than cause
it.
The intracellular
thiol concentration (mainly
glutathione) increases within the first few weeks of
treatment in patients who subsequently improve.4
We have noted that during treatment with sodium
aurothiomalate most of the gold present in the red
cell is in the membrane rather than the cytosol,
particularly in non-smokers,5 6 and that in the in
vitro experiments with penicillamine some peni-
cillamine crossed the membrane but much of it
was absorbed or reacted causing changes in cellular
function.7 8
Likely sites for these therapeutic agents to react
on the membrane are the available exofacial thiols,
and in this preliminary study we have investigated
the question of how such groups are altered in
rheumatoid arthritis. This initial study was carried
out with patients who were not being treated
with disease modifying agents as these drugs
may further affect the thiol status of the cell
membrane.5-7
Materials and methods
All reagents used in this study were commercially
obtained unless otherwise stated. Whole blood was
collected in heparinised anticoagulant tubes from
10 healthy normal volunteers and 10 patients with
classical or definite rheumatoid arthritis as defined
by the American Rheumatism Association criteria.
The
latter were not
receiving any prescribed
second line or cytotoxic drugs.


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Samples were centrifuged at 3000 rpm for 10
minutes and the supernatant fluid and the buffy
white coat removed by suction. The resulting cells
were washed three times in 0-125 M Na2HPO4;
0 15 M NaCl buffer (adjusted to pH 7-4 with
concentrated
hydrochloric
acid).
The
samples
were divided into two roughly equal volumes and
stored in buffer and buffer/glucose (5 mM) over-
night at 4°C in sealed containers (about 16 hours)
to produce glucose depleted and activated cells.
After this the cells were washed with their respec-
tive media: phosphate/saline, with or without 5
mM glucose. The supernatants were carefully
checked for lysis by eye and by spectrophotometry
(k= 541 nm). Lysed samples were discarded.
The number of cells in a given volume of pellet
were counted, and 0 5 ml was transferred to a
sterile container with 8-5 ml of the respective
buffers and 1-0 ml of 10-3 M Ellman's solution.
The container was gently mixed and allowed to
react for 30 minutes. The suspensions were then
centrifuged (3000 rpm for 10 minutes), the super-
natant removed, and the visible spectrum (600-
350 nm) recorded. The concentration of Ellman's
anion was calculated using Beer's law (molar
absorbance Emax for ES
13600 cm2/mol, 412
nm, and used in the equation below to give the
total number of thiols present on the surface of the
membrane.
Total number of thiols
present on the
erythrocyte membrane
[ES - I x volumex Avogadro's No
number of cellsx 1000
where [ES-
is the concentration of ElIman's
anion in solution calculated using Beer's law.
The change in sulphydryl group concentration
on the membranes of depleted cells as a result of
glucose addition is monitored by adding sugar to a
depleted erythrocyte suspension and then sampl-
ing at specific time intervals before addition of
Ellman's reagent.
Results and Discussion
ElIman's reagent (ESSE) is an accepted method of
assaying biological fluids'1'2 and membranes'3 14
668


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Annals ofthe Rheumatic Diseases 1990; 49: 757-762
Specificity of rheumatoid factors in relation to the
disease state in rheumatoid arthritis
M G Jones, M E Shipley, J P Hearn, F C Hay
Abstract
Rheumatoid factors found in patients with
rheumatoid arthritis react with human IgG
and with IgG from some other species. The
levels of rheumatoid factor give some indi-
cation of prognosis, albeit a rather poor one in
this highly variable disease. The high degree
ofvariability may, in part, be due to differences
in the fine specificity of the rheumatoid factor
in each individual patient, leading to differ-
ences in the types ofimmune complex formed.
To study this hypothesis the fine specificity of
rheumatoid factors of the IgM, IgA, and IgG
classes for IgG from human, baboon, orang-
utan, macaque, owl monkey, gorilla, marmo-
set, cow, pig, sheep, goat, horse, mouse, and
chicken was examined. Differential reactivity
for these species was found and associations
between the presence of rheumatoid factor
and the development of moderate or severe
erosions.
Immunology Departnent,
St George's Hospital
Medical School,
Cranmer Terrace,
Tooting, London
SW17 ORE
M G Jones
F C Hay
Departments of
Immunology and
Rheumatology Research,
University College and
Middlesex School of
Medicine, Arthur Stanley
House, 40-50 Tottenham
Street, London WIP 9PG
M E Shipley
MRC/AFRC
Comparative Physiology
Research Group,
Institute of Zoology,
Regent's Park,
London NW1 4RY
Correspondence to:
Dr Jones.
Accepted for publication
20 November 1989
Rheumatoid factors bind to normal human IgG
and IgG from many different species.' It is long
established
that
raised
levels
of such
anti-
globulins
are
associated
with
rheumatoid
arthritis and that this antiglobulin activity is
found in IgM, IgA, and IgG classes.2 The
degree of binding of rheumatoid factor to IgG of
different
species
is
controversial,
as
is
the
clinical significance of the spectrum of such
polyspecific rheumatoid factor activity. Some
have shown that rheumatoid factors binding


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with rabbit IgG are more specific for rheumatoid
arthritis than are those reactive with human
IgG.3 4 In contrast, others suggest that human
rather than rabbit IgG is a better discriminator
between patients with rheumatoid arthritis and
controls.5 It has been shown that rheumatoid
factor binding to human IgG has similar bind-
ing activity with the primate rhesus monkey
IgG, but lower binding with non-primate rabbit,
horse, sheep, and cow.6
The aim of this study was to examine the fine
specificity of rheumatoid factors in each of the
three main classes, IgM, IgA, and IgG, and to
compare these results with various measures of
disease process and outcome (particularly degree
of erosiveness and presence of extra-articular
manifestations) to see if any can be linked to a
particular isotype or specificity of rheumatoid
factor. The specificities were measured with a
panel of IgGs from a variety of primate and non-
primate species and their relation
to disease
activity at the time of drawing the blood was
established. To discover whether the pattern of
reactivity with
the panel of IgGs had any
predictive value stored sera from patients with
rheumatoid arthritis were studied and the results
compared with
their outcome
at long term
follow up.
Materials and methods
IgG PREPARATION
IgG from
various
species
was
purified
by
passing the serum over a DEAE-cellulose column
and eluting with a gradient ofincreasing molarity
of 0-017 M to 0-5 M phosphate buffer pH 8-0.
SOLID PHASE RADIOIMMUNOASSAY
Ninety six well, flexible plates were coated with
100
1t of a
10
[ig/ml protein
solution
in
phosphate buffered saline (PBS) pH 7-2 and
incubated for one hour at 37°C and overnight at
4°C. Unbound proteins were removed by three
washes with cold PBS.
Serum (1 1d) was added to each well with 100
1t 0 05 M TRIS HCI pH 8-0 containing 0 05%
Tween 20 (TRIS HCI Tween), and incubated
for one hour at 37°C (serum dilution 1:100).
The plates were then washed three times with
cold TRIS HCI Tween.
Depending on the class of rheumatoid factor
being


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being
detected,
100
1t
of
5
,tg/ml
rabbit
antihuman IgM, IgA, or Fd of IgG in TRIS
HCI Tween were added and incubated for one
hour at 37°C. The plates were then washed
three times with cold TRIS HCI Tween. A final
layer of 1 ,ug 1251 labelled goat antirabbit IgG
was added in 100
,tl TRIS HC1 Tween and
incubated for one hour at 37°C. The plates were
washed three times with cold TRIS HC1 Tween
and counted in a gammacounter.
SOLID PHASE CAPTURE ASSAY
Ninety six well, flexible plates were coated with
100 [tl of a 10 [tg/ml protein solution in PBS and
incubated for one hour at 37°C and overnight at
4°C. Unbound proteins were removed with
three washes of cold PBS.
Serum (1 ,ul) was added to each well with 100
,ul TRIS HC1 Tween and incubated for one hour
at 37°C (serum dilution 1:100). The plates were
then washed three times with cold TRIS HCI
Tween.
One hundred
microlitres of 5
,tg/ml
1251
aggregated IgG was added in TRIS HC1 Tween
and incubated for four hours at 37°C. The plates
were washed three times with cold TRIS HCI
Tween and counted in a gammacounter.
757


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INTRODUCTION
The incidence of malignant tumors in Japanese 
young men is 86.2 in 100,000 adolescents and young 
adults, affecting approximately 7,600 people per year [1]. 
Katanoda et al [2] investigated the incidence of malig-
nant disease among young people in Japan from 2009 
to 2011. Per million population, there were 120.2 aged 
0–14 years, 130.5 aged 15–19 years, 229.0 aged 20–29 
years, and 537.3 males aged 30–39 years. This data was 
similar to that of the United States and World data 
(World Health Organization) [2]. As in other developed 
countries, advances in treatment have significantly 
improved outcomes for young patients with cancer. For 
advanced testicular cancer, the 3-year survival rates for 
good-, intermediate-, and poor-risk patients are 94.7%, 
Received: May 17, 2022  Revised: Aug 30, 2022  Accepted: Sep 22, 2022  Published online Jan 1, 2023
Correspondence to: Yasushi Yumura  
 https://orcid.org/0000-0003-0909-478X
Reproduction Center, Yokohama City University Medical Center, Urafune-cho 4-57, Minami-ku, Yokohama, Kanagawa 232-0024, Japan.
Tel: +81-45-261-5656, Fax: 81-45-253-1962, E-mail: yumura@yokohama-cu.ac.jp
Copyright © 2023 Korean Society for Sexual Medicine and Andrology
Long-Term Fertility Function Sequelae in Young 
Male Cancer Survivors
Yasushi Yumura , Teppei Takeshima , Mitsuru Komeya , Jurii Karibe, Shinnosuke Kuroda ,  
Tomoki Saito
Reproduction Center, Yokohama City University Medical Center, Yokohama, Kanagawa, Japan
With advances in cancer treatment, such as cytotoxic chemotherapy and radiotherapy, grave new sequelae of treatment have 
emerged for young cancer survivors. One sequela that cannot be overlooked is male infertility, with reportedly 15% to 30% 
of cancer survivors losing their fertility potential. Cytotoxic therapy influences spermatogenesis at least temporarily, and in 
some cases, permanently. The degree of spermatogenesis impairment depends on the combination of drugs used, their cumu-


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lative dose, and the level of radiation. The American Society of Clinical Oncology has created an index to classify the risks to 
fertility based on treatment. Medical professionals currently use this risk classification in fertility preservation (FP) programs. 
FP programs are currently being promoted to prevent spermatogenesis failure resulting from cancer treatment. For patients 
who are able to ejaculate and whose semen contains sperm, the semen (sperm) is cryopreserved. Moreover, for patients who 
lack the ability to ejaculate, those with azoospermia or severe oligozoospermia, and those who have not attained puberty (i.e., 
spermatogenesis has not begun), testicular biopsy is performed to collect the sperm or germ cells and cryopreserve them. This 
method of culturing germ cells to differentiate the sperm has been successful in some animal models, but not in humans. FP 
has recently gained popularity; however, some oncologists and medical professionals involved in cancer treatment still lack 
adequate knowledge of these procedures. This hinders the dissemination of information to patients and the execution of FP. 
Information sharing and collaboration between reproductive medicine specialists and oncologists is extremely important for 
the development of FP. In Japan, the network of clinics and hospitals that support FP is expanding across prefectures.
Keywords: 
Keywords: Cancer; Chemotherapy; Fertility preservation; Male infertility; Radiotherapy; Sperm cryopreservation
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) 
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Review Article
pISSN: 2287-4208 / eISSN: 2287-4690
World J Mens Health 2023 Apr 41(2): 255-271
https://doi.org/10.5534/wjmh.220102
Male reproductive health and infertility


