Spontaneous Pathology of the Laboratory Non-human Primate [1 ed.] 0128130881, 9780128130889

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Spontaneous Pathology of the Laboratory Non-human Primate

Edited by Alys E. Bradley Charles River Laboratories, Edinburgh, United Kingdom

Jennifer A. Chilton Charles River Laboratories, Reno, NV, United States

Beth W. Mahler Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-12-813088-9 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Stacy Masucci Acquisitions Editor: Wendi Baker Editorial Project Manager: Sam Young Production Project Manager: Punithavathy Govindaradjane Cover Designer: Christian J. Bilbow Typeset by TNQ Technologies

Contributors Roger Alison, Roger Alison Ltd, Lampeter, United Kingdom

Elizabeth H. Hutto, Hutto Pathology Services, Hopkinton, MA, United States

James E. Baily, Charles River Laboratories, Edinburgh, United Kingdom

Kaori Isobe, Charles River Laboratories Edinburgh Ltd., Tranent, United Kingdom

Alys E. Bradley, Charles River Laboratories, Edinburgh, United Kingdom

William O. Iverson, Iverson Consultancy, LLC, Faber, VA, United States

Agathe Bédard, Charles River Laboratories, Montreal, QC, Canada

Robert A. Kaiser, XenoTherapeutics, Boston, MA, United States

Maurice Cary, Pathology Experts GmbH, Witterswil, Switzerland

Kevin A. Keane, Blueprint Medicines, Cambridge, MA, United States

Ronnie Chamanza, Janssen Pharmaceutical Companies of Johnson & Johnson, High Wycombe, United Kingdom

M. Kelly Keating, Animal Dermatology Group, Inc., Las Vegas, NV, United States

Jennifer A. Chilton, Charles River Laboratories, Reno, NV, United States

Shyamesh Kumar, Vertex Pharmaceuticals, Boston, MA, United States

J. Mark Cline, Pathology/Comparative Medicine, Wake Forest School of Medicine, WinstoneSalem, NC, United States

Elizabeth F. McInnes, Syngenta Ltd., Bracknell, United Kingdom

Karyn Colman, Translational Medicine/PreClinical Safety, Novartis Institutes for BioMedical Research, Cambridge, MA, United States

Jagannatha V. Mysore, Department of Pathology, Nonclinical Safety, Bristol Myers Squibb, New Brunswick, NJ, United States

Sarah Cramer, StageBio, Frederick, MD, United States

Stuart W. Naylor, Charles River Laboratories, Edinburgh, United Kingdom

Eveline P.C.T. de Rijk, Charles River Laboratories, ‘sHertogenbosch, the Netherlands Edward Dick, Texas Biomedical Research Institute, San Antonio, TX, United States

Anne-Marie Mølck, Novo Nordisk A/S, Måløv, Denmark

Michael Owston, Charles River Laboratories, Ashland, OH, United States

Pierluigi Fant, Charles River Laboratories, Lyon, France

Ingrid D. Pardo, Biogen, Inc., Cambridge, MA, United States

Thierry D. Flandre, Novartis Pharma AG, Basel, Switzerland

George A. Parker, Charles River Laboratories, Durham, NC, United States

Begonya Garcia, Charles River Laboratories, Evreux, France

Nicola M.A. Parry, Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA, United States

Olga Gonzalez, Texas Biomedical Research Institute, San Antonio, TX, United States Margarita M. Gruebbel, Experimental Pathology Laboratories, Inc., Sterling, VA, United States

Alessandro Piaia, Switzerland

Novartis

Pharma

AG,

Basel,

Solomon Haile, Charles River Laboratories, Montreal, QC, Canada

Florence M. Poitout-Belissent, Clinical Pathology, Charles River Laboratories, Montreal ULC, Senneville, QC, Canada

Warren Harvey, Nonclinical Consulting, Drug Development Solutions, ICON plc, Dublin, Ireland

Shari A. Price, Charles River Laboratories, Frederick, MD, United States

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Contributors

Shannon R. Roff, Charles River Laboratories, Frederick, MD, United States

William Siska, Clinical Pathology, Charles River Laboratories, Reno, NV, United States

Petrina Rogerson, Charles River Laboratories, Tranent, United Kingdom

Ingrid Sjögren, Novo Nordisk A/S, Måløv, Denmark

Aaron M. Sargeant, Charles River Laboratories, Spencerville, OH, United States

Justin D. Vidal, Charles River Laboratories, Ashland, OH, United States

Melissa M. Schutten, Genentech, Safety Assessment, One DNA Way, South San Francisco, CA, United States

Angela L. Wilcox, Clinical Pathology, Charles River Laboratories, Reno, NV, United States

Julie A. Schwartz, Charles River Laboratories, Reno, NV, United States

Zbigniew W. Wojcinski, Toxicology & Pathology Consulting, LLC, Hillsborough, NC, United States

Heather A. Simmons, Wisconsin National Primate Research Center, University of WisconsineMadison, Madison, WI, United States

Jayne A. Wright, Jayne Wright Ltd., Hereford, United Kingdom

Inger Thorup, Novo Nordisk A/S, Måløv, Denmark

Introduction The use of non-human primates (NHPs) in biomedical and toxicological research has always been a conundrumdboth blessing and curse. The blessing comes in generally highly reliable safety data when extrapolated to human exposures. The curses lie in the ethical dilemma and in the knowledge base of spontaneous findings that occur in NHPs. While these blessings and curses will remain with us for some time to come, it is hoped that the latter will be somewhat alleviated through this publication.

for toxicologic studies. The information has been gleaned from numerous journal articles, books, case reports, control animal historical data, and from colony cases obtained from the editor’s and author’s facilities. Thus, the text is the sum of the authors’ experiences and builds on the knowledge of NHP findings previously published. This book is not intended to provide indepth information of these topics, but to provide a starting point for identification of findings and basic knowledge of their etiologies.

With few exceptions, NHPs are not pathogen free and are not free of background inflammatory or congenital lesions. They are susceptible to environmental toxins, pathogens, and other conditions that influence findings within organs. Contained in the pages of this book are compilations of known and some not previously published, incidental, idiopathic, and infectious agent findings in NPHs commonly utilized

No work of this scope is completed without extensive participation by knowledgeable individuals; therefore, sincere thanks are due to the many authors ensconced in the toxicology and pathology disciplines who provided their expertise constructing the chapters of this book. It is hoped that the contents herein provide valuable consolidated references, and the text becomes a reliable source for those working with NHPs.

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Chapter 1

Choice of the non-human primate for biomedical research Jagannatha V. Mysore1, Karyn Colman2, Warren Harvey3 and Robert A. Kaiser4 1

Department of Pathology, Nonclinical Safety, Bristol Myers Squibb, New Brunswick, NJ, United States; 2Translational Medicine/PreClinical Safety,

Novartis Institutes for BioMedical Research, Cambridge, MA, United States; 3Nonclinical Consulting, Drug Development Solutions, ICON plc, Dublin, Ireland; 4XenoTherapeutics, Boston, MA, United States

1. Introduction Whether research is focused on troops of free-roaming primates in the wilderness or on captive-bred animals in laboratory environments, studies involving non-human primates (NHPs) have answered critical questions from wide-ranging disciplines, such as human evolution, sociobiology, conservation biology, biomedical sciences, and drug development. Other laboratory animals (mice, rats, beagle dogs, etc.) are utilized to address similar questions as NHPs; however, these research models often have notable limitations. For example, rats lack foveal vision and continuous binocular fusion of the vision during free movement that is inherently present in humans and most NHPs, and which make the non-human primate a more suitable subject for much of ocular research.1 In many research areas, NHPs are the more favored animal models due to the very fact of phylogenetic proximity and greater genetic homology to humans. At times, multiple NHP models are studied as a necessity for understanding the intricacies of ecological, behavioral, physioanatomical, molecular interactions or therapeutic strategies for a given human disease, a foremost example being NHP models of HIV/AIDS.2e4

1.1 Non-human primate models in biomedical research NHPs are generally considered excellent animal models for addressing specific research questions because of their phylogenetic proximity resulting in physiological and anatomic similarity to humans. This similarity between humans and NHPs means there is often greater validity and translatability of the data obtained from primate models as opposed to other animal models (e.g., reproduction and pregnancy, cognition and cognitive aging). This

physiological similarity also means that one can address questions using NHP models that cannot be adequately addressed using other species, specifically in disease etiology and progression (e.g., models of AIDS, lung disorders, and drug metabolism). For these reasons, NHPs stand as the choice for much of the forefront of biomedical research.

2. Non-human primates as disease models The translational insights obtained from the NHP disease models for noninfectious or infectious diseases are often very relevant to humans. Information gained from these models can be readily applied to the development of diagnostic tools, to validation of targets for therapeutic applications, to biomarker identification, and to vaccination strategies.1

2.1 Non-human primates as infectious disease models From avian influenza to zika virus, NHP models (and approximately 70 other infectious disease models) continue to contribute toward the understanding of disease morbidity, transmission, pathogenesis and immunology.5,6 NHP modes serve to help identify disease vectors and currently, NHP models infected with the novel coronavirus (SARS-CoV-2) have been being utilized to elucidate various facets of the pandemic beginning in 2020.7,8 Additionally, NHP models are essential in understanding more complex infectious disease processes that come under the umbrella of “Molecular Koch’s postulates” such as bovine spongiform encephalopathy caused by

Spontaneous Pathology of the Laboratory Non-human Primate. https://doi.org/10.1016/B978-0-12-813088-9.00009-4 Copyright © 2023 Elsevier Inc. All rights reserved.

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prion protein, hantavirus pulmonary syndrome, and ehrlichiosis.9e11 Studies in NHP models have contributed much to our understanding of the early events of HIV infection and its pathogenic mechanisms. There is currently no experimental animal model that can capture the full spectrum of HIV infection in humans and its clinical sequelae. Despite its limitation as a surrogate model, Rhesus macaques currently represent the most relevant animal model for HIV/AIDS research.12e15 Human herpes simplex virus (cold sores), measles, and tuberculosis can potentially transmit from human carriers to NHPs (reverse zoonosis), as well as vice versa. A thorough understanding of the natural history of “reverse zoonosis” is vital for the conservation of NHPs in the wild, in addition to understanding the potential impact of such transmission on results produced in experimental work.16,17

2.2 Non-human primates as noninfectious disease models In research areas such as neurocognitive disorders, neurodegenerative diseases, obesity, atherosclerosis, pulmonary dysfunction, and transplantation immunopathology, etc., the NHP models have unparalleled utility when compared to other laboratory animal models. This exceptionality is well documented in the voluminous body of literature; and continues to contribute toward understanding of the physiology and underlying pathologies of these conditions in humans.18e21 Collectively, NHPs exhibit features of lung architecture and immunity that make them highly appropriate for elucidating novel therapeutic approaches to treat chronic lung disease in humans.22,23 These features have significantly impacted our understanding of the origins and treatment of chronic lung disease in multiple age groups. Macaques are a well-established model of diet-induced coronary artery atherosclerosis, as they develop arterial lesions similar in terms of etiology and characteristics of arterial pathology to those seen in humans.24 There are many possibilities for establishing models for human genetic diseases using NHPs and, with continued advances in molecular, genetic, and embryo technologies, their role in biomedical research will likely increase despite a climate of increased scrutiny and regulation.25 Laboratory rodents and dogs often fail to answer research questions in the area that involve higher intellectual functions of the prefrontal cortex and complex motor skills of humans, whereas NHP’s similarities in these areas make them irreplaceable.26 A global initiative is underway to map the circuitry of the human brain in a similar approach to that utilized by the Human Genome Organization (HUGO-http://www.hugo-international.org/).27 In support of this initiative, Japan has launched a research program called as “Japan Brain/MINDS” (Brain/Mapping

by Innovative Neurotechnologies for Disease Studies. https://brainminds.jp/en/) and in this ambitious multiinstitutional program, the NHP model of choice is the common marmoset (Callithrix jacchus).28 A similar initiative has been taken up in the USA by the Texas Biomedical Research Institute (Southwest National Primate Research Center) to map the brain at the single cell levels in 3D using the direct comparisons between humans and NHPs (https://www.newswise.com/articles/building-a-3dbrain-atlas).

3. Non-human primates as models of aging and reproduction NHPs have made unique contributions to our understanding of cognitive aging and to the search for possible methods to prevent cognitive declines with age.18 The common marmoset is frequently used to assess age-related neurogenesis, vision, neuroanatomy, cognition, and the pathogeneses of neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases.29e33 Further insight into the aging process is now expected with the successful creation of transgenic marmosets.34 Reproductive senescence research and metabolic changes (e.g., insulin-resistance, diabetes, and obesity) associated with aging also utilize common marmosets.35e38 Despite many basic similarities in the endocrine regulation of reproduction that are common among mammals, NHPs exhibit characteristics not common among other mammals which make them valuable models of specific relevance to humans39e44 Endometriosis is common in NHPs, as it is in humans, making non-human primates good natural models of this disease. Baboons and macaques have been excellent NHP models for the study of endometriosis’ pathogenesis, pathophysiology, spontaneous evolution, and have been essential to the development of new medical treatment options.45

4. Non-human primates in drug and biopharmaceutical discovery and development Non-human primates play a pivotal role, in conjunction with other laboratory animal species, during the discovery and development of new molecular entities (NMEs) or investigational medicinal products (IMPs) that have potential human therapeutic properties. In the recent past, demand for non-human primates has surged significantly over other laboratory animals due to the fast-paced increase in humanized therapeutic monoclonal antibodies (mAbs) and other innovative biotherapeutic modalities that rely on genetic similarity, or even identity, for translatable

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preclinical modeling. These new biologic modalities rely on homologous targets in the NHP for testing, as the lack of homology in other animal species results in excessive and often lethal immune reactions. Undoubtedly, no one species of animal can answer all the questions posed at the time of discovery concerning safety and risk assessments. The selection of the most appropriate species is often made on a case-by-case basis, in the context of the costs involved (both monetary and ethical), historical or known data of the target, route of administration, biophysical properties of NMEs and indications, among other considerations. Following a candidate molecule selection during the discovery phase, the lead compound enters the development phase for safety screening in preclinical species. During the safety assessment, use of a non-rodent species (dogs, minipigs, NHPs) in addition to rodents is mandated by regulatory agencies and limits the uncertainties of the safety data of an Investigational New Drug (IND). At this stage of development, the scientific rigor comprising of a correct choice of species, dose selection, experimental design, etc., are crucial for the success in First-in-Human (FIH) clinical trials. Regulatory agencies (WHO, FDA, EMEA, MHLW, etc.) worldwide accept the NHP models when they represent the most relevant species for the given compoundclass, modality, or indication (see Chapter 2).46,47 When compared to other most commonly used nonrodent species (dogs, minipigs), the following attributes of NHPs distinguish them as a suitable “translationalbridge” between rodent studies and clinical studies in humans.2,3,48e53 l

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Role of social rank (dominant vs. subordinates) that affects physiology, behavior and effects of drugs; this trait is advantageous in assessment of psychiatric drugs. Well-developed prefrontal cortex, cognitive functions and complex motor skills; this feature is very useful to assess neurocognitive and neurodegenerative disorders. NHPs are the species with a homologous immune system to humans; humanized monoclonal antibodies (mAbs) and other large molecule modalities (Fabs, peptides, conjugated proteins, etc.) possess high specificity and limited cross-reactivity to the target. Functionally, the MHC-1 allelic repertoire of macaques parallels that of humans despite sequence differences. Interplay between the MHC-1 mediated antigen presentation and CD8þ T cells is an important attribute to understand the pathogenesis/immunobiology of intracellular pathogens, organ transplantation studies, and to validate immunomodulatory biologics or vaccines. Major site of de novo lipogenesis is the liver over adipose tissue; This is ideal for obesity research. The lung architecture and pulmonary immunology is more similar to humans as compared to other species.

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Frontally placed eyes, binocular vision with color discrimination/trichromatic vision, histology of retina (macula lutea/fovea) resemble that of humans. Blood coagulation pathways of NHPs are similar to that of humans. Placentation is similar to that of humans (chorioallantoic, bidiscoid, villous, deciduate, hemomonochorial) NHPs are less susceptible to vomiting than dogs, therefore overall determination of dosimetry and plasma exposure of the test-article is more straightforward in oral administration studies.

5. Challenges for the use of non-human primates in research Despite some major advantages over rodents or other nonrodent laboratory animals (dogs, minipigs), NHPs present their own challenges and caveats for use in research. The following list summarizes the major issues.2,54e56 l

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Overall, studies involving NHPs are very expensive in terms of cost and resources when compared to other laboratory animals. Due to ethical and cost considerations when using NHPs, numbers should be kept as low as possible and this can result in reduced statistical power in studies. Individual animal variation in responses and/or background observations can interfere with safety assessment endpoints. Cytochrome P450s of Cynomolgus monkeys are similar to humans; however, there are species differences in their substrate specificity and inhibitor selectivity.1 There may be long waiting times to obtain adequate numbers of juveniles, neonates, adults, or reproductively mature females. Importation of wild-caught and captive-bred NHPs may carry primary and opportunistic infections that can interfere with safety assessment, of which some are zoonotic and pose risk to laboratory staff. In the United States of America, commercial airlines will not transport NHPs.

6. The future of non-human primates in biomedical research The future of NHPs in biomedical research may involve several cutting-edge technologies and strategies: CRISPR/ Cas9-mediated transgenic modeling, stem cellebased regenerative medicine, and utilization of metabolic disease models in comparative systems biology.

1. Commonly used NHP species in drug development.

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6.1 Transgene models Thus far, generation of transgenic NHPs for human diseases is inefficient or unsuccessful despite several attempts by employing techniques like retroviral vector, pronuclear microinjection, somatic cell nuclear transfer, etc.57 With the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas9 endonucleasesprecise gene-editing techniques-generation of knock-in and knock-out models are being achieved at a relatively faster rate when compared to older techniques.58 Nevertheless, genetically modified monkey models have been generated for Huntington’s disease, autism, and microcephaly with some success.2 Recently, the dystrophin gene (responsible for Duchenne muscular dystrophy) and the beta gene of hemoglobin were modulated by using CRISPR/Cas9 technique in rhesus macaques.59,60

6.2 Stem cellebased regenerative models Pluripotent stem cells derived from the embryonic stem cells or somatic cells from skin (fibroblasts), blood, umbilical cord, etc., can be “reprogrammed” via viral vectors/CRISPRCas9 genome editing technology to convert them into induced-pluripotent cells (iPSCs). The conversion is accomplished by manipulating key transcription factors.61 In a Parkinson’s disease NHP-model, autologous iPSCs derived from skin fibroblasts were modulated to dopamine secreting neurons in-vitro and autotransplanted into the brain. In this study, treated cynomolgus monkeys exhibited functional recovery of motor deficits up to 2 years. Histologically, there was survival of dopaminergic neurons (iPSCs) and extensive outgrowth in the putamen with no immune reaction.62 In this kind of proof-of-concept study using a novel therapeutic strategy in a neurodegenerative disease, NHP models remain irreplaceable since the clinical parameters, comparative neuroanatomy of humans (neuronal circuitry of putamen/caudate nucleus, location of substantia nigra pars compacta dopamine neurons) are not equivalent to other laboratory animals such as rodents.63

6.3 Comparative systems biology models Biomedical research has progressed from an “-omics” (genomics, proteomics, transcriptomics, metabolomics etc.) era to a more holistic approach known as “Systems Biology.” This interdisciplinary methodology takes all “-omics”, organs, systems, and phenotypes into consideration and computes the data using bioinformatics tools. The main advantage of this approach is a comprehensive understanding of the complex biology of diseases like type 2 diabetes, obesity, nonalcoholic fatty liver disease, cardiovascular diseases, etc.64,65

In the future, simulation studies for this complex interdisciplinary approach will be in need of a laboratory animal species that is as close as possible to humans in their biology and responses. Metabolic disease models in nonhuman primates (in particular cynomolgus and/or rhesus macaques) are very promising for the “simulation studies” compared to other laboratory animal models.

7. Conclusion This chapter collates a “bird’s eye view” of the importance and contributions of NHP models in biomedical research and sets the stage for succeeding specialized chapters in this book. The NHP models continue to contribute immensely in basic and applied aspects of biomedical research including toxicologic pathology, despite being expensive. It is noteworthy that the phylogenetic proximity of NHP models to humans stands out as a translational-bridge to human diseases and often with no alternative laboratory animals. During the last two decades, the demand for NHP models continues to grow because of the increased and successful entry of protein- and genome-based therapeutics and diagnostic markers in the biopharmaceutical sector. Frequently in this class of NMEs, the NHP models are preferred as the most relevant for PK/PD, tissue crossreactivity, and safety evaluation studies. Recently, multiple NHP models underscored the invaluable role during the “warp-speed” development of therapies and vaccine modalities for the pandemic caused by SARS-CoV-2. Furthermore, mechanistic research questions on certain tissue types/functions (prefrontal cortex, foveal vision, color perception) and diseases (Parkinson’s disease, endometriosis) in humans necessitate the NHP models to obtain pertinent answers. Recent developments in the areas such as systems biology, brain mapping, stem cell biology, bioinformatics, and transgene techniques have furthered the unique potential for NHP models to understand complex diseases.

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December 2017;58(2):160e71. ISSN 1930-6180. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/29228218. Newman C, Friedrich TC, O’connor DH. Macaque monkeys in Zika virus research: 1947-present. Curr Opin Virol August 2017;25:34e40. ISSN 1879-6265. Disponível em: https://www.ncbi. nlm.nih.gov/pubmed/28750247. Zhang K, et al. Experimental infection of non-human primates with avian influenza virus (H9N2). Arch Virol 2013;158:2127e34. Chandrashekar A, et al. SARS-CoV-2 infection protects against rechallenge in rhesus macaques. Science August 2020;369(6505):812e7. ISSN 1095-9203. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/ 32434946. Singh DK, et al. Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets. Nat Microbiol January 2021;6(1):73e86. ISSN 2058-5276. Disponível em: https:// www.ncbi.nlm.nih.gov/pubmed/33340034. Gardner MB, Luciw PA. Macaque models of human infectious disease. ILAR J 2008;49(2):220e55. ISSN 1084-2020. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/18323583. Herzog C, et al. Tissue distribution of bovine spongiform encephalopathy agent in primates after intravenous or oral infection. Lancet February 2004;363(9407):422e8. ISSN 1474-547X. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/14962521. Sironen T, et al. Pathology of Puumala hantavirus infection in macaques. PLoS One August 2008;3(8):e3035. ISSN 1932-6203. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/18716663. Evans DT, Silvestri G. Nonhuman primate models in AIDS research. Curr Opin HIV AIDS July 2013;8(4):255e61. ISSN 1746-6318. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/23615116. Lifson JD, Nl H. Lessons in nonhuman primate models for AIDS vaccine research: from minefields to milestones. Cold Spring Harb Perspect Med 2012;2(6):a007310. Van Rompay KK. The use of nonhuman primate models of HIV infection for the evaluation of antiviral strategies. AIDS Res Hum Retrovir January 2012;28(1):16e35. ISSN 1931-8405. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/21902451. Veazey RS. Animal models for microbicide safety and efficacy testing. Curr Opin HIV AIDS July 2013;8(4):295e303. ISSN 1746-6318. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/23698560. Mätz-Rensing K, et al. Outbreak of tuberculosis in a colony of rhesus monkeys (Macaca mulatta) after possible indirect contact with a human TB patient. J Comp Pathol 2015 ;153(2e3):81e91. ISSN 1532-3129. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/ 26166434. Messenger AM, Barnes AN, Gray GC. Reverse zoonotic disease transmission (zooanthroponosis): a systematic review of seldomdocumented human biological threats to animals. PLoS One 2014;9(2):e89055. ISSN 1932-6203. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/24586500. Didier ES, et al. Contributions of nonhuman primates to research on aging. Vet Pathol March 2016;53(2):277e90. ISSN 1544-2217. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/26869153. Havel PJ, et al. Use and importance of nonhuman primates in metabolic disease research: current state of the field. ILAR J December 2017;58(2):251e68. ISSN 1930-6180. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/29216341. Mattison JA, et al. Caloric restriction improves health and survival of rhesus monkeys. Nat Commun January 2017;8:14063. ISSN 2041-1723. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/28094793.

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21. Stouffer RL, Woodruff TK. Nonhuman primates: a vital model for basic and applied research on female reproduction, prenatal development, and women’s health. ILAR J December 2017;58(2):281e94. ISSN 1930-6180. Disponível em: https://www.ncbi.nlm.nih.gov/ pubmed/28985318. 22. Plopper CG, Hyde DM. The non-human primate as a model for studying COPD and asthma. Pulm Pharmacol Ther October 2008;21(5):755e66. ISSN 1094-5539. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/18339566. 23. Plopper CG, et al. Lung effects of inhaled corticosteroids in a rhesus monkey model of childhood asthma. Clin Exp Allergy July 2012;42(7):1104e18. ISSN 1365-2222. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/22702509. 24. Jokinen MP, Clarkson TB, Prichard RW. Animal models in atherosclerosis research. Exp Mol Pathol February 1985;42(1):1e28. ISSN 00144800. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/3881275. 25. Chan AW. Progress and prospects for genetic modification of nonhuman primate models in biomedical research. ILAR J 2013;54(2):211e23. ISSN 1930-6180. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/24174443. 26. Verdier JM, et al. Lessons from the analysis of nonhuman primates for understanding human aging and neurodegenerative diseases. Front Neurosci 2015;9:64. ISSN 1662-4548. Disponível em: https:// www.ncbi.nlm.nih.gov/pubmed/25788873. 27. Yuste R, Bargmann C. Toward a global BRAIN initiative. Cell March 2017;168(6):956e9. ISSN 1097-4172. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/28256259. 28. Lin MK, et al. A high-throughput neurohistological pipeline for brain-wide mesoscale connectivity mapping of the common marmoset. Elife February 2019;8. ISSN 2050-084X. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/30720427. 29. Engelberth RC, et al. Morphological changes in the suprachiasmatic nucleus of aging female marmosets (Callithrix jacchus). BioMed Res Int 2014;2014:243825. ISSN 2314-6141. Disponível em: https:// www.ncbi.nlm.nih.gov/pubmed/24987675. 30. Geula C, Nagykery N, Wu CK. Amyloid-beta deposits in the cerebral cortex of the aged common marmoset (Callithrix jacchus): incidence and chemical composition. Acta Neuropathol January 2002;103(1):48e58. ISSN 0001-6322. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/11837747. 31. Lacreuse A, et al. Oestradiol modulation of cognition in adult female marmosets (Callithrix jacchus). J Neuroendocrinol May 2014;26(5):296e309. ISSN 1365-2826. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/24617856. 32. Mccormack AL, Mak SK, DI Monte DA. Increased a-synuclein phosphorylation and nitration in the aging primate substantia nigra. Cell Death Dis May 2012;3:e315. ISSN 2041-4889. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/22647852. 33. Ridley RM, et al. Very long term studies of the seeding of betaamyloidosis in primates. J Neural Transm September 2006;113(9):1243e51. ISSN 0300-9564. Disponível em: https:// www.ncbi.nlm.nih.gov/pubmed/16362635. 34. Colman RJ. Non-human primates as a model for aging. Biochim Biophys Acta, Mol Basis Dis 09 2018;1864(9 Pt A):2733e41. ISSN 0925-4439. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/ 28729086. 35. Nyirenda MJ, et al. Prenatal programming of metabolic syndrome in the common marmoset is associated with increased expression of 11beta-hydroxysteroid dehydrogenase type 1. Diabetes December

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51.

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2009;58(12):2873e9. ISSN 1939-327X. Disponível em: https:// www.ncbi.nlm.nih.gov/pubmed/19720800. Ross CN, et al. Relation of food intake behaviors and obesity development in young common marmoset monkeys. Obesity September 2013;21(9):1891e9. ISSN 1930-739X. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/23512878. Tardif SD, et al. Characterization of obese phenotypes in a small nonhuman primate, the common marmoset (Callithrix jacchus). Obesity August 2009;17(8):1499e505. ISSN 1930-7381. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/19325546. Rutherford JN, et al. Developmental origins of pregnancy loss in the adult female common marmoset monkey (Callithrix jacchus). PLoS One 2014;9(5):e96845. ISSN 1932-6203. Disponível em: https:// www.ncbi.nlm.nih.gov/pubmed/24871614. Ferin M. Neuroendocrine control of ovarian function in the primate. J Reprod Fertil September 1983;69(1):369e81. ISSN 0022-4251. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/6411915. Karsch FJ, et al. Neuroendocrine basis of seasonal reproduction. Recent Prog Horm Res 1984;40:185e232. ISSN 0079-9963. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/6385166. Plant T, et al. Puberty in nonhuman primates and humans. In: Plant TEAZ, editor. Knobil and Neill’s physiology of reproduction. 4th. San Diego, Ca, USA: Elsevier; 2006. p. 2177e230. Chellman GJ, et al. Developmental and reproductive toxicology studies in nonhuman primates. Birth Defects Res B Dev Reprod Toxicol December 2009;86(6):446e62. ISSN 1542-9741. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/20025046. Weinbauer GF, et al. Physiology and endocrinology of the ovarian cycle in macaques. Toxicol Pathol December 2008;36(7S):7Se23S. ISSN 1533-1601. Disponível em: https://www.ncbi.nlm.nih.gov/ pubmed/20852722. Dettmer AM. The integrative biology of reproductive functioning in nonhuman primates. Am J Primatol March 2013;75(3):197e201. ISSN 1098-2345. Disponível em: https://www.ncbi.nlm.nih.gov/ pubmed/22826005. D’hooghe TM, et al. Nonhuman primate models for translational research in endometriosis. Reprod Sci February 2009;16(2):152e61. ISSN 1933-7205. Disponível em: https://www.ncbi.nlm.nih.gov/ pubmed/19208783. Greaves P, Williams A, Eve M. First dose of potential new medicines to humans: how animals help. Nat Rev Drug Discov March 2004;3(3):226e36. ISSN 1474-1776. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/15031736. Orsi A, et al. Overview of the marmoset as a model in nonclinical development of pharmaceutical products. Regul Toxicol Pharmacol February 2011;59(1):19e27. ISSN 1096-0295. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/21156195. Anderson DJ, Kirk AD. Primate models in organ transplantation. Cold Spring Harb Perspect Med September 2013;3(9):a015503. ISSN 21571422. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/24003248. Buckley LA, et al. Considerations regarding nonhuman primate use in safety assessment of biopharmaceuticals. Int J Toxicol October 2011;30(5):583e90. ISSN 1092-874X. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/22013138. Furukawa S, Kuroda Y, Sugiyama A. A comparison of the histological structure of the placenta in experimental animals. J Toxicol Pathol April 2014;27(1):11e8. ISSN 0914-9198. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/24791062. Mustari MJ. Nonhuman primate studies to advance vision science and prevent blindness. ILAR J December 2017;58(2):216e25. ISSN 19306180. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/28575309.

52. Maness NJ. The importance of understanding MHC-I diversity in nonhuman primate models of human infectious diseases. Toxicol Pathol January 2017;45(1):157e60. ISSN 1533-1601. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/27729588. 53. Kawamura S. Color vision diversity and significance in primates inferred from genetic and field studies. Genes Genomics 2016;38:779e91. ISSN 1976-9571. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/27594978. 54. Sasseville VG, Diters RW. Impact of infections and normal flora in nonhuman primates on drug development. ILAR J 2008;49(2):179e90. ISSN 1084-2020. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/18323580. 55. Sasseville VG, Mansfield KG. Overview of known non-human primate pathogens with potential to affect colonies used for toxicity testing. J Immunot 2010 ;7(2):79e92. ISSN 1547-6901. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/19909217. 56. Yoda N, et al. Characterization of intestinal and hepatic P450 enzymes in cynomolgus monkeys with typical substrates and inhibitors for human P450 enzymes. Xenobiotica August 2012;42(8):719e30. ISSN 1366-5928. Disponível em: https://www.ncbi.nlm.nih.gov/ pubmed/22324396. 57. Chen Y, Niu Y, Ji W. Transgenic nonhuman primate models for human diseases: approaches and contributing factors. J Genet Genomics June 2012;39(6):247e51. ISSN 1673-8527. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/22749011. 58. Chen Y, Niu Y, Ji W. Genome editing in nonhuman primates: approach to generating human disease models. J Int Med September 2016;280(3):246e51. ISSN 1365-2796. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/27114283. 59. Chen Y, et al. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9. Hum Mol Genet July 2015;24(13):3764e74. ISSN 1460-2083. Disponível em: https:// www.ncbi.nlm.nih.gov/pubmed/25859012. 60. Midic U, et al. Quantitative assessment of timing, efficiency, specificity and genetic mosaicism of CRISPR/Cas9-mediated gene editing of hemoglobin beta gene in rhesus monkey embryos. Hum Mol Genet 07 2017;26(14):2678e89. ISSN 1460-2083. Disponível em: https:// www.ncbi.nlm.nih.gov/pubmed/28444193. 61. Daadi MM, et al. Nonhuman primate models in translational regenerative medicine. Stem Cell Dev December 2014;23(Suppl. 1):83e7. ISSN 1557-8534. Disponível em: https://www.ncbi.nlm.nih.gov/ pubmed/25457970. 62. Hallett PJ, et al. Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson’s disease. Cell Stem Cell March 2015;16(3):269e74. ISSN 1875-9777. Disponível em: https://www. ncbi.nlm.nih.gov/pubmed/25732245. 63. Grow DA, Mccarrey JR, Navara CS. Advantages of nonhuman primates as preclinical models for evaluating stem cell-based therapies for Parkinson’s disease. Stem Cell Res 09 2016;17(2):352e66. ISSN 1876-7753. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/ 27622596. 64. Fendt SM, Maranas CD. Editorial overview: systems biology: advances diseases understanding and metabolic engineering. Curr Opin Biotechnol August 2015;34:vevi. ISSN 1879-0429. Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/26096133. 65. Kadarmideen HN. Systems biology in animal production and health. Cham: Springer; 2016. 2 volumes ISBN 9783319433332 (v. 1 alk. paper) 9783319433301 (v. 2 alk. paper). Disponível em: https://link. springer.com/book/10.1007/978-3-319-43335-6. https://link.springer. com/book/10.1007/978-3-319-43332-5.

Chapter 2

Regulations and ethics concerning the use of non-human primates in research Warren Harvey1 and Robert A. Kaiser2 1

Nonclinical Consulting, Drug Development Solutions, ICON plc, Dublin, Ireland; 2XenoTherapeutics, Boston, MA, United States

1. Introduction Non-human primates (NHPs) are used in many areas of biomedical research where their similarities to humans make them exclusively valuable animal models. NHPs are used in the regulatory safety evaluation of potential therapeutics, particularly when other species are not relevant, as is the case with many large biological molecules that rely on close sequence homology to human cell proteins and receptors. Indeed, NHP use is expected to increase due to the growth in the development of biotherapeutics with specific targets that are not compatible in lower species. Research with NHPs has contributed to medical and scientific advances in our understanding in many disease indications including cancer, HIV/AIDS, Alzheimer, Parkinson, infectious and inflammatory diseases and metabolic disorders,1e5 just to name a few, and more recently in the development of vaccines and antibody testing for COVID-19.6,7 NHP use accounts for approximately one-half of 1% of animals in current medical research, but their use presents a contentious issue that raises ethical concerns.8 This chapter will briefly discuss the regulatory guidance supporting NHP use in research and drug development and the ethical issues of NHP use in biomedical and biological research.

2. Regulatory considerations for the use of non-human primates in research The strict regulatory system that exists in Europe, the United States, and most other developed nations is the very embodiment of principles aimed to guide decisions on when and how to conduct studies using NHPs. Some countries have specific regulations surrounding non-human primate research (e.g., the United Kingdom considers

NHPs specially protected species and researchers must explain why no other species can be used instead).9 The European Commission has produced a directive that lays out a framework for animal welfare legislation (2010/63/EU).10 The Directive sought to improve the controls on the use of laboratory animals, set minimum standards for housing and for the training of those handling animals and supervising the experiments. The Directive also aimed to reduce the numbers of animals used for experiments by requiring that an animal experiment should not be performed when an alternative method exists, and by encouraging the development and validation of alternative methods to replace animal methods. In the United States, currently there are several layers of oversight on research with animals guided by a complex set of federal and state laws, regulations, and guidelines. Additional institutional policies have been implemented to systematically address federal requirements and as the result of a voluntary international accreditation process. All non-human primate research is governed by the Animal Welfare Act of 1966 and subsequent amendments (enforced by the US Department of Agriculture (USDA)).11 Research receiving federal funds will be subject to the Public Health Service Policy on Humane Care and Use of Animals (PHS Policy; enforced by the Office of Laboratory Animal Welfare, or OLAW at the National Institutes of Health).12 The PHS Policy also endorses the US Government Principles for the Utilization and Care of Vertebrate Animals Use in Testing, Research, and Training, which forms the foundation for ethical and humane care and use of laboratory animals in the United States. Scientists must also comply with guidelines set forth in the Guide for the Care and Use of Laboratory Animals (also known as “the Guide”).13 Each research facility must maintain an Institutional Animal Care and Use Committee (IACUC) and

Spontaneous Pathology of the Laboratory Non-human Primate. https://doi.org/10.1016/B978-0-12-813088-9.00003-3 Copyright © 2023 Elsevier Inc. All rights reserved.

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Spontaneous Pathology of the Laboratory Non-human Primate

report whether they have the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) accreditation. Regulatory controls on the use of animals in research have existed in the United Kingdom since 1876. These were revised and strengthened with the introduction of the Animals (Scientific Procedures) Act (ASPA) in 1986.9 In 2013, ASPA was amended to bring United Kingdom law into line with the requirements of EU Directive 2010/63,10 and new codes of practice and guidance have been introduced. The United Kingdom regulations are widely regarded as some of the strictest in the world. Research involving animals can only be undertaken when all three of the licenses are granted: establishment license (research can only take place in research institutes or companies which have appropriate animal accommodation and veterinary facilities); project license (research can only be done as part of an approved research or testing program); personal license (research can only be carried out by people with sufficient training, skills, and experience).9 All international NHP imports and exports require permit or certificate documents. The Convention on International Trade in Endangered Species (CITES) permit

system is the backbone of the regulation of trade in live NHPs and all NHP-derived biological specimens. The document is the confirmation by the issuing authority that the conditions for authorizing the trade are fulfilled. This means that the trade is legal, sustainable, and traceable in accordance with the Convention. The transportation of NHPs to and from the United States is integrated into law under the Lacey Act and the Endangered Species Act of 1973 (ESA), which implements the CITES.14 There are guidance documents for developing preclinical safety programs to support human clinical trials. In particular, the International Conference on Harmonization (ICH) M3(R2) titled Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals provides recommendations of what ICH regulatory authorities expect for a safety evaluation program,15 which is applicable in the United States, the European Union, and Japan. Specialized ICH guidelines (ICH S1A-S12) are also available that detail the requirements for types of nonclinical studies or specifying programs for certain compound classes or patient populations (Table 2.1). There is also an expectation that the internationally agreed-upon Organisation for Economic

TABLE 2.1 Specialized International Conference on Harmonization (ICH) guidelines. Code

Document title

Date

M3(R2)

Guidance on the nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals

2009

S1A

Guideline on the need for carcinogenicity studies of pharmaceuticals

1995

S1B(R1)

Testing for carcinogenicity of pharmaceuticals

2022

S1C(R2)

Dose selection for carcinogenicity studies of pharmaceuticals

2008

S2(R1)

Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use

2011

S3A

Note for guidance on toxicokinetics: the assessment of systemic exposure in toxicity studies

1994

S3B

Guidance for repeated dose tissue distribution studies

1994

S4

Duration of chronic toxicity testing in animals (rodent and non rodent toxicity testing)

1998

S5(R3)

Detection of reproductive and developmental toxicity for human pharmaceuticals

2000

S6(R1)

Preclinical safety evaluation of biotechnology-derived pharmaceuticals

2011

S7A

Safety pharmacology studies for human pharmaceuticals

2000

S7B

The nonclinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals

2005

S8

Immunotoxicity studies for human pharmaceuticals

2005

S9

Nonclinical evaluation for anticancer pharmaceuticals

2009

S10

Photosafety evaluation of pharmaceuticals

2013

S11

Nonclinical safety testing in support of development of paediatric pharmaceuticals

2020

S12

Nonclinical biodistribution considerations for gene therapy products

2021

Guidelines available at www.ich.org/products/guidelines.htmlguidelines.

Regulations and ethics concerning the use of non-human primates in research Chapter | 2

Co-operation and Development (OECD) guidelines be considered, where possible.16 This background of regulatory expectations is helpful to keep in mind to develop a preclinical safety testing strategy tailored for the particular needs of a drug development program (i.e., modality and indication). Issued by the US FDA in 2002, the Animal Efficacy Rule (“Animal Rule”)17 expedites the development of new drugs and biologics that can act as countermeasures against biological, chemical, radiological, or nuclear substances of toxic potential. The rule applies exclusively to new agents for which definitive human efficacy studies cannot be conducted as it would be unethical to introduce the disease in humans to then test the safety and efficacy of a product. The US FDA can grant marketing approval to new products that have been demonstrated in well-controlled animal studies to be safe and capable of producing clinical benefit in humans. This type of approval is indicated for diseases such as smallpox, anthrax, botulism and plague.18

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3. Ethical considerations for the use of non-human primates in research Worldwide animal testing for biomedical research on the discovery and development of new medical entities such as drugs, biological therapeutics, medical devices, and innovative medical procedures remains a sensitive issue and continues to raise concerns with the general public. Although researchers would favor alternative in vitro models to using animals, the many simultaneous and interrelated biological, chemical, and physiological processes only available in whole-animal models provide a pivotal contribution to medical advances in both human and veterinary health (Fig. 2.1). Experiments on NHPs are only to be performed when information cannot be obtained using in vitro systems or other animal species. Their use is limited to scientific procedures investigating potentially life threatening and debilitating diseases. NHPs are therefore instrumental for

FIGURE 2.1 Whole animal models are required to capture the integrated physiology compounded from multiple inputs with effects on a snapshot of an individual animal’s basic biochemistry, such as (clockwise from top left) hormonal cycles, diurnal and seasonal rhythms, effects of food consumption, geographic source/location/climate, and genetic heterogeneity (here represented by the impact of liver-metabolic variation). Currently, in vitro systems cannot adequately simultaneously model integration of these and other factors that will contribute to biologic responses to manipulations or test article administrations, especially when accounting for genetic and epigenetic interanimal variability particularly present in outbred models. Although this heterogeneity constitutes a challenge to reproducibility of individual studies it is of paramount importance in translatability to human subjects.

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Spontaneous Pathology of the Laboratory Non-human Primate

the promotion of human health and play a critical role in the advancement of various areas in biomedical research. NHPs remain essentially the nonrodent species of choice in some areas of biomedical and biological research and for the regulatory safety assessment of biopharmaceuticals.19,20 Their use within the biotechnology and pharmaceutical industries is growing in keeping with the increasing number of very specific biopharmaceuticals entering the drug pipeline that are not active or relevant in lower species, but this raises important scientific and ethical issues.21 In all research institutions, researchers must assure a group of independent individuals that the proposed scientific procedures are justified, not redundant with previous studies, and will be performed appropriately, generating valid data that will address a legitimate scientific question. Ethical review committees function separately from the scientific team and any funding agency and can restrict or stop any primate research that the committee considers unnecessary, inappropriately designed, or inadequately justified given the potential effects on the study animals. Importantly, they are generally comprised of individuals familiar with every angle of the research involved (research, veterinary, regulatory, etc.), as well as individuals to represent community and lay concerns. Some would argue that humans, rather than NHPs, are the more appropriate and ethically preferable subjects for biomedical research.22 The logic behind this is that despite the similarities between humans and NHPs, small but significant biological differences do exist. Administration of TGN1412 is a striking example. TGN1412, a CD28 superagonist antibody administered to humans during a clinical trial, led to six human volunteers with life-threatening conditions involving multiorgan failure. The preclinical studies of TGN1412 in NHPs failed to predicate the massive and uncontrolled release of proinflammatory cytokines (cytokine storm) that occurred in humans.23 It was established retrospectively that cynomolgus monkey T-cell biology is different from humans in a way that dramatically reduces the susceptibility of monkey T effector memory cells to respond to TGN1412 in a manner similar to humans.24 Therefore, conclusive results cannot always be obtained from NHP models, and so the ethically preferable choice would be to experiment on a limited number of humans. However, it is essential for the protection of humans that prior research be conducted on animals as a result of the Nuremberg Code25 that defines a set of research ethics for human experimentation and states animal studies must precede research on humans.26 Despite all models being inherently imperfect, appropriately executed testing in representative preclinical species provides the most effective layer of safety available prior to initial human use. In addition to their utility in properly identifying a real risk of an experimental drug,

device, or procedure to human patients and preventing clinical harm, animal studies admittedly have the potential to generate both false negative and false positive results. As such, the imperfection of these models leaves the possibilities of either (1) incorrectly stopping development of an intervention due to a species-specific effect that is actually irrelevant in humans, or worse (2) advancing dangerous interventions into human trials due to lack of sensitivity or relevance to detect the risk preclinically. Opponents to NHP testing that employ the argument of the false negative approach should consider that developers are also accepting this false positive possibility, and the use of NHPs is therefore not self-serving, efficient, or cheap.27 NHPs are simply the best available model with the highest likelihood of providing human-translatable data in efforts to prevent human suffering and promote human treatments. Directive 2010/63/EU,10 legislation adopted by Europe, on the protection of animals used in scientific purposes, specifically recognizes the highly developed social and cognitive capacities of the non-human primate, with specific requirements for their housing and care, and specific measures for natural behavioral environmental and social needs. Protecting the welfare of animals is multifactorial and includes an understanding of the ethical basis in costbenefit scenarios (the costs to the animal being weighed against the benefits to humankind, as well as to animals). Methods to promote the well-being of animals, and appropriate oversight of animal care and use are defined in the Guide for the Care and Use of Laboratory Animals.13

3.1 Implementation of the “3Rs” There may come a time when NHPs use in biomedical research will no longer be necessary, such as in the advent of at least equally reliable in vitro or in silico alternate models or in humanized models in lower species. Until then researchers are accountable to deliver the highest possible standards of animal welfare. This introduces the concept of the “3Rs,” Replacement, Reduction and Refinement, first developed by two British scientists, William Russell and Rex Burch in 1959, in their publication The Principle of Humane Experimental Technique.28 This initiative set out to minimize the use of animals in research and any associated suffering without affecting the quality of scientific work.29 The “3Rs” principle can be summarized as: l

l

l

Replacement of animals with nonanimal methods when available, Reduction of the number of animals used to obtain information of a given amount and precision, Refinement of scientific procedures and husbandry to minimize harm (e.g., pain, suffering, distress) and improve animal welfare.

Regulations and ethics concerning the use of non-human primates in research Chapter | 2

Animal welfare regulations and guidelines for the conduct of safety studies in animals vary around the world, but all share a commitment to the “3Rs”.10,13,15,30 To effectively achieve implementation requires active commitment and collaboration within and between all stakeholders (researchers, veterinarians, and animal care staff) as well as research sponsors/funders and regulatory authorities. Since it is clear that NHPs are still essential in biomedical research, it is the responsibility of the scientific community to (1) ensure potential benefits arising from their use outweigh the burden placed on the animal while establishing a boundary of acceptable animal use; (2) strive to achieve the highest level of well-being in animals used within the scope of the study objectives. It is important to balance these concepts appropriately. The regulatory implementation of the “3Rs” has ensured the study design considers and minimizes impact on the animals enrolled in them.

3.2 “3Rs” and the opportunities for the use of non-human primates in research The majority of NHPs used in research are for pharmaceutical research and development31 and the growing pipeline of biopharmaceuticals, such as monoclonal antibodies (mAbs) and precise gene-targeting technologies, is driving an increase in NHP use worldwide.32 ICH guidelines provide flexibility and support to adopt a case-by-case approach to nonclinical testing and incorporating best-practice for the appropriate use of NHPs for development of biopharmaceuticals. For example, ICH S6(R1) guideline titled Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals33 identifies approaches to minimize the use of NHPs in development of mAbs when there is scientific rationale and knowledge for doing so. These guidelines may include the following: l

l

l

l

l

The acceptance of in vitro alternatives for some types of molecules/therapeutic indications. Eliminate the need for studies with nonrelevant species simply to examine off-target toxicity. Elimination of stand-alone safety pharmacology studies unless there is specific cause for concern. Incorporating safety pharmacology endpoints into repeated-dose toxicity studies, with the most widely implemented technologies/methods including jacketed telemetry for cardiovascular assessment.34 Evaluate the potential of using a single species for longer-term toxicology studies if the toxicity profile in the short-term studies is the same in a second relevant species. Conduct a maximum of two general toxicology studies (1 month or longer)done to support first-in-human (FIH) trials and one to support registration.

l

l

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Include recovery of recovery animals in fewer dose groups. Expand prenatal and postnatal study design to combine reproductive and developmental toxicity studies that minimize the number of animals used in developmental and reproductive toxicity (DART) studies.

These approaches to refinement and reduction focus on principles of good experimental design, and better interpretation and reporting of studies, which help to improve the quality and reproducibility of animal experiments.35,36 The net result is smaller-scale studies, improved utility of data generated from each study, and/or less studies required in specific drug development programs. The European Commission’s Scientific Committee on Health, Environmental and Emerging Risks (SCHEER) in 2017 published a new Opinion on the need for NHPs in biomedical research, production, and testing of products and devices.3 The SCHEER working group conducted a focused, informed, and factual analysis of the status of alternatives to NHPs and the available opportunities for applying all “3Rs”. The SCHEER Opinion clearly identified ongoing progress in applying all “3Rs” to different areas of NHP research. Most of the advances in science and technology that have contributed to the “3Rs” have come from researchers working within their specialist fields, with much of this activity being catalyzed by the UK’s National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs). Approaches that are contributing to the “3Rs” include greater use of human volunteers; development of noninvasive technologies such as imaging; substitution of NHP models with genetically altered mice; advancement of in vitro and in silico methods; development of stem cell models; more efficient design of studies; increased application of training techniques such as positive reinforcement.37

3.3 NHP housing and care management Driven by the guidelines for accommodation and care of animals presented in Appendix A of the European Convention ETS 12338 and Directive 2010/63/EU10 regulations, a switch to European-style pen enclosures for housing NHPs has increased significantly, replacing the more outdated, traditional one-over-one caging units. The changing expectations of pharmaceutical companies and clients of contract research organizations, in line with their global animal welfare policies, have seen substantial improvements in housing conditions and creating a large variety of environmental enrichments, which significantly improve the well-being of captive NHPs. Depending on circumstances (e.g., age of the animals), the minimum mandatory space allocation for macaques under the EU regulations38 can be 5e12 times larger than those in the ILAR Guide13 used in the USA and elsewhere.

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Double-tier cages are not permitted, and enclosures for Old World monkeys, such as macaques, must be 1.8 m high (equivalent to 5.9 feet), recognizing the importance of the vertical dimensions for primates, which flee upwards when alarmed. The minimum enclosure volume for Old World monkeys is 3.5 cubic meters (equivalent to 127 cubic feet), in which up to three macaques under 3 years of age, or two macaques over 3 years of age can be housed. In this way, the EU regulations38 encourage pair- and group-housing and maximize the available room space. The enclosure space should allow animals to walk, climb, jump, and in general to express the largest variety of their specific behavioral repertoire. To better fulfill this requirement, environmental enrichment plays a pivotal role. Foraging devices can be used to encourage NHPs to leverage their cognitive and motor skills by exploring their environment in search of food. Wooden structures, ropes, and swings can induce monkeys to climb and explore the environment from elevated positions, which positively contributes to the animals’ feeling of security. Providing coarse grain sawdust bedding or bark substrates where food items may be placed encourages foraging activities that NHPs would typically perform in a natural environment.21,39 Interaction with staff using positive reinforcement techniques to desensitize and habituate animals to handling and technical procedures is a major refinement goal.40 For rhesus and cynomolgus monkeys, single housing induces behavioral abnormalities which include autism-like behaviors (stereotypic behaviors) and depression-like behaviors (sitting in a hunched posture, head lower than shoulders), while group housing decreases such abnormal behaviors.41 In addition, there is evidence to suggest that the immunological responses of natural killer cell activity and interferon production are affected by housing conditions.42 These behavioral and physiological abnormalities are undesirable for achieving biomedical research objectives and for improving animal welfare, indicating the importance of providing social environmental enrichment to animals. Thus, data from experiments carried out on well-balanced, healthy, and calm animals are more likely to be consistent and meaningful, whereas poor welfare can lead to a variety of physiological and psychological responses that will affect interpretation of experimental results.39 NHP diet is also an important factor to be considered, which should be varied and scattered to encourage foraging.38 NHPs are generally fed commercial primate diets in the form of extruded pellets or biscuits. This is supplemented with daily fresh vegetables, fruits, and nuts for enrichment, but more importantly, to ensure that animals receive a balanced diet with sufficient quantities of proteins, vitamins, and minerals, some of which may be deficient in captive primate diets. In particular, due to

limitations in caging and provided diet formulation, the diet fed to marmosets unlikely recapitulates their natural diet. Marmosets have a dietary requirement for vitamin D3, and inadequate dietary levels can result in osteomalacia or other bone pathology. Marmosets lack gulonolactone oxidase and are subsequently susceptible to scurvy; therefore, vitamin C is an essential dietary nutrient required for collagen cross-linking and for preservation of their health.43

4. Conclusion NHPs are phylogenetically the most closely related preclinical species to humans, with many distinctive and unique similarities not found in other animal models. However, tragic events in drug development history should also serve as a constant reminder to nonclinical safety scientists that close phylogenetic proximity of the NHP and humans do not automatically imply predictability and translational relevance of the model. Their use remains necessary in the safety evaluation testing of many new pharmaceuticals, mainly biologicals and in several areas of biomedical research, such as research on diabetes, obesity, lung disorders, cardiac and vascular injury. Regulatory bodies and guidance documents are available to support researchers in drug development and biomedical research. These guidance documents implement the “3Rs” approach to ensure all studies are conducted to best practice incorporating up-to-date scientific knowledge, while minimizing the use of the NHP and/or optimizing the procedures that are required in this most translatable, precious, and ethically sensitive animal model.

References 1. Friedman H, Ator N, Haigwood N, et al. The critical role of nonhuman primates in medical research - white paper. Pathog Immun 2017;2(3):352e65. 2. Phillips KA, Bales KL, Capitanio JP, et al. Why primate models matter. Am J Primatol 2014;76(9):801e27. 3. SCHEER (Scientific Committee on Health, Environmental and Emerging Risks). Final Opinion on the need for non-human primates in biomedical research, production and testing of products and devices (update 2017). 2017. 4. Hobson W. Safety assessment studies in nonhuman primates. Int J Toxicol 2000;19(2):141e7. 5. Weatherall D. The use of non-human primates in research. London: Academy of Medical Sciences; 2006. 6. Yee JL, Van Rompay K, Carpenter AB, et al. SARS-CoV-2 surveillance for a non-human primate breeding research facility. J Med Primatol 2020;49(6):322e31. 7. Muñoz-Fontela C, Dowling WE, Funnell SGP, et al. Animal models for COVID-19. Nature 2020;586:509e15. 8. Chatfield K, Morton D. The use of non-human primates in research. In: Schroeder D, et al., editors. Ethics dumping. Springer Briefs in Research and Innovation Governance; 2018.

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9. United Kingdom animals (scientific procedures) act 1986 amendment regulations 2012 (the Act). 10. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes; 2010. 11. Animal welfare act. 7 U.S.C. xx 2131e2159. 12. Office of laboratory animal welfare public health Service policy on humane care and use of laboratory animals; 2015. 13. National Research Council (US). Committee for the update of the guide for the care and use of laboratory animals. Guide for the care and use of laboratory animals. 8th ed. Washington (DC): National Academies Press (US); 2011. 14. Kreger M, Farris M. National Research Council (US) Institute for Laboratory Animal Research. International perspectives: the future of nonhuman primate resources. Washington (DC): National Academies Press (US); 2003. International transportation of nonhuman primates: US fish and wildlife service perspective. 15. ICH (International Conference on Harmonization M3(R2). Guidance on nonclinical safety studies for the conduct of human clinical trials and marketing authorisation for pharmaceuticals. 2009. 16. OECD guidelines for the testing of chemicals, Section 4. https:// www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testingof-chemicals-section-4-health-effects_20745788. 17. U.S. Department of health and human services, food and drug administration, center for drug evaluation and research (CDER)/ center for biologics evaluation and research (CBER). Product development under the animal rule, Guidance for Industry; October 2015. 18. Allio T. The FDA animal rule and its role in protecting human safety. Expet Opin Drug Saf 2018;17(10):971e3. 19. Chapman KL, Pullen N, Graham M, et al. Preclinical safety testing of monoclonal antibodies: the significance of species relevance. Nat Rev Drug Discov 2007;6:120e6. 20. Chapman K, Pullen N, Coney L, et al. Preclinical development of monoclonal antibodies: considerations for the use of non-human primates. mAbs 2009;1(5):505e16. 21. Chapman K, Bayne K, Couch J, et al. Opportunities for non-human primates testing implementing the 3Rs in drug development and safety assessment studies using nonhuman primates. In: Bluemel J, et al., editors. The nonhuman primate in drug development and safety assessment. Massachusetts, USA: Elsevier; 2015. 22. Quigley M. Non-human primates: the appropriate subjects of biomedical research? J Med Ethics 2007;33:655e8. 23. Attarwala H. TGN1412: from discovery to disaster. J Young Pharm 2010;2(3):332e6. 24. Horvath CJ, Milton MN. The TeGenero incident and the duff report conclusions: a series of unfortunate events or an avoidable event? Toxicol Pathol 2009;37(3):372e83. 25. United States. Nuremberg code. Trials of war criminals before the Nuremberg military tribunals under control council law No. 10. Washington, D.C.: U.S. Government Printing Office; 1947.

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26. Ghooi RB. The Nuremberg codeea critique. Perspect Clin Res 2011;2(2):72e6. 27. Bailey J. Non-human primates in medical research and drug development: a critical review. Biog Amines 2005;19(4e6):235e55. 28. Russell W, Burch R. The principles of humane experimental technique. London, UK: Methuen & Co. Ltd.; 1959. 29. Graham ML, Prescott MJ. The multifactorial role of the 3Rs in shifting the harm-benefit analysis in animal models of disease. Eur J Pharmacol 2015;759:19e29. 30. Vasbinder MA, Locke P. Introduction: global laws, regulations, and standards for animals in research. ILAR J 2016;57(3):261e5. 31. Buckley LA, Chapman K, Burns-Naas LA, et al. Considerations regarding nonhuman primate use in safety assessment of biopharmaceuticals. Int J Toxicol 2011;30(5):583e90. 32. Sewell F, Edwards J, Prior H, et al. Opportunities to apply the 3Rs in safety assessment programs. ILAR J 2016;57(2):234e45. 33. ICH S6(R1). Preclinical safety evaluation of biotechnology-derived pharmaceuticals. 2011 [cited 2017 Sep 6]. Available from: http:// www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/ Safety/S6_R1/Step4/S6_R1_Guideline.pdf. 34. Kaiser R, Tichenor S, Regalia D, et al. Telemetric assessment of social and single housing: evaluation of electrocardiographic intervals in jacketed cynomolgus monkeys. J Pharmacol Toxicol Methods 2015;75:38e43. 35. Parker R, Browne W. The place of experimental design and statistics in the 3Rs. ILAR J 2014;55:477e85. 36. Prior H, Sewell F, Stewart J. Overview of 3Rs opportunities in drug discovery and development using non-human primates. Drug Discov Today Dis Models 2017;23:11e6. 37. Burm SM, Prins JB, Langermans J, et al. Alternative methods for the use of non-human primates in biomedical research. ALTEX 2014;31(4):520e9. 38. Council of Europe. European convention for the protection of vertebrate animals used for experimental and other scientific purposes. ETS No.123 Appendix A guidelines for accommodation and care of animals (Article 5 of the Convention). 2006. 39. Jennings M, Prescott MJ. Refinements in husbandry, care and common procedures for non-human primates: ninth report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Lab Anim 2009;43(1):1e47. 40. Bonini L. Refinement techniques in non-human primate neuroscientific research. Ann Ist Super Sanita 2019;55(4):408e12. 41. Koyama H, Tachibana Y, Takaura K, et al. Effects of housing conditions on behaviors and biochemical parameters in juvenile cynomolgus monkeys (Macaca fascicularis). Exp Anim 2019;68(2):195e211. 42. Schapiro SJ, Nehete PN, Perlman JE, et al. A comparison of cellmediated immune responses in rhesus macaques housed singly, in pairs, or in groups. Appl Anim Behav Sci 2000;68(1):67e84. 43. Kramer JA. Diseases of the gastrointestinal system. The common marmoset in captivity and biomedical research. 2019. p. 213e30.

Chapter 3

Infectious diseases of non-human primates Warren Harvey1, Elizabeth H. Hutto2, Jennifer A. Chilton3, Ronnie Chamanza4, Jagannatha V. Mysore5, Nicola M.A. Parry6, Edward Dick7, Zbigniew W. Wojcinski8, Alessandro Piaia9, Begonya Garcia10, Thierry D. Flandre9, Ingrid D. Pardo11, Sarah Cramer12, Jayne A. Wright13 and Alys E. Bradley14 1

Nonclinical Consulting, Drug Development Solutions, ICON plc, Dublin, Ireland; 2Hutto Pathology Services, Hopkinton, MA, United States; Charles River Laboratories, Reno, NV, United States; 4Janssen Pharmaceutical Companies of Johnson & Johnson, High Wycombe, United

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Kingdom; 5Department of Pathology, Nonclinical Safety, Bristol Myers Squibb, New Brunswick, NJ, United States; 6Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA, United States; 7Texas Biomedical Research Institute, San Antonio, TX, United States; 8Toxicology & Pathology Consulting, LLC, Hillsborough, NC, United States; 9Novartis Pharma AG, Basel, Switzerland; 10Charles River Laboratories, Evreux, France; 11Biogen, Inc., Cambridge, MA, United States; 12StageBio, Frederick, MD, United States; 13Jayne Wright Ltd., Hereford, United Kingdom;

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Charles River Laboratories, Edinburgh, United Kingdom

1. Introduction Non-human primates (NHPs), primarily cynomolgus macaques (Macaca fascicularis) and to a lesser extent rhesus macaques (Macaca mulatta), are routinely used in safety evaluation assessment studies because of their high specificity for biopharmaceuticals and relevant pharmacology.1 The common marmoset (Callithrix jacchus), a New World species, although infrequently used in safety studies, are commonly used in research, neurobiology research in particular.2 Unlike other nonrodent laboratory animal species, elimination of infectious diseases from NHP colonies has proven to be challenging. Certain pathogens continue to persist within NHP colonies with the potential to adversely affect the general health of animals on safety evaluation studies and confound study findings. A common awareness and appreciation of NHP husbandry conditions within research facilities over the years has seen preventative measures to limit infectious disease outbreaks and progress toward the elimination of specific pathogens from captive NHP colonies.3 However, the increased worldwide demand for macaques in biomedical research, especially the well-established Mauritius-origin cynomolgus monkeys, has resulted in the shortage of these animals. As an alternative source, researchers have turned to macaques from China and Southeast Asia. However, importation of NHPs from different geographic areas with varying primary and opportunistic pathogenic

potential may predispose them to new or different infections or serve as the origin of the spread of infectious agents to other indigenous or specific pathogen-free (SPF) research colonies, potentially affecting animal health and study outcomes.3 Moreover, NHPs of varying origin may pose a risk to the human populations by providing exposure to potential zoonotic pathogens.3 The coevolution of host and pathogen allows for the propagation and persistence of microbes within the animal, often with a limited detrimental effect to the host. In contrast, when the host’s normal immune response is impaireddfrom environmental stress, experimental or natural infection with immunosuppressive viruses, or safety evaluation testing of immunomodulatory agentsdthe hostpathogen balance is disturbed and may cause potentially confounding disease.4e6 For example, nearly all Old World and New World NHPs harbor infectious agents such as cytomegaloviruses (CMVs) and Lymphocryptoviruses (LCVs) as lifelong, persistent, and typically asymptomatic infections.7 During immunosuppression, these latent viruses may recrudesce, causing progressive cytolytic or lymphoproliferative diseases, respectively. In some viral infections, the clinicopathologic presentations of primary infection differ from the reactivation of latent infection, confounding accurate diagnosis and proper interpretation of study findings. Clearly, infectious diseases may adversely affect research programs and the interpretation of study findings. Therefore, strategies to tackle microbial quality

Spontaneous Pathology of the Laboratory Non-human Primate. https://doi.org/10.1016/B978-0-12-813088-9.00020-3 Copyright © 2023 Elsevier Inc. All rights reserved.

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control in NHPs include evaluation of source colonies and animals, strict quarantine measures, infectious disease preventive health programs, and eradication and control of specific infectious agents, as well as occupational health and safety programs for the animal handlers. This chapter briefly describes the common and opportunistic infectious agents that may affect animal health or confound the interpretation of study findings involving macaques and common marmosets assigned to toxicologic studies. Readers are referred to other seminal and comprehensive works that cover the infectious diseases in more completeness, including the following references; Abee, Mansfield, Tardif, Morris, Eds., Nonhuman Primates in Biomedical Research: Diseases, 2012; Sasseville et al., Meeting Report: Spontaneous Lesions and Diseases in Wild, Captive-Bred, and Zoo-Housed Nonhuman Primates and in Nonhuman Primate Species Used in Drug Safety Studies, 2012; Saravanan, et al., Research Relevant Conditions and Pathology in Nonhuman Primates, 2020; Saravanan et al., Nonhuman Primate Diseases of Relevance in Drug Development and their Impact on the Interpretation of Study Findings in The Non-human Primate in Nonclinical Drug Development and Safety assessment, 2015; Mansfield and Kemnitz, Challenges in Microbial Quality Control for Nonhuman Primate, 2008.8e11

2. Viruses 2.1 Retroviruses The family Retroviridae contains viruses that have had, and continue to have, substantial impact on the health of animals as well as untold effects on research and safety outcomes. There are two subfamilies within the Retroviridae; the Orthoretrovirinae and the Spumaretrovirinae. The Orthoretrovirinae contains the genera Deltavirus, Lentivirus, Gammavirus, and Betavirus (among others that do not affect NHPs), while the Spumaretrovirinae contains only one genus, Spumavirus.

2.1.1 Betaretroviruses Simian type D retroviruses (SRV) are members of the genus Betaretrovirus, family Retroviridae of which eight distinct serotypes (SRV-1 to -7 and SRV-T) have been identified based on virus neutralization studies and genetic divergence.11,12 SRV was originally isolated from a mammary neoplasm in a female rhesus macaque and was definitively identified and linked to the development of immunosuppressive disease in macaques in the 1980s.12 Cynomolgus monkeys are more commonly infected with SRV-2, while SRV-1 is more common among rhesus monkeys.11 If housed together, macaques may share serotypes as the animals often mouth, groom, or bite one

another providing the viral inoculum.12,13 The geographic origin of macaques may influence the SRV-seropositive rates as there is approximately a 50% prevalence among Indonesian animals as compared to the virtually virus-free animals of Mauritius origin.11 SRV infection affects both humoral and cell-mediated immune responses, which contribute to a plethora of (SRV)-associated opportunistic infections.13e15 SRV has had significant impact on research and pharmaceutical safety studies due to the potential for animals to be seronegative for years yet shed virus, thus transmitting the virus throughout a colony, allowing the SRV to escape detection and elimination for years.16 Laboratory facilities therefore either maintained SRV-positive animals or reinfected colonies up until the late 1990s when virus isolation techniques, in conjunction with serologic screening and SRVPCR techniques, helped to establish SRV-free colonies.3 SRV infects immune cells (CD4þ and CD8þ T lymphocytes and B lymphocytes), macrophages, epithelial cells, and choroid plexus cells. Animals infected with SRV may have varied outcomes clinicallydthey may clear the infection, become a carrier, or succumb to fatal disease, often with severe secondary infections. Asymptomatic animals may have nothing more than generalized lymphoid hyperplasia while symptomatic animals usually have marked lymphoid depletion within the spleen, lymph nodes, and thymus and often have bone marrow suppression with peripheral anemia.11 The lymphocytes and plasma cells within the lymph node paracortex may be replaced by histiocytes and contain hyalinised arterioles.17 SRV-2 has been associated with retroperitoneal fibromatosis in dual infection with Retroperitoneal Fibromatosis Herpesvirus (see Rhadinoviruses).

2.1.2 Lentiviruses-simian immunodeficiency viruses (SIV) Simian immunodeficiency viruses (SIV) are among the most thoroughly studied viruses of NHPs. SIV induces a disease spectrum in macaques that is remarkably similar to the disease spectrum of HIV/AIDS in humans, including profound immunosuppression and development of opportunistic infections.18 There are 40 distinct SIVs identified that are native to African species of NHPs, including the Sooty Mangabey and African green monkey, which are considered “natural” hosts for the virus.16 The SIVs are well adapted to their natural hosts and rarely cause overt disease in these species. However, cross-species transmission can occur, and Asian macaques are highly susceptible to infection, either through direct exposure to African NHPs, exposure to tissues/bodily fluids harboring SIV, or through experimental inoculation.19 SIV infection

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in macaques results in variably progressive immune deficiency characterized by profound depletion of CD4þ T lymphocytes, development of opportunistic infections, an increased incidence of tumors, and lentiviral-associated inflammatory lesions.20,21 Modern husbandry and breeding practices have virtually eliminated SIV from macaque colonies used for biomedical research and drug safety testing.3 Though virus may not be demonstrated in the lesion, pulmonary arteriopathy in large and medium-sized vessels has been observed in macaques infected with SIV or simmian-human immunodeficiency virus (SHIV). In rhesus monkeys the lesion is characterized by intimal thickening due to increased presence of smooth muscle cells, intracellular matrix, collagen, and macrophages. Thrombosis may also be observed. Interestingly, in macaques infected with SHIV containing the HIV nef gene, intimal and medial lesions like the arteriopathy seen in humans have been observed. Similar lesions have been seen in pigtailed macaques infected with SIV and SHIV, while systemic arteriopathy in SIV-infected rhesus macaques, most likely caused by disseminated CMV infection, has also been observed.10 SIV infection in macaques causes disease that is very similar to HIV in humans, and macaques are a commonly used model for studying HIV-associated encephalitis as well as peripheral neuropathy.22e25 Numerous models have been developed using multiple macaque species (including cynomolgus, rhesus, and pigtail), multiple viral strains, and development of accelerated models.26 In macaque models, encephalitis typically develops in approximately 25% of animals over a time course of one to three years.25 In humans, HIV encephalitis is characterized by neuronal loss, astrocytosis, microglial nodules, and formation of multinucleated giant cells. Experimental SIV in macaques causes similar encephalitic lesions though SIV-induced disease may be more severe than HIV encephalitis.25 SIV encephalitis is characterized by SIVþ macrophages that infiltrate meninges, form perivascular cuffs and nodules, and sometimes infiltrate brain parenchyma.24,25,27 Astrocytes and microglia are activated, and microglial nodules may form.25 Multinucleated giant cells form frequently, not only in the brain, but also within the lungs and lymphoid tissues (Fig. 3.1A and B).23,27,28 Due to immunosuppression, secondary opportunistic infections may occur (Fig. 3.1C). Experimental SIV infection of macaques to model peripheral neuropathy has shown ganglionitis with neuronophagia and nodules of Nageotte, secondary to neuronal loss, in the trigeminal and dorsal root ganglia.29 Experimental models of SIV in rhesus macaques have produced cutaneous maculopapular exanthema similar to the condition in HIV patients. Histopathology was characterized by dermal perivascular lymphoplasmacytic and histiocytic infiltrate.30 Experimental SIV infection also caused decreased epidermal nerve fiber density.23,24

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FIGURE 3.1 Simian immunodeficiency virus (SIV) infection: (A) SIV giant cell pneumonia is characterized by interstitial inflammation, type II pneumocyte hypertrophy and hyperplasia with or without alveolar fluid accumulation, and formation of multinucleated giant cells (arrow). (B) Multinucleated cells (arrow) are found within the brain of an SIV infected macaque. (C) Pancreas: there are characteristic adenovirus inclusion bodies of the cytoplasm or nucleus (arrows) in tissues from an SIV infected macaque with secondary adenoviral infection. (A) Courtesy of M. Kelly Keating; (B-C) Courtesy of Dr. Andres Mejia. Wisconsin National Primate Research Center. University of WI. Madison WI.

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2.1.3 Deltaretrovirus-simian T-lymphotropic viruses (STLV) The primate T-lymphotropic viruses include the Simian Tlymphotropic viruses (Simian T-Cell Leukemia Virus (STLV) STLV-2, STLV type L, STLV-3) and have antigenic relationships with the human T-cell-lymphotropic viruses. Non-human primates from the continents of Africa and Asia may carry STLV asymptomatically. In some cases, the animals develop lymphoproliferative disease or lymphoma.10,12 Experimental models in which young rhesus macaques were infected with STLV-3 resulted in maculopapular, erythematous rash throughout sparsely haired skin (trunk, groin, medial thigh, and facial skin) characterized by mononuclear perivascular dermal inflammation, mild acanthosis, spongiosis and, orthokeratotic hyperkeratosis.31 Because infection with STLV may cause changes in immunotoxicology endpoints, screening for the viruses prior to study and within colonies is recommended.10,27,32

2.2 Paramyxoviruses (measles virus) Measles virus is an enveloped, single-stranded, negative sense, RNA morbillivirus in the Paramyxoviridae family. This virus is highly contagious among many NHPs and usually finds its way into a colony from contact with an infected human. Measles usually causes mild or asymptomatic disease unless animals are immunodeficient. If immunocompromised, monkeys can develop secondary viral, fungal, or bacterial infections or neurologic disease and there may be virus-induced abortion in pregnant females.6,33,34 Measles-infected animals have been reported with recrudescence of cytomegalovirus (CMV), Macacine herpesvirus 3, or adenovirus.34 The hallmark lesions of measles in NHPs include the maculopapular exanthema of glabrous or sparsely haired skin, Koplik’s spots (white spots) of the buccal mucosa, and variable degrees of lymphoid depletion. Particularly in macaques and marmosets, there may be severe gastroenteritis leading to hemorrhagic diarrhea.6,35 Koplik’s spots are often considered the earliest indication of measles infection in humans and usually occurs prior to skin lesions, but reference to these lesions as precursor to overt disease in NHPs is anecdotal in the literature, and the spots have currently only been reported following the appearance of skin lesions.36 Histologically, Measles virus infection can result in marked thymic atrophy and lymphoid depletion or necrosis in other lymphoid organs (Fig. 3.2B).29 In the lymphoid organs, there are often antigen-positive multinucleated syncytial cells, also known as Warthin-Finkeldey giant cells, in the germinal centers. Similar syncytial cells may be

noted in the proliferative bronchointerstitial pneumonia of the lung (Fig. 3.2A) or a number of other tissues of epithelial origin. A necrotizing, often hemorrhagic, gastroenteritis is not uncommon in NHPs with severe cases (Fig. 3.2D). The skin lesions have multinucleated keratinocytes associated with necrotic cells and may develop acanthosis, parakeratosis, and hyperkeratosis. The characteristic brightly eosinophilic, single to multiple, variably sized intracytoplasmic and/or intranuclear inclusions may be noted in nearly any epithelial tissue (Fig. 3.2C), glial cells, or neurons. Additional findings may include hypercellularity of bone morrow, liver necrosis and perivascular cuffs of lymphocytes and plasma cells in the brain with or without demyelination. Animals that survive measles infections will have lifelong immunity and vaccination programs may prevent colony outbreaks.

2.3 Herpesviruses Herpesviruses are large double-stranded DNA viruses that are prevalent throughout the animal kingdom. Both Old World monkeys (OWMs) and New World monkeys (NWMs) are primary hosts to a number of simian herpesviruses that generally bear significant homology to human herpesviruses, and often cause similar, if not identical, disease manifestations.37 As with other herpesviruses in general, they often cause a mild or inapparent disease in their natural host but may be associated with severe disease when transmitted to other species.38 This well-described spontaneous interspecies transmissibility of human and simian herpesviruses is responsible for the high zoonotic risk of these viruses. As in humans, simian herpesviruses are subdivided into three distinct subfamilies: alpha (Alphaherpesvirinae), beta (Betaherpesvirinae), and gamma (Gammaherpesvirinae) herpesviruses. Simian alpha herpesviruses of importance include the B virus (Macacine herpesvirus 1, formerly Cercopithecine herpesvirus 1), herpes simplex 1 (Human herpesvirus 1), Herpes T (Saimiriine herpesvirus 1, formerly Herpesvirus tamarinus), and simian varicella virus (Cercopithecine herpesvirus 9); while beta herpesviruses include the cytomegalovirus (Macacine herpesvirus 3, formerly Cercopithecine herpesvirus 8). Gamma herpesviruses are lymphotrophic viruses that are responsible for lymphoproliferative disease and other neoplasia. They are divided into two genera: lymphocryptoviruses and rhadinoviruses, and herpesvirus saimiri (Saimiriine herpesvirus 2), and simian lymphocryptoviruses (LCV) are of particular significance in laboratory NHP. Simian LCV are members of the lymphocryptovirus

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FIGURE 3.2 Measles (Morbillivirus sp.) infection: (A) Immunohistochemistry for measles virus antigen in of the lung of a cynomolgus monkey: The typical measles virus respiratory disease is characterized by interstitial pneumonia and type II pneumocyte hyperplasia, hypertrophy, and syncytia (arrow) which are antigen positive for measles virus. (B) Spleen of a measles-infected macaque: There is profound lymphoid depletion and necrosis of the lymphoid component due to measles infection. (C) Mammary gland from a cynomolgus macaque: The characteristic intracytoplasmic and/or intranuclear inclusions of measles-infected epithelial cells are variable in size and number but brightly eosinophilic. (D) Ileum of a measles-infected macaque: A fulminant, necrotizing, and hemorrhagic enteritis is a common finding in macaques during measles outbreaks.

genus which contains more than 50 distinct viruses isolated from a host of OWM and NWM species.39 The viruses are closely related to EBV (Human herpesvirus 4), the single known human LCV, which is considered the type species of the genus Lymphocryptovirus, and can cause infectious mononucleosis or lymphoma under certain conditions.39,40 Simian LCVs that infect OWM are more closely aligned with human EBV than are those of NWM species, and they show remarkable similarity in genomic organization and biologic properties that translate to very similar pathological manifestation and epidemiology in humans and NHP hosts.7,39,41 In addition, laboratory NHPs develop a clinical syndrome similar to that seen with EBV in both immunocompetent and immunocompromised hosts.41e43 These viruses often show no strict host specificity, and with

several examples of severe disease resulting from crossspecies transmission. Almost all herpesviruses are known for causing typical vesiculoulcerative lesions in and around the mouth, face, skin, or genitals. Microscopically, multifocal necrosis in the liver and other tissues, cowdry type A intranuclear inclusions, and multinucleated syncytial cells, are some of the most frequently observed features. Herpesviruses are also known for their interspecies transmissibility and ability to establish latent infections that endure for the life of the host with no clinically apparent signs. In response to various stimuli such as immunosuppression, latent viruses in their natural host can reactivate and undergo lytic replication resulting in shedding of infectious virus that can then be transmitted to a naïve host.38

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2.3.1 Macacine herpesvirus 1 (B virus) and saiminiine herpesvirus 1 (Herpes T) Macacine herpesvirus 1 (McHV1), commonly known as herpes B, or Cercopithecine herpesvirus 1, is a member of the Simplexvirus genus of the subfamily Alphaherpesvirinae and is closely related to the human herpes simplex viruses 1 and 2 (HSV-1 and HSV-2). McHV1, like other members of this genus, is spread through direct contact of mucosal surface or injured skin of a naïve animal with infected bodily secretions during active shedding of the virus. Following replication in epithelial cells, virions travel retrograde along sensory nerve axons to the sensory ganglia (trigeminal and/or sacral ganglia). Latent infection is established within neurons of the sensory ganglia and is maintained for life. Viral reactivation can occur with stress, during the breeding season, coinfection with immunosuppressive pathogens, and with administration of immunosuppressive agents.37,44 Prevalence of McHV1 is very high in captive macaque colonies in which the virus has not been eliminated through screening. Infection rate among captive animals increases with age, with the majority of animals over 3 years of age being infected.45 Active infection is uncommon and is usually mild and self-limiting. Lesions occur on the oral or genital mucosa and are characterized by vesicular lesions which evolve to erosions and ulcerations (Fig. 3.3A). Disseminated infection is rare and is usually fatal. Disseminated disease may occur in young animals or may occur in immunocompromised or stressed adults and can affect the nervous system as well as the lung, liver, and kidneys. Lesions associated with systemic infection include interstitial nephritis, ulceration and necrosis of multiple mucosal surfaces, and necrosis in multiple organs including liver (Fig. 3.3B), lymphoid tissue, adrenals, and pancreas. There is typically syncytia formation and intranuclear inclusions in epithelial cells.11,38 Nervous system lesions are typically noted in the brainstem and trigeminal and facial ganglia and nerves.46 Changes may include meningoencephalomyelitis, with neuronal degeneration or necrosis, gliosis, lymphocytic perivascular infiltrates (cuffing), and intranuclear amphophilic to basophilic inclusion bodies inside neurons and glial cells. McHV1 poses a serious risk of zoonotic infection in humans. Since the discovery of the virus, approximately 51 cases of McHV1 infection in humans have been reported and with high mortality rate.47 Infection is most commonly acquired from asymptomatic animals with no visible lesions of disease. A single case of human to human transmission has been published.48 Modes of transmission to humans include ocular and mucous membrane splashes, needlestick, bites, and scratches. McHV1 causes severe encephalomyelitis in humans and has a mortality rate of greater than 80% in the untreated.47 Antiviral treatments

FIGURE 3.3 Macacine Herpesviruses 1 (B virus; McHV1): (A) In its mildest form, McHV1 presents with vesiculation of the epithelium of skin or mucosal surfaces. Vesicles may contain hemorrhagic fluid and acantholytic keratinocytes, which may contain intranuclear inclusions. Histologically, it may be indistinguishable from Simian Varicella Virus (SVV) and ancillary means of diagnosis is required to define the infection. (B) In uncomplicated disseminated disease, the liver may have apoptotic or necrotic cells (arrows) and rare intranuclear inclusions.

can effectively control progression of disease but cannot eliminate it. Saimiriine herpesvirus (SaHV1), previously known as Herpes T virus, causes asymptomatic infection in Saimiri sciureus (squirrel monkeys) but severe disseminated infection and development of T-cell lymphoma and lymphocytic leukemia in Aotus trivirgatus (owl monkeys), Saguinus oedipus (tamarins), and Callithrix jacchus (common marmosets).48e53

2.3.2 Herpesvirus 2: HVP-2: cercopithecine herpesvirus 16; (previously cercopithecine SA8) This is a neurotropic virus with latency in ganglia. This virus causes subclinical natural disease in Cercopithecus aethiops (African green monkeys) and Papio anubis (olive or anubis baboons).51 Experimental infection in baboons leads to disseminated disease in the nervous system and other organs.52 Zoonotic transmission has not been described for HVP-2.