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INTRODUCTION
Infertility is a problem affecting one in six couples, 
which is approximately 15% of the population wishing 
to start a family. In 30% of infertile couples, the male 
factor is a major cause [1,2]. A man is considered in-
fertile if he is not able to induce a pregnancy after at 
least 12 months of regular intercourse without contra-
ception [3]. In 30% to 45% of infertile men, the cause of 
the abnormal semen parameters is not identified (idio-
pathic male infertility) [4]. The term idiopathic is used 
only if defined causes of male infertility can be exclud-
ed, such as: sperm autoimmunity, sexual and/or ejacu-
latory dysfunction, cryptorchidism, acquired testicular 
Received: Sep 14, 2022   Accepted: Oct 15, 2022   Published online Jan 4, 2023
Correspondence to: Tanja Kunej  
 https://orcid.org/0000-0002-0465-1762
Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Domžale 1230, Slovenia.
Tel: +386-13203890, Fax: +386-17241005, E-mail: tanja.kunej@bf.uni-lj.si
*These authors contributed equally to this work as co-first authors.
Copyright © 2023 Korean Society for Sexual Medicine and Andrology
Towards a Multi-Omics of Male Infertility
Ana Ogrinc Wagner* , Aleksander Turk* , Tanja Kunej
Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Domžale, Slovenia
Infertility is a common problem affecting one in six couples and in 30% of infertile couples, the male factor is a major cause. 
A large number of genes are involved in spermatogenesis and a significant proportion of male infertility phenotypes are of 
genetic origin. Studies on infertility have so far primarily focused on chromosomal abnormalities and sequence variants in 
protein-coding genes and have identified a large number of disease-associated genes. However, it has been shown that a 
multitude of factors across various omics levels also contribute to infertility phenotypes. The complexity of male infertility has


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led to the understanding that an integrated, multi-omics analysis may be optimal for unravelling this disease. While there is a 
vast array of different factors across omics levels associated with infertility, the present review focuses on known factors from 
the genomics, epigenomics, transcriptomics, proteomics, metabolomics, glycomics, lipidomics, miRNomics, and integrated 
omics levels. These include: repeat expansions in AR, POLG, ATXN1, DMPK, and SHBG, multiple SNPs, copy number vari-
ants in the AZF region, disregulated miRNAs, altered H3K9 methylation, differential MTHFR, MEG3, PEG1, and LIT1 meth-
ylation, altered protamine ratios and protein hypo/hyperphosphorylation. This integrative review presents a step towards a 
multi-omics approach to understanding the complex etiology of male infertility. Currently only a few genetic factors, namely 
chromosomal abnormalities and Y chromosome microdeletions, are routinely tested in infertile men undergoing intracyto-
plasmic sperm injection. A multi-omics approach to understanding infertility phenotypes may yield a more holistic view of 
the disease and contribute to the development of improved screening methods and treatment options. Therefore, beside dis-
covering as of yet unknown genetic causes of infertility, integrating multiple fields of study could yield valuable contributions 
to the understanding of disease development. Future multi-omics studies will enable to synthesise fragmented information 
and facilitate biomarker discovery and treatments in male infertility.
Keywords: 
Keywords: DNA methylation; Epigenetics; Expression; Male infertility; Multi-omics; Non-coding RNAs (ncRNAs)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) 
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Review Article
pISSN: 2287-4208 / eISSN: 2287-4690


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World J Mens Health 2023 Apr 41(2): 272-288
https://doi.org/10.5534/wjmh.220186
Male reproductive health and infertility


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Annals ofthe Rheumatic Diseases 1992; 51: 399-401
Immunoadsorbent plasmapheresis for a patient with
antiphospholipid syndrome during pregnancy
S Kobayashi, N Tamura, H Tsuda, C Mokuno, H Hashimoto, S Hirose
Abstract
The case of a 34 year old woman with
systemic lupus erythematosus with a history
of three previous recurrent abortions and
lupus anticoagulant and anticardiolipin anti-
bodies is reported. Immunoadsorbent plasma-
pheresis with a dextran sulphate column was
used to remove lupus anticoagulant,
anti-
cardiolipin antibodies, and antibodies to DNA
during her fourth pregnancy in combination
with low doses of aspirin and prednisolone.
Although
during
the
course
of treatment
prednisolone was transiently increased to 30
mg/day owing to an asymptomatic increase of
lupus anticoagulant and anticardiolipin anti-
bodies, the
levels of lupus anticoagulant,
anticardiolipin antibodies, and antibodies to
DNA were decreased by immunoadsorbent
plasmapheresis and a baby girl was delivered
successfully by caesarean section. Therefore,
immunoadsorbent plasmapheresis with dex-
tran sulphate seems to reduce the risk of
recurrent abortion in patients with the anti-
phospholipid syndrome.
Department of
Rheumatology,
Juntendo University
School of Medicine,
Tokyo,
Japan
S Kobayashi
N Tamura
H Tsuda
C Mokuno
H Hashimoto
S Hirose
Correspondence to:
Dr Hiroshi Hashimoto,
Department ofRheumatology,
Juntendo University,
School of Medicine,
2-1-1 Hongo,
Bunkyo-ku,
Tokyo 113,
Japan.
Accepted for publication
16 April 1991
Lupus anticoagulant and anticardiolipin anti-
bodies are high risk factors for repeated abortion
and intrauterine death.'13 These factors have
been
associated
with
arterial
or
venous
thrombosis,
or
both.
Lupus
anticoagulant
interferes
at
the
level
of the prothrombin
activation complex of the clotting cascade4 and
may interfere with the production ofprostacyclin
by the vessel wall and other tissues, including
the myometrium, during pregnancy.5
It was noted that treatment with high dose


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prednisolone (40-60 mg/day) in combination
with aspirin (75 mg/day) improved fetal survival
in pregnant women with lupus anticoagulant
and poor obstetric history.' 6 Such treatment
for several months sometimes causes serious
maternal adverse reactions, however, and the
effect of high dose prednisolone on the fetus is
unknown. In addition, therapeutic benefits are
not always obtained and, therefore, alternative
treatments should be evaluated.
Recently, treatment with immunoadsorbent
plasmapheresis using a dextran sulphate column
was introduced for patients with systemic lupus
erythematosus to remove antibodies to DNA,7
and we found that anticardiolipin antibodies
and lupus anticoagulant bind with dextran
sulphate by anionic charge.8 Here, we would
like to report the case of a patient with systemic
lupus erythematosus with lupus anticoagulant,
anticardiolipin antibodies, and recurrent spon-
taneous abortion who was treated witir the new
approach of immunoadsorbent
during pregnancy.
plasmapheresis
Materials and methods
PLASMA SEPARATOR AND ADSORBENT COLUMN
As a plasma separator, hollow fibre membrane
filters of polysulphone with an average pore size
of 0-2 ,tm and effective surface area of 0-5 m2
(SF 05; Kanegafuchi Chemical Industry, Tokyo,
Japan), were used together with Liposorber LA
40 (Kanegafuchi Chemical Industry), in which
dextran sulphate cellulose beads (350 ml) were
packed and used as the sorbent gel for lupus
anticoagulant, anticardiolipin antibodies, and
antibodies to DNA.
PROCEDURE FOR PLASMAPHERESIS
Blood was withdrawn at a rate of 50 ml/min
by a peristaltic pump through a double lumen
catheter inserted into the femoral vein. Plasma
was separated at a speed of 20 ml/min and
passed through the Liposorber in a downward
flow. The filtrate was infused into the patient
with the portion rich in blood cells. The total
extracorporeal
circulation volume was about
400 ml. For anticoagulant, nafamostat mesilate9
(FUT-175; Torni Co, Tokyo, Japan) was used at
an initial dose of 20 mg and then continuously


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infused at a rate of 20 mg/h. In this patient one
litre of plasma was treated for each procedure.
This procedure was carried out two to three
times a week. Nafamostat mesilate, with a half
life of eight minutes, prolongs activated partial
thromboplastin time (APTT), and therefore
APTT, anticardiolipin antibodies, and lupus
anticoagulant were measured before plasma-
pheresis and one day after treatment.
MEASUREMENT OF LUPUS ANTICOAGULANT,
APTT, ANTICARDIOLIPIN ANTIBODIES, AND
ANTIBODIES TO DNA
The APTT was assessed in our laboratory.
Automated APTT reagent10 (General Diagnos-
tics, Morris Plains, NJ, USA) which contained
rabbit brain cephalin and colloidal silica was
used, and all assays were performed on a Coag-
A-Mate/Single Channel (General Diagnostics).
The normal range for APTT is 26-40 seconds
(mean (2SD)). The sample containing lupus
anticoagulant showed a prolonged APTT, which
was not corrected by adding normal plasma.
Anticardiolipin antibodies were measured by
enzyme linked immunosorbent assay (ELISA)
as described." The results were expressed as
units, each unit equivalent to one standard
deviation above the mean of results for 100
serum samples from apparently healthy blood
399


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Annals of the Rheumatic Diseases 1994; 53: 406-420
REVIEW
Animal models for osteoarthritis:
processes, problems and prospects
Kenneth P H Pritzker
Model systems are systems that are used to
simplify complex phenomena and to under-
stand otherwise elusive processes.' An animal
model for human disease can be defined as
a homogenous
set
of animals which have
an inherited,
naturally acquired, or experi-
mentally induced biological process, amenable
to scientific investigation, that in one or more
respects resembles the disease in humans.2 3
Elusive and complex as a precise under-
standing may be, virtually all definitions of
osteoarthritis express the disease as transient
and progressive
structural changes
in
joint
tissues,
principally
articular
cartilage,
sub-
chondral bone, synovium, and synovial fluid.4
Articular cartilage oedema,
fibrillation, and
erosion accompanied
by
chondrocyte
pro-
liferation and decreased
staining of matrix
proteoglycans, subchondral bone thickening,
deformation of the articular surface, osteo-
phyte formation, synovial intimal cell hyper-
plasia, and synovial fibrosis are some of the
standard
features
associated
with
osteo-
arthritis. These changes result from a poorly
understood but very large series of functional
events which may occur over a large fraction
of the life of each affected individual. Bio-
chemical, cell or organ culture models can
provide information and insight concerning
the mechanisms for functional events within
cartilage, bone or synovium or selected cells
within these tissues. These ex vivo models,
inherently more highly controlled than animal
models,
are most useful for understanding
short
term
biological
events,
and
events
isolated from the physiological influences of
adjacent structures and general metabolism.
As yet, ex vivo models cannot simulate the
structural changes which occur in joint tissues
in
animals
over months
to
years.
Hence,
animal models for osteoarthritis are required to
study how the complex structural changes in
tissues
evolve


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evolve
over time, spontaneously
or
following experimental injury, and to deter-
mine how constitutive, environmental or bio-
mechanical risk factors4 may initiate, promote,
or otherwise regulate these changes (table 1).
Animal models are also needed to show how
therapeutic strategies may ameliorate, resolve
or prevent osteoarthritis.6 8
The decision
to use
a particular animal
model to study osteoarthritis is governed by
criteria
of relevance,
appropriateness,
and
availability.3 9 10
Relevance
refers
to
the
comparability of the phenomenon studied in
animals to the corresponding process in human
disease. Animals with characteristics similar to
Table I
Osteoarthritis: constitutional and eznvironmiiental
factors
Constitutional factors
*
Genetics
*
Age
*
Sex, Parity
*
Hormone and metabolic factors
*
Social rank
Environmental factors
*
Nutrition
*
Exercise
*
Drugs
Biomechanical forces
*
Joint instability
*
Displaced joint loads
*
Joint overloading
human disease as well as animals with features
that differ in a known specific manner from
human
osteoarthritis may
provide
relevant
models. Appropriateness refers first to the need
to use animals rather than simpler models to
investigate the process in question and second
to the specific criteria that make one set of
animals more appropriate than others for the
experimental goals and objectives. Simplicity,
predictability
in
reproducing
circumscribed
aspects of the disease, as well as the capacity
to induce new insight, are appropriate factors
when
animal models
are
considered.9
For
example, to test drug effects on an animal
model, simplicity and predictability are para-
mount whereas to understand pathogenesis, a
more complex model that more closely mimics
human
disease may be more
appropriate.
Availability criteria refers to practical factors
such
as
adequacy
of animal
supply,
the
presence of controls, the ease of handling and
environmental maintenance, as well as costs
(table 2).
Table 2
Animal models for osteoarthritis: availability
criteria
0
0
0
S0
0
S0
0
0
S


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string_value:
S0
0
0
S
Genetic homogeneity or heterogeneity
Availability and homogeneity of nutrition and exercise
history
Life span of animal, time span of disease, age of onset
Disease frequency
Availability of matched control population
Anatomical and physiological attributes
Quantitative
*
Joint tissues sufficient for studies
*
Adequate access to synovial fluid
Qualitative
*
Resemblance to human tissues
*
Weight bearing vs non weight bearing joints
Availability of experimental techniques for model
Supply of animals-standardisation
Ease of handling
Access of model to experimental manipulation
*
Drugs
*
Physical intervention
Previous research in model (background knowledge,
peer acceptance)
Costs
Connective Tissue
Research Group,
Mount Sinai Hospital,
Toronto, and
Departments of
Pathology and
Surgery,
University ofToronto,
Toronto, Canada
K P H Pritzker
Correspondence to:
Dr K P H Pritzker,
Department of Pathology,
Mount Sinai Hospital,
600 University Avenue,
Toronto, Ontario,
Canada M5G IX5
406


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Reconstitution of the alternative pathway of complement by plasma infusions given patient with SLE-like syndrome associated with hereditary C3 dysfunction


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Annals of the Rheumatic Diseases 1994; 53: 691-694
CONCISE REPORTS
Reconstitution of the alternative pathway of
complement by plasma infusions given to a patient
with an SLE-like syndrome associated with a
hereditary C3 dysfunction
Bo Nilsson, Ulf R Nilsson, Alex Karlsson-Parra, Gunilla Sjolin-Forsberg, Roger Hallgren
Abstract
Objective-To reconstitute a dysfunctional
form ofcomplement factor C3 in a patient
with
a
systemic
lupus
erythematosus
(SLE) -like syndrome.
Methods-The
propositus
was
treated
with plasma infusions during five sessions
over a period of eight months.
Results-The
alternative
pathway
was
reconstituted
to
normal
levels
for
approximately two to three days after each
infusion. C3 fragments were incorporated
into previously detected deposits of IgG
and IgM at the dermal-epidermal junction
and the immune complex levels gradually
decreased during the whole treatment
period.
Conclusion-The reconstitution appears
to result in the solubilisation of tissue
immune
complexes
and
a
subsequent
transportation to the fixed macrophage
system.
(Ann Rheum Dis 1994; 53: 691-694)
dysfunctional
C3
in
this
autoimmune
condition.
Patient and methods
CASE REPORT
The patient received plasma from her husband,
who had plasmapheresis before each session.
Both the patient and her husband were 0
Rhesus positive. The husband had not received
blood transfusions or any other blood products
which reduced the possibility that he was
immunised against leukocyte antigens. In vitro
analyses confirmed that the plasma contained
no such antibodies (agglutination and cyto-
toxic assays). The plasma was always given
through a leukocyte depletion filter to avoid
adverse reactions and immunisations against
leukocyte antigens. The study was approved by
the Ethical Committee of the Medical Faculty,
Uppsala
University. The patient gave
her
informed consent to participate in the study.
The plasma treatment was performed during
five periods (table).
University Hospital,
Uppsala, Sweden
Department of Clinical
Immunology and
Transfusion Medicine


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B Nilsson
U R Nilsson
A Karlsson-Parra
Department of
Internal Medicine,
Section of
Rheumatology
R Hallgren
Department of
Dermatology
G Sj6lin-Forsberg
Correspondence to:
Dr Bo Nilsson,
Department of Clinical
Immunology and
Transfusion Medicine,
University Hospital,
751 85 Uppsala, Sweden.
Accepted for publication
11 May 1994
The strong association between deficiencies of
the complement system and the occurrence of
immune
complex
associated
inflammatory
conditions
such
as
systemic
lupus
erythe-
matosus (SLE),lA
emphasises the important
role of complement in immune complex (IC)
metabolism.
We recently reported of a patient with an
SLE-like syndrome which was associated with
a dysfunction of C3.5 The propositus suffered
from fatigue, swollen fingers, arthralgia and
Raynaud's
syndrome
combined
with
the
presence of anti-centromere antibodies. The
dysfunction was a result of a double deficiency
with a null allele originating from the father
and one allele for the dysfunctional C3 from
the mother. The haemolytic function by the
alternative pathway (APW) was shown to be
completely eliminated while the CPW function
was reduced by 75%. The reason for this was
that the dysfunctional C3, especially in the
APW, was not cleaved into C3a and C3b due
to an inability of the C3 to interact properly
with the convertases.
We report the results of repeated plasma
infusions into the C3 dysfunctional patient
in
an
attempt
to
clarify
the
role
of the
Session 1
The first dose was taken well but six hours after
the second dose the patient developed fever,
nausea and diarrhoea which continued until
the next day. There was also transient mild
haematuria
(1+)
and
proteinuria
(1+). A
Coombs
test showed no erythrocyte-bound
IgG or C3d before or after the transfusions.
SESSIONS 2-5
A minute inflammatory reaction was observed
after the second, third and fourth treatment
period in that the fingers were more swollen
and the arthralgia was worse.
The
treatment
during
the
eight month
period resulted in no obvious clinical improve-
ment
of the


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of the
propositus
clinical
condition
except
that
the
patient's
Raynaud's
phe-
nomena seemed
to disappear. The disease
activity assessed by the SLE Disease Activity
Index (SLEDAI)6 remained at the same level
before and after the treatment period (score 6).
However, the patient has subjectively improved
and
the
fatigue
has
vanished.
Before
the
treatment the propositus was unable to work
for five years but she has now returned to her
previous occupation as a physiotherapist.
691


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Annals of the Rheumatic Diseases 1995; 54: 237-243
REVIEW
Osteoarthritis and magnetic resonance imaging:
potential and problems
C W Hutton, W Vennart
Magnetic resonance imaging (MRI) is in its
infancy.
Despite widespread
clinical
appli-
cations it has enormous unrealised potential.
Unlike
all
other imaging
modalities,
it
is
intrinsically a varied technology that has to be
engineered
for
each
problem
investigated.
Present imaging using whole body systems
lacks specificity and resolution, but this should
eventually be overcome using
tailor made
dedicated
equipment
such
as
specialised
surface coils and targeted gradient coils. MRI
offers
potential
for
in
vivo
non-invasive
measurement and imaging that may revol-
utionise the understanding of osteoarthritis
(OA). Currently, there are no known physio-
logical hazards associated with the magnetic
field and gradient strengths used in routine
MRI systems, and there
is no other non-
invasive method that allows repeat imaging of
specific aspects of the joint without the use of
ionising radiation and hence promises insight
into the interactions between the
different
structures or the processes that are involved in
joint physiology and pathology. Understanding
the potential ofMRI to investigate OA requires
an insight into the physics of the technique.
Mount Gould Hospital,
Plymouth, United
Kingdom
C W Hutton
Department of
Physics, University of
Exeter, Exeter, United
Kingdom
W Vennart
Accepted for publication
10 October 1994
Basic physics ofMRI
Nuclear
magnetic
resonance
(NMR)
was
developed as a method for studying the atomic
nucleus
and
chemical
composition
of
materials.' 2 With the development of methods
of localising nuclear signals, it was extended to
imaging.' MRI
is primarily concerned with
mapping the distribution of protons within
hydrogen atoms.4 When protons, which spin
on their axes and possess a magnetic moment,
are immersed in a homogeneous magnetic field
(B.)
they
precess
around
the
field
with
orientations allowed by quantum mechanics


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that are parallel and anti-parallel to the field
corresponding to low and high energy states.
The frequency of the precession is given by
wo = -yB0, where -y is the ratio of the magnetic
moment
of
the
nucleus
to
its
angular
momentum.
The
majority
of
commercial
imaging systems operate in the range of values
of Bo= 0-5-1-5 Tesla (T), the corresponding
proton precessional frequencies being 20-60
MHz.
If the protons are exposed to a pulse of
radiofrequency radiation in a plane perpen-
dicular
to
B.,
they
are
displaced
from
equilibrium and precess around B0 in the plane
of the
radiofrequency
radiation.
After
the
radiofrequency
pulse
is
switched
off,
the
protons return to equilibrium with a process
that involves the dephasing of the spinning
protons relative to each other, and one that
restores the equilibrium of low and high energy
states. The former process, spin-spin relax-
ation, is determined by a constant, T2; the
latter, spin-lattice relaxation, by a constant,
TI. Both TI and T2 vary and depend on a set
of parameters such as tissue composition, field
strength, and temperature. As the spinning
protons
relax
back
to
equilibrium,
their
associated magnetisations vary in time and
space. This can induce a current in a coil of
wire wound round the sample. To derive data
for MRI experiments, this signal is amplified
and
its
amplitude,
frequency
and
phase
measured. Figure 1 illustrates this process.
The
proton
precessional
frequency
(the
Larmor equation)
is influenced by different
chemical
environments
which
produce
a
shielding effect on the proton, such that the
Larmor equation becomes w = -yBO(I -8); B. is
the applied field and 8 is a shielding factor. The
shielding factor gives information about the
chemical composition of the material and is the
basis of magnetic resonance spectroscopy.
The position of the protons giving rise to the
MRI
signal can be
identified by applying
magnetic field gradients across the sample.
The gradient causes the resonant frequency to
vary with spatial position according to the
t
t
,


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t
t
,
high energy
(b)
equilibrium
i
i i
i low energy
I
I
I
T1
(c)
t
*I
++
+
JR
RF excitation
R
e
(d)
'
v
C>
t t
*.ld
RFemission
(a)
Figure 1
(a) Water protons spinning and precessing in
equilibrium in a static magnetic field B0; protons are
quantum particles-just afew more than half will precess
with thefield (low energy state) and slightly less against the
field (high energy state). (b) This equilibrium distribution
can be represented diagramatically as two energy levels with
more protons in the lower level (t) than the upper level (1).
(c) Application ofa burst ofradiofrequency (RF) radiation
to the system excites a net number ofprotonsfrom the low to
the high energy state. (d) When the RF is removed the spins
return to equilibrium by emiting RF radiation
this is
detected in the MRI investigation.
237


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Ann. rheum. Dis. (1971), 30, 565
Antigen recognition by T-cells
and its suppression
Significance and origin of rosette-forming cells
JEAN-FRANtOIS BACH
Hopital Necker, Paris
Antigen recognition
is one of the most debated
aspects of the immune response and its mechanism
is central to most theories of antibody formation.
The clonal selection theory of Bumet (1959), which
seems to be widely accepted at the moment, holds
that lymphocyte clones are already in existence at
birth, each bearing receptors specific for one antigen.
Recent studies have shown that three cell popula-
tions are involved in immune responses, at least for
some antigens:
(1) Macrophages;
(2) Thymus-dependent (T) lymphocytes;
(3) Bone-marrow-dependent (B) lymphocytes.
The existence and mechanism of co-operation of
these three cell types are still matters of discussion.
One of the most debated points, and one which will
interest us here, is the specificity at T and B-cell
levels.
Several techniques have been proposed for study-
ing antigen binding by lymphoid cells, the most
frequently used being bacterial adherence,
auto-
radiography of cells having bound isotope-labelled
antigens and, especially, immunocytoadherence (the
rosette test), which was described by Nota, Liaco-
poulos-Briot, Stiffel, and Biozzi (1964) and Zaalberg
(1964) and developed by Biozzi, Stiffel, Mouton,
Bouthillier, and Decreusefond (1968). This antigen
recognition in vitro has been shown to be a model of
recognition in vivo. In other words, cells which bind
antigens in vitro are the same as those which bind
antigens in vivo, this being the first step in antigen
recognition (Bach, Muller, and Dardenne, 1970).
Antigen binding can be prevented in several ways,
including removal of antigen, neutralization of the
receptors responsible for the binding and membrane
modification making the receptors unavailable for
antigen.
The Clones
The clonal selection theory of Burnet (1959)
is
supported by the fact that antibody-forming cells (as


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studied by the local haemolysis in gel technique) are
restricted in specificity, class, and allotype (Dubiski
and Fradette, 1966). One particular plaque-forming
cell generally produces antibodies of one class, one
allotype, one specificity, and one affinity, with a few
exceptions (Makela and Cross, 1970; Liacopoulos,
1971). While this is suggestive of clonal populations
of lymphocytes, direct proof of the existence of
clones before immunization is needed to support the
clonal selection theory. Such direct proof has been
afforded by a number of workers who have been able
to remove one clone of a given specificity from a
population of normal lymphoid cells, leaving them
unresponsive to the particular antigen. Three main
methods have been applied for this purpose: cell
retention on antigen-coated columns, specific killing
by
isotope-labelled
antigens,
and
removal
of
rosette-forming cells by centrifugation on gradient.
The first experiments were those of Wigzell (1970),
showing that it was possible to deplete a cell popula-
tion of its antigen-sensitive lymphocytes by passing
the cells down an antigen-coated column. Cells with
high affinity are retained more easily than cells with
low
affinity. The retention
is
inhibited by the
presence of free antigen in the column. These experi-
ments were done primarily with immune cells but
have also been performed with normal cells. This
system works for both thymus-dependent antigens
(e.g. serum albumin), against which the immune
response needs the presence of the thymus, and for
thymus-independent antigens (e.g. polyvinyl pyroly-
done). The experiments of Truffa-Bachi and Wofsy
(1970), using a different type of antigen-coated
column, have confirmed those of Wigzell (1970) and
Lecture given at the VII European Rheumatology Congress, Brighton, June, 1971.