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2.3.3 Herpesvirus 6, 7, and 9: cercopithecine herpesvirus (simian varicella virus) Simian varicella virus (SVV; Cercopithecine herpesvirus 9) is a member of the genus Varicellovirus in the subfamily Alphaherpesvirus of the Alphaherpesviridae. SVV is closely related to the varicella-zoster virus of humans, which is the causative agent of varicella (chicken pox) and herpes zoster (shingles). While outbreaks of SVV in old world primates are uncommon, particularly in animals with healthy immune systems, recrudescence of latent infections is possible in animals being administered immunomodulatory agents or undergoing stress.40,41 Infections have been reported in Old World primates (superfamily Cercopithecoidea, subfamily Cercopithecinae), particularly Erythrocebus patas (patas monkeys), Chlorocebus aethiops (African green or vervet monkeys) as well as various species of macaques such as cynomolgus macaques.53,54 Zoonotic transmission has not been described for this virus. Although the natural host for this virus has not been identified, disease is more severe in African primates than Asian macaques. Wild caught cynomolgus macaques in Mauritius had an incidence of 0.8% based upon serology although the incidence increases remarkably depending on duration of which the animals were kept in a holding facility.42,53 Transmission of SVV is primarily via the aerosol route but can be spread through direct contact with vesicular fluid from cutaneous lesions. The majority of infections are subclinical. When clinical symptoms do occur, they are frequently mild and limited to a vesicular to hemorrhagic viral exanthema of haired skin and mucosal surfaces which are primarily localized to the face, thorax, and abdomen, sparing the plantar aspects of the feet and hands.53 When the immune system is compromised, viral infection of dermal or mucosal blood vessels often results in hemorrhage and dissemination to multiple organs, including the gastrointestinal tract, liver, lymphoid tissues, and lungs with the addition of more severe skin lesions.37 Cutaneous lesions are characterized by ballooning degeneration of epithelial cells and vesiculation of the epithelium. Vesicles may contain hemorrhagic fluid and acantholytic keratinocytes, which may contain intranuclear inclusions. Multinucleated syncytial cells may be present within the epidermis and intranuclear inclusions are frequently present in syncytial cells and keratinocytes. These findings may be quite severe in immunocompromised individuals (Fig. 3.4 A and B). If SVV infection is disseminated, lesions may be found in the liver, lung, and less frequently in the spleen, lymph nodes, kidney, adrenal, peripheral ganglia, and gastrointestinal tract. In the liver, there is multifocal necrosis with vacuolization and intranuclear inclusions within hepatocytes. Pulmonary lesions include multifocal hemorrhage and edema with exudation of fibrin and necrosis of alveolar walls and bronchiolar epithelium with intranuclear inclusions present in airway epithelium. In other tissues, necrosis, particularly of the spleen and lymph nodes, inflammation and intranuclear inclusions in epithelial cells may be seen.55 Encephalitis

FIGURE 3.4 Simian Varicella Virus (SVV); (A) Uncomplicated SVV usually presents with minor vesicular skin lesions; however, immunocompromised animals may present with marked proliferative, vesicular, ulcerative and necrotizing skin lesions, as in this case from a cynomolgus monkey. (B) Ballooning degeneration, multinucleated and syncytial cell formation with marked hyperplasia of the epidermis is common with SVV infections in immunocompromised animals. Intranuclear inclusions (arrows) are usually noticeably present within hyperplastic and dysplastic keratinocytes, and the lesions are commonly infiltrated by granulocytes and lymphoplasmacytic cells. SVV lesions are similar to those of other Herpesviruses; therefore, ancillary diagnostics (PCR, IHC, etc.) should be employed to differentiate the etiologic agent responsible.

may occur, and infected cells show eosinophilic intranuclear inclusions. Low level viremia results in hematogenous spread into ganglia. Transaxonal spread of virus from infected skin may also produce additional ganglionic infections. The virus can establish latency in the trigeminal and dorsal root ganglia for years, colonizing the entire neuroaxis. Experimental infections have shown SVV DNA detected in ganglia as early as 6 days post-infection.56

2.3.4 Cytomegalovirus The genus Cytomegalovirus (CMV) are members of the subfamily Betaherpesvirinae of the family Herpesviridae. CMV are enveloped, double-stranded DNA viruses and are highly prevalent in several NHP species including macaques,

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baboons, marmosets, African green monkeys, and chimpanzees.57,58 Cytomegaloviruses are species specific, with little cross-infection between species; therefore, zoonotic potential is low. The most thoroughly studied CMV of NHPs is Macacine Herpesvirus 8 (McHV8) of rhesus macaques.59 A number of other CMVs have been isolated from other NHPs, including cynomolgus macaques. CMV infection usually occurs early in life, is typically subclinical, and persists in a latent state for life.60 Immunosuppression or deficiency are also known to play a role in the pathogenesis of this disease, especially upon manifestation of clinical disease. Most juvenile macaques are serologically positive by 6 months of age. Asymptomatic infection is common in immunocompetent macaques, and the virus is shed in body secretions including urine, milk, semen, and saliva (saliva is the typical mode of transmission). CMV infection is highly prevalent in rhesus macaque colonies, with seropositivity approaching 100% in sexually mature animals.61 Persistently infected animals continue to shed virus during periods of active virus replication throughout life, thus continuing the spread of virus throughout the colony. CMV can be found in a wide variety of tissues in infected animals, but has a particular predilection for replication and persistence in cells of myeloid origin, smooth muscle cells, and endothelial cells,48 and occurs even in the immunocompetent host. In immune suppressed macaques, often animals infected with SIV, CMV can cause widespread, systemic disease that may be fatal.51e53 Lesions caused by active CMV infection are commonly found in the lung, kidneys, intestines, brain, lymphoid tissue, sacral spinal cord, spinal and facial nerves, liver, testes and arteries.51 It has been identified in bone marrow which serves as a reservoir for infection. Histologically, CMV-associated lesions are characterized by suppurative inflammation associated with cytomegaly as well as intracytoplasmic and intranuclear inclusion bodies (Fig. 3.5A and B). Changes in the nervous system consist of hemorrhagic meningoencephalitis/meningomyelitis with necrosis, edema, and meningeal inflammation with perivascular cuffs containing lymphocytes, neutrophils, and macrophages. These findings are amplified in animals with immunocompromised conditions, such as SIV infection.62 Immunohistochemistry maybe required to confirm the diagnosis in subtle cases or to differentiate lesions from other herpesviruses.

2.3.5 Gammaherpesviruses The subfamily Gammaherpesvirinae of the family Herpesviridae is comprised of two genera, the lymphocryptoviruses (gamma-1 herpesviruses) and the rhadinoviruses (gamma-2 herpesviruses).58 Both genera contain viruses of importance in humans, Old World and New World primates. Similar to other herpesviruses, gammaherpesviruses are well adapted to their natural hosts, in which they rarely cause more than

FIGURE 3.5 Cytomegalovirus (CMV): (A) Peripheral Nerve: CMVassociated lesions are characterized by suppurative inflammation associated with cytomegaly as well as typical herpes-type intracytoplasmic and intranuclear inclusion bodies (arrows). (B) Peripheral nerve: The microscopic lesions of CMV may resemble other herpes viral infections; therefore, immunohistochemical labeling of CMV antigen (arrows) may be required for diagnosis. Images courtesy of M. Kelly Keating.

transient, mild clinical disease. However, when inadvertent or experimental trans-species infection occurs, severe disease may result.63 The gammaherpesviruses are transmitted via oral secretions and infect B lymphocytes where they establish lifelong, persistent infection. 2.3.5.1 Lymphocryptovirus The Lymphocryptovirus (LCV) genus includes the prototypical LCV and human herpesvirus 4 (HHV-4), commonly known as the Epstein-Barr virus (EBV), the etiologic agent of mononucleosis (International Committee on the Taxonomy of Viruses).58 There are many LCVs of Old World and New World species of NHP, including macaques (Cercopithecine herpesvirus 15 or rhesus LCV, RhLCV) and common marmosets (Callittrichine herpesvirus 3).7 Zoonotic transmission of LCV to humans has not been reported for this virus.

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RhLCV is closely related to HHV-4, and HHV-4 infection in macaques closely parallels that of RhLCV in humans. Seroprevalence of LCV approaches 100% in both human and macaque populations.7 LCV infection occurs primarily in young animals and is transmitted through oral secretions. Primary infection of immunocompetent macaques is typically asymptomatic but when experimentally inoculated in rhesus macaques with SIV a transient lymphadenopathy accompanied by atypical lymphocytes in the peripheral blood with evidence of activated B lymphocytes was observed.42 Administration of immunomodulatory test articles to cynomolgus macaques can cause recrudescence of latent LCV infection, giving rise to lymphadenopathy, nodular hyperplasia in the spleen or lymphoid hyperplasia of lymph node follicles (Fig. 3.6D), lymphoid hyperplasia (with follicle formation) in several organs such as kidney and liver, and lymphocytosis in peripheral blood. These entities are most commonly B-cell in origin and similar to human non-Hodgkin’s lymphoma associated with EBV infection. Patients immunosuppressed by HIV infection develop proliferative plaquelike lesions of the tongue and esophagus termed oral leukoplakia, that is caused by EBV infection.64 SIV-infected macaques develop comparable lesions that have been associated with LCV infection.7 Histologically, the lesions are characterized by parakeratotic hyperkeratosis and epithelial hyperplasia of the tongue, esophagus, and skin of the penis and thorax. There is ballooning degeneration of epithelial cells with intranuclear inclusions typical of herpesviruses present in epithelial cells.7,65 Non-Hodgkins lymphoma (NHL) is one of the most common AIDS defining diseases in HIV-infected patients.66 NHL is a B-cell lymphoma that is caused by LCV infection that develops late in HIV infection. Similar to HIV-infected people, NHL develops in late-stage SIV infection in macaques and is associated with depletion of CD4þ T lymphocytes.7 Interestingly, the incidence of NHL in SIV-infected cynomolgus macaques is substantially higher at near 40% compared to that in SIV-infected rhesus macaques at closer to 15%.7 NHL occurs preferentially at extranodal sites, including the central nervous system, gastrointestinal tract, heart, kidneys, and nasal cavity.42,67 A case of LCV-associated lymphoma is reported here (Fig. 3.6 A-C). LCV has also been associated with posttransplant lymphoproliferative disorder (PTLD) in rhesus and cynomolgus macaques that have received immunosuppressive regimens following solid organ and bone marrow transplants.67 A retrospective analysis of cynomolgus macaques that received renal transplants demonstrated that 5.6% had evidence of PTLD characterized by atypical proliferations of CD20þ B lymphocytes in multiple nonlymphoid organs.67

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FIGURE 3.6 Lymphocryptovirus (LCV): (A) Stomach: B-cell lymphoma caused by LCV infection in late-stage SIV infection in macaques may present in nearly any organ-here the gastric mucosa is thickened and effaced by multiple neoplastic foci (arrows). (B) Immunohistochemistry for CD20 antigen confirmed B-cell lineage of the foci in Fig. 3.6A. (C) Cytological evaluation of the foci in Fig. 3.6A: There are numerous, uniform, neoplastic lymphocytes composing the cell population of the neoplasm. (D) Administration of immunosuppressing agents may result in recrudescence of LCV as a lymphadenopathy composed of proliferative, occasionally bizarre, lymphocytes accompanied by cytolysis and there may be intranuclear inclusions noted (arrow). (AeC) Courtesy of Andres Mejia, Wisconsin National Primate Research Center.

Callitrichine Herpesvirus 3 (CaHV3) was first isolated from common marmosets with lymphoproliferative disease and lymphoma.68,69 Similar to other LCVs, CaHV3 can be detected in sera of healthy animals. However, CaHV3 prevalence in captive and wild marmosets is much lower

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than LCV prevalence in humans and macaques, at roughly 40%e60%.68 Clinical signs include weight loss, diarrhea, lack of appetite and lymphoproliferations especially in the GALT and mesenteric lymph nodes. 2.3.5.2 Rhadinoviruses The Rhadinovirus genus belongs to the gamma-2herpesvirus subfamily of the Herpesviridae. The best characterized rhadinovirus is human herpesvirus 8 (HHV8) which is the causative agent of Kaposi’s sarcoma seen in AIDS patients.70,71 There are two distinct lineages of rhadinoviruses identified in macaques: the Retroperitoneal Fibromatosis Herpesvirus (RFHV), which is classified in the Rhadinovirus-1 (RV1) lineage with the closely related HHV-8, and the Rhesus Rhadinovirus (RRV) which is classified in the Rhadinovirus-2 (RV2) lineage. Both viruses are endemic within certain breeding colonies of macaques.72 The rhadinoviruses, like other herpesviruses, are persistent infections and shedding of virus can occur through oral secretions without symptoms of disease being present. RFHV was first identified with PCR on samples from macaques with retroperitoneal fibromatosis.73 Retroperitoneal fibromatosis (RF) is a highly vascular, fibroproliferative disease that occurs in macaques immune suppressed due to SRV-2 infection. RF is characterized by nodular proliferations of fibrovascular tissue involving the mesentery, mesenteric lymph nodes, and serosal surfaces of abdominal viscera (Fig. 3.7A and B).73 Subcutaneous nodules of proliferative fibrovascular tissue occur less frequently.74 The incidence of RF has substantially decreased in recent years, likely due to elimination of SRV2 through the implementation of specific pathogen free (SPF) colonies. Due to the lack of a serologic test for RFHV, the actual prevalence of the virus within colonies is unknown. RRV was identified independently at both the New England Primate Research Center and at the Oregon National Primate Research Center.75,76 Serologic surveys indicate that greater than 90% of animals over 2 years of age are seropositive. Virus can be found in oral swabs, indicating that oral fluids are a likely source of infection.76 RRV infects CD20þ B lymphocytes in which persistent infection is established. The majority of RRV infections are asymptomatic. Experimental inoculation with RRV has been shown to induce B-cell lymphoproliferative disease similar to multicentric Castleman’s Disease.77 RRV has also been associated with the development of nonHodgkins lymphoma in animals experimentally coinfected with SIV.77 Saimiriine herpesvirus 2 (SaHV2) is a rhadinovirus that naturally infects squirrel monkeys. Squirrel monkeys are infected through saliva, usually before 2 years of age.

FIGURE 3.7 Retroperitoneal Fibromatosis Herpesvirus (RFHV): (A) Retroperitoneal fibromatosis (RF) is a highly vascular, fibro-proliferative disease that occurs in macaques coinfected with RFHV and immunosuppressive SRV-2. RF is characterized by nodular proliferations of fibrovascular tissue involving the mesentery, mesenteric lymph nodes, and serosal surfaces of abdominal viscera. (B) Higher magnification of Fig. 3.7A: The RF nodules are composed of highly vascular fibromatous tissue forming variably dense sheets and streams.

SaHV2 is highly endemic in wild populations of squirrel monkeys and is usually asymptomatic.78 However, crossspecies infection of other NHP species, including tamarins, marmosets, howler monkeys, and spider monkeys, results in the development of lymphoma or leukemia.79e82 Transmission of infection to these species has largely been experimental, as natural infection is rare.

2.3.6 Herpes simplex: (human herpesvirus 1 and 2; herpes simplex virus 1) Experimental and natural herpes simplex virus (HSV-1; HSV-2) can cause epizootics with high morbidity and mortality in New World monkeys and subclinical disease in Old World monkeys.38,83 In New World monkeys, HSV-1 and HSV-2 can cause disseminated disease in multiple organs including the nervous system.38 Clinical signs

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include versicular to ulcerative dermatitis and stomatitis, serous nasal and ocular discharges, tongue ulcers, anorexia, and depression. Histologically there are subcorneal pustules with acantholytic cells, and coagulative epithelioid necrosis in the skin. Brain lesions are characterized by nonsuppurative meningitis, perivascular cuffs, neuronophagia, neuronal degeneration with satellitosis, syncytia formation, gliosis, and neuronal basophilic intranuclear inclusion bodies.

2.4 Hepatitis viruses The hepatitis virus group consists of five viral classes of zoonotic potential belonging to five different families: Hepatitis A (HAV), B (HBV), C (HCV), delta (HDV), and E (HEV). Hepatitis A and E are transmitted by the oralfecal route (enterically transmitted), and only cause acute self-limiting infections in both New World and Old World monkeys; whereas HBV, HCV, and HDV, which are transmitted via blood and other body fluids (parenterally transmitted), can cause serious liver disease leading to cirrhosis or hepatocellular carcinoma. Parenterally transmitted HBV, HCV, and HDV are not reported as a cause spontaneous disease in NHPs other than chimpanzees and a few species of Old World monkeys. However, since the NHP is the only available model for studying these viruses, a large body of work on the hepatic pathology of these viruses has been compiled from experimental work done in laboratory NHP.84,85 Parenterally transmitted viruses do not appear to affect NWM, macaques, and other laboratory

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NHP used in toxicity studies, and the chimpanzees have been the only models used to study these viruses.86,87 The chimpanzee is the only experimental animal that can attain HCV infection and was the primary source of much current information on infection and treatment in humans.88 The HBV (family Hepadnaviridae) has been recovered and demonstrated in cynomolgus monkeys.89 Experimental studies in NHP are also carried out to evaluate the efficacy of potential hepatitis vaccines, as well as to evaluate the zoonotic potential of different various hepatitis virus strains.84,90e92

2.4.1 Hepatitis A virus (infectious hepatitis) Outbreaks of hepatitis A virus (HAV) in colonies of macaques and African green monkeys occur sporadically, usually finding its way into a colony via contaminated feeds or by the fecal-oral route within colonies. HAV is an RNA virus of the genus Picornavirus, family Picornaviridae and related to the human hepatitis A virus. In general, HAV is a mild disease or subclinical in NHPs, producing typical periportal inflammation with individual cell necrosis or apoptosis (councilman bodies) (Fig. 3.8A and B). In more severe or persistent cases, there are usually alterations of liver enzyme assays, and there may be progression to more severe chronic inflammation and portal or bridging fibrosis of the liver.92e94 On study, infected animals may experience far more severe hepatitis when administered immunomodulatory agents or hepatic-directed test articles.94 Vaccination programs may eliminate or control HAV within colonies.

FIGURE 3.8 Hepatitis A virus (HAV): (A) HAV is a mild disease or subclinical in NHPs, producing typical periportal inflammation. (B) Individual cell necrosis and apoptosis in the liver of HAV infected macaques produces characteristic “Councilman bodies” most prominently in the periportal regions (arrow).

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2.4.2 Hepatitis E Liver lesions similar to those observed with spontaneous or experimental HAV infections are also observed with experimental HEV (family Herpesviridae; genus Orthohepevirus) in several NHP species, including, cynomolgus and rhesus macaque, African green monkeys, owl monkeys, tamarin, and squirrel monkeys.84 Like HAV, experimental HEV in macaques is usually mild or subclinical and the clinical response may be measured by ALT elevation in liver enzyme assays. HEV is a generally noncytopathogenic virus with a pathogenesis that is likely centered on the host’s immune response to infection.95 Minor differences to HAV infection exist, and include a longer incubation period and a dose-dependent clinical presentation with HEV infection. In experimental infections of NHPs with HEV, the clinical presentation and severity of findings are directly related to the infectivity titer of challenge virus.91

2.5 Adenovirus The family Adenoviridae contains the genus Mastadenovirus, which includes the adenoviruses of primates. Adenovirus is primarily spread via fecal-oral transmission, but infection can occur through aerosolization and inhalation in some instances. Prevalence in asymptomatic animals can be very high in some colonies.96 Adenovirus has been cultured from animals with and without diarrhea, but generally adenoviral infection is asymptomatic in immunocompetent adult animals. Immunosuppressed or immunodeficient individuals may experience more severe disease, affecting organs outside the intestinal tract, including necrotic liver foci or pneumonia.97 The characteristic, large basophilic intranuclear inclusions (Fig. 3.9A and B) are usually identified but may be misinterpreted as those of other viruses (CMV, herpes B virus, SV40, and measles virus) or protozoa. Adenoviral infection of macaques presents a potential for zoonotic infection.98 Some adenoviruses isolated in fecal cultures from macaque colonies are closely related to human isolates associated with respiratory and gastrointestinal disease.99

2.6 Polyomaviruses (SV40, SV12, CPV) Polyomaviruses are nonenveloped, circular, double-stranded DNA viruses of the Polyomaviridae family. Simian virusV40 (SV40) and the related cynomolgus polyomavirus (CPV) are opportunistic and cause common and persistent infections of many species of macaques.33 Seven viruses have been reported in NHPs: SV40 in macaques, SV12 in baboons, B-lymphotropic papovirus (LVP) in African green monkeys, polyomavirus papionis 2 in baboons, cynomolgus polyomavirus (CPV), SV40-CAL in marmosets, and

FIGURE 3.9 Adenovirus: (A) Adenovirus often has a characteristic appearance in the intestinal tract, with large amphophilic inclusions visible even at lower magnification. (B) The characteristic large amphophilic to basophilic nuclear inclusions of adenoviral infection in a macaque.

chimpanzee polyomavirus.33,100e107 Zoonotic infection rates are relatively high, although the clinical course of long-term infection in otherwise healthy humans is unknown.54 Serologic evidence indicates that both SV40 and CPV are readily transmitted in breeding colonies of macaques. Colonies have been reported with seropositive prevalence rates of 90%e100%.100 Even with high prevalence, there is rarely clinical disease associated with these viruses and recrudescence is usually in conjunction with immunosuppression. SIV infected animals may develop CNS lesions that resemble progressive multifocal leukoencephalopathy (PML) in human AIDS patients.101 Infection of the kidney with SV40 results in chronic nonsuppurative interstitial nephritis centered on the renal medulla, often with hyperplasia and dysplasia of collecting duct epithelium and accompanied by enlarged tubular epithelial cells containing intranuclear inclusions.100 Immunohistochemistry and in situ hybridization are localization techniques that reveal viral inclsions and often identify far more infected cells than would be found by routine stains. The enlarged nuclei of collecting duct cells

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with intranuclear polyomavirus inclusions should be differentiated from the incidental multinucleate cells of the collecting ducts (see Chapter 9).

2.7 Simian parvovirus Simian parvovirus (SPV) is a member of the Parvoviridae family, recognized as infecting both cynomolgus and rhesus macaques. Colony surveillance may require molecular diagnostics in conjunction with serology in order to detect viraemic antibody-negative and nonviraemic antibodypositive states.33,108 The virus is normally quiescent in healthy NHPs; however, immunosuppressed individuals may suffer anemia due to viral replication in erythrocytic cells of the bone marrow, leading to cellular depletion.100 Bone marrow may have histologic evidence of viral activation, with dyserythropoiesis or decreased erythrocytic progenitor cell content and the presence of intranuclear inclusions.

2.8 Papillomavirus Papillomaviruses are a diverse group with high genetic variability and complex host interactions, some of which are carcinogenic, that occur naturally in wild and captive populations of macaques and other NHPs.109 These are double-stranded DNA viruses with capsids that are transmitted among sexually mature animals, usually without clinical ramifications in healthy individuals.33 When lesions occur, they are usually firm, exophytic masses of skin and often described as “cauliflower-like.”110 Sasseville et al. described the histologic features as “characterised by massive hyperplasia of the stratum spinosum and corneum . Basophilic intranuclear inclusions may be observed.”10 The lesions may regress spontaneously; however, persistence of infection has been associated in female macaques with cervical dysplasia and both sexes have developed carcinoma (cervical/penile) associated with the virus.111

2.9 Lymphocytic choriomeningitis virus (Callitrichid Hepatitis virus) Lymphocytic choriomeningitis virus (LCMV) is a member of the Arenaviridae family. LCMV was formerly named Callitrichid Hepatitis virus, due to its propensity to cause severe hepatic disease in these species of New World monkeys, including common marmosets. Mice are the natural reservoirs for LCMV and the virus is spread through their urine. LCMV is transmitted to primates through the ingestion of infected wild rodents or through feeding of infected baby mice.112 LCMV has been responsible for epizootics in New World monkeys with high morbidity and mortality. After an incubation period of 1e2 weeks, animals develop clinical signs of hepatic

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failure including jaundice, coagulopathy, and elevations in liver enzymes and bilirubin.112 Some animals die peracutely without developing clinical signs. Gross necropsy findings include hepatosplenomegaly, jaundice, subcutaneous and intramuscular hemorrhage, and pleural and pericardial effusions. Microscopically, hepatic lesions are characterized by multifocal, random hepatocellular necrosis, degeneration, and apoptosis, the latter of which appear as distinctive acidophilic bodies (councilman bodies), and mild lymphocytic inflammation.112,113 Histologic findings may also include mononuclear infiltrates in the meninges, with or without perivascular cuffing by mononuclear cells within the cerebral neuropil.113 LCMV is a zoonotic infection which can cause meningitis or meningoencephalitis in humans.114,115

2.10 Flaviviruses 2.10.1 West Nile virus Infection with these tick or mosquito-borne RNA viruses is rare and typically occurs under laboratory conditions, although asymptomatic cases in captive monkeys were reported in the United States during wild bird epizootics of West Nile virus in 2002.116,117 Nervous system macroscopic findings have only been described in experimental infection of non-human primates used in neurovirulence experiments and vaccine development and/or efficacy studies.118e120

2.10.2 GB agent viruses The GB agent viruses (GB viruses A, B, and C) are members of the Hepacivirus genus, family Flaviviridae. The GB viruses were first discovered following inoculation experiments in tamarins in which the animals had been inoculated with serum from a human patient (identified in the study as “G.B.”) with hepatitis.121,122 GBV-A is known to infect a number of New World primate species, but there is no known disease association with infection. Surveys of domestic colonies of common marmosets have demonstrated viremia with GBV-A in up to 20% of animals.123 GBV-C is frequently found in the human population in association with other viruses and is closely related to GBV-A.122 GBV-C has been demonstrated to alter the course of HIV infection in coinfected individuals and reduce mortality rate.124 Whether a similar situation might occur with GBV-A infected NHPs is unknown. GBV-B is closely related to the human hepatitis C virus (HCV) and shares 25%e30% homology with HCV at the amino acid level.125,126 The natural host for GBV-B is unknown but several species of New World primates are susceptible to infection including the tamarin and the common marmoset.110 Inoculation studies demonstrate that the common marmoset is highly susceptible to infection

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with GBV-B and develops characteristic hepatic lesions following inoculation. Histologically, liver lesions are characterized by multifocal nonsuppurative inflammation in the parenchyma, portal lymphocytic hepatitis, and piecemeal necrosis.111 Thus, the common marmoset and tamarin are utilized as models for HCV.127

2.11 Parainfluenza viruses Parainfluenza viruses (PIV) have a world-wide distribution, are highly infectious, and infect numerous species including humans and NHPs. Parainfluenza viruses (PIV) are members of the Paramyxoviridae family. PIV-1 and PIV-3 are members of the Respovirus genus, while PIV-2 is within the Rubulavirus genus. Outbreaks of severe respiratory disease associated with PIV-1 or a Sendai-like virus have been reported in common marmosets.128 Infected animals may present with ocular and nasal discharge, sneezing, coughing, and dyspnea. The pathology of PIV in marmosets has not been well described, but acute interstitial pneumonia is most commonly reported.129 Secondary bacterial infections with pathogens such as Klebsiella pneumoniae may complicate pneumonia. Syncytial cells and cells containing intranuclear and intracytoplasmic inclusions may be evident within lesions.129

Similar in presentation to SHF is Reston virus (RESTV) related to Ebola virus, and a member of the Flaviviridae family. A fatal outbreak of RESTV occurred in macaques of the United States of America in the late 1980s following importation of infected monkeys from the Philippines. Humans exposed to infected animals and who had positive antibody titers remained asymptomatic and the infected colony of NHPs in the Philippines was eventually depopulated.133 Likewise, the clinical and pathological presentation of SHF in macaques mimics that of hemorrhagic viruses in humans and macaques develop similar lesions when experimentally infected with Ebola and Marburg (Fig. 3.10A and B) viruses.134,135 This stresses the importance of macaques as models of human diseases and the need for biosecurity vigilance for imported animals.

2.13 Lyssavirus (rabies virus) Infection with rabies, an enveloped, single-stranded, negative sense, RNA virus, is very rare in New World and Old World non-human primates, especially in captive-bred

2.12 Simian hemorrhagic fever viruses Simian hemorrhagic fever (SHF) is caused by a singlestranded, enveloped RNA virus within the family Arteriviridae that is highly lethal among Asian macaques in captivity. The natural hosts have persistent nonclinical infections and include baboons, patas monkeys, and African green monkeys. SHF in macaques presents as an acute febrile disease dominated by impairment of hemostasis and coagulation leading to fatal hemorrhagic diathesis. In the 1960se1970s, SHF was responsible for outbreaks and high mortality in US and USSR macaque colonies.130,131 Initially, the only abnormal parameters observed are increased body temperature and measurable fibrin degradation products. As the disease progresses, serum enzymes such as lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, and creatine kinase start to increase. Hematomas may be seen at the site of venepuncture. There is thrombocytopenia, prolonged thromboplastin times, and disseminated intravascular coagulation. The clinical syndrome is characterized by fever, anorexia, facial edema, depression, photophobia, ataxia, cyanosis, dehydration, and epistaxis. Gross lesions are generally only observed in the final 24 h before death if at all. Gross and microscopic lesions include congestion, hemorrhage, microthrombosis, necrosis of lymphoid tissues, and proximal duodenal mucosa, hemorrhagic ulcers throughout the gastrointestinal tract and splenomegaly.12,132

FIGURE 3.10 Experimental Marburg virus in macaques: (A) Marburg infection produces lesions that are virtually indistinguishable from other hemorrhagic viruses. Characteristics of Marburg infection include lymphocytolysis, necrosis, and hemorrhage of the spleen and (B) hepatocellular swelling, vacuolation, degeneration, necrosis and apoptosis with pleomorphic intracytoplasmic inclusions (arrows). Images courtesy of M. Kelly Keating.

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cerebral motor cortex.50,139 Microscopic findings included meningitis and perivascular cuffing with mononuclear cells, neuronal cell swelling and chromatolysis, neuronophagia, intranuclear inclusion bodies, and axonal degeneration as sequelae to destruction of lower motor neurons. Neuronal swelling is especially pronounced in the dentate nucleus, facial and vestibular nuclei, reticular formation, cerebellar nuclei, and spinal motor neurons.146

2.15 Encephalomyocarditis virus

FIGURE 3.11 Rabies virus: Non-human primates housed in outdoor facilities have a low risk of exposure to rabies virus infected wildlife. This neurotropic virus has characteristic, variably sized, eosinophilic, intracytoplasmic inclusions (arrows) within CNS neurons of the infected animals.

laboratory animals. Monkeys in outdoor enclosures exposure to native wildlife may be at risk of exposure. However, rabies infection has been reported in NHPs (common marmosets and rhesus macaque) and they can be the source of transmission to humans.136e138 Typical microscopic findings with rabies infection consist of glial nodules, perivascular mononuclear infiltrates, and neuronal degeneration with intracytoplasmic viral inclusions referred to as Negri bodies (Fig. 3.11) in the brain.139

2.14 Enteroviruses (poliovirus) Poliovirus, an RNA virus of the genus Enterovirus, family Picornaviridae, is very uncommon in NHPs. Poliovirus colonizes the gastrointestinal tract, specifically the oropharynx and intestines. There are two serotypes: PVI is most encountered in man, and PVII most encountered in monkeys. Poliovirus can be transmitted by close contact from an infected human to Old World non-human primates such as chimpanzees, gorillas, and orangutans.140,141 Verschoor et al. reported molecular characterization of the first polioviruses from a New World squirrel monkey.142 Experimental oral and/or intracranial inoculation of poliovirus has been reported in Macaca fascicularis (cynomolgus macaques), Macaca mulatta (rhesus macaques), and Cercopithecus aethiops (African green monkeys).143e145 Animals developed paralysis of one or more extremities and/or paresis of the trunk and thoracic muscles. Pronounced hyperemia of the meninges may be seen at necropsy. As in affected humans, microscopic changes were most prominent in the gray matter of the ventral horn of the spinal cord, medulla oblongata, cranial nerve nuclei in the brainstem, cerebellar nuclei, and the

Encephalomyocarditis virus is a non-enveloped RNA virus within the Picornaviridae, genus Cardiovirus of which there are myocardiotropic and neurotropic variants. Six cases of epizootic myocarditis associated with encephalomyocarditis virus were reported in a group of juvenile rhesus macaques at the New England Primate research Center at Harvard Medical School.147 There was acute onset dyspnea, pulmonary congestion, pleural and pericardial effusion, hepatomegaly, and multifocal pale foci throughout the myocardium. These pale foci comprised lymphoplasmacytic and histiocytic infiltrates with admixed necrotic and degenerate myofibers, and frequent mineralization.

2.16 Monkeypox Monkeypox is of the genus Orthopoxvirus known to infect humans and other animals. Several outbreaks have been reported in Old World and New World monkeys. It was first described in a cynomolgus macaque in Denmark in 1958, and first described in humans living in the Democratic Republic of Congo in 1970. Recently, there have been outbreaks in humans in the USA, Europe and other continents (2022-2023). The natural reservoir of the virus is unknown but there are a wide range of permissive hosts and NHPs that can harbor the virus. Transmission is by direct contact. In experimental exposure to cynomolgus monkeys, the pathogenesis resembles that in humans.148 Clinical signs are papules of proliferating acanthocytes which progress to vesicles, then become umbilicated with a necrotic center. Eventually they slough leaving a scar. Intracytoplasmic eosinophilic inclusion bodies may be seen on histopathology within acanthocytes. Few infections lead to multiorgan necrosis and death, especially when secondary infections occur.148

3. Bacteria Non-human primates can host a variety of bacteria, some that are potentially patholgenic and that can jeopardize the interpretation of experimental results. Under stressful conditions, these bacteria may manifest as disease in the animals; therefore, animals showing signs of delayed development or poor weight gain should carefully be

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evaluated prior to use in research studies. Summarized below are the more commonly encountered bacteria noted in the non-human primate and the disease states that may ensue from their presence.

3.1 Shigella Shigella spp. are among the most common Gram-negative enteric pathogens found in macaques with S flexneri the most frequent isolate, including serotypes la, 2a, 3, 4, 5, 6, and 15, and to a lesser extent S. sonnei and S. boydii.149,150 Endemic infections within colonies are maintained by asymptomatic carriers.149,151 Infection is spread by the fecal-oral route among NHPs housed within the same social group, by shared use of equipment, by movement of animals between groups, and through importation of new NHPs into an existing colony. Disease is manifested by diarrhea or dysentery (bacillary dysentery), usually when endemically infected animals experience a stressful event such as a change in hierarchy within a well-established social group or transport to a new facility. Indian macaques up to 3-years-old are most susceptible, and more susceptible than Chinese macaques. A great many animals are asymptomatic carriers; however, dysentery is rare in marmosets and baboons.152 Juan-Salles et al. reported a case of shigellosis in a squirrel monkey presenting with diarrhea.153 A foul-smelling, liquid stool containing mucus, frank blood, and/or mucosal fragments is observed with bacillary dysentery. Anal atonia and rectal prolapse may also be observed. Affected monkeys are very weak, moderately to severely dehydrated, and require rapid veterinary fluid treatment and electrolyte supplements. Bacillary dysentery can produce an epizootic of high morbidity and mortality when introduced into a naive population. A more common form of shigellosis is a subacute to chronic diarrhea with liquid to semisoft stool that may occur in colonies in which enzootic infections develop. Diarrhea is intermittent to episodic; occasionally animals will have firm, mucus-laden feces with streaks of fresh blood. These animals do not appear clinically ill, and clinical episodes may resolve spontaneously. Enteric shigellosis lesions occur primarily in the cecum and colon. The colonic mucosa is usually covered with a fibrinopurulent exudate that can progress to a pseudomembranous enterocolitis, and the intestinal wall is edematous and hemorrhagic with focal areas of ulceration (Fig. 3.12A). Microscopically, there is erosion and ulceration of the mucosa with mats of bacteria filling the defects, as well as hemorrhage and edema (Fig. 3.12B). Neutrophilic infiltrates are commonly observed, and in severe cases, vasculitis may be present. Luminal contents can vary from fluid and mucus with fibrin and cellular debris to frank hemorrhage and mucus in the lumen. Intussusception of the

FIGURE 3.12 Shigella sp. infection, colon of a macaque: (A) Grossly, shigella causes fibrinopurulent and pseudomembranous colitis with mural thickening due to edema and hemorrhage, often with ulceration. (B) Microscopically, pseudomembranes consisting of fibrin and bacterial colonies cover necrotic, ulcerative, mucosal foci that extend into the submucosa.

small intestine, rectal prolapse, splenomegaly, and mesenteric lymphadenopathy may occur. Gross lesions in NHPs are confined to the cecum and colon and consist of multifocal ulceration and petechiation in the mucosa, with blood and fluid feces found throughout the large bowel.154 Grossly, the lesions of shigellosis cannot be differentiated from those of other enteric pathogens, such as Salmonella sp. or Campylobacter sp., therefore, ancillary diagnostic procedures, such as culture or PCR, are required for definitive diagnosis. Shigella may cause the initial oral necrotizing lesion in which anaerobic bacteria may grow and produce necrotoxins that lead to progression of the lesion resulting in gangrenous oral necrosis (cancrum oris) or noma.155 Shigella spp. are fastidious, Gram-negative, nonmotile, facultative anaerobic bacteria that do not survive for prolonged periods between collection and culture, so it is important to use transport medium under refrigerated or

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frozen conditions.156,157 Since shigella are produced segmentally in the lower gastrointestinal tract, single time point cultures can produce false negative results. Therefore, it is recommended to collect samples for culture over three consecutive days. While Shigella spp. are considered primary pathogens, they can asymptomatically colonize up to 20%e25% of clinically normal NHPs.150 Shigellosis is a zoonotic disease and multiple antibiotic-resistant strains are commonly encountered.158

3.2 Campylobacter Campylobacter spp., Gram-negative, microaerophilic, motile bacteria, are one of the most common bacterial causes of gastroenteritis in humans worldwide.157 Campylobacter spp. colonize the intestinal tract of NHPs, but infection is not always associated with clinical signs of disease.159,160 The species most commonly isolated from humans and non-human primates are C. jejuni and C. coli; other species like C. fetus are less commonly found.157 Recurring enterocolitis caused by Campylobacter spp. is often the main cause of morbidity in colonies of rhesus monkeys held in captivity.159 The animals act as reservoirs without clinical manifestations, and rhesus monkeys are a potential source of contamination both for healthy animals of the colony and for the professionals working in direct contact with them.161 Similar to other enteric bacteria, Campylobacter sp. are fastidious, requiring special handling for culture, but PCR assays are more commonly employed for identification.162

3.3 Salmonellosis Within the family Enterobacteriaceae, there are two recognized species within the genus, S. enterica and S. bongori, with six main subspecies of S. enterica: enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV), and indica (VI). Historically, serotype (V) was bongori, which is now considered its own species.163,164 There are over 2500 serovars of Salmonella allowing members of the genus to be differentiated from one another based on the KauffmannWhite classification scheme.165 Using the historical naming scheme, the frequently reported serovars of Salmonella spp. from non-human primates have been S. typhimurium, S. choleraesuis, S. anatum, S. stanley, S. derby, and S. oranienburg, all of which have been reclassified as subspecies of S. enterica.166 Salmonella sp. are Gram-negative, facultative anaerobic bacteria.157 Infection generally occurs by fecal-oral transmission, and infection can result from ingestion of contaminated food, water, or fomites. Salmonella sp. can survive and multiply for relatively long periods in the environment.157 Rhesus monkeys inoculated orally with S. typhimurium developed diarrhea, which peaked in

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severity at 48e72 h post-inoculation.167 Mild morphologic changes occurred in the colon and ileum in animals with diarrhea, including shortening of villi, villous edema, mild elongation of crypts, reduction of mucus content in goblet cells, and an increase of mononuclear cells in the epithelium. Additionally, there were lesions in the liver consisting of multifocal necrosis and lymphocyte infiltration, and in the lymph nodes consisting of granulomas with multinucleated cells.167 Reports of salmonella infection resulting in severe disease within established primate colonies are rare but chronic carriers and severe outbreaks have been described in cynomolgus macaques.168 Macaques are highly resistant to salmonellosis; however, if resistance is lowered, either via stress or chemical immunosuppression, severe disease can develop.169 Clinical signs in more severe cases of enteric salmonellosis include watery diarrhea, sometimes with hemorrhage or mucus and are often the animals are pyrexic. In more severe cases, enteric salmonellosis may be grossly characterized by edema, hyperaemia, and rare mucosal ulceration in the ileum and colon. Enlargement of the spleen and mesenteric lymph nodes may occur. Microabscesses are rarely found in colonic lymphoid tissue. In cases of septicaemia, areas of focal necrosis can be found in the liver and spleen. There may be hypersecretion of mucus in the stomach and intestines.169,170

3.4 Helicobacter spp. Helicobacter pylori and H. heilmannii bacteria can lead to a variety of human gastrointestinal disorders such as gastric inflammation (gastritis), peptic ulcer disease, distal gastric adenocarcinoma, and gastric mucosal-associated lymphoid tissue (MALT) lymphoma.171 H. pylori is common in rhesus monkeys and has also been isolated from cynomolgus monkeys.172,173 One study demonstrated the presence of H. heilmannii in cynomolgus monkeys from Mauritius, the Philippines, and Vietnam without a direct correlate with the severity of gastric inflammation; however, another study found that rhesus macaques with natural Helicobacter sp. infection had increased gastric inflammation and increased IgG ratios as compared to noninfected animals.173,174 H. cinaedi frequently infects asymptomatic rhesus monkeys, and there has been a report of it in association with colitis and hepatitis.175 In most cases from the editor’s facilities, there has been no clear correlation of the presence of Helicobacter sp. bacteria and gastric inflammation. The Helicobacter sp. organisms may be identified within gastric glands as spiral forms (Fig. 3.13). A novel Helicobacter sp. identified in cotton-top tamarins and was associated with progressive colitis mimicking features of ulcerative colitis in humans.176 A novel Helicobacter species, “H. jaachi,” was isolated from tissues, including feces, of clinically normal common marmosets.177

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involving thoracic and abdominal organs; signs of debilitation tend to appear only shortly before death. Animals with advanced tuberculosis may have developed a cough; loss of appetite or weight; and/or have enlarged or draining lymph nodes as well as unhealed skin wounds, or abdominal masses. Gross lesions can include caseous granulomas (“tubercles”) in the tracheobronchial or hilar lymph nodes and lungs that may become confluent, liquefy, or cavitate. Granulomas in the liver, spleen, and other organs are common in the disseminated disease.140 Histologically, granulomas have central areas of caseous necrosis surrounded by epithelioid macrophages, Langhans-type giant cells, and lymphocytes (Fig. 3.14A and B). A border of lymphocytes and fibrous connective tissue tends to FIGURE 3.13 Helicobacter species, gastric gland in the stomach of a macaque: Helicobacter sp. organisms may be identified within gastric glands of NHPs as spiral forms (arrows) and are commonly noted incidentally in macaques, with or without associated inflammation.

3.5 Mycobacterium spp. Macaques and marmosets are susceptible to infection by Mycobacterium sp. Commonly identified species include M. tuberculosis and M. bovis. Mycobacterium avium complex (MAC) consists of bacteria known to infect humans, but are also recorded for NHPs, that may not consistently produce tubercles. MAC includes M. intracellulare and M. avium.178

3.5.1 Mycobacterium tuberculosis Mycobacterium tuberculosis has been, and continues to be, an infectious and zoonotic agent of concern for NHP colonies, thus the Centers for Disease Control and Prevention (CDC) directed quarantine and surveillance are compulsory in colonies.179 Negative intradermal antigen tests are routine for animals prior to study start; however, false negatives occur, and false positives may occur with infection by environmental mycobacterial species. M. tuberculosis is most commonly reported in macaques, but has also been noted in the common marmoset.180 Marmosets exhibit a striking susceptibility to mycobacteria and are able to replicate some of the same clinical and pathological manifestations as humans.180e182 M. tuberculosis rapidly spreads by the aerosol route and is zoonotic; therefore, it is a well-recognized pathogen used to define a pathogen-free macaque colony.183 Cynomolgus monkeys from the Philippines are reported to develop chronic tuberculosis with meningitis and ocular disease.184 Clinical signs do not provide reliable indication of the severity of disease caused by M. tuberculosis in monkeys as many infected animals may be asymptomatic. A monkey can appear healthy but may have extensive miliary disease

FIGURE 3.14 Mycobacterium tuberculosis (TB): (A) TB granulomas in the tracheobronchial lymph nodes have central areas of caseous necrosis surrounded by epithelioid macrophages, Langhans-type giant cells, and lymphocytes that typically enlarge and efface the lymph node. (B) The lung is a common, nonlymphoid location for TB granulomatous inflammation. (C) Acid-fast staining of a lymph node: Multiple sections of TB granulomas often must be examined to identify the pathogen as the number of organisms that may be observed microscopically with acid fast staining may be relatively low.

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surround the mature lesion. Acid-fast bacilli may be found within the caseous core or within the surrounding macrophages (Fig. 3.14C). Multiple sections and granulomas often must be examined to identify the pathogen as the number of organisms that may be observed microscopically with acid fast staining (Ziehl-Neelsen technique) may be relatively low.135 The intradermal Mantoux tuberculin skin test (TST), using mammalian old tuberculin (MOT) by intradermal injection into the eyelid, remains the mainstay of NHP tuberculosis routine surveillance and antemortem diagnosis for NHPs. Unfortunately, there may be false positive and false negative reactions, so combination with other tests such as the interferon (IFN) gamma releasing assay (PRIMAGAM; Prionics AG, Zurich, Switzerland), Quantiferon Gold Test (Cellestis, QIAGEN, Venlo, Netherlands), or detection of antibodies to several mycobacterial proteins (PrimaTB STAT-PAK; Chembio Diagnostic Systems, Medford, NY) can resolve equivocal results.141 These tests all depend on the functional immune system; therefore, to eliminate false positive results culture of the mycobacteria remains the gold standard for diagnosis.

3.5.2 Mycobacterium avium complex (MAC) Mycobacterium avium complex (MAC) consists of nearly ubiquitous environmental organisms M. avium and M. intracellular that are usually nontuberculous. These bacteria are normal inhabitants of soil and water and are opportunistic pathogens of humans and NHPs.185 Freeranging macaques have tested positive for MAC in Malaysia and infection has been documented in captive animals immunocompromised by SIV.186,187 A case from the editor’s facility occurred in an animal with a positive intradermal tuberculin test noted during prestudy screening. Gross examination of the animal was without findings; however, microscopically there were microabscesses in multiple organs and acid fast organisms were abundantly present within the lesions (Fig. 3.15A and B). Tissues submitted for PCR analysis were positive for M. avium.

3.5.3 Mycobacterium leprae The spectrum of disease seen with M. leprae depends on the degree of cell mediated immunity the host is able to mount. Natural infections of the lepromatous form, indicating no cell mediated immunity, have occurred in chimpanzees and sooty mangabeys (Cercocebus torquatus atys).188 Naturally occurring leprosy in a cynomolgus macaque obtained from the Philippines was reported in 1990 and subsequent genomic evaluation indicated a human to primate infection had occurred.189,190 Case image below was from an animal housed in an outdoor pen in Louisiana with suspected exposure to armadillo feces. Grossly there was nodular thickening of the skin, and deformity of the

FIGURE 3.15 Mycobacterium avium complex (MAC): (A) MAC granulomas in the lung of a macaque. MAC are environmental mycobacterial species that occasionally cause caseous abscesses in non-human primates and that are microscopically indistinguishable from those of M. tuberculosis. (B) MAC organisms (arrows) are usually abundant within macrophages of the abscesses when evaluated by Acid-fast staining techniques. This organism was PCR positive for M. avium sp.

hands and feet. Microscopically, there was nodular histiocytic infiltrate with variable numbers of lymphocytes and plasma cells (Fig. 3.16A and B). Large numbers of Acidfast bacilli were demonstrable with Fite-Faraco acid-fast stain (case materials supplied courtesy of Gary B. Baskin).

3.6 Moraxella (Branhamella; Neisseria) catarrhalis Moraxella catarrhalis is the currently accepted nomenclature for the organism responsible for “bloody nose syndrome” in macaques. It’s a Gram-negative, oxidasepositive diplococcus and is a common colonizer of the upper respiratory tract of humans and NHPs.191,192 While these bacteria cause otitis media in children and pulmonary

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FIGURE 3.16 Mycobacterium leprae: (A) The spectrum of disease seen with Mycobacterium leprae depends on the degree of cell-mediated immunity the host is able to mount. Natural infections of the lepromatous form, indicating no cell-mediatted immunity, result in nodular histiocytic infiltrate that effaces the subcutis. (B) The lesion in Fig 3.16A had dense infiltrates of variable numbers of lymphocytes and plasma cells. Globi accumulations of bacteria (arrows) were visible on H&E-stained sections. Images courtesy of Gary B. Baskin.

FIGURE 3.17 Moraxella catarrhalis is the currently accepted nomenclature for the organism responsible for “bloody nose syndrome” in macaques: (A) Infections with M. catarrhalis typically result in congestion and hemorrhage of the nasal cavity mucosa. (B) Immunocompromised animals may have disseminated disease in which the Gram-negative diplococci bacteria may be identified by staining techniques (Gram Stain).

3.7 Escherichia coli disease in human adults, they primarily cause hemorrhagic rhinitis in captive NHPs.191,192 Humidity levels within animal compounds has impact on the pathologic presence of the organism, as its been reported most commonly in the autumn and winter months, and may be precipitated by reduction in ambient humidity of less than 45%.193 More severe disseminated infections in NHPs are usually in animals with immunosuppressive conditions or receiving immunomodulatory agents. Clinically, macaques may present with sneezing and bloody discharge from the nose, with or without periorbital skin edema. In one report, 64% of monkeys with epistaxis were positive for M. catarrhalis.193 Microscopically, there is engorgement of mucosa and submucosal blood vessels with perivascular hemorrhages, often with low numbers of granulocytes and mononuclear cells present (Fig. 3.17A). In disseminated disease, Gram-negative organisms may be identified in inflammatory lesions (Fig. 3.17B).

According to Mansfield et al. and Sasseville and Mansfield, there are “six categories of diarrheagenic E. coli are defined, based on the underlying mechanism of disease pathogenesis, in vivo and in vitro growth characteristics, and the presence of specific genes encoding virulence factors. These include enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EaggEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), and diffuse adherent E. coli (DAEC).”33,194 These authors state that “A systematic approach including the use of adhesion assays, biopsies, and/or molecular identification of virulence genes is required for a definitive identification.”194 The bacteria are spread via fecal-oral route and in most cases, infection is clinically silent or causes mild diarrhea. In more severe cases, E. coli infection usually presents as persistent diarrhea that may progress to a hemorrhagic diarrhea. Some authors describe microscopic features that are considered “pathognomonic” for the attaching and

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and interstitial epithelial cells, and sparse fibrin. Fibrinous pleuritis may occur as the lesion progresses from caudal to cranial and the upper lung lobes are rarely affected.30,33,196

3.9 Bordetella bronchiseptica Bordetella bronchiseptica is a Gram-negative, aerobic coccobacilli that inhabits the upper respiratory tract of a number of species, including a number of NHP species, such as the common marmoset.197 During an outbreak of disease in one colony of common marmosets, bilateral mucopurulent nasal discharge was observed in animals of all ages. Death occurred in animals less than 1 year of age, frequently without preceding clinical signs. Gross lesions included pneumonia, pleurisy, and pericarditis. Microscopically, the pneumonia was characterized by suppurative bronchopneumonia with necrosis of the bronchiolar epithelium.197 FIGURE 3.18 Escherichia coli infection: (A) Brain from an immunocompromised infant rhesus monkey that was found dead. The meninges and brain surface have purulent exudate (arrows) and there are congested cerebral blood vessels with multifocal hemorrhages. (B) Microscopically there is suppurative inflammation of the brain and meninges with vascular fibrinoid necrosis. Images courtesy of Dr. Andres Mejia. Wisconsin National Primate Research Center. University of WI. Madison WI.

effacing bacteria, which include an irregular colonic epithelium coated by bacilli intimately associated with the apical membrane of the colonic mucosal epithelium.33 With colonic mucosal compromise or in immunocompromised animals, the bacteria may disseminate resulting in sepsis and multiorgan necrohemorrhagic lesions (Fig. 3.18A and B). In severe cases, animals become anemic, dehydrated, and hypovolemic, leading to clinical decline or death. In both Old World and New World primates, a chronic and persistent form is also recognized.194

3.8 Rhodococcus equi Rhododococcus equi is a Gram-positive, facultative anaerobic intracellular pathogen commonly found in soil. It has close relationship with Mycobacterium sp. and Corynebacterium sp. and was formerly known as Corynebacterium equi.195 It finds reservoir in the intestinal tract of many herbivores and is passed in feces.195 Immunocompromised NHPs are at risk of contracting R. equi infections, which primarily cause necroulcerative and pyogranulomatous gastrointestinal lesions.33 Bronchopneumonia may develop as a secondary complication in juvenile/baby monkeys with R. equi infection, and is seen especially in the lower lobes of the lungs as panbronchitis, atelectasis/hepatization and areas containing purulent exudate.30,196 The pulmonary exudate may contain neutrophils, erythrocytes, desquamated alveolar

3.10 Klebsiella pneumoniae Klebsiella pneumoniae is a Gram-negative, nonmotile, encapsulated rod-shaped bacteria that can be found in the nasopharynx and gastrointestinal tract of humans and NHPs.157 K. pneumoniae has been associated with pneumonia, meningitis, septicemia, peritonitis, sinusitis, and otitis in NHPs.198e201 Klebsiella pneumonia and septicemia occur more commonly and are associated with higher mortality in young animals than in adults and are frequently associated with stress.200 Pulmonary lesions are characterized by a suppurative bronchopneumonia with abscessation and fibrinosuppurative pleuritis. The inflammatory infiltrate is composed predominantly of neutrophils as well as macrophages in more chronic lesions. In inflammed areas, neutrophilic infiltrates contained large numbers of small bacilli surrounded by clear halos. In peracute septicemia of the young, bacteria may be evident in blood vessels with limited or no associated inflammation. Wadsworth reported K. pneumoniae-induced meningitis and cerebellar abscess as the causes of death in a small but significant number of marmosets in the breeding unit of ICI Pharmaceuticals. These animals had purulent leptomeningitis throughout the brain, often associated with pneumonia or acute purulent otitis media.202 In recent years, highly virulent hypermucoviscosity (HMV) strains of K. pneumoniae have become important emerging pathogens of humans and NHPs.201,203 HMV infection has been associated with liver abscesses, bacteremia, meningitis, and endophthalmitis in humans and with multisystemic abscesses in African green monkeys.201 Subclinically infected rhesus and cynomolgus macaques were a potential source of infection for susceptible African green monkeys.201

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3.11 Yersinia spp. Yersinia enterocolitica and Y. pseudotuberculosis are Gram-negative, facultative anaerobic bacilli and both cause fulminating enteric and systemic diseases in NHPs. Infections are acquired by the fecal-oral route. Bacteria adhere to the cell membrane of intestinal epithelial cells and are subsequently ingested by bacterial endocytosis with vacuole formation.204 It is difficult to identify any marked differences in the clinical syndromes associated with these two agents in NHPs.205,206 Both cause diarrhea, often with blood, dehydration, anorexia, and weight loss. Most infections are self-limiting, but debilitated or immunocompromised animals are at increased risk for severe clinical disease. Y. enterococolitica and Y. pseudouberculosis can be isolated from rectal swabs or blood from septicemic animals, although culturing requires selective conditions for extended periods. PCR analysis of stool samples may prove beneficial in suspected outbreaks of Yersinia infection.207 Infection with either species of Yersinia produces similar gross and histopathological lesions, including edema, ulceration, congestion, and thickening of the intestinal wall. Mucosal ulceration of the gastrointestinal tract can occur in any part or throughout; intestine may also be congested or hemorrhagic and may have visible necrotizing or granulomatous foci that resemble intestinal tuberculosis (Fig. 3.19).150 Hepatomegaly, splenomegaly, and lymphadenopathy may also be evident. Microscopically, epithelioid granulomas within both the intestinal wall and lymph nodes and inflammatory cell infiltration consisting of

various cell types including eosinophils within the muscularis propria and serosa are observed in affected tissues. Another noticeable feature is marked lymphoid hyperplasia in the intestinal submucosa. Coccobacilli are usually prominently observed within the necrotic foci of the affected tissue. Yersinia sp. infection in the NHP is frequently identified in an advanced stage of disease; as such, treatment is often difficult. Aggressive treatment with systemic antibiotics may be therapeutic in early stages of disease combined with fluid therapy.205

3.12 Chromobacterium violaceum Cases of Chromobacterium violaceum infection have been observed in aged rhesus monkeys resulting in vasculitis and thrombosis, associated with lung abscessation and pleuritis. The Gram-negative bacillus bacterium can be observed within the lesions. Low CD4 T cell levels support a role for immunocompromise in the pathogenesis of this disease. Compromise of the immune system is also supported by the presence of hemolysins, colicins, and detoxification enzymes that block the destruction of the bacteria by host defense systems.10 Infection usually results in septicemia and may be evident in lung, liver, spleen, lymph nodes, and kidney. Affected organs are swollen and congested with scattered petechial hemorrhages and contain variably sized areas of necrosis which may be cavitated and contain tan, semifluid exudate. Large numbers of Gram-negative bacilli can be seen in the areas of necrosis on histopathology.10

3.13 Francisella tularensis Francisella tularensis is a zoonotic infectious disease in North America, Europe, and Asia, and may cause acute pulmonary perivascular lesions in rhesus monkeys.208 Acid-fast and Giemsa stains may demonstrate these bacteria in lesions; however, in chronic lesions bacteria may not be identified histologically.209

3.14 Corynebacterium spp.

FIGURE 3.19 Yersiniosis sp. cecum of a macaque: Infection with either Y. enterocolitica or Y. pseudotuberculosis produces similar gross and histopathological lesions, including edema, ulceration, congestion, and thickening of the intestinal wall. Mucosal ulceration of the gastrointestinal tract can occur in any part or throughout; intestine may also be congested or hemorrhagic and may have visible necrotizing or granulomatous foci that resemble intestinal tuberculosis (arrows).

Corynebacterium species are one of the most common causes of opportunistic ascending urinary tract bacterial infections and may be a significant cause of the high incidence of interstitial nephritis in young cynomolgus monkeys.210,211 Under laboratory conditions, ascending infections may be associated with urolithiasis or a history of urinary catheterization, while infections via the hematogenous route may occur via septic emboli from indwelling vascular catheters or from bite wound innoculation. In continuous intravenous infusion studies, chronic catheter-associated interstitial nephritis may be further complicated by glomerulonephritis as a result of microembolization and immune complex deposition.212 Species

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of Corynebacterium identified as NHP pathogens includes Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, and Corynebacterium renale.210 SRV viremic animals are more susceptible to infection by pyogenic bacteria such as Rhodococcus equi (previously Corynebacterium equi).213

3.15 Streptococcus spp. These Gram-positive, encapsulated bacteria are present in clinically healthy NHPs and humans in the nasopharyngeal cavity. Bacteria can be spread by aerosol or contaminated fomites to cause respiratory disease, otitis media, and meningitis. Stressed and/or immunocompromised animals develop more severe disease. Disease is typically characterized by fibrinopurulent leptomeningitis containing degenerate neutrophils, macrophages, and fewer lymphocytes. Inflammation can extend into the adjacent parenchyma including blood vessels. When vessels are affected, this can lead to fibrinous thrombosis and necrotizing vasculitis with associated ischemic necrosis.54,214

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There is a well-established link between streptococcal infections in the rhesus monkey and glomerulonephritis.212,215 Most of the glomerular lesions are characterized by mesangioproliferative glomerulonephritis, and immunostaining of the tissues shows that they contain antigen IgM complexes and several components of complement.216 In laboratory NHPs, these findings are usually not associated with a clinical disease or clinical pathology findings, and are generally considered as incidental findings, particularly in macaque monkeys.

3.16 Staphylococcus aureus Staphylococcus aureus is a Gram-positive, facultative anaerobic skin bacteria that may cause meningoencephalitis in Old and New World NHPs including chimpanzees, squirrel monkeys, and cynomolgus monkeys.217e219 The bacteria may be spread by bite wounds, endocarditic emboli, otitis media extension, or septicemia. Cases at the editor’s facility often had vertebral, paravertebral, or paraspinal abscesses (Fig. 3.20AeD) and usually had skin lesions identified that were considered the entry points for the

FIGURE 3.20 Staphylococcus aureus infection: (A) Dissemination of S. aureus to the brain and spinal cord resulted in paralysis with anisocoria in a macaque. (B) The paravertebral abscess (arrow) from the animal in Fig. 3.20A was grossly visible at necropsy. (C) The animal in Fig. 3.20A had necrotizing inflammatory lesions in multiple organs, including lung. (D) Cocci bacteria were identified within vascular lumen in the brain of the animal shown in Fig. 3.20A.

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Spontaneous Pathology of the Laboratory Non-human Primate

infections. Experimental manipulations such as implants, cerebral cannulas, and in-dwelling catheters can also cause spread of this bacterium, including to the brain.54,214 With S. aureus infection of the brain, yellow exudate can be observed over the sulci, abscesses may form, and brain tissue may be hyperemic. Microscopically, the meninges may be infiltrated with many neutrophils and fewer macrophages that frequently extend into the adjacent parenchyma and are accompanied by multiple areas of necrosis and microgliosis.218 In recent years, methicillin-resistant strains of S. aureus (MRSA) have gained notoriety as persistent infections in humans and have been diagnosed in macaques as well.220 Under colony conditions, the bacteria are carried in approximately 6% of the populations that have been studied.221 In most cases, the disease state is mild with rashlike skin lesions that may progress to pustules and that heal poorly or fail to heal with standard antimicrobial therapy. Disseminated bacterial disease is rare in healthy animals but has been diagnosed on occasion in macaques at the editor’s facility with fulminant necrohemorrhagic lesions in multiple organs (Fig. 3.21AeC). Some evidence suggests that transmission of MRSA between wild populations of macaques and humans may occur, such as at feeding stations.220 Zoonotic potential is therefore assumed to be possible within the captive situation as well.

3.17 Listeria monocytogenes Listeria monocytogenes is a Gram-positive, facultative anaerobic, motile, intracellular, rod-shaped bacteria. Listeria has been reported to cause septicemia and meningitis in neonatal Cynomolgus macaques, in which the infection was believed to be transmitted at birth. In these cases, meninges contained purulent material and fibrin and ventricles were filled with fibrinopurulent exudate.222 Microscopically, there was suppurative meningoencephalitis and occasional neutrophilic infiltrates into adjacent white matter.222 L. monocytogenes can also cause foodborne disease in immunocompromised adult non-human primates.54

3.18 Morganella morganii Within the genus Morganella, there are two subspecies defined: M. sibonii and M. morganii. M. morganii was previously classified under the genus Proteus as Proteus morganii. M. morganii is a Gram-negative rod commonly found in the environment and in the intestinal tracts of humans and NHPs as normal flora. It can cause enteritis chiefly involving the stomach and small intestine in immunocompromised animals.196 Grossly there is edema with many punctate or larger hemorrhages and a large amount of mucus. The contents of the large intestine are

FIGURE 3.21 Methicillin-resistant Staphylococcus aureus (MRSA) infection: (A) With progression, the papillary rash characteristic of MRSA may ulcerate, may be poorly responsive to antimicrobial therapy, and may fail to heal. (B) MRSA sepsis in a macaque resulted in necrohemorrhagic pyelonephritis. (C) Microscopic image of the kidney in Fig. 3.21 B: There were necrohemorrhagic abscesses with MRSA-positive colonies effacing much of the renal MRSA was identified by tissue culture.

liquid or semiliquid. Microscopically these hemorrhages are spread throughout the mucosa and submucosa with necrosis and desquamation of the epithelium extending into crypts. There is often lymphoid hyperplasia of Peyer’s

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patches and increased germinal centers in the mesenteric lymph nodes and tonsils. Bronchopneumonia may arise as a secondary complication.196

3.19 Nonpathogenic bacteria commonly noted in non-human primate tissue For completeness, there should be mention of bacteria that are commonly noted in NHP tissues and that are without known pathologic significance. Large rod- or filamentous-shaped organisms are quite common in the stomach of macaques and have been identified as “megabacterium”, but are not actually bacteria (see Opportunistic fungal infections). There may commonly be colonies of nonpathogenic oral bacteria that adhere to the tongue or labial mucosa of NHPs and that are captured in histologically tissue section (Fig. 3.22). Likewise, the large intestinal lumen and mucosal surface may have remnants of normal bacteria that are present in tissue section.

4. Parasites Parasites are quite commonly identified in tissues from NHPs utilized in toxicological research. The vast majority are identified incidentally during routine evaluation of the tissues. Fibrous or mineralized nodular foci are the remnants of larval or adult parasites that die in situ and subsequently undergo dystrophic calcification. They may be noted in nearly any tissue but are very common for the pleura of the lung, the mesentery, and the liver (Fig. 3.23A and B). In the thorax, these foci may cause pleural adhesions to the body

39

wall or adjacent lung lobes. Less commonly, larger foci are noted during physical exam or radiography. Adult and larval worms (strongyles, cestodes, and other helminths) have been found in the thoracic and abdominal cavities of animals at the editor’s facility (Fig. 3.24AeC).

4.1 Protozoa 4.1.1 Cryptosporidium sp. Cryptosporidium parvum is a common parasite of NHPs in captivity that is generally clinically silent. The organism is environmentally persistent, residing in soil and water, and may be transmitted via the fecal-oral route among NHPs. In one study, over 95% of colony macaques were seropositive by 2e3 years of age.33 Severe diarrhea and weight loss have been observed in immunosuppressed or immunodeficient animals or in juveniles infected with C. parvum. C. parvum is commonly found in the colon with associated inflammation in these cases, and the organism may disseminate to other organs, including hepatic biliary ducts, small intestine, lung, trachea, or conjunctiva.223,224 Roughly spherical, approximately 3e4 mm diameter, basophilic bodies may be noted to adhere to the apical surface of colonic mucosal epithelium (Fig. 3.25A). Bile ducts may have extensive epithelial necrosis and/or hyperplasia with associated inflammation and annular fibrosis (Fig. 3.25B). In the lung, it has been the designated causative agent of necrotizing bronchiolitis.224 It should be noted that individual hyperplastic bile ducts have been noted in cynomolgus monkey livers, and for which extensive work up failed to confirm C. parvum present. The significance of these cases is not yet known (see Chapter 7).

FIGURE 3.22 Oral bacterial organisms: There may commonly be colonies of nonpathogenic oral bacteria that adhere to the tongue or labial mucosa of NHPs, and that are captured in histologically tissue section.

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Spontaneous Pathology of the Laboratory Non-human Primate

4.1.2 Giardia intestinalis Giardia intestinalis (Syn. G. lamblia, G. duodenalis) is a flagellate protozoan parasite that is found worldwide and infects humans and a number of animal species, including NHPs.226,227 Infective cysts of G. intestinalis are passed in the feces of the infected host and can persist in the environment for extended periods of time.226 Transmission of G. intestinalis occurs via the fecal-oral route when contaminated water or food is ingested by a potential host. Once ingested, cysts are excysted following exposure to the acidic stomach environment and trophozoites are released to infect and replicate in the small intestine.226 Infection with Giardia in NHPs has been associated with watery to hemorrhagic diarrhea and may potentially be a contributing factor in Marmoset Wasting Syndrome, a chronic condition of common marmosets.228 Histologically, intestinal lesions associated with Giardia infection can be mild and include mononuclear cell infiltration of the lamina propria and blunting of the intestinal villi.7 Crescent- or pear-shaped organisms may be seen attached to the intestinal mucosa or within the lumen of the intestine. Giardia can be detected in the feces of NHPs without evidence of disease.229 Surveys of colonies of common marmosets have demonstrated that up to roughly 40% of animals’ fecal samples may test positive for Giardia and may not be associated with clinical disease.228 Infected animals serve as a potential source for zoonotic transmission of Giardia when in close contact with humans, such as their human handlers.229

4.1.3 Trypanosoma spp. FIGURE 3.23 Parasitic nodules: (A) Mineralized and fibrotic nodular foci are usually the remnants of larval or adult parasites that die in situ and subsequently undergo dystrophic calcification, here in the liver of a macaque. Nodular remnants of parasites may be noted in nearly any tissue but are very common for the pleura of the lung, the mesentery, and the liver. (B) Small intestine of a macaque: Fibrosis of a parasite or previously parasitized blood vessel results in a distinct nodule in the mesentery (arrow).

In general, diagnosis of C. parvum infection can be made by fecal floatation and evaluation, biopsy, antigen capture assay, or immunohistochemistry (IHC). Cryptosporidium spp. also are commonly identified within the gastric fundus of cynomolgus monkeys as opportunistic infections.225 Investigation by Dubey et al. indicate these cryptosporidians occur in immunosuppressed individuals and are similar to C. muris, a common protozoal parasite in the stomach of rodents.225 Similar cryptosporidium have been identified in otherwise healthy cynomolgus monkeys at the editor’s facility (Fig. 3.26); therefore, immunosuppression may only exacerbate a background finding in these animals.

Non-human primates are susceptible to infection by the trypanosomes present in Africa and South America. Trypanosoma cruzi (Chagas disease) is transmitted by the feces of insects from the genus Triatoma and is occasionally recognized in monkeys, usually as a nonpathogenic entitiy. In the United States of America, molecular techniques (PCR, ELISA) indicated an 8.5% prevalence of infection in cynomolgus macaques in an outdoor-housed colony in south Texas.230 Likewise, characterization of T. cruzi by discrete typing units (DTU) in a Texas colony of rhesus monkeys indicated low concentrations of circulating T. cruzi DNA present, while local wildlife hosts had high concentrations and likely served as the reservoir.231 While this infection typically targets the heart and gastrointestinal tracts, it has been seen to affect a cardiac ganglion, causing inflammation. Damage to the cardiac autonomic nervous system has been proposed as a potential cause of clinical disease.232 The organism may induce inflammation in additional sites similar to that noted in human cases of Chagas disease, including the peripheral nerves (Fig. 3.27B). T. cruzi may

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FIGURE 3.24 Aberrant helminth migration: (A) Radiograph of a macaque with a large, opaque parasitic nodule adhered to the lung (arrow). (B) Lung with large parasitic nodule from the macaque depicted in Fig. 3.25Adthe nodule contained partially degraded and mineralized helminths. Note: there are pleural adhesions between lung lobes adjacent to the parasitic nodule, a common sequelae to parasites of the thoracic cavity and lung (C). An unidentified helminth recovered from the pleura of a macaque.

produce a chronic mononuclear cell myocarditis in marmosets and macaques.233,234 The amastigote stages are found within myocardial fibers (Fig. 3.27A) and only appear to incite an inflammatory response when the sarcoplasmic membranes rupture exposing the parasite and its byproducts. Other nonpathogenic trypanosomes include T. minasense in marmosets, T. saimirii in squirrel monkeys, T. diasi in capuchins, and T. primatum in African apes.230,231,235

4.1.4 Sarcocystis spp. Sarcocystosis is caused by intracellular apicomplexan parasites of the genus Sarcocystis. There are more than 120 recognized species in the genus, and the parasites usually develop in an obligatory heteroxenous predator-prey twohost life cycle, cycling between the definitive host (often a predator species) and the intermediate host (a respective prey species).236 The cystic phase of this parasite has been described in skeletal muscle fibers and occasionally in

cardiac or smooth muscle fibers in a wide variety of animals throughout the world.236,237 Sarcocystis infection is common in the skeletal muscle of NHPs (Fig. 3.28).238 Sarcocystis kortei (thick-walled) and S. nesbitti (thinwalled) have been described in the rhesus monkey.239 Sarcocysts were present in muscle sections from 79 (21%) of 375 wild-caught Old and New World monkeys examined by one author, comprising 14 species, whereas none of 369 laboratory-born monkeys had sarcocysts.238 In most cases, the cysts are without inflammation associated; however, infiltrates of lymphocytes, plasma cells, and eosinophils may be associated secondary to degeneration of the cysts within the muscle fibers.240

4.1.5 Plasmodium spp. Malaria (Plasmodium spp.) is an infection of NHPs in colonies, mostly those imported from malaria-endemic areas, such as Southeast Asia, where macaques serve as a

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Spontaneous Pathology of the Laboratory Non-human Primate

FIGURE 3.26 Cryptosporidium species, stomach of a macaque: Cryptosporidium spp. are commonly identified within the gastric fundus of cynomolgus monkeys as opportunistic infections. Here multiple stages of Cryptosporidium sp. are visible within the gastric gland of a macaque, including schizonts (S), oocysts (O), and male gamonts (MG).

FIGURE 3.25 Cryptosporidium species infection: (A) Roughly spherical, approximately 3e4 mm diameter, C. parvum may be noted adhere to the apical surface of colonic mucosal epithelium (arrows). (B) C. parvum may disseminate to other organs, including hepatic biliary ducts where they incite extensive epithelial necrosis and/or epithelial hyperplasia with associated inflammation and annular fibrosis. Fig. 3.25A courtesy of Gary B. Baskin.

reservoir.241,242 Animals from Mauritius continue to be free of plasmodium infection. Species of Plasmodium identified in macaques include P. coatneyi, P. fragile, P. knowlesi, P. inui, and P. cynomolgi and clinical presentation varies by species.33 In most cases, the infection is silent; however, reactivation of latent infection is not uncommon following splenectomy, during stressful conditions or following administration of immunomodulatory agents.33 Recrudescence of Plasmodium sp. infection may be noted on blood smear evaluation with the identification of various parasite stages within red blood cells (Fig. 3.29AeC). Both latent and active Plasmodium infections can cause alterations in cytokine profiles making the organism of concern when present in animals included in preclinical studies.33,243 Clinical cases in macaques may include fever, depression, anorexia, weight loss, anemia, leukopenia, and diarrhea. The onset of fever coincides with rupture of infected erythrocytes.244 Typical Plasmodium infection

FIGURE 3.27 Trypanosoma cruzi, infection: (A) Heart: T. cruzi amastigote stages are found within myocardial fibers. Once the fibers rupter, a chronic mononuclear cell myocarditis ensues in NHPs. (B) Peripheral Nerve: T. cruzi infection in NHPs may induce inflammation in additional sites including the autonomic nervous system.

causes hepatosplenomegaly and multifocal hepatitis with intraerythrocytic merozoites, trophozoites, and schizonts. Myeloid hyperplasia of the bone marrow may be seen in conjunction with erythropoiesis.244 Rarely, there may be cerebral malaria in macaques with regions of necrosis or thrombosis as the parasitized red cells are sequestered in microvessels (Fig. 3.30A and B).245,246 Malarial hemazoin pigment is an insoluble crystalline substance produced by the parasites during detoxification when they digest hemoglobin.247 The pigment is readily observed in Kupffer cells, bone marrow macrophages, and

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FIGURE 3.28 Sarcocystis species, skeletal muscle: Sarcocystis spp., an intracellular apicomplexan/coccidian protozoan parasite, invades skeletal muscle cells in NHPs to form cysts filled with bradyzoites. Note the striated cyst wall (arrow) visible in this H&E-stained tissue section.

red pulp of the spleen where it is sequestered within cytoplasmic vacuoles (Fig. 3.29B).248 This pigment is birefringent under polarized light (Fig. 3.29C). There is a well-established link between chronic parasitism, such as with the malarial parasites, causing antigen overload and glomerulonephritis in macaques.249,250 Most of the glomerular lesions are characterized by mesangioproliferative glomerulonephritis, and immunostaining of the tissues shows that they contain antigen IgM complexes and several components of complement.250 In laboratory NHP, these findings are usually not associated with a clinical disease or clinical pathology findings, and are generally considered as incidental findings.251 Malaria infection does not occur in marmosets under natural conditions, but can be experimentally maintained in several species of callitrichids.233 Standard thin blood smears do not routinely identify latent infections due to the low level of parasitemia, but are standard procedure once there is evidence of recrudenscence.251

4.1.6 Balantidium coli Balantidium coli is a large, ovoid (50e130 mm long by 20e70 mm wide), ciliated protozoa that inhabits the lumen of the cecum and colon of normal New and Old World NHPs. The trophozoites (100e300 mm) contain a distinct elongated, kidney or bean shaped macronucleus with a micronucleus lying within the notch of this macronucleus. The trophozoites normally reside in the lumen of the large intestine and are readily identified by routine histology (Fig. 3.31A) but are generally not associated with diarrhea or alterations of the intestinal mucosa. B. coli is regarded as

a commensal, since it is found in many clinically normal primates. The parasitic load and incidence of B. coli as reported by Drevon-Gaillot et al. varies according to the source of macaques, with incidences of 13% reported in cynomolgus macaques from Mauritius and up to 75% in cynomolgus macaques from Vietnam and the Philippines.252 B. coli may be increased in number in animals suffering gastrointestinal disease, inflammation, or alterations in normal gut transit times and may be invasive when the opportunity presents for mucosal penetration, such as ulceration, or as a sequela to administration of some immunomodulatory agents.33 If mucosal invasion occurs, these lesions are often associated with a mild to severe enterocolitis (Fig. 3.31B). One fatal invasive infection has been reported and protozoal invasion has occurred at the editor’s facility, with colonic perforation and peritonitis.253 The protozoa may form intramural abscesses, invade capillaries and lymph ducts, emigrate to draining lymph nodes and spread to distant organs, most commonly the liver. Diagnosis is based on clinical signs, post-mortem evidence of an ulcerative condition of the large intestine, and the presence of large numbers of invasive B. coli trophozoites.253

4.1.7 Trichomonas spp. Trichomonas are common commensal flagellates found in the small and large intestine of macaques and, in most cases, are not associated with disease and are without accompanied inflammation. In SIV-associated immunodeficient macaques, the parasite may inhabit the gastric glands with associated gastritis, and disease has occasionally been

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FIGURE 3.30 Cerebral malaria, cerebellum: (A) Plasmodium species infection in a macaque with dissemination to the brain resulted in regionally extensive necrosis and inflammation of the cerebellum and meninges with vascular congestion. (B) Higher magnification of Fig. 3.30A: The cerebellar architecture is distorted and effaced and there is marked inflammation of the tissues due to cerebral Plasmodium infection.

observed with propria.254,255

parasitic

invasion

into

the

lamina

4.1.8 Toxoplasma gondii

FIGURE 3.29 Plasmodium species infection: (A) Recrudescence of Plasmodium sp. infection may be noted on blood smear evaluation by identification of various parasite stages that stain deeply basophilic within red blood cells following Giemsa stain proceedures. (B) Malarial hemazoin pigment, an insoluble crystalline substance produced by the parasites when they digest hemoglobin, is readily observed in red pulp of the spleen as dark, brown pigmented material sequestered in phagocytic cells. (C) Hemazoin pigment within the liver is birefringent under polarized light. Fig. 3.29A and C Courtesy of William Iverson.