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The Journal of Clinical Investigation   
R E S E A R C H  A R T I C L E
1
Introduction
The liver is a highly structured tissue where oxygen-rich blood 
enters the hepatic lobule at peripheral portal triads and drains 
out through the central vein (reviewed in ref. 1). As blood flows, 
oxygen, nutrients, and hormones are taken up and metabolized 
by hepatocytes that actively shape their microenvironment and 
create a gradient along the lobular axis. In turn, this gradient, 
together with local morphogens, shapes the cellular identity and 
subsequent functional heterogeneity of cells within the lobule. 
Consequently, hepatic metabolic functions are nonuniformly dis-
tributed along the lobular axis, a phenomenon called liver zona-
tion (reviewed in ref. 1). Energy-demanding functions, such as 
protein synthesis and secretion or de novo glucose production, are 
assigned to the periportal layers, where oxygen from arterial blood 
is abundant. Mid-lobular hepatocytes, on the other hand, special-
ize in the secretion of iron-regulating hormones, whereas pericen-
tral hepatocytes preferentially engage in xenobiotic metabolism, 
bile acid biosynthesis, and glycolysis (reviewed in ref. 2).
Acute liver failure, characterized by sudden and severe hepat-
ic injury, is a life-threatening condition (3). Drug-induced hepato-
toxicity is the leading cause of acute liver failure in North Amer-
ican and European countries (3). Liver transplantation remains 
the most effective treatment for acute liver failure, but the lack 
of donor organs limits its availability. The survival rate of acute 
liver failure has stagnated in recent years, and novel treatment 
options are urgently needed to improve the overall outcome of 
patients (4, 5). Liver zonation also has a role in this process, as it 
can explain zonated damage during acute liver injury. The zonat-
ed nature of xenobiotic metabolism, for instance, is responsible 
for the observed pericentral damage upon drug overdose. This is


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due to the accumulation of toxic intermediates in the hepatocytes 
expressing the detoxification enzymes, including cytochrome 
P450 2E1 (CYP2E1) and cytochrome P450 1A2 (CYP1A2) (6, 7), 
and is exemplified in the carbon tetrachloride (CCl4) murine mod-
el of liver injury (8, 9). Although molecular CCl4 is not toxic, hep-
atotoxicity develops following its metabolic activation by CYP2E1 
enzymes and the formation of the highly reactive trichloromethyl 
(CCl3) radicals. This highly reactive metabolite triggers oxidative 
damage to proteins, DNA, and lipids in pericentral CYP2E1-posi-
tive hepatocytes and culminates in liver injury and cell death (10).
Hepatic glutamine metabolism represents another highly 
zonated process where glutamine synthesis occurs through the 
catalytic action of glutamate-ammonia ligase (GLUL, also known 
as glutamine synthetase, [GS]), an enzyme whose expression is 
confined to a layer of hepatocytes surrounding the central veins, 
known as scavenger cells (reviewed in ref. 11). While glutamine 
synthesis from glutamate appears to be an efficient and required 
mechanism to capture ammonia (12), the fate of glutamine synthe-
sized at high rates in the pericentral zone is unclear. In this study, 
we demonstrate that a pericentral enzyme, asparagine synthe-
tase (ASNS), colocalizes with GLUL and converts its main prod-
uct, glutamine, into asparagine. We show that during acute liver 
injury, pericentrally expressed ASNS is highly induced through 
the enhanced recruitment of the nuclear receptor liver receptor 
homolog 1 (LRH-1; NR5A2) to its promoter region. This transcrip-
tional process is part of a noncanonical adaptive mechanism to 
The nonessential amino acid asparagine can only be synthesized de novo by the enzymatic activity of asparagine synthetase 
(ASNS). While ASNS and asparagine have been implicated in the response to numerous metabolic stressors in cultured cells,


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the in vivo relevance of this enzyme in stress-related pathways remains unexplored. Here, we found ASNS to be expressed 
in pericentral hepatocytes, a population of hepatic cells specialized in xenobiotic detoxification. ASNS expression was 
strongly enhanced in 2 models of acute liver injury: carbon tetrachloride (CCl4) and acetaminophen. We found that mice 
with hepatocyte-specific Asns deletion were more prone to pericentral liver damage than their control littermates after 
toxin exposure. This phenotype could be reverted by i.v. administration of asparagine. Unexpectedly, the stress-induced 
upregulation of ASNS involved an ATF4-independent, noncanonical pathway mediated by the nuclear receptor, liver receptor 
homolog 1 (LRH-1; NR5A2). Altogether, our data indicate that the induction of the asparagine-producing enzyme ASNS acts as 
an adaptive mechanism to constrain the necrotic wave that follows toxin administration and provide proof of concept that i.v. 
delivery of asparagine can dampen hepatotoxin-induced pericentral hepatocellular death.
Asparagine protects pericentral hepatocytes during 
acute liver injury
Yu Sun, Hadrien Demagny, Adrien Faure, Francesca Pontanari, Antoine Jalil, Nadia Bresciani, Ece Yildiz, Melanie Korbelius, 
Alessia Perino, and Kristina Schoonjans
Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Authorship note: YS and HD contributed equally to this work.
Copyright: © 2023, Sun et al. This is an open access article published under the terms 
of the Creative Commons Attribution 4.0 International License.
Conflict of interest: The authors have declared that no conflict of interest exists.
Submitted: July 12, 2022; Accepted: January 27, 2023; Published: April 3, 2023.
Reference information: J Clin Invest. 2023;133(7):e163508. 
https://doi.org/10.1172/JCI163508.


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Monographic section
205
Sex-based differences  
in autoimmune diseases
Elena Ortona1, Marina Pierdominici1, Angela Maselli2, Caterina Veroni1, Francesca Aloisi1 
and Yehuda Shoenfeld3
1Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Rome, Italy 
2Dipartimento del Farmaco, Istituto Superiore di Sanità, Rome, Italy 
3Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center (affiliated to Tel-Aviv University), 
Tel-Hashomer, Israel
Ann Ist Super Sanità 2016 | Vol. 52, No. 2: 205-212
DOI: 10.4415/ANN_16_02_12
INTRODUCTION
Autoimmune diseases include more than 80 chronic 
disorders that affect nearly 5% of the population in 
Western countries. They are characterized by an exag-
gerated immune response leading to damage and dys-
function of specific or multiple organs and tissues. The 
aetiology of autoimmune diseases is still unknown, but 
the available evidence points to an interaction between 
genetic, environmental and life style factors in disease 
development. Autoimmune diseases are typically more 
prevalent in women than in men and are considered 
the fourth leading cause of disability for women [1]. 
The strongest sex bias is observed in Sjogren’s syn-
drome, systemic lupus erythematosus (SLE), autoim-
mune thyroid disease and scleroderma where the ratio 
of women to men is 7:1-10:1. In rheumatoid arthritis 
(RA), multiple sclerosis (MS) and myasthenia gravis, 
the female to male ratio is 2:1-3:1, while there is little 
or no sex bias in inflammatory bowel diseases and type 
1 diabetes [2-4]. Symptom severity, disease course, re-
sponse to therapy and overall survival may also differ 
between males and females with autoimmune diseases. 
In general, women mount stronger humoral and cellu-
lar immune responses than men and this is believed to 
have an effect on the different susceptibility to develop 
autoimmune diseases. The main factors affecting the 
differences between female and male immune systems 
are the sex hormones, the presence of two X chromo-


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somes versus one X and one Y chromosome, and the 
different response to environmental factors, such as 
microbial exposure and diet [5]. Sociological differenc-
es between genders may also affect the development of 
autoimmunity. However, the discussion of this topic is 
beyond the scope of this article and readers can refer 
to recent reviews [6]. Here, we briefly review the role 
of sex-related factors in autoimmunity and then high-
light key discoveries and controversies on the role of 
sexual dimorphism in three paradigmatic autoimmune 
diseases: SLE, RA and MS.
SEX HORMONES IN AUTOIMMUNITY
Due to the presence of hormone receptors on im-
mune cells [7], sex hormones, such as estrogens, pro-
gesterone, androgens and prolactin, can influence 
different aspects of immune system function and po-
tentially affect the risk, activity and progression of au-
toimmune diseases. Generally, estrogens, in particular 
17-β estradiol (E2) and prolactin, act as enhancers at 
least of humoral immunity, and testosterone and pro-
gesterone as natural immunosuppressants [3]. Sex hor-
mones have different effects depending not only on the 
concentration but also on the type of target cell and 
the receptor subtype expressed on a given cell type. At 
periovulatory to pregnancy levels [8], E2 has mainly 
anti-inflammatory effects, by inhibiting production and 
signaling of pro-inflammatory cytokines, such as tumor 
Address for correspondence: Marina Pierdominici, Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Viale Regina Elena 
299, 00161 Rome, Italy. E-mail: marina.pierdominici@iss.it.
Key words
•  sex 
•  autoimmunity 
•   systemic lupus 
erythematosus 
•  rheumatoid arthritis 
•  multiple sclerosis
Abstract
Autoimmune diseases are characterized by an exaggerated immune response leading to 
damage and dysfunction of specific or multiple organs and tissues. Most autoimmune 
diseases are more prevalent in women than in men. Symptom severity, disease course,


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response to therapy and overall survival may also differ between males and females with 
autoimmune diseases. Sex hormones have a crucial role in this sex bias, with estrogens 
being potent stimulators of autoimmunity and androgens playing a protective role. Accu-
mulating evidence indicates that genetic, epigenetic and environmental factors may also 
contribute to sex-related differences in risk and clinical course of autoimmune diseases. 
In this review, we discuss possible mechanisms for sex specific differences in autoimmu-
nity with a special focus on three paradigmatic diseases: systemic lupus erythematosus, 
rheumatoid arthritis, and multiple sclerosis.


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The Journal of Clinical Investigation   
R E S E A R C H  A R T I C L E
1
Introduction
The hypothalamic AGRP/NPY neuron is well known to be orexi-
genic (1, 2), opposing the action of its counterpart, the hypothalamic 
anorexigenic POMC neuron (3, 4). However, constitutive absence 
of AGRP or NPY has little or no impact on feeding or body weight 
gain on normal chow (5). Only mice with adult ablation of AGRP/
NPY neurons show hypophagia (6, 7), indicating that developmen-
tal compensation occurs to normalize food intake in the absence 
of either orexigenic peptide. We have shown that the constitutive 
absence of AGRP does have effects on glucose homeostasis and the 
reproductive system (8, 9). Agrp-KO mice have improved glucose 
tolerance if they are obese genetically as a result of leptin signaling 
deficiency. Agrp-KO females also have earlier puberty onset, while 
ob/ob Agrp-KO females are fertile. In addition, mammary gland 
development is restored in ob/ob Agrp-KO females. These were sur-
prising results since severe hypoleptinemia is associated with infer-
tility and failure to undergo puberty (10–12).
We have previously described a gut-to-brain axis (13, 14) that 
comprises meal-associated bile acid secretion and FGF15/19 
signaling which regulates the expression and activity of AGRP- 
expressing cells. This signaling pathway could constitute a physi-
ological role for AGRP in glucose homeostasis. This pathway may 
also be relevant to bariatric surgery (15), wherein procedures result 
in altered bile flow, increased FGF19 concentrations, and improved 
glucose control, sometimes with remission of preexisting type 2 dia-
betes mellitus (16, 17). In this report, while searching for potential 
sites of regulation within the Agrp upstream regulatory region, we 
found that public databases had identified multiple AGRP mRNA 
variants. The mouse Agrp gene has 3 widely separated transcription 
start sites that result in 3 different transcripts (AGRP-A, AGRP-B,