T. gondii causes toxoplasmosis, a common pathogen of New and Old World primates. Tamarins and marmosets are particularly sensitive to toxoplasmosis.185 Animals may be found dead without previous signs of illness and appear in good physical condition at death, or solitary tissue cysts may be found as incidental findings in the absence of other lesions.256 Symptomatic animals typically have fever, with progression to cough, anorexia, or neurologic signs. Lungs are consistently involved in fatal disease and key morphologic findings in severe disease include necrosis with or without inflammation in the lungs, liver, kidney,

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4.1.9 Amebae

FIGURE 3.31 Balantidium coli, colon: (A) The trophozoites of the commensal protozoa B. coli normally reside in the lumen of the large intestine and are readily identified by routine histology. (B) B. coli may be increased in number in animals suffering gastrointestinal disease or alterations in normal gut transit times and may be invasive when the opportunity presents for mucosal penetration. If mucosal invasion occurs, the organisms may invade into or through the large intestinal smooth muscle layers (arrow), with associated inflammation and in rare cases, peritonitis.

skeletal muscle, pancreas, spleen, mesenteric lymph node, heart, and adrenal. At necropsy, lungs appear firm, dark red, and are congested and edematous when sectioned. The trachea frequently contains red tinged froth. Tissue cysts contain bradyzoites and free forms (tachyzoites) may be present at the periphery of the necrotic lesions.256 Toxoplasmosis affecting the nervous system has been reported in SIV-infected macaques.232 Microscopic findings included necrosis, hemorrhage, gliosis, and inflammatory infiltrates of neutrophils, mononuclear cells, and eosinophils. Tachyzoites were frequently present within various cells including endothelial cells. Tachyzoites are banana-shaped, 2  6 mm, Gram-negative, acid-fast negative, and have pointed ends with an eccentric nucleus and occasional halo. Cysts are ˂ 60 mm in diameter.232 Special stains and immunohistochemistry may assist in recognizing and identifying the organisms. The natural hosts for Toxoplasma sp. are felines, and rodents and cockroaches serve as a vectors to NHPs.

Infections of the central nervous system (CNS) by amebae, such as Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris, are rare in NHPs, but more commonly noted in animals with access to the outdoor environment, particularly in warmer climates, where the organisms live in soil and water. Amebae can cause two types of CNS disease in human and animal hosts: primary amebic meningoencephalitis (PAM) and granulomatous amebic encephalitis (GAE). PAM is primarily associated with N. fowleri infection and may occur in immunocompetent individuals, whereas GAE is primarily associated with Acanthamoeba spp. and B. mandrillaris, and is usually a slow, progressive disease of immunocompromised animals and humans.257 For example, an SIV-infected rhesus macaque was reported to have developed meningoencephalitis due to Acanthamoeba sp.258 B. mandrillariseassociated meningoencephalitis has been observed in a number of New and Old World primates.259 Microscopically, lesions are characterized by multifocal to coalescing, necrotizing, suppurative to granulomatous meningoencephalitis with intralesional amebic trophozoites (Fig. 3.32). Identification of trophozoites in areas of inflammation aid in the diagnosis and immunofluorescence and PCR can be used to identify the genus.257 Intestinal amebae such as Entamoeba sp. are common in NHPs but are generally nonpathogenic. They may increase in number under favorable conditions in similar manner to, or accompanied by increased numbers of, Balantidium coli (Fig. 3.33). The organism may become pathogenic in the immunosuppressed individual. For instance, The Southwest National Primate Research Center reported a case in a 16-year-old female baboon where

FIGURE 3.32 Granulomatous amebic encephalitis (GAE): The opportunistic, pathogenic amoeba may infect NHPs exposed to the natural environment, including soil and water in temperate to warm climates, in which the organisms live. Infection results in severe granulomatous and necrotizing encephalitis with amoebic trophozoites (arrows) usually identified in the lesions. Balmuthia sp. was confirmed by fluorescent immunohistochemistry in this case.

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FIGURE 3.33 Entameba species, colon: Intestinal amoebae such as Entamoeba sp. (arrows) are common in the large intestine of NHPs, but are generally nonpathogenic. They may increase in number under favorable conditions in similar manner to or accompanied by increased numbers of Balantidium coli.

persistent moisture of the perineum and fecal contamination due to nonhealing dermatitis led to an amebic skin infection.10 Entamoeba histolytica infection has been reported in Old World monkeys as nervous system abscesses containing pale eosinophilic organisms with lightly basophilic nuclei that resembled macrophages.259 Periodic-acidSchiff, Masson Trichrome, or Giemsa stains help to visualize the ameba.

4.2.1.2 Oesophagostomum spp. Oesophagostomum sp. “nodular worms” are common parasitic nematodes in the small and large intestine of macaques from India (O. apiastomum, O. stephanostomum), Africa (O. bifurcatum), and the Far East (O. aculeatum, O. apiastomum, O. stephanostomum). Necropsy examination on 697 wild-caught macaques showed Oesophagostomum, to be one of the most common patent infections.260 L3 larvae migrate deep into the wall of the cecum and cause nodule formation; then emerge as L4 larvae to mature in the lumen of the large intestine. Diarrhea may be coincident with emergence of the L4 larvae but often infections are asymptomatic. Occasionally animals exhibit exhaustion due to chronic debilitation from protein loss and leakage of blood through damaged mucosa. Heavy infections by any of the Oesophagostomum species may cause ulcerative enteritis. Cases of pyogranulomatous arteritis and nephritis have been reported associated with nodular worms.261 The larvae of Oesophagostomum sp. are usually noted encased in fibrous tissue with variable numbers of infiltrating lymphocytes, plasma cells, macrophages, and granulocytes. Multinucleated giant or epithelioid macrophages and eosinophils may predominate the lesion (Fig. 3.34). With time these parasitic foci may degenerate, and form mineralize nodules.

4.2 Metazoa 4.2.1 Nematodes 4.2.1.1 Trichospirura leptostoma T. leptostoma is a spirurid nematode that infects the pancreas of the common marmoset.164 Infected animals shed embryonated eggs in the feces 8e9 weeks after infection. The intermediate hosts are common species of cockroaches in which embryonated eggs develop to infective L3 larvae.165 The patent period lasts 2 years, after which eggs can no longer be detected in the feces of infected animals, despite the continued presence of adult parasites within the pancreas. Infection with T. leptostoma has been associated with wasting syndrome in the common marmoset.166 Clinical manifestations of infection can range from asymptomatic to symptoms of pancreatic insufficiency, secondary to chronic pancreatitis, with weight loss.150 Histologically, pancreatic exocrine atrophy and fibrosis are commonly associated with infection.167 Parasites can be identified within pancreatic ducts in tissue sections. Adults have typical features of spirurid nematodes, including a segmented esophagus, thick shelled eggs containing toothed-larvae within uteri of females, and unequal spicules in the male.

FIGURE 3.34 Oesophagostomum species: “nodular worms” are common parasites in the gastrointestinal tract of NHPs. L3 larvae migrate deep into the wall of the cecum and cause nodule formation then emerge as L4 larvae and mature in the lumen of the large intestine. The larvae of Oesophagostomum sp. are usually noted encased in fibrous tissue with variable numbers of infiltrating lymphocytes, plasma cells, macrophages, and granulocytes. They are characterized by large platymyarianmeromyarian musculature, vacuolated lateral cords, an intestine lined with few multinucleated cells with a dense microvillar layer and brush border, and a paired reproductive tract.

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4.2.1.3 Strongyloides sp. Rhabditoidea are a primitive group of nematodes in which Strongyloides is the only known genus of veterinary importance. Only females are parasitic. S. stercoralis and S. fuelleborni (synonym S. simiae) infect NHPs. Completely parasitic and free-living life cycles occur. Larvated eggs are produced via parthenogenesis by females buried in the mucosa of the small intestine. Eggs hatch and first stage larvae are passed in the feces. After hatching larvae can develop to produce L3 parasitic larvae which penetrate the skin, mucosa of the mouth or esophagus, which can lead to systemic migration (visceral larval migrans). Larvae may enter the bloodstream, where they are carried to the lungs. Here they enter alveoli, migrate to the trachea and to the esophagus or are coughed-up and swallowed to mature in the intestine (homogonic cycle). Alternatively, in favorable environmental conditions, larvae can develop through four stages to produce free-living male and female adults which subsequently produce parasitic larvae (heterogonic cycle). Skin penetration may cause a transient erythematous reaction. Most infections are asymptomatic and cross-sections of larvae or parasitic females are not an infrequent finding in the stomach and intestines of captive bred macaques. 4.2.1.4 Nochtia nochti Nochtia nochti is a trichostrongylid “threadworm” that is found in the stomach of NHPs on occasion, primarily noted in macaques. The worm induces exophytic masses in the gastric wall, usually at the junction of the pylorus and fundus.262 Microscopically, the lesions are proliferative and characterized by hyperplastic mucous cells lining tortuous gastric glands supported by a fibrous stalk with associated chronic inflammation. Both adult and morulated eggs may be present in or near the lesions. The nematode adult has a pseudoceolom, platymyarian-meromyarian musculature and a large intestine with multinucleated cells. The adults have reproductive tracts distinct from females (ovary present) and males (testis present).263 4.2.1.5 Angiostrongylus sp. Angiostrongylus (Parastrongylus) cantonensis is the metastrongyloid rat lungworm for which rats are the definitive hosts and snails the intermediate hosts.264 Natural and/or experimental cerebrospinal angiostrongyliasis has been reported in Old and New World primates.265e267 Non-human primates are typically exposed by eating snails, rats, or food contaminated with rodent feces. In NHPs, Angiostrongylus sp. have aberrant migration through the CNS tissues causing encephalomalacia and eosinophilic meningoencephalitis. Nematode larvae can be observed in the subarachnoid space adjacent to cerebral vessels in association with multifocal hemorrhages and necrosis.265 Parasitic

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migration likely causes damage due to both direct and mechanical trauma and due to release of metabolic products and/or antigens from living and dead parasites.268 4.2.1.6 Lungworms Asymptomatic filariasis is a common finding in imported and wild caught South American monkeys (callitrichids, squirrel monkeys, howler monkeys); however, lungworms and heartworms of the genera Filaroides and Filariopsis and Dirofilaria sp. are generally not noted in captive-bred monkeys raised in the laboratory environment due to a general lack of arthropod vectors.269,270 Nonetheless, case reports of infection in Old World monkeys have been reported. A case of Dirofilaria immitis has been reported in a macaque from Louisiana and a case of Edesonfilaria malayensis has been reported in a cynomolgus monkey.271 Clinically, symptomatic animals infected with lungworms have respiratory signs, including persistent cough or hemoptysis.272 Both lungworms and heartworms may induce eosinophilic vasculitis in monkeys. 4.2.1.7 Gongylonema sp. Gongylonema pulchrum is unusual among the spirurid worms in having a wide final host range. The intermediate hosts are coprophagous beetles and cockroaches, and infection in NHPs is usually via ingestion of infected beetles. Adults and larvated eggs may be seen in the mucosa of the tongue or esophagus of macaques and other NHPs, and intestines or pancreas of callitrichids. Less commonly, they are found in the stomach or bronchi. Infections are usually subclinical but callitrichids can develop a debilitating intestinal burden with catarrhal gastritis and fibrous reaction around the parasitic nodules in the wall of the gastrointestinal tract.260 The pancreatic form is much milder. Histologically, there are usually cross-sections of parasites within an epithelium, as the worms burrow through the tissue. There is usually little or no inflammatory response to the presence of these worms in the tissue. Gonglyonema spp. have coelomyarian musculature, a pseudocoelom, large lateral cords, and an intestine lined by columnar epithelium. Males have distinct cuticular lateral alae that are lacking in the females, and the worms are dimorphic, with females having a uterus, often with embryonated eggs, and the males having testes (Fig. 3.35A and B). Diagnosis is by detection of the characteristic oval, thick shelled larvated eggs in feces or tongue scrapes. 4.2.1.8 Trichuris sp. The whipworm, Trichuris trichiura, infects man and NHPs.260 Eggs are ingested and larvae hatch and invade the anterior small intestine where they remain for 2e10 days before moving to the cecum and developing to adults. Infections may be asymptomatic or produce acute or chronic

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NHPs.274 Rarely, there may be infestation of periocular tissues. There is usually little inflammation, and worms and eggs are found in sections of nasal epithelium or the skin of face, hands, and feet. Eggs are operculate and embryonated and are deposited in nasal or cutaneous stratified squamous epithelium. Shedding of the epithelium releases the eggs either into the environment or to be swallowed and excreted in feces. Whether transmission is direct, without intermediate host, via ingestion, or through an intermediate host is still uncertain.275 The organism has zoonotic potential and zoonoses have been reported among humans.275,276 Diagnosis is usually via histopathology or swab evaluation from the nasal cavity. Microscopically, the worms have coelomyarianpolymyarian musculature, prominent stichocytes where the esophagus is contained, and have characteristic paired bacillary bands. Females have a single uterine tube that may contain embryonated eggs (Fig. 3.37). 4.2.1.10 Capillaria spp.

FIGURE 3.35 Gonglyonema pulchrum, esophagus: (A) Gonglyonema pulchrum infection in NHPs is usually via ingestion of infected intermediate hosts, such as beetles and cockroaches. Adults and larvated eggs may be seen in the mucosa of the or esophagus. (B) Cross-sections of parasites within the tongue epithelium usually have little or no inflammatory response. Gonglyonema spp. have coelomyarian musculature, a pseudocoelom, large lateral cords, and an intestine lined by columnar epithelium. Note: Both sexes are represented in this tissue section. Males have distinct cuticular lateral alae (LA) that are lacking in the females, and the worms are dimorphic, with females having a uterus, often with embryonated eggs (U), and the males having testes.

inflammation, especially in the cecum. Trichuris sp. are blood feeders and use a mouth stylet to enter blood vessels or lacerate tissue to create a pool of blood which they then ingest.273 Heavy burdens can be detected as blood in feces and rarely cause anemia. Histologically, Trichuris sp. have polymyariancoelomyarian musculature, a pseudocoelom, a nucleated hypodermis, and characteristic hypodermal bacillary bands (Fig. 3.36A and B).273 4.2.1.9 Anatrichosoma spp. Anatrichosoma spp. are parasitic nematodes in the family Trichiuridae. The worms are occasionally noted in the upper respiratory or alimentary tract tissues of humans and

Capillaria spp. are well known parasites of NHPs. C. hepatica (also Calodium hepaticum) is a parasite of rodents and carnivores, which serve as primary hosts. NHPs primarily become infected via fecal-oral route (rodent feces), or by consumption of infected rodents. C. hepatica has a direct life cycle and no intermediate host. Embryonated eggs in the carcass of the infected are ingested and mature to larval worms in the intestines, penetrate the intestinal wall to gain vascular access, and migrate to the liver. Adult worms form in the liver of monkeys where worm mating occurs, and eggs are laid. There is no natural access for the eggs to exit the body; therefore, the eggs remain in the liver, inciting granulomatous inflammation and fibrosis in NHPs (Fig. 3.38A). In the wild, the eggs within the liver of the infected animals would be consumed by a scavenger or predator and passed in predator feces into the environment, where they would embryonate and infect the next host. Microscopically, it is rare to see a C. hepatica adult worm, as animals are routinely administered antihelmintics and short life-span of the helminth, but occasionally the remains of an adult may be encountered as a mineralized focus encased in fibrous tissue. When adults are noted, they have characteristic dark bacillary bands, and stochocytes around the esophagus. Males have coiled testes. The eggs of C. hepatica are not uncommonly encountered in liver sections. The eggs are barrel-shaped and have prominent bipolar plugs and striated shells (Fig. 3.38B). Infections are rarely fatal and most are discovered incidentally at routine necropsy. Intestinal capillariasis is caused by C. philippinensis. The life cycle of C. philippinensis differs from that of C. hepatica, in that the females produce both unembryonated eggs that pass in feces, and embryonated eggs that

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FIGURE 3.36 Trichuris trichiura, large intestine: (A) The whipworm, Trichuris trichiura, infects the large intestine of NHPs. Eggs are ingested and larvae hatch and invade the anterior small intestine where they develop to adults. Infections may be asymptomatic or produce acute or chronic inflammation, especially in the cecum. Trichuris sp. are blood feeders and use a mouth stylet to enter blood vessels or lacerate tissue. (B) Trichuris sp. have polymyariancoelomyarian musculature, a pseudocoelom, a nucleated hypodermis, and characteristic hypodermal bacillary bands (BB).

hatch and develop inside the host. Once ingested, the infection is self-sustaining, with worm reproduction and development in the small intestinal mucosa. Intestinal capillariasis has been a serious human health issue in tropical regions of the world but has only rarely been reported in captive NHPs.277 4.2.1.11 Baylisascaris sp.

FIGURE 3.37 Anatrichosoma species, nasal cavity: Anatrichosoma spp. are parasitic nematodes in the family Trichiuridae. The worms are occasionally noted in the upper respiratory or alimentary tract of NHPs. There is usually little inflammation associated with worms and eggs. Anatrichosoma spp. have coelomyarian-polymyarian musculature, prominent stichocytes where the esophagus is contained, and have characteristic paired bacillary bands. Females have a single uterine tube that may contain embryonated eggs (U).

Baylisascaris sp. are common intestinal parasites of raccoons (B. procyonis) and skunks. Members of the genus are known aberrant pathogens of humans and NHPs, such as B. columnaris. In intermediate hosts, such as NHPs, Baylisascaris sp. larvae migrate through nervous system tissues and other organs. Neural larval migration (cerebrospinal nematodiasis) has been reported in rhesus macaques and golden lion tamarins, cynomolgus macaques, and other captive NHPs.278e280 Affected animals do not usually show clinical signs. Typically, Baylisascaris sp. are identified as an incidental microscopic finding with eosinophilic granulomatous inflammation containing cross-sections of larvae (Fig. 3.39). Identifying characteristics of the larvae include prominent lateral cuticular alae, conical lateral excretory columns, and a centralized intestine.279

4.2.2 Acanthocephala The thorny-headed worms belong to the phylum Acanthocephala and are pathogenic parasites of NHPs. They have primarily been reported in squirrel monkeys and

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FIGURE 3.39 Baylisascaris species, cerebellum: Baylisascaris sp. are common intestinal parasites of raccoons (B. procyonis) and skunks and are aberrant pathogens of NHPs. Baylisascaris sp. larvae migrate through nervous system tissues and other organs of NHPs. Typically, Baylisascaris sp. are identified as an incidental microscopic finding with eosinophilic granulomatous inflammation containing cross-sections of larvae. Identifying characteristics of the larvae include prominent lateral cuticular alae, conical lateral excretory columns, and a centralized intestine with multinucleated cells. Image courtesy of Julie Schwartz.

region that is used to bore into the host’s intestinal mucosa where they absorb nutrients through their body wall, as there is no oral appendage (Fig. 3.40A and B).282 The worms are sexually dimorphic with the gonads floating within the pseudocoelom. In NHPs, the parasites are often noted grossly as firm, tan to white nodules in tissues, or more commonly, in the mesentery or serosa and adventitia of organs.

4.2.3 Cestodes 4.2.3.1 Taenia spp. FIGURE 3.38 Capillaria species, liver: (A) Capillaria spp. are known parasites of NHPs. NHPs primarily become infected via fecal-oral route (rodent feces), or by consumption of rodents. Capillaria hepatica has a direct life cycle and no intermediate host. Embryonated eggs in the environment are ingested by the host and mature to larval worms in the intestines, penetrate the intestinal wall to gain vascular access, and migrate to the liver. Adult worms form in the liver of monkeys where worm mating occurs, and eggs are laid. In monkeys, there is no natural access for the eggs to exit the body; therefore, the eggs remain in the liver, inciting granulomatous inflammation and fibrosis. (B) The eggs of C. hepatica are barrel-shaped with prominent bipolar plugs and striated shells.

marmosets, but are identified in macaques as well.281 There are a number of mammals that may serve as definitive hosts, and aberrant hosts include NHPs and humans. Infection is usually acquired via ingestion of the intermediate hostsdearthworms, cockroaches, crustaceans, and other invertebratesdin which the eggs hatch and develop into the infective cystacanth stage. Acanthocephalans have a pseudocoelom and the body is divided into the anterior region and the trunk or soma. The adult worms have a hook-covered proboscis on the anterior

The tapeworms, Taenia solium and Taenia saginata, are rare intestinal parasites causing taeniasis in NHPs. Taeniasis is without clinical manifestation in healthy animals. More commonly noted are the encysted forms of Taenia spp. (Cysticercus cellulosae) found in various tissues other than the intestine and referred to as cysticercosis (Fig. 3.41AeC). These encysted forms are occasionally noted in the mesentery or the serosa of the intestines. One case was diagnosed in the liver of a macaque at the editor’s facility. Spontaneous neurocysticercosis has been diagnosed in a rhesus monkey.283 A similar case presented in a cynomolgus monkey at the editor’s facility (Fig. 3.42A and B). CNS involvement of these parasitic cysts can produce neurological disorders similar to that noted in humans, based on results from experimental infections in rhesus monkeys; however, the spontaneous case presented in the cynomolgus monkey was asymptomatic, and those from the study required high parasitic loads to induce clinical neurologic signs.283,284 As long as the cysticercus larva remains viable, there is relative host immune tolerance. It is

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FIGURE 3.40 Acanthocephala species, mesentery: (A and B) The thorny-headed worms belonging to the phylum Acanthocephala are aberrant pathogenic parasites of NHPs. Infection is usually acquired via ingestion of the intermediate hostseearthworms, cockroaches, crustaceans, and other invertebrates-in which the eggs hatch and develop into the infective cystacanth stage. The worms have a characteristic thin cuticle (C), subtended by a syncytial epidermis (E) and thick hypodermis (H) that has lacunar spaces, longitudinal muscle layer (LM) and circular muscle layer (CM), a proboscis armed with spines (P) is retracted in Fig. 3.40B. Unique to Acanthocephalans, there are lemnisci (Li) that are continuous tubelike structures surrounded by compressor muscles. The parasite is typically encased in a pseudocyst (Pc) and there may be granulomatous inflammation (Gi) including multinucleate giant cells.

only when the parasite dies that massive antigen exposure occurs, with intensification of the immune response and inflammatory reaction and the appearance or worsening of symptoms.174 4.2.3.2 Echinococcus sp. Echinococcus granulosus is the causative agent of hydatidosis in NHPs. Adult cestodes are found in the small intestine of carnivores, which shed embryonated eggs in their feces into the environment. The intermediate hosts are herbivores that ingest the infective eggs, which mature to oncospheres and pass through the intestinal wall. These encysted cestode larvae may be found in a wide variety of ungulates and non-human primates, usually in the liver or lungs, but occasionally in other organs (Fig. 3.43A and B).

FIGURE 3.41 Tania species, mesentery: The tapeworms, Taenia solium and Taenia saginata, are rare intestinal parasites in NHPs. Most commonly noted are the encysted forms found in various tissues other than the intestine and referred to as cysticercosis. (A and B) Characteristic for cysticercosis, the larva is within a fluid filled thin-walled cyst (C) and has a scolex (Sx) with birefringent hooklets and suckers and a convoluted spiral canal (Sc). (C) Common to cestodes, cystocerca larvae have calcareous corpuscles.

The unilocular hydatid cyst develops over several months and is composed of a thick outer concentrically laminated membrane, and within, a granular germinal membrane. Brood capsules containing protoscolices (Fig. 3.43C) develop from this membrane around 5 months after

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FIGURE 3.42 Neurocysticercosis, brain: (A) Gross examination of the brain of a cynomolgus monkey in which a well-circumscribed cavitary region contains a centralized, tan nodule. (B) Histologic section of the nodule in Fig. 3.42A:dthere is a thin-walled cyst containing the larval cysticerci. Features of the parasite include a scolex with sucker and hooks (Sx) and a spiral canal (Sc) in section.

infection and may become detached and float free in the cyst fluiddso called “hydatid sand.” Not all hydatid cysts develop brood capsules and sterility of the cyst is dependent on the age of the host at infection. Rupture of a cyst may lead to anaphylactic shock. Released brood capsules and protoscolices may develop into numerous daughter cysts. The pathogenesis of hydatidosis depends on the severity of the infection and the organs affected. Cysts in the lung and liver are well tolerated. Echinococcus multilocularis mainly infects rodent but primates are susceptible. Adults occur in the intestine and hydatids mainly occur in the liver.

4.3 Arthropods

FIGURE 3.43 Echinococcus species: (A and B) Echinococcus granulosus is the most common causative agent of hydatidosis in NHPs. Grossly, the hydatid cysts appear as thick wall, fluid-filled structures containing sandlike material, which are the free-floating protocolices. (C) Transmission electron microscopy of a hydatid protoscolex: the head of the developing tapeworm-visible is the rostellum (R) encircled by the hooks (H).

At necropsy, lungs show multifocal gray cystic lesions up to 2 mm diameterd“mite houses.” On histopathology there is yellowish brown iron positive intracellular pigment distributed perivascularly or peribronchiolar within the lungs (Fig. 3.44A). The parasite is often visible in sections (Fig. 3.44B). Foci of dark pigment with no identifiable acariasis are seen in some monkeys. The lesions of bronchiolitis, mixed cell infiltrate with bronchiectasis or eosinophilic granulomatous inflammation occur throughout the lungs. Bronchial lymph nodes are deeply pigmented due to deposition of mite pigment.

4.3.1 Pneumonyssus

4.3.2 Skin mites

Pneumonyssus simicola is a parasitic mesostigmated mite of the family Halarachnidae. It inhabits the respiratory tract of macaques, but is rarely seen now in captive bred animals kept without an environmental access (outdoor housing) and due to the routine treatment with ivermectin for prophylactic prevention.

Various genus of skin mites have been identified in NHPs, including Demodex spp., Audycoptes spp., Prosarcoptes spp. and Fonsecalges sp.262,285 The reaction to these parasites is highly variable, from no reaction to inflammation and epidermal acanthosis or hyperkeratosis (Fig. 3.45).262,286 With rupture of follicles there may be furunculosis.

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5. Opportunistic fungal infections Primary fungal infections in healthy NHPs are rarely observed, however, animals that are immunosuppressed, such as occur with immunomodulatory test articles or immunosuppressive viral infections, may develop opportunistic fungal infections. Opportunistic pathogenic fungi commonly encountered in NHPs are described briefly below.

5.1 Pneumocystis carinii

FIGURE 3.44 Pneumonyssus simicola, lung: (A) Pneumonyssus simicola is a parasitic mesostigmated mite of the family Halarachnidae, recognized histologically by the presence of yellowish-brown iron positive intracellular pigment distributed perivascularly or peribronchiolar within the lungs. (B) Pneumonyssus simicola mites are occasionally noted in the affected lung tissue.

P. carinii, an extracellular obligate lung pathogen of NHPs that has characteristics of a fungal organism, sharing RNA homology with Saccharomyces sp.287 Infection of neonatal macaques is generally asymptomatic, with infected animals clearing the organism.288 Persistently infected animals may become carriers; however, fulminant disease is usually restricted to immunodeficient macaques.289 Immunosuppressed macaques with protracted disease have respiratory symptoms, including dyspnea and tachypnea. Microscopically, in otherwise healthy individuals, P. carinii appears as pale pink foamy material filling alveolar spaces, with minimal inflammation. More severe cases may have Type 2 pneumocyte hyperplasia (Fig. 3.46A and B). For immunosuppressed animals, pneumocystis can induce a nodular or interstitial pneumonia with vascular involvement and dissemination to the regional lymph nodes.191,290 Gomori’s methenamine silver (GMS) stain and immunohistochemistry can be used to identify P. carinii.

5.2 Candida albicans Candida albicans is a saprophytic fungus that is a common member of the normal skin and mucosal flora. The fungus is an opportunistic pathogen of immunosuppressed NHPs and humans, and the causal agent of “thrush,” a plaque-like lesion of the oral cavity. The organism is generally a pathogen of mucosa, but a case of invasive Candida infection in a pre-term neonatal macaque has been reported, a feature of the pathogenic state in common with death in pre-term human infants.291 Histologically, the organisms invade the epithelium and both yeast forms and fungal hyphae are usually present (Fig. 3.47). Similar to other fungi, this organism is positive when Grocott-Gomori’s (or Gömöri) methenamine silver or periodic acid Schiff stains are applied to tissue sections.

5.3 Cryptococcus neoformans FIGURE 3.45 Follicular mites, genus unknown, skin: Various genus of skin mites have been identified in NHPs, including Demodex spp., Audycoptes spp., Prosarcoptes spp. and Fonsecalges sp. The reaction to these parasites is highly variable.

Cryptococcus neoformans is a saprophytic fungus that may cause disease in New and Old World monkeys. Pulmonary cryptococcosis is the most common presentation, but disseminated disease involving the nervous system and

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FIGURE 3.47 Candida albicans, tongue: Candida albicans is a saprophytic fungus that is a common member of the normal skin and mucosal flora. The fungus is an opportunistic pathogen of immunosuppressed NHPs. The organisms invade the epithelium and both yeast forms and fungal hyphae are usually present (arrows). FIGURE 3.46 Pneumocystis carinii, lung: (A) P. carinii is an extracellular obligate lung pathogen of NHPs that has characteristics of a fungal organism, sharing RNA homology with Saccharomyces sp. Fulminant disease is usually restricted to immunodeficient animals. Microscopically, P. carinii appears as pale pink foamy material filling alveolar spaces. (B) In more severe cases, there may be interstitial pneumonia with Type II pneumocyte hyperplasia associated with P. carinii infection.

other tissues has been reported.292,293 Grossly, lesions may have a gelatinous consistency, due to the abundant fungal organisms. Microscopically, lesions consist of minimal inflammation surrounding 2e20 mm, round, narrow-based budding yeast that are surrounded by a thick polysaccharide capsule, giving the tissues a characteristic “soap bubble” appearance (Fig. 3.48).

5.4 Coccidioides sp. Coccidioidomycosis, also known as valley fever, California fever, or San Joaquin Valley fever, is caused by a dimorphic fungus found in the soil of specific regions. Two nearly identical species are recognized, Coccidioides immitis and Coccidioides posadasii.294 These fungi retain their parasitic spherule form in tissues, multiplying by the production of uninucleate endospores that mature in tissue to spherules. The saprophytic hyphal (mold/mycelial) form

FIGURE 3.48 Cryptococcus neoformans, brain: Cryptococcus neoformans is an opportunistic saprophytic fungal pathogen of NHPs. Pulmonary disease is most common; however, the fungus may disseminate to the CNS. C. neoformans has a characteristic “soap bubble” appearance in tissue sections due to the thick polysaccharide capsule surrounding the thin-walled yeast body (arrows).

occurs in soil, where production of the infectious arthroconidia occurs. The form the fungus adopts is dependent upon the temperature and salinity in the environment, hence the spherules in tissue; however, mycelia have been noted

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FIGURE 3.49 Coccidioidomycosis: Coccidioidomycosis, also known as valley fever, California fever, or San Joaquin Valley fever, is caused by a dimorphic fungus, Coccidioides spp., found in the soil of specific regions for which the diseases are named. These fungi retain their parasitic spherule form (micrometer bar) in tissues.

in tissue for which the animal had been dead for some time and natural body temperature was lost. Initial infection is by inhalation of environmental arthroconidia. Clinical signs included coughing, tachypnea, labored breathing, urinary incontinence, subcutaneous edema, diarrhea, dehydration, hind-limb paresis to paralysis and weakness or reluctance to move. Gross lesions are most commonly seen in the thoracic organs and less often in the vertebral column and abdominal cavity. Histologic findings have been similar in all organs for all species and are characterized by multifocal areas of granulomatous inflammation composed of numerous multinucleated giant cells admixed with lymphocytes, plasma cells, histiocytes, and viable and degenerate neutrophils and eosinophils. Fungal spherules are 25e30 microns in diameter with a 2to 3-micron-thick wall (Fig. 3.49). Bone lesions affected the vertebrae, adjacent soft tissue, and spinal cord.

5.5 Dermatophytosis Macaques are susceptible to infection by Dermatophytes (Microsporum spp., Trichophyton spp.) and there are reports of similar infections in chimpanzees, gibbons and other NHPs.295 Animals present with multifocal, roughly spherical, erythematous and crusting lesions of the skin (Fig. 3.50). There is usually alopecia of the lesions in regions of haired skin. Pruritis is a prominent feature, and animals may have more severe skin lesions due to scratching and self-trauma, which increases the risk for secondary infections. Microscopically, there are usually abundant arthrospores and fungal hyphae located in hair follicles and these elements are commonly noted in skin

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FIGURE 3.50 Dermatophytosis, skin: NHPs are susceptible to infection by dermatophytes such as Microsporum spp., or Trichophyton spp. The animals usually present with multifocal, roughly spherical, erythematous, and crusting lesions of the skin.

scrapings. Diagnosis may be made from skin scrapings of the lesions implanted in selective media for dermatophytes.

5.6 Rare opportunistic fungal infections Opportunistic fungal infections by other, rare genera are occasionally reported in NHPs, usually as single occurrences and primarily in animals that are immunocompromised. Blastomycosis has been reported as the cause of fatal multi-systemic granulomatous disease in a rhesus macaque.296 A case of Talaromyces sp. was reported in a cynomolgus monkey administered an immunomodulatory agent, with resultant nodular granulomatous disease of the spleen (Fig. 3.51A) and other organs.297 Microscopically this fungus exhibits characteristic reproduction by binary fission in sections of tissue stained by Gomori Methenamine Silver (GMS) method (Fig. 3.51B). Pulmonary diaspiromycosis has occurred in a macaque at the editor’s facility, and is caused by the soilborne dimorphic fungi Emmonsia crescens and Emmonsia parva.298 Similar to other pathogenic saprophytic fungi, they maintain a hyphae form in soil, and infection is via inhalation of the infectious stage. The fungus converts to its diaspore morphology at body temperatures near 37 C. Diaspores are 35e40 mm thick walled, encapsulated structures (Fig. 3.51C).299 The spores from Emmonsia spp. must be differentiated from those of Coccidioides (Emmonsia sp. case materials supplied courtesy of Keven McDorman). Other mycotic infections that have been reported in NHPs in the research setting include Aspergillus spp. and Histoplasm sp.

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5.7 Nonpathogenic fungal organisms-gastric megabacteria Large rod- or filamentous-shaped organisms are quite common in the stomach of macaques and have been identified as “megabacterium”, although more formally, the organism is reported as an anamorphic ascomycetous yeast of the genus Macrorhabdus.300,301 The fungi form growth mats that, in vivo, are attached to the mucosal surface but dislodge easily and are most commonly noted free within the gastric lumen histologically (Fig. 3.52A and B). No pathology has been attributed to these organisms in NHPs, although overgrowth has been reported under study conditions following rapamycin treatment.300

FIGURE 3.51 Rare opportunistic fungal infections in non-human primates: (A) A case of Talaromyces sp. infection in a cynomolgus monkey administered an immunomodulatory agent was reported by Iverson et al. to produce nodular granulomatous disease of the spleen. (B) GrocottGomori’s methenamine silver stain (GMS) highlights the characteristic binary fission by which the fungus, Talaromyces sp., replicates in tissue (arrow). (C) Pulmonary adiaspiromycosis is caused by the soilborne dimorphic fungi Emmonsia crescens and Emmonsia parva. The fungus converts to its diaspore morphology at body temperatures near 37 C, evidenced as 35e40 mm thick-walled, encapsulated structures in tissue sections. Fig. 3.51C courtesy of Kevin McDorman.

FIGURE 3.52 Macrorhabdus sp. (megabacteria), stomach: (A) Large rod- or filamentous-shaped organisms commonly found in the gastric lumen of macaques have been previously called “megabacterium”; however, the organism is an anamorphic ascomycetous yeast of the genus Macrorhabdus. (B) Macrorhabdus sp. in the macaque stomach form a dense mat attached loosely to the mucosal surface, with the rods generally perpendicular to the mucosal surface, and that are easily dislodged during tissue collection, resulting in the more commonly noted luminal aggregates. There is currently no inflammation or tissue alterations associated with this yeast.

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6. Microsporidians 6.1 Enterocytozoon bieneusi The microsporidian Enterocytozoon bieneusi is a common asymptomatic enteric infection among colony NHPs and a potential zoonotic disease for humans.302,303 The organism is environmentally stable and is persistent in soil or water. Healthy individuals rarely have illness from E. bieneusi, but immunocompromised individuals may develop sclerosing cholangiohepatitis or proliferative serositis.303e305 Histologically, there is a sclerosing of the gallbladder and bile ducts and spores may be visible in the distorted and sloughed luminal epithelial cells of bile ducts or gallbladder.304

6.2 Encephalitozoon cuniculi This Gram-positive, acid-fast positive, microsporidian parasite can be observed in tissue as rod-shaped, 1.5e3 mm long spores and/or 60e120 mm, thin-walled pseudocysts. It has been reported to cause mortality in New World monkey neonates and is zoonotic.306e309 Congenital transmission has been reported in squirrel monkeys and captive cotton tamarins, and it has been associated with stillbirths and abortions.308e310 Spreading hematogenously, Encephalitozoon cuniculi can cause vasculitis, perivasculitis, and granulomatous inflammation in the nervous system and less commonly in the kidneys, heart, lung and liver. Encephalitozoon cuniculi may cause nonsuppurative meningitis and changes in neural endothelial cells, ependymal cells, and macrophages.308,310,311

7. Other pathogens of non-human primatesdPentastomida Armillifer is a genus of obligate parasites belonging to the Pentastomida, order Porocephalidae. Pentastomes are now regarded as a group of modified parasitic crustaceans. They are transmitted to primates by handling or eating infected snakes, which are the definitive hosts, or oral uptake of environmental ova. Monkeys act as the intermediate host for Armillifer agkistrodontis and case reports of massive parasite loads have been reported.312 Adults are found in the lung, trachea, and nasal passages of snakes. After ingestion of the infective parasite eggs, larvae migrate to various organs and develop into nymphs that are commonly noted in the mesentery or organ serosa of NHPs. The nymphs are recognized by their characteristic C-shape (Fig. 3.53A). Visceral pentastomiasis is usually asymptomatic. Microscopically, pentastomid nymphs have characteristic features, including a thick cuticle with eosinophilic, refractile pores; skeletal muscle, and sickle-shaped cephalic hooks (Fig. 3.53BeC).

FIGURE 3.53 Pentastomiasis; Armillifer species: Armillifer is a genus of obligate parasites belonging to the Pentastomida, order Porocephalidae, and are now regarded as a group of modified parasitic crustaceans. (A) After ingestion of the infective parasite eggs, larvae migrate to various organs and develop into nymphs that are commonly noted in the mesentery of NHPs, recognized by their characteristic C-shape (arrow). (B and C) Microscopically, pentastomid nymphs have characteristic features, including a thick cuticle (C) with eosinophilic, refractile sclerotized pores and annular bands (A); skeletal muscle (SM), and sickle-shaped cephalic hooks (CH).

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8. Conclusion Infectious diseases still and will continue to be a risk to NHP colonies and safety assessment toxicology programs. An infectious agent can directly impact health leading to illness and potentially death of animals in captive colonies or on drug safety studies. Nonpathogenic agents on the other hand may affect the ability to complete all experimental endpoints on a toxicology study or lead to zoonotic disease following human contact. Microbes may have limited disease potential in an animal of normal immunologic status but could be pathogenic in an immunocompromised animal. Moreover, determining the etiology of an infectious disease during a drug safety study as test item or nontest item-related can be a challenge for the toxicologist and pathologist. Therefore, researchers should be knowledgeable and aware of common disease signs caused by these agents to ensure colony health and appropriate interpretation of clinical and microscopic findings. It is not possible to eliminate all of the infectious agents from NHP colonies, but with implementation of enhanced biosafety measures to produce pathogen-free animals, the impact of these infectious agents on NHP colonies, as well as zoonotic risk to the personnel handling animals, can be minimized. It is now recognized that all NHP facilities should have standardized and comprehensive microbial quality control programs in place and disease surveillance monitoring procedures established.

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267. Weinstein PP, Rosen L, Laqueur GL, Sawyer TK. Angiostrongylus cantonensis infection in rats and rhesus monkeys, and observations on the survival of the parasite in vitro. Am J Trop Med Hyg 1963;12:358e77. https://doi.org/10.4269/ajtmh.1963.12.358 (From NLM). 268. Lin RJ, He JW, Chung LY, Lee JD, Wang JJ, Yen CM. Angiostrongylus cantonensis (Nematode: Metastrongiloidea): in vitro cultivation of infective third-stage larvae to fourth-stage larvae. PLoS One 2013;8(8):e72084. https://doi.org/10.1371/journal.pone.0072084 (From NLM). 269. Lowenstine LJ, Osborn KG. Chapter 9: Respiratory system diseases of nonhuman primates. In: Abee CR, Mansfield K, Tardif S, Morris T, editors. Nonhuman primates in biomedical research. 2nd ed. Academic Press; 2012. p. 413e81. 270. Kornegay RW, Giddens Jr WE, Morton WR, Knitter GH. Verminous vasculitis, pneumonia and pulmonary infarction in a cynomolgus monkey after treatment with ivermectin. Lab Anim Sci 1986;36(1):45e7. 271. Baskin GB, Eberhard ML. Dirofilaria immitis infection in a rhesus monkey (Macaca mulatta). Lab Anim Sci 1982;32(4):401e2. 272. Wolff PL. Parasites of new world primates. Zoo Wild Anim Med: Curr Ther (Phila) 1993;3:378e89. 273. Rivero J, Cutillas C, Callejón R. Trichuris trichiura (Linnaeus, 1771) from human and non-human primates: morphology, biometry, host specificity, molecular characterization, and phylogeny. Front Vet Sci 2020;7:626120. https://doi.org/10.3389/fvets.2020.626120 (From NLM). 274. Conrad HD, Wong MM. Studies of Anatrichosoma (Nematoda: Trichinellida) with descriptions of Anatrichosoma rhina sp. n. and Anatrichosoma nacepobi sp. n. from the nasal mucosa of Macaca mulatta. J Helminthol 1973;47(3):289e302. https://doi.org/10.1017/ s0022149x00026584 (From NLM). 275. Eberhard ML, Mathison B, Bishop H, Handoo NQ, Hellstein JW. Zoonotic anatrichosomiasis in an Illinois resident. Am J Trop Med Hyg 2010;83(2):342e4. https://doi.org/10.4269/ajtmh.2010.100144 (From NLM). 276. Eberhard ML, Hellstein JW, Lanzel EA. Zoonotic anatrichosomiasis in a mother and daughter. J Clin Microbiol 2014;52(8):3127e9. https://doi.org/10.1128/jcm.01236-14 (From NLM). 277. Brack M, Gass H, Stirnberg E. Intestinal capillariasis in new world monkeys. J Med Primatol 1994;23(1):37e41. https://doi.org/ 10.1111/j.1600-0684.1994.tb00093.x. 278. Gozalo AS, Maximova OA, StClaire MC, Montali RJ, Ward JM, Cheng LI, Elkins WR, Kazacos KR. Visceral and neural larva migrans in rhesus macaques. J Am Assoc Lab Anim Sci 2008;47(4):64e7 (From NLM). 279. Shoieb A, Radi ZA. Cerebral Baylisascaris larva migrans in a cynomolgus macaque (Macaca fascicularis). Exp Toxicol Pathol 2014;66(5e6):263e5. https://doi.org/10.1016/j.etp.2014.03.004 (From NLM). 280. Johnson-Delaney CA. Parasites of captive nonhuman primates. Vet Clin North Am Exot Anim Pract 2009;12(3):563e81. https://doi.org/ 10.1016/j.cvex.2009.07.002 (From NLM). 281. Dunn FL. Acanthocephalans and cestodes of South AMERICAN monkeys and marmosets. J Parasitol 1963;49:717e22 (From NLM). 282. Herlyn H, Taraschewski H. Evolutionary anatomy of the muscular apparatus involved in the anchoring of Acanthocephala to the

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intestinal wall of their vertebrate hosts. Parasitol Res 2017;116(4):1207e25. https://doi.org/10.1007/s00436-017-5398-x (From NLM). Johnston JM, Dyer CD, Madison-Antenucci S, Mergen KA, Veeder CL, Brice AK. Neurocysticercosis in a rhesus macaque (Macaca mulatta). Comp Med 2016;66(6):499e502 (From NLM). Chowdhury N, Saleque A, Sood NK, Singla LD. Induced neurocysticercosis in rhesus monkeys (Macaca mulatta) produces clinical signs and lesions similar to natural disease in man. Sci World J 2014;2014:248049. https://doi.org/10.1155/2014/248049 (From NLM). Guerim L, Gazêta GS, Serra-Freire NM, de Sá LM, Catão Dias JL. Cebus apella (Primata: Cebidae) as a new host for Fonsecalges johnjadini (Acari: Psoroptidae, Cebalginae) with a description of anatomopathological aspects. Mem Inst Oswaldo Cruz 2001;96(4):479e81. https://doi.org/10.1590/s0074-02762001000400007 (From NLM). Starost MF, Karjala Z, Brinster LR, Miller G, Eckhaus M, Bryant M, Hoffman V. Demodex spp. in the hair follicles of rhesus macaques (Macaca mulatta). J Med Primatol 2005;34(4):215e8. https:// doi.org/10.1111/j.1600-0684.2005.00112.x (From NLM). Stringer SL, Stringer JR, Blase MA, Walzer PD, Cushion MT. Pneumocystis carinii: sequence from ribosomal RNA implies a close relationship with fungi. Exp Parasitol 1989;68(4):450e61. https:// doi.org/10.1016/0014-4894(89)90130-6 (From NLM). Matsumoto Y, Yamada M, Tegoshi T, Yoshida Y, Gotoh S, Suzuki J, Matsubayashi K. Pneumocystis infection in macaque monkeys: Macaca fuscata fuscata and Macaca fascicularis. Parasitol Res 1987;73(4):324e7. https://doi.org/10.1007/BF00531086. Vogel P, Miller CJ, Lowenstine LL, Lackner AA. Evidence of horizontal transmission of Pneumocystis carinii pneumonia in simian immunodeficiency virus-infected rhesus macaques. J Infect Dis 1993;168(4):836e43. https://doi.org/10.1093/infdis/168.4.836 (From NLM). Board KF, Patil S, Lebedeva I, Capuano 3rd S, Trichel AM, Murphey-Corb M, Rajakumar PA, Flynn JL, Haidaris CG, Norris KA. Experimental Pneumocystis carinii pneumonia in simian immunodeficiency virus-infected rhesus macaques. J Infect Dis 2003;187(4):576e88. https://doi.org/10.1086/373997 (From NLM). Pecoraro HL, Berg MR, Dozier BL, Martin LD, McEvoy CT, Davies MH, Ducore R. Candida albicans-associated sepsis in a preterm neonatal rhesus macaque (Macaca mulatta). J Med Primatol 2019;48(3):186e8. https://doi.org/10.1111/jmp.12401 (From NLM). Pal M, Dube GD, Mehrotra BS. Pulmonary cryptococcosis in a rhesus monkey (Macaca mulatta). Mycoses 1984;27(6):309e12. https://doi.org/10.1111/j.1439-0507.1984.tb02035.x. . [Accessed 14 August 2022]. Normile TG, Bryan AM, Del Poeta M. Animal models of cryptococcus neoformans in identifying immune parameters associated with primary infection and reactivation of latent infection. Front Immunol 2020;11:581750. https://doi.org/10.3389/ fimmu.2020.581750 (From NLM). Sharpton TJ, Stajich JE, Rounsley SD, Gardner MJ, Wortman JR, Jordar VS, Maiti R, Kodira CD, Neafsey DE, Zeng Q. Comparative genomic analyses of the human fungal pathogens Coccidioides and their relatives. Genome Res 2009;19(10):1722e31. M.G M, Ovchinnikov R, A.N P. Therapy of dermatophytosis in Japanese macaques (Macaca fuscata). Med Mycol 2004;11:65.

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296. Wilkinson LM, Wallace JM, Cline JM. Disseminated blastomycosis in a rhesus monkey (Macaca mulatta). Vet Pathol 1999;36(5):460e2. https://doi.org/10.1354/vp.36-5-460 (From NLM). 297. Iverson WO, Karanth S, Wilcox A, Pham CD, Lockhart SR, Nicholson SM. Talaromycosis (Penicilliosis) in a cynomolgus macaque. Vet Pathol 2018;55(4):591e4. https://doi.org/10.1177/ 0300985818758468. 298. England DM, Hochholzer L. Adiaspiromycosis: an unusual fungal infection of the lung. Report of 11 cases. Am J Surg Pathol 1993;17(9):876e86 (From NLM). 299. Takeshige A, Nakano M, Kondoh D, Tanaka Y, Sekiya A, Yaguchi T, Furuoka H, Toyotome T. Adiaspore development and morphological characteristics in a mouse adiaspiromycosis model. Vet Res 2020;51(1):119. https://doi.org/10.1186/s13567020-00844-3. 300. Stone D, Kenkel EJ, Loprieno MA, Tanaka M, De Silva Feelixge HS, Kumar AJ, Stensland L, Obenza WM, Wangari S, Ahrens CY, et al. Gene transfer in adeno-associated virus seropositive rhesus macaques following rapamycin treatment and subcutaneous delivery of AAV6, but not retargeted AAV6 vectors. Hum Gene Ther 2021;32(1e2):96e112. https://doi.org/10.1089/ hum.2020.113 (From NLM). 301. Schulze C, Bilk S, Schüle A, Kutzer P, Ochs A. Fungal colonization of the stomach with Macrorhabdus-like ascomycetous yeasts in two Folivorous Colobine primates from a German zoological Garden. J Comp Pathol 2016;154(1):117. https://doi.org/10.1016/ j.jcpa.2015.10.150. 302. Karim MR, Wang R, Dong H, Zhang L, Li J, Zhang S, Rume FI, Qi M, Jian F, Sun M, et al. Genetic polymorphism and zoonotic potential of Enterocytozoon bieneusi from nonhuman primates in China. Appl Environ Microbiol 2014;80(6):1893e8. https://doi.org/ 10.1128/aem.03845-13 (From NLM). 303. Mansfield KG, Carville A, Hebert D, Chalifoux L, Shvetz D, Lin KC, Tzipori S, Lackner AA. Localization of persistent Enterocytozoon bieneusi infection in normal rhesus macaques (Macaca mulatta) to the hepatobiliary tree. J Clin Microbiol 1998;36(8):2336e8. https:// doi.org/10.1128/jcm.36.8.2336-2338.1998 (From NLM). 304. Chalifoux LV, MacKey J, Carville A, Shvetz D, Lin KC, Lackner A, Mansfield KG. Ultrastructural morphology of Enterocytozoon

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bieneusi in biliary epithelium of rhesus macaques (Macaca mulatta). Veterinary pathology 1998;35(4):292e6. Chalifoux LV, Carville A, Pauley D, Thompson B, Lackner AA, Mansfield KG. Enterocytozoon bieneusi as a cause of proliferative serositis in simian immunodeficiency virus-infected immunodeficient macaques (Macaca mulatta). Arch Pathol Lab Med 2000;124(10):1480e4. https://doi.org/10.5858/2000-124-1480ebaaco (From NLM). Davis MR, Kinsel M, Wasson K, Boonstra J, Warneke M, Langan JN. Fatal disseminated encephalitozoonosis in a captive, adult Goeldi’s monkey (Callimico goeldii) and subsequent serosurvey of the exposed conspecifics. J Zoo Wildl Med 2008;39(2):221e7. https://doi.org/ 10.1638/2007-0114r.1 (From NLM). Guscetti F, Mathis A, Hatt JM, Deplazes P. Overt fatal and chronic subclinical Encephalitozoon cuniculi microsporidiosis in a colony of captive emperor tamarins (Saguinus imperator). J Med Primatol 2003;32(2):111e9. https://doi.org/10.1034/j.16000684.2003.00016.x (From NLM). Zeman DH, Baskin GB. Encephalitozoonosis in squirrel monkeys (Saimiri sciureus). Vet Pathol 1985;22(1):24e31. https://doi.org/ 10.1177/030098588502200104 (From NLM). Juan-Sallés C, Garner MM, Didier ES, Serrato S, Acevedo LD, Ramos-Vara JA, Nordhausen RW, Bowers LC, Parás A. Disseminated encephalitozoonosis in captive, juvenile, cotton-top (Saguinus oedipus) and neonatal emperor (Saguinus imperator) tamarins in North America. Vet Pathol 2006;43(4):438e46. https://doi.org/ 10.1354/vp.43-4-438 (From NLM). Anver MR, King NW, Hunt RD. Congenital encephalitozoonosis in a squirrel monkey (Saimiri sciureus). Vet Pathol 1972;9(6):475e80. https://doi.org/10.1177/030098587200900607 (From NLM). Reetz J, Wiedemann M, Aue A, Wittstatt U, Ochs A, Thomschke A, Manke H, Schwebs M, Rinder H. Disseminated lethal Encephalitozoon cuniculi (genotype III) infections in cotton-top tamarins (Oedipomidas oedipus)da case report. Parasitol Int 2004;53(1):29e34. https://doi.org/10.1016/j.parint.2003.10.003 (From NLM). Mätz-Rensing K, Lampe K, Rohde G, Roos C, Kaup FJ. Massive visceral pentastomiasis in a long-tailed macaquedan incidental finding. J Med Primatol 2012;41(3):210e3. https://doi.org/10.1111/ j.1600-0684.2012.00544.x (From NLM).

Chapter 4

Clinical examination of the non-human primate William O. Iverson1 and Nicola M.A. Parry2 1

Iverson Consultancy, LLC, Faber, VA, United States; 2Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA,

United States

1. Introduction The successful conduct of any scientific experiment requires that only healthy animals be included as test subjects. In this discussion, the non-human primates (NHPs) being considered as potential test subjects are thoroughly examined by performing a physical examination and conducting laboratory tests to evaluate critical body systems, parasite burden, and serological status to viruses of interest. In addition, prophylactic anthelmintics and vaccinations are usually administered. These important preliminary procedures are essential to ensure the health of the colony and provide the best chance for a valid and successful study.

2. The clinical exam of the non-human primate 2.1 The physical exam As with other species, clinical examination of the nonhuman primate begins with a thorough, complete, and systematic examination of the entire body.1 With nonhuman primate species, this requires mild sedation to enable thorough evaluation of the eyes and mouth. However, before sedation, the veterinarian should observe the animal in its home enclosure, including its interactions with other individuals if group housed. This can help the veterinarian recognize certain health problems. For example, an animal’s tendency to avoid using an arm, hand, leg, or foot can indicate injury, and warrants further investigation under sedation. In most cases, the combination of a low dose of ketamine hydrochloride and help from a trained assistant to provide physical restraint is sufficient to allow complete examination. This also provides an opportunity to obtain prestudy blood samples or to repeat any intradermal tuberculin tests that may be scheduled.

It is important to follow a systematic process for examination of the entire body by using a checklist (Table 4.1).2 This allows for consistency and ensures no abnormalities are overlooked. The authors prefer to begin at the head of the animal and proceed along the entire body to the tip of the tail. Throughout the examination, the hair and skin over the entire body should be examined and palpated to detect lesions such as alopecia, masses, or scabs. The eyes, ears, and nose should be examined using a penlight to look for any discharge or other abnormalities. The lips should be everted to examine the mucosa, and the gums should also be examined. After opening the mouth, a piece of clean gauze should be used to gently hold the tip of the tongue while maneuvering it to examine all surfaces, paying particular attention to lesions such as blisters, erosions, or ulcers. Maneuvering the tongue in this way also allows the veterinarian to examine the rest of the oral mucosa. Capillary refill time is easily evaluated by applying pressure to the gum above the canine teeth. Once the tissue has blanched, the veterinarian removes pressure and notes the time for return to the normal pink color. If the animal is involved in a study that targets lymphoid tissues, the veterinarian should examine the oropharynx and tonsils using a laryngoscope. The physical exam should progress from head to tail. The regional lymph nodes in the head and neck should be palpated. Next, the axillary lymph nodes, and both arms and hands are examined. The skin of the thorax and abdomen are then examined, followed by auscultation of the thorax to ensure that the heart sounds and lung sounds are normal, and that the heart rate and respiratory rate are appropriate for the species. The skin on the back between the scapulae may be pinched and released to look for evidence of dehydration. The abdomen should be palpated to detect lesions such as enlarged organs or masses.

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TABLE 4.1 Components of the non-human primate physical examination checklist. Animal identifying features: Tattoo number Sex Body weight Species Examination findings: Head, face, neck regions Skin and haircoat Ocular motility Pupillary reflexes Dilate eyes Direct ophthalmoscopy Mouth, tongue, teeth, throat Capillary refill time Nose, nostrils Ears, otoscopic exam Thoracic region Skin and haircoat Auscultate lungs Auscultate heart Upper extremities, hands Abdominal, pelvic, inguinal, perineal regions Skin and haircoat External genitalia Upper extremities, feet Tail Fecal staining (perianal, tail regions) Miscellaneous Disposition, character Temperature Skin hydration Blood pressure Palpate lymph nodes (e.g., cervical, axillary, femoral, popliteal) ECG Femoral pulse rate Palpate spine Fascial muscle tone Miscellaneous reflexes (e.g., palmar, plantar, patellar, superficial abdominal, auditory-palpebral) Check for hernia Urine Feces Adapted from Kessler MJ, et al. Selection and pairing of normal rhesus monkeys (Macaca mulatta) for research: Procedures, techniques, and observations. J Med Primatol. 1979;8:365e371.

Clinical examination of the non-human primate Chapter | 4

The inguinal area includes lymph nodes, external genitalia, and perineum, all of which should be examined to ensure they are within normal limits. The anal area and the overlying ventral surface of the tail should be checked for signs of fecal staining. This is also an opportune time to obtain and record rectal temperature using a digital thermometer. Each rear limb and foot is then examined, including popliteal lymph nodes. Finally, a complete examination of the tail, all the way to the tip, is performed. Body weight is usually measured and recorded either now or at the beginning of the examination. Minor abnormalities discovered during the physical examination may not preclude the use of an animal for a specific study, but accurate and complete documentation of any lesions before enrollment in a protocol is critical. Frequently, a complete physical examination may also be included near the end of a study, to identify any treatmentrelated effects.

2.2 Screening procedures 2.2.1 Clinical pathology Generally, the same tests to be used in the study protocol will be the tests run during screening (see Chapter 21). This is to ensure all animals entering the study are healthy, and that they will meet the specific requirements for the subject study if they are selected for the study. This will include a complete blood count (CBC), and clinical chemistries for liver enzymes, proteins (albumin, globulin, etc.), and renal functional parameters. Also essential for renal assessment is a urinalysis from a good quality urine sample.

2.2.2 Radiology The only radiologic evaluation usually needed during screening would be chest imaging to help eliminate preexisting lung conditions, and to serve as an extra step to detect any Mycobacterium tubercles that may be present. However, addition of the abdominal radiograph privides additonal information that may be of value. For instance, kidney agenesis is rare, but occurs as an incidental finding that is usually clearly identified by radiography.

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stress during a study may precipitate diarrhea due to these organisms.

2.2.4 Serology The study protocol will specify which viruse screening the animals should undergo by serological testing. This could be for as few as 5 or as many as 10 different agents, but it should always include a test for Macacine herpesvirus 1, commonly referred to as B virus. Even if serologically negative, all macaques should be considered potential carriers of this virus, and appropriate action should be taken if bites or scratches to staff occur.

3. Prophylactic therapy Even if all parasite tests are negative, all animals are frequently treated with ivermectin because of its extreme efficacy against both internal and external parasites. The only routine vaccination given to animals housed indoors is for measles virus. However, this may be unnecessary if all staff that come into contact with the animals are tested and have protective titers against the virus.

4. Conclusion Of paramount importance in the conduct of any experiment or toxicology study using NHPs, is a healthy and wellcharacterized test systemdthe animals themselves. This is ensured using both a thorough physical examination and a combination of laboratory tests that are specified in the Study Protocol or Study Plan. In addition, administration of some medications yields an animal that is free of most troublesome parasites. Animals meeting these predefined study requirements can then be considered for inclusion in the experimental groups. Other criteria for inclusion include age and body weight. Overall, these important preliminary steps ensure healthy animals will participate in the study and that the results will reflect only Test Articleerelated effects.

References 2.2.3 Parasitology and bacteriology Parasite examination will include both a fecal flotation for eggs and a blood film examination for circulating protozoa such as malaria. Fecal cultures for Shigella spp. and Campylobacter jejuni are also prudent to perform because

1. Ephraim GP, Barrows SZ. Clinical aspects of primate medicine. Iowa State Univ Vet 1988;50(1):9e10. 2. Kessler MJ, Kupper JL, Grissett JD, et al. Selection and pairing of “normal” rhesus monkeys (Macaca mulatta) for research: procedures, techniques, and observations. J Med Primatol 1979;8:365e71.

Chapter 5

Routes of administration for the non-human primate Warren Harvey Nonclinical Consulting, Drug Development Solutions, ICON plc, Dublin, Ireland

1. Introduction Selection of the appropriate administration route for a non-human primate (NHP) study is an important factor in the safety assessment of a test substance. In general, as the objective of a safety evaluation study is to mimic human exposure, the route of administration of a test substance to the test species such as the NHP should correlate with normal circumstances encountered for human exposure. The most common routes of administration used in NHP research are oral (gavage or capsule), inhalation, and parental administration by intravenous, subcutaneous, or intramuscular routes. It is not the intention of the author to describe each administration route in detail but to provide an introduction into various common routes used in NHP research. For more detail on general routes administration, see “General Principles of Xenobiotic Disposition” (Volume 1, Chapter 1) in Haschek and Rousseaux’s Handbook of Toxicologic Pathology, 4thh Ed.1

2. Selecting the right dose route Substances are administered to rhesus or cynomolgus macaques and common marmosets by a wide variety of dose routes. As for any test species, the selected route of dosing in NHP safety evaluation studies will be determined by the proposed route of exposure in humans. A key factor in determining the route selected is whether the test substance is being administered for a local or systemic effect. Test substances administered locally are generally applied externally or topically on the surface of the area where the therapeutic effect of the test substance is desired, i.e., do not require absorption into the systemic blood circulation to be effective. Systemic effect is achieved by enteral or parenteral routes. Enteral route (e.g., oral,

sublingual, and rectal) refers to the absorption and passage of a test substance through the gastrointestinal tract. Parenteral routes (e.g., subcutaneous, intramuscular, and intravenous) circumvent the alimentary canal and involve the direct entry of the test substance into the blood stream through injection or infusions. Parenteral administration methods typically produce the highest bioavailability of substances because these methods avoid the first-pass effect of hepatic metabolism, which commonly occurs with orally administered substances. Parenteral routes also circumvent some of the unpredictability associated with enteral absorptive processes. The selection of a particular dose route will depend on the objectives and purpose of the study being conducted. Certain special routes may be used to provide better therapeutic outcomes: intra-arterial route in cancer chemotherapy and intrathecal route for central nervous system infections or for spinal anesthesia. These routes are much more hazardous and would obviously require greater skills and care in administering the drug. The administration route used to dose animals on a preclinical safety study should be the same or closely resemble the intended human route of administration.2 The route of administration may also be influenced by the physicochemical properties of the test substance which could adversely affect animals. These include: the dose formulation, solubility, viscosity, pH, biocompatibility, purity, stability, standardization, and microbial contamination. Background data on such properties together with any extraneous effects (e.g., irritancy), should always be investigated before dosing an NHP. The expected effect may be altered by concentration and dose volume, and this should also be evaluated. The ideal vehicle can be administered via the intended clinical route, and provides suitable stability and bioavailability of the test substance. It is critical that biological

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effects produced by vehicles be minimized or at the very least be well understood.3 Unanticipated vehicle-related effects can compromise the integrity of safety studies.4 Administration of a test substance to the NHP requires a level of restraint, sedation, or general anesthesia. The use of such manipulations should be considered when selecting

the administration route to refine procedures so that they are less invasive or aversive to the animals. In addition, each route has advantages and disadvantages (Table 5.1) that should be considered depending on the final effect to be achieved, and ultimately the route selected will markedly affect the pharmacokinetics of the test substance.

TABLE 5.1 Advantages and disadvantages of common routes of administration for non-human primates. Route

Advantages

Disadvantages

Oral (PO)

Relatively safe. Sterility is not required. Has minimal risk of acute drug reaction.

Variable absorption (potentially slow, erratic, and incomplete). First pass effect may be significant. Ingestion of drug could cause gastric irritation and vomiting. Complexes formed with ingesta could interfere with drug absorption. There are potential effects of low gastric pH or by the digestive and liver enzymes on the drug before entry into the circulation. Requires skill in delivery to avoid lung instillation or injury to the stomach or esophagus.

Rectal (PR)

Partially avoids first-pass effect. Avoids destruction by gastric acid and digestive enzymes.

Possible rectal mucosal irritation or injury. Rectum is devoid of villi, so absorption is often slow or incomplete. Not a well-accepted route of administration.

Intravenous (IV)

Absorption is circumvented by direct vascular access. Rapid onset of action. Suitable for large administration volumes. Irritating and nonisotonic solutions may be administered since the intima of the vein is insensitive.

Shorter duration of action. Thrombophlebitis of veins is a risk, as is necrosis of adjoining tissue. May have severe adverse effect especially when organs such as liver, heart, brain are involved in toxicity. Not suitable for oily solutions or poorly hydrophilic substances.

Intramuscular (IM)

Absorption can be bespoke: fast from aqueous solution or slow and sustained from repository formulations. Suitable for oily and irritating excipients. Convenient for animals that are difficult to restrain. Muscles are highly vascularized thus, drugs are absorbed hematogenously or through the lymphatic fluid.

Injection into fascia might lead to erratic absorption. Possibility of improper deposition in nerves, fats, blood vessels, or between muscle bundles in connective sheaths.

Subcutaneous (SC)

Absorption can be bespoke: Fast from aqueous solution or slow and sustained from repository formulations. Suitable for poorly soluble suspensions and for instillation of slow-release implants.

Not suitable for large volumes or irritating substances. Possibility of necrosis or sloughing of the skin from irritating substances.

Inhalation

Instantaneous absorption through the lung and rapid onset of drug effects. Avoids hepatic first-pass effect. Provides localized effect to lungs with minimal systemic side effect.

Regulating target doses may be difficult. Specialized equipment is required for drug delivery.

Transdermal

Provides a sustained effect. Avoids hepatic first-pass effect.

Usually very slow onset of drug effects. May have enhanced absorption and toxicity with inflamed or abraded skin. Substance must be highly lipophilic.

Routes of administration for the non-human primate Chapter | 5

3. Commonly used routes of administration in NHP research 3.1 Enteral administration (oral route) Oral administration of substances directly into the mouth (tablet or capsule) or by orogastric or nasogastric gavage (as a solution or suspension) is a common route used in NHP research. Oral administration is a desirable route of administration in NHPs for testing compounds that are ultimately intended for human oral administration. Tablets or gelatin capsules when delivered directly into the mouth should be placed at the back of the mouth so the animal swallows, to ensure full dose administration. The number and size of capsules or tablets administered should be proportional to the size of the animal being dosed to minimize regurgitation. Gavage (esophageal or gastric), accomplished through the use of flexible cannulas to minimize potential esophageal damage, is often used in research settings instead of mixing substances in water or food, to ensure precise and accurate dosing of animals. Oral administration of substances typically requires restraint. Habituation or positive reinforcement training to restraint may reduce the stress associated with the procedure.5,6 Repeated administration of large volumes of substances by orogastric or nasogastric gavage should be avoided as it can cause stress due to gastric distension leading to vomiting. Therefore, the smallest volume possible is recommended for the oral route of administration. Administration of large volumes by gavage is possible and tolerated by the animal when delivered at a slower rate. Limitations of oral dosage may include a slower onset of action compared with parenteral delivery, and a potentially significant first-pass effect by the liver for those substances metabolized through this route resulting in reduced efficacy. Additional limitations include lack of absorption of substances due to chemical polarity or interference with absorption by ingesta or local irritation, lack of systemic absorption from the digestive tract, degradation of substances by digestive enzymes and acid, and absorption differences based on fasting or nonfasting states.7e9

3.2 Local administration (topical route) The NHP is phylogenetically close to humans but there are some differences in skin anatomy between monkeys and humans; monkeys are covered with a dense coat of pelage as apposed to the thinly dispersed hair of humans; their epidermis has little undersculpture; they have numerous apocrine glands over nearly the entire hairy skin; monkeys have fewer sebaceous glands that directly open to the skin surface.10 NHPs are rarely used in the preclinical safety assessment of a new topical drug candidate. The minipig would be considered the first choice for evaluation of dermal tolerance.11,12 On occasions when macaques are

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used, the animals are jacketed to prevent the animals from interfering with the application site. Macaque skin is not as good a model for human skin, especially in the pharmacokinetic aspects, as is pig skin. Therefore, unless there are distinctive characteristic properties of the test substance that require safety evaluation testing in an NHP species, the NHP would not be considered a suitable nonrodent model for topical studies.

3.3 Parenteral administration 3.3.1 Intravenous route The intravenous route of delivery is the most efficient means of delivering substances to animals and humans because it bypasses the need for solute absorption.13 With this method, substances are administered as a bolus or infusion directly into blood vessels on either an acute or chronic basis. This often depends on the pharmacokinetics of the test substance, as well as the maximum tolerated dose, the time interval over which delivery is required (referred to as dosing intensity), and the need to minimize variations in peak and trough blood levels in the test substance being administered. Short-term bolus administrations are limited to approximately 5 min as manual dosing (e.g., for volumes up to 10 mL/kg) or as chairrestrained infusion (e.g., up to 4 h) using precision electronic infusion pumps equipped with alarms to indicate flow interruptions in habituated monkeys. When continued infusion (24 h) is required, an implanted port catheter system, together with ambulatory infusion would be the method of choice. Infusion techniques for NHP species are also well established for the intrathecal route; however, the use of this route is limited in macaques. Details on the various techniques, equipment, and refinements for using catheters and vascular access ports in animals have been published.14e16 Test substances administered intravenously should be isotonic (the same concentration as the blood), close to physiologic pH (6.8e7.2), sterile, and delivered aseptically. Otherwise, injected formulations containing particulate material or of low pH can precipitate when mixed with blood resulting in vascular occlusion, emboli, and thrombosis of local and distant capillary beds such as those found in the ears, tail, toes, or lungs.17 Aseptic preparation of the skin overlying the injection site and use of sterile technique for needle insertion are critical for success and animal recovery. Procedural-related histopathological findings of the administration site vein (Fig. 5.1A), such as endothelial hyperplasia, intimal thickening and thrombi, and rarely, interstitial pneumonitis in lungs, are generally the most frequent reported changes.18,19 Additional histological changes that occur spontanously at administration sites include localized hemorrhage, edema, fibrosis or fiboplasia (Fig. 5.1B-C), and occasionally localized degenerative changes (necrosis). Small accumulations of hemosiderin may be present. These findings may be due to mechanical

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The position of the catheter within the blood vessel is an important consideration when conducting a continuous infusion study. The local concentration of the test substance within the cannulated vessel can be higher close to the catheter insertion site for a longer period of time when low flow rates are used. In combination with the mild local inflammation that is typically associated with the implanted catheter, this higher concentration may result in phlebitis and vascular thrombosis.19

3.3.2 Subcutaneous route Nonirritating, isotonic substances may be given subcutaneously, using aseptic peri-injection techniques, which represents a rapid, inexpensive, and simple method of parenteral substance administration. Substances administered subcutaneously often are absorbed at a slower rate compared with other parenteral routes, providing a sustained effect20 and have a low oral bioavailability.21 The exact mechanism of absorption is not completely understood but is thought to be due to uptake of macromolecules within the subcutis by small capillaries underling the skin, with minimal lymphatic absorption.22 Substances delivered subcutaneously can be aqueous or oily fluids, depots of oily materials for slow absorption, solid pellets, or injected into suitably sized osmotic minipumps or other implantable pumps, which subsequently are surgically inserted into a subcutaneous pocket.23,24 One frequent area of administration in NHPs is the dorsum, utilizing the region between the shoulders to the hips; however, any area possessing loose skin can be utilized. The subcutaneous space is an excellent site for large volume fluid delivery in small or dehydrated animals, avoiding technical difficulties and problems sometimes seen with direct intravenous

FIGURE 5.1 Procedural-related histopathological findings at intravenous administration sites. (A) Following intravenous access, the injection vein often exhibits intimal thickening (star), endothelial hyperplasia (asterisk) or mural defects (arrow) where penetration by the catheter occurred. (B) There may be subcutaneous hemorrhage following catheterization, due to minor blood extravasation. (C) Minor injury to the subcutis or dermis may result in localized fibroplasia or fibrosis (asterisk) near the injection vessel.

injury, or minor extravasation of non-isotonic or irritating test article materials. On rare occasions, needle tracts may appear in tissue section (Fig. 5.2) (see also Chapter 13).

FIGURE 5.2 The introduction of needles or catheters into the skin may result in microscopically visible needle tracts that vary in characteristics, based on age of the lesion and degree of healing, but are generally linear and perpendicular to the epidermis. Here a needle tract has healed by fibrosis.

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administration, such as fluid overload and pulmonary edema, because excess subcutaneous fluid is excreted rapidly by the kidneys. Inadvertent subcutaneous administration is a common complication of intradermal injections, and small, sharp needles are required for success with intradermal delivery.25

3.3.3 Intramuscular route An intramuscular injection is usually given in the thigh muscle and is a common parenteral route in NHPs and humans. Generally, intramuscular injections of test substances in an aqueous solution results in uniform and rapid absorption of the substance, because of the abundant vascular supply in muscle tissue.22 Smaller volumes are administered intramuscularly than for subcutaneous delivery. Substances that are irritating via intramuscular injection or inadvertent injection of nerves may result in paresis, paralysis, muscle necrosis, and localized muscle sloughing (See Chapter 12). Repeated injections may result in muscle inflammation and necrosis.26

3.3.4 Intranasal route Intranasal administration is a noninvasive, rapid, and efficient route of drug delivery27 used for a variety of therapeutics, including vaccines, chemotherapies, and analgesics. Intranasal administration circumvents hepatic first-pass metabolism, avoids gastrointestinal decomposition, and provides a rapid onset of action as the nasal mucosa that lines the nasal cavity is richly supplied with blood vessels.28 Intranasal administration has been shown to deliver drugs from the nose to the brain in minutes along the olfactory and trigeminal nerve pathways, bypassing the bloodebrain barrier.29 In humans and NHPs, drug delivered into the nasal cavity is primarily deposited in the respiratory zone located between the inferior and middle turbinate, and is absorbed into the systemic circulation.26 Blood concentrations for substances administered intranasally may approach concentrations seen following intravenous administration, and small, lipophilic molecules are absorbed more rapidly by the intranasal route than large molecular weight or highly polar substances.30 NHPs generally are sedated or anesthetized31 for the intranasal delivery, to minimize struggling and sneezing. Volumes administered intranasally are small compared with those of other routes to minimize the potential for suffocation and death.

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Inhalational delivery typically uses vapors (for example, volatile anesthetic gases) or aerosols of nebulized particles in solution.32 Animals are conscious with this delivery method and are restrained with a specialized nose mask to optimize delivery. Substances administered by aerosol are deposited by gravitational sedimentation, inertial impaction, or diffusion. As a rule of thumb, larger particles are deposited in the airways by gravitational sedimentation and inertial impaction, whereas smaller particles make their way into distal alveolar spaces by diffusion. Particles less than 3 mm in diameter penetrate the alveoli, and those that are 3e5 mm in diameter distribute uniformly throughout the lung. Materials deposited in the oropharynx, proximal trachea, or airways will be transported up the trachea by the mucociliary apparatus, into the mouth, and swallowed with subsequent first pass-effect after absorption.33 Inhalational administration is a highly complex technique requiring specialized equipment and knowledge, and it is beyond the scope of this chapter to discuss this methodology in further detail34,35 Substances administered by this route should be nonirritating to minimize pharyngeal edema, bronchial spasm, anaphylaxis, peracute death, and chronic pulmonary fibrosis. Animals should be conditioned to restraint devices and nose masks prior to experimental initiation.

4. Other routes of administration 4.1 Rectal route Rectal administration can deliver both systemic and local effects. Rectal administration via suppositories (a solid drug formulation) or enema (liquid drug formulation) to produce a systemic effect is useful in situations in which the patient is unable to take medication orally (e.g., is unconscious, vomiting, convulsing). Rectal administration of test substances by enema or suppository is less common in NHPs than in humans. Test substances are absorbed through the rectal mucosa. In view of the anatomy of the venous drainage of the rectum, approximately 50% of the dose bypasses the portal circulation, which is an advantage if the drug has low oral bioavailability.36 However, drug absorption via this route tends to be incomplete and erratic, in part because of variability in drug dissociation from the suppository. Rectal administration is also used for local topical effects (e.g., antiinflammatory drugs in the treatment of colitis).

3.3.5 Inhalation administration

4.2 Buccal/sublingual route

Inhaled delivery of a substance directly to the lungs provides many advantages for the treatment of respiratory diseases. A high local concentration of a therapeutic drug in the lungs offers a significant advantage that minimizes dose and systemic exposure, and maximizes efficacy.32 The lung has a large surface area, which is supplied by a dense capillary network, making absorption from the lungs rapid.