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and AGRP-C), which differ in their 5′-untranslated regions (5′-
UTRs), implying the existence of 3 different promoters/enhancers 
and varying modes of regulation for each of the transcript variants. 
In addition, the cell type specificities of these promoters/enhancers 
are likely to be different. We show that the AGRP-B mRNA variant is 
suppressed by bile acids but remains unaffected by fasting. In con-
trast, the AGRP-A variant, the most proximal variant, is regulated 
by fasting but remains unaffected by bile acid treatment. Previous-
ly, we had shown that bile acids and FGF19 both affected glucose 
metabolism by improving insulin sensitivity and glucose-stimu-
lated insulin secretion (GSIS) in lean mice on normal chow, DIO 
(diet-induced obesity) mice, and obese leptin-deficient (ob/ob) mice 
(13, 14). This improved glucose metabolism is dependent on FGFR1 
signaling in AGRP-expressing cells as well as downstream MC4R 
(13), since Cre-mediated deletion of Fgfr1 in AGRP-expressing cells 
abolished the glucose homeostatic response to taurocholate.
In order to determine whether AGRP-B–expressing cells are 
involved in glucose homeostasis, we generated a knockin/knock-
out allele of the mouse Agrp gene that expresses FLP recombinase 
from the Agrp-B promoter, Agrp-B-FLP. Using the Agrp-B-FLP 
allele in combination with an FLP-dependent tdTomato reporter as 
a lineage tracker, we found that the Agrp-B promoter is expressed 
in endocrine, non-neuronal cells of the pars tuberalis, colocalizing 
Glucose homeostasis can be improved after bariatric surgery, which alters bile flow and stimulates gut hormone 
secretion, particularly FGF15/19. FGFR1 expression in AGRP-expressing cells is required for bile acids’ ability to improve 
glucose control. We show that the mouse Agrp gene has 3 promoter/enhancer regions that direct transcription of each 
of their own AGRP transcripts. One of these Agrp promoters/enhancers, Agrp-B, is regulated by bile acids. We generated


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an Agrp-B knockin FLP/knockout allele. AGRP-B–expressing cells are found in endocrine cells of the pars tuberalis and 
coexpress diacylglycerol lipase B — an endocannabinoid biosynthetic enzyme — distinct from pars tuberalis thyrotropes. 
AGRP-B expression is also found in the folliculostellate cells of the pituitary’s anterior lobe. Mice without AGRP-B were 
protected from glucose intolerance induced by high-fat feeding but not from excess weight gain. Chemogenetic inhibition 
of AGRP-B cells improved glucose tolerance by enhancing glucose-stimulated insulin secretion. Inhibition of the AGRP-B 
cells also caused weight loss. The improved glucose tolerance and reduced body weight persisted up to 6 weeks after 
cessation of the DREADD-mediated inhibition, suggesting the presence of a biological switch for glucose homeostasis 
that is regulated by long-term stability of food availability.
The gut signals to AGRP-expressing cells  
of the pituitary to control glucose homeostasis
Shun-Mei Liu,1 Bruno Ifebi,1 Fred Johnson,1 Alison Xu,1 Jacquelin Ho,1 Yunlei Yang,1,2 Gary Schwartz,1,2 Young Hwan Jo,1,3  
and Streamson Chua Jr.1,2
1Department of Medicine, 2Department of Neuroscience, and 3Department of Molecular Pharmacology, Albert Einstein College of Medicine, New York, New York, USA.
Conflict of interest: The authors have declared that no conflict of interest exists.
Copyright: © 2023, Liu et al. This is an open access article published under the terms 
of the Creative Commons Attribution 4.0 International License.
Submitted: August 3, 2022; Accepted: February 7, 2023; Published: April 3, 2023.
Reference information: J Clin Invest. 2023;133(7):e164185. 
https://doi.org/10.1172/JCI164185.


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Ann. rheum. Dis. (1976), 35, 415
Fc-rosette inhibition by hypocomplemen-
taemic systemic lupus erythematosus sera
TADAO MORITO, KIYOAKI TANIMOTO, YOSHIMI HASHIMOTO,
YOSHIHIKO HORIUCHI, AND TAKEO JUJI*
From the Department ofInternal Medicine and Physical Therapy, and Department of Transfusion,* School of
Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo, Tokyo, Japan
Morito, T., Tanimoto, K., Hashimoto, Y., Horiuchi, Y., and Juji, T. (1976). Annals of
the Rheumatic Diseases, 35, 415-421. Fc-rosette inhibition by hypocomplementaemic
systemic lupus erythematosus sera. Human red cells sensitized with one of the Rh
antisera (Ripley) form rosettes (Fc-rosette) with human B lymphocytes and the rosettes
are well inhibited by aggregated human IgG. Since sera of hypocomplementaemic
patients with systemic lupus erythematosus (SLE) have frequently been reported to
contain immune complexes, they were used for the inhibition of Fc-rosette formation
in this study.
The results of Fc-rosette inhibition rates of the sera were inversely correlated with the
serum CH,0 levels. When the sera were separated into top, middle, and bottom fractions
by ultracentrifugation, the bottom fractions showed more effective inhibitions than the
others. Similarly, the strongest inhibition was found in the void volume of the serum
separated by Sephadex G200 gel filtration. Reduction and alkylation of IgG resulted
in the loss of reactivity with Fc receptor of B lymphocytes, and the rosette inhibiting
activities of the SLE sera were markedly reduced after reduction and alkylation. Some of
anti-HLA sera were inhibitory for the Fc-rosette formation, while the tested sera did
not contain anti-HLA activity assessed by the microcytotoxicity test. These results
indicated that circulating immune complexes contained in the sera inhibit the rosette
formation, and that the Fc-rosette inhibition test is a simple and relatively sensitive
method for the detection of circulating immune complexes.


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Antinuclear antibody activities of the sera were tested by the indirect immuno-
fluorescent method; however, clear correlations were not obtained between Fc-rosette
inhibition rates and staining patterns of antinuclear antibodies. On the other hand,
the positive groups of LE-test exhibited slightly greater inhibition rates of the rosette
than the negative groups.
A previous report (Morito and others, 1976) showed
that Fc-receptors mainly on human B lymphocytes
are detected by the rosette formation (Fc-rosette)
using the human red cells sensitized with one of the
Rh-antisera (Ripley), and that the Fc-rosette
is
strongly inhibited by aggregated human IgG. This
suggested that circulating immune complexes
in
systemic lupus erythematosus (SLE) sera will inhibit
the Fc-rosette formation when the complexes contain
aggregated IgG. In the present study it was found
that all the tested hypocomplementaemic SLE sera
inhibited the Fc-rosette formation. Analysis of the
sera suggested that the inhibition was caused mainly
by the immune complexes present in the sera.
Accepted for publication February 11, 1976.
Correspondence to Dr. K. Tanimoto.
Materials and methods
SLE SERA
Eleven sera from patients with SLE who fit the criteria of
the American Rheumatism Association (Cohen and
others, 1971) were used after heat inactivation at 56°C
for 30 minutes for the inhibition of Fc-rosette formation.
Serum CH50 levels of these sera were < 20 units. 7 normal
human sera and 8 normocomplementaemic SLE sera
were used as controls.
PREPARATION OF AGGREGATED HUMAN IgG
Human Cohn Fraction II (purchased from Midori Juji,
Tokyo) was used as human IgG, and 10 mg/ml diluted
in 0-15 mol/l phosphate-buffered saline, pH 7 4, were
heat aggregated at 63'C for 20 minutes. Under this
condition, IgG showed moderate
turbidity
but
no


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Annals of the Rheumatic Diseases, 1977, 36, 3-12
Rubella virus and rheumatoid arthritis
HELENA HART AND B. P. MARMION
From the Department ofBacteriology, Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG
sumMARY
A collection of synovial fibroblasts from 19 patients with rheumatoid arthritis (RA) and
12 patients with osteoarthrosis or other non-RA disease has been examined for rubella virus antigens
by immunofluorescence and radioimmunoassay with negative results. Eluates of synovial membrane
prepared under conditions likely to dissociate antigen-antibody complexes have shown no rubella
antibody. A serological survey of RA patients and those with other forms of arthritis has shown no
differences in the frequency
or levels of rubella haemagglutination-inhibiting antibody. These
results provide little support for various hypotheses that a persistent infection with rubella virus
underlies or initiates the rheumatoid process.
The
phenomenon
of
persistent
infection
with
rubella virus is familiar from the congenital rubella
syndrome and from some patients with a variety of
subacute
sclerosing
panencephalitis
(Townsend
et al., 1975; Weil et al., 1975), and also from persis-
tent infection of cultured cells in vitro. The tendency,
taken with the observation of a self-limiting synovitis
as an occasional complication of acute rubella, lends
attraction to a hypothesis that a persistent infection
of synovial cells with rubella virus might be the
ultimate antigenic stimulus in juvenile (JRA) or
adult rheumatoid arthritis (RA).
To
test
this
hypothesis
numerous
workers
have compared the frequency, or mean titre, of
rubella antibody in rheumatoid patients with that
in control groups of healthy persons, or patients
suffering from other forms of arthritis (Chandler
et al., 1971; Laitinen et al., 1972; Simsarian et al.,
1970; Deinard et al., 1974). These studies have gener-
ally failed to show a difference except for those
described by Patterson et al., (1973 and personal


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communication) and Deinard et al. (1974). Other
workers have examined antibody from RA synovial
fluid cryoprecipitates, or eluates from RA synovial
membranes without, however,
detecting
striking
amounts of rubella antibody (Cremer et al., 1974;
Zvaifler, 1973).
However, the resolution of rubella virus infection
may be protracted in some rheumatoid patients.
Thus Ogra,
et al.
(1975) observed a prolonged
infection with rubella virus in some JRA patients,
Accepted for publication 27 April, 1976
Correspondence to Professor B. P. Marmion
3
as judged by high and persistent levels of IgM and
IgG antibody to rubella virus and the demonstration
of rubella antigen by immunofluorescence in smears
of synovial fluid cells. These effects may be related
to the higher prevalence ofhistocompatibility antigen
HLA-B27 in JRA patients (Racheleksfy et al., 1974),
with its postulated immunological implications.
An association of persistent rubella infection and
adult RA appears to be much less common. A
serologically documented case of rubella arthritis
progressing to, or associated with, classical adult
RA was described by Martenis et al. (1968), who
mentioned
other
patients.
More
recently,
in
Edinburgh, a 26-year-old woman has been observed
who had an illness resembling rubella and subse-
quently developed classical RA with positive latex
and SSCT tests and a raised erythrocyte sedimenta-
tion rate. A raised titre (1/2048-1/4096) of rubella
antibody by haemagglutination-inhibition in both
the IgG and IgM fractions of her serum was de-
tected early in the illness. The rheumatoid state
persisted for 18 months after onset of symptoms
when there was a resolution of the symptoms and
signs accompanied by a fall in the level of IgM
rubella antibody (Duthie et al., unpublished).
Other investigators have approached the problem
by trying to show rubella virions or other gene pro-
ducts, or interference by rubella virus, in RA synovial
cells, usually fibroblasts. Thus Grayzel and Beck
(1970), Smith and Hamerman (1969), and Ford and


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Oh (1965) found that RA synovial fibroblasts were
more resistant to infection with rubella or Newcastle
disease viruses than non-RA fibroblasts. This was
construed as an
intrinsic
viral interference,
but


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Annals of the Rheumatic Diseases, 1977, 36, 34-38
Is persisting antigen responsible for the chronicity
of experimental allergic arthritis?
A. FOX AND L. E. GLYNN
From the MRC Rheumatism Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berkshire
SL6 OHN
SUMMARY
Animals were injected intra-articularly with antigen after prior immunization with that
antigen in Freund's incomplete adjuvant in order to precipitate immune complexes in the surfaces
of menisci, ligaments, and cartilage. On reimmunization with antigen in Freund's complete adjuvant
10 weeks later, generally an arthritis limited to the intercondylar fossa developed; but on intra-
articular injection of antigen a second time a widespread arthritis developed in that joint. Thus
immune deviation had not occurred and animals were in an immunological condition such as to be
capable of developing widespread arthritis given the correct intra-articular stimulus. It is concluded
that antigen, persisting as immune complexes, plays no part in maintaining widespread monarthritis,
presumably owing to its inability to participate in a delayed hypersensitivity reaction as a result of
sequestration.
Experimental allergic arthritis (EAA) is a chronic
disease which histologically resembles rheumatoid
arthritis. It is produced in rabbits by immunizing
them with a soluble antigen in Freund's complete
adjuvant (FCA) and then injecting the antigen intra-
articularly. The substitution of Freund's incomplete
adjuvant (FIA) for FCA results only in an acute
inflammation
on
joint
challenge
with
antigen
(Dumonde and Glynn, 1962; Consden et al., 1971).
It has been shown that in EAA antigen persists as
immune complexes in the superficial layers of men-
isci, ligaments, and cartilage (Cooke et al., 1972; Hol-
lister and Mannik, 1974) (but not in synovial fluid:
Belovic and Kinsella, 1973; or synovium: Doble et
al., 1973), and it was suggested that slow release of
antigen from them continuously activates comple-
ment which in turn causes the persisting inflamma-