The oral cavity has been used as a site for local and systemic drug delivery.37 Sublingual (under the tongue) or buccal (between gum and cheek) administration does not lead to the drug entering the gastrointestinal tract, it is placed under the tongue, and therefore, it is considered oral and enteral. This route is advantageous for test substances that have low oral availability because the mucous

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membranes of the oral cavity have a very rich blood supply, thus providing a highly vascular absorptive surface. These vascular areas are ideal for lipid-soluble drugs that would be metabolized in the gut or liver, since the blood vessels in the mouth bypass the liver (do not undergo first pass liver metabolism), and drain directly into the systemic circulation. Buccal formulations can provide extended-release options to provide long-lasting effects. This route is usually reserved for test substances soluble in saliva and active in very small concentrations, e.g., cardiovascular drugs, steroids, barbiturates, enzymes, antiemetics, vitamins, minerals, and vaccines.38e40 However, conducting repeated dose preclinical studies by the buccal or sublingual routes in NHPs is not feasible.41

4.3 Intraosseous route Intraosseous infusion of fluids may be used in hypotensive animals when intravenous access is limited22 and rarely used in NHP safety evaluation studies. Intraosseous access is used most frequently for emergency care of critically ill infants and children when intravenous access cannot be rapidly achieved.42 The fluids directly enter the central venous circulation from the medullary venous sinuses. Potential complications include osteomyelitis, iatrogenic fracture, and growth plate injury.22

4.4 Epidural and intrathecal route The spinal cord rests in a medium of cerebrospinal fluid (CSF) and is contained by a series of protective membranes, known as the meninges: the pia mater, arachnoid mater, and dura mater. The pia mater covers the spinal cord and the arachnoid mater lies closely adherent to the outer, tough, dura mater. The epidural space lies outside all three membranes. The contents of the epidural space include a rich venous plexus, spinal arterioles, lymphatics, and extradural fat. The intrathecal space lies between the arachnoid mater and pia mater and contains the CSF. Epidural delivery is the administration of substances (e.g., analgesics) into the epidural space either as a single injection or as a continuous infusion via an indwelling catheter. Intrathecal administration is direct delivery of substances into the CSF in the intrathecal space of the spinal cord and therefore, circumvents the bloodebrain barrier.43e45 The technique requires animals to be sedated heavily and given a local anesthetic block over the spinal needle insertion site; alternatively animals can undergo general anesthesia prior to implementation. Intrathecal or epidural administration of substances requires considerable technical skill and in-depth knowledge of anesthesia, analgesia, and spinal cord and vertebral column anatomy. These techniques should be performed only by well-trained

personnel. Additionally, these routes are not without consequences that may be noted histologically (see Chapter 10). Spinal delivery of substances to NHP in safety evaluation assessment studies is rarely conducted. NHP models to ascertain mechanism of action of drugs that follow intrathecal delivery are available in the rhesus46 and cynomolgus monkey.47

4.5 Ocular route The monkey eye closely resembles the human eye with regard to anatomy and physiology, including presence of a macula.48 Intravitreal administration procedures and assessments of ocular toxicity in cynomolgus monkeys are well established.49e51 The common marmoset provides a potential alternative to the cynomolgus monkey for ocular toxicity testing.52 There are available ocular drug delivery systems.53

4.6 Intratracheal route Intrapulmonary delivery is accomplished by either intratracheal instillation or inhalation. Intratracheal instillation is an easier delivery method requiring less specialized equipment and provides instantaneous delivery of a known amount of test substance, suspended in a small volume of vehicle, directly to the lung, or even to a single lobe within the lung of anesthetized animals. Because it administers the material directly to the lower respiratory tract, it avoids deposition in the nasal passages.54 Volumes administered by the intratracheal route must be small to avoid suffocation. The NHP is an excellent model of human tuberculosis after direct intratracheal instillation of M. tuberculosis, in large part because they recapitulate the full spectrum of infection outcome and pathology seen in humans55 and use in vaccine research.56

4.7 Intraperitoneal route Intraperitoneal injection of substances into the peritoneal cavity is commonly used in laboratory rodents but rarely is used in NHPs.22 It can be used in smaller primate species to administer large volumes of fluid safely when intravenous administration is challenging or not possible. Absorption of substances delivered intraperitoneally is characteristically faster than subcutaneous administration and much slower than for intravenous injection. Although intraperitoneal delivery is considered a parenteral route of administration, the pharmacokinetics of substances administered intraperitoneally are more similar to those seen after oral administration, because the primary route of absorption is into the mesenteric vessels, which drain into the portal vein and pass through the liver. Therefore substances administered

Routes of administration for the non-human primate Chapter | 5

intraperitoneally may undergo hepatic metabolism before reaching the systemic circulation.

5. Conclusion Selecting the most suitable route of administration depends on the potential human therapeutic target and physiochemical properties of the test substance. There are many different routes by which a test substance can enter the body, each with its advantages and disadvantages, and the route has a large impact on how fast the test substance is taken up and how much of it arrives at its target in an active form. The administration of test substances to NHPs is a key component of many scientific projects. Researchers should consider all opportunities, where possible, to experimental refinement to minimize adverse effects on animals.

References 1. Lehman-McKeeman L, Armstrong LE. General Principles of xenobiotic disposition. In: Haschek WM, Rousseaux CG, Wallig MA, Bolon B, editors. Haschek and Rousseaux’s handbook of toxicologic pathology. 4th ed., vol. 1. San Diego: Elsevier. Manuscript Submitted for Publication; 2020. 2. ICH (International Conference on Harmonization S6 R1). Preclinical safety evaluation of biotechnology-derived pharmaceuticals. 2011. 3. Thackaberry EA, Kopytek S, Sherratt P, Trouba K, McIntyre B. Comprehensive investigation of hydroxypropyl methylcellulose, propylene glycol, polysorbate 80, and hydroxypropyl-beta-cyclodextrin for use in general toxicology studies. Toxicol Sci 2010;117(2):485e92. 4. Gad SC, Cassidy CD, Aubert N, Spainhour B, Robbe H. Nonclinical vehicle use in studies by multiple routes in multiple species. Int J Toxicol 2006;25(6):499e521. 5. Ruys JD, Mendoza SP, Capitanio JP, Mason WA. Behavioral and physiological adaptation to repeated chair restraint in rhesus macaques. Physiol Behav 2004;82(2e3):205e13. 6. Zhang S, Ye B, Zeng L, Chen Y, He S, Wang C, et al. DrugContaining gelatin treats as an alternative to gavage for long-term oral administration in rhesus monkeys (Macaca mulatta). JAALAS 2012;51(6):842e6. 7. Kondo H, Shinoda T, Nakashima H, Watanabe T, Yokohama S. Characteristics of the gastric pH profiles of unfed and fed cynomolgus monkeys as pharmaceutical product development subjects. Biopharm Drug Dispos 2003;24(1):45e51. 8. Becker DE. Drug therapy in dental practice: general Principles: part 1-pharmacokinetic considerations. Anesth Prog 2006;53(4):140e6. 9. Chen EP, Mahar Doan KM, Portelli S, Coatney R, Vaden V, Shi W. Gastric pH and gastric residence time in fasted and fed conscious cynomolgus monkeys using the Bravo pH system. Pharm Res (N Y) 2008;25(1):123e34. 10. Bernstein JA, Didier PJ. Nonhuman primate dermatology: a literature review. Vet Dermatol 2009;20(3):145e56.

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28. Fortuna A, Alves G, Serralheiro A, Sousa J, Falcão A. Intranasal delivery of systemic-acting drugs: small-molecules and biomacromolecules. Eur J Pharm Biopharm 2014;88(1):8e27. 29. Johnson NJ, Hanson LR, Frey WH. Trigeminal pathways deliver a low molecular weight drug from the nose to the brain and orofacial structures. Mol Pharm 2010;7(3):884e93. 30. Illum L. Nasal drug delivery: new developments and strategies. Drug Discov Today 2002;7:1184e9. 31. Saccone PA, Lindsey AM, Koeppe RA, Zelenock KA, Shao X, Sherman P, et al. Intranasal opioid administration in rhesus monkeys: PET imaging and antinociception. J Pharmacol Exp Therapeut 2016;359(2):366e73. 32. Patil JS, Sarasija S. Pulmonary drug delivery strategies: a concise, systematic review. Lung India 2012;29(1):44e9. 33. Fernández Tena A, Casan Clarà P. Deposition of inhaled particles in the lungs. Arch Bronconeumol 2012;48(7):240e6. 34. Paranjpe M, Müller-Goymann CC. Nanoparticle-mediated pulmonary drug delivery: a review. Int J Mol Sci 2014;15(4):5852e73. 35. Tepper JS, Kuehl PJ, Cracknell S, Nikula KJ, Pei L, Blanchard JD. Symposium summary: “breathe in, breathe out, its easy: what you need to know about developing inhaled drugs”. Int J Toxicol 2016;35(4):376e92. 36. de Boer AG, Moolenaar F, de Leede LG, Breimer DD. Rectal drug administration: clinical pharmacokinetic considerations. Clin Pharmacokinet 1982;7(4):285e311. 37. Chinna Reddy P, Chaitanya KSC, Madhusudan Rao Y. A review on bioadhesive buccal drug delivery systems: current status of formulation and evaluation methods. Daru 2011;19(6):385e403. 38. Bind AK, Gnanarajan G, Kothiyal P. A review: sublingual route for systemic drug delivery. Int J Drug Res Tech 2013;3(2):31e6. 39. Choi JH, Jonsson-Schmunk K, Qiu X, Shedlock DJ, Strong J, Xu JX, et al. A single dose respiratory recombinant adenovirus-based vaccine provides long-term protection for non-human primates from lethal ebola infection. Mol Pharm 2015;12:2712e31. 40. Aravantinou M, Mizenina O, Calenda G, Kenney J, Frank I, Lifson JD, et al. Experimental oral herpes simplex virus-1 (HSV-1) co-infection in simian immunodeficiency virus (SIV)-infected rhesus macaques. Front Microbiol 2017;8:2342. 41. Greaves P. Histopathology of preclinical toxicity studies. Interpretation and relevance in drug safety evaluation. 4th ed. Academic Press; 2012. 42. Tobias JD, Kinder Ross A. Intraosseous infusions: a review for the anesthesiologist with a focus on pediatric use. Anesth Analg 2010;110:391e401. 43. Calias P, Papisov M, Pan J, Savioli N, Belov V, Huang Y, et al. CNS penetration of intrathecal-lumbar idursulfase in the monkey, dog and

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mouse: implications for neurological outcomes of lysosomal storage disorder. PLoS One 2012;7(1):e30341. Farquhar-Smith P, Chapman S. Neuraxial (epidural and intrathecal) opioids for intractable pain. Br J Pain 2012;6(1):25e35. Duarte RV, Lambe T, Raphael JH, Eldabe S, Andronis L. Intrathecal drug delivery systems for the management of chronic non-cancer pain: protocol for a systematic review of economic evaluations. BMJ Open 2016;6(7):e012285. Hartvig P, Gustafsson LL, Bergström K, Moström U, Pontén U, Lindberg B, Lundqvist H. Positron emission tomography: an animal model of spinal distribution of drugs after intrathecal administration. Ups J Med Sci 1987;92(2):205e13. Felice BR, Wright TL, Boyd RB, Butt MT, Pfeifer RW, Pan J, et al. Safety evaluation of chronic intrathecal administration of idursulfaseIT in cynomolgus monkeys. Toxicol Pathol 2011;39:879e92. Short BG. Safety evaluation of ocular drug delivery formulations: techniques and practical considerations. Toxicol Pathol 2008;36:49e62. Niggemann B, Korte SH, Srivastav S. Ocular toxicity testing in nonhuman primates. In: Weinbauer GF, Vogel F, Waxmann, editors. Novel approaches towards primate toxicology. Münster, Germany: Verlag GmbH; 2006. p. 202e17. Gokulgandhi MR, Vadlapudi AD, Mitra AK. Ocular toxicity from systemically administered xenobiotics. Expet Opin Drug Metabol Toxicol 2012;8(10):1277e91. Thackaberry EA, Farman C, Zhong F, Lorget F, Staflin K, Cercillieux A, et al. Evaluation of the toxicity of intravitreally injected PLGA microspheres and rods in monkeys and rabbits: effects of depot size on inflammatory response. Invest Ophthalmol Vis Sci 2017;58:4274e85. Korbmacher B, Atorf J, Fridrichs-Gromoll S, Hill M, Korte S, Kremers J, et al. Feasibility of intravitreal injections and ophthalmic safety assessment in marmoset (Callithrix jacchus) monkeys. Primate Biol 2017;4:93e100. Kuno N, Fujii S. Recent advances in ocular drug delivery systems. Polymers 2011;3:193e221. Osier M, Oberdorster G. Intratracheal inhalation vs intratracheal instillation: differences in particle effects. Fund Appl Toxicol 1997;40:220e7. Scanga CA, Flynn JL. Modeling tuberculosis in nonhuman primates. Cold Spring Harb Perspect Med 2014;4(12):a018564. DiNapoli JM, Yang L, Samal SK, Murphy BR, Collins PL, Bukreyev A. Respiratory tract immunization of non-human primates with a Newcastle disease virus-vectored vaccine against Ebola virus elicits a neutralizing antibody response. Vaccine 2010;29(1):17e25.

Chapter 6

The alimentary system of the non-human primate Jagannatha V. Mysore1, Nicola M.A. Parry2 and Jennifer A. Chilton3 1

Department of Pathology, Nonclinical Safety, Bristol Myers Squibb, New Brunswick, NJ, United States; 2Division of Comparative Medicine,

Massachusetts Institute of Technology, Cambridge, MA, United States; 3Charles River Laboratories, Reno, NV, United States

1. Introduction The principal function of the alimentary tract, this tubular to saccular organ system, is mastication of food, followed by digestion with the assistance of glandular secretions (e.g., salivary glands, liver, and pancreas), absorption of the nutrients, and excretion of the fecal material. Additionally, this organ system is symbiotically dependent on multiple species of bacteria (nonpathogenic species from the family Enterobacteriaceae) and a few protozoan organisms for digestion and synthesis of certain nutrients. Furthermore, evolutionary adaptation for foraging in their respective habitats (arboreal vs. terrestrial) has resulted in subtle structural differences in this organ system between the Old World and New World monkeys. Spontaneous and toxicologically induced lesions of the alimentary tract can have significant consequences on the health of the non-human primates (NHPs), which are summarized in this chapter.

2. Anatomy and histology of the alimentary system 2.1 The oral cavity and salivary glands The three pairs of major salivary glands are the parotid (serous), submandibular (seromucinous), and sublingual (seromucinous); and minor glands are distributed in the buccal cavity, palates, and tongue. The parotid duct enters the oral cavity lateral to the second upper molar tooth. The gross and light microscopic features of the three pairs of major salivary glands of Old World monkeys are similar to those of the salivary glands in humans (Figs. 6.1 and 6.2).1,2,48 Teeth perform the mechanical actions of peeling and piercing of food, as well as mastication assisted by salivary lubrication. In nature, dentition of NHPs has evolved based on foraging habits. Macaques are omnivores and

FIGURE 6.1 (A) The submandibular salivary gland is composed of secretory units with acini that surround duct systems in a lobular array (asterisk) and that drain into the interlobular ducts (black arrow). (B) There are both serous-type acini (black arrow) characterized by abundant dark basophilic cytoplasmic secretory granules within acinar cells, and mucous-type acini (white arrow) surrounding a system of intercalated ducts (asterisk) that join the larger interlobular duct within the lobule. Cynomolgus macaque, Hematoxylin and Eosin (H&E).

Spontaneous Pathology of the Laboratory Non-human Primate. https://doi.org/10.1016/B978-0-12-813088-9.00010-0 Copyright © 2023 Elsevier Inc. All rights reserved.

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FIGURE 6.2 Salivary glands within the tongue may be either mucinous, serous, or mixed. Mucous-type acini (asterisk) are characterized by irregular aggregation and are connected by a duct (black arrow) to the lingual surface. Cynomolgus macaque, Hematoxylin and Eosin (H&E).

predominantly feed on plants (fruits, leaves, and stems) and invertebrates (crabs, insects, and shrimp), and their dentition supports piercing and peeling of fruits and the chitinous layers of invertebrates. Marmosets predominantly feed on plant exudates (gum, sap, latex, and resin) and insects, and have dentition that supports gnawing. Similar to humans, NHPs have two sets of teethdthe deciduous dentition (milk teeth) and permanent dentition (successor teeth). Major differences between the teeth of Old World monkeys and humans include the presence of sharp canines and a triangular-shaped mandibular first premolar that occludes with the maxillary canine in the NHPs (Fig. 6.3). The general dental formulas for macaques (Old World monkeys) and marmosets (New World monkeys) are: Dental formula for macaques: Upper and lower arcaded2/2 incisors, 1/1 canine, 2/2 premolars, 3/3 molars Dental formula for marmosets: Upper and lower arcaded2/2 incisors, 1/1 canine, 3/3 premolars, 2/2 molars The tongue is a solid muscular organ that assists with mastication. It also plays an important role in gustation and has four different types of papillae (filiform, foliate, fungiform, and circumvallate) (Figs. 6.4 and 6.5) in the mucosa. The filiform papillae are widely distributed across the entire tongue, are nongustatory, and sense texture. Although these are referred to as “filiform” they are often platform-shaped in the macaque rather than conical shaped as in humans. The other papillae discern different tastes and assist in the choice of food. The tip and anterior two-thirds of the tongue have numerous fungiform papillae that sense sweet taste.1e3 In the subfamily Cercopithecidae (macaques, mangabeys, and patas), the cheeks extend bilaterally as buccal pouches that open into the floor of the oral vestibule and

FIGURE 6.3 The dental arcade of the adult cynomolgus macaque has 4 incisors (a), 2 canines (b), 2 premolars (c) and 3 molars (d) that are of equal number in the mandible and maxilla for a total of 32 teeth. Old World monkeys such as macaques have a distinctly triangular first mandibular premolar (black arrow).

extend into the neck region. The mucous membranes of the cheek pouches are the continuum of oral mucosa with minor salivary glands. The buccal pouches are useful for temporary storage of forage at the time of rapid collection.1

2.2 Esophagus and stomach As is the case with dentition, the remaining gastrointestinal system has evolved to adapt to the natural foraging habits of Old World and New World monkeys. Morphologically, in NHPs, the pathway from mouth to stomach is almost a direct line, whereas in humans the pharynx is at a right angle to the oral cavity.1 The basic esophageal anatomy of macaques and marmosets is similar to humans. The wall of the uppermost (cervical) segment is chiefly composed of striated muscles with an upper esophageal sphincter (UES) encompassing the cervical esophagus at the level of the cricoid cartilage. The major components of the UES are made up of thyropharyngeus and cricopharyngeus muscles in association with outer longitudinal and inner circular esophageal muscles. The wall of the middle (thoracic) segment is composed of a mixture of striated and smooth muscles (transitional zone) and the lower (abdominal) segment including the lower esophageal sphincter (LES) at

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FIGURE 6.4 (A) The tongue of a macaque with thick dorsal mucosa filled with papilla and thinner ventral mucosa without papilla. (B) Fungiform papillae have a mushroom like shape, and often contain taste buds (black arrow). (C) Filiform papillae are the most abundant of the macaque tongue and are highly keratinized. Cynomolgus macaque, Hematoxylin and Eosin (H&E).

the entry into the gastric cardia is solely composed of smooth muscles.4 Histologically, the esophagus distinguishes itself from the rest of the gastrointestinal tract by the absence of serosal covering in the cervical and thoracic segments. However, the abdominal segment is covered by the serosa.5,6 The anatomy of the stomach in monkeys is comparable to that in humans with respect to segmentation, except in colobines (colobus monkeys, langurs, and proboscis monkeys) that have a sacculated stomach that allows bacterial fermentation of plant fibers. In nature, colobines forage leaves, fruits, and seeds exclusively, whereas rhesus and cynomolgus monkeys are omnivores.7 In comparison with the human stomach, the stomach of the cynomolgus monkey has a more prominent gastric fundus (the dome-shaped portion that extends above the gastroesophageal junction) that extends to the level of the gastroesophageal junction and differs from the gastric body (corpus). Histologically, the fundus contains prominent gastric pits, lacks parietal cells (oxyntic cells that produce hydrochloric acid and intrinsic factor), and has extensive mucus-secreting

cells, and a basal layer of chief cells (the cells that produce pepsinogen) (Figs. 6.6 and 6.7). Similar features are also present in the stomach of rhesus monkeys. Further aborally, the parietal cells are present in fewer numbers at the transition zone between the fundus and the body, and they increase in density toward the body. The presence of specific glandular cell populations may be better elucidated following special staining or immunohistochemistry (IHC) for cell markers. For example, IHC for Hþ/Kþ ATPase will elucidate parietal cells more clearly.8 In the mucosa of the body, a zone of parietal cells extends from beneath the mucosal surface toward the basal aspect. In addition, the fundic and antral mucosa contain variably sized lymphoid aggregates and lymphofollicular foci within the lamina propria. Occasionally, these lymphoid aggregates extend through the muscularis mucosae and into underlying submucosa.5,8,9,172 The stomach has key physiological functions in the body centered on mechanical and secretory roles in digestion. The main functions include transitory storage of food, maceration, and secretion of digestive enzymes

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FIGURE 6.6 Subgross longitudinal sections of the stomach (cardia, fundus, body, antrum, and pylorus) of the macaque: The gastroesophageal junction (GE) has an abrupt change from stratified squamous epithelial mucosa of the esophagus to glandular mucosa of the cardia (C). Mucosae of the fundus (F) and the body (B) contain numerous deep invaginations (gastric pits/foveolae) forming distinct mucosal folds. The antral (A) mucosal folds are of a lesser degree bordering the pyloric sphincter (P). The pyloric sphincter is a circumferential invagination toward the lumen formed by the mural inner circular smooth muscle. The lumen continues toward the duodenum (D), where the mucosa has distinct small intestinal villi. Cynomolgus macaque, Hematoxylin and Eosin (H&E).

FIGURE 6.5 (A) The circumvallate papilla is covered by a stratified squamous epithelium with numerous taste buds on the either side of the cleft. The lamina propria is interspersed with nerve fibers and frequently contains mononuclear cells. (B) The taste buds are embedded within the squamous mucosa of the circumvallate papilla and contain basal cells, spindle-shaped taste receptor (gustatory) cells and sustentacular (supporting) cells. Cynomolgus macaque, Hematoxylin and Eosin (H&E).

(pepsinogen and lipase), intrinsic factor, hydrochloric acid, and hormones (e.g., gastrin, histamine, endorphins). The mucin produced by the gastric mucous neck cells and intestinal goblet cells provides lubrication for the ingesta and protects the epithelial lining of the gastrointestinal mucosa.10

2.3 The small and large intestines The small and large intestines form one of the largest organs in NHPs and have several key physiological functions in the body: mechanical and secretory roles in digestion; absorption of macronutrients and micronutrients; excretion of digestive waste and metabolic waste products; body fluid and electrolyte homeostasis; and immunological defense. They also provide a significant surface area of specialized epithelium that serves as a selectively permeable barrier for absorption of nutrients, water, and electrolytes, while

functioning as a barrier to toxins, pathogens, and other antigens. This specialized epithelium also plays significant roles in digestion and metabolism.10,172 The small intestine (Fig. 6.8) is suspended by the mesentery and can vary in length among the different species of NHPs. Differences in digestion and absorption of nutrients account for these relative differences in length. For example, the length of the small intestine in faunivorous NHPs tends to be short, while herbivorous species have a comparatively longer small intestine. It comprises the duodenum, jejunum, and ileum, and is relatively simple in all NHPs; however, many prosimian NHPs do not have these distinct subdivisions. The gross and histological appearances of the different regions of the small intestine are more uniform in prosimian species. The inner wall of the small intestine in NHPs is also covered by numerous mucosal folds known as plicae circulares. These circular folds form ridges that project into the lumen of the small intestine and increase its surface area.11 Microscopically, the layers of the small intestine comprise the mucosa, submucosa, muscularis mucosae, muscularis externa, and serosa.10 The surface area of the small intestine is markedly increased by mucosal projections known as villi. These finger like projections are covered by a layer of simple columnar epithelial cells (enterocytes) with a brush border

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FIGURE 6.7 (A) The gastroesophageal junction has an abrupt change from stratified squamous epithelial mucosa of the esophagus to glandular mucosa of the cardia. Frequently, lymphoplasmacytic aggregates or wellformed lymphoid follicles (black arrow), often with well-formed germinal centers, are noted at the junction. (B) The pyloric antrum terminates at the pyloric sphincter (asterisk) formed by the inner circular smooth muscles of the gastric wall adjacent to the duodenum (b) where the mucosa has distinct small intestinal villi and mucinous Brunner’s gland (BG) in the submucosa. Cynomolgus macaque, Hematoxylin and Eosin (H&E).

and interspersed goblet cells that help trap bacteria. However, villi are typically absent beyond the level of the ileocecal valve. Below the epithelial layer, the lamina propria comprises loose connective tissue that forms the cores of the intestinal villi. It contains a rich vascular and lymphatic network which functions in absorption of digestive products. The lamina propria contains a central lacteal which is involved in lipid absorption, as well as vascular capillaries which are fenestrated to help absorption. The muscularis externa is composed of two layers of smooth muscledthe inner circular and outer longitudinal layersdwith ganglion cells and nerve fibers of the myenteric (Auerbach’s) plexus are found in between.10 Brunner’s glands are more numerous in the proximal duodenum,

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FIGURE 6.8 (A) The duodenum is lined by mucosal villi (V) subtended by the Brunner’s glands (BG) that drain into the crypts of Lieberkuhn (CK). The small intestine has numerous folds known as plicae circulares (PC). (B) The deep epithelium of the duodenum contains the crypts of Lieberkuhn (CK), and Brunner’s glands (BG). The crypts are lined by stratified glandular epithelium and contain Paneth cells (P) with bright eosinophilic cytoplasmic granules. Prominent infiltrates of lymphocytes and plasma cells (LP) are present within the lamina propria of the mucosa representing one form of gut-associated lymphoid tissue (GALT). Cynomolgus macaque, Hematoxylin and Eosin (H&E).

beginning just distal to the pylorus, and their numbers reduce along the middle and distal regions of the duodenum. The glands secrete alkaline fluid that helps to protect the duodenal mucosa from acid damage.11 The large intestine (Fig. 6.9 A and B) is variable among the different species of NHPs, although typically it comprises the colon, cecum (in most species), and appendix (in some species). All primates have a cecum and a colon that are relatively larger than that of other mammals. Similar to humans, NHPs cannot digest cellulose, but the fiber content in their feed facilitates the digestion and transit time of ingesta.1,7 New World monkeys and prosimians tend to lack an appendix but have a large cecum. In contrast, Old World monkeys have a more recognizable appendix and a cecum.

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In anthropoid apes, the appendix is well developed, but the cecum is absent. In general, the length of the cecum relative to that of the colon tends to be greater in prosimians than in monkeys and apes; conversely, the appendix is larger in monkeys and apes than in prosimians.11 Old World monkeys have a colon with taeniae coli, haustra, and a sigmoid flexure, but no vermiform appendix. Taeniae coli are the three prominent and distinct bands of smooth muscle that are part of the muscularis externa and run along the entire length of the colon and cecum (Fig. 6.9A). Taeniae are visible as whitish bands of tissue on the external surface of the colon and cecum. Because the taeniae coli are shorter than the large intestine, they contract lengthwise, causing the colon and cecum to become sacculated between the taeniae into pouches known as haustra. New World monkeys have no sigmoid flexure or haustra in the colon. Prosimians also have no sigmoid flexure, and no taeniae or haustra in the colon.12

FIGURE 6.9 (A) Low magnification and (B) higher magnification of the colon: The colon is comprised of the mucosa (M) subtended by the submucosa (SM) and muscularis externa (ME). Taenia coli (TC) are bands of smooth muscle that run longitudinally along the colon and may be seen in cross-section. The muscularis mucosa (MM) is a small band of smooth muscle that lies between the mucosa and submucosa. The lamina propria (LP) between the epithelial layers of the mucosa contain abundant lymphocytes and plasma cells. Cynomolgus macaque, Hematoxylin and Eosin (H&E).

Microscopically, the layers of the large intestine are similar to those of the small intestine, containing mucosa, submucosa, muscularis mucosae, muscularis externa, and serosa. Below the epithelial layer, the lamina propria contains loose connective tissue with the vascular and lymphatic network. The colon contains no villi or crypts of Lieberkühn. Its mucosa is lined by a simple columnar epithelium with a thin brush border. Interspersed goblet cells are present and are more numerous than in the small intestine (Fig. 6.9B). Paneth cells are typically absent in the large intestine. Otherwise, the lamina propria in the colon is mostly like its counterpart in the small intestine.1 The appendix is a variably distinct entity in macaques, but more commonly described in marmosets. The mucosa appears like the mucosa in the colon, but the muscularis externa is like its counterpart in the small intestine but without taeniae. Moving aborally along the colon, the colonic crypts become shorter and eventually disappear near the rectoanal junction. The muscularis mucosae also become tattered and disappear, and the lamina propria merges with the underlying submucosa in this region.2,6 At the rectoanal junction (Fig. 6.10), the simple columnar epithelium of the intestine transitions to a layer of keratinized stratified squamous epithelium where the skin lines the anal region. The dermis of the skin in this region contains sebaceous glands, mucous-secreting anal glands, apocrine circumanal glands, and hair follicles. The macaque has anal glands (Fig. 6.11) that consist of ducts surrounded by tubuloalveolar glands. Large amounts of smooth and skeletal muscle are also present, forming the internal and external anal sphincters.11

FIGURE 6.10 Rectoanal junction of the macaque: The colonic crypts become shorter and eventually disappear near the rectoanal junction as the colonic mucosa (CM) merges into the squamous epithelium (SE) of the anus. The muscularis mucosa also becomes tattered and disappears, and the lamina propria merges with the underlying submucosa. The junction continues until it joins with the perianal skin (PS) where adnexa become a prominent feature. Cynomolgus macaque, Hematoxylin and Eosin (H&E).

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FIGURE 6.11 The anal gland of the macaque is tubuloalveolar in structure and generally surrounds a large, single duct leading to the rectoanal junction. Cynomolgus macaque, Hematoxylin and Eosin (H&E).

The intestinal tract is also a lymphoid organ, by virtue of its mucosa-associated lymphoid tissue (MALT). This comprises a collection of aggregates of lymphoid tissue that are present at various locations throughout the intestinal tract (including the tonsils in the oral cavity, the intestines, and the appendix). MALT consists of a diffuse collection of immune cells (including lymphocytes, plasma cells and macrophages) that provide localized defense against foreign antigenic substances in various submucosal locations throughout the body, including in the gastrointestinal tract. Gut-associated lymphoid tissue (GALT) is a specific type of MALT, and is predominantly located in the lamina propria of the intestinal mucosa. It comprises a diffuse infiltrate of immune cells, as well as discrete aggregates of immune cells, such as Peyer’s patches that can be seen microscopically as submucosal lymphoid nodules. They may disrupt the muscularis mucosae and extend into the mucosa, sometimes almost to the luminal surface.6,10,12,13

3. Embryology of the non-human primate alimentary tract The gastrointestinal tract is one of the first organ systems to develop in the primate embryo, developing as a diverticulum of the yolk sac.14 During the process of gastrulation, the single-layered blastula becomes a three-layered structure known as the gastrula. It comprises the three germ layers, which are known as endoderm, mesoderm, and ectoderm. All three layers contribute to the development of the gastrointestinal tract.15 The primitive gut arises when the flat embryonic disc folds, and the inner endodermal layer forms the gut tube as a diverticulum of the yolk sac. The gut tube communicates ventrally with the yolk sac and is surrounded by lateral plate splanchnic mesoderm, which eventually forms the lamina propria, submucosa, and mucosa of the

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gastrointestinal tract. The tube is the intraembryonic part of the yolk sac and extends from the cephalic end to the caudal end of the embryo.15 Endoderm produces the epithelium lining of the intestinal tract, while ectoderm gives rise to the epithelial lining of the proctodeum (the caudal aspect of the anal canal) and the stomodeum (the mouth and some of the salivary glands). Splanchnic mesoderm forms most of the connective tissue and smooth muscle components; it gives rise to the smooth muscles of the intestines and lower esophagus. Neural crest cells migrate to the gut tube and form the components of the enteric nervous system, which is part of the autonomic nervous system.14,15 Within the gut tube, three distinct regionsdforegut, midgut, and hindgutdextend the entire length of the embryo and ultimately give rise to different components of the tract. The large midgut arises when lateral folding of the embryo pinches off a pocket of the yolk sac. As embryonic folding continues and the gut tube lengthens, the ventral connection between the midgut and yolk sac narrows and elongates to form the vitelline duct through which the midgut and yolk sac communicate. In the embryo, the foregut ultimately becomes the proximal duodenum (as well as the pharynx, esophagus, stomach, liver, and pancreas). The midgut becomes the remainder of the small intestine, cecum, appendix, ascending colon, and part of the transverse colon and the hindgut becomes the remainder of the transverse colon, the descending colon, the sigmoid colon, the rectum, and most of the anal canal.15 The yolk sac is a significant structure in the embryo and contains its own blood supplydthe vitelline arteries, which arise from the abdominal aorta. The gastrointestinal tract and its associated organs develop from the yolk sac, and the vitelline arteries persist in the adult to supply the digestive tract. The structures that arise from the foregut, midgut, and hindgut are supplied by the celiac, cranial mesenteric, and caudal mesenteric arteries, respectively.14

4. Congenital lesions of the alimentary tract Data analysis from the California Regional Primate Center’s database on the frequency of spontaneous congenital defects in rhesus and cynomolgus monkeys over a 14-year period recorded only a small number of defects in the gastrointestinal system. Spontaneous congenital abnormalities of any kind are reported much less frequently in laboratory-raised NHPs than in humans. In Old World monkeys, reported rates range from 0.3% to 1.6%.16e19 A review of such lesions during a 14-year period at the California Regional Primate Research Center found a 0.3% (3/ 965) rate in cynomolgus macaques, and a 0.9% (40/4390)

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rate in rhesus macaques.16 In contrast, the rate of spontaneous congenital abnormalities in humans is estimated to be approximately ten times greater at 3%e6%.20

4.1 Congenital lesions of the teeth and oral cavity In general, the incidence of developmental anomalies of dentition is low in Macaca mulatta. In captive capuchin monkeys, congenital lesions such as supernumerary teeth, teeth inversion, and dental crowding are of low incidence (1%).21 The sporadic incidence of cleft palate (palatoschisis), with or without brachygnathia and mandibular aplasia, is documented in neonates and in a stillborn animals, respectively.16 Cleft palate is frequently reported in squirrel monkeys (Saimiri spp.) and has also been reported in other Old World and New World monkeys. Cleft palate develops during the first trimester of pregnancy due to the failure of fusion of lateral palatine processes.22 It can arise as a central, unilateral, or bilateral defect in the hard and/or soft palate. Newborn animals with this condition are at risk of nasal regurgitation that can lead to recurrent upper respiratory infections and aspiration pneumoniadthe latter of which is often fatal.23

4.1.1 Oral salivary gland hamartoma A hamartoma is a focal, benign, congenital lesion usually consisting of mature cells that grow in a disorganized mass. They can arise from any one of the germ cell layers (ectoderm, endoderm, or mesoderm). Intraoral salivary gland hamartomas are rare in NHPs, reported once on the mucosal surface of the lower lip in a cynomolgus monkey. Histologically, the mass consisted of well-circumscribed, irregular lobules of hyperplastic to normal-appearing mucinous acini and ducts separated by thick connective tissue septae. A traumatic injury was ruled-out for the finding in this case, and the lesion was attributed to a likely developmental anomaly.24 The case presented below occurred in the caudal aspect of the tongue of a young (1 year).110 The type of infiltrate/inflammation will vary depending upon the agent used. Care should be taken to distinguish between a true drug/vaccine effect, background findings, and procedure-related effects (e.g., repeated injections). Infiltrates may be accompanied by other changes associated with inflammation such as fibrosis, edema, and hemorrhage. Inflammation of blood vessels within the dermis might also occur.

10.2 T celledependent antibody response (TDAR) model induction site Protocols for the use of NHPs as models of T celledependent antibody response (TDAR) have been developed.111 Evidence of cyclosporine-induced immunosuppression was observed in a cynomolgus monkey model of TDAR where anti-keyhole limpet hemocyanin IgM and IgG responses to drug treatment were determined, and the subsequent amelioration by cyclosporine confirmed the utility of this model for immunotoxicity evaluation.111 The site at which the induction agent, keyhole limpet hemocyanin (KLH), is administered normally acquires localized inflammation with occasional ulceration present. The degree of inflammation induced by KLH is highly variable between animals, but generally there is a robust inflammatory response (Fig. 13.28A and B) with or without edema and hemorrhage. With severe inflammation the sites often heal with fibrosis present.

10.3 Administration sites for biologic test articles Subcutaneous administration of biologic test materials to NHPs may produce a variety of findings at the administration site, dependent on the properties of the delivery agent or diluent and based on the immunogenicity or pharmacology of the test article. These findings in NHPs may not always be predictive of the findings in human

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FIGURE 13.27 Delayed-type hypersensitivity (DTH) features in skin of a cynomolgus macaque with histologic severity grading: Severity Grade 0: Normal skin without inflammation, not pictured. (A) Severity Grade 1: Minimal perivascular inflammation of the subcutis and/or dermis is present but generally limited to scattered dermal vessels with small collections of perivascular inflammatory cells (black arrows). The inflammatory cells consist predominantly of mononuclear cells with fewer granulocytes. Minimal acanthosis and hyperkeratosis of the epidermis (green arrow) may be present, but the architecture is well preserved. (B) Severity Grade 2: Mild perivascular inflammation of the subcutis and/or dermis is present with easily identifiable perivascular cuffs of inflammatory cells (black arrows) and limited infiltration into surrounding tissues and may also have inflammatory cell cuffing of adnexa and minimal to mild acanthosis and hyperkeratosis of the epidermis (green arrow). Ulceration is rarely present, and the architecture is well preserved. (C) Severity Grade 3: Moderate perivascular inflammation of the subcutis and dermis is present. Consistently includes large collections of inflammatory cells that cuff and invade adnexa and multifocally replace or obscure adipose tissue (black arrows). The subcutis is consistently affected, commonly with small areas of effacement in the superficial muscle layer, may have minimal to mild edema, hemorrhage, and/or necrosis of the subcutis and/or dermis (red arrow), and may have minimal to moderate acanthosis and hyperkeratosis of the epidermis and ulceration may be present (green arrow). (D) Severity Grade 4: Marked inflammation of the subcutis and/or dermis. The architecture is partially or completely effaced by the inflammation (black line), and may have minimal to marked edema, hemorrhage, and/or necrosis of the subcutis and/or dermis. Moderate acanthosis and hyperkeratosis of the epidermis and ulceration are common (green arrow); Hematoxylin and Eosin (H&E). Reproduced with permission from Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition; Animal Models in Toxicologic Research: Nonhuman Primate. J.A. Chilton, S.T. Laing and A. Bradley, Elsivier Inc. 2021.

clinical trials; however, NHPs remain one of the best indicators of the risk of such reactions.112 In many cases, the presence of biological materials in the subcutis will result in minimal inflammation that is not due to pharmacology or immune-mediated processes, but due to the physical presence of the material in the subcutis or procedurally related (Fig. 13.29A).113 The importance of control animals for

determination of test article-associated skin inflammation that may occur during preclincial studies cannot be over emphasized. When immunogenicity is a compounding factor, the inflammation may be more severe in animals receiving the test article as compared to those administered the diluent or carrier substance. When immunogenicity is the sole inciting factor, control animals may have no

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FIGURE 13.28 (A) Injection of keyhole limpet hemocyanin (KLH) normally induces localized inflammation, the degree of which is highly variable between animals, ranging from minimal inflammation (arrows) to (B) a robust inflammatory response with or without edema and hemorrhage of the muscle and/or subcutis (arrows).

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The gross appearance of the SR-Bup administration site reaction varies from a firm swelling to mild erosions or ulcerations overlying the inflammatory focus. Ulceration is more likely if the SR-Bup is administered in a location easily reached by the animal due to self-induced trauma/ scratching. Histologically, the inflammation varies by timing from administration to examination, but a common feature is the presence of clear to pale basophilic vacuoles of various sizes encased in the inflammation. Eventually, the inflammation will resolve; however, the vacuoles may remain embedded in fibrous tissue formed during the healing process (Fig. 13.30AeD). Additionally, on rare occasions, the SR-Bup may distribute to the lymph node draining the dermatome in which it was administered, appearing with similar clear vacuolation and inflammation as the original administration site (Fig. 13.31). Because of the inflammatory response that may occur with administration of SR-Bup, clear records should be kept on where and when the medication was administered. Administration should avoid the regions utilized for testarticles or delayed-type hypersensitivity (DTH) evaluation sites, as the inflammation from the SR-Bup may interfere with evaluation of test-article effects in nearby skin.

inflammation at the administration site. In other cases, the properties of the diluent, carrier substance, or active portion of the material may induce inflammation due to physical properties. For example, administration of products with pH ranges either above or below physiologically neutral may induce administration site inflammation (Fig. 13.29B).

10.4 Sustained-release buprenorphine administration site reaction Pain management for NHPs is of upmost importance for animal welfare and best study-related outcomes. Sustainedrelease buprenorphine hydrochloride (SR-Bup) has proven invaluable in this regard; however, as in other animal species reported, administration of this medication often results in dermal or subcutaneous inflammation localized to the administration site in the macaque.114,115,116 The matrix in which the active ingredient resides is a DL-lactide-cocaprolactone copolymer with N-methyl-2-pyrrolidone (NMP) as a solvent, which is hydrophobic. This matrix forms a gel for slow-release of the active ingredient once injected into the skin of the NHP, but this also promotes foreign body reaction at the injection site. It has recently been reported up to 3% of rhesus monkeys administered SR-Bup might develop reactions, that there is a doseresponse relationship to these reactions, and that there may be specific MHC alleles associated with the reactions.115

FIGURE 13.29 (A) The presence of biological materials in the subcutis may result in minimal inflammation (arrow) due to immunogenicity, due to the physical presence of a material in the subcutis or due to procedural manipulation. In the latter case, the inflammatory infiltrates are usually present in control animals as well. (B) Administration of products with pH ranges either above or below physiological neutral may induce administration site inflammation, such as this case of an acidic (pH 5.5) diluent that had minor extravasation during intravenous infusion to a control animal.

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FIGURE 13.30 Administration of sustained-release buprenorphine hydrochloride (SR-Bup) to macaques: (A) There is typically a foreign body reaction surrounding the injected SR-Bup (visible as variably sized clear vacuoles) within the dermis and/or subcutis of the administration site. (B) In the acute phase of the reaction there are significant granulocytes present (arrow) in addition to the macrophages surrounding an accumulation of pale basophilic SRBup within its vacuole. (C) With time, the inflammation becomes increasingly granulomatous with multinucleated giant cells (G) and fibroplasia/fibrosis begins to form at the margins of the vacuoles (F). (D) In the final healing stages, inflammation resolves but vacuoles may remain embedded within fibrous tissue.

10.5 Carcinogen-induced neoplasia

FIGURE 13.31 Sustained-release buprenorphine hydrochloride (SRBup) administered in the scapular region of a macaque has distributed to the axillary lymph node draining the associated dermatome. Similar clear vacuoles and inflammation (arrow) as at the original administration site lesion may be noted.

In addition to the nonneoplastic lesions described, any component of the integument may respond to carcinogenic compounds resulting in cutaneous neoplasia. Carcinogenic studies using NHPs are less common than those using rodents because of the increased time required for primates to develop neoplasia, but some studies have been performed.117,118 A variety of suspected and known carcinogens have been tested in non-human primates in studies over a 32-year period, funded by the National Cancer Institute, though skin tumors were not detected in these studies.118 However, when cutaneous applied dimethylbenzanthracene was combined with UV radiation or dodecylbenzene it resulted in papillomas, basal cell tumors, and mesodermal sarcomas in rhesus macaques.119

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10.6 Steroid-induced dermal atrophy Atrophy of the skin components may occur without known etiology in some NHPs, but administration of steroid compounds is not an uncommon cause. With chronic administration of dexamethasone, for example, there may be reduction in size and number of follicular and pilosebaceous units and thinning of the overlying epidermis (Fig. 13.32). If severe, there may be grossly evident alopecia. When evaluating NHP skin for atrophy, the collection site must be considered. For example, skin of the macaque thorax may normally have thinner epidermis and fewer follicles and other adnexa in general as compared to other body regions.

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utilized toxic “warhead” for ADCs and causes acanthosis, hyperkeratosis, parakeratosis, and single-cell necrosis of the epidermis, and other epithelial tissues (Fig. 13.33).120,121 ADCs targeting the epithelial growth factor receptor (EGFR) pathway may have significant skin toxicity in the macaque and lesions may vary from acanthosis with hyperkeratosis to epidermal ulceration (Fig. 13.34AeC). These forms of toxicity have translated to the clinical setting for some ADCs.122 For ADCs linked to DNA alkylating toxin PBD or duocarmycin, there may be increased pigmentation of skin as a finding in NHPs.120,123 The change in skin pigmentation is often noted grossly and during the cage-side observations over time, an important notation since some animals have darker skin naturally. Histologically, there is

10.7 Antibody-drug conjugate skin toxicity The NHP has provided a good model of skin toxicity for antibody-drug conjugates (ADCs) in many cases and is often the only animal model suitable for testing toxicity of these biologics. The toxin conjugated to the antibody may have epidermal toxicity. For instance, pyrrolobenzodiazepine (PBD), a DNA alkylating agent, is a commonly

FIGURE 13.32 Chronic administration of dexamethasone to a cynomolgus macaque resulted in atrophy of dermal appendages, thinning of the epidermis and degeneration with calcification of the subcutis. Image courtesy of David Calise.

FIGURE 13.33 Pyrrolobenzodiazepine (PBD), a DNA alkylating agent that is a commonly utilized toxic “warhead” for antibody-drug conjugates (ADCs) may cause acanthosis, hyperkeratosis, parakeratosis and singlecell necrosis of the epidermis of the skin. The toxic insult to the epidermis is usually accompanied by inflammation.

FIGURE 13.34 Antibody-drug conjugates (ADCs) targeting the epithelial growth factor receptor (EGFR) pathway may have significant skin toxicity in the macaque: (A) Grossly, skin ulcers and flaking of skin are commonly noted. (B) Microscopically, there may be epidermal clefting and ulceration. (C) ADC targeting EGFR may induce acanthosis with hyperkeratosis with clefting and associated inflammation at the dermalepidermal interface.

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increased pigment within the epidermis and superficial dermis of the skin (Fig. 13.35A and B).

11. Fish skin grafting for non-human primate skin lesions Management of skin lesions in NHPs has always presented great challenges. Acellular fish skin grafting is a recently developed, engineered, regeneration technique that has been applied to NHP skin lesions and surgical sites with increasing success. The graft is composed of cod skin that is processed to remove all but the scaffold proteins, which are rich in type 1 collagen and omega-3 fatty acids (Fig. 13.36A).124,125 Once applied to a wound, the graft is infiltrated by host cells, and the collagen is remodeled by fibroblast activity, followed by autologous cell infiltration and stem cell migration.125 The graft material may remain visible microscopically for some time within a lesion before finally being resorbed and tissue has remodeled or regenerated (Fig. 13.36B and C).

12. Conclusion The integumentary system of NHPs responds to exogenous and endogenous factors similar to that observed in other

FIGURE 13.36 (A) Acellular fish skin graft material has a distinctive collagen scaffolding that may be identified microscopically. (B) When applied to an ulcerative lesion in a macaque, the healing process is accelerated, and the graft (arrows) eventually is replaced by normal host cellular and matrix components with re-epithelialization of the epidermis and normal sloughing of the overlying epithelial crust. (C) Remnants of the fish skin graft may be visible as a fine, collagen meshwork (arrow) within the healing wound.

FIGURE 13.35 (A) Histologically, normal macaque skin has very little intradermal pigment. (B) For antibody-drug conjugates (ADCs) linked to DNA alkylating toxins pyrrolobenzodiazepine (PBD) or duocarmycin, there may be increased pigment within the epidermis (arrows) and superficial dermis of the skin. Note the acanthosis and hyperkeratosis in this section of skin, also common skin features of alkylating agents.

species. Skin lesions may occur from topical or systemic exposure to chemicals or drugs and manifest as direct toxicity, immune-mediated reactions, or photosensitization. Conditions causing compromise to the protective integument barrier such as thermal injury, radiation, or immunosuppression predispose the animal to secondary/ opportunistic infections. Many dermatological lesions, including infectious diseases of the skin, can manifest as a result of toxicity. Other factors including sex, age, hormonal or nutritional status, and genetic background contribute to skin disease in monkeys. Although relatively few in number, familiarity with spontaneous lesions of the skin in NHPs is important to adequately assess toxicity and overall animal health status.

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J Immunot 2005;2(4):191e6. https://doi.org/10.1080/15476910 500362838. Engelhardt JA. Predictivity of animal studies for human injection site reactions with parenteral drug products. Exp Toxicol Pathol 2008;60(4e5):323e7. https://doi.org/10.1016/j.etp.2008.04.003. Thomaidou E, Ramot Y. Injection site reactions with the use of biological agents. Dermatol Ther 2019;32(2):e12817. https:// doi.org/10.1111/dth.12817. Nunamaker EA, Stolarik DF, Ma J, Wilsey AS, Jenkins GJ, Medina CL. Clinical efficacy of sustained-release buprenorphine with meloxicam for postoperative analgesia in beagle dogs undergoing ovariohysterectomy. J Am Assoc Lab Anim Sci 2014;53(5):494e501. Haertel AJ, Schultz MA, Colgin LM, Johnson AL. Predictors of subcutaneous injection site reactions to sustained-release buprenorphine in rhesus macaques. J Am Assoc Lab Anim Sci 2021;60(3):329e36. https://doi.org/10.30802/AALAS-JAALAS-20-000118. Molter CM, Barbosa L, Johnson S, Knych HK, Chinnadurai SK, Wack RF. Pharmacokinetics of a single subcutaneous dose of sustained release buprenorphine in northern elephant seals (Mirounga angustirostris). J Zoo Wildl Med 2015;46(1):52e61. https://doi.org/ 10.1638/2014-0115R.1. Sieber SM, Correa P, Dalgard DW, McIntire KR, Adamson RH. Carcinogenicity and hepatotoxicity of cycasin and its aglycone methylazoxymethanol acetate in nonhuman primates. J Natl Cancer Inst 1980;65(1):177e89. Thorgeirsson, U. P.; Snyderwine, E. G.; Gomez, D. E.; Adamson, R. H. Dietary heterocyclic amines as potential human carcinogens: experimental data from nonhuman primates. In Vivo 1996, 10 (2), 145-152. Engelhardt JA, Fant P, Guionaud S, Henry SP, Leach MW, Louden C, Scicchitano MS, Weaver JL, Zabka TS, Frazier KS, et al. Scientific and regulatory policy committee points-to-consider paper*: drug-induced vascular injury associated with nonsmall molecule therapeutics in preclinical development: part 2. Antisense oligonucleotides. Toxicol Pathol 2015;43(7):935e44. https:// doi.org/10.1177/0192623315570341. Thorgeirsson UP, Dalgard DW, Reeves J, Adamson RH. Tumor incidence in a chemical carcinogenesis study of nonhuman primates. Regul Toxicol Pharmacol 1994;19(2):130e51. https://doi.org/ 10.1006/rtph.1994.1013. Palotay JL, Adachi K, Dobson RL, Pinto JS. Carcinogen-induced cutaneous neoplasms in nonhuman primates. J Natl Cancer Inst 1976;57(6):1269e74. https://doi.org/10.1093/jnci/57.6.1269. Pillow TH, Schutten M, Yu SF, Ohri R, Sadowsky J, Poon KA, Solis W, Zhong F, Del Rosario G, Go MAT, et al. Modulating therapeutic activity and toxicity of pyrrolobenzodiazepine antibodydrug conjugates with self-immolative disulfide linkers. Mol Cancer Therapeut 2017;16(5):871e8. https://doi.org/10.1158/1535-7163. MCT-16-0641. Mecklenburg L. A brief introduction to antibody-drug conjugates for toxicologic pathologists. Toxicol Pathol 2018;46(7):746e52. https:// doi.org/10.1177/0192623318803059. Tijink BM, Buter J, de Bree R, Giaccone G, Lang MS, Staab A, Leemans CR, van Dongen GA. A phase I dose escalation study with anti-CD44v6 bivatuzumab mertansine in patients with incurable squamous cell carcinoma of the head and neck or esophagus. Clin Cancer Res 2006;12(20 Pt 1):6064e72. https://doi.org/10.1158/ 1078-0432.CCR-06-0910. Sauter, A.; Kloft, C.; Gronau, S.;

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Bogeschdorfer, F.; Erhardt, T.; Golze, W.; Schroen, C.; Staab, A.; Riechelmann, H.; Hoermann, K. Pharmacokinetics, immunogenicity and safety of bivatuzumab mertansine, a novel CD44v6-targeting immunoconjugate, in patients with squamous cell carcinoma of the head and neck. Int J Oncol 2007, 30 (4), 927-935. 123. Elgersma RC, Coumans RG, Huijbregts T, Menge WM, Joosten JA, Spijker HJ, de Groot FM, van der Lee MM, Ubink R, van den Dobbelsteen DJ, et al. Design, synthesis, and evaluation of linkerduocarmycin payloads: toward selection of HER2-targeting antibody-drug conjugate SYD985. Mol Pharm 2015;12(6):1813e35. https://doi.org/10.1021/mp500781a. 124. Magnusson S, Baldursson BT, Kjartansson H, Rolfsson O, Sigurjonsson GF. Regenerative and antibacterial properties of acellular fish skin grafts and human amnion/chorion membrane:

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implications for tissue preservation in combat casualty care. Mil Med 2017;182(S1):383e8. https://doi.org/10.7205/milmed-d-16-00142. From NLM. 125. Kotronoulas A, Jónasdóttir HS, Sigurðardóttir RS, Halldórsson S, Haraldsson GG, Rolfsson Ó. Wound healing grafts: omega-3 fatty acid lipid content differentiates the lipid profiles of acellular Atlantic cod skin from traditional dermal substitutes. J Tissue Eng Regen Med 2020;14(3):441e51. https://doi.org/10.1002/term.3005. From NLM. 126. Colman K. International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): non-proliferative and proliferative lesions of the non-human primate (M. fascicularis). Journal of Toxicologic Pathology 2021;34(3):1Se182S. https://doi.org/ 10.1293/tox.34.1S.

Chapter 14

The mammary gland of the non-human primate Shari A. Price1, Shannon R. Roff1, Karyn Colman2 and Petrina Rogerson3 1

Charles River Laboratories, Frederick, MD, United States; 2Translational Medicine/PreClinical Safety, Novartis Institutes for BioMedical Research,

Cambridge, MA, United States; 3Charles River Laboratories, Tranent, United Kingdom

1. Introduction The mammary gland can be an important tissue target in toxicology studies concerning reproductive hormones or involving therapeutic candidates that might have hormonal or proliferative effects. This is particularly important given that breast cancer is the most common neoplasia in women with an incidence of one in eight developing breast cancer in their lifetime.1,2 The ability of the mammary gland to proliferate, differentiate, and regress repeatedly makes it particularly sensitive to hormones, compounds that mimic hormone activity, or compounds that may have proliferative effects through other mechanisms. There are several similarities between human and macaque mammary tissues that support use of these nonhuman primates (NHPs) as models for mammary gland toxicology studies. The mammary glands of humans and macaques share similar anatomy and histology as well as patterns of hormone receptor expression, cytokeratin immunophenotype, and proliferation.3e5 Human and macaque reproductive physiology are also similar with macaques having a 28-day menstrual cycle similar to women with similar patterns of estrogen and progesterone fluctuation during the cycle.2,6e8 In addition, aging macaques experience declining ovarian function and menopause similar to that in humans.2 Further, humans and macaques have nearly identical milk composition and lactate during similar developmental periods for their offspring.4,9,10 Therefore, the laboratory macaque provides a useful model for mammary gland development and toxicology. The following sections provide a brief overview of normal macaque mammary gland anatomy, histology, and developmental stages; but detailed information is available in previously published documents.3,4

2. Anatomy, histology, and embryology of the non-human primate mammary gland 2.1 Gross anatomy The mammay gland of the macaque has been thoroughly reviewed by Cline, et al, in The Mammary Glands of Macaques, 2011, and the reader is refered to the article for more information.4 Adult macaques have two mammary glands in the pectoral region. The mammary consists of the skin, the parenchyma and the stroma. The skin has a indistinct areola, and the majority of the parenchymal (glandular) and stromal tissue is located below and lateral to the nipple, extending into the axilla. The mammary gland of the adult macaque is more flattened than that of humans but is histologically very similar. There is a thin but distinct mammary fat pad.3,4 Innervation consists of sensory nerves of the nipple, major lactiferous ducts, and some terminal ductal lobular units similar to humans.4,8 Five to seven lactiferous ducts exit from the nipple. The mammary gland becomes more pronounced during pregnancy and lactation. Primate mammary tissue can be studied at the subgross level using whole mount technique. The male NHP has a less developed mammary, comprised of a small nipple and a rudimentary ductal tree and lobular network of glandular tissue as compared to the female (Fig. 14.1A and B).3,4,11

2.2 Microscopic anatomy Histologically, the mammary glands of macaques and humans are very similar. The mammary gland of the nonlactating adult is made up of a system of branching ducts which end with terminal ductal lobular units (TDLU)

Spontaneous Pathology of the Laboratory Non-human Primate. https://doi.org/10.1016/B978-0-12-813088-9.00017-3 Copyright © 2023 Elsevier Inc. All rights reserved.

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2.3 Embryology and developmental stages

FIGURE 14.1 (A) The subgross anatomy of the female macaque as compared to the (B) male macaque mammary gland and nipple captured by whole slide imaging: Notice that the male has a less developed mammary comprised of a small nipple and a rudimentary ductal (black arrow) and lobular (green arrow) glandular network as compared to the female. Images courtesy of Jennifer Chilton.

(Fig. 14.2A).3 The terminal ductal lobular units are comprised of a terminal intralobular duct and surrounding alveoli (Fig. 14.2B). The ducts and terminal secretory alveoli (Fig. 14.3) are lined by two layers of simple epithelium. Cells lining the lumens of ducts vary from cuboidal to columnar and cells lining the lumen of alveoli are cuboidal.3,12 The layer of cells that lies beneath the luminal layer adjacent to the basement membrane are flattened myoepithelial cells. These myoepithelial cells produce the basement membrane surrounding ducts and alveoli and contract to facilitate milk ejection during lactation.12 The remainder of the mammary gland is made up of stroma containing fibroblasts and adipocytes. In the nonlactating gland, 95% of the volume is fat, fibrous connective tissue, and vascular and nervous supply, with the glandular tissue making up the other 5%.3 In addition to lactiferous ducts, there may also be a few lobular units in the nipple. The lactiferous ducts, which are plugged by keratin in the nonlactating animal, are lined with stratified squamous epithelium as they approach the surface. In the resting mammary gland of the macaque, there are often intraepithelial fat droplets that are not seen in human mammary gland.3,4 The male macaque mammary gland is similar to that of the female but is much less developed with a small nipple and a rudimentary ductal/lobular network approximately 5 mm in diameter.4,13

Embryonic development of the mammary gland in the macaque is similar to that of humans with breast primordia developing along the “mammary line,” which runs bilaterally and parallel to the midline of the embryo.4 The primary breast bud, which forms early in gestation, consists of a solid mass of epithelial cells which express cytokeratin 17 throughout, cytokeratins 14 and 19 basally and is continuous with overlying skin.4,14 A primordial system of ducts grows and extends out from the primary breast bud during fetal development so that a small branching ductal system is present at birth (Fig. 14.4). The fetal mammary gland is likely insensitive to hormone exposure occuring in the mother that drives maternal mammary development, but sex steroid receptors of the fetal mammary gland are poorly understood currently.4 During puberty in macaques (around 2e3 years for rhesus monkeys and slightly later for cynomolgus monkeys), nipple development precedes menstruation by several months, similar to the phenomenon in humans.3,15 The rudimentary ductal tree rapidly elongates and extensively branches to form a dense arborizing structure which differentiates centrifugally with the most mature structures near the nipple. Terminal end buds composed of an outer layer of cytokeratin 14-expressing myoepithelial or “cap” cells surrounding an inner core of cytokeratin 18/16-expressing luminal columnar cells form the leading edge of mammary gland growth in both humans and macaques.4,16,17 These highly invasive structures, which express estrogen and progesterone receptors, invade the surrounding stromal/connective tissue. Primitive ducts lined with multiple layers of epithelium and nascent lobuloalveolar units are also present and prominent in the developing mammary gland (Fig. 14.5A).4 There is a high degree of individual variation in pubertal mammary gland development where some early pubertal macaque mammary glands already consist of welldifferentiated, densely packed type I (less developed) and type 2 (maturing) lobuloalveolar units while others show substantially less development. Therefore, developmental stage and morphology of the breast should be taken under consideration and care should be taken to avoid interpretations of hyperplasia or neoplasia at this highly variable stage. Further, this individual variability might hamper or preclude animal-to-animal comparisons at this stage. There has been little description of pubertal mammary development in male macaques, but transient gynecomastia has been described.4 The nonlactating adult mammary gland consists of a fully branched ductal tree and a homogenous pattern of type 2 lobules with no apparent centrifugal differentiation like that of puberty so that there is little regional variation in mammary gland histology in the adult macaque (Fig. 14.5B).4,18

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FIGURE 14.2 (A) The mammary gland of the non-lactating, nulliparous adult female consists of a system of branching ducts (black arrow) which end with terminal ductal lobular units (TDLUs) (green arrows). (B) The TDLUs are composed of a terminal intralobular duct (arrowhead) surrounded by alveoli (asterisk). Images courtesy of Jennifer Chilton.

FIGURE 14.3 The resting glandular epithelium lining the mammary gland alveoli is composed of cuboidal cells (arrowheads) that often have intraepithelial fat droplets. The layer of cells beneath the luminal layer adjacent to the basement membrane are flattened myoepithelial cells (black arrow). The remainder of the mammary gland is made up of glandular tissue (black asterisk) and stromal support tissues (green asterisk) containing fat, fibrous connective tissue, and vascular and nervous supply to the gland. Image courtesy of Jennifer Chilton.

Sex steroid receptor expression within the NHP mammary gland is similar to that observed in humans and may be evaluated by immunohistochemistry (see Chapter 22).19 Proliferative effects during the menstrual cycle appear to be minimal but compartmental, where ductal tissues appear to be more proliferative during the luteal phase and lobuloalveolar tissues being more proliferative during the late follicular phase.20 It should be noted that rhesus macaques breed seasonally, while cynomolgus macaques do not. Seasonal changes in macaque mammary glands have not been reported.4,21 The proliferative and secretory changes induced during pregnancy and lactation signal the final and complete stage of mammary gland differentiation and function.4 Under high exposure to a variety of hormones and growth factors, the macaque mammary gland undergoes extensive growth and

FIGURE 14.4 A primordial system of ducts grows and extends out from the primary breast bud during fetal development so that a small branching ductal system is present at birth consisting of glandular and ductal tissues (arrowheads) often with adnexal structures such as hair follicles (black arrows) nearby. Image courtesy of Jennifer Chilton.

differentiation.22 The volume of glandular tissue increases between 10 and 20-fold due to epithelial proliferation and secretory distention of the ductal/lobular system.4 Lobuloalveolar units increase significantly in both number and size with the majority being mature and secretion of milk protein, immunoglobulins, and fats begins. At this stage, glandular tissue occupies the majority of the fat pad (Fig. 14.6).3,4 After birth, lactation occurs for about 12 months in the mother. Macaque milk composition is similar to that of humans but with slightly higher protein content.9,10 Postlactational involution in macaques has not been well described. However, mammary tissue from primiparous or multiparous animals examined years after last lactation appears to be generally similar to that of nulliparous animals.4 After menopause, the macaque mammary gland regresses to a ductal network with significant ductal atrophy and little proliferative activity. It should be noted that there is

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FIGURE 14.5 (A) The typical peripubertal mammary gland of a macaque has ducts lined with multiple layers of epithelium (black arrow) and nascent lobuloalveolar units (green arrows). (B) The nonlactating adult mammary gland of a macaque consists of a fully branched ductal tree and a homogenous pattern of lobules (asterisk) with little regional variation in mammary gland histology. Images courtesy of Jennifer Chilton.

should be taken not to mistake heterogeneous maturation for focal hyperplasia or carcinoma in situ.16

3. Congenital lesions of the non-human primate mammary gland

FIGURE 14.6 The proliferative and secretory changes induced during pregnancy and lactation result in extensive growth and differentiation, and the glandular tissue increases between 10 and 20-fold due to epithelial proliferation and secretory distention of the ductal/lobular system. Lobuloalveolar units increase significantly in both number and size with the majority being secretory, producing milk protein, immunoglobulins, and fats. Image courtesy of Jennifer Chilton.

individual variation in the amount of mammary tissue remaining at this life stage.4,23 Of note, the senescent mammary gland appears to remain responsive to steroid hormones.4,6,13,24 The most common observation in non-human primate toxicologic studies is immaturity of the mammary gland, due to the young age of most animals assigned to studies (usually less than 4 years of age). Although not a pathologic finding, variability in maturation and the common use of young individuals in toxicologic studies can hamper interpretation in the uninitiated. Identification of normal maturation can be differentiated from abnormal hyperplastic or neoplastic lesions by location near the margin as well as the presence of immature or undeveloped glandular tissue adjacent to the area of maturation. Therefore, care

Congenital lesions in the mammary gland of non-human primates are uncommon with supernumerary nipples (polythelia) being the primary reported finding (Fig. 14.7).25 Supernumerary nipples most commonly present as a single extra nipple on the ventral thorax aligned with the ipsilateral nipple. A high incidence of neonatal deaths in a single colony of bonnet macaques was associated underdeveloped rudimentary nipples with no known cause for the abnormality26; however, polythelia reported in other species is considered an incidental finding.25 Although other congenital lesions of mammary tissue (polymastia, amastia) have been described in humans,27 these lesions have not been described in non-human primates.

4. Degenerative lesions of the nonhuman primate mammary gland Degeneration of the mammary gland has not been reported in young primates on toxicity studies, although age-related atrophy may be expected in older animals.4,12 Single cell necrosis is not infrequently observed in macaques as part of the normal mammary gland cyclic pattern of change. There is some variation in the diameter of lactiferous ducts, which is partly dependent on the plane of section, but occasionally dilatation is excessive. Dilatation or cystic change, sometimes with accumulation of proteinaceous material, is more frequently observed in aging primates and may occur in male animals as well (Fig. 14.8A).4,12 With immature animals, there is often increased loose fibrous

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typically underrecorded due to their small size and inactive appearance (Fig. 14.9A). Rarely, physical injury to the mammary tissue may lead to mild inflammation (Fig. 14.9B). In breeding colonies, mastitis might be expected to occur occasionally in lactating females. Care should be taken when evaluating pubertal females. Terminal end buds of sprouting mammary tissue stimulate the development of loose connective tissue around the developing glands. This may cause confusion since it mimics the edematous appearance of the early phase of acute inflammation.

6. Hyperplastic and neoplastic lesions of the non-human primate mammary gland FIGURE 14.7 Supernumerary nipples (polythelia) is the most reported congenital lesion of mammary gland of non-human primates. The nipples most commonly present as a single extra nipple on the ventral thorax aligned with the ipsilateral nipple; however, the case presented has multiple, asymmetrical redundant nipples (black arrows) on the thorax and midline of the body. Image courtesy of Jennifer Chilton.

tissue that encircles the mammary tissue which should be recognized as a normal variation (Fig. 14.8B).

5. Inflammatory and vascular lesions of the non-human primate mammary gland Vascular lesions have not been reported in the mammary glands of young non-human primates on toxicity studies. Small foci of inflammatory cells occur spontaneously in mammary tissue, as at other sites, and may be regarded as physiological. Most often these infiltrates are composed of lymphocytes and/or other mononuclear cells, and they are

Hyperplastic and neoplastic lesions occur spontaneously in non-human primates with an estimated lifetime incidence of 5%e6%.28 The most commonly reported neoplasms are carcinomas, although other epithelial and spindle cell tumors have also been described.29 Classification of neoplastic and hyperplastic lesion in laboratory monkeys is most significant in chronic toxicity or carcinogenicity studies and much work has been done in classifying their histologic features and biological characteristics as potential models of human breast cancer.28e30 Some authors have estimated rates of spontaneous mammary neoplasms in macaques to be 6.1%,30 which is comparable to an incidence of 4%e8% reported in low risk populations of women.29 This low risk is most likely due to management and use of non-human primates in biomedical research, as the majority of study animals are premenopausal, nulliparous, have a controlled diet (limiting obesity), or are ovariectomized; these factors all contribute to lower breast cancer rates in women.30,31 However, the usefulness of the non-human primate model for human breast cancer has been challenged because of the heterogeneity of lesions, low spontaneous incidence, and feasibility of study parameters

FIGURE 14.8 (A) Dilatation of the mammary ducts (asterisk) is more frequently observed in aging primates and has been noted in both male and female animals. (B) Increased loose fibrous tissue that encircles the mammary glands is considered a normal variation in young animals. Images courtesy of Jennifer Chilton.

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6.1 Lobular hyperplasia of the mammary gland Lobular hyperplasia of the mammary gland can present as either focal or multifocal, with or without atypia. Focal lobular glandular hyperplasia most often presents as enlarged to expansile lobular unit(s) surrounded by hypoplastic or atrophic adjacent lobules. Hyperplastic glandular lobules are hypercellular with normal cellular phenotypes, minimal cellular and structural atypia, maintenance of normal lobular structure, and are surrounded by scant fibrous stroma.11,34 Focal hyperplasia is more commonly recognized in macaques and is observed in quiescent, nonpregnant, nonpostpartum females that are not receiving exogenous hormone treatment.11,34 Atypical lobular hyperplasia (Fig. 14.10) consists of nonuniform and irregular enlargement of acini with anisocytosis, anisokaryosis, piling of the epithelium (with at least two layers of epithelium present), loss of cellular polarity and variably cystic dilation of glands containing secretory material.12,34 These lesions have been reported in rhesus macaques and have occured in cynomolgus macaqeus at the editor’s facility. The focus can present grossly as multiple firm subcutaneous nodules.28,35 Atypical lobular hyperplasia resembles lobular carcinoma in situ (LCIS); however, in atypical lobular hyperplasia, 50% of acini are composed of atypical cells in LCIS.12,16,28,34

FIGURE 14.9 (A) Incidental small foci of inflammatory cells (black arrow) occurring in mammary tissue are composed of lymphocytes and other mononuclear cells and are without tissue disruption. (B) True inflammation is rare in NHP mammary glands, but presents with either acute or chronic inflammatory infiltrates (green arrow) and loss or disruption of tissue architecture. The common cause is trauma to the mammary gland. Images courtesy of Jennifer Chilton.

compared to the reproducible specific tumor types produced in rodent models.28 Regardless of their utility as a breast cancer model, classification of hyperplastic and neoplastic lesions of the mammary gland remains an important aspect of safety assessment in chronic non-human primate studies. Hyperplasia of the mammary gland can refer to either an enlargement of the gland in response to hormonal stimulation or reflect an abnormal proliferation of the ducts or lobules within the tissue.12 In either case, studies indicate that hyperplastic lesions exist on a continuum of severity and are often considered precursors to development of neoplastic lesions.12,32,33 Therefore, characterization of lesions in non-human primate mammary glands is an important component of human risk assessment.12

FIGURE 14.10 Atypical lobular hyperplasia consists of nonuniform and irregular enlargement of acini with anisocytosis, anisokaryosis, piling of the epithelium with at least two layers of epithelium present, loss of cellular polarity, and variable cystic dilation of glands that occurs in less than 50% of the glandular tissue. Lobular carcinoma in situ (LCIS) resembles atypical lobular hyperplasia but is differentiated by exhibiting a greater proportion (>50%) of atypical acini within the gland. Image courtesy of Jennifer Chilton.

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6.2 Ductal hyperplasia Ductal hyperplasia is a common finding in both rhesus and cynomolgus macaques and reported up to a 42% incidence in control animals in chronic studies.12,16,25,30 Ductal hyperplasia can arise from the epithelium of either the inter- or intra-lobular ducts and can induced in intact and hormone-treated ovariectomized individuals.12 Histologically, lesions are characterized by three or more layers of both glandular and myoepithelium which may form either solid or papillary structures.12,34 Atypical ductal hyperplasia is characterized by ductal hyperplasia with either cellular or architectural atypia including anisocytosis, anisokaryosis, piling of the epithelium (with at least three layers of epithelium present), and loss of cellular polarity (Fig. 14.11).12,30 Atypical ductal hyperplasia occurs at a higher prevalence in estrogen-treated animals and may resemble ductal carcinoma in situ (DCIS) but is more confined with less nuclear and cellular atypia.30,34

7. Neoplastic lesions of the non-human primate mammary gland The continued development and analysis of non-human primate models in biomedical research have produced many comprehensive reviews of mammary neoplasms in the macaque.28e30 The most commonly reported neoplasms are ductal and lobular carcinomas although squamous cell, myoepithelial, nipple adenoma (erosive adenomatosis), and spindle cell tumors have also been described.29 Morphologically, neoplastic mammary lesions in non-human primates have a high degree of heterogeneity with the most commonly reported being ductal carcinoma in situ, lobular carcinoma in situ, and invasive ductal carcinoma.25,34 In addition, several immunohistochemical similarities exist between mammary neoplasia in non-human primates and

FIGURE 14.11 Ductal hyperplasia with atypia is characterized by three or more layers of both glandular and myoepithelium which may form either solid or papillary structures and has architectural atypia, piling of the epithelium, and loss of cellular polarity. Image courtesy of Jennifer Chilton.

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women. These include an increased expression of Ki67, a loss of estrogen and progesterone receptors in a subset of carcinomas, a decrease in E-cadherin expression in lobular carcinoma and an overexpression of HER2/new oncogene in higher grade lesions.25,30 This further supports evaluation and classification of mammary neoplasms encountered in order to aid risk assessment in chronic studies using nonhuman primates. While the literature contains primarily examples of mammary neoplasia in female animals, it occurs rarely in males and should be a differential for male mammary enlargement (see Fibroadenoma).

7.1 Fibroadenoma Fibroadenomas are classified as a proliferation of the epithelium of the mammary ducts accompanied by an exuberant, concentric proliferation of fibrous connective tissue.34 Neoplastic cells are well differentiated and arranged as islands of tubular or alveolar nodules separated by abundant fibrous connective tissue.12 Fibroadenomas can be either intraductal, where the surrounding fibrous connective tissue invaginates between glandular islands, or periductular, where the glandular architecture is maintained. The associated fibrous stroma may be edematous, myxomatous, or dense fibrous.34 Fibroadenomas in macaques have been further described to include fibroadenomatous change (FAC) which includes a nodular expansion, but lacks the proliferative stroma and circumscription associated with a typical fibroadenoma.30 The example provided was from a male animal with unilateral breast enlargement (Fig. 14.12).

7.2 Ductal carcinoma in situ (DCIS) Ductal carcinoma is classified as a proliferation of neoplastic ductal epithelial cells with malignant cellular and

FIGURE 14.12 Fibroadenomas have proliferation of the epithelium of the mammary ducts accompanied by an exuberant, concentric proliferation of fibrous connective tissue. Neoplastic cells are well differentiated and arranged as islands of tubular or alveolar nodules separated by abundant fibrous connective tissue. The example provided occurred in a young male macaque (50%) of atypical acini. LCIS maintains an acinar pattern with monomorphic lobular cells that expand and often distort the acinus.12,30 Common features of malignancy (cellular atypia, nuclear pleiomorphism, mitotic figures) are minimal or absent.12

7.5 Infiltrating lobular carcinoma Similar to ductal carcinoma, lobular carcinoma may become infiltrative, entering into the surrounding mammary tissue. Classification for infiltrating lobular carcinomas based on the human literature include several patterns: classic, alveolar, solid, and tubuloalveolar. However, the most commonly encountered pattern is the classic in which tumor cells form linear arrays interspersed within the stroma. The cells usually form a concentric pattern around surviving ductular structures (Fig. 14.15).

In some cases, apical cytoplasmic blebbing and small amounts of intraluminal proteinaceous material can also be seen. This is in contrast with the human nonlactational breast tissue.4,34

8. Miscellaneous lesions of the nonhuman primate mammary gland

9. Toxicologic lesions of the nonhuman primate mammary gland

In nonlactating, mature cynomolgus macaques, evidence of a secretory change can be seen, most frequently observed as intracytoplasmic vacuoles or lipid droplets (Fig. 14.16).

Toxicologic lesions in the laboratory monkey mammary gland are often hormonally induced changes. Mammary gland enlargement can be seen in non-human primates as a response to exogenous hormonal influences and frequently resembles normal mammary development during pregnancy and/or lactation.3 These same changes (enlargement and glandular development) in males are frequently referred to as gynecomastia (Fig. 14.17A and B). In addition, some other compounds that bind to steroid receptors (e.g., spironolactone, an aldosterone antagonist or anabolic steroids) can induce similar changes.37e39 Extended or prolonged administration of combined estrogenic and progestogenic steroids has been shown to result in cystic changes and/or enhanced secretory activity, with or without obvious hyperplasia, in rhesus and cynomolgus macaques.23,40 In contrast, a reduction in mammary gland tissue (atrophy) may be observed in mature non-human primates if the administration of a xenobiotic results in inhibition of hormonal influences; however, this is not frequently reported. Single cells necrosis/apoptosis can be exacerbated by the administration of some novel anticancer therapeutics.

FIGURE 14.15 The classic pattern of infiltrating lobular carcinoma in which tumor cells form single files interspersed within the stroma. Image courtesy of Jennifer Chilton.

FIGURE 14.16 Secretory change may occur in the mammary gland of mature cynomolgus macaques, noted as intracytoplasmic vacuoles or lipid droplets, some with apical cytoplasmic blebbing and small amounts of intraluminal proteinaceous material. Images courtesy of Jennifer Chilton.

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FIGURE 14.17 (A) Mammary glandular development (black arrow) in males is termed gynecomastia and may be induced by hormone-altering chemicals or drugs, but also occurs spontaneously without known etiology on rare occasion. (B) Histology of the mammary tissue from the macaque in Fig. 14.17A: NHPs with gynecomastia often have well-developed mammary glandular tissues that cannot be distinguished histologically from those of females. Images courtesy of Jennifer Chilton.

10. Conclusion A functional understanding of the development, cyclic differentiation, normal and pathologic involution, and proliferative lesions of the non-human primate mammary gland is essential for interpretation of pathologic changes in toxicology studies. The dynamic nature of this tissue makes it a prominent target in toxicology studies, particularly for proliferative and/ or neoplastic changes. A variety of useful techniques including whole mount, standard H&E, and immunohistochemical stains can be used to elucidate the nature of any observed pathologic changes in mammary tissue.

References 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics, 2008. CA Cancer J Clin March 2008;58(2):71e96. 2. Appt SE. Usefulness of the monkey model to investigate the role soy in postmenopausal women’s health. ILAR J 2004;45(2):200e11. 3. Cline JM. Assessing the mammary gland of nonhuman primates: effects of endogenous hormones and exogenous hormonal agents and growth factors. Birth Defects Res B Dev Reprod Toxicol April 2007;80(2):126e46. 4. Cline JM, Wood CE. The mammary glands of macaques. Toxicol Pathol December 2008;36(7):134se41s. 5. Tsubura A, Hatano T, Hayama S, Morii S. Immunophenotypic difference of keratin expression in normal mammary glandular cells from five different species. Acta Anat 1991;140(3):287e93. 6. Cline JM, Soderqvist G, von SE, Skoog L, von SB. Effects of hormone replacement therapy on the mammary gland of surgically postmenopausal cynomolgus macaques. Am J Obstet Gynecol January 1996;174(1 Pt 1):93e100.

7. Uno H. Age-related pathology and biosenescent markers in captive rhesus macaques. Age (Omaha ) January 1997;20(1):1e13. 8. Macpherson EE, Montagna W. Proceedings: the mammary glands of rhesus monkeys. J Invest Dermatol July 1974;63(1):17e8. 9. Jenness R. The composition of human milk. Semin Perinatol July 1979;3(3):225e39. 10. Lonnerdal B, Keen CL, Glazier CE, Anderson J. A longitudinal study of rhesus monkey (Macaca mulatta) milk composition: trace elements, minerals, protein, carbohydrate, and fat. Pediatr Res September 1984;18(9):911e4. 11. Cameron AM, Faulkin Jr LT. Subgross evaluation of the nonhuman primate mammary gland: method and initial observations. J Med Primatol 1974;3(5):298e310. 12. Vidal J, Mirsky M, Colman K, Whitney K, Creasy D. Reproductive system and mammary gland. In: Sahota P, Popp J, Hardisty J, Gopinath C, editors. Toxicologic pathology: nonclinical safety assessment. CRC Press; 2017. p. 717e830. 13. Perry DL, Spedick JM, McCoy TP, Adams MR, Franke AA, Cline JM. Dietary soy protein containing isoflavonoids does not adversely affect the reproductive tract of male cynomolgus macaques (Macaca fascicularis). J Nutr June 2007;137(6):1390e4. 14. Jolicoeur F. Intrauterine breast development and the mammary myoepithelial lineage. J Mammary Gland Biol Neoplasia July 2005;10(3):199e210. 15. Golub MS, Hogrefe CE, Germann SL, Lasley BL, Natarajan K, Tarantal AF. Effects of exogenous estrogenic agents on pubertal growth and reproductive system maturation in female rhesus monkeys. Toxicol Sci July 2003;74(1):103e13. 16. Wood CE, Hester JM, Cline JM. Mammary gland development in early pubertal female macaques. Toxicol Pathol October 2007;35(6):795e805. 17. Howard BA, Gusterson BA. Human breast development. J Mammary Gland Biol Neoplasia April 2000;5(2):119e37.