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tion. However, it has also been shown that immune
complexes persist in similar locations in similar
amounts in thejoints ofanimals injected with antigen
after prior immunization with that antigen in FIA.
Since these joints recover completely within a few
weeks from the induced arthritis, it was concluded
that
something
more
than
immune
complexes
activating complement is required (Fox and Glynn,
1975).
Accepted for publication April 30, 1976
Correspondence to Mr. A. Fox
It was therefore suggested that slow release of
antigen from complexes could continuously activate
a delayed hypersensitivity (DHS) reaction. The need
for a DHS reaction to antigen which is demonstrably
not produced on immunization with antigen
in
FIA but requires FCA (Audibert et al., 1976), has
been stressed (Glynn, 1968). In addition the pro-
duction of a macrophage migration inhibition factor
(MIF) by cultures of synovia from EAA joints
suggests a continuing DHS reaction (Stastny, et al.,
1973). Also injection of MIF into joints results in
arthritis (Andreis et al., 1974).
We present evidence that if animals are immunized
with antigen in FIA and then injected intra-articularly
with that antigen in order to precipitate complexes
in collagenous surfaces, on reimmunization with
antigen in FCA 10 weeks later they do not develop
a widespread chronic
arthritis although a mild
inflammation does develop which is restricted to the
intercondylar
fossa.
However,
on
intra-articular
reinjection of antigen unequivocal
arthritis does
develop throughout
the joint, showing that this
immunization schedule does not induce immune
deviation and that the animals are in an immuno-
logical condition to develop arthritis given the correct
intra-articular stimulus. We conclude that antigen
persisting as immune complexes is not involved in
maintaining widespread arthritis, presumably as a
result of inability to participate in a DHS reaction.
34


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Annals ofthe Rheumatic Diseases 1996; 55: 411-420
REVIEW: PAIN
Series editor: Bruce L Kidd
Cerebral mechanisms operating in the presence
and absence of inflammatory pain
Anthony K P Jones, Stuart W G Derbyshire
During episodes ofinflammatory arthritic pain,
the brain has the task of integrating new
information from receptors of muscle, tendon,
and skin afferents with nociceptive information
from the joint capsule and, possibly, peri-
articular bone. This integration will incorpor-
ate motivational, affective, discriminative, at-
tentional, and motor responses that may result
in changes of behaviour and modification of
central nervous system (CNS) processing of
pain. It is as yet unclear what the nature of this
CNS modification may be, and suggested
influences
on
pain
related
affective-motor
responses or the inflammatory process itself
remain largely speculative.
It is likely to be some time before we begin
to have a proper understanding of the complex
integrated
processes
that
operate
during
arthritic pain and other chronic pains. How-
ever, we are now able to measure aspects of
those processes in terms of measurement of
behavioural and cerebral responses to pain.
These measurements are providing early clues
to the possible underlying CNS mechanisms
and their relation to changes
in
affective-
motivational behaviour. This paper will review
the normal brain mechanisms of the conscious
appreciation of pain, together with working
hypotheses for how these mechanisms
are
altered during inflammatory and other types of
clinical pain syndromes.
Human Physiology and
Pain Research
Laboratory,
Manchester University
Rheumatic Diseases
Centre, Clinical
Sciences Building,
Hope Hospital,
Salford M6 8HD,
United Kingdom
A K P Jones
S W G Derbyshire
Correspondence to:
Dr Jones.
Accepted for publication
14 March 1996
Normal mechanisms: anatomy and
physiology
It is perhaps surprising that there should still
be debate about which areas of the brain might
be involved
in the processing of such
an


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an
important and commonplace sensation as pain.
This has come about largely because of the
understanding of pain as an experience involv-
ing sensory, cognitive-evaluative, and affective-
motivational components' which defies simple
explanations
involving
pain
centres'
and
specific pathways.2
Whatever areas of the brain are involved in
pain processing, integration of the different
components
must be accommodated.
This
implies that there is unlikely to be any final
common area of the brain that processes pain.
Indeed, it is perhaps the search for a single
dominant 'pain centre' that has done more to
impair our understanding of the functional
anatomy of pain than anything else. Parallel
processing of the conscious appreciation of
primary sensations such as vision and touch3
are now well documented, and it would be
surprising if such parallel processing did not
also exist for non-primary sensations such as
pain. There is now growing evidence that pain
is processed by a network or 'matrix' of struc-
tures in the brain (fig 1), some components of
which could be considered to comprise parallel
but integrated processing.
NEUROANATOMY OF NOCICEPTION IN ANIMALS
Studying pain in animals presents an insoluble
problem. Human beings can
call out and
express their pain in ways that are commonly
understandable
to other members of their
species; unfortunately, there is no equivalent
frame of reference when dealing with animals.
Increased
blood
pressure,
movements
of
withdrawal, dilatation of the pupil, increased
depth of ventilation, attacking the source of
noxious stimulation, and cries may be common
to all mammals in the face of seemingly 'pain-
ful' stimulation, but all such responses can be
elicited
after the cerebral cortex has been
destroyed, in the probable absence of any
subjective experience. Furthermore, the sub-
jective experiences of an animal, if it has any,
may be totally different from those of humans.
For these reasons, human interpretation of
what is observed in another species cannot be


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by extrapolation from human experience.
This is not to suggest that all 'pain' research
in animals is valueless: the common ancestry
between humans and animals suggests that
some of the underlying biological processes
involved in human pain experience are likely to
be shared with other animals. A distinction is
therefore drawn between 'nociception' and
pain-which
should
not
be
used
inter-
changeably-the term nociception being used
to refer to the biological response of receptors
in tissue to potentially damaging stimuli such
as intense heat or cold, chemical irritation, or
intense pressure. The study ofnociceptive pain
avoids the fact that pain in man can occur
without any evident nociceptor activation, as in
peripheral and central deafferentation pain,
and may also occur without any evidence of
spinothalamic tract activation, as in psycho-
genic pain.
The spinothalamic tract has been demon-
strated to be 'both necessary and sufficient' for
nociception in most species studied.4 It pro-
vides the major direct nociceptive input from
the spinal cord to the thalamus. As a conse-
quence, the spinothalamic tract ascending in
the anterolateral cord is widely considered as
the 'pain pathway'.
41


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Site of vulnerability on SARS-CoV-2 spike induces broadly protective antibody against antigenically distinct Omicron subvariants


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The Journal of Clinical Investigation   
R E S E A R C H  A R T I C L E
1
Introduction
Since the start of the pandemic in December 2019, the severe acute 
respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has led to 
over 660 million cases of coronavirus disease 2019 (COVID-19) 
and over 6.5 million deaths globally. Although the rapid develop-
ment and distribution of vaccines and therapeutics have curbed 
the effect of COVID-19 to a large extent, the emergence of cir-
culating variants of concern (VOCs) continues to represent a 
major threat due to the potential for further immune evasion 
and enhanced pathogenicity. The D614G variant was the earliest 
variant to emerge and became universally prevalent thereafter. 
Compared with WT, the D614G variant exhibited increased trans-
missibility rather than increased pathogenicity and was therefore 
unlikely to reduce efficacy of vaccines in clinical trials (1). Between 
the emergence of D614G to October of 2021, 4 additional signif-
icant VOCs evolved worldwide, including Alpha, Beta, Gamma, 
and Delta. Among these variants, Delta became a serious global 
threat because of its transmissibility, increased disease severity, 
The rapid evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants has emphasized 
the need to identify antibodies with broad neutralizing capabilities to inform future monoclonal therapies and vaccination 
strategies. Herein, we identified S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) 
that was derived from an individual previously infected with WT SARS-CoV-2 prior to the spread of variants of concern 
(VOCs). S728-1157 demonstrated broad cross-neutralization of all dominant variants, including D614G, Beta, Delta, Kappa, 
Mu, and Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/BL.1/XBB). Furthermore, S728-1157 protected hamsters against in vivo 
challenges with WT, Delta, and BA.1 viruses. Structural analysis showed that this antibody targets a class 1/RBS-A epitope


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in the receptor binding domain via multiple hydrophobic and polar interactions with its heavy chain complementarity 
determining region 3 (CDR-H3), in addition to common motifs in CDR-H1/CDR-H2 of class 1/RBS-A antibodies. Importantly, 
this epitope was more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike 
constructs, as compared with diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential and may 
inform target-driven vaccine designs against future SARS-CoV-2 variants.
Site of vulnerability on SARS-CoV-2 spike induces 
broadly protective antibody against antigenically 
distinct Omicron subvariants
Siriruk Changrob,1 Peter J. Halfmann,2 Hejun Liu,3 Jonathan L. Torres,3 Joshua J.C. McGrath,1 Gabriel Ozorowski,3 Lei Li,1  
G. Dewey Wilbanks,1 Makoto Kuroda,2 Tadashi Maemura,2 Min Huang,1 Nai-Ying Zheng,1 Hannah L. Turner,3 Steven A. Erickson,4 
Yanbin Fu,1 Atsuhiro Yasuhara,1 Gagandeep Singh,5,6 Brian Monahan,6,7 Jacob Mauldin,6,7 Komal Srivastava,6,7 Viviana Simon,5,6,7,8,9 
Florian Krammer,5,6,7 D. Noah Sather,10,11,12 Andrew B. Ward,3 Ian A. Wilson,3,13 Yoshihiro Kawaoka,2,14,15,16 and Patrick C. Wilson1
1Drukier Institute for Children’s Health, Department of Pediatrics, Weill Cornell Medicine, New York, New York, USA. 2Influenza Research Institute, Department of Pathobiological Sciences, School of 
Veterinary Medicine, University of Wisconsin–Madison, Madison, Wisconsin, USA. 3Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA. 
4University of Chicago Department of Medicine, Section of Rheumatology, Chicago, Illinois, USA. 5Department of Pathology, Molecular and Cell Based Medicine, 6Department of Microbiology, 7Center for 
Vaccine Research and Pandemic Preparedness, 8The Global Health and Emerging Pathogens Institute, and 9Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai,


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New York, New York, USA. 10Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA. 11Department of Pediatrics and 12Department of Global Health, 
University of Washington, Seattle, Washington, USA. 13The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA. 14Division of Virology, Department of Microbiology 
and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan. 15The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo, 
Japan. 16Pandemic Preparedness, Infection and Advanced Research Center (UTOPIA), University of Tokyo, Tokyo, Japan.
 
 Related Commentary: https://doi.org/10.1172/JCI168080
Authorship note: SC, PJH, HL, and JLT contributed equally to this work.
Conflict of interest: The University of Chicago has filed a patent application on No-
vember 11, 2021, relating to anti-SARS-CoV-2 antibodies with PCW and SC as inventors 
(application number PCT/US22/79916). Some of the mAbs in this study are being 
considered for the development of therapeutic antibodies. The Icahn School of Med-
icine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological 
assays (U.S. provisional application numbers: 62/994,252; 63/018,457; 63/020,503; and 
63/024,436) and NDV-based SARS-CoV-2 vaccines (U.S. provisional application number: 
63/251,020), which list FK as a coinventor. VS is listed on the serological assay patent 
application as a coinventor. Mount Sinai founded a company, Kantaro, to market sero-
logical tests for SARS-CoV-2. FK has consulted for Merck and Pfizer, before 2020, and is 
currently consulting for Pfizer, Seqirus, Third Rock Ventures and Avimex. The Krammer 
laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2.
Copyright: © 2023, Changrob et al. This is an open access article published under the 
terms of the Creative Commons Attribution 4.0 International License.