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18. Cline JM, Soderqvist G, von SB, Skoog L. Regional distribution of proliferating cells and hormone receptors in the mammary gland of surgically postmenopausal macaques. Gynecol Obstet Invest 1997;44(1):41e6. 19. Khan SA, Bhandare D, Chatterton Jr RT. The local hormonal environment and related biomarkers in the normal breast. Endocr Relat Cancer September 2005;12(3):497e510. 20. Stute P, Wood CE, Kaplan JR, Cline JM. Cyclic changes in the mammary gland of cynomolgus macaques. Fertil Steril October 2004;82(Suppl. 3):1160e70. 21. Dukelow WR, Grauwiler J, Bruggemann S. Characteristics of the menstrual cycle in nonhuman primates. I. Similarities and dissimilarities between Macaca fascicularis and Macaca arctoides. J Med Primatol 1979;8(1):39e47. 22. Neville M. Regulation of mammary development and lactation. In: Neifert M, editor. Lactation: physiology, nutrition, and breastfeeding. Boston, MA: Springer; 1983. p. 103e40. 23. Cline JM, Soderqvist G, von SE, Skoog L, von SB. Effects of conjugated estrogens, medroxyprogesterone acetate, and tamoxifen on the mammary glands of macaques. Breast Cancer Res Treat April 1998;48(3):221e9. 24. Wood CE, Register TC, Franke AA, Anthony MS, Cline JM. Dietary soy isoflavones inhibit estrogen effects in the postmenopausal breast. Cancer Res January 15, 2006;66(2):1241e9. 25. Cline J, Brignolo L, Ford E. Urogenital system. In: Abee C, Mansfield K, Tardif S, Morris T, editors. Nonhuman primates in biomedical research. 2nd ed. Elsevier, Academic Press; 2012. p. 483e562. 26. Sesline DH, Simpson J, Henrickson RV. Neonatal deaths in bonnet monkeys born to dams with rudimentary papillae mammae. Lab Anim Sci October 1983;33(5):467e8. 27. Caouette-Laberge L, Borsuk D. Congenital anomalies of the breast. Semin Plast Surg February 2013;27(1):36e41. 28. Tarara RP. Review of mammary gland neoplasia in nonhuman primates. Breast Dis 2007;28:23e7.

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29. Cooper TK, Gabrielson KL. Spontaneous lesions in the reproductive tract and mammary gland of female non-human primates. Birth Defects Res B Dev Reprod Toxicol April 2007;80(2):149e70. 30. Wood CE, Usborne AL, Starost MF, Tarara RP, Hill LR, Wilkinson LM, et al. Hyperplastic and neoplastic lesions of the mammary gland in macaques. Vet Pathol July 2006;43(4):471e83. 31. McPherson K, Steel CM, Dixon JM. ABC of breast diseases. Breast cancer-epidemiology, risk factors, and genetics. BMJ September 9, 2000;321(7261):624e8. 32. Allred DC, Mohsin SK, Fuqua SA. Histological and biological evolution of human premalignant breast disease. Endocr Relat Cancer March 2001;8(1):47e61. 33. Arpino G, Laucirica R, Elledge RM. Premalignant and in situ breast disease: biology and clinical implications. Ann Intern Med September 20, 2005;143(6):446e57. 34. Cline JM, Wood CE, Vidal JD, Tarara RP, Buse E, Weinbauer GF, et al. Selected background findings and interpretation of common lesions in the female reproductive system in macaques. Toxicol Pathol December 2008;36(7):142se63s. 35. Nelson LW, Shott LD. Mammary nodular hyperplasia in intact rhesus monkeys. Vet Pathol 1973;10(2):130e4. 36. Hubbard GB, Wood DH, Butcher WI. Mammary carcinoma with metastasis in a rhesus monkey (Macaca mulatta). Vet Pathol September 1984;21(5):531e3. 37. Lumb G, Newberne P, Rust JH, Wagner B. Effects in animals of chronic administration of spironolactone–a review. J Environ Pathol Toxicol May 1978;1(5):641e60. 38. Wu FC. Endocrine aspects of anabolic steroids. Clin Chem July 1997;43(7):1289e92. 39. Kicman AT. Pharmacology of anabolic steroids. Br J Pharmacol June 2008;154(3):502e21. 40. Tavassoli FA, Casey HW, Norris HJ. The morphologic effects of synthetic reproductive steroids on the mammary gland of rhesus monkeys. Mestranol, ethynerone, mestranol-ethynerone, chloroethynyl norgestrel-mestranol, and anagestone acetate-mestranol combinations. Am J Pathol May 1988;131(2):213e34.

Chapter 15

The respiratory system of the non-human primate Alessandro Piaia1, Begonya Garcia2, Thierry D. Flandre1 and Jennifer A. Chilton3 1

Novartis Pharma AG, Basel, Switzerland; 2Charles River Laboratories, Evreux, France; 3Charles River Laboratories, Reno, NV, United States

1. Introduction The respiratory system, which extends from the nasal cavity to the alveolus of the lung, is covered by a large mucosal surface that is 25 times larger than the surface covered by the skin. The extensive alveolar capillary surface is a direct interface between air and circulating blood, and as such, is prone to injury from both airborne and bloodborne toxicants. Airborne substances can harm different levels of the respiratory system, by virtue of their ability to reach a site of deposition via inhalation and diffuse into the tissues and/or bloodstream. Aerodynamic factors can determine the site of deposition, with particles larger than 10 mm usually deposited at the nasal passages, particles between 2 and 10 mm deposited on the mucus covering of the bronchial tree, and smaller molecules depositied as far as the alveoli.1e3 The knowledge of respiratory pathology associated with toxicities of drugs or other substances has been limited. Previous literature on inhaled small molecules pertaining to non-human primates (NHPs) was scarce and related to toxicants (i.e., smoke, ozone, or dust) rather than drugs.4 The proper selection of the animal species for modeling the human pulmonary disease process must consider anatomical and functional similarities and differences in both upper5,6 and lower7,8 respiratory tracts across the species. For this reason, NHPs offer a more appropriate model for studying the toxic effects of inhaled substances on the nasal passages and for extrapolating the findings to humans.9 NHPs are also the better species for the evaluation of biological agents by virtue of the close homology of NHP immune system to the human immune system.10,11 Pathology related to drug toxicity in the respiratory tract of the non-human primate is an evolving field. Before emergence of biologics, NHPs were not the primary large species of choice for respiratory toxicology studiesdthat

honor went to the dog. However, with the growing development of biologic therapeutics, also called large molecules (e.g., antibodies, proteins, oligonucleotides), the use of NHPs as test subjects is expanding, including for applications that are respiratory system targeted.

2. Anatomy and histology of the respiratory system The respiratory system is summarized here. It is composed by a conduction system (airways) and a parenchyma with specific blood-air interface for gas exchange. A unique characteristic of the conductive system is that the epithelial lining is quite thin and is kept moist by a constantly renewed mucus layer. These two features act as a barrier to particulate matter and passageway for the air flow. The mucus moves orally along the mucosal lining, and is eventually removed by expectoration or is swallowed. Air moves freely and rhythmically bidirectionally within the system.12,13 By convention, the respiratory system is divided into an upper portion, (including nasal cavities, pharynx, larynx, and trachea), and a lower portion, (including bronchial tree and lung parenchyma).9 This basic structure of the respiratory system is common among mammal species. The respiratory system is complex and present with numerous species-specific adaptations in the architecture and in the cell composition.3 In many NHPs, including macaques, there are laryngeal diverticula, also known as air sacs or laryngeal sacs.14

2.1 Nasal cavity NHPs are a oronasal-breathing species and are regarded as microsmatic (poor sense of smell), due to the less complex architecture of the turbinates and smaller size of brain

Spontaneous Pathology of the Laboratory Non-human Primate. https://doi.org/10.1016/B978-0-12-813088-9.00021-5 Copyright © 2023 Elsevier Inc. All rights reserved.

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olfactory bulbs, as compared to other laboratory species (dogs and rodents). This is a reflection of lesser olfactory capabilities in these species.6,15,16 However, this concept has recently been challenged for some species, such as the common marmoset (Callithrix jacchus) which has a larger extension of olfactory epithelium in the nasal cavity and is considered to be macrosmatic.16,17 The nasal cavity has a complex anatomical organization that is relatively similar among great apes, macaques, and marmoset.5,6,9,18 The nasal cavity is divided into multiple regions. The nasal vestibule (Fig. 15.1) includes the frontal opening with the nares, followed by the proper nasal cavity filled by the nasal turbinates, and then the posteriorly located nasopharynx. The vestibulum of the nose is a relatively wide cavity in all primate species that is usually empty space in laboratory monkeys with the exception of the marmoset, in which a projection of the maxillary turbinates extends to the vestibule. The nasal septum divides the nasal cavity into two chambers, and has a dorsal thickening as a characteristic feature of primates.5,18,19 Some structures usually seen in other laboratory species (Massera body, swell bodies, and/or lateral nasal glands) are not present in the primate species; however, macaques possess a nasopalatine duct connecting the vestibule to the oral cavity.5,6,20 The vomeronasal organ is a tubular, epithelial covered structure present in the common marmoset and located on the base of the nasal septum. It connects to oral and nasal cavities via the nasopalatine duct. Histologically this organ is characterized by a specialized olfactory nonciliated neuroepithelium.21 Intraepithelial ducts are also present across the thickness of the olfactory

FIGURE 15.1 The nasal cavity at the level of the vestibule in a macaque: Posterior to the nares, the nasal cavity (NC) is divided by the cartilaginous septum (S) covered by respiratory epithelium. At this level, the residual crowns of the teeth (RC) and maxillary sinuses (MSs) are visible within the maxilla and the roof of the oral cavity (OC) forms the ventral border of the tissue section.

epithelium, connecting the submucosal glands (gland of Jacobson) to luminal aspect of the vomeronasal organ.22 Receptor-free epithelium may be observed and may increase in distribution with aging.23 In NHPs, the nasal turbinates are usually paired, and comprised of the maxilloturbinate and the ethmoturbinate. They are characterized by a simple scroll shape without the complexity usually seen in other laboratory animal species.6,24 The maxilloturbinate lies ventrally, close to the base of the nasal cavity and extends from the vestibule to the nasopharynx. The ethmoturbinate, arising from the ethmoidal bone, lies dorsal of the maxilloturbinate. Histologically, four types of epithelium are found in the nasal cavities of laboratory macaques, marmosets and rats, and have been well described by Harkema, et al. and Wako, et al., and consist of the following: The squamous epithelium (SE), the transitional epithelium (TE), the respiratory epithelium (RE), and the olfactory epithelium (OE) (Diagram 1). The distribution of the four types of epithelium is generally similar across NHPs used in the research setting with some differences mainly in the SE and TE distribution in the macaque and the marmoset species. The SE is a nonkeratinized to slightly keratinized stratified epithelium, mainly present on the dorsal part of nasal vestibule in the macaque species, while in the common marmoset it is mainly present ventrally in the vestibule.5,6,18 The interface with TE is abrupt and the TE is characterized by multilayered cuboidal to low-cylindrical epithelium. TE epithelium is

DIAGRAM 1 Distribution of nasal epithelium on the lateral wall of the nasal cavity in the macaque (A) and rat (B): Four distinct epithelial cell populations line both mammalian speciesdOE, olfactory epithelium; RE, respiratory epithelium; SE, squamous epithelium; TE, transitional epithelium. However, considerably more OE line the intranasal surface of the rat, compared to the monkey. Other structures depicted: B, brain; et, ethmoturbinate; it, incisor tooth; mt, maxilloturbinate; na, naris; N, nasal associated lymphoid tissue (NALT); nt, nasoturbinate. Reproduced with permission from Harkema JR, Carey SA, Wagner JG. The nose revisited: a brief review of the comparative structure, function, and toxicologic pathology of the nasal epithelium. Toxicol Pathol 2006;34(3):252e69.

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DIAGRAM 2 Diagram of the epithelial population in the nasal cavity of the common marmoset, lateral wall of the right nasal fossa: e, ethmoturbinate; EB, ethmoidal bone; m, maxilloturbinate; n, naris; NTE, nasal transitional epithelium; OE, olfactory epithelium; RE, respiratory epithelium; SE, squamous epithelium. Reproduced with permission from Wako K, Hiratsuka H, Katsuta O. Anatomical structure and surface epithelial distribution in the nasal cavity of the common cotton-eared marmoset (Callithrix jacchus). Exp Anim 1999 Jan;48(1):31e6.

microscopically composed of a visible basal lamina with up to 4 types of cells: mucus cells, luminal nonciliated cells with few to no secretory granules, small mucus granule cells and basal cells, as described in the bonnet monkey (Macaca radiate).5,25 The OE has variable distribution in the macaque as compared to the common marmoset (Diagram 2). In the macaque, the OE is distributed roughly diagonally from the pavement of vestibule to the floor of the nasal cavity, while in the common marmoset it originates from the roof of the vestibule diagonally and caudally to the pavement of the nasal cavity.15,18 The RE is the most abundant epithelium in the nose and is diffusely present over nasal cavities and turbinates. The transition from TE to the RE is rather gradual, and not abrupt as seen between TE and SE; however, the RE is still readily recognizable by a thicker basal lamina under the epithelial layer.5 The RE is regarded as pseudostratified epithelium, due to presence of basal cells, cylindrical ciliated cells, elongated mucus cells spanning up to the epithelial surface, and small mucus granule cells present in the middle of the epithelium. The arrangement gives a global effect of nuclear stratification.5,18,19 The OE is limited to the dorsal aspect of the nasal cavity, covering the dorsal posterior nasal cavity dome and partially distributed over the dorsal attachment of ethmoturbinate and the upper part of the nasal septum and lateral wall.5,26 The OE in the common marmoset is slightly more widely distributed than in the macaque, being mainly present along the nonturbinal surfaces, anteriorly on the nasal septum and posteriorly on the small cupular recess.16,17,27 The transition of the OE with RE is not well defined. Histological organization of the OE is characterized by a highly specialized cell population, namely olfactory sense neurons, sustentacular cells, and basal cells. These unique cells are arranged in a pseudostratified epithelium, laying on a thick submucosa where subepithelial glands (Bowman’s glands) and dendrites of the olfactory sense neurons can be seen.2,5,26

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Paranasal sinuses are variably present in different species of primates and are valuable features utilized for phylogenic analysis of primates.28 Interestingly, macaques are the only cercopithecoid monkeys with a maxillary sinus which is relatively small and restricted to the molar region of the maxilla.29,30 On the contrary, platyrrhine monkeys usually have a well-developed maxillary sinus. In the common marmoset it is located in the medial part of the maxilla.31 All sinuses are covered by simple respiratory epithelium. Nasal associated lymphoid tissue (NALT) has a characteristic lymphoepithelium covering the lymphoid aggregate and may be found on both lateral and septal aspect of the nasopharynx. This is equivalent to the human Waldeyer ring, which is made up of adenoid, bilateral tubal, palatine, and lingual tonsils.2,15,26

2.2 Larynx The larynx (Fig. 15.2) is the first site of constriction of the airflow in the respiratory tract and therefore, a site of deposition of inhaled, larger particulate material. The anatomical organization is less complex than the one found in the nasal cavities, and the species differences less remarkable. The main difference between rodent, dog and monkey is the position of the laryngeal diverticula. In the nonrodent species the diverticula are paired and extend laterally while in the rodent the diverticulum is a single ventral invagination (ventral pouch). In the monkey the diverticula are lined by respiratory epithelium. Three types

FIGURE 15.2 Histologic section at the level of the proximal lower respiratory tract of the macaque in the sagital plane: The oropharynx (OP) and nasopharynx (NP) meet at the larynx. Ingesta diverges into the esophagus (E) assisted by the epiglottis (EG) and air moves into the larynx. The vocal cords (VC) are skeletal muscle and contingent with the areolar support tissue and the vocal folds (VF). The other laryngeal muscles extend to the trachea (T), which is also delineated by the presence of tracheal cartilage (TC) and by thyroid gland tissue (TG). Laryngeal glands (LG) are distributed through the lamina propria of the larynx and associated with the laryngeal sinus (LS) and vocal folds.

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of epithelium can be found in the larynx-squamous and respiratory. The squamous epithelium extends over the vocal cords and the transition with the respiratory epithelium occurs at the opening of the diverticula.32 Below this point and entering the trachea, nonsquamous respiratory epithelium is the only epithelial type lining the remaining of the conductive system of the respiratory tract.

2.3 Tracheobronchial tree The tracheobronchial tree begins distal to the larynx, conducting air to the lower respiratory system (lung). It is relatively similar among non-human primates used in research, and it takes the pattern of irregularly dichotomous branching that is similar, but not identical to, the symmetric dichotomous branching of the human tracheobronchial tree.33e36 Cartilage rings in the trachea (Fig. 15.3A and B) and cartilage plaques in the bronchial tree follow the airways toward regions of increased peripheral branching and

to the proximal nonrespiratory (terminal) bronchioles. Monkeys have relatively few nonrespiratory bronchioles and several respiratory bronchioles before the alveolar ducts and alveolar acini.8,36 The tracheobronchial airways are lined by a continuous pseudostratified respiratory epithelium, which begins in the proximal trachea and extends progressively lower to the bronchial ramification. There are several publications describing the different types of epithelium in primates and other laboratory species;7,8,36e38 however, in the macaque, the epithelium consists of 4 cell types: ciliated cells, mucous or goblet cells, small mucous granule cells, and basal cells.37 The Club cells (nonciliated bronchiolar epithelial cells, formerly known as Clara cells39) are described in the macaque and mainly found in the terminal bronchioles.7,37 In the common marmoset, the distribution is similar, with minor cell type variation present in the trachea.40

2.4 Lung Macroscopically, the macaque and marmoset lungs have two lobes on the left and four lobes on the right as compared to humans with two left lobes and three right lobes.41 In the macaque, the left cranial lung lobe has a distinct fissure that extends from near the bronchial bifurcation and that gives the appearance of separate lobe (Fig. 15.4A and B). The terminal section of the airway tree is the structural and functional anatomical unit of the lung-the pulmonary acinus (Fig. 15.5). The lung is composed of nonrespiratory bronchioles that branch into several respiratory bronchioles, which in turn open into alveolar ducts and alveoli. It’s lined by epithelial cells of the bronchioles and the alveolar cell types.8 With age, there are decreases in alveolar number in macaques that mirrors the phenomenon in humans.42

3. Congenital lesions of the respiratory system

FIGURE 15.3 (A) In cross-section, the normal macaque trachea is delineated by hyaline cartilaginous “rings” (C) and encased in an adventitia (A). (B) The lumen of the trachea is lined by pseudostratified ciliated respiratory epithelium (EP) subtended by prominent basal lamina (BL) and lamina propria (LP). Tracheal glands (TG), smooth muscle (M), and lymphoid tissue (not pictured) lie within the submucosa (SM). In the macaque, there may be little delineation between the lamina propria and submucosa of the trachea.

Congenital lesions are rarely reported in non-human primates and they represent a minority of anecdotal references in the macaque, especially when associated with healthy status impairment or death of the subject. For example, congenital cystic adenomatoid malformation (CCAM) has been described in a neonatal cynomolgus monkey, which died 17 days after the birth.43 Bronchiectasis can occur with low incidence in cynomolgus monkeys, and it may be of congenital origin in the majority of the cases. A common finding in premature or aborted macaque offspring is lung immaturity (Fig. 15.6) as the primate lung completes maturation shortly before birth, approximately Day 168 of the normal gestation period.44 The immature lung is characterized by cuboidal alveolar epithelium and widened alveolar septa. Atelectasis indicative of in utero death is also a common feature of still-born infants in both macaque and marmoset species.14,45

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FIGURE 15.5 Histology of the normal macaque lung: Nonrespiratory bronchiole (NRB) is surrounded by smooth muscle (SM) and branches into several respiratory bronchioles (RBs), which in turn open into alveolar ducts and alveoli (A). Vascular structures (V), nerves, and lymphoid tissue (L) are also present at the level of the terminal bronchus and bronchioles.

FIGURE 15.4 The macaque lung dorsal (A) and ventral (B) views: Left cranial lobe (1), left caudal lobe (2), right cranial lobe (3), right middle lobe (4), right caudal lobe (5), and accessory lobe (6). Note: there is a distinct fissure (arrows) in the left cranial lung lobe that should not be misinterpreted as a seperate lung lobes.

4. Degenerative lesions of the respiratory system Degenerative lesions of the lung are infrequent and usually isolated spontaneous findings in non-human primates. They usually occur associated with other findings or as toxic lesions, especially in association with inflammatory changes. Any inflammation in the nasal cavity is usually accompanied with transient degenerative lesions in the most sensitive olfactory and the transitional-cell epithelium.

4.1 Degeneration of the respiratory epithelium Degenerative changes in the olfactory epithelium occur secondary to inflammation or mechanical insult, and may include cellular vacuolation, necrosis, fibrosis, or atrophy.26

FIGURE 15.6 Macaque lung, gestation day number 159: The alveoli are lined by primarily cuboidal epithelium and the alveolar septa are wide. Specimen was inflated with formalin for preservation. Image courtesy of David Calise.

For instance, mucosal erosion of the turbinates may occur due to nasogastric catheterization.46 More severe lesions may be associated with either ulceration or hyperplastic changes. These changes must be differentiated from those with an infectious or toxic etiology (see Toxicologic lesions of the respiratory tract).

4.2 Pleural or interstitial fibrosis and adhesions Foci of fibrosis, either pleural (Fig. 15.7) or interstitial, may be noted in the lungs of NHPs. These foci are usually characterized by areas of increased mature collagen, with occasional fibroblasts/fibroplasia in chronic-active lesions. There may be low numbers of inflammatory cells associated with the fibrosis. The alveoli adjacent to pleural fibrotic foci may be distorted, with fibrosis of the associated

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at any level of the respiratory system and may be observed in up to 100% of NHPs utilized for toxicology studies.47 Spontaneous vascular lesions of the lung are more rare in NHPs, and when present, are usually focal and of minimal severity. Under most circumstances, the microscopic evaluation of the respiratory system is limited to the trachea, bronchus, bronchioles, and alveoli. On occasion, the larynx, pharynx, and nasal cavity may also be examined, but these last tissues are rarely included for routine toxicologic studies unless indicated as target tissues of the test substance.

5.1 Mixed or mononuclear cell infiltrates of the respiratory system FIGURE 15.7 Foci of pleural fibrosis (arrows) are not uncommonly noted in the lungs of NHPs and my range in character from loose fibrous to dense fibrous tissue depending on the maturity of the collagen. Low numbers of inflammatory cells (arrow heads) are often associated with fibrosis. Both fibrosis and inflammatory cell infiltrates may extend into the adjacent lung interstitium.

FIGURE 15.8 Pleural adhesions (white arrow) between lung lobes or between lung and thoracic wall or diaphragm are frequently noted on the pleural surface of the NHP lung, due to variety of etiologies, including pleuritis or parasite migration. The case presented was due to parasitic cysts (black arrows) on the pleural surface of the lung.

septa, while the mesothelium is usually normal or mildly hypertrophic. The foci may be the result of focal pleuritis that has resolved, or the migration of parasites through the pleura, and may be associated with pleural adhesions to the body wall or diaphragm (Fig. 15.8).

5. Inflammatory and vascular lesions of the respiratory system Inflammatory cells, from minor infiltrates to fulminant inflammation, are the most commonly encountered findings

Inflammation in the respiratory system is usually of minimal mononuclear to mixed-cell type which can occur at any location as a spontaneous finding. It accounts for roughly half of the inflammatory changes that can be encountered in the respiratory system and should be differentiated from the normal respiratory lymphoid tissues commonly observed in the respiratory system of NHPs. In the nasal cavities, cellular infiltrates may be observed, occasionally in association with degenerative changes including ulceration of the mucosa. The most common locations within the nasal cavity are the midseptal and medial aspect of the vestibulum, at the transition between squamous to transitional-cell epithelia, or between transitional-cell and respiratory epithelia.5 A collection of inflammatory cells, mainly granulocytes, may be admixed with the mucous on the epithelial surface of the nasal cavities.5 In the lung, inflammation may occur at interstitial, alveolar, and/or bronchial/brochioloalveolar locations, with variable tissue reaction. Interstitial inflammation is mainly centered around vessels and alveolar septa, with occasional effects in the alveolar spaces (Fig. 15.10A and B). It is occasionally associated with minor degenerating inflammatory cells, increased alveolar macrophages, minor hemorrhage and/or alveolar epithelial hyperplasia. The lesion is usually focal or multifocal, limited in severity, and very commonly noted at the tips or margins of the lung lobes (Fig. 15.9).45 On occasion, the mixed-cell inflammation may resemble a poorly organized abscess (Fig. 15.10C) with increased neutrophils and hemorrhage. At bronchioalveolar location, the lesion may have hypertrophy/ hyperplasia of the bronchiolar epithelium, variable thickening and distortion of the septa by fibrotic alteration, and accumulation of inflammatory cells. Inflammation at bronchial level is very rare48; however, very low numbers of neutrophils are common in the submucosa of the trachea. Alveolitis (Fig. 15.11) is also quite rare, but is noted as an incidental finding in NHPs, likely representing a low-level infection of unknown etiology. Aggregation of alveolar macrophages (Fig. 15.12) may be focal or multifocal in the alveolar space, usually spanning over a couple of alveolar lumina. The change is relatively frequent. Macrophages may adhere to one

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FIGURE 15.9 Lung inflammatory cell infiltrates are usually of chronic nature and focal or multifocal in NHPs. There may be perivascular, alveolar, and/or interstitial distribution, but the lesion is usually limited in severity and distribution. It is commonly noted at the tips or margins of the lung lobes (arrow).

another and their cytoplasm varies in appearance from a diffusely pale eosinophilic to foamy or microvesiculated

5.2 Eosinophilic airway inflammation Although rare, there are spontaneous cases of eosinophilic to lymphoplasmacytic airway inflammation resembling allergic bronchitis noted in macaques (Fig. 15.13).49,50 The lesions are characterized by variable numbers of eosinophils commonly admixed with lymphocytes and plasma cells. The infiltrates are predominantly located within and/ or subtending the respiratory epithelium of the bronchus, bronchioles, and to lesser extent, the trachea. Similar lesions have been induced in NHPs by the respiratory administration of various allergens, including dust mites51,52; however, the allergens related to the spontaneous cases have not been identified.

5.3 Foreign body granulomas and aspirated materials Foreign body granulomas or granulomatous inflammation may occasionally be seen in the NHP lung, most often related to aspiration food but occasionally other foreign substances are noted (Fig. 15.14 A and B). There may be regurgitation and aspiration of test article during orogastric or nasogastric intubation studies. Depending on the physical properties of the test article, such as pH and viscosity, there may be significant injury and inflammation to the lung parenchyma (Fig 15.15). Hair fragments are rarely noted in

FIGURE 15.10 (A) Interstitial inflammation is usually centered around vessels and alveolar septa, with occasional effects in the alveolar spaces. It may be associated with minor degenerating inflammatory cells, increased alveolar macrophages, minor hemorrhage (arrow), and/or alveolar epithelial hyperplasia. (B) Increased numbers of alveolar or interstitial macrophages (arrow) may be noted in inflammatory foci of the lung. (C) Rarely, the infiltration may appear abscess-like with loss of tissue architecture, increased neutrophils, and hemorrhage. Infectious or toxic agents should be suspected with these foci.

the lung following grooming and inhalation of the hair. Granulomas tend to have a circular morphology, sharply demarcated from the surrounding tissue. The granuloma cellularity may vary from giant cells with few lymphocytes

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FIGURE 15.11 Alveolitis is quite rare, but is rarely noted as an incidental finding in NHPs, likely representing a low-level infection of unknown etiology.

FIGURE 15.12 A small dense aggregation of alveolar macrophages with eosinophilic cytoplasm fill a cluster of alveolar spaces, a commonly encountered finding in NHP lungs.

FIGURE 15.13 Spontaneous eosinophilic airway disease in NHPs is occasionally noted. The specific allergens have thus far not been identified. The finding is characterized by eosinophils usually admixed with mononuclear cells that surround or permeate the respiratory epithelium of small airways.

FIGURE 15.14 (A) Foreign body granulomas may occasionally be present in the lung of NHPs, most often related to aspiration of food and remnants of plant material (arrow) may be noted. Some lesions may have significant mixed cell infiltrates and localized tissue injury, likely due to the introduction of bacteria with the foreign material. (B) Other foreign substances of unknown origin (arrow) that have been inhaled may lodge in the airways or alveoli leading to granulomatous inflammation.

FIGURE 15.15 Aspiration of test articles during oral administration may occur. Many oral test articles have acidic pH, which may result in significant tissue injury and inflammation in the lung, including epithelial degeneration or necrosis and septal fragmentation and blunting.

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to a mixture of giant cells, lymphocytes, macrophages, and granulocytes. The granulomatous inflammation may have a more infiltrative pattern, associated with variable fibroblastic proliferation and hyperplasia or metaplasia of the alveolar epithelium. In some instances, foreign materials induce a more suppurative reaction, due to contamination with bacteria or due to acute injury.

5.4 Vasculitis Inflammation of vessels (vascular inflammation, synonym vasculitis/arteritis/periarteritis) is a very rare event in the respiratory system as a spontaneous finding. While some cases are focal in the lung, many cases have multifocal lesions in various organs due to spontaneous polyarteritis nodosa.53,54

5.5 Thrombosis and thromboembolism Thrombosis (Fig. 15.16 A and B) of lung vessels is occasionally observed in NHPs, most commonly with intravenous administration of test articles. Vascular thrombosis, in association with interstitial lung inflammation, edema, and perivascular inflammation, consistent with focal infarct, have been reported with the incidence of these findings associated with the incidence of venous inflammation and thrombus formation at the infusion site.47,55 The lung thrombi may increase in incidence with continuous infusion time length and may be more prevalent with substances that contain particulates or have a low pH.56 The character of the thrombus is determined by chronicity and extent of tissue injury associated with the lesion. The lung of NHPs may have various materials or tissues that become lodged in the small vasculature (see Chapter 19). Hair fragments that embolized following intravenous injection are not uncommon and some catheters may shed materials that lodge in the lung vessels (Fig. 15.17). Bone marrow emboli (Fig. 15.18) and fat emboli are both reported in NHP lungs secondary to bone fractures resulting in escape of marrow content into the vasculature.

6. Hyperplastic, metaplastic, and neoplastic lesions of the respiratory system In the nasal cavities, hyperplastic and metaplastic changes are not infrequent. The transitional-cell epithelium is the most sensitive, and hyperplastic or metaplastic changes may be observed near the transition zones between epithelial cell types.5

6.1 Goblet cell hyperplasia and metaplasia Mucous cell (goblet cell) hyperplasia and/or metaplasia can occur in the transitional-cell and/or respiratory epithelium (Fig 15.19). Recommendations for proliferative lesions of the respiratory tract are currently described in NHPs from the International Harmonization of Nomenclature and

FIGURE 15.16 (A) Acute thrombosis is characterized by intralumenal fibrin and blood cells adhered to the vessel wall. They are occasionally observed in studies with intravenous administration of test articles and other substances. The character of the thrombus is determined by chronicity and extent of tissue injury associated with the lesion. (B) With chronicity, the thrombus usually has associated inflammatory cell infiltrates and may either reorganize and resolve or form a permanent fibrous plug.

Diagnostic Criteria (INHAND): Non-proliferative and Proliferative Lesions of the Non-human Primate (M. Fascicularis). J Toxicol Pathol. 2021;34 (3Suppl).57 These recommendations for description and morphologic diagnosis of proliferative lesions of the respiratory tract are: (a) metaplasia for an increased number of mucous cells in the transitional-cell epithelium; (b) hyperplasia for increased number/density of mucous cells in respiratory epithelium; (c) hyperplasia/metaplasia when the mucosal folding and pseudoglands or intraepithelial crypt formation are seen.57

6.2 Osseous or cartilaginous metaplasia of the lung Osseous metaplasia and cartilaginous metaplasia are known incidental findings in the common marmoset lung, although at relative low incidence (2.0%).58 Metaplastic bone and cartilage also are occasionally noted in the macaque lung (Fig 15.20).

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FIGURE 15.17 Rarely there may be shedding of catheter sheath material from the infusioin site into the blood stream. These fragments may lodge in the lung vasculature, forming a characteristic hyaline, crescent-shaped embolus (arrow) that attracts inflammatory cells, including foreign body-type macrophages.

FIGURE 15.19 Metaplastic change from transitional epithelium to goblet (mucous) cells (arrows) may be observed in the respiratory epithelium of the upper respiratory tract. With increased number of goblet cells there may be pseudogland or intraepithelial crypt formation.

FIGURE 15.20 Small foci of well-formed metaplastic cartilage are occasionally noted within the lung of NHPs. Similar metaplastic foci of bone are also occasionally noted in NHP lungs. FIGURE 15.18 A rare consequence of bone fracture is the intravascular bone marrow and/or fat embolus in the lung. These tissues may escape into the vasculature following a fracture and lodge in various small vessels of the lung.

6.3 Alveolar epithelial hyperplasia Hyperplasia of the alveolar epithelium (Fig 15.21) may be observed focally or multifocally, and it is often accompanied by increased interstitial fibrous tissue.46 The finding is characterized by a proliferation of Type II alveolar epithelial cells. These foci presumably occur in areas of previous insult to the Type I epithelium and represent a form of healing. Alveolar epithelial hyperplasia is characterized by compactly spaced cuboidal to columnar cells with scant cytoplasm lining the alveoli or protruding on short stalks of collagen as small papillary structures. As

FIGURE 15.21 Alveolar epithelia hyperplasia is characterized by small fronds of cuboidal to columnar epithelium with scant cytoplasm lining the alveoli or protruding on short stalks of collagen as small papillary structures. Note the lack of tissue compression or cellular atypia.

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with humans and other animals, cellular atypia, multinucleation, or increased mitoses with compression of adjacent tissue would signal a transition from hyperplasia to an adenoma in the lung.

6.4 Smooth muscle hypertrophy and hyperplasia Less commonly identified is smooth muscle hypertrophy or hyperplasia of the NHP lung (Fig 15.22A and B). This may be a consequence of allergic airway disease, in which case there are usually significant numbers of eosinophils; however, it may be noted without significant inflammation associated. Whether this represents a spontaneous smooth muscle hypertrophy or a case of paucigranulocytic asthma similar to that reported for humans is not known.59,60

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et al.61,62 These lesions included nasal cavity adenoma, pulmonary squamous cell carcinoma, bronchioalveolar carcinoma (Fig. 15.23A and B), bronchiolar papilloma, and chondromatous hamartoma. Almost all of the reported neoplastic lesions were present in relatively young subjects of 6 years or less. Lung alveolar adenoma seems to be slightly more frequent in aging rhesus monkeys (Macaca mulatta).63 There are anecdotal reports present for papillary adenoma in macaques63 and primary squamous cell carcinoma in the rhesus monkey.64 For the common marmoset, there are relatively rare instances of lymphosarcoma and fibrosarcoma described; however, these were metastases of the tumors from primary locations in other organs.45

7. Miscellaneous lesions of the respiratory system

6.5 Pulmonary neoplasia

7.1 Pulmonary mineralization

Neoplastic lesions are rarely reported in the non-human primates utilized for toxicologic studies. There are individual cases of neoplasia observed in the respiratory system of cynomolgus monkeys, nicely reviewed by Kaspareit

Mineralization of the lung vessels and/or the lung parenchyma are casual observations in the lung of cynomolgus monkeys, but not reported in the literature as common lung change. Mineralization is far more common in the common

FIGURE 15.22 (A) Smooth muscle hypertrophy occurs rarely in NHPs as a spontaneous finding and is usually focal to multifocal. (B) There may be low numbers of inflammatory cells with minor epithelial hypertrophy or hyperplasia overlying a core of hypertrophic or hyperplastic smooth muscle (SM). Note the accumulation of pigmented material associated with the mononuclear cell infiltrate (arrow). Images courtesy of Rebekah Keesler.

FIGURE 15.23 A case of bronchioalveolar adenocarcinoma in an aged macaque: (A) The tumor is highly invasive, effacing, and replacing the pulmonary architecture. (B) The tumor cells are composed of highly pleomorphic, poorly differentiated epithelial cells forming sheets and occasional alveolar-like spaces interspersed with a dense collagenous stroma and including multifocal necrosis and hemorrhage.

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marmoset lung, usually multifocal and either interstitial or subpleural45 or affecting the lung vessels. While the lesion is usually regarded as dystrophic in origin in the cynomolgus monkey, in the common marmoset it has to be carefully assessed, as metastatic mineralization may potentially be linked with overdose of vitamin D.65

7.2 Extramedullary hematopoiesis Megakaryocytes are resident cells in the lung as a primary site of terminal platelet production66 and may occasionally be observed in the lungs of common marmoset.58

7.3 Pulmonary pigments Pigment-laden macrophages containing endogenous pigment and exogenous particulate material can be commonly found in the lung of NHPs, usually within the alveolar/interstitial macrophages. They may accumulate in perivascular and peribranchial locations; however, in more severe cases they can be found widespread in the interstitium. Unless special stains or electronic microscopy (EM) analysis is applied, it may be difficult to clearly differentiate whether the deposits are composed of hemosiderin, lipofuscin, other endogenous materials, or are composed of exogenous materials. Hemosiderin deposits are typically red-brown, granular deposits that are predominantly perivascular and may be widespread in the lungs of macaques, leading to grossly observable brown foci in lungs (Fig. 15.24A and B). It has been proposed that they most often represent hemosiderin deposition after vascular blood leakage.67 Fine black pigments, either free in tissue or phagocytosed by macrophages and deposited in the interstitium at the bronchial level, are usually examples of anthracosis (Fig. 15.25). These deposits are without tissue reaction or excessive inflammation noted.46,47

FIGURE 15.24 (A) Hemosiderin deposits may be widespread in the lung, leading to macroscopically observable brown foci in NHP lungs. (B) Microscopically, hemosiderin typically forms perivascular red-brown, granular deposits that may also be present in alveolar macrophages.

7.4 Continuous infusion pneumonitis A consequence of long-term continuous infusion in macaques may be interstitial to alveolar pneumonitis.68 The lesions in the lung may include thrombosis, multifocal edema and hemorrhage, pleural thickening, or regions of necrosis (Fig. 15.26).

8. Toxicologic lesions of the respiratory system 8.1 Upper respiratory tract response to toxic injury Inflammatory cell infiltration or inflammation, epithelial degeneration, epithelial hyperplasia, epithelial erosion and/ or metaplasia are findings that may be observed in nasal cavities or larynx following inhalation of test articles or

FIGURE 15.25 Anthracosis is exceptionally common in NHP lungs and often presents as macrophages containing pigmented exogenous particulate material within the alveoli or interstitium. These materials often have birefringence under polarized light.

toxins; however, these changes are not specifically toxininduced and are occasionally noted as incidental in NHPs during routine evaluation of tissues, therefore, the underlying etiology, toxic or otherwise, should be determined by

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8.2 Toxic inflammation and hemorrhage of the lung

FIGURE 15.26 A consequence of long-term continuous infusion in macaques may be interstitial to alveolar pneumonitis with thrombosis, edema, hemorrhage, pleural thickening, or regions of necrosis. These findings are often most severe in lung lobe tips and in subpleural regions.

weight of evidence. Changes noted include squamous cell metaplasia (Fig. 15.27A), or mucous cell metaplasia of the respiratory epithelium. Vacuolar degeneration (Fig. 15.27B), single cell necrosis, and erosion/ulceration of the epithelium may be observed as either direct exposureeinduced epithelial cell damage or mechanical epithelial cell damage, dependent on physicochemical and toxic properties of the material inhaled.32

Bronchioalveolar or interstitial inflammation is one of the most common lung findings observed in resperatoryfocused toxicology studies conducted with NHPs. It is characterized by inflammatory cell infiltration (e.g., mononuclear or mixed inflammatory cells, alveolar macrophages, giant multinucleated cells) in the alveolar lumen and/or septa. It may be associated with pneumocyte type II hyperplasia and/or material accumulation in the alveolar lumen (e.g., fibrin, hemorrhage, or edema) (Fig. 15.28). With increased vascular permeability there may be increased alveolar fluid and red cell accumulation (Fig. 15.29). Alveolar macrophage aggregation or histiocytic infiltrate is an accumulation of macrophages, vacuolated (foamy) or not, within alveoli, occasionally infiltrating alveolar septa and sometimes associated with eosinophilic proteinaceous material and/or mixed inflammatory cells. The distribution of this finding tends to be multifocal to diffuse when it is treatment related. Histiocytic infiltrate is likewise a nonspecific physiological response to inhaled foreign proteins or oligonucleotides through phagocytosis and clearance of inhaled material by alveolar macrophages.69e71

8.3 Lung findings due to PEGylation of test articles Alveolar macrophage aggregation can be seen with systemic administration of therapeutic proteins coupled with high molecular weight polyethylene glycol (PEG). These PEGylated proteins will accumulate in macrophages and are visible as vacuolation of these cells in multiple organs including lung (Fig. 15.30).72

FIGURE 15.27 (A) Squamous metaplasia is a consequence of epithelial abrasion or insult, and may occur following administration of intratracheal test articles due to repeated intubation. The respiratory epithelium is replaced by a keratinizing squamous epithelium of 2e5 layers thickness (arrow). (B) Vacuolation (arrows) with minimal degeneration of the olfactory epithelium in the nasal cavity may occur with products that target or disrupt the epithelium. In this case the cynomolgus monkey was treated with an inhaled biologic agent.

FIGURE 15.28 Bronchoalveolar and/or interstitial inflammation is one of the most common lung findings observed in respiratory-focused toxicology studies conducted with NHPs. Here it is characterized by inflammatory cell infiltration in the alveolar lumen and septa with Type II pneumocyte hypertrophy/hyperplasia, fluid accumulaton and test material accumulation in the alveolar lumen.

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FIGURE 15.29 Variable degrees of alveolar hemorrhage may occur with drug-related insult to the pulmonary microvasculature.

FIGURE 15.30 Alveolar macrophage aggregation may be noted with systemic administration of therapeutic proteins coupled with high molecular weight polyethylene glycol (PEG). PEGylated proteins are visible in macrophages as cytoplasmic vacuolation (arrows).

8.4 Acute respiratory distress syndrome Macaques are susceptible to acute respiratory distress syndrome (ARDS), due to infection, trauma, or toxic insult. It has been documented as a postoperative complication in rhesus macaques with sepsis or neurogenic trauma.73 Acutely, there is an exudative phase of the condition, characterized neutrophil and macrophage activation, leading to fluid and/or hemorrhage in the airways and pulmonary atelectasis. Hyalin membranes (Fig. 15.31) form in the alveoli and there may be suppurative inflammation.74 Within approximately 48 hours the fibrinoproliferative phase begins, with hyperinflated lungs, fibrinous thrombosis, and vascular necrosis.75

FIGURE 15.31 Hyaline membranes (arrows) in the lung of a macaque with the early phase of respiratory distress syndrome (ARDS).

antidrug antibody (ADA) development. Due to the foreign protein structure of many biologics, the NHP often develops a Type III hypersensitivity reaction in which ADAs complex with the drug, eventually depositing into vascular walls. In macaques, ICs may deposit in the lung vasculature, resulting in IC vascular inflammation (Fig. 15.32). Additionally, there may be perivascular inflammation, thrombosis, hemorrhage, and/or edema in the lung.76,77 Immunohistochemical detection of complement C3, monkey IgM, IgG and humanized proteins may clarify IC deposition as granular deposit in the tunicas of the arteries.77,78 Electron microscopy may help in discerning electron-dense deposits representing immunoglobulins at the surface of the endothelial cells or within the intima of the arteries. Clinical signs (decrease activity, skin reddening, rapid breathing), clinical pathology (decrease in red blood cell mass, platelet and decreased/increased white blood cell), exposure (accelerated clearance), and immunogenicity (presence of antidrug antibodies) are also key elements to the overall weight of evidence approach to

8.5 Drug-induced hypersensitivity reactions 8.5.1 Immune complex vasculitis A common complication of biologic administration to NHPs is immune complex (IC) deposition secondary to

FIGURE 15.32 Immune complex vasculitis in the lung of a macaque: Due to the foreign protein structure of biologic agents, NHPs often develop a Type III hypersensitivity reaction with drug complexes depositing into vascular walls, leading to vascular inflammation that is often transmural.

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confirm an IC-mediated hypersensitivity reaction.79 When IC deposition is considered the cause of the vascular inflammation, care must be taken to differentiate IC secondary to ADA development from that of primary test article associated IC formation.80

8.5.2 Anaphylaxis and anaphylactoid reactions Anaphylaxis is a Type I hypersensitivity reaction with rapid immediate degranulation of mast cells and basophils. Release of mediators is mediated by immunoglobulins (IgE). Anaphylactoid reactions trigger degranulation of basophils and mast cells without immunoglobulin mediation. Both reactions may result in acute respiratory distress in NHPs, often accompanied by emesis, skin flushing or rash, blood pressure and body temperature decline, and weakness. Collapse and death are not uncommon in the most severe cases. At necropsy, the lungs may be reddened grossly. Microscopically, there may be variable numbers of inflammatory cells, primarily within the interstitium, and in more severe cases, the alveoli may be filled with fluid and/ or blood (Fig. 15.33).

8.6 Antibody drug conjugates Antibody drug conjugates (ADCs) are biologics in which a toxin (the warhead or payload) is linked to monoclonal antibody (mAb). These target-specific ADCs carry the toxic payload directly to the target and have gained popularity as anti-cancer therapeutics, particularly against the difficult to treat solid tumors. However, the warhead may have offtarget toxicities resulting in lung lesions. For example, pyrrolobenzodiazepine (PBD) has been a popular warhead that produces DNA damage leading to cell death. Changes in the lung may include cellular alterations of the alveolar type II cells with or without cellular sloughing, cell

FIGURE 15.34 (A) Pyrrolobenzodiazepine (PBD) has been a popular cellular toxin linked to an antibody to form an antibody-drug conjugate (ADC) therapeutic agent. It induces a variety of cellular alterations of the alveolar type II cells such as nuclear and cellular atypia and may include significant tissue necrosis. (B) With chronicity, ADC-induced lesions may heal by fibroplasia and/or fibrosis of the lung.

necrosis, or alveolar macrophage infiltrates (Fig. 15.34A). With chronicity, these lesions may heal by fibroplasia and/ or fibrosis of the lung (Fig. 15.34B).

8.7 Increased leukocyte trafficking

FIGURE 15.33 Anaphylaxis is a Type I reaction mediated by IgE and resulting in rapid degranulation of mediator cells (mast cells and basophils) often leading to respiratory distress. Microscopic lesions, when present, include interstitial inflammation and alveolar fluid accumulation.

Test articles administered to NHPs that induce immune or tissue responses and that include elevations of inflammatory cytokines may result in visibly increased leukocytes in the fine vasculature of organs. Common organs involved include adrenal glands, kidneys, liver, and lung. It is not uncommon to note increased numbers of leukocytes within the pulmonary capillaries with elevations of TNFa, INFƔ, and MCP-1 (Fig. 15.35). Animals with increased leukocyte trafficking within microvasculature may or may not have additional findings or more severe outcomes related to infusion reaction or cytokine release syndrome.

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FIGURE 15.35 Administration of a biologic resulted in increased cytokine release in the form of interferon gamma (INFY) and tumor necrosis factor alpha (TNFa) in this macaque. The increased cytokines were associated with increased leukocytes within capillaries of the lung and other organs that were visible by light microscopy.

9. Non-human primate models of respiratory disease NHPs have been, and continue to be, models of various inflammatory lung diseases. From infectious agents to environmental and idiopathic inflammatory diseases, there are NHP models currently in use or being developed.

9.1 Macaque model of SARS-CoV-2 infection During the 2020e21 pandemic of SARS-CoV-2 (COVID 19), there was urgent need for NHP models of the disease. The macaque was relatively abundant in supply and had proven to be a good model of the acute severe respiratory syndrome disease (SARDS) as it affected the human respiratory system.81e83 Some research also indicated the common marmoset as a good model of COVID 19.82 These models provided valuable insight into the pathogenesis of COVID 19 and served as test systems for various therapies in development.84e86 In macaques, older animals had increased inflammation present in bronchioalveolar lavage samples and reduced anti-SARS-CoV-2 IgG levels following infection as compared to younger animals which appeared to recapitulate the disease in humans.83 Typical of COVID-19 infection, there is primarily an interstitial pneumonia characterized by thick alveolar septa with Type II pneumocyte hypertrophy and hyperplasia. There are usually accumulations of alveolar edema and fibrin and variable numbers of alveolar macrophages, multinucleated cells, neutrophils, and eosinophils (Fig. 15.36). There may be alveolar necrosis. Inflammation may extend to the upper airways and be associated with squamous metaplasia of the respiratory epithelium81.

FIGURE 15.36 The lung of a macaque experimentally infected with COVID-19: There are infiltrates of mononuclear cells with rare granulocytes within the interstitium, Type II pneumocyte hypertrophy and hyperplasia and occasional alveolar multinucleated cells (arrows) with increased numbers of alveolar macrophages are noted. Image courtesy of Jerald Ward.

9.2 Models of airway hypersensitivity and asthma Respiratory hypersensitivity and asthma are common afflictions in humans. Hypersensitivity pneumonitis is typically a Type III allergic reaction mediated by T1 cells and IgG secreting plasma cells that may occur due to inhalation exposure to a variety of allergens, including dust mites, feed products, animal dander, plant pollens, and fungal spores.87,88 Asthma is a type I allergic reaction mediated by T2 helper-cells, mast cells, and eosinophils leading to exaggerated airway inflammation and bronchoconstriction. While most humans have low-level responses that may be unrecognized, others have fulminant, life threatening hypersensitivity responses, or debilitating asthma. In some cases, the diagnosis between asthma and hypersensitivity pneumonitis is difficult to make, as one may contain elements of the other, such as neutrophilic infiltrates.88 Although mouse models of asthma have been used for some time, they have limitations in translation to humans. Some argue that mouse models fail to fully recapitulate the pathogenesis in humans and that NHP models of asthma would be comparatively more complete for evaluation of immune regulation, effector function, and for development of therapies.41,89 Hypersensitivity pneumonitis may be induced in macaques by the administration of Ascaris sp. and dust mite antigens. 52,90e92 These allergens may be administrated by intravenous route as well. For example, the asthma models in rhesus and cynomolgus monkey may be induced by

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inflammatory as well as to proliferative. This chapter provides a basic overview of the anatomy of the respiratory system of the NHP, as well as covering the spontaneous and toxic lesions of the airway.

References

FIGURE 15.37 Experimentally induced respiratory hypersensitivity model in a macaque: the airway is infiltrated by a mixed cell population with prominent eosinophils (arrows), which is a typical hypersensitivity response of cynomolgus monkeys following sensitization airway by challenge with a variety of materials as a model of respiratory hypersensitivity disease. Similar lesions have been occasionally noted as incidental in control or colony macaques.

administration of ovalbumin, house dust mite, or soluble forms of Ascaris suum.51,52,92e94 Airway eosinophilic cell infiltration (Fig. 15.37) has been reported in the literature as a typical hypersensitivity response of cynomolgus monkeys following sensitization airway challenge to a variety of materials as a model of allergic airway disease.89,94 Lipopolysaccharide (LPS) challenge by inhalation is used in marmoset to acute inflammation as a model of neutrophilic airway disease associated with airway hyperresponsiveness.95

10. Conclusion In conclusion, an understanding of the anatomy and spontaneous pathology of the NHP respiratory system is vital for the complete evaluation of the organism, particularly in toxicology studies. The respiratory system is a complex association of anatomic structures, responsible for the vital function of gas exchange, bringing oxygen to the blood and removing the carbon dioxide from it. This complexity is more evident in the upper part of the respiratory system with a large diversity in anatomical structures across species; further down into the bronchi and lung, species-specific anatomical variations are less evident, yet still present. The knowledge of those differences is important, when dealing with toxicological changes in the respiratory system, and of paramount importance in inhalation studies. The use of non-human primates (NHPs) in toxicology has been increasing in the past decades, in line with the expansion of biologics in the therapeutic area of inhalation, which often requires the NHP as the pharmacologically relevant species to be tested. The exposure to the external environment and to the internal blood circulation makes the respiratory system particularly prone to develop lesions spontaneously or due to administered toxicants. The lesions can range from degenerative to

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16. Smith TD, Bhatnagar KP, Tuladhar P, Burrows AM. Distribution of olfactory epithelium in the primate nasal cavity: are microsmia and macrosmia valid morphological concepts? Anat Rec A Discov Mol Cell Evol Biol 2004;281A(1):1173e81. 17. Smith TD, Eiting TP, Bonar CJ, Craven BA. Nasal morphometry in marmosets: loss and redistribution of olfactory surface area. Anat Rec 2014;297(11):2093e104. 18. Wako K, Hiratsuka H, Katsuta O, Tsuchitani M. Anatomical structure and surface epithelial distribution in the nasal cavity of the common cotton-eared marmoset (Callithrix jacchus). Exp Anim 1999;48(1):31e6. 19. Harkema JR, Plopper CG, Hyde DM, Wilson DW, St George JA, Wong VJ. Nonolfactory surface epithelium of the nasal cavity of the bonnet monkey: a morphologic and morphometric study of the transitional and respiratory epithelium. Am J Anat 1987;180(3):266e79. 20. Ankel-Simons F. Primate anatomy: an introduction. Elsevier Science; 2010. 21. Bhatnagar KP, Meisami E. Vomeronasal organ in bats and primates: extremes of structural variability and its phylogenetic implications. Microsc Res Tech 1998;43(6):465e75. 22. Taniguchi K, Matsusaki Y, Ogawa K, Saito TR. Fine structure of the vomeronasal organ in the common marmoset (Callithrix jacchus). Folia Primatol Int J Primatol 1992;59(3):169e76. 23. Moriya-Ito K, Hayakawa T, Suzuki H, Hagino-Yamagishi K, Nikaido M. Evolution of vomeronasal receptor 1 (V1R) genes in the common marmoset (Callithrix jacchus). Gene 2018;642:343e53. 24. Mills RP, Christmas HE. Applied comparative anatomy of the nasal turbinates. Clin Otolaryngol Allied Sci 1990;15(6):553e8. 25. Harkema JR, Plopper CG, Hyde DM, St George JA. Regional differences in quantities of histochemically detectable mucosubstances in nasal, paranasal, and nasopharyngeal epithelium of the bonnet monkey. J Histochem Cytochem 1987;35(3):279e86. 26. Chamanza R, Taylor I, Gregori M, Hill C, Swan M, Goodchild J, et al. Normal anatomy, histology, and spontaneous pathology of the nasal cavity of the cynomolgus monkey (Macaca fascicularis). Toxicol Pathol 2016;44(5):636e54. 27. Rossie JB. Ontogeny and homology of the paranasal sinuses in Platyrrhini (Mammalia: Primates). J Morphol 2006;267(1):1e40. 28. Rossie JB. The phylogenetic significance of anthropoid paranasal sinuses. Anat Rec Adv Integr Anat Evol Biol 2008;291(11):1485e98. 29. Koppe T, Rae TC, Swindler DR. Influence of craniofacial morphology on primate paranasal pneumatization. Ann Anat Anatomischer Anzeiger 1999;181(1):77e80. 30. Rae TC, Koppe T, Spoor F, Benefit B, McCrossin M. Ancestral loss of the maxillary sinus in old world monkeys and independent acquisition in Macaca. Am J Phys Anthropol 2002;117(4):293e6. 31. Nishimura TD, Takai M, Tsubamoto T, Egi N, Shigehara N. Variation in maxillary sinus anatomy among platyrrhine monkeys. J Hum Evol 2005;49(3):370e89. 32. Renne RA, Gideon KM. Types and patterns of response in the larynx following inhalation. Toxicol Pathol 2006;34(3):281e5. 33. Phalen RF, Oldham MJ. Tracheobronchial airway structure as revealed by casting techniques. Am Rev Respir Dis 1983;128(2 Pt 2):S1e4. 34. Patra AL. Comparative anatomy of mammalian respiratory tracts: the nasopharyngeal region and the tracheobronchial region. J Toxicol Environ Health 1986;17(2e3):163e74. 35. Curths C, Knauf S, Kaup F-J. Respiratory animal models in the common marmoset (Callithrix jacchus). Vet Sci 2014;1(1):63.

36. Plopper CG, Hyde DM. Chapter 7depithelial cells of the bronchiole. In: Parent RA, editor. Comparative biology of the normal lung. 2nd ed. San Diego: Academic Press; 2015. p. 83e92. 37. Plopper CG, Heidsiek JG, Weir AJ, George JA, Hyde DM. Tracheobronchial epithelium in the adult rhesus monkey: a quantitative histochemical and ultrastructural study. Am J Anat 1989;184(1):31e40. 38. Reynolds SD, Pinkerton KE, Mariassy AT. Chapter 6depithelial cells of trachea and bronchi. In: Parent RA, editor. Comparative biology of the normal lung. 2nd ed. San Diego: Academic Press; 2015. p. 61e81. 39. Winkelmann A, Noack T. The Clara cell: a “Third Reich eponym”. Eur Respir J 2010;36(4):722e7. 40. Seidel V, Hoffmann R, Braun A, Seehase S, Knauf S, Kaup FJ, Bleyer M. Distribution and morphology of Clara cells in common marmosets (Callithrix jacchus). J Med Primatol 2013;42(2):79e88. 41. Miller LA, Royer CM, Pinkerton KE, Schelegle ES. Nonhuman primate models of respiratory disease: past, present, and future. ILAR J 2017;58(2):269e80. 42. Herring MJ, Avdalovic MV, Quesenberry CL, Putney LF, Tyler NK, Ventimiglia FF, et al. Accelerated structural decrements in the aging female rhesus macaque lung compared with males. Am J Physiol Lung Cell Mol Physiol 2013;304(2):L125e34. 43. Okabayashi S, Ohno C, Kato M, Nakayama H, Yasutomi Y. Congenital cystic adenomatoid-like malformation in a cynomolgus monkey (Macaca fascicularis). Vet Pathol 2008;45(2):232e5. 44. Palmer S, Morgan TE, Prueitt JL, Murphy JH, Hodson WA. Lung development in the fetal primate, Macaca nemestrina. II. Pressurevolume and phospholipid changes. Pediatr Res 1977;11(10 Pt 1):1057e63. 45. Bleyer M, Kunze M, Gruber-Dujardin E, Mätz-Rensing K. Spontaneous lung pathology in a captive common marmoset colony (Callithrix jacchus). Primate Biol 2017;4(1):17e25. 46. Sato J, Doi T, Kanno T, Wako Y, Tsuchitani M, Narama I. Histopathology of incidental findings in cynomolgus monkeys ( macaca fascicularis ) used in toxicity studies. J Toxicol Pathol 2012;25(1):63e101. 47. Chamanza R, Marxfeld HA, Blanco AI, Naylor SW, Bradley AE. Incidences and range of spontaneous findings in control cynomolgus monkeys (Macaca fascicularis) used in toxicity studies. Toxicol Pathol 2010;38(4):642e57. 48. Syre MA, Bradley AE, Marel J. Spontaneous histopathological lesions of the respiratory tract in the laboratory cynomolgus monkey (Macaca fascicularis). In: Charles River Laboratories Preclinical Services, E., editor. 25th STP annual meetingdtoxicology pathology of respiratory system; 2006. Vancouver. 49. Shirai N, Geoly FJ. Eosinophilic airway inflammation in a cynomolgus monkey. Vet Pathol 2010;47(2):318e21. 50. Sakaguchi M. Analysis of Japanese monkeys (Macaca fuscata) and dogs with naturally occurring Japanese cedar (Cryptomeria japonica) pollinosis. Allerg Immunol (Paris) 2000;32(3):97e8. 51. Schelegle ES, Gershwin LJ, Miller LA, Fanucchi MV, Van Winkle LS, Gerriets JP, et al. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae). Am J Pathol 2001;158(1):333e41. 52. Gundel RH, Gerritsen ME, Gleich GJ, Wegner CD. Repeated antigen inhalation results in a prolonged airway eosinophilia and airway hyperresponsiveness in primates. J Appl Physiol 1985;68(2):779e86.

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53. Porter BF, Frost P, Hubbard GB. Polyarteritis nodosa in a cynomolgus macaque (Macaca fascicularis). Vet Pathol 2003;40(5):570e3. 54. Albassam MA, Lillie LE, Smith GS. Asymptomatic polyarteritis in a cynomolgus monkey. Lab Anim Sci 1993;43(6):628e9. 55. Chamanza R. Non-human primates: cynomolgus (Macaca fasicularis) and rhesus (Macaca mulatta) macaques and the common marmoset (Callathrix jacchus). In: McInnes EF, Mann P, editors. Background lesions in laboratory animals. W.B. Saunders; 2012. p. 1e15. 56. Turner PV, Brabb T, Pekow C, Vasbinder MA. Administration of substances to laboratory animals: routes of administration and factors to consider. J Am Assoc Lab Anim Sci 2011;50(5):600e13. 57. Colman K, Andrews RN, Atkins H, Boulineau T, Bradley A, Braendli-Baiocco A, Capobianco R, Caudell D, Cline M, Doi T, Ernst R, van Esch E, Everitt J, Fant P, Gruebbel MM, Mecklenburg L, Miller AD, Nikula KJ, Satake S, Schwartz J, Sharma A, Shimoi A, Sobry C, Taylor I, Vemireddi V, Vidal J, Wood C, Vahle JL. International harmonization of nomenclature and diagnostic criteria (INHAND): non-proliferative and proliferative lesions of the non-human primate (M. fascicularis). J Toxicol Pathol 2021;34(3 Suppl). 58. Kaspareit J, Friderichs-Gromoll S, Buse E, Habermann G. Background pathology of the common marmoset (Callithrix jacchus) in toxicological studies. Exp Toxicol Pathol 2006;57(5e6):405e10. 59. Carr TF, Zeki AA, Kraft M. Eosinophilic and noneosinophilic asthma. Am J Respir Crit Care Med 2018;197(1):22e37. 60. Ikeda T, Go T, Kadota K, Ibuki E, Yokomise H. Two cases of nodular smooth muscle proliferation suspected of primary lung cancer from preoperative images: a case report. J Cardiothorac Surg 2020;15(1):179. 61. Kaspareit J, Friderichs-Gromoll S, Buse E, Korte R, Vogel F. Spontaneous pulmonary neoplasms in cynomolgus monkeys (Macaca fascicularis)da report of two cases. Exp Toxicol Pathol 2001;53(4):267e9. 62. Kaspareit J, Friderichs-Gromoll S, Buse E, Habermann G. Spontaneous neoplasms observed in cynomolgus monkeys (Macaca fascicularis) during a 15-year period. Exp Toxicol Pathol 2007;59(3):163e9. 63. Simmons HA, Mattison JA. The incidence of spontaneous neoplasia in two populations of captive rhesus macaques (Macaca mulatta). Antioxidants Redox Signal 2011;14(2):221e7. 64. Jean SM, Morales PR, Paul K, Garcia A. Spontaneous primary squamous cell carcinoma of the lung in a rhesus macaque (Macaca mulatta). JAALAS 2011;50(3):404e8. 65. Patrick DJ, Rebelatto MC. Chapter 12dtoxicologic pathology and background lesions of nonhuman primates. In: Bluemel J, Korte S, Schenck E, Weinbauer GF, editors. The nonhuman primate in nonclinical drug development and safety assessment. San Diego: Academic Press; 2015. p. 235e56. 66. Lefrançais E, Ortiz-Muñoz G, Caudrillier A, Mallavia B, Liu F, Sayah DM, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature 2017;544(7648):105e9. 67. Yamakawa Y, Ide T, Mitori H, Oishi Y, Matsumoto M. Accumulation of brown pigment-laden macrophages associated with vascular

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83. Singh DK, Singh B, Ganatra SR, Gazi M, Cole J, Thippeshappa R, et al. Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets. Nat Microbiol 2021;6(1):73e86. 84. Zheng H, Li H, Guo L, Liang Y, Li J, Wang X, et al. Virulence and pathogenesis of SARS-CoV-2 infection in rhesus macaques: a nonhuman primate model of COVID-19 progression. PLoS Pathog 2020;16(11):e1008949. 85. Mercado NB, Zahn R, Wegmann F, Loos C, Chandrashekar A, Yu J, et al. Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques. Nature 2020;586(7830):583e8. 86. Chandrashekar A, Liu J, Martinot AJ, McMahan K, Mercado NB, Peter L, et al. SARS-CoV-2 infection protects against rechallenge in rhesus macaques. Science 2020;369(6505):812e7. 87. Spagnolo P, Rossi G, Cavazza A, Bonifazi M, Paladini I, Bonella F, et al. Hypersensitivity pneumonitis: a comprehensive review. J Investig Allergol Clin Immunol 2015;25(4):237e50 (quiz follow 250). 88. Bogaert P, Tournoy KG, Naessens T, Grooten J. Where asthma and hypersensitivity pneumonitis meet and differ: noneosinophilic severe asthma. Am J Pathol 2009;174(1):3e13. 89. Coffman RL, Hessel EM. Nonhuman primate models of asthma. J Exp Med 2005;201(12):1875e9.

90. Weiszer I, Patterson R, Pruzansky JJ. Ascaris hypersensitivity in the rhesus monkey. I. A model for the study of immediate type thypersensitity in the primate. J Allergy 1968;41(1):14e22. 91. Yasue M, Nakamura S, Yokota T, Okudaira H, Okumura Y. Experimental monkey model sensitized with mite antigen. Int Arch Allergy Immunol 1998;115(4):303e11. 92. Van Scott MR, Hooker JL, Ehrmann D, Shibata Y, Kukoly C, Salleng K, et al. Dust mite-induced asthma in cynomolgus monkeys. J Appl Physiol 2004;96(4):1433e44. 93. Hart TK, Cook RM, Zia-Amirhosseini P, Minthorn E, Sellers TS, Maleeff BE, et al. Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys. J Allergy Clin Immunol 2001;108(2):250e7. 94. Kraneveld AD, Folkerts G, Van Oosterhout AJ, Nijkamp FP. Airway hyperresponsiveness: first eosinophils and then neuropeptides. Int J Immunopharm 1997;19(9e10):517e27. 95. Curths C, Wichmann J, Dunker S, Windt H, Hoymann HG, Lauenstein HD, et al. Airway hyper-responsiveness in lipopolysaccharide-challenged common marmosets (Callithrix jacchus). Clin Sci (Lond., Engl.: 1979) 2014;126(2):155e62.

Chapter 16

The hematolymphoid system of the non-human primate Ronnie Chamanza1, Stuart W. Naylor2 and Jennifer A. Chilton2 Janssen Pharmaceutical Companies of Johnson & Johnson, High Wycombe, United Kingdom; 2Charles River Laboratories, Reno, NV, United States

1

1. Introduction The hematopoietic and lymphoid system is comprised of organs in which cells of the acquired and innate immunity are generated, mature, maintained, conduct immune surveillance, interact with antigens, and mount immunologic reactions.1,2 Immune system cells include lymphocytes (Bcells, T-cells, and natural killer cells), plasma cells, monocytes, macrophages, dendritic cells, mast cells, and granulocytes. The “lymphoid” component of the hematolymphoid system comprises organs which are classified as primary and secondary lymphoid organs. Primary or central lymphoid organs are the bone marrow and thymus where lymphocyte proliferation and maturation take place independent of stimulation by exogenous antigens.2 Secondary lymphoid organs include the spleen, lymph nodes, mucosal-associated lymphoid tissue (MALT), and serosaassociated lymphoid clusters (SALCs). The lymphoid system also includes tertiary lymphoid structures (TLS), which are ectopic tertiary lymphoid tissues that are induced to develop in nonlymphoid organs in a variety of pathophysiological situations, including autoimmune and infectious diseases, transplanted organs, inflammatory disorders, and tumors.3 Some hematolymphoid organs such as the bone marrow and spleen are also involved in the production (hematopoiesis) of nonimmune cells that carry blood gases (erythrocytes) and maintain vascular integrity (megakaryocytes and platelets),2 hence the term “hematolymphoid.” In the macaque monkey, it is mainly the bone marrow that is involved in hematopoiesis of erythrocytes and megakaryocytes. In addition, the bone marrow is the site of origin of the pluripotent stem cell, a self-renewing cell from which all other hematopoietic cells are derived.1 However,

in depth details of the hematopoietic function of this primary lymphoid organ is beyond the scope of this chapter. The focus of the chapter is on the physiology, anatomy, pathology, and spontaneous findings of the hematolymphoid system of the non-human primate (NHP). All lymphoid organs share the same basic functional and morphological features that enable them to conduct immune surveillance, interact with antigens, and mount immunologic reactions that facilitate the removal of harmful agents in a coordinated fashion. Each organ has a reticular meshwork of stroma that facilitates and regulates immune functions, hematopoietic cells that interact with antigens and mount an immune response, and a vascular network that allows trafficking of immune cells. The main functions of the immune system are to defend the individual against a multitude of pathogens, (viruses, bacteria, fungi, and parasites), carry out destruction of aberrant cells (neoplastic cells or transplanted tissue), and establish immune tolerance.4 Disorders of the immune system include: loss or absence of physiological tolerance to “self” antigens leading to the immune system response against itself (autoimmune disorder); failure or reduced ability of the immune system to generate appropriate immune responses against invading microorganisms (immunodeficiency or immunosuppression); and generation of an excessive immune response to antigens (hypersensitivity reactions and inappropriate inflammation). In addition, cells of the immune system can also undergo uncontrolled proliferation that can lead to cancer of circulating blood cells, particularly in immunosuppressed NHPs or humans. As is true for diseases affecting other body systems, disorders of the immune system result from a complex interplay of genetic predisposition, developmental defects, and

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environmental influences, including exposure to pathogens, toxic drugs or chemicals.

2. Anatomy of the hematolymphoid system 2.1 Development of the hematolymphoid system of non-human primates Dysfunction of the developing immune system, such as aberrant activation, can result in many life-threatening autoimmune disorders in both early and later life, while suppression of the immune system during development can lead to decreased host resistance to infection.4 Therefore, an understanding of the development of the immune system is critical in understanding and developing drugs that combat immune system disorders or modulate the immune system. To this end, the study of the immune system ontogeny, known as developmental immunotoxicology (DIT), is emerging as an important discipline with the potential to characterize critical windows during the development of the immune system. DIT may help identify changes that can lead to adverse health outcomes related to immune system dysfunction that occur in both animal models and humans.5 The development and establishment of a functional immune system in the laboratory NHP has been characterized in the rhesus and cynomolgus macaques.4,6,7 Apart from minor differences in the timing of development of various key components of the immune system, it is largely similar to that of humans, and macaques are considered an appropriate model for the human immune system.6 Furthermore, given that mammalian species with relatively long gestation periods (for example, the rhesus macaque has an approximately 5 month gestation period) have a more mature immune system at birth than those with shorter gestational period, and that the fetal development in the NHP is very similar to that in the human, the development of the immune system in the NHP unsurprisingly mirrors that in humans.6,8,9 The ontogeny of the

hematolymphoid system is a complex multistage process that occurs in a unique, coordinated, sequential, and temporal sequence, and involves multiple organs and sites during development and in the adult life. The three main groups of anatomical sites that have a permanent or transient immune function during development include: transiently active embryofetal organs; organs with primarily immune functions; and multifunctional organs with partial immune functions.7,10 The anatomical sites of these three groups are listed in Table 16.1. Early development begins with the appearance of primitive and definitive hematopoietic stem cells (HSC) that have the ability to self-renew and differentiate into all components of the immune system, and all blood cell lineages.4,11 The process involves at least two waves of hematopoiesis that include primitive or extraembryonic hematopoiesis, which occurs in extraembryonic tissues composed of visceral endoderm and mesoderm, and generates transitory hematopoietic cell populations, and permanent or definitive hematopoiesis, which arises later in development from definitive multipotent HSC and starts in the aorta-gonad mesonephros region (AGM). Although, the exact origin of HSC precursors is not known, the yolk sac is believed to be the only source, and therefore, the site of primitive hematopoiesis. In the NHP, the process begins during the third week of development, before the establishment of blood flow. Definitive hematopoiesis, which occurs within the embryo proper (intraembryonic hematopoiesis), starts in the AGM, from definitive HSCs, which are believed to originate from a different subset of mesodermal cells to those that initiate primitive hematopoiesis, indicating that the AGM may have the autonomous capacity to generate these hematopoietic progenitors. From the AGM, definitive HSC seed into the embryonic liver in an orderly migration that results in the liver taking on the role of hematopoiesis.4,10 From the fetal liver, hematopoietic cells migrate to the thymus, spleen, lymph nodes, and gut-associated lymphoid tissue (GALT), where additional expansion, differentiation, and maturation occurs.4,12e14 Pools of progenitor cells are formed and expanded in the bone marrow and spleen for myeloerythroid

TABLE 16.1 The three main groups of anatomical sites that have a permanent or transient immune function. Hematolymphoid organs during development Group

Organ/Anatomical site

Transiently active embryofetal organ

Intra- and extra-embryonic hematopoietic islets, yolk sac, placenta, the aortagonad mesonephros region (AGM), liver

Primary immune function organs

Thymus, lymph nodes, mucosal-associated lymphoid tissue (MALT)

Multifunctional with partial immune function

Spleen, bone marrow, liver, skin

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lineages, in the bone marrow for B-cell lineages, and in the thymus for T-cell lineages.

2.1.1 Chronology of lymphoid tissue development The ontogeny of the myeloid-lymphoid system in macaques is similar in temporal and anatomical sequence to that of humans.4,6 In both NHPs and humans, the liver is the major site of hematopoiesis from the mid-first trimester until the mid-second trimester (Fig. 16.1A and B) when the bone marrow hematopoiesis is established. The structural development of the liver and thymus are more advanced in the first trimester than that of the spleen, axillary and mesenteric lymph nodes, GALT, and bone marrow.4 Demarcation of the thymus into cortex and medulla (Fig. 16.2), and the spleen into red and white pulp (Fig. 16.3A and B) is completed by mid-gestation, followed by the presence of B- and T-cells within Peyer’s patches prior to the third trimester.4 Lymph node morphogenesis, including formation of germinal centers is completed prior to birth in both species (Fig. 16.4).

FIGURE 16.1 In non-human primates, the liver is the major site of hematopoiesis from the mid-first trimester until the mid-second trimester at which time the bone marrow hematopoiesis is established. (A) In late gestation, hematopoietic cells are clearly evident as clusters of dark nuclei scattered throughout the fetal liver. (B) Hematopoietic cells (arrows) are common in the liver to varied degree until late gestation and the perinatal period.

FIGURE 16.2 Late gestation fetal thymus of a macaque: Demarcation of the thymus into cortex (C) and medulla (M) is completed by mid-gestation with thymocytes present; however, the fetal thymus is hypolobulated until after birth. By late gestation, the thymus has migrated to its permanent location in the cranial mediastinum. Mesenchymal vasculature, stromal tissues and vagal nerve fibers have invaded the gland in such a way as to produce its lobulated appearance and with the reticular epithelial cell framework in place, the thymus continues to grow and develop until puberty.

FIGURE 16.3 Late gestation fetal spleen of a macaque: (A) Demarcation of the NHP spleen into red pulp (RP) and white pulp (WP) is completed by mid-gestation. Visually, the spleen lacks well-formed follicles; however, primary follicles are present and organization into specific T- and B-cell areas has occurred. (B) Similar to other organs, the splenic red pulp is a site of hematopoiesis in the fetus (arrows).

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CD20þCD5þ phenotype.6 In the adult human and NHPs, CD5 is expressed by most T-cells and is therefore a T-cell marker, while only a small subset of B-cells remains CD5þ. Therefore, lymphocyte subset differentiation occurs by the early to mid-second trimester in the macaque, with increasing lymphoid tissue organization into specific T- and B-cell areas in peripheral lymphoid organs, which coincides with the presence of immunoglobulin and cytokinesecreting cells in these tissues.

FIGURE 16.4 Late gestation fetal mesenteric lymph node of a macaque: The lymph node morphogenesis, including formation of germinal centers, is completed prior to birth in macaques. (M) medulla, (C) cortex, and (PC) paracortex are delineated. Germinal centers of lymphoid follicles (F) are formed prior to birth; however, germinal centers are generally difficult to see in hematoxylin and eosin (H&E) stained sections of fetal lymph nodes. Note the exramedullary hematopoiesis present in the fetal lymph node (oval).

2.1.2 Lymphocyte development In macaques as in humans, T-cells appear earlier than Bcells, with CD3 positive cells appearing in the thymus, and to a lesser extent the spleen during the first trimester.4 By GD40, in the late first trimester (GD40), CD68þ macrophages may be detected in the liver near the blood islands, along with CD117þ stem cells, while HLA-DR immunepositive cells are first seen among primordial lymphoid tissue at GD45.7 Cells expressing B-cell markers, CD20 or CD79, are first observed in the mid-second trimester in the liver, spleen, thymus, lymph nodes, bone marrow, and GALT,4,6 while CD4þ and CD8þ also first occur in the second trimester in the thymus and spleen. However, cells expressing B-cell markers and MHC class II molecules appear in larger numbers early in the development of peripheral lymphoid organs than CD3 positive T-cells, which are rarer early in the second trimester but increase in number with advancing fetal age.6 As in humans and other animals, the early fetal B-cells are characterized by coexpression of CD20 and CD5 in the spleens, mesenteric lymph nodes, and small intestines of fetuses during the early second trimester.4,6 This population of CD5 positive B-cells is designated B-1a cells to distinguish them from a subset of CD5- B-1b cells with similar attributes, and from B-2 cells of bone marrow origin.15e17 In the rhesus macaque fetus of between 65d100 GD, approximately 95% of the B-cells in the spleen, lymph nodes, and intestinal lymphoid aggregates are CD5þ and CD20þ, with nearly all CD20 positive cells also expressing CD5, while CD5B-cells make up only 1% of the B-cells in all tissues of fetuses during this period.6 By day 145, the frequency of CD20þ B-cells that are negative for CD5 increases in the developing follicles, while CD5þ cells decrease with fetal age, although some of the B-cells are still of the

2.1.3 Development of populations of antigen presenting cells and their tissue distribution Antigen-presenting cells (APCs) are a heterogenous group of immune cells that process and present antigens within an antigen-binding cleft of class II MHC molecules for recognition by T-cells, and include dendritic cells, macrophages, Langerhans cells, and B-cells.18,19 Besides class I and class II MHC molecules, they also express several important receptors that facilitate their function, including receptors specific for the Fc region of IgG molecules and the third complement component.19 MHC polymorphisms have a profound impact on several features of the immune system, such as disease susceptibility and response to organ transplantation. In humans and cynomolgus macaques, the MHC gene complex is located on chromosome 6 and 4, respectively, and comprises genes that code for human leukocyte antigens (HLAs) in humans, with Class I MHC molecules having three main genes in humans HLA-A, HLA-B, HLA-C, and Class II MHC molecules six main genes, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLADQB1, HLA-DRA, and HLA-DRB1.19e22 The MHC of cynomolgus (Mafa MHC) and rhesus (Mamu MHC) macaques are organized in the same way as that of humans and differ only by their high degree of classical class I gene duplication.20,21 As in humans, NHP orthologs of the HLADR gene are expressed primarily on APCs such as B-cells, monocytes, macrophages, thymic epithelial cells, and activated T-cells HLA-DR is critical for the presentation of peptides to CD4þ T-cells. In the developing cynomolgus macaque, HLA-DR positive cells are first seen on GD45 among primordial lymphoid tissue at the level of the cisterna chyli,7 and by GD50, they are present in the liver, lymph nodes, lung, thymus, and the surrounding thymic mesenchyme. In the rhesus fetus, macrophages are first detected during the late first trimester in the developing liver, spleen, thymus, and lymph nodes, and in the intestines by the early second trimester.4,6 During the first trimester, the CD68þ macrophages are observed only in blood islands of the developing liver, with no positive cells noted among surrounding hepatocytes, and their numbers increase until the early second trimester when they start decreasing with the disappearance of blood islands.