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Submitted: November 2, 2022; Accepted: February 28, 2023; Published: April 17, 2023.
Reference information: J Clin Invest. 2023;133(8):e166844. 
https://doi.org/10.1172/JCI166844.


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ARTICLE
A cell-free antigen processing system informs HIV-1
epitope selection and vaccine design
Srona Sengupta1,2,8, Josephine Zhang1*, Madison C. Reed1*, Jeanna Yu1*, Aeryon Kim3, Tatiana N. Boronina4, Nathan L. Board1,
James O. Wrabl5, Kevin Shenderov1, Robin A. Welsh2, Weiming Yang2, Andrew E. Timmons1, Rebecca Hoh6, Robert N. Cole4,
Steven G. Deeks6, Janet D. Siliciano1, Robert F. Siliciano1,7, and Scheherazade Sadegh-Nasseri2
Distinct CD4+ T cell epitopes have been associated with spontaneous control of HIV-1 replication, but analysis of antigen-
dependent factors that influence epitope selection is lacking. To examine these factors, we used a cell-free antigen processing
system that incorporates soluble HLA-DR (DR1), HLA-DM (DM), cathepsins, and full-length protein antigens for epitope
identification by LC-MS/MS. HIV-1 Gag, Pol, Env, Vif, Tat, Rev, and Nef were examined using this system. We identified 35
novel epitopes, including glycopeptides. Epitopes from smaller HIV-1 proteins mapped to regions of low protein stability and
higher solvent accessibility. HIV-1 antigens associated with limited CD4+ T cell responses were processed efficiently, while
some protective epitopes were inefficiently processed. 55% of epitopes obtained from cell-free processing induced memory
CD4+ T cell responses in HIV-1+ donors, including eight of 19 novel epitopes tested. Thus, an in vitro processing system utilizing
the components of Class II processing reveals factors influencing epitope selection of HIV-1 and represents an approach to
understanding epitope selection from non–HIV-1 antigens.
Introduction
CD4+ T cells connect the humoral- and cell-mediated arms of the
immune system, both of which are vital for vaccine responses
against chronic viral infections. Elegant analyses of CD4+ T cell
responses to HIV-1 by Walker and colleagues have provided
insights into protective HLA-DR alleles and viral epitopes asso-
ciated with control of viral replication. The breadth and magni-


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tude of Gag-specific CD4+ T cell responses are associated with
anti-Env neutralizing antibodies (Ranasinghe et al., 2015) and
inversely correlated with viral load (Laher et al., 2017; Ranasinghe
et al., 2012; Ranasinghe et al., 2013). Three CD4+ T cell epitopes in
Gag are associated with spontaneous viral control (Ranasinghe
et al., 2012, 2013). However, it remains unclear why few in-
dividuals develop these protective responses and whether this is
influenced by antigen-processing mechanisms.
The antigen-processing pathway for major histocompatibility
complex Class II (MHC-II)–restricted CD4+ T cell epitopes begins
with the endocytosis of exogenous antigens or autophagy of
intracellular contents (Unanue et al., 2016). Full-length protein
antigens bind to MHC-II molecules in the late endosomal MHC-II
compartment (MIIC) of professional APCs, with subsequent
cleavage/trimming of the exposed protein around the bound
region (Kim et al., 2014). Epitope selection by MHC-II is facili-
tated by chaperones HLA-DM (DM) and HLA-DO. DM recognizes
structurally flexible conformations of peptide:MHC-II (pMHC-
II) complexes (Chou and Sadegh-Nasseri, 2000) due to partially
filled or unfilled P1 pockets in the peptide binding groove
(Anders et al., 2011; Chou and Sadegh-Nasseri, 2000) and in-
duces dissociation of poorly bound peptides such as the class II–
associated invariant chain (CLIP). The resulting open MHC-II
groove is peptide-receptive and can rapidly scan for the best fit-
ting sequences (Chou and Sadegh-Nasseri, 2000; Natarajan et al.,
1999; Rabinowitz et al., 1998), generating tightly formed pMHC-II
complexes that are no longer recognized by DM (Narayan et al.,
2007, 2009) and that would likely have a longer half-life on the
surface of APCs (Nelson et al., 1994). DO works cooperatively with
DM to promote its function in B cells, where it is primarily ex-
pressed (Poluektov et al., 2013; Welsh et al., 2019, 2020).
CD4+ T cell epitopes are often defined using overlapping


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synthetic peptides. While this approach provides a broad survey
of the T cell epitopes within proteins of interest, it does not
.............................................................................................................................................................................
1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA;
2Department of Pathology, Johns Hopkins University School of Medicine,
Baltimore, MD, USA;
3Department of Inflammation and Oncology and Genome Analysis Unit, Amgen Research, Amgen Inc., South San Francisco, CA, USA;
4Department of
Biological Chemistry, Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, MD, USA;
5Department of Biology, Johns
Hopkins University, Baltimore, MD, USA;
6Department of Medicine, University of California, San Francisco, San Francisco, CA, USA;
7Howard Hughes Medical Institute,
Baltimore, MD, USA;
8The Graduate Program in Immunology and Medical Scientist Training Program, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
*J. Zhang, M.C. Reed, and J. Yu contributed equally to this paper. Correspondence to Scheherazade Sadegh-Nasseri: ssadegh@jhmi.edu; Robert F. Siliciano: rsiliciano@
jhmi.edu.
© 2023 Sengupta et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/
4.0/).
Rockefeller University Press
https://doi.org/10.1084/jem.20221654
1 of 24
J. Exp. Med. 2023 Vol. 220 No. 7
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Journal of Medical Genetics (1975).
12, 135.
Amniotic fluid macrophages and the antenatal
diagnosis of anencephaly and spina bifida
GRANT R. SUTHERLAND,* D. J. H. BROCK, and J. B. SCRIMGEOUR
Department of Pathology, Royal Hospital for Sick Children and University of Edinburgh, and the
Departments of Human Genetics and Obstetrics and Gynaecology, University of Edinburgh
Summary.
The macrophage content of amniotic fluid has been measured and
the upper limit of normal on an arbitrary scale is 41.
Amongst 65 amniotic fluids
collected for antenatal diagnostic studies before 22 weeks' gestation there were eight
which had macrophage counts ranging from 82 to 6226, three of these were shown
to have anencephaly and two spina bifida.
The reasons for three apparently false
positives are as yet undetermined.
Rhesus iso-immunized amniotic fluids were
found to have macrophage counts of up to 276 and a possible explanation for this is
considered.
It is argued that an elevated amniotic fluid macrophage count may
indicate a CNS defect or possibly other fetal abnormality.
The finding by Sutherland, Brock, and Scrim-
geour (1973) of large numbers of macrophages in
the amniotic fluid from two cases ofanencephaly has
been used by Nelson, Ruttiman, and Brock (1974)
to make the antenatal diagnosis of a further case.
The macrophage content of spina bifida and anen-
cephalic amniotic fluids has been quantitated and
compared with that from normal pregnancies.
Materials and methods
Amniotic fluids were obtained by amniocentesis for
antenatal chromosome studies, a-fetoprotein estimation,
during the management of Rhesus iso-immunized preg-
nancies and at induction of labour in cases where the
fetus was known to have anencephaly.
Amniotic fluid cultures were established as previously
described (Sutherland, Grace, and Bain, 1973).
The
culture vessel was a 50 mm Petri dish which contained
five or six 6 x 22 mm glass coverslips.
The culture
medium was Ham's F10 supplemented with 30% fetal
calf serum.
The day after cultures had been set up


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(usually about 20 hours later) one of the coverslips was
aseptically removed from the Petri dish, washed well in
phosphate buffered saline, fixed with methanol, stained
with Giemsa, and mounted on a slide.
The number of
glass adherent cells in 10 low power fields ( x 100) were
counted and this number was adjusted to relate to 10 ml
of amniotic fluid, ie, if the cells from 5 ml of amniotic
fluid had been inoculated into the Petri dish then the
number was multiplied by two.
The resultant macro-
phage count (glass adherent cells/10 low power fields/
10 ml of amniotic fluid) has been used to compare dif-
ferent samples of amniotic fluid.
Amniotic fluid a-
fetoprotein was measured according to Brock and Sut-
cliffe (1972).
Results
The macrophage counts for the 94 fluids studied
are shown in Fig. 1.
All the fluids classed as 'nor-
mal' had low macrophage counts (range 0-41), and
there was no reason to suspect fetal CNS abnor-
mality either because the infant had been delivered
or because the amniotic fluid a-fetoprotein levels
were not elevated.
Four amniotic fluids from cases
of hydramnios of unknown aetiology had macro-
phage counts within the normal range.
A number
of samples of amniotic fluid showing different de-
grees of blood staining had normal macrophage
counts.
Rhesus iso-immunized amniotic fluids had
highly variable macrophage counts ranging up to
276.
Where serial samples were obtained from
these pregnancies the macrophage counts tended to
decrease as gestation progressed.
Details of the cases with anencephaly or spina
bifida are shown in Table I.
In all except one of
these fluids the macrophage count was elevated,
135
Received 21 June 1974.
* Present address and reprint requests to: Cytogenetics Unit,
Department of Pathology, The Adelaide Children's Hospital, North
Adelaide 5006, Australia.


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Review article
Journal of Medical Genetics (1975).
12, 174.
Cell membrane receptors for serological reagents
G. W. G. BIRD
West Midlands Regional Blood Transfusion Service, Vincent Drive, Edgbaston, Birmingham B15 2SG
Many useful genetic markers are cell surface struc-
tures.
Furthermore, the cell surface is intimately
involved in cellular immunity, and often in humoral
immunity.
It is therefore important, both in gene-
tics and immunogenetics, to understand cell surface
structure.
Although the cell membrane has been actively
studied for many years, progress in this field is
understandably slow.
It is generally agreed that
the cell membrane is fundamentally a phospholipid
bilayer with inner and outer hydrophilic areas and a
central hydrophobic region.
Besides lipids, the
membrane contains proteins and carbohydrates; the
carbohydrates occur as glycolipids or glycoproteins.
Whereas the protein and lipid portions of the cell
membrane occupy the plane of the membrane, the
carbohydrate chains of the glycolipid and glycopro-
tein components of the cell membrane project out-
wards, so that their N-terminal ends are exposed
above
the
membrane
surface.
Recent
work,
chiefly by Singer and Nicolson (1972) and Nicolson
(1973), on the structural organization ofmembranes,
in terms of the thermodynamics of macromolecular
systems, has led to the conclusion that the cell mem-
brane is a mosaic ofalternating globular proteins and
phospholipid units, in which the phospholipid units
represent the bricks and the globular protein the
mortar (Fig. 1).
The mosaic structure is dynamic
rather than static, with considerable lateral move-
ment of the phospholipid and protein components.
Different membrane components move laterally in
the plane of the membrane at different rates, de-
pending on restraints applied from outside or inside
the cell.
The movement may account for certain
apparent changes on the cell surface, such as the
appearance of 'new' HL-A specificities on cultured
cells (Mackintosh, Hardy, and Aviet, 1971), or for


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changes observed after treatment of intact cells by
Received 26 June 1974.
enzymes.
Indeed, it has been suggested (Singer
and Nicolson, 1972) that after proteolysis or malig-
nant transformation, agglutinin-binding sites diffuse
and aggregate in clusters (Fig. 2).
This might
explain, for example, the very strong agglutination
of papain-treated A2
cells by the virtually A1-
specific agglutinin obtained from the seeds of Doli-
chos biflorus.
Although this lateral movement may
sometimes affect the 'display' of surface compo-
nents, molecular orientation is generally maintained.
Celi surface carbohydrates
We know more about the structure of erythro-
cytes than about any other cells, and more about
those antigens which are carbohydrate than those
which are lipid, protein, or lipoprotein.
There are only seven monosaccharide components
of the human red cell membrane (Winzler, 1969):
galactose, mannose, fucose, glucose, acetylgalactos-
amine, acetylglucosamine and N-acetylneuraminic
(sialic) acid.
All human erythrocyte membrane
carbohydrate chains are therefore combinations of
these seven sugars, and differ from one another in
the serial arrangement of the sugar components, and
the nature of the chemical linkages between them.
There are therefore structural similarities in recep-
tor sites for various serological reagents, so that it is
not surprising that they crossreact; what is surpris-
ing therefore is that many reagents are very highly
specific.
The ABH and Lewis systems
The ABH and Lewis blood group antigenic de-
terminants are the carbohydrate moieties of glyco-
proteins or glycolipids.
Part of the work which led
to the elucidation of ABH and Lewis structure was
done, not on red cells, but on blood group specific
substances present in soluble form in certain body
fluids,
eg,
saliva
of secretors, pseudomucinous
174


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Journal of Medical Genetics (1975).
12, 390.
45,X Turner's syndrome in monozygotic twin sisters
G. PESCIA,* P. E. FERRIER, D. WYSS-HUTIN, and D. KLEIN
Department of Pediatrics and Genetics, University of Geneva, Switzerland
Summary.
A 7-year-old girl was admitted to the hospital for anaemia, secon-
dary to intestinal blood loss (melaena).
She was found to have 45,X Turner's
syndrome.
Her identical twin sister also had Turner's syndrome with a 45,X
chromosome complement.
According
to various
criteria the probability of
monozygosity was 0.9905.
Although the incidence of twinning is greater than
usual in families of patients with Turner's syndrome, affected cases have only
been observed in twin sisters on six occasions.
It seems therefore that the 45,X
chromosome complement itself is not a factor predisposing to twinning, but that
in some families, a factor is at play, which causes either twinning or the 45,X
aneuploidy, or both.
Turner (1938) in his original publication reported
that one of his patients with the triad 'infantilism,
pterygium colli and cubitus valgus' had a normal
twin sister.
Since Turner's original paper, the
association of Turner's syndrome and twinning in
the same family has been recorded on 28 occasions.
The purpose of the present paper is to report the
case of two monozygotic sisters with non-mosaic
45,X Turner's syndrome.
Case reports
The proposita, the second born twin, a 7-year-old-
girl, was admitted to our paediatric service for investiga-
tion of hypochromic anaemia due to recurrent melaena.
Family history was negative for consanguinity, small
stature, sterility, or anaemia.
Twinning has occurred
on two occasions on the matemal side (see pedigree,
Fig. 1).
Pregnancy was complicated by protracted vomiting
during the first 4 months, and by a threatened abortion at
3 months.
Delivery occurred on 15 June 1966, in the
estimated 30th week of gestation.
V.1 weighed 1950 g
at birth, and V.2 (propositus) 1720 g.
Placenta was
unique.
The mother was 22 years old and the father 23.


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The neonatal period was uneventful.
Psychomotor
development of both twins was slow, they walked at 18
months, and began to speak only at 4 years.
Longi-
tudinal growth was regular but slower than that of other
children.
V.2 experienced her first episode of melaena, followed
by palor and fatigue, at the age of 4 years.
A second
episode occurred at the age of 6 years, and a third, at the
age of 7 years, prompted the hospital admission.
V.1
never experienced any gastrointestinal bleeding.
On physical examination (Fig. 2), the proposita (V.2)
was a shy, slightly retarded (2 years retardation) 7 year-
old girl.
Height was 117 cm (10th to 25th centile) and
weight 2400 g (50th centile).
Skin was pale, with several
pigmented naevi on the trunk and a few areas of hyper-
pigmentation on the
extremities.
Palpebral
fissures
were slanted downwards, and a divergent squint was
(fO
Twins
OI
(9 3
Monozyqotic twins
Miscarriage
II
/
Proposita
0
45,X Turner's
syndrome
III
IIIv
1943
944
V
FIG. 1.
Family pedigree.
Received 4 November 1974.
* Present address: Clinique Universitaire de PNdiatrie, 30 bd. de la
Cluse, 121 1-Geneva 4, Switzerland.
390


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Journal of Medical Genetics (1976).
13, 1-8.
Aspects of pre-eclamptic toxaemia of pregnancy,
consanguinity, and twinning in Ankara
ALAN C. STEVENSON,* BURHAN SAYj,t SEDAT USTAOGLU4, and
ZIYA DURMUSt
Summary.
It appears that women classed as having pre-eclamptic toxaemia
are less frequently consanguineous with their husbands than all other mothers and
in particular those mothers classed as having pregnancies complicated by chronic
hypertensive disease.
Search revealed no evidence for possible biases which could
have simulated such findings.
Further evidence is advanced suggesting that, though pre-eclamptic toxaemia is
more common in all types of twin pregnancies than in single births, it is more com-
mon where the twins are dizygous than where they are monozygous.
It is pointed out that both these findings would be expected ifthere was a contri-
bution to the aetiology of pre-eclamptic toxaemia by maternal/fetal immunological
incompatibility.
However, if such a mechanism exists it is not always determined
at the same gene locus.
Starting in 1971 a special study was made of all
women
delivered
in the Dogum Evi Hospital,
Ankara.
From data accumulated after the first
5286 mothers who had been delivered, Stevenson
et al (1971) concluded that the proportion of women
who had pre-eclamptic toxaemia who were related
to their husbands was significantly less than in the
other patients.
By early 1973, 23 416 mothers with single preg-
nancies and 271 with twin pregnancies had been
studied.
Basic
information
concerning
these
patients is set out in Table I.
Records in respect of
58 of the patients with single pregnancies were in-
complete in respect of one or more of the variables
presented in subsequent tables, so these forms were
ignored and the total in subsequent tables is 23 358.
Ten women (none classified as having pre-eclamptic
toxaemia) were identified as having been admitted
for two pregnancies during the period of the study.
The data from both admissions have been included.
Received 16 December 1974.


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* MRC Population Genetics Unit, Old Road, Headington, Oxford.
Present address: Southern District-Highland Health Board, Old
Laboratories, Royal Northern Infirmary, Inverness IV3 5SF.
t Department of Paediatrics, the Hacettepe University, Ankara,
Turkey.
t Dogum Evi Hospital, Ankara, Turkey.
TABLE I
OUTLINE OF DATA
Single births
Males
12455
Females
10 961
Total (births)
23 416
Twin births
MM pairs
80
MF pairs
112
FF pairs
79
Total (pregnancies)
271
Total (births)
542
Grand total (pregnancies)
23 687
Grand total (births)
23 958
Frequency of 'true' pre-eclamptic toxaemia per 1000 total
births
2.4
Stillbirth rate per 1000 total births
19.3
Live births-died before leaving hospital per 1000 total
births
17.0
'Perinatal' mortality per 1000 total births
36.3
Proportion of mothers in relationship with their husbands:
16
132 (fi-st cousins one removed)
2.8%
164 or less (second cousins or less) 0.8%
Any consanguinity
17.0%
* These coefficients are really those of the offspring of such
marTiages, but as a convenience (with many precedents!) they are
applied in this and other Tables to the marriages.
1


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Journal of Medical Genetics (1976).
13, 30-33.
Blood groups and other polymorphisms
in multiple sclerosis
J. L. MacDONALD, D. F. ROBERTS, D. A. SHAW, and M. SAUNDERS
Department of Human Genetics, and the Department of Neurology, University of Newcastle upon Tyne
Summary.
A comparative study was made of blood groups and other poly-
morphic systems in 136 patients with multiple sclerosis in the Newcastle area.
The
majority of systems showed no difference from normal frequencies, but differences
in the ABO and rhesus systems are suggested.
The demonstration of an association of multiple
sclerosis
with
the
histocompatibility
antigens,
HL-A 3 of the LA series, and HL-A 7 and W-18 of
the FOUR series (Arnason et al, 1972; Naito et al,
1972;
Bertrams,
Kuwert,
and
Liedtke,
1972;
Jersild et al, 1973; Smeraldi, 1973), has reopened the
question of genetic susceptibility to the disease, and
in particular whether patients represent a subsection
of the population that is genetically identifiable.
It appears worth while, therefore, to re-examine the
association of the disease with blood groups, which
has remained controversial throughout the 10 years
since it was first suggested (McAlpine, Lumsden,
and Acheson, 1965).
This paper reports a com-
parison of 8 blood group and 5 other polymorphic
systems in multiple sclerosis patients in the New-
castle area of the north-east of England.
Subjects and methods
The subjects were 136 multiple sclerosis patients, diag-
nosed and accepted as such in the Neurology Department
of the Royal Victoria Infirmary, Newcastle.
None of
the patients was included in the earlier series of Simpson
et al (1965) as they had all presented after that date.
From each patient a blood and saliva specimen was
obtained.
The blood specimens were tested by standard
serological procedures with the following antisera: anti
A, Al, B, A+B, C, c, D, E, e, M, N, S, s, K, k, P, Jka,
Fya.
The salivas were tested with anti-H lectin and
where necessary with A and B antisera for secretor
status.


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status.
For identification of the various isoenzymes, horizontal
starch gel electrophoresis was performed, using gels of
9% BDH hydrolysed starch.
Adenylate kinase was
examined by the method of Fildes and Harris (1966), acid
Received 28 January 1975.
30
phosphatase by the discontinuous buffer system as given
by Hopkinson, Spencer, and Harris (1964), and phospho-
glucomutase was typed using double the molar con-
centration of all ingredients of the buffer given by
Spencer, Hopkinson, and Harris
(1964)
at pH
7.4.
Haptoglobin was typed using the discontinuous buffer
system of Poulik and Smithies (1957) at 450 volts for 3 to
4 hours in a cold room at 40 C, and staining with
benzidene to visualize the phenotypes.
Comparative figures were obtained in two ways.
The
first
series
(referred to
as the population
series)
is
derived from the frequencies regarded as normal for
the population.
For the ABO and red cell enzyme
systems, frequencies specific to the north of England
are available (Papiha and Roberts, 1976), and for hapto-
globins (Kopec, 1970; Papiha, 1973).
For the other
blood groups, however,
the MN,
Ss, rhesus,
Kell,
Duffy, Kidd, and secretor frequencies were taken from
Race and Sanger (1968, pages 91, 92, 180, 267, 332,
348, and 294, respectively), and the P frequencies from
Mourant (1954, page 366, line 1).
Such comparative
data are not entirely satisfactory,
first because they
ignore the possible existence of local genetic variation in
the populations of Britain; and secondly because there
may be some laboratory variation in technique.
Both of
these could promote differences between our observed
and the 'expected' frequencies.
To overcome these two
possible objections, as a second control series, specimens
were
taken from
136
individuals
without
multiple
sclerosis and tested at the same time by the same pro-
cedures in this laboratory.
These controls included 98
individuals with minimal dermatoses attending the out-
patient clinics of the dermatology department, and 38


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normal parents of a series of healthy children.
Results
In Table I are set out the results on the multiple
sclerosis
patients, compared
with
the numbers
expected
in
each
system from
the
population


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Review article
Journal of Medical Genetics (1976).
13, 81-90.
Genetic markers in atherosclerosis: A review*
NEWTON E. MORTON
Population Genetics Laboratory, University of Hawaii, Honolulu
Summary.
There is a growing number of lipoprotein markers recognized by
immunological, electrophoretic, and other biochemical methods, and a beginning
has been made on studying their modes of inheritance and linkage relations.
Suggestive but inconclusive evidence of a relation between the cerumen polymor-
phism and arteriosclerosis has been published.
Associations of the ABO blood
groups with cardiovascular disease and serum lipid levels have been established,
but the exact relation to lipoproteins and atherosclerosis remains to be determined.
The subject of genetic markers in atherosclerosis
is at the intersection of genetics, medicine, epide-
miology, immunology, and biochemistry.
The ob-
servations are scattered, sometimes preliminary or
conflicting, and any review must soon be obsolete.
Recognizing these limitations, I shall discuss the
present state of our knowledge on antigenic poly-
morphisms of lipoproteins (Ag, Lp, and Ld), the
controversial evidence of a relation between Lp and
the HL-A locus, the cerumen polymorphism (W),
electrophoretic variants of lipoproteins,
available
evidence on linkage of lipoprotein markers, and
associations of the ABO blood groups with cardio-
vascular diseases and lipoproteins.
Citation in this
review does not imply credence, and my comments
in the last section are deliberately speculative.
Antigenic polymorphisms
Three genetic polymorphisms of ,B-lipoprotein
have been reported on the basis of the Ouchterlony
method of double diffusion in agar gel.
Allison
and Blumberg (1961)
discovered
in
a multiply
transfused patient (C. de B.) an antiserum which
they attributed
to
a polymorphic factor Ag( +),
later designated Ag(a).
Subsequently, Hirschfeld
(1963) showed that anti-Ag(a) is a mixture of three
specificities, anti-Ag(al), anti-Ag(x), and anti-Ag(z),
which behave as a factor-union system.