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The CD68þ macrophages become infrequent in the late gestation and postnatal liver of the rhesus macaques. In contrast, CD55þ and CD205þ dendritic cells (DC) are first observed in the lymphoid tissues of the rhesus fetus around GD65, which is in the second trimester. At this period, CD68 positive macrophages are numerous in the spleen, where they are mainly localized within the red pulp (Fig. 16.5A), and in scarce amounts in the lymph nodes, thymus (Fig. 16.5B), and intestines, where they are mainly scattered within the lamina propria.6 In contrast, intestinal CD55 positive DC are more numerous than CD68þ macrophages at GD65, and keep increasing so that by GD100, DCs in the spleen are more common than macrophages, with a ratio of macrophages to DC of 1:2, which remains the case throughout gestation.6 Most splenic DCs in the rhesus fetus are localized in the white pulp within the T-cell compartment.

2.1.4 Development, subgross anatomy, and histology of lymph nodes Lymph nodes are strategically situated along the lymphatic vasculature network and at major vascular junctions,23 as

FIGURE 16.5 (A) CD68 antigen positive macrophages are detected by IHC and are numerous in the NHP spleen, where they are mainly localized within the red pulp. (B) CD68 antigen positive macrophages appear in low quantities in the adult macaque thymus.

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this strategic position enables filtration of antigens, as well as provides a site for antigen presentation to lymphocytes and induction of adaptive immune responses. Lymph node development starts with the formation of a lymph node anlagen that consist of mainly 2 cell types, lymphoid tissue inducer (LTi) cells, which are hematopoietic precursor cells, and lymphoid tissue organizer (LTo) cells, which are nonhematopoietic stromal or mesenchymal cells involved in the activation and recruitment of LTi cells.23 A series of cross-talk interactions between these cell populations appear to be crucial to lymph node development. The lymphatic system, on the other hand, develops from lymph sacs, the formation of which is considered to be tightly linked to the development of the lymph nodes.23,24 The cell of origin of the lymph sac is disputed, but progenitor lymphatic endothelial cells (LEC) are thought to form the lymphatic sac as well as play a major role in the lymphoid tissue organization and an additional LTo.23,24 Therefore, early lymph node organogenesis depends on tight interactions between the budding endothelium, surrounding layers of mesenchymal cells that invade them, and the hematopoietic-derived LTi cells. The LECs form the subcapsular sinus and induce the rapid growth of the blood vascular tree that includes primordial HEV structures expressing adhesion molecules.25 The differentiating mesenchymal LTo cells give rise to dedicated fibroblastic reticular cell (FRC) subsets that include follicular dendritic cells (FDC) and reticular cells.23e25 The gross anatomy and architecture of normal lymph nodes of NHPs and other animals is variable depending on the location of the lymph node, age of animal, and activation status or antigen stimulation of the node.26e28 As one of the larger laboratory species, NHP lymph nodes are more numerous, larger, and organized into more complex chains, than those of smaller laboratory animals.26 For instance, while the lung of the rat is drained by two posterior mediastinal nodes, that of macaques is drained by more than five lymph nodes that includes tracheobronchial and mediastinal lymph nodes at the tracheal bifurcation. Comparatively, humans have 35 or more tracheobronchial nodes classified into five separate groups.26,29 Subgrossly, the lymph node is organized into the following main structures, a fibrous capsule, a subcapsular sinus, a cortex with B-cell rich follicles and germinal centers, a T-cell rich paracortex, medullary sinuses, medullary cords, and the hilus where efferent veins enter the node (Fig. 16.6A and B).30 The lymph node cortex of NHPs and humans is subdivided into 10 or more lobules separated by fibrous trabeculae.31 NHPs also have an increased number of anastomoses of afferent lymphatic vessels as compared to rodents. The connective tissue capsule is covered by either loose or dense connective tissue in which adipose tissue can be found. In NHPs, the thickness and density of connective tissue of the capsule

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FIGURE 16.6 (A) The normal NHP lymph node has an outer cortex (Cx) composed of B-cells organized into primary or secondary follicles (SF) and a deeper paracortex (PC) composed of T-cells. The central medulla (M) has the medullary cords that contain plasma cells, B-cells, and macrophages, and medullary sinuses that contain histiocytes and sinus fibroblastic reticular cells. The connective tissue capsule (C) has connective tissue extensions or trabeculae (circle), which provide support for blood vessels entering the nodes and extend radially toward the center of the lymph node. The hilus (H) is where efferent veins enter the node. (B) Germinal centers (GC) of follicles have dark areas (D) containing centroblasts that proliferate, switch immunoglobulin (Ig) class, and increase affinity for antigens, then populate the light areas (L) with centrocytes; However, recent evidence suggest that germinal center B-cells are able to cycle back and forth between the dark and light zones, and represent different states of activation of the same cell population. The lymph node is encased in a connective tissue capsule (C) which has trabeculae extension that provides support for blood vessels entering the nodes and extend radially toward the center of the lymph node. Subtending the capsule is the subcapsular or marginal sinus (oval) which contains sinus macrophages that provide first-line antigen recognition or surveillance.

varies with the type or location of the lymph node and disease status. The capsule has connective tissue extensions or trabeculae, which provide support for blood vessels entering the nodes and extend radially toward the center of the lymph node. On either side of the trabecular are small cortical sinuses or trabecular sinuses. The subcapsular sinus (or marginal sinus), lies immediately below the capsule. It

receives afferent vessels and is continuous with the trabecular sinuses and joins the medullary sinus in the medulla. It also contains a layer of subcapsular sinus macrophages that strategically line the sinus and are directly exposed to the afferent lymph. Subcapsular sinus macrophages are the frontline of immune defense that interact with viral, bacterial, or tumor lymph-borne antigens. The histomorphology of normal NHP lymph nodes is also highly variable (Fig. 16.7AeC). Because lymph nodes do not have a simple, consistent shape, serial sections of a lymph node can reveal markedly different proportions of cortex, paracortex, and medulla.26 Therefore, pathologically altered lymph nodes can be difficult to distinguish from normal or reactive lymph nodes. In addition, NHPs tend to have larger, more numerous, or variable numbers of secondary follicles, when compared to rodents because of increased environmental pathogen or antigen exposure.26,28 Due to variations in cell populations between controls and treated animals, immunohistochemistry (IHC) and image analysis may often provide a tool for both subjective and objective analysis and interpretations (Fig. 16.8AeF). The basic histology of lymph nodes of NHP is similar to that described for other laboratory species, and has been extensively covered elsewhere.2,30,32 Briefly, the lymph node consists of various subtypes of lymphocytes embedded within a fine reticular stroma composed of a network of FRCs and extracellular matrix. While the role of lymphocytes as the parenchymal tissue that interacts with antigens to mount an immune response is well-known, the role of FRCs in regulating the immune functions has only been realized recently.33,34 The lymph node cortex has an outer region composed of B-cells organized into primary or secondary follicles, depending on the presence of germinal centers, and a deeper T-cell region paracortex or deep cortical unit (which is discussed in detail further below). Primary follicles are the quiescent follicles that have not been exposed to antigens and therefore lack germinal centers. They are composed of distinct, small, densely staining resting lymphocytes.35 Secondary follicles, in contrast, contain germinal centers which are composed of large, pale staining, activated, proliferating B-cells representing centroblasts, and smaller centrocytes, which are nonproliferating progeny of centroblasts. Germinal centers have dark and light zones surrounded by the mantle zone, and form or proliferate in stimulated lymph nodes.26,32,35 Centroblasts are located in the dark zone, where they proliferate at an unparalleled rate in mammalian tissues, switch Ig class, and increase affinity for the antigen by somatic hypermutation of their antibody variable region genes.36,37 Having undergone somatic hypermutation, they continue to proliferate and eventually give rise to smaller, nondividing cells called centrocytes, which are located in the light zone of the germinal center. However, it is worth noting that

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FIGURE 16.7 Variation in germinal centers and paracortices in the mandibular lymph nodes of macaques: (A-C) The size and number of follicular germinal centers (GCs) may be increased in some individuals and the paracortex (Px) may be promininat (Fig. 16.7A) as a normal variation based on antigenic stimulation, while in others the size and number of GCs may appear decreased (Fig. 16.7B). Similar variations of the size of the paracortex (Px) may be noted in individual animals, occasionally with very thin paracortex tissue (Fig. 16.7C).

FIGURE 16.8 Evaluation of lymphoid cell populations in macaque lymph nodes by immunohistochemical methods (IHC): (A) A lymph node with typical hyperplasia of the germinal centers of follicles stained with hematoxylin and eosin (H&E). (B) CD20 positive B-cell and (C) CD3 positive T-cell populations in the hyperplastic follicles from Fig. 16.8A. Notice that although the T-cells and B-cells are highly concentrated in the paracortex and cortical germinal centers, respectively, there are lesser populations of the cells in other regions of the lymph node. (D) Detection of the cell marker Ki67 delineates the highly replicative B-cell population primarily within the hyperplastic follicles of Fig.16.8A. (E) IHC detects CD 138 antigen positive plasma cells and (F) CD68 antigen positive histiocytic cells concentrated within the medullary chords and the medullary sinus, respectively. Images courtesy of Shantel Bouknight.

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recent evidence appears to show that germinal center Bcells are able to cycle back and forth between the dark and light zone, which suggests that centroblasts and centrocytes may simply represent different states of activation of the same cell population.37,38 Regardless, germinal center Bcells expressing high-affinity antibodies develop and differentiate into antibody-secreting plasma cells and memory B-cells that mediate and sustain protection against invading pathogens. Memory B-cells ultimately locate in the mantle zone. T-cells represent a minor, but heterogenous population of between 5% and 20% in germinal centers, but have an essential function in its maintenance.39,40 One population in particular, the follicular helper T-cell, has a T-helper (Th) immunophenotype, and is positive for CD4, CXCR5 (required for follicular homing), CD40L (CD154), IL-21, and CD57; whereas others (CD8þ Th17 cells, and FOXP3þ regulatory cells) play functional roles in regulating the germinal center reaction.37,39,40 The cortex also contains the FDC, which is an important cell of stromal origin that resides in the follicular area, and is responsible for trapping and presenting antigens to B-cells, and thus helps shape the B-cell antigenome (the sum of all B-cell antigens). The FDC, which unlike the DC is of mesenchymal origin, and is therefore not derived from the HSC, is a highly specialized cell that plays a crucial role in B-cell activation, affinity maturation of antibodies, and memory B-cell development.31 Although it has a dendritic morphology, the FDCs are nonphagocytic and nonmigratory cells which are completely distinct from the bone marrowederived classical DC found in T-cell rich area. Thus, while DCs are critical for T-cell responses, the FDC is important for B-cell responses. The FDC binds immune complexes via Fcg (FcgRIIB or CD32) and complement receptors (CR1 or CD35 and CR2 or CD21), internalizes and retains them in their native form for long periods of time, which is crucial for antigen concentration, memory Bcell development, and efficient germinal center reaction.41 Since it lacks phagocytic activity, the FDC traps immune complexes in recycling endosomal compartments, thereby protecting the antigen from degradation. The paracortex or deep cortex is located between the superficial cortex, lymphoid follicles, and the medullary sinuses. It is a T-cell rich area, but also contains DCs of the interdigitating dendritic subtype, FRCs, scattered large Bcells, plasmacytoid dendritic cells, and high endothelial venules (HEVs). T-cells migrate to and locate in the paracortical zone where they participate in both cell-mediated and humoral-mediated immune responses. The paracortex is where they encounter APCs such as interdigitating DC. The medulla, which is the innermost layer, contains large blood vessels, medullary sinuses, and medullary cords. The medullary cords contain plasma cells, B-cells, and macrophages, while medullary sinuses contain sinus histiocytes

and sinus FRCs. Medullary sinuses are vessel-like spaces that separate the medullary cords, and receive lymph from the trabecular sinuses and cortical sinuses. The medullary sinus drains the lymph into the efferent lymphatic vessels.

2.1.5 Functional anatomy of the lymph node: cell trafficking and antigen presentation One of the main challenges of the immune system is to bring rare antigen-specific B-cells together with rare antigen-specific T-cells and antigen-charged APC in a naïve subject. In particular, T-helper cells are needed to generate high affinity antibodies with memory against most protein antigens. It is the primary role of the lymph node and other secondary lymphoid tissues to facilitate these interactions. As discussed above, the FDC of the B-cellrich follicles facilitates efficient B-cell maturation, somatic mutation and selection of high affinity B-cells, while the Tcell-rich paracortex contains large numbers of DC that are potent APCs for T-cell activation. While APC and dendritic cells migrate into the lymph nodes via afferent lymphatics, most circulating naïve T-cells enter the lymph node from the blood via HEVs.42,43 Lymph node and MALT HEV are vascular sites that efficiently extract naïve T- and B-cells from the circulation into the lymphoid organ, a role similar to that of the marginal sinus in the spleen. The homing of naïve T-cells and migratory DCs to the lymph node paracortex is mediated by CCR7, a chemokine receptor on DC and naïve T-cells, and by the ligands CCL19 and CCL21 expressed by FRCs and HEVs.43 Overall, well-coordinated processes and programmed release of distinct chemokines within the lymph node and other secondary lymphoid tissues orchestrate the coming together of antigen-responsive B-cells and T-cells, followed by the migration of the activated B-cells and selected T-cells to the FDC clusters where they form germinal centers.

2.1.6 Development, anatomy, and function of the spleen In NHPs as in other animals, the spleen represents the largest single aggregation of lymphoid tissue in the body. Its main function is to filter the blood and present antigens to the immune system. As a secondary lymphoid tissue, it also contributes to phagocytosis and orchestration of humoral and cellular immunity. Its nonimmunologic function includes the removal of senescent and deformed erythrocytes. Additionally, the spleen serves as the differentiation site for platelets, reticulocytes, and monocytes, as well as as the reservoir for granulocytes and erythrocytes. The spleen also plays a role in embryogenesis of the pancreas.7 Embryologically, the splenic primordium appears during the first trimester in humans (fifth week) and late first trimester (by GD55) in macaques as a mesodermal proliferation along the left side of the mesogastrium, dorsal to the

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stomach.44 At this point, the splenic mesenchyme is loosely organized, with the majority of the lymphocytes being CD20þ B-cells, and only rare CD3 positive cells present.4,6 By the beginning of the second trimester in macaques (GD56-65) all of the CD20þ cells observed in the spleen also express CD5, and are randomly dispersed throughout the spleen, occasionally forming loose aggregates. The second trimester also marks the first evidence of organization and demarcation of the splenic mesenchyme into distinct red and white pulp area regions, with the appearance of clusters of lymphoid cells around central arterioles as early periarterial lymphoid sheath (PALS).4,6 Nearly all of the cells surrounding the central arterioles are CD3þ, with less frequent CD4þ and CD8þ cells near white pulp borders.4 Anatomically, in the early stages of splenic development, the counterclockwise rotation of the stomach and duodenum swings the mesogastrium and mesoduodenum to the left, positioning the pancreatic tail and spleen in the left upper quadrant. This causes the spleen, stomach, and pancreas to be spatially associated with each other, even though they are derived from different embryonic germ layers, mesoderm for the spleen, and the endoderm for the stomach and pancreas.45 Due to this close association between the splenic and the dorsal pancreatic mesenchyme, and the expression of a number of similar markers between the spleen and pancreas, the spleen is implicated in the development of the pancreas and is considered a likely source of islet precursor cells. In addition, it is not uncommon to observe one or more remnants of splenic tissue or accessory spleens in or around the pancreatic tail in both humans and NHP, as a result of their close association during development. In laboratory NHPs, the average splenic weight, expressed as a proportion of body weight, is approximately 0.15% which is toward the lower end when compared to other laboratory animal species and humans (1% for dogs, 0.7% for mice, and 0.5% for rats and humans).46 Laboratory NHPs have a lowly contractile and nonstorage-type, defensive spleen that shows little to no variability in relative weights due to either contraction or congestion.26,27,47 In contrast, animal species with highly contractile or storage type spleens, such as the dog, have an abundance of smooth muscle in the splenic capsule and trabeculae, and show higher relative splenic weights. Storage spleens also show greater variability in splenic weights at necropsy, due to congestion after standard euthanasia,26,27 while animals with thinner capsules, and trabecular tissue that contain less smooth muscle, such as the laboratory NHP and humans, show less or no evidence of contraction or reservoir function.27,46 Therefore, splenic weight changes in NHP appear to be a more reliable indicator of treatment effects than in the dog.

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The histology of the spleen in laboratory animals has been reviewed extensively.26,27,48 The spleen of laboratory NHP is an oblong-shaped organ situated on the left side of the abdomen, dorsal to the stomach. Externally, it suspended within the abdominal cavity and covered by peritoneum (serous membrane), which is adherent to the splenic fibrous capsule (connective tissue), and forms several ligaments to surrounding visceral tissues. Of great importance is the gastrosplenic ligament which contains the splenic vessels.44 Microscopically, as in humans and other animals, the NHP spleen (Fig. 16.9) is composed of red and white pulp.44 The white pulp contains the lymphoid components (lymphoid follicles periarteriolar lymphoid sheaths (PALS)). At the interface between the follicular mantle zone and the red pulp, the marginal zone may be visible as pale basophilic cells, which are primarily comprised of marginal B-cells, dendritic cells and macrophages. The red pulp, which makes up the majority of the volume of the spleen (75%e80% in humans) is comprised of capillaries, and venous sinuses within a loose reticular meshwork and often contains varilable numbers of immune amd hematopoietic cells.46,49 The reticular tissue contains many macrophages, lymphocytes, polymorphonuclear cells and platelets or megakaryocytes, while plasma cells are found along the sinusoids, trabeculae, and marginal zones.50 The strands of reticular tissue between the sinusoids are also called cords of Billroth, while the venous sinuses are lined by an endothelium that express endothelial markers such as Von

FIGURE 16.9 Normal spleen of a macaque: The spleen contains red pulp (R) which consists of venous sinuses and splenic cords that serve to filter blood passing through the organ and returning to venous circulation. The white pulp consists of the B-cell rich follicles (F) and T-cell rich periarteriolar lymphoid sheaths (PALS) and represents the lymphoid component of the spleen. Note that the follicles here have a light staining germinal center, dark staining mantle zone and peripheral lighter marginal zone, giving these structures a layered appearance. The capsule (C) has ligamentous attachments (L) to the mesentery and stomach and has fibromuscular trabecula (T) that extend into the deep tissue of the spleen.

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Willebrand factor.44 The white pulp consists of lymphoid follicles, containing mostly B-cells, and the PALS, made up of mostly T-cells (Fig. 16.10AeC). The structure and organization of lymphoid follicles is similar to those of lymph nodes and MALT. The structural organization of the spleen can also be viewed from its complex microcirculation. From this view point, the spleen can be considered as a branching tree of arterial vessels in which the white pulp forms a sheath of lymphoid tissue (PALS) surrounding the arteries after they leave the trabeculae, and is thickened in places, forming lymphoid follicles.46,51 While the white pulp is intimately associated with the arterial tree, the red

FIGURE 16.10 Lymphoid components of the macaque spleen evaluated by immunohistochemistry: (A) the white pulp lymphoid follicles contain abundant CD20 antigen positive B-cells. Note: the amorphous hyaline material filling the center of this hyperplastic germinal center. (B) Periarteriolar lymphoid sheaths consist primarily of CD3 antigen positive T-cells. (C) CD138 antigen positive plasma cells are scattered through the red pulp, and to lesser extent, lymphoid follicles and marginal zones.

pulp is similarly associated with the venous system that drains the organ.46 Most of or all capillaries do not lead to venules, but empty into the labyrinthine reticular meshwork of the red pulp before draining into the venous system (open circulation), which means the reticular meshwork constitutes an intermediate circulation between the capillaries and venous channels.46 To date, the existence of a closed circulation in human and NHP spleens remains controversial.46,49,52 The spleen receives approximately 5% of the cardiac output via the splenic artery, which enters the spleen at the hilus and progresses through small arterioles to capillaries terminating either in the lymphoid follicles of the white pulp, or to capillaries leading into the red pulp. The splenic artery branches divide into segmental arteries that enter along the splenic trabeculae, and since there is little collateral circulation at this level, occlusion of one of these arteries is usually associated with infarction of the corresponding region in the spleen, which is a frequent observation in thromboembolic diseases. The segmental arteries give rise to trabecular arteries, which branch into central arteries that become ensheathed with T-cells to form PALS. The branches of the central arteries that supply the red pulp are known as the penicillar arteries, while arterioles and capillaries supplying the lymphoid follicles and marginal zone in the NHP spleen arise from a follicular artery branch of the central artery.53 Considerable thickening and hyalinization of the walls of these arteries and arterioles is not uncommon in the spleen of aged macaques. Similar to the lymphoid content in lymph nodes, the adult spleen may have considerable white pulp variation of size in normal animals (Fig. 16.11AeD). Comparatively, the size and morphology of some components of splenic lymphoid follicles differ between NHP and other laboratory animals such as rodents. First, as a immuno-defensive organ, the spleen of NHPs has much more numerous wellformed primary and secondary lymphoid follicles with germinal centers than in the dog or rodents,26,27 although both wide inter-animal and intra-animal variations in the size and shape of lymphoid follicles and germinal centers also exist.27 Additionally, in some macaques, large, misshapen, and coalescing lymphoid follicles with irregular and bizarre germinal centers, often resulting in compression of the surrounding tissue, may be seen, and various theories have been offered as to their cause or etiology.26,28,54,55 The first and most espoused theory is that they represent nodular hyperplasia of the white pulp, and to support this theory is the frequent presence of hyalinized germinal centers (hyaline material, amorphous eosinophilic material) in the same spleens, which are considered to represent antigen-antibody complex deposition, possibly as a result of persistent antigenic stimulation (Fig. 16.12A and B).26,28 However, others consider these to be developmental in origin, and the current proposed term “compound follicles”

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FIGURE 16.11 Morphological variations of the normal macaque spleen: (A) Most commonly, the spleen of the macaque has representative numbers of average-sized lymphoid follicles and periarteriolar lympphoid sheaths (PALS). (B) Similar to changes noted in lymph nodes, there may be increased size or number of lymphoid follicles in the normal spleen. (C) Occasionally, there may be animals with scant follicular tissue identified. (D) There may be similarly varied lymphoid content of the periarteriolar lymphoid sheaths (PALS), here with increased cellularity.

is recommended to describe this common observation in the macaque spleen (Fig. 16.13).27,56 Another slight difference between the spleen of laboratory NHP and that of rats is that the marginal zone is usually not as conspicuous and distinct in the NHP, and the marginal sinus that separates the marginal and mantle zones is even less so, compared to the rat.26,27 Although the PALS in the NHP spleen are not as prominent as in the rat, and less obvious compared to the prominent B-cells follicles and germinal centers, they can be easily appreciated, particularly in sections where the central artery has been exposed.

2.1.7 Development and functional anatomy of the monkey thymus The thymus is a primary lymphoid organ of dual embryonic origin, starting as an epithelial organ in early prenatal life, and later populated by lymphocytes. It develops from the epithelium of the ventral diverticula of the third pharyngeal

pouch, along with the thyroid and parathyroid gland.57 At this early stage the thymus is located in the neck, before migrating downward and medially into the cranial mediastinum as epithelial tubules, plugs, or cords. The epithelial cells proliferate in the mediastinum, and the thymus loses its connection with the branchial clefts. Eventually the cells become surrounded and invaded by the mesoderm that includes vascular mesenchymal tissue, vagal nerve fibers, and hematopoietic precursor cells from the bone marrow that become the lymphoid component of the thymus, or thymocytes.57e59 Mesenchymal vascular and stromal tissues invade the gland in such a way as to produce its lobulated appearance.57 They become unified with the reticular epithelial cell framework of the thymus, and the organ continues to grow and develop until puberty. In the macaque, the thymus primordium is first apparent around GD35 at the topographical level of the thoracic inlet and is in close contact with the cranial part of the pericardium.58 By GD40, it becomes elongated and slightly

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FIGURE 16.13 A compound follicle within the spleen of a macaque: These compound follicles are occasionally noted in the otherwise normal NHP spleen. Whether these are hyperplastic foci or congenital anomalies is not known.

FIGURE 16.12 (A) Hyperplastic lymphoid follicles may contain accumulations of hyaline, eosinophilic material (asterisk), which are considered to represent antigen-antibody complexes. (B) Higher magnification of hyaline materials accumulated in the germinal center of an atrophic lymphoid follicle, which may represent persistence of the matrerial from a previous hyperplastic phase.

lobulated containing polymorphic, densely packed thymocytes embedded in a loose mesenchyme, and covered with a distinct layer of surface epithelial cells.4,58 The first immunoreactivity in the macaque thymus is present at GD50 with positive staining for HLA-DR which is first confined to the medulla.58 Positively immunoreactive CD3 cells first appear toward the end of the first trimester and are apparent by GD60.4,58 The CD3þ thymocytes are initially found predominantly in the medulla but increase in the cortex with further organ development. CD4þ and CD8þ cells appear in the early second trimester in the outer cortex, medulla, and interlobular septal regions, a pattern that continues throughout development.4 Hassall’s corpuscles appear around the same time and increase in number until the early postnatal period, then decrease with adulthood.4,58 FoxP3 expression follows a similar pattern to CD3,

appearing in the first trimester with strong positive staining of nearly all thymocytes, particularly in the medulla. Unlike T-cells markers, the expression of B-cell markers, CD20 or CD79, in the developing macaque thymus starts in the second trimester, is lower in frequency, and restricted to the corticomedullary junction, with rare cells in the medulla.4,58 Therefore, by early second trimester or approximately GD60, the macaque thymus is functionally mature, with a morphologically distinct cortex, medulla, and Hassall’s corpuscles, although positive immunoreactive CD68 cells appear around GD70, with a scattered even distribution between the medulla and cortex.58 In the cynomolgus macaque, maximal thymus weight relative to body weight occurs at birth, while maximal absolute thymus weight occurs near sexual maturity.60 The beginning of thymus involution in macaques coincides with the onset of sexual maturity, which is estimated to occur between 3½ and 4½ years in cynomolgus macaques (Fig. 16.16 A and B).61 The thymus of post-natal NHP is a bilobed organ with an elongated, leaflike shape (from where it derives its name due to its resemblance to a leaf of the thyme plant),62,63 that is located in the cranial mediastinum in the thoracic cavity. Both lobes contain an outer and darker staining cortex and inner, lighter staining medulla, and are surrounded on the outside by a thin fibrous capsule composed of an outer and inner layer of collagen and reticular fibers (Fig. 16.14AeC). The inner layer of the capsule gives rise to septae, which partially subdivide the thymic cortex into interconnecting lobules of various sizes and orientations.64 Occasionally,

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lymphocytes can be observed in between the two layers of the capsule. Besides the septae, trabeculae also extend from the capsule or septae into the center of lobules. However, compared to other lymphoid tissues, there is very little supporting connective tissue in the thymus. The bulk of the supporting framework is composed of a network of reticular fibers and epithelial reticular cells joined by simple contacts and specialized junctions.57,64 The lymphoid tissue is mostly composed of thymocytes (T-cells) which are more numerous and closely packed in the cortex (Fig. 16.15). The medulla contains fewer lymphoid cells and the characteristic Hassall’s corpuscles. The connective septa separating adjacent lobules contain blood vessels, nerves, and efferent lymphatics. Intriguingly, the thymus medulla of the laboratory NHP, human, and other animals, particularly in the young, may also contain “myoid” cells, which bear a resemblance to striated muscle, but whose functional significance is not fully known. Thymic myoid cells are thought to play a role in the regulation of processes of thymocytes proliferation, differentiation, and selection during the development. In humans, thymic myoid cells may be involved in the pathogenesis of myasthenia gravis.65,66 The postnatal thymus is composed of six different types of epithelial cells and several stromal cells of mesodermal origin. Stromal cell types have a special function in providing the proper environment for the maturation and/or selection of antigen specific T-cells, and development and expression of T-cell antigen receptors.67 The current understanding of the role of thymic epithelial cells (TEC) in the maturation or education of thymocytes and involution of the thymus has greatly evolved in recent years. It is now well-appreciated that the maturation of thymocytes requires cell-to-cell interactions between thymocytes and endothelial cells, mesenchymal fibroblasts, and the two types of TECs, cortical and medullary.68 The two TEC types contribute to the education of developing T-cells. Whereby cortical TECs mediate the selection of T-cells expressing functional receptors (positive selection), medullary TECs

FIGURE 16.14 (A) the normal NHP thymus prior to the start of involution is composed of multiple lobules (oval) divided by septa (S) and has a prominent dark basophilic cortex (Cx) and light basophilic to eosinophilic medulla (M) when viewed on low magnification (2 objective). (B) The thymus is encased in a thin fibrous capsule (C) composed of an outer and inner layer of collagen and reticular fibers. The inner layer of the capsule gives rise to septae (S), which partially subdivide the thymic cortex into interconnecting lobules of various sizes and orientations. The differential staining of the cortex (Cx) and the medulla (M) is prominent. (C) Higher magnification of the macaque thymus: The lymphoid tissue is mostly composed of T-cells, also known as thymocytes, that are numerous and closely packed in the cortex (Cx). The medulla (M) contains fewer lymphoid cells and the characteristic Hassall’s corpuscles (HC). The connective septa (CS) separating adjacent lobules contain blood vessels, nerves, and efferent lymphatics.

FIGURE 16.15 Evaluation of T-cell content in an adolescent macaque thymus by immunohistochemical methods (IHC): The increased distribution of T-cells in the cortex (Cx) as compared to the medulla (M) of the fully formed thymus may be visualized via IHC for CD3 antigen.

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deplete those with potential specificity to self-antigens (negative selection).68e70 Further, medullary TEC contribute to the development of T-cell tolerance by expressing and presenting tissue-restricted antigens (TRA), so that developing T-cells can assess the self-reactivity of their antigen receptors prior to leaving the thymus.70 The thymus undergoes age-related involution at a much more accelerated rate than any other tissue, and TECs are thought to play a role in the process.68,69 This thymic regression includes reductions in thymic mass, loss of thymic structure, disorganization to thymic architecture, and replacement by mature adipose tissue (Fig. 16.16A and B), which results in diminished thymocyte numbers and reduced naïve T-cell output.68,71 This postnatal thymic

involution is now known to be attributable to TECs rather than thymocytes.69 The understanding of the role of TEC in age-related thymus changes has bearing on the development of novel strategies to counter thymic atrophy in relevant clinical scenarios.68

3. Congenital lesions of the hematolymphoid system Congenital anomalies of the lymphoid system, other than cysts, ectopic tissue, and hypoplasia, are relatively uncommon in the NHP.72 While in humans, the most clinically important congenital lesions of the lymphoid system are malformations of the lymphatics, which can lead to primarily head, cervical, or thoracic lymphangiomas, lymphangiectasis, lymphangiomatosis, or lymphatic dysplasia syndrome,73,74 such congenital errors of lymphatic development have thus far not been reported in the NHP.

3.1 Thymus ectopia and parathyroid gland ectopia within the thymus The most commonly encountered congenital anomaly of the NHP is thymic ectopia. Ectopic thymic tissue may be encountered nearly anywhere within the thoracic cavity, but is very commonly reported in the thyroid and parathyroid glands (Fig. 16.17), which is not surprising, as they have close proximity in the early embryonic stage, and migrate to their respectively locations in the cervical region at approximately the same time. Conversely, ectopic parathyroid glands may occur in the thymus as a result of their common embryological origin in the third pharyngeal pouch.75 Other locations reported for ectopic thymic tissue include the heart, esophagus, and pharynx.

FIGURE 16.16 The thymus of the NHP undergoes rapid growth during development, peaks in size around adolescence, and begins involution as early as birth and no later than the onset of puberty. (A) Involution is characterized by reduction in thymic mass, loss of thymic structure, and disorganization to thymic architecture. Notice the replacement of thymus by well-formed adipose tissue, a hallmark of normal involution. (B) As the thymus further involutes, there is near complete replacement of the lymphoid tissue by well-formed lobules of mature adipose tissue. Note that the leaf-shape of the thymus is recapitulated in the shape of the adipose replacement with remnant thymic septa visible as connective tissue between adipose lobules.

FIGURE 16.17 The most commonly encountered congenital anomaly of the NHP is thymic ectopia within the thyroid gland (TG) or parathyroid gland (PT). The thymic tissue may have cortex (Cx), medulla (M), or both present as an ectopic focus, and there may be well-formed substructures, such as Hassall’s corpuscles (HC), or septa (S) present.

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3.2 Congenital thymic cysts Congenital thymic cysts may represent cystic remnants of the thymopharyngeal ducts (Fig. 16.18AeC), or dilatation of thymic tubular structures. Both are frequently observed in the thymus of laboratory macaques and marmosets.28,54,75

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They are usually thin walled, and lined by thymic epithelial cells, or other types of epithelia that vary from flattened to columnar or ciliated cells, or thymic tissue without an epithelial lining.76 Congenital cysts may be unilocular, or multilocular, often contain floccular eosinophilic proteinaceous material,75 and may become more prominent with thymic involution or atrophy. The thymic cysts observed within the medulla in laboratory NHPs may also have thymic myoid cells or skeletal muscle tissue, which have been reported as ectopic muscle, and considered to be hamartomatous or aberrant muscle tissue.28,75 Similarly, prominent myoid cells with characteristics corresponding to those of skeletal muscle cells have been reported in the thymic medulla in several animals, including humans with myasthenia gravis, thymoma, or multilocular cysts.77e80

3.3 Ectopic spleen Accessory or ectopic spleens are not uncommon in NHPs, particularly in marmosets.28,54,81,82 They mostly occur as incidental findings of no clinical significance, and are mostly observed in the tail of the pancreas or surface of the stomach, although other locations, including the hilar region of the spleen, the omentum, or pelvic region, have been reported in humans and have been noted by the editor in NHPs.83 In macaques, accessory spleens have been found in the mesentery or attached to the serosal surfaces of mesenteric organs (Fig. 16.19A). Ectopic spleen which is embeded within the pancreas should be differentiated from splenosis (See Miscellaneous findings of the hematolymphoid system). In human pathology, accessory spleen is mostly reserved for congenital foci of healthy splenic tissue which are separate from the main body of the spleen.83 Histologically, ectopic or accessory spleens may be differentiated from splenosis, by their well-defined capsule, hilus, trabeculae, red pulp, and white pulp with follicles and central arterioles (Fig. 16.19B). Similar foci may occur within the pancreatic tissue and may not have a well-defined capsule. These ectopic splenic foci appear to retain much of the function and responses to stimuli as that of the spleen. For instance, follicular hyperplasia or lymphoid depletion, when noted in the spleen, will often also be present in the ectopic splenic focus of an individual animal. FIGURE 16.18 Congenital thymic cysts in non-human primates: (A) congenital cysts within the thymus are not uncommon, appearing as irregular, unilocular, or multilocular spaces or dilated ductal spaces within the tissue. (B) Congenital cysts may be filled with mucin, floccular eosinophilic proteinaceous material, or cell debris (asterisk) and may be difficult to differentiate from degenerative or acquired cysts, but generally are thin-walled and lined by squamous, glandular, or ciliated epithelium (arrows). (C) The presence of ciliated epithelium lining the cyst (arrows) is characteristic of the thyropharyngeal duct from which congenital cysts originate.

3.4 Ectopic salivary gland in the mandibular lymph node Ectopic salivary gland may occur in three forms: accessory gland, heterotopic gland or gland associated with branchial cleft anomalies.84 Accessory salivary gland tissue is wellformed with a thick external capsule and usually embedded within the submandibular lymph node

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FIGURE 16.20 Accessory salivary gland is characterized by wellformed salivary gland tissue with a thick external capsule embedded within the submandibular lymph node. This finding is without associated pathology in NHPs.

specific etiology and interrelationship of benign vascular proliferative lesions of lymph nodes, such as lymphangiomatosis, angiomyomatous hamartoma, or vascular transformation of sinuses, have not been elucidated, developmental errors in lymphatic organization in the lymph nodes are suspected to play a role, at least in some of the cases.87e89 Hypoplasia of the spleen, thymus, bone marrow, and lymph nodes has been reported at a low incidence in baboons.90

4. Degenerative lesions of the hematolymphoid system of the non-human primate FIGURE 16.19 (A) Congenital accessory spleens are usually identified in proximity to the spleen, but less common sites include the serosal surfaces of abdominal cavity, such as this nodule of splenic tissue on the small intestine of a macaque. (B) Congenital accessory spleens usually have multiple distinct microscopic features including, a well-defined capsule, red pulp with trabeculae and appropriate cell populations, and white pulp with follicles and central arterioles surrounded by PALS.

(Fig. 16.20). The diagnosis of accessory salivary gland is considered most accurate when there are no other abnormalities associated with the ectopic glandular tissue. Accessory parotid salivary gland has been reported in approximately 21% of the human population, while submandibular accessory tissue is much rarer.84,85

3.5 Other congenital findings in non-human primates Other congenital anomalies in NHPs have been sparsely reported. An angiomyomatous hamartoma of the lymph node was reported in the cynomolgus monkey.86 While the

Lymphoid tissue tends to react to pathogens and other environmental stimuli by lymphoid hyperplasia, atrophy, or depletion, or by the advent of inflammation in response to the pathogens. Therefore, spontaneous degenerative lesions are very limited, particularly in the younger age group of laboratory NHPs used in toxicology studies.

4.1 Capsular and trabecular fibrosis of the spleen Rarely, there may be segmental to diffuse, capsular fibrosis of the spleen noted as an incidental finding in NHPs (Fig. 16.21).28,54 Although an underlying cause for the capsular fibrosis may not be identified, this change is a known consequence of trauma to the splenic surface and may be an indication of previously insult. Additionally, thickening or increase in the fibrous component of the capsule may be observed in spleen with long standing hemorrhage or inflammation with splenic congestion due to viral infections.

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FIGURE 16.21 A rare finding in NHPs is capsular fibrosis of the spleen characterized by increased thickness of the capsule with hypercellularity (arrows). Although an underlying cause for the capsular fibrosis may not be identified, this change is a known consequence of trauma to the splenic surface and may be an indication of previously insult.

4.2 Degenerative cysts of the thymus Similar to their congenital counterpart, degenerative cysts appear as unilocular or multilocular spaces; are typically lined by flat, cuboidal or columnar epithelium; and often contain floccular eosinophilic proteinaceous material; however, these cysts do not have ciliated epithelium and may have much thicker fibrous walls with inflammation, in contrast to the congenital cysts.75 Regardless of the congenital or degenerative nature, the recommended morpholigic diagnosis for data entry is simply “cyst, epithelial.”56

4.3 Stress-induced changes in the hematolymphoid system Stress is perhaps one of the most commonly encountered issues that presents during studies with associated tissue changes in hematolymphoid organs (Fig. 16.22AeC). Stress-induced lymphoid depletion occurs quite commonly in NHPs and may be virtually indistinguishable from test articleeinduced lymphoid depletion (See Toxicological findings of the hematolymphoid system). In the lymph nodes and spleen, decreased lymphoid cellularity commonly presents as loss or reduction of germinal centers in lymphoid follicles, while in the thymus it is often associated with thinning or loss of thymocytes that begins with cortical lymphocyte apoptosis or lymphocytolysis, progresses to cortical thinning and eventually may lead to near complete loss of the organ. Stress-induced lymphoid depletion in the thymus is often without the normal adipose tissue replacement noted for involuted thymuses. In severe cases, stress may be accompanied by serous atrophy of the bone marrow adipose tissue. Localized or systemic inflammation as a result of infection may lead to secondary inflammation or inflammatory infiltrates in the lymph node sinuses, or splenic red pulp, which can confuse the picture in otherwise stressed or immunosuppressed animals. If

FIGURE 16.22 (A) Animals experiencing stress may have generalized serous atrophy of body adipose tissue noted, including that of the bone marrow. (B) Acute severe stress may result in splenic contraction, noted microscopically as wrinkling of the capsule, contraction of the splenic muscular septae and decreased red pulp content. The case presented had had intestinal entrapment through the inguinal ring. (C) Acute stress produces loss of lymphoid cells, noted microscopically as increased apoptosis with increased tingible body macrophages (lymphocytolysis), giving the “starry sky” appearance to the cortex of the thymus.

examined proximal to death, animals with extreme stress may have contraction of the spleen histologically visible as wrinkling of the capsule, contraction of the splenic muscular septae and decreased red pulp red cell content. The contribution of stress on toxicology studies has been extensively reviewed by Everds et al and others.91

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4.4 Other degenerative findings of the hematolymphoid system Other spontaneous degenerative findings include mineralization in the thymus,28 lipid granuloma in the spleen,92 pigment accumulation, and lymphangiectasis (Fig. 16.26). Different types of pigment may be observed within sinus macrophages of various lymph nodes in macaques and marmosets. Animals with moderate to marked pneumoconiosis or anthracosis may have dark brown pigment in the bronchial and mediastinal lymph nodes that drain the lung. In the mesenteric lymph nodes of NHPs, up to mild grades of sinus histiocytosis can be considered a normal background finding and these macrophages often contain endogenous pigments (e.g., lipofuscin) or exogenous pigments reflecting uptake of dietary exogenous material from the digestive tract. Similar pigment may be observed in the macrophages in the lamina propria of the small intestines. In the bone marrow, spleen, or lymph nodes, pigment accumulation may also represent hemosiderin, and occur as focal to multifocal, goldenebrown globular, intracellular pigment. Hemosiderin deposits represent the degradation of heme-based endogenous materials such as lysed red blood cells or heme-scavenging/iron reclamation processes. In macaques from Southeast Asia regions where malaria is endemic, malarial pigment may be observed as a dark brown to blackish pigment within reticuloendothelial cells throughout the splenic red pulp,27 and often in association with reticuloendothelial hyperplasia (see Chapter 3).27

eosinophils, noted in lymphoid tissues of healthy NHPs (Fig. 16.23A and B). In mandibular lymph nodes and to a lesser extent, other lymph nodes, increased granulocytes may occur alone or in conjunction with low-level extramedullary hematopoiesis. Similarly, increased numbers of granulocytes are occasionally noted within the splenic red pulp, particularly in regions subtending the capsule.92 Granulocytic infiltrates and macrophage aggregates have been reported in the spleen of control animals without other obvious disease conditions.28 Neutrophils may be rapidly recruited to lymph nodes in response to various stimuli, where they may interact with the resident immune system populations, primarily lymphocytes, as part of the innate and adaptive immune mechanisms.93 Eosinophils may predominate in the lymph nodes and splenic red pulp when parasite load, either in the gastrointestinal tract or elsewhere, has increased above tolerance thresholds. With intestinal parasitic nematode infections (most commonly Oesophagostomum), a substantial amount of eosinophilic infiltrates may be observed in mesenteric lymph nodes.27 Therefore, the increased number of granulocytes in the NHP lymph nodes and spleen may be a surveillance

5. Inflammatory and vascular lesions of the hematopoietic system 5.1 Inflammatory cell infiltrates Spontaneous inflammation or inflammatory infiltrates may occur in local lymph nodes draining a site of inflammation or site of necrosis (such as administration sites), proximal to neoplastic foci, or as part of a generalized inflammatory disease process. Inflammatory cells can also occur within a lymph node in response to primary lymphocyte necrosis or following profound lymphoid depletion induced by stress or background viruses.30 However, spontaneous alterations of inflammatory cell populations in the lymphoid system are of important notation for the purposes of comparative analysis when they are encountered on study or in otherwise normal tissues of untreated young cynomolgus macaques.

5.1.1 Increased granulocyte content of lymphoid tissues Sporadically, there may be noticeably increased numbers of granulocytesdprimarily neutrophils and/or occasional

FIGURE 16.23 (A) Within the spleen, there may be noticeably increased numbers of granulocytes in the subcapsular region of the red pulp as compared to other splenic regions. (B) Increased numbers of eosinophils in the mesenteric lymph node of a macaque, due to increased parasite load in the intestinal tract infected with Oesophagostomum sp. nematodes.

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population expansion. Granulocytic infiltrates should be distinguished from conditions such as extramedullary hematopoiesis (EMH) and granulocytic leukemia.

5.1.2 Lymph node sinus histiocytosis A relatively common finding among NHPs is lymph node sinus histiocytosis (Fig. 16.24A and B). The sinuses may be filled with bland histiocytic cells containing finely granular cytoplasm. Very rarely, the histiocytic infiltrate may be of such magnitude as to induce grossly enlarged lymph nodes (Fig. 16.25). Although this finding is considered a lymphadenopathy of unknown etiology in humans (RosaiDorfman disease), there is no current evidence sinus histiocytosis has similar significance in NHPs. In NHPs, sinus histiocytosis may be identified in animals with minor infections, but is frequently noted in normal animals with no evidence of disease, and thus it is often considered an incidental finding of no clinical relevance.28,94,95

5.1.3 Lymph node lymphoplasmacytosis Lymphoplasmacytosis of the lymph nodes is occasionally noted as an incidental finding in NHPs. It is most commonly identified within the medullary cords in NHPs,

FIGURE 16.25 On very rare occasions, spontaneous sinus histiocytosis is of a magnitude that produces generalized lymph node enlargement in an otherwise normal lymph node.

although nearly any lymph node compartment may be infiltrated, and the cells may form islands and clusters within lymph node compartments. There may be enlarged medullary cords, or generalized lymph node enlargement, due to increased numbers of cells that are primarily plasmacytoid. These lymphoid cells are without atypia, which helps differentiate the cell population from malignancies; however, IHC and/or flow cytometry for polyclonality would be confirmatory.96 The increased numbers of these lymphoid cells may be due to underlying inflammatory conditions, antigenic stimulation, or may be an exaggeration of the germinal center oligoclonal nature.96,97 In the spleen there may also be cases of increased lymphoplasmacytic cells within the red pulp.

5.1.4 Lymph node inflammation

FIGURE 16.24 (A) Sinus histiocytosis in the NHP is characterized by sinuses filled with bland histiocytic cells containing finely granular to vacuolated cytoplasm. (B) The histiocytic nature of the cell population within the lymph node sinuses may be visualized by immunohistochemical detection of cell antigen CD 68.

With infectious disease, the type of lymphadenitis can vary depending on the pathogenic nature of the inciting factor (bacterial, viral, fungal, or parasitic), and the response can vary from acute to granulomatous. In acute lymphadenitis, neutrophils predominate, but other cell types such as plasma cells that are normally resident in the medullary sinuses may increase. For instance, in both macaques and marmosets, gingivitis and periodontal disease can occur spontaneously98 leading to lymphadenitis and inflammatory lesions in the mandibular lymph nodes, which mainly involve plasma cell infiltrates. With intestinal parasitic nematode infections (most commonly Oesophagostomum), a substantial amount of eosinophilic infiltrates may be observed in mesenteric lymph nodes,27 and up to marked pyogranulomatous inflammation in the GALT is more frequent with these aberrant parasites. Lymphoid follicular hyperplasia may also occur in association with nodal inflammation, particularly in acute inflammatory lesions,

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while moderate to marked atrophy or lymphoid depletion may be associated with a pyogranulomatous inflammation or macrophage or plasma cell infiltration in the lymph node sinuses or red pulp in the spleen. The latter is commonly seen in viral infections that cause lymphoid depletion such as measles or retroviruses. Inflammation due to primary or opportunistic bacterial infection can cause abscesses or pyogranulomatous inflammation in lymph nodes, spleen, or bone marrow and are typically associated with a generalized systemic infection, affecting other organs. Although any systemic bacterial, protozoal, or fungal infection can potentially cause abscess or pyogranulomatous inflammation in the spleen or lymph nodes, the most commonly encountered bacterial infections that frequently affect the spleen or lymph nodes in both captive and wild NHP are Yersinia and Mycobacteria (see Chapter 3).

5.2 Lymph node sinus erythrocytosis and erythrophagocytosis Erythrocytes transported from extranodal sites of hemorrhage via lymphatic drainage may be present within lymph node sinuses, either extracellularly (erythrocytosis) or within macrophages (erythrophagocytosis).30 It is not uncommon to see erythrocytosis in the lymph node draining the administration sites for animals from preclinical studies (Fig. 16.26).

FIGURE 16.27 Spontaneous thrombosis is exceptionally rare within lymphoid tissue; however, small vessels may experience thrombosis secondary to the infusion process.

identified as rare, single entities when they occur in animals assigned to studies. Because the splenic arteries and its branches are end arteries with reduced to no collateral circulation, occlusion of one of these arteries by a thrombus is usually associated with infarction of the corresponding region of the spleen, which is may be observed in thromboembolic diseases.

5.3 Thrombosis Spontaneous thrombosis is exceptionally rare within lymphoid tissue; however, similar to other organs, small vessels may experience thrombosis secondary to the infusion process (Fig. 16.27). These thrombotic foci are usually

5.4 Hemorrhage Small focal hemorrhages are not uncommonly noted in a variety of lymphoid organs. The most common locations include the thymus and lymphoid follicles (Fig. 16.28A and B). The etiology of these small hemorrhages is unclear but they may represent agonal vascular leakage. Larger hemorrhages may occur on rare occasion due to trauma.

6. Hyperplastic and neoplastic lesions of the hematolymphoid system A significant proportion of neoplastic and proliferative diseases reported in NHPs is associated with various infectious agents and is therefore described elsewhere (see Chapter 3). This section describes reports of neoplastic findings where there was no compelling evidence of an infectious agent involvement. FIGURE 16.26 Erythrocytes transported from extranodal sites of hemorrhage via lymphatic drainage may be present within lymph node sinuses (asterisk), either extracellularly (erythrocytosis) or within macrophages (erythrophagocytosis). It is not uncommon to see erythrocytosis in the lymph node draining the administration sites for animals from preclinical studies, as hemorrhage is a common sequela of the injection process. Note: the medullary sinus and vascular spaces (arrow) are dilated due to draining of fluid from the administration site at the dermatome associated with this lymph node.

6.1 Hyperplastic and neoplastic diseases of the thymus 6.1.1 Thymoma Two cases of thymoma have been reported in NHPs. One in a 4-year-old male cynomolgus macaque was reported by

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6.2 Hyperplastic and neoplastic lesions of the spleen 6.2.1 Reticuloendothelial hypertrophy or hyperplasia of the splenic red pulp Reticuloendothelial hyperplasia of the red pulp occurred in 7% control cynomolgus macaques in one retrospective study.28 This spontaneous finding in the spleens of macaques is characterized by plump (hypertrophic) endothelial cells, occasionally in multiple layers (hyperplasia) above prominent, hyalinized, eosinophilic basement membranes (Fig. 16.29). This finding may be striking microscopically, with prominent vascular profiles of the red pulp. It may be more prevalent or more severe secondary to various infections that involve the splenic red pulp, such as Plasmodium sp. and other bloodborne pathogens. Reticuloendothelial hyperplasia is also noted in some nodular lesions of the spleen (see Nodular Lesions of the Spleen).

6.2.2 Nodular and mass-like lesions of the spleen

FIGURE 16.28 Small focal hemorrhages are not uncommonly noted in a variety of lymphoid organs: (A) minor hemorrhage in the splenic lymphoid follicle and (B) thymus. The etiology of these small hemorrhages is unclear; however, they are acute in morphologic features consistent with onset near the time of euthanasia.

Kotani et al.99 It was diagnosed as a mixed type (type AB) based on the WHO classification system for humans. It consisted of thymic cortex-like CD3 positive lymphoid cells, with nests of medulla type cells, and areas of fascicular proliferation of elongated spindle cells in a sporadic storiform pattern that stained for cytokeratin. Schwarz et al. described a thymoma in a two year-old female cynomolgus macaque composed of dense sheets of vimentin positive spindle-shaped cells and large cystic cavities separated by thick connective tissue stroma. Hassall’s corpuscles, individual and small clusters of mature small lymphocytes, scattered eosinophils, and large multinucleated giant cells were also observed, which varied widely in size and nuclear number. Rare spindle and Hassall’s corpuscle cells were cytokeratin positive. Based on the WHO classification system this would be described as a medullary thymoma (type A, spindle cell thymoma).100

Nodular foci in the spleens that range in size from microscopic to grossly visible have been identified in cynomolgus and rhesus macaques at the editor’s facilities. They have been identified in colony animals, control animals, and as isolated cases in animals receiving test articles in which the nodules were considered preexisting. These foci have variable microscopic features, but share tumoral features, such as adjacent tissue compression and tissue architecture replacement or effacement. Most of the nodules have been discovered as incidental lesions identified during standard histologic evaluation of the spleen, but larger nodules have been grossly visible as distorted,

FIGURE 16.29 Reticuloendothelial hyperplasia of the spleen is characterized by plump (hypertrophic) endothelial cells, occasionally in multiple layers or with multinucleated epithelial cells (hyperplasia) above prominent, hyalinized, eosinophilic basement membranes.

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nodular spleens. There are morphologic variations of the nodules. In one variation, the nodular proliferative lesion was composed of polygonal to elongated cells forming densely packed and poorly organized vascular channels. These nodules were accompanied by mixed leukocytes embedded in eosinophilic background material that formed an unencapsulated mass. Within the mass there were no white pulp elements identified. There was compression of adjacent splenic tissue and mitotic figures were present in low numbers (Fig. 16.30AeD). This form of splenic nodular lesion was found to be multifocal within the spleen and occasionally, had significant sclerosis. In some cases, there was diffuse reticuloendothelial cell hyperplasia adjacent to the nodular lesions or throughout the remaining splenic red pulp. A second variation of nodular lesion of the spleen in macaques contained numerous vascular channels lined by

hypertrophic endothelial cells with little or no atypia, and usually were accompanied by mixed cell infiltrates or extramedullary hematopoiesis (Fig. 16.31A and B). These nodular lesions were usually multifocal or coalescing within the spleen and they contained no white pulp components. Mitotic figures were not a feature of these nodules. Both morphologic variations of the nodules have been evaluated with IHC methods (Fig. 16.32AeF). Commonly, the nodules were devoid of organized lymphoid tissue. Of those nodules evaluated by IHC, the cellular components within the masses were CD31 positive, confirming endothelial origin. Additionally, the nodules had variably positive results for CD8 and vimentin; however, this IHC was not performed on all specimens. In general, the nodules are considered likely sclerosing angiomatoid nodular transformation (SANT), a poorly described rare, benign, incidental vascular entity confined

FIGURE 16.30 Nodular masses of the spleen of macaques: (A) Within the spleen there is an unencapsulated, expansile mass compressing the adjacent splenic parenchyma. No white pulp elements are present within the mass. (B) The nodule from Fig 16.30A is composed of polygonal to elongated cells forming densely packed and poorly organized vascular channels. The nodule is accompanied by mixed leukocytes embedded in eosinophilic background material that form an unencapsulated mass and rare mitotic figures (arrow) are noted. (C) A small mass in the spleen of a macaque: Note the reticuloendothelial hyperplasia (asterisk) of the remaining splenic red pulp in this animal. (D) Higher magnification of the nodule in Fig. 16.30C has densely packed and poorly organized vascular channels with mixed leukocytes and rare mitotic figures (arrow).

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than a neoplastic process.104 Although these nodular lesions in macaques have similarities with SANT, further investigation is needed to clearly define them.

6.2.3 Neoplastic lesions of the spleen

FIGURE 16.31 Nodular mass of the spleen of macaques: (A) A large nodule within the spleen of a cynomolgus macaque compresses the adjacent tissue. The nodule is composed of vascular channels lined by hypertrophic endothelial cells and has mixed cell infiltrates and abundant extramedullary hematopoiesis (EMH) present. (B) Higher magnification of the nodule in 16.31A: Portions of the nodule contain increased fibrous stroma. Vascular channels are poorly organized and lined by plump endothelial cells. Within the vascular channels are accumulations of red cells. Multifocal clusters of endothelial-like cells form irregular islands, and the tissue contains abundant EMH admixed with mature leukocytes.

to the spleen and reported in humans and other NHPs.101,102 These nodules are reported with variable morphologies as described above, and have differential diagnoses of splenic hamartoma, splenoma, inflammatory pseudotumor, littoral cell angioma, hemangioma or hemangiosarcoma. In humans there has not been a consensus on the nomenclature, and not all authors agree on the morphologic diagnosis of SANT versus the differential diagnoses listed above for these lesions. The diagnosis remains reliant on IHC panels to elucidate the cell populations which are rarely performed in NHPs in the preclinical setting.103 As reported in humans, SANT is currently considered a polyclonal, reactive lesion rather

Primary neoplasia of the spleen is exceptionally rare in NHPs. A few currently reported cases include an angioleiomyoma in an owl monkey (Aotus trivirgatus),105 and a littoral cell angioma of the spleen in a Japanese macaque (Macaca fuscata).106 More recently, a multilobular splenic mass was identified in a young (10 ng/ mL circulating progesterone indicating ovulation.48 The uterus becomes digitally palpable measuring approximately 0.5 cm by 1 to 1.5 cm long at maturity.35 The age at the first reproduction is typically 17e20 months if the female is removed from the family group and paired.19 The appearance of the external genitalia changes from small protruding vulva to a more prominent vulva surrounded by pale pebbled dermis in the mature female (Fig. 17.17AeC). The clitoris is not externally visible in the marmoset.8 Aged female marmosets do not undergo menopause. There is ageassociated reduction in litter sizes and intermittent to absent ovarian cycles although the ovaries are hormonally active.25

2.5 Social and seasonal effects 2.5.1 Macaque species Rhesus macaques have a distinct period of reproductive inactivity, which occurs in the summer in the Northern Hemisphere. During this time, follicle development arrests and the uterus becomes quiescent (Fig. 17.15).49 Menstrual

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removed or becomes ill, ovulation may be observed in a previously subordinate animal within 11e40 days.48 If a cycling subordinate female is returned to a family group, progesterone levels will typically fall below 10 ng/mL within w5e10 days although there is almost always fighting between females within the group to reestablish the order of dominance.48

3. Congenital lesions of the female reproductive system 3.1 Ectopic ovarian tissue

FIGURE 17.15 Photomicrograph of the uterus and ovaries of a rhesus macaque in a period of seasonal reproductive inactivity: Note the very thin attenuated endometrium within the uterus (U) as well as the absence of follicular activity in both ovaries (O).

Ectopic ovarian tissue is a common finding on the ovarian adnexae and surface of the uterus in cynomolgus macaques16; one retrospective study in cynomolgus macaques found a prevalence of 7%.51 It has also been reported in the broad ligament.

3.2 Para-ovarian cysts Cysts of mesonephric or paramesonephric origin are common in macaques (Fig. 17.16A and B). These are most

cycle records, age, and date of tissue collection rather than histology should be used to differentiate between reproductive inactivity and immature animals. Cynomolgus macaques are less seasonal, but have a slight suppression of ovarian function in the summer months.10 Social effects on reproductive cyclicity in macaques are profound, and the effects of moving the animals or rearranging social groups may persist for months.10,15

2.5.2 Marmoset species Marmosets live in extended family units with the dominant female producing young and the dominant male and older (immature and sexually mature) siblings assisting in the rearing of offspring.22,50 Ovulation is suppressed in subordinate females as evidenced by the age of first ovulation which occurs at 13e14 months in nonfamilial groups and pairs, and at 18e20 months for daughters living within families. Approximately 82% of daughters in familial groups have no evidence of ovulation as characterized by inhibition of GnRH secretion from the hypothalamus, insufficient LH stimulation of ovaries, and measurable plasma progesterone levels of less than 10 ng/mL.48 This may decline as daughters age and become less-subordinate or non-subordinate. A small subset of subordinate marmosets may have ovarian cycling, although these cycles are often inadequate with a shortened luteal phase.22,48 Ovulation may be suppressed for months to years when within a stable family group.24 Conversely, dominant females may retain reproductive seniority for many years within a stable family group. When subordinate females are removed from the family group or if the dominant female is

FIGURE 17.16 (A) Gross specimen from a rhesus macaques with a right, a thin-walled para-ovarian cyst filled with clear fluid (C). Note the left ovary (L) and uterus (U) are present in the image. (B) Photomicrograph of multiple para-ovarian cysts (C) with thin walls lined by attenuated epithelium adjacent to the ovary (O) and fimbria (F) in a cynomolgus monkey.

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often para-ovarian, but cystic mesonephric duct remnants within the wall of the vagina are also seen. There is one report of a clinical para-ovarian cyst in a geriatric marmoset in the Wisconsin National Primate Research Center database.

DSD56 and is characterized by the presence of both ovarian and testicular tissue in the gonads. This condition is exceptionally rare in NHPs, and has been reported in a rhesus macaque.57 Several cases in young cynomolgus monkeys have been noted at the editor’s facilities.

3.3 Disorders of sexual development

3.4 Imperforate vagina and fused labia

Disorders of sexual development (DSD) is the current terminology used to describe defective gonadal/genital development and has three classifications described in humans, based on morphologic features and chromosomal evaluation,52,53 but for NHPs, chromosomal evaluation is rarely performed. Nonetheless, DSD has been reported in NHPs on occasion.32,54,55 An XY female marmoset was identified with atypical external genitalia without evident vulva or testes, female plasma testosterone concentration of 2 nmol/L, normal ovaries with developing follicles, a uterus, and a microscopically normal vagina.32 DNA sequencing of hair from the marmoset demonstrated two bands corresponding to both the X-linked ZFP gene and the Y-linked ZFP gene as well as Y-linked SRY chromosome sequences.32 Male pseudohermaphroditism, now termed “XY DSD,” has been reported in two cynomolgus macaques in which the gonads were of only female origin but external genitalia was of male orientation.54 True hermaphrodism has currently been reclassified ovotesticular

Imperforate vagina has been reported in two common marmosets.52,58 Congenital fused labia (CFL) have been noted in captive or colony-bred marmosets (Fig. 17.17E and F); most commonly in prepubescent females. CFL varies from complete fusion of the labia majora with only urethral patency to minimal fusion that would not be associated with mechanical infertility.59 There is a report of spontaneous CFL resolution and pregnancy at sexual maturity in a marmoset female after pairing with a male.60 The etiology is currently unknown for functional CFL infertility, due to the reduced vaginal opening, although it has been strongly associated with specific family lines in some captive populations.61

3.5 Mullerian duct anomalies Uterine didelphys and bicornuate uteri are not described in the literature for macaques or marmosets but cases have been noted by the authors during gross examinations

FIGURE 17.17 External genitalia of common marmosets: (A) The normal appearance of 1-month-old female marmoset. (B) A normal 7-month-old female marmoset. (C) A normal adult female marmoset. (D) a normal 1-month-old male marmoset. (E) a 7-month-old female marmoset with moderate congenital labial fusion and a reduced vaginal opening. (F) An adult female marmoset with severe labial fusion no vaginal opening; however, the urethra was patent in this animal. Image assistance Megan Sosa.

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Similar to the condition in humans, the cause is not known.

3.7 Uterocervical malformation Very rarely there may be malformations other than those previously mentioned that effect the uterus and/or cervix. The case presented had cervical elongation with spiral deviation (Fig. 17.21).

4. Degenerative lesions 4.1 Ovarian follicular mineralization Ovarian follicular mineralization (Fig. 17.22) is a common finding among cynomolgus and rhesus macaques.16,65 The author notes follicular mineralization is rarely noted in marmosets. FIGURE 17.18 Macaque bicornuate uterus with an ovarian cyst: Partial failure of Mullerian duct fusion has resulted in two abnormally thin uterine horns (U) with a cyst (C) in one ovary.

(Fig. 17.18 and Fig. 17.19A and B). These are uncommon congenital Mullerian duct abnormalities with partial failure of Mullerian duct fusion resulting in two uterine lumina in a bicornuate uterus.62,63 Complete failure of Mullerian duct fusion occurs in uterine didelphys and results in a bicornuate uterus, two uterine lumina, and two cervices.64

3.6 Uterine hypoplasia Failure of complete development of the uterus is rarely noted in otherwise healthy macaques (Fig. 17.20A and B).

4.2 Polyovular follicles Polyovular follicles are characterized by two or more oocytes within a developing follicle and are a common finding in young adult rhesus macaques16 and are reported in cynomolgus macaques (Fig. 17.23).

4.3 Ovarian follicular cysts and rete ovarii cysts Cystic follicles are not uncommon in macaques (Fig. 17.24); however, persistent solitary luteal cysts within the stroma of the ovary proper are not reported in macaques. A syndrome of polycystic ovaries with suppressed ovulation and endometrial hyperplasia has been reported rarely as a spontaneous finding in female cynomolgus macaques,66 and may

FIGURE 17.19 Macaque uterine didelphys: (A) There are two complete uterine bodies with cervical juncture at a common vagina. Note one ovary is associated with each uterine body. (B) Longitudinal incisions on both uterine bodies and the vagina reveal a separate cervix for each uterine body and a common vagina.

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FIGURE 17.21 A cervical malformation in a macaque is characterized by elongation and spiraling deviation. FIGURE 17.20 Uterine hypoplasia: (A) The uterus (U) of this macaque is smaller with incomplete development when compared to the adjacent pelvic structures. (B) Histologically, the uterus (U) is rudimentary with no glandular development, reduced endometrial stroma, and nearly absent uterine musculature. The adjacent cervix (C-circle) also has poorly developed features. Note: Compare Fig. 17.20B to the normal macaque uterus and cervix in Fig. 17.2B.

accompany naturally occurring androgen exposure during gestation,67 or be associated with high circulating testosterone levels in adulthood.67 High circulating serum testosterone in the female rhesus macaque is defined as > or equal to 0.31 ng/ml and is often accompanied by increased levels of androstenedione, 17-hydroxyprogesterone, estradiol, cortisol, corticosterone, antimullerian hormone and luteinizing hormone.67 The polycystic ovary is characterized by an overabundance of follicles in similar progression, usually antral follicles, that enlarge the ovary (Fig 17.25A).66 The diagnosis of polycystic ovary is made in conjunction with the age and endocrine status of the female macaque, as peripubertal animals often have multiple follicles of relatively uniform progression (Fig. 17.25B), but do not have the adult age status (usually over 4 years of

FIGURE 17.22 Photomicrograph of a macaque ovary with multiple very dark basophilic mineralized follicles within the superficial cortex.

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FIGURE 17.23 Macaque polyovular ovary: There are two ova (arrows) within a single developing follicular structure.

FIGURE 17.24 Photomicrograph of a large thin-walled ovarian cyst replacing a follicle within the ovary.

age), even though they can exhibit high circulating testosterone levels. With progression, the polycystic ovary may have marked reduction of luteal or follicular tissue and increased cortical fibrosis (Fig. 17.25C). Although well described in humans and other laboratory animals, rete ovarii cysts are rarely noted in macaques (Fig. 17.26).65,68 Rete ovarii develops from cells of mesonephric origin and cysts are often also referred to as cystadenomas; however, there is debate as to the neoplastic nature of these lesions with most recent literature classifying the lesion as a nonneoplastic process.

4.4 Uterine endometrial cysts and cervical cysts Similar to other secretory epitheliums of the female reproductive tract, small- to medium-sized cysts occasionally form in the uterine endometrium and cervix (Fig. 17.27A and B).

FIGURE 17.25 Macaque polycystic ovaries as compared to a peripubertal female macaque: (A) Polycystic ovary from an 8.9-year-old multiparous rhesus macaque on gestational day 71 with high circulating testosterone levels: There are multiple, primarily antral follicles that enlarge the ovary. Most of the follicles are devoid of ova and some have degradation and sloughing of the granulosa cell layer. Multiple foci of a corpul luteum are present (asterisks). (B) Ovary from a 3.8-year-old peripubertal nulliparous female macaque: There are numerous antral folllicles with viable ova within the ovary, but no luteal tissue is identified. (C) Polycystic ovary from an adult multiparous female macaque: Progressive changes in the polycystic ovary include continued loss of granulosa cells and ova, leaving cystic spaces sparsely lined by or devoid of follicular epithelium and increased cortical fibrous tissue. Case material for (A) courtesy of David H. Abbott and Danielle Bellino.

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primarily around myometrial veins and consists of thickening of the vascular wall and adventitia by pale eosinophilic extracellular matrix, presumably the residuum of marked uterine vascular enlargement during pregnancy. When present, this change has strong positive predictive value, but absence is not assurance that an animal has never been pregnant.16

5.2 Uterine infarction

FIGURE 17.26 Photomicrograph of a rete ovarii cyst within the hilus of the ovary. Rete ovarii cysts develop from cells of mesonephric origin.

Uterine infarction involving the endometrium and myometrium is rarely seen in cynomolgus macaques, with a necropsy prevalence of around 2% in a large retrospective survey.69 The lesion consists of coagulative necrosis in a midline “watershed” distribution, in animals with other major acute morbidities, and is suspected to be the result of disseminated intravascular coagulopathy (DIC). A single case of uterine infarction was noted in a pregnant rhesus macaque with a uterine diverticulum during an infectious disease study (author’s unpublished observation). DIC was not noted in the dam. A single case of a uterine infarction was diagnosed in a marmoset at the Wisconsin National Primate Research Center, secondary to a dissecting vascular aneurism in the wall of the uterus. The animal survived for several years after hysterectomy.

5.3 Serosal hemorrhage of the uterus On occasion, small hemorrhages are noted on the serosal surface of the uterus in macaques (Fig. 17.28). These are considered to be incidental findings.

6. Inflammatory lesions 6.1 Ovary and oviducts In the absence of infection, endometriosis, or experimental manipulation, inflammatory lesions of the ovaries or oviducts (uterine tubes) are rare. Both macaques and marmosets have been used as a model for genital chlamydial infection.70,71

6.2 Uterus FIGURE 17.27 Macaque uterus and cervix: (A) Photomicrograph of a uterine endometrial cyst and (B) a cervical cyst in a cynomolgus macaque.

5. Vascular lesions 5.1 Pregnancy-associated vascular change This physiologic change is not strictly speaking a lesion, but it can be used to infer reproductive history. This change occurs

Endometritis in macaques, especially those housed in indoor colonies, is most commonly associated with pregnancy, pregnancy loss due to infectious causes, or experimental pregnancy research (fetectomy, cesarean section or infectious disease). Endometritis has been noted in a small number of marmosets. Two of three cases in the marmoset breeding colony at the Wisconsin National Primate Research Center were associated with pregnancy.

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FIGURE 17.29 Mucosa-associated lymphoid tissue (MALT) with a distinct lymphoid follicle (arrow) is subjacent to the keratinized vaginal epithelium and is a normal constituent of the female reproductive tract.

FIGURE 17.28 There are numerous small serosal hemorrhages on the anterior serosal surface of the uterus. These spontaneous microhemorrhages may be noted on occasion in macaques.

6.3 Cervix, vagina, and vulva The cervix and vagina of macaques normally have mucosaassociated lymphoid tissue (Fig. 17.29), which may become activated by infection, leading to the formation of lymphoid follicles. Lymphoid tissue in the marmoset cervix and vagina is much less prevalent and prominent than in the macaque. The lymphoid tissues should not be misinterpreted as inflammation. Likewise, during normal cycling in the macaque, there may be significant numbers of neutrophils within the cervical lumen and secretions that may extend into the superficial mucosa (Fig. 17.30). True inflammation is most often associated with bacterial infections and contains abundant granulocytes, with or without macrophages, and usually has associated tissue injury.

FIGURE 17.30 Normal menstrual cycling leads to significant numbers of neutrophils within the cervical lumen and mucosa in macaques.

6.3.1 Condylomatous eosinophilic vulvovaginitis Harari et al. reported a distinctive inflammatory polypoid vulvovaginitis in Mauritian-origin cynomolgus macaques, with an eosinophilic inflammatory component, and molecular evidence of lymphocryptovirus infection.72

7. Hyperplastic and neoplastic lesions Papillomavirus infections are common in macaques, and in the female genital tract they are associated with intraepithelial dysplastic and neoplastic lesions, as well

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as invasive malignancies.73 The most clearly oncogenic of the macaque papillomaviruses, rhesus papillomavirus D, infects both rhesus and cynomolgus macaques74 (see Chapter 3).

7.1 Ovaries and oviducts 7.1.1 Ovarian surface epithelial hyperplasia Papillary hyperplasia of the ovarian surface epithelium is reported in macaques.16 The ovarian surface epithelium (OSE) varies from squamous to columnar and OSE cells are classified by their morphology.75 OSE cell types are defined based on the cell height with Type 1 measuring 7.5 m with w65% of cells having apical projections.75 Density of OSE cells varies as well, with Type I having significantly less cell density than Type III.75 There are shifts in OSE cell type distribution on the ovary in reference to the primary follicle and the expression of cadherin, estrogen receptor alpha, progesterone, and caspase-3 vary over the course of the menstrual cycle.75

7.1.2 Teratoma/dermoid cyst Ovarian teratomas and dermoid cysts, are tumors of germ cell origin and are one of the most common neoplasms of the macaque ovary. Although the terms “teratoma” and “dermoid cyst” both refer to the same developmental type of phenomenon, “dermoid cyst” is usually applied when only components of skin are contained in the tumor (Fig. 17.31A and B). Although not previously published, ovarian epidermoid cysts have been diagnosed in several marmosets at the Wisconsin National Primate Research Center. Teratoma is the term used when components of multiple tissue types, mature or immature, are identified within the tumor (Fig. 17.32A-C). They are more often diagnosed in young animals than other types of ovarian neoplasms.16,76 There is one case report of an ovarian teratoma in a common marmoset.77

FIGURE 17.31 (A) Photomicrograph of a macaque ovary with a dermoid cyst (C). (B) Higher magnification photomicrograph of the cyst in Fig. 17.31A: The cyst is lined by squamous epithelium with central keratin debris. Image courtesy of Mathew Smith.

the majority of the ovarian parenchyma with small projections of neoplastic cells protruding into cystic lumina.65 The subtle histologic differences between the benign cystadenoma and cystadenocarcinoma were not described, although the cystadenocarcinoma was cytokeratin positive as well as smooth muscle actin positive. Immunohistochemical staining of the cystadenoma was not determined.65 Descriptions of cystadenocarcinomas in the human literature include the presence of solid areas within the tumor, an abundance of delicate papilla protruding into the cystic cavity, and often, neoplastic cellular projections on the surface of the cystadenocarcinoma.80

7.1.3 Ovarian epithelial carcinoma Among rhesus macaques, ovarian surface epithelial neoplasms are commonly reported as adenomas, adenocarcinomas, cystadenomas, and cystadenocarcinomas with serous, mucinous, or papillary features.65,78,79 Descriptions of these neoplasms are scant within the NHP literature, thus comparative pathology practices assume histologic features comparable to human examples for each neoplasm. A cyst adenoma and cystadenocarcinoma have been described in a survey of ovarian pathology in rhesus macaques.65 Both neoplasms were described as multilocular masses effacing

7.1.4 Sex cord stromal tumors Sex cord stromal tumors may be benign or malignant and include granulosa cell tumors, granulosa-theca cell tumors, thecomas, fibrothecomas, Sertoli-leydig tumors, and luteomas. Granulosa cell tumors are relatively common in rhesus macaques65 and may be hormonally active, producing estrogens and associated endometrial hyperplasia.16 Histology and IHC of four granulosa cell tumors in a rhesus macaque revealed unencapsulated expansile neoplasms composed of sheets of round to polygonal cells with Cal-

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FIGURE 17.33 Granulosa cell tumor of a marmoset ovary: (A) There is a large granulosa cell tumor present, with a small rim of remnant ovary (O) at the periphery. (B) The granulosa cell tumor is characterized by ribbons, trabecula, sheets, and nests of fusiform cells containing scant cytoplasm. There are regions of neoplastic cells with fibrovascular stromal cores.

FIGURE 17.32 Teratoma in the ovary of a macaque: (A) The subgross image of the teratoma characterized by the presence of multiple somatic tissues, including (I) intestine, (D) dental structures, (N) nervous tissue, (S) skin and adnexa, (E) esophagus, (L) and lung, Remains of an ovarian follicle (RO) are in section. (B) Higher magnification of the neural tissue present in the teratoma. (C) higher magnification of the (I) intestine and (L) lung tissue in the teratoma.

Exner bodies (proteinaceous eosinophilic concretions) that stained positively for cytokeratin, vimentin, inhibin, and negatively for smooth muscle actin.65 A single granulosa

cell tumor of the ovary was diagnosed in a marmoset at the Wisconsin National Primate Research Center (Fig. 17.33A and B).

7.1.5 Trophoblastic tumors Germ cell neoplasms are rarely reported in the ovary of cynomolgus macaques and include mixed trophoblastic tumors, an ovarian epithelioid trophoblastic tumor, and choriocarcinomas.81e84 Rhesus macaque neoplastic literature includes an ovarian dysgerminoma.85 A single ovarian

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germinal inclusion cyst was diagnosed in a marmoset at the Wisconsin National Primate Research Center.

7.1.6 Ectopic oviduct epithelium Endosalpingosis, ectopic oviduct epithelium, has been noted in common marmosets at the Wisconsin National Primate Research Center.

7.2 Uterus 7.2.1 Epithelial plaque response Epithelial plaque responses usually consist of monomorphic proliferations of trophoblastic cells present on both sides of the uterus at the beginning of pregnancy. Rarely they may occur in nulliparous animals. They are benign and regress spontaneously in non-human primates.16,86 They may also be experimentally induced by trauma to the endometrium and are progesterone dependent.87 Lesions often present only at the surface. Lesions extending deeper into the endometrium are more likely to be trophoblastic neoplasms, the most invasive variant being choriocarcinoma. In some cases, the proliferation of trophoblastic cells may be pleomorphic with multinucleated forms and cells with nuclear enlargement and centronuclear vacuolization (Arias-Stella reaction Fig. 17.34A and B).

FIGURE 17.34 Macaque uterus with an epithelial plaque: (A) Photomicrograph of atypical proliferative lesions in the endometrium of a nulliparous cynomolgus. Benign epithelial plaque response is present at the surface on both sides of the uterine lumen (circle) in a luteal phase endometrium. Lesions extending deeper into the endometrium are more likely to be trophoblastic neoplasms, the most invasive variant being choriocarcinoma. (B) Higher magnification of the proliferations of trophoblastic cells that are highly pleomorphic with frequent multinucleated forms and cells with nuclear enlargement and centronuclear vacuolization (Arias-Stella reaction-arrows).

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7.2.2 Decidual change/deciduosis Deciduosis is characterized by the presence of endometrial stroma in ectopic locations, such as on the serosal surface of the uterus, ovaries, and other organs within the peritoneal cavity. Deciduosis differs from endometriosis in that it is restricted to only one cell typedstromaldwhile endometriosis contains both stroma and glandular elements. The condition has been described in rhesus macaques,88 cynomolgus macaques88 and common marmosets.89 Decidualization of the endometrium (Fig. 17.35A) also occurs in macaques and is characterized by proliferation of endometrial stroma cells with distinct decidual cell morphology. These cells are round to polygonal, with variably distinct cell borders, moderate amounts of pale eosinophilic highly vacuolated or granular cytoplasm, and centrally placed

FIGURE 17.35 Uterus of a nulliparous female cynomolgus macaque with decidual change: (A) There is luminal hemorrhage and sloughing of the superficial endometrium consistent with menses. Within the endometrium, there are multiple aggregates of pale staining decidual cells (arrows). (B) The decidual cells are round to polygonal with distinct cell borders and moderate amounts of clear cytoplasm. The cells have eosinophilic cytoplasmic granules and centrally placed round to oval nuclei with finely stippled chromatin and usually a single basophilic nucleolus.

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round to oval nuclei with finely stippled chromatin and a single basophilic nucleolus (Fig. 17.35B).90 Proliferative decidual cells develop during pregnancy and serve to provide nutritive and immunoprivileged matrix for the developing embryo.91 These cells may also develop in the presence of endometriotic cysts in the macaque when exposed to exogenous progestin (medroxyprogesterone acetate for endometriosis control), aromatase inhibitors, or other progestinlike substances16,88; however, the case provided occurred in a nulliparous female spontaneously. Such a condition is similarly described in humans as an adaptive response in which decidual cells are initiated whether a conceptus is present or absent.91

7.2.3 Endometrial polyps Endometrial polyps are masses of irregular endometrial tissue with, stroma, endometrial glands (normal, cystic, or hyperplastic) with or without small amounts of smooth muscle.92 When large, they may completely fill the uterine lumen (Fig. 17.36). They may be sessile (Fig. 17.37) or pedunculated.93 Endometrial polyps are often associated with heavy and/or abnormal uterine bleeding. Polyps in macaques may be spontaneous but are also associated with unopposed estrogen94 and long-term administration of Tamoxophen,95 There is one report of endometrial polyps in a common marmoset.92

7.2.4 Leiomyomas

FIGURE 17.36 The appearance of a large endometrial polyp (P) expanding the lumen of a thin-walled uterus (U) transected in the coronal plane. The colon (C) and a large leiomyoma (L) are also present in the section. Image courtesy of Puja Basu.

Endometrial leiomyomas are the most common neoplasm of the uterus in macaques.96 These well-differentiated smooth muscle tumors are often fibrotic, and generally lie within the myometrium, but may expand the serosal surface or extend into the endometrium and cause marked uterine distortion (Fig. 17.38A and B). They may be associated with dysmenorrhea and infertility. Microscopically, leiomyomas consist of spindle shaped to markedly elongated neoplastic cells with eosinophilic cytoplasm forming bundles and fascicles; however, there may be variants with scant cytoplasm (Fig. 17.39A and B). There are no reports of leiomyomas in marmosets in published literature or in the Wisconsin National Primate Research Center database.

7.2.5 Uterine hemangiomas Uterine hemangiomas are rare neoplasms of the myometrium in macaques,16 consisting of multilocular blood-filled spaces lined by well-differentiated endothelium. There are no reports of uterine hemangiomas in common marmosets in the published literature.

7.2.6 Endometrial carcinoma Endometrial carcinomas are rarely reported in macaques (Fig. 17.40A and B).16 The Wisconsin National Primate

FIGURE 17.37 Photomicrograph of a sessile endometrial polyp characterized by focal irregular glandular proliferation within the uterine endometrium (circle).

Research Center archives have an endometrial adenocarcinoma arising in the uterus of a 26 year-old rhesus macaque with a chronic history of endometriosis. Evaluation of the uterus after hysterectomy revealed a firm white 1.5  2 cm mass in the anterior uterine wall 1 cm distal to the fundus. The unencapsulated mass was composed of

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sheets and packets of polygonal to spindle-shaped neoplastic epithelial cells with pale eosinophilic cytoplasm, round nuclei, finely stippled to vesicular marginated chromatin, and single eccentric nucleoli. Neoplastic cells effaced and replaced the uterine endometrium and extended shallowly into the myometrium. Necropsy 3 months later revealed incomplete excision of the neoplasm with extension into the adjacent periuterine connective tissue. A transmural uterine endometrial adenocarcinoma was diagnosed in a marmoset at the Wisconsin National Primate Research Center (Fig. 17.41A-C). The unencapsulated mass was composed of nests, islands, and sheets of polygonal neoplastic cells that effaced and replaced the endometrium, myometrium, and focally extended into the peri-uterine connective tissue. There was moderate nuclear pleomorphism and 0e1 mitoses per high power field.

7.2.7 Trophoblastic neoplasms

FIGURE 17.38 Macaque uterus with multiple leiomyomas: (A) The serosal surface of the uterus is expanded and distorted by multiple leiomyomas (L) including a pedunculated leiomyoma. (B) Sagittal section of a uterus with multiple leiomyomas (L) in the wall of the uterus causing distortion and deviation of the uterine lumen as well as an endometrial polyp (P) expanding and filling the uterine lumen. Image (a) courtesy of Puja Basu.

Uterine neoplasms arising from trophoblasts reported in the macaque may be benign or malignant and are considered forms of gestational trophoblastic disease (Fig. 17.42A-D).97 They may be comprised of implantation-type intermediate trophoblasts classified as placental site trophoblastic tumors; chorionic-type intermediate trophoblasts that form nests, cords, and/or solid masses with extracellular matrix and necrosis characteristic of epithelioid trophoblastic tumors; or dimorphic populations of cytotrophoblastlike cells with multinucleate populations of syncytiotrophoblastlike cells

FIGURE 17.39 Photomicrographs of two uterine leiomyomas from macaques: (A) A typical leiomyoma has pale bundles and whorls of smooth muscle cells with elongate nuclei and moderate amounts of pale eosinophilic cytoplasm. (B) A less typical variant of leiomyoma characterized by scant cellular cytoplasm resulting in a densely packed basophilic appearance.

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FIGURE 17.40 (A) Photomicrograph of an endometrial carcinoma in a macaque (EC) infiltrating into the myometrium (M). There is focal adenomyosis (AM) surrounded by lymphocytic inflammation within the myometrium adjacent to the carcinoma. (B) Neoplastic epithelial cells of the endometrial carcinoma are poorly differentiated and vary from polygonal to elongate with pale eosinophilic vesicular cytoplasm.

FIGURE 17.41 Marmoset uterus with endometrial carcinoma: (A) The uterus is enlarged with multiple pale nodular foci expanding the myometrium and serosa. (B) There is marked variation in the thickness of the endometrium and myometrium with luminal pyometra. (C) Neoplastic cells form nests, islands and acini supported by scant stroma that efface and replace the endometrium and myometrium with moderate nuclear pleiomorphism and mitoses noted.

and intermediate trophoblastlike cells consistent with choriocarcinomas.86 One case of gestational trophoblastic disease, not further classified, was noted in a common marmoset at the University of WisconsineMadison.

7.3 Cervix, vagina, and vulva 7.3.1 Cervical squamous metaplasia Squamous metaplasia of the endocervical glands is a common estrogen-dependent finding in peripubertal macaques, and in macaques given exogenous estrogens.16 The metaplastic cells typically undermine the normal goblet cells of the normal endocervical epithelium, forming small islands of squamous epithelium (Fig. 17.43).

7.3.2 Intraepithelial neoplasia (papillomavirus) Papillomavirus-associated cervical dysplasia and neoplasia (cervical intraepithelial neoplasiaor CIN) occur in female macaques with a history of breeding activity.73,74 As in women, CIN lesions in female macaques are associated with infection with genital papillomavirus (PV). Polymerase chain reaction screening has identified a background genital PV prevalence as high as 35% in colony-acquired adult female cynomolgus macaques,74 a prevalence similar to that for human PV infection in sexually active younger women.98 The spectrum of lesions induced by PVs in macaques includes benign papillomas, CIN grades I, II, and III, and invasive carcinoma. As in women, higher-grade lesions are most common in the cervical transformation zone, and

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FIGURE 17.42 Trophoblastic neoplasm of the Macaque uterus: (A) Photomicrograph of an invasive trophoblastic neoplasm composed of multiple pale unencapsulated expansile nests of trophoblasts (arrows) within the uterine endometrium. (B) Higher magnification of pleomorphic trophoblasts with round nuclei, abundant pale cytoplasm, and moderate numbers of multinucleate cells. (C) Neoplastic cells have cytoplasmic cytokeratin that is highlighted red with immunohistochemistry. (D) Triple immunostaining demonstrating glandular and trophoblast positivity for EGFR (brown), stromal positivity for estrogen receptor alpha (red), and nuclear Ki67 (blue) positivity in proliferating cells.

are characterized by an increase in the thickness of basal epithelial cell layers, with increasing atypia. Papillomavirusassociated, warty, exophytic lesions are also seen, and it is likely because differing viral subtypes are associated with progressive and incidental benign lesions.

7.3.3 Squamous cell carcinoma

FIGURE 17.43 Macaque cervical squamous metaplasia: Photomicrograph of metaplastic cells at the base of cervical glands forming small islands of squamous (S) epithelium.

Squamous cell carcinomas occur at the higher end of the intraepithelial neoplasia spectrum, described above in Section 7.3.2, with dysplastic keratinizing epithelial cells invading the basement membrane and forming irregularly sized and shaped nests within the underlying stroma in both the cervix (Fig. 17.44) and vagina of macaques.73 Keratin accumulation is often noted within foci of neoplastic cells and the formation of one keratin pearl is considered sufficient for diagnosis.99

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FIGURE 17.44 Macaque cervical squamous cell carcinoma: Photomicrograph of dysplastic and neoplastic squamous cells forming irregular nests within the cervix. Note the keratin pearl (P).

8. Miscellaneous spontaneous, experimental and iatrogenic lesions of the female reproductive system

FIGURE 17.45 Endometriosis in a macaque: The large dark fluid-filled cyst or endometrioma (E) is adjacent to the uterus (U) and urinary bladder (UB) within the abdomen of a macaque.

8.1 Endometriosis Endometriosis is defined as the presence of hormonally responsive endometrial tissue, both glandular and stromal, outside the uterine lumen. It is a common disorder of macaques, occurring with an incidence as high as 30% in sexually mature females in some colonies of rhesus100 and cynomolgus101 macaques. Endometriosis may occur spontaneously in animals with no history or surgical or hormonal manipulation but risk factors in captive NHP include: hysterotomy, cesarean section, and long-term estrogen treatment.102 Grossly, lesions may be cystic, erythematous, or fibrous; they usually but not always contain brown turbid fluid. In severe cases, fibrous adhesions between the viscera form, or the omentum becomes entwined around the organs afflicted with endometriosis and there may be cysts of varied color (Fig. 17.45). Cysts have been described as white, red, or brown based on grossly noted color associated with the presence or absence of blood content. Rarely, cysts may rupture spontaneously causing life-threatening hemoabdomen. The most common sites of endometriosis are the ovaries and the caudal cul-desac between the uterus and the colon (Fig. 17.46).103 Histologically, endometriosis consists of glandular epithelium resembling that of the eutopic endometrium, surrounded by characteristic densely cellular endometrial stroma (Fig. 17.47A), along with evidence of current or

FIGURE 17.46 Endometriosis in a macaque: The serosal surface of the uterus (U) is adhered to the colon (C) at the dotted line. A large focus of endometriosis (E) consisting of irregularly sized and shaped glands with multiple hemorrhages (H) forms a plaque on the serosa of the adhered colon.

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FIGURE 17.47 Macaque endometriosis, endometrial glands (A) and endometriosis with progestin influence (B). Macaque endometriosis: (A) Photomicrograph of a focus of endometriosis in a macaques that has glands with central secretions surrounded by collagenous stroma and densely cellular endometrial stroma (S). (B) Endometriosis of the serosal surface of the uterus (U) with glands surrounded by progestin (P) influenced endometrial stroma.

past hemorrhage. There may be cystic variants of the glands present (Fig. 17.48). Endometriotic lesions vary in size and degree of hemorrhage and inflammation with the menstrual cycle, peaking in size and clinical symptoms in the menstrual phase. Decidualized variants of endometriosis have been described,88 most commonly after treatment with progesterone progestins. Spontaneous endometriosis does not occur in the common marmoset, which does not menstruate. Abdominal laparotomy of w250 adult female marmosets (between 2 and 12 years) at the German Primate Center Gottingen between 1990 and 2002 did not reveal endometriotic lesions.104 Einspanier et al. did successfully induce endometriosis in 20/29 common marmosets using two methods of uterine flushing to mimic retrograde menstruation. Endometriosis cases in marmosets at the WNPRC were very rare and associated with experimental reproductive manipulations only (Fig. 17.48), comparable to identified risk factors in macaques.

8.2 Adenomyosis Adenomyosis is the presence of endometrial tissue within the myometrium (Fig. 17.49A), in a location clearly distinct from the endometrium. Small clusters of a few isolated endometrial glands surrounded by scant endometrial stroma are common incidental findings in macaques; however, more extensive lesions may also occur, displacing and deforming the myometrium. Rarely, there is cystic adenomyosis in which there is cavitation of the glandular tissue, often with luminal fluid accumulation (Fig. 17.49B). Adenomyosis may occur concurrently with endometriosis since

FIGURE 17.48 Marmoset uterus with endometrioisis: Photomicrograph of a marmoset uterus with cystic endometriosis. Irregular endometrial cystic glands with central hemorrhage are noted. Endometriosis was induced experimentally in this animal as spontaneous endometriosis is not recorded for marmosets.

both are estrogen-dependent conditions.95 Chronic estrogen exposure may increase the prevalence of adenomyosis.94 Adenomyosis has been identified in 10 marmosets in the Wisconsin National Primate Research Center pathology data base. Review of these cases indicated that all animals had experimental hormonal manipulations. Cloprostenol sodium (Estrumate) is commonly used to regulate estrous cycles for

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FIGURE 17.49 Uterine adenomysois in macaques: (A) Photomicrograph of endometrial glands (G) within the inner and outer layers of the myometrium. Note adenomyosis may occur concurrently with endometriosis. (B) Cystic adenomyosis with markedly ectatic fluid-filled endometrial glands within the myometrium of a macaque.

research purposes as well as for population control because marmosets are most typically housed in male-female pairs or in family groups. All animals received between 6 and 73 doses of Estrumate, while 2/10 were ovariectomized and 5/10 underwent oocyte aspiration or embryo collections with varying hormonal protocols. Cloprostenol sodium has been administered to hundreds of marmosets for many years suggesting that the development of adenomyosis is likely a multifactorial in the common marmoset.

8.3 Hydrosalpinx Hydrosalpinx may develop in experimentally ovariectomized marmosets (Fig. 17.50). The incidence in one study involving ovariectomy with hormonal implants was 66%, with unilateral hydrosalpinx in 13% of subjects and 46% developing bilateral hydrosalpinx. Hydrosalpinx is characterized by marked ectasia of the fallopian tubes with clear fluid, often causing them to appear as thin-walled clear cysts on the serosal surface of the uterine fundus.

8.4 Obstetric fistulas Macaques that live in semi-free ranging or pen conditions may have reproductive trauma that is not diagnosed until the time of physical examination or necropsy. Obstetric fistulas develop in individuals that have prolonged labor where the infant’s head compresses the vaginal wall against the pelvis, leading to pressure necrosis and fistula formation between the vagina, rectum (Fig. 17.51), urinary bladder, or ureter.105

FIGURE 17.50 Common marmoset hydrosalpinx of the uterus and oviducts: There is marked bilateral expansion of the oviducts (O) with clear fluid following ovariectomy.

8.5 Vaginal prolapse Vaginal prolapse is most common in multiparous females, but on rare occasions, has been noted in nulliparous macaques. The condition occurs as the vaginal wall weakens,

allowing abdominal organs to move into the vaginal vault forcing the vagina to displace and protrude from the body (Fig. 17.52A and B).

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FIGURE 17.51 Reproductive tract and rectum of a macaque with rectovaginal fistula: The chronic rectovaginal fistula (F) has a distinct junction between the vaginal mucosa (VM) and the rectal mucosa (RM) allowing fecal material to enter the vaginal vault.

8.6 Endometrial pigmentation On occasion, pigment or pigmented cells may be noted as incidental in the endometrial stroma of macaques. The morphology of the pigmented cells may vary from small cells with indistinct nuclei to large cells with abundant

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FIGURE 17.53 Endometrium of a macaque: Individual and small clusters of dark brown pigmented cells consistent with melanophages are present within the endometrial stroma.

pigmented cytoplasm and prominent single or multiple nuclei consistent with melanophages (Fig. 17.53).

8.7 Ovarian smooth muscle metaplasia Smooth muscle metaplasia within the ovary of aged cynomolgus monkeys has been reported, likely originating from metaplastic ovarian stromal cells or ectopic endometrial stromal cells.16

9. Toxicologic lesions 9.1 Estrogen, progestogen, and androgen effects on the female reproductive system The effects of hormones, both endogenous and exogenous, are markedly variable depending on the age of the individual at the time of exposure. The rhesus macaque has been a popular model for numerous endocrine studies. The effects of exogenous estrogens in the adult female macaque reproductive tract are proliferative and comparable to the follicular phase of the menstrual cycle, while progestin effects are more secretory as noted during the luteal phase of the menstrual cycle.95 Androgen effects on the adult macaque ovary include increased numbers of primary and antral follicles with increased numbers of granulosa cells within affected follicles. Androgen effects on the uterine endometrium are typically negligible although there may be metabolic conversion into estrogens.95

FIGURE 17.52 Vaginal prolapse in a cynomolgus macaque: (A) The vagina (V) is prolapsed between the vaginal labia. (B) The pelvis has been removed to expose the uterus (U), mucocutaneous junction of the labia (L), and prolapsed vagina (V) with vaginal mucosa everted.

9.1.1 Prenatal testosterone (T) effects on the hypothalamic-pituitary-gonadal axis (HPG) Prenatal testosterone (T) treatment of female rhesus macaques in utero leads to reduced negative feedback in the hypothalamus and pituitary gland.106e108 This very

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important model of polycystic ovary syndrome leads to ovaries with increased numbers of primary, preantral, and small antral follicles in ovarian sections that often do not progress to ovulatory follicles in adult animals.106 Although not a specific reproductive pathology, these animals have increased visceral fat with diminished insulin sensitivity and/or insulin secretion depending on the gestational age of T exposure.106

9.1.2 Bisphenol A Another important rhesus model involves Bisphenol A exposure, which alters fetal oogenesis in the second trimester and follicle formation during third trimester with increased numbers of multioocyte follicles.109

9.1.3 Clitoromegaly Clitoral size is variable among individual rhesus macaques.110 Significant clitoral growth and clitoromegaly may be induced by androgenic treatments that cause increased circulating levels of testosterone in both female macaques and marmosets during the first 6 months of life, especially the first 2 months after birth.111,112

9.1.4 Endometrial stromal hyperplasia Endometrial stromal hyperplasia will occur in cynomolgus macaques treated with both conjugated equine estrogens and medroxyprogesterone acetate.95

9.1.5 Endometrial glandular hyperplasia Endometrial hyperplasia seldom occurs naturally in macaques, but is readily induced by treatment with exogenous estrogens. This effect is antagonized by the addition of a progestogen, and has been used as a model of estrogeninduced endometrial cancer.113

9.2 Teratogenic research models Marmosets have been investigated as a model for teratogenic studies.35 Radiation at gestational day 25 resulted in craniorachisis, limb reduction, palatoschisis, micrognathia, and auricular abnormalities while exposure to thalidomide during pregnancy resulted in meromelia, phocomelia, amelia, auricular abnormalities and mandibular defects similar to both man and macaques. Human and marmoset twins and triplets exposed to thalidomide developed differing severities of teratogenic lesions.35

10. Conclusion The species-specific anatomy, biology, spontaneous diseases, and images contained in this chapter are designed to provide basic information, a starting point, for the evaluation of the female reproductive system in the three

non-human primate species typically used in research. The authors encourage use of additional resources for more detailed scientific and diagnostic evaluation of specific reproductive tissues.

Acknowledgments The authors gratefully acknowledge the valuable contributions of all previous pathologists of the Wisconsin National Primate Research Center as well as the following individuals: Dr. David Abbott for sharing many years of marmoset research, beautifully summarized, and for very critical editorial review. Ms. Megan Sosa for sharing her observational knowledge and marmoset handling for photography. Dr. Casey Fitz and Dr. Puja Basu for radiographs and images, respectively.

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87. Ghosh D, De P, Sengupta J. Effect of RU 486 on the endometrial response to deciduogenic stimulus in ovariectomized rhesus monkeys treated with oestrogen and progesterone. Hum Reprod 1992;7(8):1048e60. 88. Atkins HM, Lombardini ED, Caudell DL, Appt SE, Dubois A, Cline JM. Decidualization of endometriosis in macaques. Vet Pathol 2016;53(6):1252e8. 89. Mohle U, Heistermann M, Einspanier A, Hodges JK. Efficacy and effects of short- and medium-term contraception in the common marmoset (Callithrix jacchus) using melengestrol acetate implants. J Med Primatol 1999;28(1):36e47. 90. Beck AP, Erdelyi I, Zeiss CJ. Endometrial decidualization and deciduosis in aged rhesus macaques (Macaca mulatta). Comp Med 2014;64(2):148e56. 91. Gellersen B, Brosens JJ. Cyclic decidualization of the human endometrium in reproductive health and failure. Endocr Rev 2014;35(6):851e905. 92. Bennett MW, Dick Jr EJ, Schlabritz-Loutsevitch NE, LopezAlvarenga JC, Williams PC, Sharp RM, Hubbard GB. Endometrial and cervical polyps in 22 baboons (Papio sp.), 5 cynomolgus macaques (Macaca fascicularis) and one marmoset (Callithrix jacchus). J Med Primatol 2009;38(4):257e62. 93. Tabrizi AD. Histologic features and differential diagnosis of endometrial polyps; an update and review. IJWHR 2016;4(4). 152-15. 94. Baskin GB, Smith SM, Marx PA. Endometrial hyperplasia, polyps, and adenomyosis associated with unopposed estrogen in rhesus monkeys (Macaca mulatta). Vet Pathol 2002;39(5):572e5. 95. Cline JM, Soderqvist G, Register TC, Williams JK, Adams MR, Von Schoultz B. Assessment of hormonally active agents in the reproductive tract of female nonhuman primates. Toxicol Pathol 2001;29(1):84e90. 96. Simmons HA, Mattison JA. The incidence of spontaneous neoplasia in two populations of captive rhesus macaques (Macaca mulatta). Antioxidants Redox Signal 2011;14(2):221e7. 97. Stevens FT, Katzorke N, Tempfer C, Kreimer U, Bizjak GI, Fleisch MC, Fehm TN. Gestational trophoblastic disorders: an update in 2015. Geburtshilfe Frauenheilkd 2015;75(10):1043e50. 98. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet 2007;370 (9590):890e907. 99. Maniar K, Wei J. Pathology of cervical carcinoma. In: The global library of women’s medicine; 2017. 100. Zondervan KT, Weeks DE, Colman R, Cardon LR, Hadfield R, Schleffler J, Trainor AG, Coe CL, Kemnitz JW, Kennedy SH.

101. 102.

103.

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Familial aggregation of endometriosis in a large pedigree of rhesus macaques. Hum Reprod 2004;19(2):448e55. Ami Y, Suzaki Y, Goto N. Endometriosis in cynomolgus monkeys retired from breeding. J Vet Med Sci 1993;55(1):7e11. Hadfield RM, Yudkin PL, Coe CL, Scheffler J, Uno H, Barlow DH, Kemnitz JW, Kennedy SH. Risk factors for endometriosis in the rhesus monkey (Macaca mulatta): a case-control study. Hum Reprod Update 1997;3(2):109e15. Fanton JW, Hubbard GB, Wood DH. Endometriosis: clinical and pathologic findings in 70 rhesus monkeys. Am J Vet Res 1986;47(7):1537e41. Einspanier A, Lieder K, Bruns A, Husen B, Thole H, Simon C. Induction of endometriosis in the marmoset monkey (Callithrix jacchus). Mol Hum Reprod 2006;12(5):291e9. Miller S, Lester F, Webster M, Cowan B. Obstetric fistula: a preventable tragedy. J Midwifery Wom Health 2005;50(4):286e94. Dumesic DA, Abbott DH, Padmanabhan V. Polycystic ovary syndrome and its developmental origins. Rev Endocr Metab Disord 2007;8(2):127e41. Dumesic DA, Patankar MS, Barnett DK, Lesnick TG, Hutcherson BA, Abbott DH. Early prenatal androgenization results in diminished ovarian reserve in adult female rhesus monkeys. Hum Reprod 2009;24(12):3188e95. Abbott DH, Vepraskas SH, Horton TH, Terasawa E, Levine JE. Accelerated episodic luteinizing hormone release accompanies blunted progesterone regulation in PCOS-like female rhesus monkeys (Macaca mulatta) exposed to testosterone during early-to-mid gestation. Neuroendocrinology 2018;107(2):133e46. Hunt PA, Lawson C, Gieske M, Murdoch B, Smith H, Marre A, Hassold T, VandeVoort CA. Bisphenol A alters early oogenesis and follicle formation in the fetal ovary of the rhesus monkey. Proc Natl Acad Sci USA 2012;109(43):17525e30. Goldschmidt B, Cabello PH, Kugelmeier T, Pereira BB, Lopes CA, Fasano DM, Andrade MC, Santos JS, Marinho AM. Variation in clitoral length in rhesus macaques (Macaca mulatta). J Am Assoc Lab Anim Sci 2009;48(5):482e485. Brown GR, Nevison CM, Fraser HM, Dixson AF. Manipulation of postnatal testosterone levels affects phallic and clitoral development in infant rhesus monkeys. Int J Androl 1999;22(2):119e28. Abbott DH. Differentiation of sexual behaviour in female marmoset monkeys: effects of neonatal testosterone or a male co-twin. Prog Brain Res 1984;61:349e58. Foth D, Cline JM. Effects of mammalian and plant estrogens on mammary glands and uteri of macaques. Am J Clin Nutr 1998;68(6 Suppl. l):1413se7s.

Chapter 18

The male reproductive system of the non-human primate Justin D. Vidal1, Petrina Rogerson2 and Eveline P.C.T. de Rijk3 Charles River Laboratories, Ashland, OH, United States; 2Charles River Laboratories, Tranent, United Kingdom; 3Charles River Laboratories, ‘s-

1

Hertogenbosch, the Netherlands

1. Introduction

2. Anatomy and histology

Test article-related effects on the male reproductive system can have a profound impact on pharmaceutical development. As with other organ systems, an understanding of normal physiology and common patterns of spontaneous and induced lesions is critical to the evaluation of the reproductive tissues and formulation of a robust risk assessment. However, the male reproductive system has several unique features that differ from other organ systems, including complex hormonal regulation; puberty and onset of sexual maturity; sex-specific characteristics such as spermatogenesis; pronounced species differences; and evaluation occurring in the context of both repeated-dose general toxicology studies and stand-alone developmental and reproductive toxicology studies. Due to the relatively late onset of sexual maturity, most non-human primates (NHPs) in general toxicology studies are immature, or peripubertal animals, which limits the assessment of the reproductive system. However, in recent years, there has been increased interest in the assessment of the male reproductive system in NHPs as biopharmaceuticals are often tested in NHPs as the only species. This has led to an increased use of sexually mature NHPs and the pathologist’s evaluation and interpretation of the organ weight and microscopic data are critical, as they are often the only available information about potential effects of a test article on the male reproductive system. This chapter provides the pathologist with an overview of the normal anatomy, histology, and endocrinology of the male reproductive system along with detailed descriptions and photomicrographs of common spontaneous and xenobioticinduced changes in order to provide a guide for both new and experienced pathologists tasked with evaluating this complicated organ system.

2.1 Testis, rete testis The testis and epididymis are covered by a serosal lining called the tunica vaginalis, which is contiguous with the peritoneum. The spermatic cord contains the vas deferens, the spermatic artery and the pampiniform plexus, nerves, and lymphatics. It is attached to the abdominal wall at the inguinal canal. The mature testis resides within the scrotum and is covered by the thick, fibrous tunica albuginea. The testis is divided into tubular and interstitial compartments. The seminiferous tubules are tightly packed loops arranged into multiple lobules, which are separated by prominent fibrous septa. Each end of a given seminiferous tubule joins the epithelial-lined channels of the rete testis, which resides within the mediastinum testis and is continuous with the efferent ducts and epididymis (Fig. 18.1). Seminiferous tubules are lined by Sertoli cells, which have a large basally oriented oval nucleus and are surrounded by a basement membrane and an outer layer of contractile myoid cells. Sertoli cells are responsive to follicle stimulating hormone (FSH) and androgens and play an essential role in supporting spermatogenesis. They provide support for the development of germ cells (spermatogonia, spermatocytes, and spermatids), maintenance of the blood-testis/tubule barrier (BTB), secretion of seminiferous tubular fluid, production of secretory components (including anti-Müllerian hormone, androgenbinding protein, inhibin, and growth factors), release of mature spermatids, and phagocytosis of residual bodies and apoptotic germ cell remnants.1 The interstitium contains Leydig cells, vasculature, macrophages, and supporting connective tissue stroma. Leydig cells produce

Spontaneous Pathology of the Laboratory Non-human Primate. https://doi.org/10.1016/B978-0-12-813088-9.00012-4 Copyright © 2023 Elsevier Inc. All rights reserved.

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FIGURE 18.1 Normal microscopic anatomy of the rete testis (arrowheads), efferent ducts (arrows), and epididymis (asterisk) in a cynomolgus monkey. The efferent ducts extend from the extra-testicular portion of the rete testis, through the adjacent adipose tissue, and embed into the head of the epididymis. (H&E).

testosterone in response to luteinizing hormone (LH) leading to the high levels of intratesticular androgens that are critical for germ cell maintenance and development as well as distribution into the systemic circulation. The relative size and number of Leydig cells vary across species with macaques having relatively inconspicuous Leydig cells.2,3 Germ cells can be classified into three main categories including spermatogonia, spermatocytes, and spermatids. Spermatogonia are the diploid, mitotic population of germ cells and they reside along the basement membrane. In primates including humans, spermatogonia can be classified as type A and type B cells.4 Type A cells are the undifferentiated population and can be further subdivided into A-dark and A-pale spermatogonia with A-dark spermatogonia serving as the reserve stem cell population and the A-pale spermatogonia actively dividing during each spermatogenic cycle. Type B spermatogonia are the differentiated spermatogonia and after three rounds of mitotic division they become preleptotene spermatocytes. While spermatogonia are present in all cross-sections of seminiferous tubules, they are sometimes inconspicuous, and these classifications can be difficult to appreciate in routinely processed paraffin embedded tissues. During spermatogonial mitosis, cell division is incomplete and germ cells develop as an organized cohort with cytoplasmic bridges. The newly emerged primary spermatocytes slowly enlarge as they progress through meiosis passing through

leptotene, zygotene, pachytene, and diplotene stages until meiotic division occurs. Prior to the genetic recombination that occurs in a pachytene spermatocyte, these cells move inside the BTB. Primary spermatocytes are a long-lived cell type, lasting over 2 weeks at which point they undergo two meiotic divisions, which occur in rapid succession, to form transient diploid secondary spermatocytes and then haploid round spermatids. These newly formed round spermatids are still linked by intercellular bridges and undergo marked morphologic changes called spermiogenesis, the process of transforming round spermatids into mature elongated spermatids. Spermatids depend on Sertoli cells for successful differentiation and ultimate release into the tubular lumen. The various morphologic forms of spermatids are characterized as steps, which are 1 through 14 in the macaque5 and 1 through 12 in the marmoset.6 As the nuclear shape changes and the spermatid elongates, spermatid cytoplasm expands and then condenses. Mature spermatids line up along the luminal surface, lose their cytoplasmic connections, shed their cytoplasmic remnants called residual bodies, and are prepared for release by the Sertoli cells in the process of spermiation. The residual bodies are resorbed by Sertoli cells. Germ cells develop sequentially from spermatogonia, spermatocytes, to spermatids in an orderly process with specific cellular associations arranged along the length of the seminiferous tubule. A given set of germ cells develop in synchrony and form specific sets of cell layers lining the

The male reproductive system of the non-human primate Chapter | 18

seminiferous tubules, which creates distinctive patterns in tubular cross-sections. These patterns of cellular associations are often referred to as “stages,” which are typically designated by Roman numerals (Fig. 18.2). The time it takes to pass through all stages and return to the starting point is one cycle. It takes multiple cycles to complete the full duration of spermatogenesis. While all species go through a generally similar process of germ cell maturation, the morphologic details and timing differ, and different classification schemes are available for each species (Table 18.1). In addition, the microscopic organization of the seminiferous tubules differs across nonhuman primates and men.7 Most mammalian species, including the well-studied rat, have a segmental arrangement of stages along the length of the seminiferous tubule, which yields a single stage within a given tubular crosssection. In men and marmosets, the arrangement is more complicated. Stages of tubules are arranged in a helical arrangement, which yields multiple stages within a given tubular cross-section. The onset of sexual maturation occurs over a wide age range and has been reported from 3 to over 5 years of age in male macaques19e21 and 12e14 months in male marmosets.22,23 There are geographical/source differences in the timing of onset with Mauritian cynomolgus monkeys reaching sexual maturity almost 2 years earlier than Asian mainland animals.21 Due to this long pubertal period and the challenges of handling mature male macaques, most studies are conducted using immature animals or occasionally a mix of immature, pubertal, and/or mature animals. There is considerable variability in the microscopic appearance of the male reproductive organs during pubertal development (Figs. 18.3 and 18.4). Males are typically considered immature up until the time spermatogenesis begins. At the start of the first wave of spermatogenesis, the formation of a lumen within the seminiferous tubules and the presence of early spermatocytes can be observed. This pubertal window typically lasts until spermatogenesis is completed and there is a full complement of sperm within the tail of the epididymis and the accessory sex glands are fully developed. Weight and age generally correlate with sexual maturity, but due to the long pubertal period and high degree of inter-animal variability, age and weight alone are typically not sufficient for predicting the maturity status of an individual animal. As a result, the sexual maturity status of NHPs in nonclinical toxicity studies should be assessed and documented by the study pathologist.24 If sexually mature animals are required, the presence of sperm in an ejaculate as a baseline measurement is the most reliable method for determining sexual maturity,21 although other methods such as measuring testicular volume can be a helpful addition. Most toxicity studies are conducted using the cynomolgus monkey, which is a nonseasonal breeding

439

macaque. This allows for the assessment of the reproductive system at baseline and throughout chronic studies of 6 months or greater. In contrast, the rhesus macaque is a seasonal breeder25 and during the nonbreeding season, the testes will have varying degrees of regression of spermatogenesis with decreased sperm within the epididymis. Due to this seasonal nature and challenges in designing studies around the nonbreeding season, the rhesus macaque is generally not a recommended model to evaluate male reproductive toxicity. Under standard laboratory conditions the common marmoset does not display a seasonal pattern of reproduction.26

2.2 Efferent ducts, epididymis, and vas deferens The efferent ducts, epididymis, and vas deferens provide a conduit for sperm from the rete testis to the urethra, where they are joined with secretions from the accessory sex glands. The efferent ducts are a series of short parallel epithelial-lined channels that transport sperm to the epididymis and remove fluid to concentrate sperm (Fig. 18.1). The epididymis is attached to the testis and has three major regions called the head (caput), body (corpus), and tail (cauda). The epididymis contains a single, coiled duct and is lined by a pseudostratified epithelium with occasional ciliated cells and has both secretory and absorptive functions depending on the location. The epididymis serves as a barrier in immunological sequestration of sperm and plays a key role in sperm maturation and development. The tail of the epididymis has a larger lumen than the head or body and stores sperm prior to ejaculation. The vas deferens connects the tail of the epididymis to the urethra.

2.3 Accessory sex glands, penis, and scrotum The accessory sex glands include the prostate, the seminal vesicles, and the bulbourethral glands. The latter are small glands, which empty into the urethra caudal to the prostate. They are not routinely examined in laboratory species. The prostate in many non-human primates is divided into two parts, the cranial and caudal lobes,27 which can usually be identified grossly. The glandular prostate does not surround the urethra ventrally. The relative size of the lobes differs between species, and there are histological differences in acinar size and staining characteristics between the lobes. The paired seminal vesicles are large in macaques and consist of highly convoluted glands. Both glands are lined by cuboidal or columnar epithelium and contain eosinophilic secretion. The ducts from the seminal vesicles join the vas deferens to form the short ejaculatory duct, which enters the urethra, while the prostatic ducts enter the urethra directly.

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FIGURE 18.2 Testes from a normal sexually mature control cynomolgus monkey (Plates 1 and 2) demonstrating the normal features of spermatogenesis at low (A, C, E, G, and I) and high (B, D, F, H, and J) magnification (H&E). Although 12 stages are described in the cynomolgus monkey,5 many of these features are subtle and cannot be easily discerned in routinely processed material. However, stages can be grouped together due to the similar microscopic features and cell types present. Understanding these basic groupings allows for a robust assessment of spermatogenesis. A and B: Pachytene spermatocytes (PS), round spermatids (RSp), and elongated spermatids (ESp) are present in stages IeIV. C and D: PS, RSp, and ESp are present in stages VeVI and ESp are fully elongated with a prominent tail and are lined up along the luminal surface ready for release. E and F: Preleptotene to leptotene spermatocytes (PL/ L), PS, and RSp are present in stages VIIeIX. The RSp still appear round and the earliest signs of elongation can be observed in stage IX.

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441

FIGURE 18.2 cont’d G and H: Zygotene spermatocytes (ZS), PS, and ESp are present in stages XeXI. Note that the ESp now are clearly beginning to elongate. I and J: PS, spermatocytes undergoing meiotic division (Div), and ESp are present. Although not always easy to identify in all cross-sections, spermatogonia (arrows) and Sertoli cell nuclei (arrowheads) can be identified in any stage.

TABLE 18.1 Species comparison of spermatogenesis and epididymal transit.4e6,8e18 Number of stages

Stages in a tubular cross-section

Cycle length (days)

Total duration of spermatogenesis (days)

DSP (millions)

Epididymal transit time (days)

Cynomolgus monkey

12

Single stagea

10.5

42

_

11

Rhesus macaque

12

Single stage

10.5

44

23

10.5

Marmoset

9

Multiple stages

10

37

18.4b

_

Human

6

Multiple stages

16

64

4.0e4.5

6

Species

DSP: Daily sperm production per gram of testis. a Some reports describe an intermediate arrangement with a low incidence of tubules with multiple stages. b Based on Callithrix penicillata

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FIGURE 18.3 Testes from multiple cynomolgus monkeys demonstrating normal pubertal development at the same magnification (H&E). (A): Immature animal with seminiferous tubules that lack a central lumen and contain only Sertoli cells and quiescent spermatogonia. (B): Peripubertal animal with the start of a lumen in several tubules and early spermatocytes. (C): Peripubertal animal with a lumen present in multiple tubules along with spermatocytes and round spermatids. (D): Mature animal with all germ cell types present including mature elongated spermatids (bottom right tubule).

Non-human primates have an os penis, but there is otherwise considerable species variation in the morphology of the penis, scrotum, and perineum.

3. Physiology 3.1 Endocrinology

FIGURE 18.4 Testis from a normal peripubertal control cynomolgus monkey. During puberty, it is normal to see variable development in the testis with neighboring tubules ranging from completely immature to those demonstrating normal spermatogenesis (H&E).

Coordinated function of the male reproductive organs is mediated through the hypothalamic-pituitary-gonadal (HPG) axis. Gonadotropin-releasing hormone (GnRH) from the hypothalamus leads to increased levels of LH and FSH. LH stimulates the Leydig cells to produce androgens and FSH regulates spermatogenesis through receptors on Sertoli cells.28 Testosterone is the major androgenic steroid and circulating levels of testosterone provide support for the accessory sex organs as well as nonreproductive organs such as muscle, skin, and bone. High levels of intratesticular testosterone are required to maintain spermatogenesis and the concentration of testosterone within the testis is much greater than in the systemic circulation.

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Testosterone is locally converted to the more potent androgen dihydrotestosterone (DHT) by 5a-reductase in the epididymis and accessory sex glands, which rely on DHT. Testosterone and androstenedione can also be converted locally into estradiol and estrone, respectively, by the enzyme aromatase within the brain and testis and estrogens play a role in normal male physiology.29 Circulating testosterone, as well as the metabolites DHT and estradiol, provides feedback inhibition of GnRH and LH. LH and testosterone are both pulsatile, with a pulse of testosterone following approximately 20 min after an LH pulse.30 Macaques and marmosets have a marked circadian rhythm with the highest serum hormone pulse frequency and/or levels occurring at night, shortly after dark.30,31 Cynomolgus monkeys and marmosets have a similar pattern of secretion throughout the year, while there are pronounced seasonal differences in rhesus macaques.32 In addition, when macaques are group housed, social hierarchy can impact serum hormone levels with lower ranking males having marked decreases in serum testosterone levels and decreases in testicular volume.33,34 This social effect appears to be less of a factor in male marmosets.35

4. Congenital lesions 4.1 Hypospadias

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(Fig. 18.5). They can be observed macroscopically, particularly if torsion has occurred (Fig. 18.6), but more often are present as incidental microscopic findings.

4.4 Increased stromal collagen This finding, which is sometimes referred to as testicular fibrous hypoplasia,42 consists of variable amounts of dense collagenous connective tissue around the rete testis, which often extends to the tunica albuginea. There can be displacement and/or replacement of seminiferous tubules with mature collagen (Fig. 18.7). The cause of this finding is unclear; it has been described as an incidental and congenital lesion in the testes of cynomolgus monkeys.43e48 The finding occurs at a higher incidence in cynomolgus monkeys from China and Vietnam47 and is most commonly seen in younger animals. When observed in sexually mature animals, it can be seen with segmental dilatation of seminiferous tubules.49

4.5 Tubular hypoplasia Tubular hypoplasia consists of focal to wedge-shaped area(s) of tubules that are reduced in size with few to no germ cells (Fig. 18.8). This finding is occasionally observed in cynomolgus monkeys and typically occurs as small foci affecting one or both testes.49

Hypospadias is a congenital condition in which the urethral meatus is located on the ventral surface of the penis, or perineum. Sporadic spontaneous cases of hypospadias have been reported36 and a defect in embryonic development appears to be the cause. Since development of the external genitalia is dependent on DHT, administration of finasteride, a 5a-reductase inhibitor in utero, can lead to an increased incidence of hypospadias and other external genital abnormalities.37

4.2 Cryptorchidism Cryptorchidism is a failure of testicular descent into the scrotum. The testes usually descend into the scrotum around the time of birth, but in some species, such as rhesus macaques, the testes may be present in the abdomen or inguinal canal until puberty.38 There is limited available information on spontaneous cases of cryptorchidism in non-human primates, but several experimental models have been described.39,40

4.3 Testicular and epididymal appendages Embryologic remnants of the mesonephric and/or paramesonephric ducts are occasionally observed in macaques.41 These are often referred to as testicular or epididymal appendages and appear as cystic or pedunculated structures attached to the testis or epididymis

FIGURE 18.5 An appendix testis (arrowheads) adjacent to the body of the epididymis in a cynomolgus monkey (H&E). An appendix testis is an embryologic remnant that is often attached to the body of the epididymis and consists of a fibrovascular core with an epithelial-lined surface. There are often small projections, and it may resemble fimbria from the oviduct in females.

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Spontaneous Pathology of the Laboratory Non-human Primate

4.6 Ectopic adrenal gland Small nodules of adrenal cortical tissue can be observed within the fat and connective tissue adjacent to the testis, efferent ducts, and/or epididymis (Fig. 18.9).50

5. Degenerative lesions 5.1 Introduction Spontaneous findings, age-related changes, disruption of hormonal control mechanisms, stress, and xenobioticinduced changes can all lead to the presence of degenerative changes within the male reproductive system. This can range from degeneration (apoptosis) and/or depletion of germ cells to complete atrophy of the seminiferous tubules. Secondary or concomitant effects can often be observed within the excurrent ducts and/or in accessory sex organs.51 Spontaneous changes in non-human primates are generally limited in younger male monkeys used in toxicity studies and literature of sexually mature and/or aging monkeys is scarce.

5.2 Testes FIGURE 18.6 Macroscopic (A) and microscopic (B) appearance of an appendix testis (arrowhead) attached to the body of the epididymis in a young adult cynomolgus monkey. The appendix testis undergone torsion, resulting in congestion and hemorrhage.

5.2.1 Tubular dilatation/degeneration Focal wedge-shaped areas of variably dilated tubular crosssections with either thinning of the seminiferous epithelium

FIGURE 18.7 Right and left testes from an immature control cynomolgus monkey. Both testes have increased stromal collagen, which has replaced large portions of the seminiferous tubules in the testis on the left (H&E).

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FIGURE 18.8 Testis from a mature control cynomolgus monkey with a focal area of tubular hypoplasia (H&E).

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FIGURE 18.10 Testis from a young adult control cynomolgus monkey that has segmental tubular dilatation extending from the junction with the rete testis (arrowhead) along with sperm stasis and cellular debris. There is increased stromal collagen present surrounding the rete testis (H&E).

5.2.2 Tubular degeneration/atrophy

FIGURE 18.9 Ectopic adrenal gland (arrowheads) adjacent to the efferent ducts of the epididymis of a cynomolgus monkey (arrows) (H&E). Image courtesy of Jennifer A Chilton.

or degeneration of germ cells may be seen together with focal areas of sperm stasis near the rete testis suggesting focal outflow obstruction (Fig. 18.10). This common change is often most evident in animals going through pubertal development and may also occur in mature animals especially with increased stromal collagen.47,49,52

Occasionally, groups or lobules of tubules may show scattered germ cell degeneration and/or loss.43 This degeneration is generally nonspecific, and all types of germ cells may be affected. Other associated morphologic features such as tubular vacuolation, presence of multinucleated giant cells, or sloughed cells may be observed.43 Incidences are higher in young males that have recently reached maturity.53 In non-human primates, partial or complete atrophy of the seminiferous epithelium can occasionally be found. Histologically, tubular atrophy is characterized by partial or complete loss of germ cells leaving a focal or larger area of primarily Sertoli cells. Since there can be varying degrees of degeneration and atrophy in the same testis, a combined term of degeneration/atrophy can be used. It can occur as an age-related change and in a cohort of aged chimpanzees, 60% of the males showed tubular degeneration and atrophy varying from hypospermatogenesis to complete loss of germ cells.54 It should be noted that non-human primates that are seasonal breeders (e.g., rhesus macaques) have normal spermatogenesis during the breeding season. During the nonbreeding season, spermatogenesis is incomplete and varying degrees of degeneration and atrophy can be observed in the testes of these animals. For this reason, the rhesus macaque is a challenging model for studying potential effects of xenobiotics on the male reproductive system.53

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5.2.3 Hypospermatogenesis Hypospermatogenesis is a commonly observed finding in cynomolgus monkeys49 and consists of a multifocal loss of germ cell layers and is thought to reflect reduced efficiency of spermatogenesis due to an intermittent failure of spermatogonial division. In an affected tubule, one or more layers of germ cells will be missing or variably present (Fig. 18.11). This can range from individual tubular crosssections to multiple areas throughout the testis. The intermittent nature of this change results in a random distribution throughout the testes, although in macaques a more diffuse pattern can be observed, which may be observed during pubertal development or be a result of loss of type B spermatogonia due to transient decreases in gonadotropic and/or androgenic support of the testis and depending on the severity, this resembles maturation depletion (see Section Germ Cell Effects, below).28,49 Distinguishing hypospermatogenesis from test articlerelated effects can be difficult. Since germ cells are missing, there is overlap with the appearance of hypoplasia, depletion, seasonal involution in rhesus macaques, and/or atrophy, but in general hypospermatogenesis is less uniform and does not follow a cell or stage specificity. In addition, since hypospermatogenesis represents a reduced production of germ cell layers, there is typically no increase in germ cell apoptosis or germ cell debris in the epididymis.

different from germ cell or tubular degeneration and atrophy as it involves coagulative necrosis affecting germ cells and Sertoli cells and can also affect Leydig cells. This can impact the blood-testis barrier resulting in an inflammatory response of the affected tubules44 and/or result in fibrosis.

5.3 Efferent ducts, epididymis, and vas deferens 5.3.1 Epithelial degeneration Degeneration of the epididymal epithelium is not common in non-human primates, but when present it may be accompanied by inflammatory cell infiltrates due to disturbance of the barrier function. Histologically, the epithelial lining can appear thin or can be completely lost. When sperm are exposed to the interstitium, formation of a sperm granuloma will be the result (see Inflammation).

5.3.2 Epithelial apoptosis and/or atrophy Apoptosis within the epithelium of the epididymis occurs in response to reduced androgenic stimulation. This results in a wave of apoptosis moving from the initial segment of the epididymis distally toward the vas deferens. Epithelial apoptosis can be observed as the early stage of this process55,56 and with chronic androgen deprivation the epididymal epithelium becomes atrophic and the apoptosis is no longer evident.57

5.2.4 Tubular necrosis Tubular necrosis is an uncommon finding in non-human primates. It can occur due to ischemic conditions secondary to vascular damage or torsion. Tubular necrosis is

5.3.3 Cellular debris and reduced sperm Cellular debris and/or reduced sperm can occasionally be observed at low levels as a spontaneous change.49 Increased numbers of sloughed testicular germ cells and/or cellular debris can be observed within the lumen of the epididymis as a secondary effect to testicular toxicity; however, younger peripubertal animals will have increased levels of germ cell debris within the epididymis and this should not be mistaken for evidence of testicular toxicity (Fig. 18.12).49,52 For the seasonal breeders (e.g., rhesus monkeys), cellular debris and/or reduced sperm are normal epididymal features during the nonreproductive period.

5.4 Accessory sex glands 5.4.1 Atrophy

FIGURE 18.11 Testis from a mature control cynomolgus monkey with hypospermatogenesis. Multiple tubules are lacking elongating or round spermatids (H&E).

Acinar atrophy within the accessory sex glands can occasionally be observed in sexually mature non-human primates as a background finding and has features similar to the accessory sex glands of sexually immature animals. In mature animals, disturbance in the pituitary-gonadal axis with subsequent effects on steroid production is most likely the underlying cause and may be seen in subordinate animals.21,51 A study with castrated cynomolgus monkeys

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human primates used in toxicity studies have not been described in the literature. Degeneration of prostate epithelium due to inflammatory infiltrates has been described in aged captive chimpanzees.54

6. Inflammatory and vascular lesions 6.1 Introduction

revealed that the caudal lobe of the prostate is more susceptible to atrophy then the cranial lobe or periurethral glands58 and collection and sectioning pattern can be an important consideration.

The importance of male reproductive organs can be inferred from the existence of a delicate balance between endocrine and immunological systems, responsible for the protection of spermatogenesis and the release of mature sperm. This involves a strong innate immune system and a suppressed adaptive immune response. This is accomplished by a variety of junctions within the testes and epididymides (the BTB and Blood-Epididymis Barrier, respectively). The majority of the inflammatory and vascular changes in male reproductive organs are the consequence of disturbances in one or more systems involved in these protective barriers or associated regulating factors.59 The origin of this disturbance may be trauma, infections, or autoimmunity and determines the type of vascular changes (perivasculitis, transmural vasculitis, necrosis, loss of internal lamina, edema, hemorrhages, etc.) or cell types involved in the inflammatory process (neutrophils, macrophages, lymphocytes, granulomas, giant cells, etc.).

5.4.2 Other degenerative changes

6.2 Testis and epididymis

Except for corpora amylacea within the seminal vesicles of mature cynomolgus monkeys (Fig. 18.13),42,43,49 other spontaneous changes in the accessory sex organs of non-

6.2.1 Orchitis

FIGURE 18.12 Epididymis with luminal cellular debris from a peripubertal control cynomolgus monkey. In peripubertal and young adult animals, it is normal to see increased amounts of cellular debris within the lumen of the epididymis (H&E).

Inflammation of the testes is uncommon in non-human primates. This may be caused by trauma, bacterial infections (e.g., Staphylococcus aureus, Staphylococcus spp., Actinobacillus spp., Chryseobacterium indologenes),60 outflow obstruction or by disruption of the blood supply and/or BTB with normally sequestered sperm antigens now exposed to the immune system. In primates (including men), such an autoimmune reaction can occur secondary to vasectomy.51 After vasectomy or spontaneous outflow obstruction due to fibrosis, sperm that escape the protective barrier as a result of sperm stasis and/or backpressure can induce a granulomatous response (more frequent in the epididymides; see sperm granuloma below) and/or a systemic immune response. In addition to the granulomatous response, lymphoid infiltrates may be present and include formation of germinal centers with plasma cells and macrophages and variable degrees of degeneration and/or atrophy of the seminiferous tubules (Fig. 18.14).

6.2.2 Epididymitis and sperm granuloma FIGURE 18.13 Seminal vesicles of a mature control cynomolgus monkey with corpora amylacea (H&E).

Compromise of the epididymal barrier function can occur due to epithelial degeneration, ischemia, or rupture of ducts leading to leakage of sperm into the interstitial tissue

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FIGURE 18.14 Rete testis with sperm stasis and cellular debris from a young adult control cynomolgus monkey. There is segmental tubular dilatation extending from the junction with the rete testis (arrowhead) along with sperm stasis and cellular debris. A chronic inflammatory cell infiltrate surrounds the tubules at the junction with the rete testis. There is increased stromal collagen present surrounding the rete testis (H&E).

resulting in granuloma formation. Nongranulomatous inflammation of the epididymides in non-human primates is uncommon, but neutrophilic peritubular inflammatory cell infiltration can be the result of an initial response to sperm antigens (see also orchitis) or can be drug-induced (e.g., by epithelial hyperplasia, sperm stasis, or occlusion).61 Sperm granulomas in the efferent ducts and epididymides of non-human primates are rare compared to rats and dogs. Occasionally, small areas of outflow obstruction with subsequent inflammation can be observed within the efferent ducts (Fig. 18.15),49 although these structures are not routinely sampled. Morphologically they consist of a foreign body type of response with central accumulation of degenerating sperm surrounded by a variety of inflammatory cells, a layer of epithelioid (spermiophagic) macrophages, and a loose vascular connective tissue containing abundant lymphocytes and plasma cells. Sometimes the process is surrounded by a fibrous capsule. Sperm granuloma formation is mostly initiated by damage of the epithelium and/or loss of integrity of the tubular structures with subsequent leakage of sperm through the epithelium into the interstitium. Underlying causes are rupture of tubules or ducts due to trauma or surgical procedures such as vasectomy.62 The pathogenesis of vasectomy starts with physical obliteration or damage to the outflow tract. This causes increased intraluminal pressure upstream, leading to rupture and sperm granulomas. Although in other species (i.e., rats, mice and dogs) sperm granulomas are either

spontaneous/congenital or chemically induced, in nonhuman primates, sperm granulomas in the epididymides or vas deferens are primarily associated with vasectomy procedures.62,63 Even in aged non-human primates (chimpanzees) sperm granulomas rarely occur spontaneously.54

6.2.3 (Peri)vasculitis Vascular inflammation may involve the wall of vessels (vasculitis/arteritis) or tissues surrounding the vessels (perivasculitis/periarteritis) and can be observed as an incidental finding in cynomolgus monkeys mainly in testes and epididymides.49 The finding is characterized by the presence of mixed inflammatory cells primarily consisting of lymphocytes, plasma cells, and neutrophils. In some cases, other tissues (e.g., intestines or heart) may be involved.46 However, when the vasculitis is more systemic with transmural necrosis of the vascular wall, the histopathology resembles polyarteritis nodosa in humans.64 As mentioned above (peri)vasculitis can be found as an incidental finding in cynomolgus monkeys. This can be very problematic in toxicity studies since some drugs can induce similar changes, either as a direct vascular effect or secondary to immune-mediated mechanisms. These druginduced vascular changes are difficult to distinguish from spontaneous polyarteritis and a robust historical control database along with an understanding of the compound’s mechanism of action can help build a weight of evidence

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FIGURE 18.15 Efferent ducts from a mature control cynomolgus monkey. There is sperm stasis with intraluminal macrophages phagocytizing sperm and perivascular lymphocytic inflammatory cell infiltrate (arrowheads) (H&E).

approach to determining the potential drug-relatedness of the effect.

6.3 Accessory sex glands Acute and chronic inflammation (prostatitis) can be found in the prostate gland of rats, often in association with inflammation in other accessory glands or urinary tract. Whether this is also true for non-human primates is not clear; however, the non-human primate has been used as an experimental animal model to study bacterial-induced prostatitis.65 In toxicology studies, inflammatory cell infiltrates are regularly observed in the prostate of non-human primates as a background finding (Fig. 18.16).50,49 The infiltrates are often nonspecific with lymphocytes located around small vessels and extending into the interstitium. Occasionally, neutrophilic infiltrates can be observed within the glandular lumen with/without lymphocytic infiltrates.

7. Hyperplastic and neoplastic lesions 7.1 Testis Proliferative lesions of the testis have not been reported in laboratory species of non-human primates,66,67 but have occasionally been reported in zoo-based species. This includes single case reports of a seminoma in both an owl monkey and a howler monkey68,69; a sex cord-stromal

tumor in a cotton-top tamarin70; and a Leydig cell tumor in a gorilla.71

7.2 Accessory sex glands Prostatic hyperplasia and benign neoplasia have been reported as a common age-related change in rhesus macaques.72e74 These proliferative lesions are most common in the peripheral portions of the cranial lobe and consist of varying degrees of basal cell proliferation ranging from small hyperplastic foci to adenomas.74 Similar lesions have been reported in cynomolgus monkeys.27,75 Prostatic adenocarcinomas have occasionally been reported.72,76 Proliferative lesions of the seminal vesicles are limited to a single report of an adenoma in a cynomolgus monkey.72

7.3 Penis Proliferative epithelial lesions have been observed on the penis of rhesus macaques77 and Mauritian-origin cynomolgus monkeys.78 The lesions range from small plaques to larger condylomatous lesions and carcinomas have been described.77,79 Development of these lesions appears to have a viral etiology with papilloma virus detected in lesions from rhesus macaques,77 and macaque lymphocryptovirus but not papilloma virus in Mauritian-origin cynomolgus monkeys.78,80

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FIGURE 18.16 Prostate glands from two control cynomolgus monkeys demonstrating a perivascular lymphocytic infiltrate (A) and a focal collection of luminal neutrophils with a surrounding lymphocytic infiltrate (B) (H&E).

8. Toxicologic lesions 8.1 Testis Microscopic assessment of the testis is considered to be the most sensitive method for detection of early test articled related effects on spermatogenesis.81 This is especially true in non-human primates, where significant interanimal variability and small group size can impact the ability to detect changes in other reproductive end points, such as organ weights, circulating endocrine biomarkers, and semen parameters.82 Early effects in the testis can be cell or stage specific and can occur by a variety of different mechanisms.83,84 By characterizing the initial pattern of change, it is often possible to gain insight into the underlying mechanism or at least, place the effects into a broad category or classification, such as germ cell effects, Sertoli cell toxicity, hormonal effects, Leydig cell changes, fluid disturbances, and vascular effects.61 With continued dosing, these initial effects on spermatogenesis may progress to tubular atrophy.61 Once diffuse tubular atrophy is present, it is no longer possible to tell the underlying mechanism or cell type affected.

8.1.1 Germ cell effects Compounds causing specific germ cell effects are typically observed as germ cell degeneration, germ cell depletion, and/or tubular atrophy.61 Depending on the timing of the

evaluation, germ cell degeneration or effects on cell division (mitotic or meiotic) may no longer be evident and the only sign of an effect is a missing germ cell layer(s). This absence of a specific cell type is termed germ cell depletion and it can affect any germ cell type. Initial lesions can be subtle, but with time, they become more obvious as more mature cells begin to drop out due to the lack of precursors. This pattern of depletion is often referred to as “maturation depletion” and the extent of the finding will vary with the original cell type affected and the length of dosing and/or off-dose period. In the case of a spermatogonial toxicant, with continued dosing, successive layers of more mature germ cells will continue to drop out (Fig. 18.17) and the lesion progresses to diffuse tubular atrophy as all germ cell types are lost. If the germ cell depletion affects more mature cell types, such as spermatids, the lesion never progresses to full tubular atrophy as spermatogonia and spermatocytes are unaffected and present. Spermatogonia are particularly prone to apoptosis associated with administration of cytotoxic agents, as they are the only mitotically active class of germ cells and they reside outside of the BTB.85 In macaques, decreases in gonadotropins and testosterone lead to a loss of type B spermatogonia, which with time can progress to tubular atrophy through maturation depletion (Fig. 18.17)28 and histologically resembles the maturation depletion seen with a direct spermatogonial toxicity. Careful evaluation of the target biology, accessory sex organ weights, epididymal

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FIGURE 18.17 Testis from a mature cynomolgus monkey with maturation depletion of germ cells following prolonged decreased gonadotropins and androgens. Only elongated spermatids remain (H&E).

histology, time of year/breeding season (rhesus macaques), and effects in other mitotic tissues are important considerations in determining which underlying mechanism might be present.

8.1.2 Sertoli cell toxicity Sertoli cell toxicity is typically manifested by loss of function rather than death and loss of Sertoli cells.86,87 Given the many roles that Sertoli cells play in supporting spermatogenesis, there are a variety of different changes that can be observed with Sertoli cell toxicity including inhibited spermiation, vacuolation, germ cell degeneration, germ cell exfoliation, and/or atrophy.61,88 Sertoli cells play a key role in supporting the conformational changes during spermatid elongation as well as the removal of the residual body and ultimate release of mature spermatids during spermiation. Retained spermatids are commonly observed in rats,61 but are not regularly observed as a specific change in the non-human primate. Sertoli cell dysfunction can result in variably sized, clear cytoplasmic vacuoles, generally near the basement due to impaired Sertoli cell fluid homeostasis or phospholipidosis (Fig. 18.18). Germ cell degeneration/depletion can leave clear spaces between Sertoli cells, giving the appearance of vacuolation, and it is common to see some degree of vacuolation in conjunction with degenerative tubular changes. Multinucleated giant cells may be observed with Sertoli cell injury due to the widening of the cytoplasmic bridges between germ cell cohorts (Fig. 18.18). These enlarged syncytial cells contain

numerous nuclei of spermatocytes or round spermatids. The presence of large numbers of multinucleated germ cells in the absence of other degenerative changes is suggestive of primary Sertoli cell injury; however, multinucleated giant cells are often a part of a spectrum of degenerative changes. Exfoliation of germ cells can be observed with Sertoli cell toxicity due to disruption of microtubular function. With longer-term exposures to Sertoli cell toxicants, disorganization of the seminiferous epithelium may occur with variable germ cell loss and may ultimately progress to diffuse tubular atrophy.53

8.1.3 Hormonal effects Alterations of the normal hormonal balance can occur by a variety of different mechanisms including decreased circulating androgens, androgen receptor agonism/antagonism, inhibition of 5a-reductase, and inhibition of aromatase. The basic mechanisms of hormonal regulation are generally similar across species, for example, decreased circulating androgen levels lead to decreased prostatic weight across all species; however, there are speciesspecific differences in the effects on spermatogenesis.28 The stage and cell specific microscopic changes described in the testis of the rat due to decreased intratesticular testosterone levels (degeneration of spermatocytes and round spermatids in stages VII/VIII and spermatid retention in stages IXeXI)89 do not occur in the non-human primate. In non-human primates, FSH plays a more prominent role in supporting spermatogenesis than in the rat and decreases

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FIGURE 18.18 Testis from a mature cynomolgus monkey demonstrating test articleerelated round spermatid degeneration/depletion with multinucleated cells and vacuolation (PAS-H).

in gonadotropins and testosterone lead to a loss of type B spermatogonia, which with time progresses to a tubular atrophy through maturation depletion as described above (Fig. 18.17).28 Decreased intratesticular levels of androgens may occur by several different mechanisms: (1) Administration of exogenous testosterone or other androgenic, estrogenic, or progestogenic xenobiotics resulting in increased negative feedback to the hypothalamus and pituitary gland leading to decreased LH release and reduced Leydig cell production of testosterone90e92; (2) Direct cytotoxicity and loss of Leydig cells as seen in the rat with ethylene dimethanesulfonate93; (3) GnRH antagonism leading to lower LH levels94; or (4) Inhibition of steroidogenic enzymes involved in testosterone biosynthesis. Decreases in accessory sex organ weight along with decreases in secretory content and/or epithelial height are a consistent effect observed with decreased androgen levels in repeated dose toxicity studies, although variability in organ weights in non-human primates may preclude detection of early changes.

8.2 Efferent ducts, epididymis, and vas deferens The efferent ducts resorb fluid produced by the seminiferous tubules and concentrate sperm.29 Xenobiotic effects

may include excessive fluid resorption resulting in sperm stasis or inhibited fluid resorption leading to dilation of the ducts. As lesions develop, the blood-epithelial barrier that separates the antigenically foreign sperm from inflammatory cells can be lost with subsequent granulomatous inflammation (sperm granuloma) and blockage of efferent ducts. There are numerous reports of xenobiotics and mechanisms leading to changes in fluid dynamics, inflammation, and/or efferent duct blockage95e102; however, these are only described in rodents and there are important species differences in efferent duct anatomy. The efferent ducts in rats and mice are tortuous and merge into a single common duct, which may make rodents much more susceptible to complete obstruction and backpressure atrophy of the testis than other mammals. Most mammals, including non-human primates and humans, have shorter efferent ducts with a parallel arrangement leading to multiple entry points into the head of the epididymis (Fig. 18.1).103 Drug-induced changes in the epididymis can be categorized into those causing effects on the epididymal epithelium and those occurring as a secondary effect within the epididymal lumen due to testicular toxicity.53,57 Epithelial apoptosis occurs within the epididymis as an early response to decreased testosterone levels, decreased conversion of testosterone to DHT, or androgen receptor

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FIGURE 18.19 Prostate of a mature cynomolgus monkey after treatment with an estrogenic test article inducing squamous metaplasia of periurethral glands. (A) The prostate was stained with PAS-H and (B) squamous cells were strongly positively stained by immunohistochemistry using an anticytokeratin 10 antibody (B).

antagonism. Epithelial apoptosis starts in the head of the epididymis and progresses distally in a wavelike fashion over time.55,56 With chronic androgen deprivation, the epididymal epithelium becomes atrophic and the apoptosis is no longer evident.57 Epithelial vacuolation within the epididymis can be seen in specific segments of the epididymis, although careful evaluation is critical as some degree of vacuolation may be present normally.43 Drugs that induce phospholipidosis can occasionally cause vacuolation in the epididymal epithelium.61 Sperm granulomas can occur as a rare spontaneous finding or as a result of xenobiotic exposure and can occur anywhere from the testis to the vas deferens. Sperm granulomas can result from local outflow obstruction as seen with ligation of the vas deferens, which results in a high incidence of epididymal sperm granulomas.104 Increased numbers of sloughed testicular germ cells and/or cellular debris can be observed within the lumen of the epididymis as a secondary effect to testicular toxicity. In young peripubertal non-human primates and in seasonal breeders (e.g., rhesus monkeys) during the nonreproductive period, there will often be increased levels of germ cell debris within the epididymis (Fig. 18.12) and this should not be mistaken for evidence of testicular toxicity.49

8.3 Accessory sex glands In rodents, decreased accessory sex organ weight with or without microscopic evidence of atrophy and/or decreased

secretory product is a commonly seen effect in the prostate and/or seminal vesicles. This can occur due to a xenobioticinduced decreased androgenic stimulation or as a secondary nonspecific stress-related effect related to decreased body weight and/or poor clinical condition.105 However, in nonhuman primates, use of mature animals is not always practical and detection of these effects in immature or peripubertal animals is not possible. When using mature animals, changes in organ weight may be detected, particularly after chronic dosing; however, organ weights are not a sensitive endpoint in non-human primates due to the large interanimal variability in organ weights, and relatively small group sizes82 and care should be exercised in not overinterpreting small differences from concurrent controls. Exogenous estrogens and aromatizable androgens can cause squamous metaplasia (Fig. 18.19) of the epithelium and stromal proliferation within the prostate of macaques.58,106

9. Conclusion Test articleerelated effects on the male reproductive system can have a profound impact on pharmaceutical development. In NHPs, the assessment of the male reproductive system often occurs by the study pathologist in the context of a repeated-dose general toxicology study rather than in stand-alone developmental and reproductive toxicology studies. This requires the pathologist to have a thorough understanding of endocrinology, spermatogenesis, spontaneous findings, and response to injury.

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18. Picut CA, Ziejewski MK, Stanislaus D. Comparative aspects of preand postnatal development of the male reproductive system. Birth Defects Res 2018;110(3):190e227. 19. Mann DR, Akinbami MA, Gould KG, Paul K, Wallen K. Sexual maturation in male rhesus monkeys: importance of neonatal testosterone exposure and social rank. J Endocrinol 1998; Mar;156(3) :493e501. 20. Smedley JV, Bailey SA, Perry RW, O’Rourke CM. Methods for predicting sexual maturity in male cynomolgus macaques on the basis of age, body weight, and histologic evaluation of the testes. Contemp Top Lab Anim Sci 2002;41:18e20. 21. Luetjens CW, Weinbauer GF. Functional assessment of sexual maturity in male macaques (Macaca fascicularis). Regul Toxicol Pharmacol 2012;63:391e400. 22. Li LH, Donald JM, Golub MS. Review on testicular development, structure, function, and regulation in common marmoset. Birth Defects Res B Dev Reprod Toxicol 2005; Oct;74(5):450e69. 23. Irfan S, Wistuba J, Ehmcke J, Shahab M, Schlatt S. Pubertal and testicular development in the common marmoset (Callithrix jacchus) shows high individual variation. Primate Biol 2015;2:1e8. 24. Vidal JD, Colman K, Bhaskaran M, et al. Scientific and regulatory policy committee best practices: documentation of sexual maturity by microscopic evaluation in nonclinical safety studies. Toxicol Pathol 2021;45(9):977e88. https://doi.org/10.1177/0192623321 990631. 25. Bansode FW, Chowdhury SR, Dhar JD. Seasonal changes in the seminiferous epithelium of rhesus and bonnet monkeys. J Med Primatol 2003;32:170e7. 26. Brand HM. Influence of season on birth distribution in marmosets and tamarins. Lab Anim 1980;14(4):301e2. 27. Mubiru JN, Hubbard GB, Dick Jr EJ, Furman J, Troyer DA, Rogers J. Nonhuman primates as models for studies of prostate specific antigen and prostatic diseases. Prostate 2008;68:1546e54. 28. Ramaswamy S, Weinbauer GF. Endocrine control of spermatogenesis: role of FSH and LH/testosterone. Spermatogenesis 2014;4(2):e996025. 29. Hess RA, Zhou Q, Nie R. The role of estrogens in the endocrine and paracrine regulation of the efferent ductules, epididymis and vas deferens. In: Robaire B, Hinton BT, editors. The epididymis: from molecules to clinical practice. New York: Kluwer Academic/Plenum Publishers; 2002. p. 317e38. 30. Steiner RA, Bremner WJ. Endocrine correlates of sexual development in the male monkey, Macaca fascicularis. Endocrinology 1981;109:914e9. 31. Kholkute SD. Diurnal and annual variations in plasma androgen levels in the adult male marmoset (callithrix jacchus). Int J Androl 1984;7(5):431e8. 32. Plant TM, Zumpe D, Sauls M, Michael RP. An annual rhythm in the plasma testosterone of adult male rhesus monkeys maintained in the laboratory. J Endocrinol 1974;62(2):403e4. 33. Czoty PW, Gould RW, Nader MA. Relationship between social rank and cortisol and testosterone concentration in male cynomolgus monkeys (Macaca fascicularis). J Neuroendocrinol 2009;21:68e76. 34. Niehoff MO, Bergmann M, Weinbauer GF. Effects of social housing of sexually mature male cynomolgus monkeys during general and reproductive toxicity evaluation. Reprod Toxicol 2010;29:57e67.

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35. French JA. The role of androgenic steroids in shaping social phenotypes across the lifespan in male marmosets (Callithrix spp.). Am J Primatol 2013;75(3):212e21. 36. Harrison RM. Hypospadias in a male rhesus monkey. J Med Primatol 1976;5(1):60e3. 37. Prahalada S, Tarantal AF, Harris GS, Ellsworth KP, Clarke AP, Skiles GL, MacKenzie KI, Kruk LF, Ablin DS, Cukierski MA, Peter CP, vanZwieten MJ, Hendrickx AG. Effects of finasteride, a type 2 5-alpha reductase inhibitor, on fetal development in the rhesus monkey (Macaca mulatta). Teratology 1997;55(2):119e31. 38. Turnquist JE, Minugh-Purvis N. Functional morphology. In: Bennett BT, Abee CR, Henrickson R, editors. Nonhuman primates in biomedical research: biology and management. London: Academic Press; 1995. p. 87e129. 39. Resko JA, Jackson GL, Huckins C, Stadelman H, Spies HG. Cryptorchid rhesus macaques: long term studies on changes in gonadotropins and gonadal steroids. Endocrinology 1980; Oct;107(4):1127e36. 40. Tao SX, Guo J, Zhang XS, Li YC, Hu ZY, Han CS, Liu YX. Germ cell apoptosis induced by experimental cryptorchidism is mediated by multiple molecular pathways in Cynomolgus Macaque. Front Biosci 2006;11:1077e89. 41. Zöller M, Friderichs-Gromoll S, Kaspareit J. Testicular and epididymal appendages in the cynomolgus macaque (Macaca fascicularis). J Med Primatol 2009;38(6):448e54. 42. Colman K, Andrews RN, Atkins H, et al. International harmonization of nomenclature and diagnostic criteria (INHAND): nonproliferative and proliferative lesions of the non-human primate (M. fascicularis). J Toxicol Pathol 2021;34(3 Suppl):1Se182S. 43. Creasy DM. Reproduction of the rat, primate, dog and pig. In: McKinnes E, editor. Background lesions in laboratory animalsda color atlas. Edinburgh: Saunders Elseviers; 2012. p. 101e10. 44. Creasy DM, Chapin RE. Male reproductive system. In: Haschek WM, Rousseaux CG, Wallid MA, Bolon B, Ochoa R, Mahler BW, editors. Haschek and Rousseaux’s handbook of toxicologic pathology. 3rd ed., vol. 3. San Diego: Academic Press; 2013. p. 2493e598. 45. Kozlosky JC, Mysore J, Clark SP, Burr NH, Li J, Aranibar N, Vuppugalla R, West RC, Mangipudy RS, Grazian MJ. Comparison of physiologic and pharmacologic parameters in Asian and Mauritius cynomolgus monkeys. Regul Toxicol Pharmacol 2015;73:27e42. 46. Patrick DJ, Rebelatto MC. Toxicologic pathology and background lesions of nonhuman primates. In: Bluemel J, Korte S, Schenck E, Weinbauer GF, editors. The nonhuman primate in nonclinical drug development and safety assessment. San Diego: Academic Press; 2015. p. 236e55. 47. Pereira Bacares M, Vemireddi V, Creasy D. Testicular Fibrous Hyoplasia in Cynomolgus monkey (Macaca fascicularis): an incidental, congenital lesion. Toxicol Pathol 2017;45(4):536e43. 48. Sato J, Doi T, Kanno T, Wako Y, Tsuchitani M, Namara I. Histopathology of incidental findings in cynomolgus monkeys (Macaca Fascicularis) used in toxicity studies. J Toxicol Pathol 2012;25:63e101. 49. Vidal JD, Bhaskaran M, Carsillo M, et al. Spontaneous findings in the reproductive system of sexually mature male Cynomolgus macaques. Toxicol Pathol 2022;50(5):660e78. https://doi.org/10.1177/ 01926233221082302.

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50. Chamanza R, Marxfeld HA, Blanco AI, Naylor SW, Bradley AE. Incidences and range of spontaneous findings in control cynomolgus monkeys (Macaca fascicularis) used in toxicity studies. Toxicol Pathol 2010;38(4):642e57. 51. Greaves P. Histopathology of preclinical toxicity studies. London: Academic Press; 2012. p. 615e66. 52. Haruyama E, Suda M, Ayukawa Y, Kamura K, Mizutamari M, Ooshima Y, Tanimoto A. Testicular development in cynomolgus monkeys. Toxicol Pathol 2012;40:935e42. 53. Vidal JD, Mirsky ML, Colman K, Whitney KM, Creasy DM. Reproductive system and mammary gland. In: Sahota PS, Popp JA, Hardisty JF, Gopinath C, editors. Toxicologic pathology, nonclinical safety assessment. Boca Raton: CRC Press; 2013. p. 717e830. 54. Chaffee BK, Beck AP, Owston MA, Kumar S, Baze WB, Magden ER, Dick EJ, Lammey M, Abee CR. Spontaneous reproductive tract lesions in aged captive chimpanzees. Vet Pathol 2016;53(2):425e35. 55. Robaire B, Fan X. Regulation of apoptotic cell death in the rat epididymis. J Reprod Fertil Suppl 1998;53:211e4. 56. Ezer N, Robaire B. Androgenic regulation of the structure and function of the epididymis. In: Robaire B, Hinton BT, editors. The epididymis: from molecules to clinical practice. New York: Kluwer Academic/Plenum Publishers; 2002. p. 297e316. 57. De Grava Kempinas W, Klinefelter GR. Interpreting histopathology in the epididymis. Spermatogenesis 2014;4(2):e979114. https:// doi.org/10.4161/21565562.2014.979114. 58. Habenicht UF, El Etreby MF. The periurethral zone of the prostate of the cynomolgus monkey is most sensitive part for an estrogenic stimulus. Prostate 1998;13:305e16. 59. Picut CA, De Rijk EPCT, Dixon D. Immunopathology of the male reproductive tract. In: Parker GA, editor. Molecular and integrative toxicology- immunopathology in toxicology and drug. Cham, Switzerland: Development Humana Press; 2017. p. 470e539. 60. Cline JM, Brignolo L, Ford EW. Urogenital system. In: Abee CR, Mansfield K, Tardif S, Morris T, editors. Nonhuman primates in biomedical research. London: Academic Press; 2012. p. 483e562. 61. Vidal JD, Whitney KM. Morphologic manifestations of testicular and epididymal toxicity. Spermatogenesis 2014; Dec 31;4(2) :e979099. 62. Chapman ES, Heidger PM. Spermatic granuloma of vas deferens after vasectomy in rhesus monkey and men. Urology 1979;6 :629e39. 63. Alexander NJ. Primates: their use in research on vasectomy. Primatology 1981;1(20):167e73. 64. Porter BF, Frost P, Hubbard GB. Polyarteritis nodosa in a Cynomolgus macaque (Macaca fascicularis). Vet Pathol 2003;40 :570e3. 65. Neal DE, Dilworth JP, Kaack MB, Didier P, Roberts JA. Experimental prostatitis in nonhuman primates: II. Ascending acute prostatitis. Prostate 1990;17(3):233e9. 66. Kaspareit J, Friderichs-Gromoll S, Buse E, Habermann G. Spontaneous neoplasms observed in cynomolgus monkeys (Macaca fascicularis) during a 15-year period. Exp Toxicol Pathol 2007;59(3e4):163e9. 67. Simmons HA, Mattison JA. The incidence of spontaneous neoplasia in two populations of captive rhesus macaques (Macaca mulatta). Antioxidants Redox Signal 2011;14(2):221e7.

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68. Gozalo A, Nolan T, Montoya E. Spontaneous seminoma in an owl monkey in captivity. J Med Primatol 1992; Jan;21(1):39e41. 69. Maruffo CA, Malinow MR. Seminoma in a howler monkey (Alouatta caraya). J Pathol Bacteriol 1966; Jan;91(1):280e2. 70. Yearley JH, King N, Liu X, Curran EH, O’Neil SP. Biphasic malignant testicular sex cordestromal tumor in a cotton-top tamarin (Saguinus oedipus) with review of the literature. Vet Pathol 2008;45(6):922e7. 71. Jones DM, Dixson AF, Wadsworth PF. Interstitial cell tumour of the testis in a western lowland gorilla (Gorilla gorilla). J Med Primatol 1980;9:319e22. 72. Lewis RW, Kim JC, Irani D, et al. The prostate of the nonhuman primate: normal anatomy and pathology. Prostate 1981;2 (1):51e70. 73. Baskerville A, Cook RW, Dennis MJ, et al. Pathological changes in the reproductive tract of male rhesus monkeys associated with age and simian AIDS. J Comp Pathol 1992;107(1):49e57. 74. McEntee MF, Epstein JI, Syring R, et al. Characterization of prostatic basal cell hyperplasia and neoplasia in aged macaques: comparative pathology in human and nonhuman primates. Prostate 1996;29(1):51e9. 75. Wakui S, Furusato M, Kato H, Nomura Y, Kano Y, Aizawa S. Prostatic basal cell hyperplasia in a cynomolgus monkey (Macaca fascicularis). Vet Pathol 1989;26(5):447e8. 76. Hubbard GB, Eason RL, Wood DH. Prostatic carcinoma in a rhesus monkey (Macaca mulatta). Vet Pathol 1985;22(1):88e90. 77. Ostrow RS, McGlennen RC, Shaver MK, Kloster BE, Houser D, Faras AJ. A rhesus monkey model for sexual transmission of a papillomavirus isolated from a squamous cell carcinoma. Proc Natl Acad Sci USA 1990;87(20):8170e4. 78. Harari A, Wood CE, Van Doorslaer K, Chen Z, Domaingue MC, Elmore D, Burk RD. Condylomatous genital lesions in cynomolgus macaques from Mauritius. Toxicol Pathol 2013;1(6):893e901. 79. Hubbard GB, Wood DH, Fanton JW. Squamous cell carcinoma with metastasis in a rhesus monkey (Macaca mulatta). Lab Anim Sci October 1983;33(5):469e72. 80. Gordon HP, Reim DA, McClain SA. Condyloma acuminatum in a cynomolgus monkey (Macaca fascicularis). Contemp Top Lab Anim Sci 2000; Mar;39(2):30e3. 81. Takayama S, Akaike M, Kawashima K, Takahashi M, Kurokawa Y. Studies on the optimal treatment period and parameters for detection of male fertility disorder in rats–introductory summary. J Toxicol Sci 1995; Aug;20(3):173e82. 82. Cappon GD, Potter D, Hurtt ME, Weinbauer GF, Luetjens CM, Bowman CJ. Sensitivity of male reproductive endpoints in nonhuman primate toxicity studies: a statistical power analysis. Reprod Toxicol November 2013;41:67e72. 83. Creasy DM. Pathogenesis of male reproductive toxicity. Toxicol Pathol 2001;29:64e76. 84. Creasy D, Bube A, De Rijk E, Kandori H, Kuwahara M, Masson R, Nolte T, Reams R, Regan K, Rehm S, Rogerson P, Whitney K. Proliferative and non-proliferative lesions of the rat and mouse male reproductive system. Toxicol Pathol 2012;40:40Se121S. 85. Meistrich ML. Components of testicular function and sensitivity to disruption. Biol Reprod 1986;34:17e28. 86. Hild SA, Reel JR, Dykstra MJ, Mann PC, Marshall GR. Acute adverse effects of the indenopyridine CDB- 4022 on the ultrastructure of Sertoli cells, spermatocytes, and spermatids in rat testes:

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comparison to the known Sertoli cell toxicant Di-n-pentylphthalate (DPP). J Androl 2007;28:621e9. Moffitt JS, Bryant BH, Hall SJ, Boekelheide K. Dose-dependent effects of Sertoli cell toxicants 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl) phthalate in adult rat testis. Toxicol Pathol 2007;35:719e27. Johnson KJ. Testicular histopathology associated with disruption of the Sertoli cell cytoskeleton. Spermatogenesis 2014;4(2) :e979106. Kerr JB, Millar M, Maddocks S, Sharpe RM. Stage-dependent changes in spermatogenesis and Sertoli cells in relation to onset of spermatogenic failure following withdrawal of testosterone. Anat Rec 1993;235:547e59. Russell LD, Malone JP, Karpas SL. Morphologic pattern elicited by agents affecting spermatogenesis by disruption of its hormonal stimulation. Tissue Cell 1981;13:369e80. Troiano L, Fustini MF, Lovato E, Frasoldati A, Malorni W, Capri M, Grassilli E, Marrama P, Franceschi C. Apoptosis and spermatogenesis: evidence from an in vivo model of testosterone withdrawal in the adult rat. Biochem Biophys Res Commun 1994;202(3) :1315e21. Beardsley A, O’Donnell L. Characterization of normal spermiation and spermiation failure induced by hormone suppression in adult rats. Biol Reprod 2003;68:1299e307. Bartlett JMS, Kerr JB, Sharpe RM. The effect of selective destruction and regeneration of rat Leydig cells on the intratesticular distribution of testosterone and morphology of the seminiferous epithelium. J Androl 1986;7:240e53. Hikim AP, Leung A, Swerdloff RS. Involvement of apoptosis in the induction of germ cell degeneration in adult rat after gonadotropinreleasing hormone antagonist treatment. Endocrinology 1995;136:2770e5. Nakai M, Hess RA, Moore BJ, Guttroff RF, Strader LF, Linder RE. Acute and long-term effects of a single dose of the fungicide carbendazim (methyl 2-benzimidazole carbamate) on the male reproductive system in the rat. J Androl 1992;13:507e18. Gotoh Y, Netsu J, Nakai M, Nasu T. Testicular damage after exposure to carbendazim depends on the number of patent efferent ductules. J Vet Med Sci 1999;61:755e60. Hess RA, Nakai M. Histopathology of the male reproductive system induced by the fungicide benomyl. Histol Histopathol 2000;15:207e24. Piner J, Sutherland M, Millar M, Turner K, Newall D, Sharpe RM. Changes in vascular dynamics of the adult rat testis leading to transient accumulation of seminiferous tubule fluid after administration of a novel 5-hydroxytryptamine (5HT) agonist. Reprod Toxicol 2002;16:141e50. Tani Y, Foster PM, Sills RC, Chan PC, Peddada SD, Nyska A. Epididymal sperm granuloma induced by chronic administration of 2-methyoimidazole in B6C3F1 mice. Toxicol Pathol 2005;33 :313e9. La DK, Creasy DM, Hess RA, Baxter E, Pereira ME, Johnson CA, Vinken P, Snook SS. Efferent duct toxicity with secondary testicular changes in rats following administration of a novel leukotriene A4 hydrolase inhibitor. Toxicol Pathol 2012;40:705e14. Seachrist DD, Johnson E, Magee C, et al. Overexpression of follistatin in the mouse epididymis disrupts fluid resorption and sperm transit in testicular excurrent ducts. Biol Reprod 2012;87(2):41.

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102. Heuser A, Mecklenburg L, Ockert D, Kohler M, Kemkowski J. Selective inhibition of PDE4 in Wistar rats can lead to dilatation in testis, efferent ducts, and epididymis and subsequent formation of sperm granulomas. Toxicol Pathol 2013;41(4):615e27. 103. Ilio KY, Hess RA. Structure and function of the ductuli efferentes: a review. Microsc Res Tech 1994;29:432e67. 104. Flickinger CJ, Howard SS. Consequences of obstruction on the epididymis. In: Robaire B, Hinton BT, editors. The epididymis: from molecules to clinical practice. New York: Kluwer Academic/ Plenum Publishers; 2002. p. 503e22.

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Chapter 19

The cardiovascular system of the non-human primate Jennifer A. Chilton1, Kevin A. Keane2, Maurice Cary3, James E. Baily4, Pierluigi Fant5 and Roger Alison6 1

Charles River Laboratories, Reno, NV, United States; 2Blueprint Medicines, Cambridge, MA, United States; 3Pathology Experts GmbH, Witterswil,

Switzerland; 4Charles River Laboratories, Edinburgh, United Kingdom; 5Charles River Laboratories, Lyon, France; 6Roger Alison Ltd, Lampeter, United Kingdom

1. Introduction Knowledge of primate-specific normal anatomic variations and a keen awareness of the spectrum of spontaneous and background lesions involving the blood vessels are critical when evaluating tissues from non-human primate (NHP) studies. This is particularly true for those with no to limited experience with NHP histopathology, where such lesions can be confounding and/or overdiagnosed. Some acceptance of spontaneous and background findings, particularly of minimal severity, is required so that the pathologist is not oversensitive to their presence (i.e., increase the diagnostic threshold). This is critical in toxicology studies using NHPs where the numbers per sex group are low (e.g., 3/sex/ group) and just two affected animals in the high dose (albeit minimal to mild) and serendipitously one or none in the control or lower dose groups. This distribution event easily appears as a treatment-related finding, where in fact only perspective from experience is required to understand the increase is only coincidental and has nothing to do with treatment. It is necessary to have thorough knowledge of spontaneous and background lesions in primate blood vessels and heart to avoid mistaking them as treatmentrelated findings. This chapter is intended to review the common spontaneous and xenobiotic-induced lesions of the cardiovascular system of captive, purpose-bred, non-human primates such as the rhesus and cynomolgus macaques, and marmosets that are used in translational research such as nonclinical safety studies. The text will also aim to aid toxicologic pathologists differentiate or identify spontaneous nonclinical disease pathology when assigning cause of death to these species in these collections. Spontaneous pathology in other NHP species that are not commonly

used in translational research have been reviewed elsewhere.1 These species are discussed when appropriate. It is well-accepted in translational research with any species of laboratory animal that the nature and incidence of spontaneous findings can vary considerably from one breeding facility to another based on both environmental and genetic factors. Likewise, this is true in cardiovascular lesions of laboratory NHPs. An example of this has been described in Mauritian-origin macaques compared to Indonesian-origin macaques which exhibit a higher incidence of spontaneous myocardial degeneration along with subendocardial hemorrhage, hemosiderin deposition, and arterial medial degeneration.2,3 Thus, it is clarified that the data presented below is only a general guide to the occurrence of these findings and the gold standard for the incidence of a spontaneous finding should be in-house generated historical control data from a single-origin or source of animals. These incidence differences will be highlighted below where possible. Additionally, it is noted that the preferred terminology used below is based on the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions of the Non-human Primate (INHAND).

2. Anatomy of the cardiovascular system In the embryo, the heart and vessels originate from progenitor cells that populate the splanchnic mesoderm as “blood islands” that eventually undergo transformation to the more mature structure of the heart tube and conduit for the systemic venous return.4 The embryological heart is

Spontaneous Pathology of the Laboratory Non-human Primate. https://doi.org/10.1016/B978-0-12-813088-9.00019-7 Copyright © 2023 Elsevier Inc. All rights reserved.

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composed of the same layers expected in the adultd endocardium, myocardium, and pericardial surface-that originate from the primitive heart tube.4 The vascular system is essential for further development of nearly all other fetal structures; therefore, forms early in embryology to provide the nutrient supply pathway to all other systems. Until birth, the blood flow to and from the fetal heart bypasses the fetal lung via the ductus arteriosus shunt between the aorta and the pulmonary artery, and via the foramen ovale between the atria, as oxygen and nutrients are provided via the maternal interface; however, once the newborn is delivered, left-to-right blood flow begins, and with increased vascular pressure the ductus arteriosus and the foramen ovale eventually close. Normal low pressure returns to the right ventricle and high pressure output from the left ventricle is initiated. This change in pressure also induces cardiomyocyte alterations leading to increased muscle mass in the left ventricle. Consistent with the gross anatomy of all mammals, the primate heart consists of four chambers divided into two atria and two ventricles (Fig. 19.1A and B). It also has fibromyxomatous valves. There are right and left atrioventricular valves consisting of two cusps that attach to chordae tendineae. Along with papillary muscles and myocardium they form the atrioventricular valvular apparatus (Fig. 19.2). Non-human primate hearts, like humans and other mammals, have sinoatrial (SA) and atrioventricular (AV) nodes for initiation of electrical impulses. The normal histologic features and functionality of the NHP heart are consistent with those observed in other mammalian species with some

exceptions. The morphology of the sinoatrial node of the Macaca fascicularis is morphologically the same as that in other mammals as well, and is characterized by unifocal impulse generation. However, it is unique with respect to other mammals in that it propagates impulses in an oblique direction toward the inferior vena cava.5 The junctional AV region of the cardia conduction system in M. fascicularis is approximately similar to the same region in the human.6 The morphology of the autonomic cardiac nervous system in the New World monkeys is like that in the Old World monkeys. They differ in that the superior cervical ganglion in the New World monkeys tends to be relatively smaller and provides a narrower contribution to the spinal nerves than in the Old World monkeys.7 The vascular system leading to and from the heart are arranged into triads of artery, vein, and an associated nerve fibers. These triads permeate the body tissues (Fig. 19.3A). Lymphatic vessels also permeate the body near the triads and may contain accumulations of lymphocytes which should not be misinterpreted as mononuclear cell infiltrates (Fig. 19.3B). Additionally, there are focal intimal thickenings at arterial forks and at the ostia of branch vessels that are circumscribed and tend to be thicker than the adjacent vessel structures (Fig. 19.4A).8 Sectioning through these arterial forks may be misinterpreted as abnormal intimal thickening and should be differentiated based on microscopic features of normal smooth muscle and endothelium present (Fig. 19.4B).

FIGURE 19.1 (A) Fetal heart of a cynomolgus monkey (Macaca fascicularis) is a formed version of the postnatal animal: (a) Left ventricle, (b) right ventricle, (c) left atrium, (d) right atrium, (e) interventricular septum, (f) auricle, (g) atrioventricular valve, (h) pulmonic valve. Note the region of the foramen ovale between the atria (circle) and the relatively similar thickness of the right ventricle vs. left ventricle in the fetus-a feature that persists into the neonatal period due to the absence of left ventricular pressure prior to birth. (B) The cardiomyocytes of the fetus and neonate have less cytoplasmic volume compared to that of adults, giving the myocardium a hypercellular appearance. With time the cells hypertrophy through normal physiological compensatory mechanisms and the left ventricle becomes increased in muscle mass vs. the right ventricle.

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FIGURE 19.2 The atrioventricular valve apparatus consists of (a) fibromyxomatous valve, (b) chordae tendinea and papillary muscles, and (c) the myocardial attachment.

FIGURE 19.3 (A) Within tissues, the arteries (a) are found in tandem with veins (b) and nerve fibers (c). Note: the venous valve (vv) consisting of two leaflets within the section of vein that ensure one directional flow of blood by passively closing when backflow occurs. (B) Normal lymph vessels within the heart may contain lymphocytes that will usually be uniform, with dark rounded nuclei and indistinct cell walls. The vessel walls may be indistinct (arrow).

FIGURE 19.4 (A) At the bifurcation of arteries, there is a normal thickened region of the vessel wall and tunica intima (arrows). (B) Sectioning through the bifurcation of blood vessels produces focal thickened regions; however, the nature of this finding as artifact is identified by the presence of normal smooth muscle and endothelium (arrows).

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Another anatomical normal variation during tumescence in NHPs is the presence of highly vascularized skin, which is edematous macroscopically; while microscopically it can resemble a hemangioma. Known as sex skin, this estrus-related swelling is observed in macaques, baboons, and chimpanzees (see Chapter 17). Florid hyperplasia has been observed in the presence of hormone-modulating compounds. These findings in older macaques can be observed in the face and chest most often but can in fact anywhere. These so-called “age spots” are benign angiomas that are typically pink to red and approximately 1e3 mm in diameter.1

3. Congenital lesions of the cardiovascular system Spontaneous cardiovascular abnormalities, alone or in different combinations, are rare but have been reported in a varitety of NHPs, including cynomolgus, bonnet, stumptailed, rhesus and Japanese macaques; African green, squirrel, talapoin and tapas monkeys; baboons, gorillas, chimpanzees, and orangutans (Table 19.1). Animals with congenital cardiovascular defects usually have associated abnormalities in heart rhythm, heart rate, conductivity or cardiac output. With valvular disfigurements, abnormal heart

TABLE 19.1 Spontaneous and chemically induced congenital cardiovascular abnormalities in non-human primates.

Cynomolgus macaque

Rhesus macaque

Latin name

Defect

References

Macaca fascicularis

Ventricular septal defects

Koie et al. (2005), Hendrickx and Binkerd, (1993), Peterson et al. (1997).

Aortic coarctation, patent ductus arteriosus, atrial/ventricular septal defects

Jerome (1987).

Macaca mulatta

Acardius acephalus

Hein et al. (1985).

Ventricular septal defects

Swindle et al. (1986b), Lapin and Yakovleva (1963), Machado et al. (1989), Krilova and Yakovleva (1972), Peterson et al. (1997).

Ventricular hypoplasia

Peterson et al. (1997).

Pulmonic stenosis

Lapin and Yakovleva (1963)

Patent ductus arteriosus

Lapin and Yakovleva (1963), Freigang and Knobil (1967).

Persistent ductus arteriosus

Brandt et al. (2002).

Valve atresia with aortic hypoplasia and left ventricle agenesis

Cukierski et al. (1986).

Atrial septal defects, persistent ductus arteriosus

Jerome (1987).

Vascular nevus

Latendresse et al. (1987)

Marmoset

Callithrix jacchus

Ventricular septal defect

Scott (1992)

Japanese macaque

Macaca fuscata

Tetralogy of Fallot

Koie et al. (2007).

Squirrel monkeys

Saimiri sciureus

Ventricular septal defects

Hendrickx and Binkerd (1993), Machado et al. (1989).

Cardiac hamartoma

Golbar et al. (2011).

Ventricular septal defects

Hendrickx and Binkerd (1993), Machado et al. (1989).

Aortic coarctation and atrial/ventricular septal defects

Jerome (1987).

Atrial septal defects, patent ductus arteriosus

Jerome (1987).

African green monkey

Stumptail (bear macaque)

Cercopithecus aethiops, Chlorocebus spp.

Macaca arctoides

Continued

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TABLE 19.1 Spontaneous and chemically induced congenital cardiovascular abnormalities in non-human primates.dcont’d Latin name

Defect

References

Bonnet macaque

Macaca radiata

Atrial septal defects

Anilkumar and Sandhyamani (1990).

Patas monkey

Erythrocebus patas

Ventricular septal defect, tricuspid dysplasia

Sao-Ling et al. (2004).

Talapoin monkey

Miopithecus talapoin

Double aortic arch

Stills et al. (1979).

Chimpanzee

Pan troglodytes

Ventricular septal defects

Binhazim et al. (1994).

Atrial septal defects

Hackel et al. (1953).

Ventricular septal defects

Hendrickx and Binkerd (1993), Machado et al. (1989), Lapin and Yakovleva (1963), Krilova and Yakovleva (1972).

Atrial septal defects

Fox et al. (2011).

Patent foramen ovale

Fox et al. (2011), Moore et al. (2007), Lapin and Yakovleva (1963).

Persistent ductus arteriosus

Fox et al. (2011).

Aortic stenosis or aneurysm

Lapin and Yakovleva (1963), Fox et al. (2011).

Ventricular septal defects

Hendrickx and Binkerd, (1993), Machado et al. (1989).

Baboon

Papio spp.

Gorilla

Gorilla gorilla

Orangutan

Pongo pygmaeus

Patent foramen ovale

Moore et al. (2007).

Ventricular septal defects

Cook et al. (1986).

Pericardial defect

De Garis (1934).

Vena cava defect

Chase and de Garis (1938).

sounds during auscultation are usually heard. Furthermore, many of these defects lead to early life physical decline; therefore, these animals usually fail prestudy examinations and are not suitable for study assignments.

stenosis and/or patent ductus arteriosus in rhesus macaques.12 Ventricular and atrial defects are the most encountered in the colonies at the editors’ facilities (Fig. 19.5). Valvular atresia associated with aortic

3.1 Congenital defects of the heart and vasculature Spontaneous cardiovascular malformations have been reviewed by Peterson et al., who reported an overall incidence of muscle malformations of 0.9% in rhesus macaques (i.e., 40 out of 4390 necropsies) and 0.3% in cynomolgus macaques (i.e., 3 out of 965 necropsies) and most defects were noted in the musculoskeletal system followed by the cardiovascular system.10 The latter include isolated cases or various combinations of atrial and ventricular septal defects, ventricular hypoplasia/agenesis, patent ductus arteriosus, and vascular agenesis.10 Similar incidences of malformations have been reported by Jerome.11 Ventricular septal defects have been sporadically reported by other authors and were occasionally associated with pulmonic

FIGURE 19.5 The heart of a cynomolgus monkey with a a full thickness congenital ventricular septal defect below the aortic valve (arrow).

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Spontaneous Pathology of the Laboratory Non-human Primate

the face, ear, chin, and neck, characterized histologically by the presence of numerous uniformly distributed thick- and thin-walled vessels filled with erythrocytes in the dermis.17

3.3 Congenital epicardial cysts and epithelial plaques

FIGURE 19.6 Heart of a young cynomolgus monkey with a large cyst on the thickened and abnormally shaped atrioventricular valve (arrow). The chordae tendinaea of the valve are fused to a single, deformed papillary muscle (arrow head), with insertion into myocardial fibrous tissue, a condition referred to as “parachute valve.”

Microscopic epithelial structures (keratinized/nonkeratinized cysts, ectopic epithelial and squamous cysts/plaques) have been occasionally reported, generally at the epicardial or subepicardial surface at the base of the heart. These epithelial foci are incidental and consistent with embryonic remnants. Epithelial plaques may present as a region of irregular squamous epithelium with variable number of layers (Fig. 19.7). The cysts may be lined by a keratinized or nonkeratinized squamous epithelium and may contain keratin debris, inflammatory cells, and cellular debris (Fig. 19.8AeC).9

hypoplasia and left ventricle agenesis has been observed in rhesus macaques and was accompanied by generalized edema and ascites (hydrops fetalis). Aortic coarctation, persistent ductus arteriosus, and atrial septal defects (the latter also observed alone) have been reported in cynomolgus, rhesus, and stumptail macaques.13 A case of tetralogy of Fallot was reported in a newborn female Japanese macaque, characterized by right ventricular hypertrophy, right atrium enlargement, ventricular septal defects, overriding aorta, stenosis of the pulmonary valve and persistent ductus arteriosus.14 A unique case of spontaneous acardiac-acephalic fetal malformation has been reported in cynomolgus monkey.15 Like man, this extremely rare congenital condition occurred in a twin gestation. The abnormal twin had a nonfunctioning heart, and several other organs were absent or abnormally formed, while the other twin fetus died before delivery. Recently, doubleoutlet right ventricle and double septal defect have been reported in a rhesus macaque.16 Atrioventricular valve cysts (Fig. 19.6) have only been reported in humans and cattle, but have been noted in an animal at the editor’s facility. This animal also had a single, disfigured papillary muscle into which the fused cordae of the valve inserted, a condition noted in humans as “parachute valve”.

3.2 Congenital proliferative vascular lesions A case of congenital vascular nevus has been reported in the skin in a female rhesus macaque. At birth, a “strawberry-colored birthmark” was noted unilaterally in

FIGURE 19.7 The epicardium of the heart of a macaque has a focus of irregularly distributed, multiple layered squamous epithelium consistent with a congenital epithelial plaque. Image courtesy of Julie Schwartz.

The cardiovascular system of the non-human primate Chapter | 19

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FIGURE 19.8 (A) A large epicardial squamous cyst forms a discrete tan nodule on the epicardial surface of the heart of a macaque. (B) Micrograph of the nodule from figure 19.8A: The cyst has an irregular, multilayered squamous epithelium and is filled with keratin debris. (C) A double cyst extending from an epithelial plaque in a NHP suggests a common congenital etiology of these lesions.

3.4 Ectopic thyroid tissue In addition to being observed in the heart, ectopic thyroid tissue has also been found to be present in the walls of the associated great blood vessels. This finding has been diagnosed in cynomolgus monkeys and common marmosets and is rare.18

4. Degenerative lesions of the cardiovascular system Degenerative findings of the heart for NHPs that are published or have been encountered by the authors are summarized here. Some degenerative lesions in the heart are relatively common as background findings in the nonhuman primate heart and generally correlate with an increase in incidence with age.19

4.1 Arteriosclerosis A rare finding in the heart of young NHPs is arteriosclerosis and includes various vascular wall lesions that lead to “hardened arteries.” Sato et al. described arterial sclerosis characterized by intimal and medial thickening of the coronary and mural arteries. Features included arterial walls with duplication of the elastic lamina which was presumed

associated with stenosis, ischemia, and infarction, though that was not directly shown.20 Atherosclerotic-type lesions are occasionally noted in colony animals or control animals assigned to studies. These lesions not only contain increased mucinous materials but may also have low to moderate numbers of inflammatory cells that expand the tunica media and tunica intima. While these lesions may be quite mild in healthy animals (Fig. 19.9AeB) they may become quite severe with age (Fig. 19.9C). It is critical to note that opinions vary regarding mucopolysaccharide deposition. Some authors consider it normal at low levels; however, there is the opinion that areas of intimal thickening with mucopolysaccharide are predisposed to develop atherosclerosis. Some authors have reported focal thickening of the intima of the blood vessels, but notably with fibrosis or mucin accumulation, led to formation of atherosclerotic lesions with near-occlusion of the affected blood vessel. These atherosclerotic lesions and intimal thickenings were described as having infiltration of the intima by smooth muscle cells, mucins, and fibrous tissue with little or no foam cells or extracellular lipids.9 Of note, atherosclerotic lesions may also be induced experimentally in cynomolgus macaques by feeding an atherogenic diet, consequently this species is a model for dietary-induced atherosclerosis research.1

Mauritius. This study states the total incidence of medial degeneration and/or hemorrhage of the coronary arteries (intramural vessels of either the right or left ventricles) was 6%. Cellular debris, hemorrhage, and/or regeneration within a focal area of the vascular smooth muscle were characteristic of this finding.2

4.3 Aortic Dissection Aortic Dissection has been reported in macaques, gorillas, owl and squirrel monkeys. This finding is characterized by a breach in the intimal lining of the aorta. Additionally, there is separation and degeneration of the underlying muscular wall of aorta forming a recess, which becomes filled with blood, fibrin, and inflammatory cells. Significantly, the wall of this recess can weaken and rupture resulting in hemopericardium or hemothorax.21

4.4 Vascular and myocardial associated pigments The accumulation of macrophages containing brown hemosiderin pigment associated with small to medium size vessels has been reported in the lungs of cynomolgus monkeys.22 These macrophages were predominantly found in the perivascular interstitium (see Chapter 15). In some cases, vessels of the heart exhibited degeneration and necrosis of mural smooth muscle cells and rupture of the internal and external elastic lamina (Fig. 19.10A). Similar

FIGURE 19.9 Arteriosclerosis in non-human primates: (A) a focal region of aortic mural expansion in a young cynomologus macaque. (B) Higher magnification of the lesion in Fig. 19.9A: The focus consists of accumulation of mucin between smooth muscle cells. The endothelium is sparse with expansion of the tunica intima by mucin and low numbers of inflammatory cells. The elastic lamina has been effaced. (C) Chronic severe atherosclerosis of the ventral cerebral artery in an aged NHP: There is extensive effacement of the tunica intima and media by loose fibrous tissue and accumulation of mucin with fat and cholesterol clefts that occludes most of the vascular lumen.

4.2 Degeneration of the arterial tunica media An investigation of spontaneous cardiac findings in Mauritian-source cynomolgus monkeys compared to Indonesian-sourced cynomolgus monkeys demonstrated that a higher incidence of arterial medial degeneration with hemorrhage was observed in animals sourced from

FIGURE 19.10 (A) An artery in the heart of a cynomolgus monkey in which there is mural degeneration and deposits of perivascular hemosiderin. (B) An artery in the brain of a cynomolgus monkey with mural degeneration, fat accumulation of the tunica intima, and perivascular hemosiderophages.

The cardiovascular system of the non-human primate Chapter | 19

perivascular accumulations of hemosiderin may be noted in other tissues, such as the brain (Fig. 19.10B). The iron-rich nature of the hemosiderin may be elucidated through Perl’s Prussian Blue staining method. Ceroid, lipofuscin, and hyaline-type intracytoplasmic granules found in cardiac muscle cells of cynomolgus monkeys were studied using histologic, histochemical, and fluorescent microscopic techniques. The studies indicated that the ceroid granules contained an insoluble lipid as well as a component that was stainable with Luxol fast blue and Mallory’s phloxine stain for hyaline. Lipofuscin granules had staining reactions characteristic of the classical agerelated pigment. Hyaline granules were devoid of lipid component and were distinctly different from either ceroid or lipofuscin. All three types of granules were located at the poles of cardiac muscle cell nuclei.23

4.5 Vascular and cardiac mineralization Dystrophic mineralization of blood vessels is a relatively common incidental finding in the NHP with lesions reported in the aorta and small arteries and arterioles of the brain.24 Vascular mineralization with no clear age dependency, growth abnormalities, weight gain, or neurologic signs has been noted in non-human primates, where males were slightly more affected than females.24 Mineralization was found in the brains of 59% of cynomolgus monkeys examined and the lesions (periodic acid-Schiff or von Kossa positive) were common in the vascular walls of the globus pallidus. The basal ganglia in rhesus monkeys were the most frequent site. Though PAS positivity suggested the presence of mucopolysaccharide the mineral deposits consisted largely of calcium, phosphorus, iron, zinc, magnesium, and aluminum. Degenerative changes preceded mineral deposition without hypercalcemia, and such mineralization was considered dystrophic. Furthermore, in the absence of neurologic or clinical signs this mineralization was considered consistent with a physiologic event. Two types were described: Type A was described as globoid bodies (also refered to as copora amylacea; see Chapter 10) with prominent concentric lamellar structures in and around the arteriolar and venular wall (Fig. 19.11A), while the authors described type B consisted of fine granules in the media of small or medium-sized arteries. Ultrastructural examination confirmed the deposits in degenerated tunica media of small or medium-sized arteries or in the thickened walls of the arterioles.24 The globus pallidus is also the location were mineralization has been observed in elderly humans, also without clinical signs. Whereas both types are observed with equal frequency in humans, type A was the most common type observed in monkeys. Other sites reported as locations for vascular mineralization in primates have been the basal ganglia, caudate nucleus, putamen, and thalamus.1,9 In monkeys degenerated vessel walls at the site of mineralization showed thickened basement membranes with prominent growth of collagen fibers. In the initial stage of both types

467

of mineralization, perpendicularly oriented needle-shaped hydroxyapatite crystals within the degenerated basement membrane or cellular debris suggested collagen fibers involvement in producing mineral crystals. Thus, it was theorized that mineralization in this location could be due to degeneration or metabolic derangement in the vascular wall.24 “Cardiac mineralization” may include any form of mineral depostion, such as vascular mural mineralization or myocardial mineralization (Fig. 19.11B).18 Dystrophic mineralization must be differentiated from metastatic mineralization when encountered. Additionally, mineralization of functional or vestigial arteries in the urinary bladder may be noted. These are considered remnants of umbilical arteries and are commonly observed in the adventitia of the bladder (see Chapter 9). Early stages of the lesions include focal degeneration, fibrosis, foreign body macrophage accumulation, and pigmentation of arterial walls.9

FIGURE 19.11 (A) Lamellar mineralization of the meningeal vessels adjacent to the optic nerve in a macaque may be due to vascular injury or age-related degeneration. (B) An animal with myocardial disease has cardiomyocyte mineralization as a sequalae.

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Spontaneous Pathology of the Laboratory Non-human Primate

4.6 Myocardial degeneration, necrosis, and fibrosis

accompanied by low numbers of mononuclear cells are seen in cynomolgus macaques with an incidence of 13% in one study of Mauritius cynomolgus monkeys, and are usually of minimal to mild severity.2 These foci are also noted in macaques of other sources, are typically small, and often included in the diagnosis of “mononuclear cell infiltrate” (Fig. 19.20). When larger foci of cardiomyocyte necrosis are present, it may be difficult to differentiate from contraction band artifact, but the presence of associated inflammatory cells or cardiac macrophages (previously known as Anitschkow cells) may help in the diagnosis.

The term “degeneration” has been variably applied by authors to describe a variety of changes noted as spontaneous occurrences in the hearts of NHPs. In some cases, “degeneration” has been limited to findings that were exclusive of hypertrophic changes, and in other cases, hypertrophy with or without other cellular changes were considered part of the spectrum of “degeneration.” The well-described entity of “cardiomyopathy” in macaques has typically included both features.9,25 Differentiation of hypertrophic conditions from that of purely degenerative conditions may not be feasible in all cases. INHAND designates karyomegaly and karyocytomegaly noted in idiopathic cardiomyopathy as degenerative changes while increased cellular diameter with enlarged, irregular shaped nuclei is categorized as cardiomyocyte hypertrophy.26 Based on these criteria, idiopathic cardiomyopathy is included in the degenerative diseases of NHPs for this text. Degenerative lesions of the heart are of particular importance, as similar changes may occur following administration of cardiotoxic substances. The incidence and severity of degenerative changes in hearts of macaques are summarized in Table 19.2.

4.6.2 Large foci of myocardial degeneration (idiopathic cardiomyopathy) Macaques are well known to have spontaneous cardiomyopathy.18,25 Typically, these lesions are focal to regionally extensive, and affect the left ventricle or interventricular septum while some are noted at the apex as well. Early lesions are characterized by nuclear vacuolation of myofibers, karyocytomegaly with anisocytosis and nuclear basophilia that centers on small to medium-sized blood vessels (Fig. 19.12AeB). As lesions progress myofiber hypertrophy is observed along with mixed inflammatory cell infiltrates, hemorrhage, fibrosis, and mineralization (Fig. 19.13AeB). These lesions may progress to frank necrosis with cellular debris, and features such as cardiomyocyte hypereosinophilia and loss of cardiomyocyte cross-striation. All areas of the heart may be affected with

4.6.1 Small foci of myocardial degeneration Foci of myocardial degeneration consisting of low numbers of degenerate or necrotic cardiomyocytes, occasionally

TABLE 19.2 Incidence of degenerative findings in the hearts of macaques. Source Sex (number examined)

China

Mauritius

Cambodia

Vietnam

Male (967)

Female (933)

Male (276)

Female (272)

Male (165)

Female (160)

Male (49)

Female (46)

Cardiomyopathy

1 (0.1%)

3 (0.3%)

e

1 (0.4%)

e

e

4 (8.0%)

3 (6.5%)

Myocardial degeneration

5 (0.5%)

5 (0.5%)

3 (1.1%)

1 (0.4%)

1 (0.6%)

2 (1.3%)

e

e

Myocardial degeneration with necrosis

5 (0.5%)

e

2 (0.7%)

1 (0.4%)

1 (0.6%)

1 (0.6%)

e

e

Necrosis

2 (0.2%)

e

e

e

e

e

e

e

Myocardial karyomegaly/ karyocytomegaly

3 (0.3%)

1 (0.1%)

1 (0.3%)

1 (0.4%)

e

e

e

e

Myocardial anisokaryosis

1 (0.1%)

e

e

1 (0.4%)

e

e

e

e

Myocardial hypertrophy

e

e

1 (0.3%)

Myocardial vacuolation

1 (0.1%)

e

e

e

e

e

e

e

Myocardial fibrosis

e

e

e

1 (0.4%)

e

e

e

e

Finding, number of animals identified, and (% affected)

Data generated on June 7, 2022 from Charles River Historical Database. e indicated the finding was not recorded for the source and sex of the animals.

The cardiovascular system of the non-human primate Chapter | 19

FIGURE 19.12 (A) Idiopathic myocardial degeneration (cardiomyopathy) is characterized by nuclear karyocytomegaly with anisocytosis and nuclear basophilia that centers on small- to medium-sized blood vessels. (B) Fibrosis is a common feature of the myocardial degeneration, here noted as blue-stained tissue radiating through the myocardium from the margins of the artery in Fig. 19.12A (Masson’s trichrome staining).

the subepicardial region of the apex, the interventricular septum, the papillary muscles of the left ventricle, and the subendocardium of the left ventricle being the most commonly reported sites in the literature.2,9 Although most of the cases encountered in young animals are of minimal to mild nature, some reach moderate to marked grade and may have associated organ weight alterations or changes in EKG parameters.

4.6.3 Cardiac fibrosis Although myocardial interstitial fibrosis is a relatively common finding in older primates of many species, it is rarely reported in the relatively young NHPs used in toxicology studies.27 Fibrosis is a feature included in idiopathic

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FIGURE 19.13 (A) progression of idiopathic myocardial degeneration is characterized by increased nuclear and cytoplasmic vacuolation, individual cell necrosis, and myofiber hypertrophy. (B) Higher magnification of Fig. 19.13A: Mononuclear or mixed inflammatory cell infiltrates are usually present in more severe cases of idiopathic myocardial degeneration and cardiomyocytes become encircled by fibrosis. Additional features may include hemorrhage, necrosis, and mineralization.

cardiomyopathy as well.25 When observed as an independent entity, fibrosis usually occurs in the papillary muscle or the free wall of the left ventricle and is characterized microscopically by the replacement of myocytes by dense, vascularized extracellular collagenous matrix (Fig. 19.14AeC). Myocardial fibrosis may be assessed by special staining techniques (Masson’s Trichrome).

4.7 Mucinous change of the myocardium and arteries A feature observed in the major blood vessels and coronary arteries is accumulation of mucopolysaccharides in the tunica intima between the endothelium and the internal elastic lamina, frequently called mucinous change (Fig. 19.15AeB). It has been described in normocholesterolemic baboons; cynomolgus, rhesus, vervet and howler monkeys.8 Multiple terms have been used to describe this feature, including nonatherosclerotic intimal thickening,

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Spontaneous Pathology of the Laboratory Non-human Primate

FIGURE 19.15 (A) Accumulation of mucopolysaccharides in the tunica intima between the endothelium and the internal elastic lamina (arrow), frequently called mucinous change, is a relatively common incidental finding in macaques. (B) Occasionally, mucinous change may affect a large portion of the tunica intima (arrows).

FIGURE 19.14 (A) On occasion there are small foci of fibrosis in the hearts of non-human primates, often noted incidentally during microscopic examination. (B) Fibrosis is relatively common in aged non-human primates and may engulf large portions of the myocardium, here noted as blue-stained tissue (Masson’s trichrome staining). Myocardial infarction is often assumed in these regionally large fibrotic foci.

spindle cell cushion, intimal pad, musculoelastic plaque, localized fibrous plaque and mucoid fibromuscular plaque.8,9 Additionally, the relationship of these benign lesions and that of the more severe atherosclerosis is debated (See Arteriosclerosis). This contrasts with a term such as vasculopathy, which should be avoided as it implies the presence of a much more serious finding and to some infers a relation to treatment. Within the interstitium of the myocardium, there is occasionally buildup of mucin that appears as pale amorphous material on hematoxylin and eosin (H&E) stained sections, usually dispersed between cardiac fibers. Rarely, larger accumulations may be noted (Fig. 19.16). The mucin may be better assessed using Alcian Blue staining method.

FIGURE 19.16 Mucinous change in the heart of a macaque: The amorphous material (mucopolysaccharides) fills the region between myofibers and contains sparse numbers of fibroblasts.

The cardiovascular system of the non-human primate Chapter | 19

4.8 Valvular myxomatous degeneration In the two main studies investigating background findings in the cardiovascular system of NHPs involving a total of over 2500 cynomolgus macaques, rhesus macaques, and common marmosets, no valve lesions were observed.28 However, valvular myxomatous degeneration has been rarely diagnosed in animals from the editor’s facility (Fig. 19.17AeB). It should be noted that these animals were young (less than 5 years old) and the changes were associated with alterations in echocardiograms in

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some animals that had valvular regurgitation. The change involves thickening of the valve by accumulations of mucopolysaccharides that may become grossly nodular over time. Older animals may be more prone to valvular degeneration, and in these cases, there may be variable numbers of inflammatory cells, fibrosis and/or hemorrhage or hemosiderin deposits (Fig. 19.17C). The rarity of this finding in young animals assigned to toxicologic studies may be due to difficulty in obtaining standard histological views of the heart valves, thus inhibiting reliable comparison between treatment groups.

4.9 Valvular vascular ectasia and hematocysts Ectasia of the lymphatics or veins of the atrioventricular valves or subtending endocardium has been noted in macaques from the author’s facility. On occasion, these dilated vessels are visible grossly as fluctuant or firm nodules on the valve leaflets or at the base of the valve. Microscopically, they may appear quiescent but markedly dilated and filled with either blood or serous fluid, or may have associated valvular edema, microhemorrhage, hemosiderin deposits, or minor inflammatory cell infiltrates (Fig. 19.18AeB). These lesions are also referred to as “hematocytsts” which have been described in multiple animal species such as mice, rats, dogs, and humans, but not previously in macaques.29e31

FIGURE 19.17 (AeB) The myxomatous change in the heart valves involves thickening of the valve by accumulations of mucopolysaccharides that result in nodular distortion, primarily within the spongiosa. (C) With age, the myxomatous degeneration of the valve becomes more fibrous and may present with microhemorrhages or accumulations of hemosiderin (arrow).

FIGURE 19.18 (A) There are multiple, blood-filled, vascular spaces (hematocysts) at the base of the valve extending into the endocardium. (B) The base of the heart valve is expanded by increased matrix and fibrous tissue surrounding the blood-filled vascular spaces. Deposits of hemosiderin (arrow) are scattered around the hematocysts (asterisks) and there is minor myxomatous change present (m).

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Spontaneous Pathology of the Laboratory Non-human Primate

4.10 Epicardial adipose atrophy A common sequalae to rapid weight loss among macaques is atrophy of the epicardial adipose tissue (Fig. 19.19AeB) characterized by decreased adipocyte size and number, or complete loss of adipocytes, occasionally with saponification, cholesterol clefts, or low numbers of inflammatory cells present. This finding is not uncommonly noted in sick or highly stressed animals.

FIGURE 19.20 Myocardial or perivascular, focal to multifocal small aggregates of mononuclear cells within the myocardium of non-human primates is rather common. These foci occur spontaneously and may have very low numbers of associated hypereosinophilic, or necrotic cardiomyocytes (arrows).

FIGURE 19.19 (A) Atrophy of the epicardial adipose tissue is characterized by decreased adipocyte size and number, or complete loss of adipocytes, occasionally with saponification, cholesterol clefts, or low numbers of inflammatory cells present. This colony animal had anorexia and severe weight loss from colitis. (B) Heart epicardium and adipose tissue of an age matched control animal at equal magnification for comparison to that of Fig. 19.19A.

5. Inflammatory and vascular lesions of the cardiovascular system 5.1 Mononuclear or mixed cell infiltrates of the heart Focal to multifocal small aggregates of mononuclear cells within the myocardium of NHPs is rather common. These foci occur spontaneously and may have very low numbers of hypereosinophilic or necrotic cardiomyocytes associated

(Fig. 19.20). In an investigation of 2464 animals, which included 2050 cynomolgus monkeys (M. fascicularis), 284 common marmosets (C. jacchus), and 130 rhesus monkeys (M. mulatta), the most common cardiac finding was typical “inflammatory cell infiltration” also called “focal myocarditis.”18 While these low-level myocardial infiltrates are common, they are also a manifestation of some toxic agents, and they may be exacerbated or increased in incidence among animals administered immunomodulatory agents; therefore, careful evaluation including control animals may be necessary to determine “weight of evidence” for study-related findings. Less commonly observed is perivasculitis or vasculitis that is localized and confined to individual organs. It is most often reported in the kidneys with the lungs being the next most common site, followed by brain and spinal cord meninges, heart, urinary bladder and sciatic nerve in that order.18 Typically, it is characterized by infiltration of the vascular wall or perivascular space by lymphocytes (Fig. 19.21A). Fibrin deposition and necrosis are not components of this background finding. While perivasculitis or vasculitis is more common in marmosets and rhesus monkeys compared to cynomolgus monkeys, perivasculitis in the meninges is common in cynomolgus monkeys (Fig. 19.21B). Typically, even when multiple meningeal blood vessels are involved the severity grade is minimal to mild. No clinical signs or alterations of clinical pathology are associated with this common finding.18 Lymphocytes can also be observed around blood vessels in the myocardium (to a lesser extent endo- and epicardium) of marmosets, rhesus, and cynomolgus monkeys. Again, the severity grade applied to such infiltrates is usually minimal to mild, and there is no associated degeneration or

The cardiovascular system of the non-human primate Chapter | 19

FIGURE 19.21 (A) perivasculitis or vasculitis that is localized has been reported in multiple organs, including the brain, and is characterized by infiltration of the vascular wall or perivascular space by lymphocytes. (B) Perivascular mononuclear cell infiltrates are common in meninges of cynomolgus monkeys and typically are minimal to mild. No clinical signs, tissue injury or alterations of clinical pathology are associated with this common finding.

necrosis. Localized vasculitis and perivasculitis should be distinguished from polyarteritis nodosa (PAN) (See Vasculitis-Polyarteritis Nodosa).

5.2 Vasculitis (Polyarteritis Nodosa-PAN) Idiopathic necrotizing vasculitis (polyarteritis nodosa or PAN) affects small-to medium sized arteries of macaques. Though well-recognized in humans, cases are rare in monkeys. It is a segmentally distributed necrotizing arteritis of varying severity reported most commonly in

473

FIGURE 19.22 (A) idiopathic necrotizing vasculitis of Polyarteritis Nodosa (PAN) affects small- to medium-sized arteries of macaques, resulting in variably severe loss of tissue architecture. (B) The inflammatory cell infiltrate is variably mononuclear cells admixed with granulocytes and tends to be accompanied by fibrinoid necrosis of the tunica media and loss of the internal elastic lamina.

vessels of multiple organs, commonly in the kidney, small intestine, colon, heart, spleen, mesentery, urinary bladder, and pancreas; however, the vasculitis may appear in nearly any organ (Fig. 19.22AeB). The inflammatory cell infiltrate is mixed and tends to be accompanied by fibrinoid necrosis of the tunica media and loss of the internal elastic lamina. Macroscopically, there may be severe subcutaneous edema and excessive fluid accumulation in body cavities.32 Additionally, the adipose tissue of the pericardium near the right coronary artery may have a low severity of granulomatous inflammation which may include lymphocytes, plasma cells, eosinophils, epithelioid macrophages, and even multinucleated giant cells33;

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Spontaneous Pathology of the Laboratory Non-human Primate

however, this type of granulomatous inflammation also occurs spontaneously without vasculitis present (See Granulomatous pericarditis). Idiopathic necrotizing vasculitis has been observed in untreated monkeys and cases have only been reported in cynomolgus monkeys, where the pattern and character of the lesions is very similar to that described for humans. It is a syndrome for which the pathogenesis is poorly understood. The presence of immunohistochemically demonstrated T lymphocytes and histiocytes suggests a cell-mediated mechanism in the pathogenesis. Others have speculated that heterogeneous immunopathogenic mechanisms are responsible, involving immune complex deposition and activation of the complement cascade, neutrophil and monocyte chemotaxis, and the release of lysosomal enzymes, oxygen-free radicals, and proinflammatory mediators. The release of vasoactive amines from platelets likely increases vascular permeability, allowing intramural deposition of immune complexes. It is important to note that the inciting antigen in most cases of idiopathic necrotizing vasculitis is unknown.32,34 Other investigators have focused on the inflammatory changes of the arterial wall and applied “arteritis” to their descriptions in various forms (e.g., endarteritis, periarteritis, or panarteritis). They correctly observed that there are multiple terms appropriate for the same lesions, but most critically observed arteritis was confined to only one or a few organs/tissues and that systemic arteritis is rare in cynomolgus monkeys. Arteritis was most frequently observed in their study in the heart, intestines and epididymis, where fibrinoid necrosis might also accompany inflammation. They also observed what they diagnosed as “panarteritis nodosa” in the intrarenal arteries, particularly the arcuate arteries, and coronary arteries of the heart in cynomolgus monkeys. Panarteritis was described as intimal thickening and infiltration of lymphocytes from the tunica intima to the adventitia. Interestingly, relative to beagles the incidence and severity grade of arteritis was observed to be much lower in cynomolgus monkeys.20 Idiopathic necrotizing vasculitis is of concern, as the microscopic findings may mimic those noted in cases of immune complex disease (ICD) induced by anti-drug antibodies that may occur following the administration of biologics to NHPs. Weight of evidence is crutial when ICD is suspected for study-assigned animals, as PAN is quite rare in macaques.

5.3 Granulomatous epicarditis On occasion, there are infiltrates of mononuclear cells, epithelioid macrophages, and multinucleated cells within

FIGURE 19.23 (A) Small aggregates of mononuclear cells and macrophages,with low numbers of degenerate adipocytes and cholesterol clefts (arrow) may be noted as spontaneous in macaques. (B) Although the epicardial adipose tissue granulomas are usually small, on occasion large aggregates of the multinucleated giant cells have been noted. (C) The larger aggregates have frequent multinucleated giant cells with cytoplasmic cholesterol clefts, mixed cell infiltrates, and fat saponification consistent with lipid degradation. These foci are commonly noted in conjunction with cases of epicardial adipose atrophy.

The cardiovascular system of the non-human primate Chapter | 19

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the adipose tissue of the epicardium in NHPs. Very low numbers of granulocytes may be admixed with mononuclear cells and there may be cholesterol clefts and/or saponification of the fat, consistent with lipid degradation (Fig. 19.23A). These inflammatory foci are generally quite small with low numbers of cells; however, the foci may reach rather large proportions in the adipose tissue in rare cases (Fig. 19.23BeC). The etiology if this finding is unknown, but it may be exacerbated by proinflammatory agents and may be more commonly noted during rapid atrophy of the heart adipose tissue during acute stress or illness.

5.4 Hemorrhage Small accumulations of red blood cells are not uncommonly noted within tissue sections. These acute foci may be noted commonly in the lamina propria or submucosa of the intestine, surrounding the vasculature of the spinal cord, within the epicardial adipose tissue, or in various other tissues (Fig. 19.24AeB). The nature of these small foci is unclear; however, their peracute nature and lack of other tissue alterations suggest they may be an agonal change or tissue handling artifact resulting in extravasation of red blood cells. Larger foci of hemorrhage that expand the perivascular tissues and are consistent with true hemorrhage may be noted rarely in organs of otherwise normal animals (Fig. 19.24C) and are of unknown etiology. The latter form is noted in the hearts of macaques as subendocardial hemorrhage and is characterized by the presence of red blood cells beneath the endocardium of the papillary muscles of the left ventricle and interventricular septum below the aortic valve (Fig. 19.25).2 Evidence of previous hemorrhage is sometimes present within the myocardium of the papillary muscles of the left ventricle and the interventricular septum and is seen as accumulates of tan to golden brown pigment (hematoidin or hemosiderin). This pigment is most commonly seen within the cytoplasm of small clusters of histiocytes but is also seen free within the extracellular space. Occasional erythrophagocytosis is present and the pigment is Perl’s stain positive for iron.2 Other hemorrhages may be traced to trauma. For instance, bruising of the limbs and head skin is not uncommonly noted either due to normal daily activity; due to restraint; or due to blood collection and venopuncture. Hematomas may form in traumatic lesions. If severe and persistent, they may require medical or surgical intervention in order to restore normal function (Fig. 19.26AeB).

FIGURE 19.24 (A) Small accumulations of red blood cells are not uncommonly noted within tissue sections of non-human primates, such as connective tissues surrounding various organs or (B) within the spinal cord. The nature of these small foci is unclear; however, their peracute nature and lack of other tissue alterations suggest they may be an agonal change or tissue handling artifact resulting in extravasation of red blood cells. (C) A large accumulation of red cells that expands the perivascular tissues within the renal papilla is consistent with true hemorrhage. Foci such as these may be noted rarely in various organs of otherwise normal animals.

6. Hyperplastic lesions of the cardiovascular system 6.1 Cardiomyocyte hypertrophy

FIGURE 19.25 Subendocardial hemorrhage is characterized by the presence of red blood cells beneath the endocardium of the papillary muscles of the left ventricle and interventricular septum below the aortic valve. Image courtesy of Julie Schwartz.

FIGURE 19.26 (A) Rupture of a subcutaneous vessel resulted in chronic, persistent hematoma of the skin of the forhead in a macaque. (B) Persistent hematomas are characterized by a fibrous capsule (a) that encompasses pools of blood crossed with strands of collagen and fibroblasts, and contains hemosiderin deposits, inflammatory cells, and irregular endothelial cell hyperplasia (b). Note the remnant of the ruptured vessel wall (c).

The hypertrophic heart may be grossly enlarged with thickened ventricular walls. Microscopic features are usually diffuse in the ventricles, and with increased severity there may be increased interstitial fibrosis and myocardial remodeling. The common features are enlarged cardiomyocytes with hypertrophic nuclei (Fig. 19.27AeB). There are multiple etiologies leading to cardiomyocyte hypertrophy. Compensatory hypertrophy of cardiomyocytes in NHPs may occur spontaneously. Cardiac hypertrophy has been also associated with functional pheochromocytomas.35 Hypertrophic cardiomyopathy (HCM) is rare, but has been reported in an 11-year-old macaque.36 Left ventricular hypertrophy has been described in rhesus macaques as well, and presented as increased risk of sudden cardiac failure in 6e-year-old-male animals.37 A case of left ventricular cardiac hypertrophy was noted at the author’s facility in a cynomolgus monkey from the colony that was less than 5 years old. The animal had diffuse hypertrophy of left ventricle and interventricular septum, but there was marked hypertrophy of the papillary muscles of the left ventricle similar to that described in human cases of papillary muscle hypertrophic cardiomyopathy (Fig. 19.28AeB; Fig. 19.29).38,39 Recent human literature indicates that papillary muscle hypertrophy is a

FIGURE 19.27 (A) Cardiomyocyte hypertrophy is characterized by enlarged cardiomyocytes with enlarged, vesicular nuclei. This change may be diffuse as in hypertrophic cardiomyopathy, may be associated with idiopathic cardiomyopathy, or may be noted sporadically as a compensatory change of unknown origin. (B) Myocardium from an age matched normal control animal at equal magnification for comparison.

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Because toxins may also induce changes similar to the spontaneous cardiac hypertrophy, careful evaluation of animals on study, along with ancillary diagnostics and data, are needed to build a “weight of evidence” for or against a test article influence.

6.2 Mesothelial hyperplasia Mesothelial hyperplasia is occasionally noted in the epicardial surface of the heart in NHPs, due to irritation or infection. It’s been noted in animals with chronic pericardial effusion or pericardial adhesions and is characterized by enlarged mesothelial cells that appear cuboidal or columnar with increased cytoplasm. The cells may be single or multilayered and may form papillary structures overlying a fibrovascular stroma (Fig. 19.30AeB).

FIGURE 19.28 Papillary hypertrophic cardiomyopathy in a young cynomolgus monkey: (A) The male macaque had a mildly hypertrophic left ventricle with markedly hypertrophic papillary muscles (asterisks). The atrioventricular valve had nodular myxomatous degeneration (oval). A small focus of subendocardial hemorrhage was noted (arrowhead). Note the pale staining, severely hypertrophic cells of the subendocardium prominently covering the papillary muscles (arrow). (B) The left ventricle of a sex and age matched control animal at equal magnification for comparison.

FIGURE 19.29 Higher magnification of the heart in Fig. 19.28A: This animal had marked hypertrophy of the papillary muscle cells that was most severe in the subendocardial region. The cardiomyocytes are markedly enlarged with pale, vacuolated cytoplasm, karyomegaly, and perinuclear clearing.

subcondition of HCM that may have an associated genetic abnormality in conjunction with secondary left ventricular pressure overload leading to the papillary muscle changes; however, no such information specific to NHPs has been published to date.37

FIGURE 19.30 (A) Mesothelial hyperplasia is occasionally noted on the epicardial surface of the heart in NHPs, due to irritation, pericardial effusion or pericardial adhesions. (B) Mesothelial hyperplasia is characterized by enlarged mesothelial cells that appear cuboidal or columnar with increased cytoplasm. The cells may be single or multilayered and may form papillary structures overlying a fibrovascular stroma. Images courtesy of Julie Schwartz.

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6.3 Vascular endothelial hypertrophy and hyperplasia A variation in vascular endothelial cell sized may be noted in NHPs. The endothelial cells may have rounded, slightly cobblestone appearance of minimal hypertrophy (Fig. 19.31AeB). This finding may be more prevalent following the intravascular administration of test articles, but has not had adverse outcomes noted. Rarely, hyperplasia of the endothelial cells of arteries occurs in NHPs, usually in response to injury or insult. The most common location is administration site veins where venipuncture has damaged the vessel, leading to reparative hyperplasia (Fig. 19.32A). The endothelial cells are enlarged with enlarged nuclei and increased cytoplasm, and

FIGURE 19.31 (A) The endothelian cells in larger blood vessels of NHPs may have a rounded “cobblestone” appearance as compared to (B) the more common, flat vascular endothelium. This finding is most common following intravenous administration of test articles, and has had no adverse outcomes noted by the author.

FIGURE 19.32 (A) The administration site vein has marked hyperplasia of the endothelium and vascular occlusion secondary to damage from venipuncture. (B) A case of hypertrophic endothelium in the myocardium of a macaque: Low numbers of arteries within the otherwise normal myocardium are hypertrophic or hypertrophied and have increased basophilia on H&E-stained section. (C) Higher magnification of the heart in Fig. 19.32A: The hypertrophic muscular tunic encloses a hypercellular tunica intima and hyperplastic endothelium.

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mitoses may be noted within the cell population. In one case, the heart and gallbladder had these types of vessels in low numbers within the otherwise normal tissues. They were characterized by hypertrophic or hypertrophied muscular tunic enclosing a hypercellular tunica intima and endothelium that was without inflammation and affected both veins and arteries (Fig. 19.32BeC). Although an unidentified insult may have resulted in the vascular changes for this animal, a congenital or arteriosclerotic etiology could not be discounted.

7. Neoplastic lesions of the cardiovascular system 7.1 Mesothelioma Mesothelioma of the pericardium has been reported in a rhesus macaque.40 This neoplasm is composed of squamous to low cuboidal epithelioid cells forming tubular and acinar structures and is an extremely rare neoplasm in primates.

7.2 Other cardiac neoplasms A thoracic schwannoma that was potentially associated with the heart has been reported in a rhesus macaque.41 An extraskeletal osteosarcoma has been reported at the heart base of a fat-tailed dwarf lemur.42 One case has been reported of fibrosarcoma arising in the interventricular septum of a middle-aged rhesus macaque.43

7.3 Hemangioma and hemangiosarcoma Hemangiomas have been reported in rhesus monkeys and other NPHs. These neoplasms are characterized by presence of multiple vascular lacunae filled by blood and lined by well-differentiated endothelial cells. These reports include a cavernous hemangioma in the ovary, an epithelioid hemangioendothelioma in the heart, and a hemangioendothelioma in the liver.44e46 Hemangiosarcomas have also been reported in NHPs. The morphology of these neoplasms is characterized by atypical endothelial cells which form vascular channels (capillary to cavernous) and solid cellular masses supported by variably developed fibrovascular stroma (Fig. 19.33). Local invasion and widespread metastasis are possible. These include a renal hemangiosarcoma, a hepatic hemangiosarcoma, and a subcutaneous hemangiosarcoma.47e49

7.4 Other vascular neoplasms Lymphangioma has been reported in a squirrel monkey.50 These are discrete lesions composed of thin-walled cysts

FIGURE 19.33 Hemangiosarcoma in a NHP: There is an unencapsulated neoplastic mass extendingt through the wall of a thrombosed ablood vessel (arrow) into the surrounding adipose tissue. The neoplastic cells have formed large, irregularly shaped vascular channels. There are multifocal solid regions of neoplastic cells supported by variably developed fibrovascular stroma. this timor was discovered as an approximately 2 cm diameter red focus adjacent to the mesenteric lymph node. No additional tumors were found during examination; therefore, the mass was considered the primary tumor.

lined by flat endothelial cells. An angioleiomyoma has been reported in the spleen of an owl monkey.51 This neoplasm is a well-circumscribed, unencapsulated lesion composed of haphazardly arranged smooth muscle bundles admixed with numerous small capillarylike structures containing blood.

8. Miscellaneous findings of the cardiovascular system 8.1 Findings associated with indwelling catheters Indwelling catheters for the daily intravenous administration of test-article are commonly used in preclinical safety assessments. Indwelling blood pressure catheters are routinely placed as part of implantable telemetry units. As such, knowledge of the local tissue changes associated with these catheters is important in distinguishing procedural related from test articleerelated findings. In a study of vascular injection sites in 84 cynomolgus monkeys’ over an 8-year period, findings were most commonly observed at the catheter tip. Within the vessel, endothelial hyperplasia and intimal thickening were observed in approximately half the animals while endothelial erosion and medial thickening were uncommon findings. Thrombi were observed relatively commonly (18% of males and 42% of females) and three types of

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thrombi were observed: organized mural thrombi; organized free thrombi around the catheter; and recently formed thrombi comprising poorly organized aggregates of fibrin. Inflammation of the vessel wall or in a perivascular location was relatively uncommon (16% in males and 27% in females) and when present was often associated with the presence of free or mural thrombi. In low numbers of animals (12.2% of males and 13.3% of females) there was necrosis of the intima and media with or without abscess formation.52 Foreign material such as shed sponge material, fragments of keratin, or hair shafts can also be observed in or near catheters in cynomolgus monkeys. These items may increase inflammatory reaction surrounding the cannulated vessels and/or in distant vessels such as in the lungs where foreign material lodges.53 Similar vascular responses and consequences are noted following placement of long-term indwelling blood pressure catchers (Fig 19.34A and B) The vascular endothelium and tunics will respond with proliferative and inflammatory reactions with nearly any foreign item, including medical devices, that remain stationary within the vessel and makes contact with the vessel wall, as will the endocardium of the heart (Fig. 19.34C).

8.2 Findings associated with continuous infusion Continuous infusion offers a means of long-term drug administration at titrated levels as opposed to bolus injections that would mimic the clinical setting more closely, primarily for biologic products. In addition to the ramification of long-term catheter placement mentioned above, there is risk of infection and thrombosis distal of the catheter, such as in lungs or other small vessels. In the lung, microthrombi may result in localized infarction (see Chapter 15).

8.3 Cardiac contraction band artifact A commonly encountered artifact in the hearts of NHPs is contraction band hypereosinophilia of cardiomyocytes. It occurs when the freshly examined heart has retained contractile energy. When these fibers are cut with metal utensils, the cardiomyocytes release the energy as an irregular, uncoordinated contraction of the heart that is often visible grossly. The activity distorts and damages the cytoplasmic and membranous components of the cardiomyocytes. These foci occur most commonly at the margins of the myocardium where the tissue has been cut in order to perform gross examination of the interior features. The finding consists of individual or sections of irregular, hypereosinophilic cardiomyocytes that may be shriveled or have cytoplasmic vacuolation (Fig. 19.35A and B) The artifact must be differentiated from acute myocardial necrosis.

FIGURE 19.34 (A) An indwelling catheter for blood pressure monitoring that remained in situ for 6 months in a cynomolgus monkey: At the insertion into the vessel, the catheters are usually secured with suture, which may be noted in the tissue sections as granulomas surrounding the suture material (visible as multipls birefringent fragments at the margins of the tissue section). There is increased fibrous tissue (f) surrounding the catheterized vessel. (B) Higher magnification of the vessel in Fig. 19.34A: There is a laminar thrombus (tb) surrounding the catheter (c) and there is neovascularization (n) and partial recanalization (rc) of the thrombus filling the vascular lumen. The vascular tunics (vt) are visible, with a discontinuous elastic lamina (el). There is also neovascularization of the tunica adventitia and increased fibrous tissue surrounding the vessel. (C) There is proliferation of the atrial endocardium in a cynomolgus monkey following misalignment of an intravascular heart rate monitor from the vena cava to the atrium. This animal was asymptomatic at 6 months following implantation.

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8.4 Perfusion fixationdinduced findings Vascular perfusion fixation is a preferred method for postmortem examination of some tissues, particularly the brain. However, this method may have cardiovascular consequences that appear in tissues when examined microscopically. The process involves an incision into the heart chamber with placement of perfusion tubing that is secured by surgical clamp. Most commonly, there is characteristic, linear contraction band artifact of the heart where the cardiac clamps are placed (Fig. 19.35B). Minor trauma to the heart may occur during the fixation process, and small fragments of cardiac muscle may get washed into the perfusion fluid and lodge in vasculature of nearly any organ (Fig. 19.36A and B). Additionally, there may be perivascular clearing in many organs, due to pressure gradient and postmortem increased permeability from the vessel to the surrounding parenchyma or connective tissue.

FIGURE 19.36 (A) A consequence of perfusion fixation is fragmentation of cardiac muscle that may be washed downstream with the perfusion fluid, forming an artificial embolus nearly anywhere in the body. Here a fragment of myocardium is lodged in the meningeal blood vessel. (B) Microembolus of cardiac muscle is an artifact consisting of well-preserved myocardium within an artery following perfusion fixation of tissues.

8.5 Fatty infiltration of the myocardium

FIGURE 19.35 (A) Contraction band artifact within the hearts of nonhuman primates is exceptionally common. It occurs when the freshly examined heart has retained contractile energy. When these fibers are cut with metal utensils, the cardiomyocytes release the energy as an irregular, uncoordinated contraction of the heart muscle that distorts and damages the cytoplasmic and membranous components, resulting in a typical hypereosinophilic region of cells (arrows). These cells are very difficult to differentiate from acute myonecrosis; however, they are without associated tissue changes such as inflammatory cell infiltrates or cardiac macrophages and are most noted at the margins of the tissue where the heart has been opened in order to grossly evaluate the internal structures. (B) During perfusion fixation, it is common to insert the perfusion pump hose into the ventricle of the heart, followed by a tight clamp to produce a seal around the tube. The clamps leave roughly linear contraction band artifact (arrows) in the heart.

Accumulations of adipose tissue within the myocardium are occasionally noted in NHPs (Fig. 19.37). Whether this represents a congenital defect, age-related change, or a form of degeneration is currently not known. No physiological alterations or study-related affects have been reported with fatty infiltration.

8.6 Intranuclear and intracytoplasmic inclusions of noninfectious nature Eosinophilic intranuclear bodies are seen occasionally in low numbers of myofibers and, as in hepatocytes, they are considered to be due to the invagination of the cytoplasm into the nuclei. Similar inclusions have been reported in human hearts undergoing hypertrophy and ultrastructurally these inclusions in humans can be divided into globular and tubular subtypes.54

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Spontaneous Pathology of the Laboratory Non-human Primate

FIGURE 19.37 Accumulations of adipose tissue within the myocardium are occasionally noted in the hearts of non-human primates. Whether this represents a congenital defect, age-related change, or a form of degeneration is currently not known.

8.7 Antidrug antibodydassociated immune complex disease One of the most commonly encountered phenomena associated with the administration of biologics to NHPs is the development of antidrug antibodies (ADA). These ADA may form complexes with the drug, and deposit in vascular walls to produce immune complex disease (IC). The severity of IC encountered on studies is variable. At the lowest severity there may be increased incidence of mononuclear or mixed cell infiltrates in dosed animals as compared to controls that are primarily vascular to perivascular in distribution and occur in many organs. At greater severity there may be frank vasculitis with mural inflammation, loss of architecture, vascular necrosis, thrombosis, tissue edema, or hemorrhage (Fig. 19.38A and B).

9. Toxicologic lesions of the cardiovascular system Xenobiotic-induced injury to cardiomyocytes, arterial or venous components varies depending on the mechanism of injury and specific component of the cell that is targeted. Lethal cell injury results in apoptosis or necrosis and ultimately cell death whilst sublethal injury results in degenerative changes, often followed by regenerative or proliferative changes during a dose-free recovery period. Injury may be a result of direct cytotoxity or due to impairment of cardiac perfusion. Toxicity may result in the exacerbation of spontaneous findings or the development of novel microscopic changes.

FIGURE 19.38 Immune complex disease (IC) in macaques administered biologic agents: (A) Severe vasculitis with mural disruption of a coronary artery due to IC. (B) Due to immune complex deposition in the vascular walls, there is vascular necrosis, thrombosis, tissue edema, and necrosis of adjacent myocytes.

9.1 Xenobiotic-induced congenital defects Xenobiotic-induced cardiovascular congenital abnormalities have been observed in NHPs, as summarized in Table 19.1, and were generally accompanied by other induced developmental changes. In baboons and rhesus and cynomolgus macaques, cardiac malformations can be caused by treatment with Bendectin, an antiemetic drug containing dicyclomine, doxylamine, and pyridoxine which was approved by FDA in late 1950s for pregnant women. Administration of high doses of Bendectin to monkeys (10- to 40-fold greater than the human dose) was associated with an increased incidence of ventricular septal defects detected prenatal. This was considered to reflect a delay in closure of ventricular septum because there were no septal defects in fetuses examined at gestational term.55

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An increased incidence of ventricular septal defects was observed after administration of 13-cis retinoid acid to pregnant cynomolgus monkeys.56 Other drugs have been associated with cardiac defects in NHPs, such as valproic acid and combinations of retinoic acid, diphenylhydantoin and phenobarbital (rhesus monkeys), and thalidomide (bonnet and cynomolgus macaques).13 Fetal development may be impaired following cocaine administration to pregnant female rhesus monkeys. Lower fetal survival rate was observed compared with controls, and the fetuses that survived did not show any modification in measured parameters such as fetal heart rate, fetal biparietal diameter; and mean gestational length was in the normal range.57

9.2 Atherosclerosis models Some investigations have reported that atherosclerosis does not occur naturally in rhesus macaques while others have reported it does have a natural occurrence in captive and free-ranging monkeys.1,58 The latter case states the findings are mild and limited to the formation of fatty streaks. Nevertheless, in naturally occurring cases increased severity may be observed in diabetic monkeys or those given atherogenic diets. Experimentally induced (i.e., dietary manipulation-high fat, high cholesterol diet) atherosclerosis in monkeys is the model of choice for studying atherosclerosis in humans. Monkeys are not only like humans with respect to susceptibility because of hyperlipoproteinemia, but the progression pattern of the disease in monkeys is like that observed in humans, where the abdominal aorta is affected first, followed by the thoracic aorta, ultimately affecting the proximal main branches of the epicardial coronary arteries, the common carotid arteries, and the cerebral arteries in that order. Even the atherosclerotic plaques observed in monkeys progress in manner like that observed in humans, i.e., hemorrhage, mineralization, stenosis, and mural thrombi.59 Several species of monkeys have been used as atherosclerosis models; however, some are more ideal than others. Cynomolgus monkeys are considered in general the model of choice.1 Overall Old World monkeys are preferred, especially when studying the influence of hormones or hormone replacement therapy on the development of atherosclerosis. Old World monkeys are unique in that their ovarian hormone profiles resemble that of humans. Additionally, they resemble women in regard to the presence of a menstrual cycle and the occurrence of menopause.59 Regression of the disease can be studied in Old World species when there is estrogen replacement immediately after ovariectomy. However, it is notable that estrogen treatment after years of estrogen deprivation has no effect on the size of the atherosclerotic plaque. In addition the influence of hormones, atherosclerosis in Old World

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monkeys has been shown to be enhanced in certain genetic lines, increasing age, dietary saturated fat (vs. unsaturated fat), social stress (subordination to dominant animals or single housing).21 One metaanalysis compared 419 animals (200 females and 219 males) derived from 11 separate investigations for the incidence of atherosclerosis.60 The results confirmed that among males, dominant individuals developed more extensive atherosclerosis than subordinates when housed in recurrently reorganized (unstable) social groups in which an estrogen-implanted female were also present. Dominant males in stable social groups tended to have less atherosclerosis than similarly housed subordinates, but this effect was not significant. On the contrary, they found that dominant females developed reliably less atherosclerosis than subordinates. New World monkeys (e.g., squirrel monkeys and cotton-top tamarins) develop vascular lesions consistent with atherosclerosis when fed high fat diets; however, they also develop kidney lesions.21,59 Specifically, they have a high frequency of chronic glomerulonephritis. Additionally, New World monkeys lack the main branch of the epicardial distributed coronary arteries. Both of course limit their usefulness as an animal model of this disease.

9.3 Anthracycline-induced cardiotoxicity Vacuolar degeneration of the myocardium is characterized by the presence of clear, nonstaining intracytoplasmic spaces of variable size (0.1e5 mm diameter) on light microscopy and which correlate with dilated sarcoplasmic reticulum ultrastructural. Classically myocardial vacuolar degeneration is associated with anthracycline toxicity and best detected microscopically in thin plastic embedded sections. In some cells the degenerative changes progress to necrosis with loss of individual fibers. The mechanisms behind anthracycline cardiotoxicity have not yet been fully elucidated but are thought to involve several pathways including oxidative stress, ion dysregulation, and alterations in cardiac-specific gene expression patterns.61 More recently vacuolar degeneration of cardiomyocytes has been reported in Rhesus macaques treated with BILN 2061, an inhibitor of hepatitis C virus NS3/NS4A serine protease. In these animals cytoplasmic vacuolation correlated with mitochondrial swelling ultrastructural.62 Myofibrillar degeneration, also known as myocytolysis, is a common expression of sublethal injury of cardiomyocytes and is frequently seen with alongside vacuolar changes in anthracycline toxicity. Microscopically myofibrillar degeneration is characterized by cytoplasmic pallor and lack of cross-striations. These findings correlate with lysis of thick myofilaments, clumps of Z-band material, and accumulation of glycogen granules ultrastructurally.

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9.4 Propofol-induced pulmonary edema Propofol may be selected as the drug of choice for many procedures in NHPs, either alone or in combination with other drugs. At increased doses, propofol may induce cardiopulmonary changes resulting in pulmonary or renal consequences noted microscopically. In this four-animal study from the author’s facility, all animals were administered midazolam and dexmedetomidine at equal doses, then propofol at ascending doses of 0.78, 2.0, 2.25, and 3.8 mg/ kg. At 3.8 mg/kg propofol, the animal developed dyspnea during postanesthesia care while all other animals had no clinical signs. Microscopically all animals had minimal to mild vascular congestion of the renal capillaries that was most severe in the 3.8 mg/kg dosed animal. At 3.8 mg/kg, there was diffuse congestion with alveolar edema (Fig. 19.39) in the dyspneic animal. Additionally, this animal had centrilobular to midzonal hepatocellular degeneration consistent with a hypoxic state. This may represent a similar process that has been noted in humans with acute pulmonary edema and other propofol-associated pathologies.63,64

veins, are generally spared. These lesions have been observed in a variety of tissues, primarily the kidney and heart in non-human primates. Early and acute lesions are generally described as mononuclear, perivascular infiltrates with only chronic lesions involving the tunica media of medium-sized vessels such as the coronary artery. The pathogenesis of these lesions is not well understood but complement activation of the alternate pathway is thought to be a key contributor.66

10. Conclusion Knowledge of non-human primate-specific normal anatomic variations and awareness of the spectrum of spontaneous and background lesions involving the heart and blood vessels is critical when evaluating preclinical or toxicologic studies. This chapter provides a review of the common spontaneous and xenobiotic-induced lesions of the cardiovascular system of captive, purpose-bred, non-human primates to aid toxicologic pathologists in the evaluation of the cardiovascular system.

References

FIGURE 19.39 Propofol-induced alveolar edema in NHPs: A cynomolgus monkey administered high dose of propofol became dyspneic shortly after recovery from the anesthesia induced by the drug. Microscopically, the lungs had multifocal, regionally extensive congestion and diffuse alveolar edema.

9.5 Antisense oligonucleotide-associated vasculitis Drug-induced vascular injury has been occasionally observed in non-human primates given RNA targeting therapeutics such as antisense oligonucleotide therapies (ASO) during chronic toxicity studies.65 The sites of predilection for ASO-associated vasculitis are small- and medium-sized arteries and arterioles. Some ASOs are visible as pale basophilic granules, usually within macrophages, cuffing blood vessels. Occasionally, small veins can also be affected, whereas large vessels, such as elastic arteries (e.g., the aorta and the pulmonary artery) and large

1. Lowenstine LJ. A primer of primate pathology: lesions and nonlesions. Toxicol Pathol 2003;31(Suppl. l):92e102. https://doi.org/ 10.1080/01926230390177668. 2. Vidal JD, Drobatz LS, Holliday DF, Geiger LE, Thomas HC. Spontaneous findings in the heart of Mauritian-origin cynomolgus macaques (Macaca fascicularis). Toxicol Pathol 2010;38(2):297e302. https://doi.org/10.1177/0192623309358906. 3. Nyska A, Gruebbel MM. Letter to the editor. Toxicol Pathol 2010;38(3):511e2. https://doi.org/10.1177/0192623310368014. author reply 513. 4. Mathew P, Bordoni B. Embryology, heart. In: StatPearls. StatPearls Publishing. Copyright © 2022, StatPearls Publishing LLC.; 2022. 5. Alings AMW, Abbas RF, de Jonge B, Bouman LN. Structure and function of the simian sinoatrial node (Macaca fascicularis). J Mol Cell Cardiol 1990;22(12):1453e66. https://doi.org/10.1016/00222828(90)90988-E. 6. Mandarim-de-Lacerda CA, Penteado CV. Topographical and morphometrical study of the atrioventricular junctional area of the cardiac conduction system in the Macaca fascicularis Raffles, 1821. Anat Anzeiger 1988;167(1):57e61 [From NLM]. 7. Kawashima T, Thorington Jr RW, Whatton JF. Comparative anatomy and evolution of the cardiac innervation in New World monkeys (Platyrrhini, e. Geoffroy, 1812). Anat Rec 2009;292(5):670e91. https://doi.org/10.1002/ar.20894 [From NLM]. 8. Stary HC, Strong JP. Coronary artery fine structure in rhesus monkeys: nonatherosclerotic intimal thickening. Primates Med 1976;9:321e58 [From NLM]. 9. Chamanza R, Marxfeld HA, Blanco AI, Naylor SW, Bradley AE. Incidences and range of spontaneous findings in control cynomolgus monkeys (Macaca fascicularis) used in toxicity studies. Toxicol Pathol 2010;38(4):642e57. https://doi.org/10.1177/ 0192623310368981.

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10. Peterson PE, Short JJ, Tarara R, Valverde C, Rothgarn E, Hendrickx AG. Frequency of spontaneous congenital defects in rhesus and cynomolgus macaques. J Med Primatol 1997;26(5):267e75. https://doi.org/10.1111/j.16000684.1997.tb00222.x. 11. Jerome CP. Congenital malformations and twinning in a breeding colony of Old World monkeys. Lab Anim Sci 1987;37(5):624e30. 12. Lapin BA, Yakovleva LA. Comparative pathology in monkeys. Springfield: Charles C. Thomas; 1963. p. 301e27. East Lawrence Avenue, Ill., U.S.A. 13. Hendrickx AG, Binkerd PE. Congenital malformations in nonhuman primates. In: Jones TC, Mohr U, Hunt RD, editors. Nonhuman primates I. Springer Berlin Heidelberg; 1993. p. 170e80. 14. Koie H, Abe Y, Sato T, Yamaoka A, Taira M, Nigi H. Tetralogy of fallot in a Japanese macaque (Macaca fuscata). J Am Assoc Lab Anim Sci 2007;46(4):66e7. 15. Hein PR, van Groeninghen JC, Puts JJ. A case of acardiac anomaly in the cynomolgus monkey (Macaca fascicularis): a complication of monozygotic monochorial twinning. J Med Primatol 1985;14(3):133e42 [From NLM]. 16. Liu D, Gilbert M, Kempf D, Didier P. Double-outlet right ventricle and double septal defects in a Rhesus macaque (Macaca mulatta). J Vet Diagn Invest 2012;24:188e91. https://doi.org/10.1177/ 1040638711425951. Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc. 17. Latendresse JR, Ngampochjana M, Ward GS. Portdwine nevusd like arteriovenous malformation in a rhesus monkey (macaca mulatta). Vet Pathol 1987;24(2):197e9. https://doi.org/10.1177/ 030098588702400219. 18. Chamanza R, Parry NM, Rogerson P, Nicol JR, Bradley AE. Spontaneous lesions of the cardiovascular system in purpose-bred laboratory nonhuman primates. Toxicol Pathol 2006;34(4):357e63. https://doi.org/10.1080/01926230600809737. 19. Qureshi SR. Chronic interstitial myocarditis in primates. Vet Pathol 1979;16(4):486e7. https://doi.org/10.1177/030098587901600413. 20. Sato J, Doi T, Kanno T, Wako Y, Tsuchitani M, Narama I. Histopathology of incidental findings in cynomolgus monkeys (Macaca fascicularis) used in toxicity studies. J Toxicol Pathol 2012;25(1):63e101. https://doi.org/10.1293/tox.25.63. 21. Sasseville VG, Hotchkiss CE, Levesque PC, Mankowski JL. Hematopoietic, cardiovascular, lymphoid and mononuclear phagocyte systems of nonhuman primates. In: Abee CR, editor. Nonhuman primates in biomedical research. Volume 2, Diseases. 2nd ed. American College of Laboratory Animal Medicine series, Elsevier/ Academic; 2012. p. 357e84. 22. Yamakawa Y, Ide T, Mitori H, Oishi Y, Matsumoto M. Accumulation of brown pigment-laden macrophages associated with vascular lesions in the lungs of cynomolgus monkeys(Macaca fascicularis). J Toxicol Pathol 2016;29(3):181e4. https://doi.org/10.1293/ tox.2015-0079 [From NLM]. 23. Jasty V, Jamison JR, Hartnagel RE. Three types of cytoplasmic granules in cardiac muscle cells of cynomolgus monkeys (Macaca fascicularis). Vet Pathol 1984;21(5):505e8. https://doi.org/10.1177/ 030098588402100509. 24. Yanai T, Masegi T, Ueda K, Manabe J, Teranishi M, Takaoka M, Matsunuma N, Fukuda K, Goto N, Fujiwara K. Vascular mineralization in the monkey brain. Vet Pathol 1994;31(5):546e52. https:// doi.org/10.1177/030098589403100506 [From NLM].

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25. Zabka TS, Irwin M, Albassam MA. Spontaneous cardiomyopathy in cynomolgus monkeys (Macaca fascicularis). Toxicol Pathol 2009;37(6):814e8. https://doi.org/10.1177/0192623309345692. 26. Colman K, Andrews RN, Atkins H, Boulineau T, Bradley A, Braendli-Baiocco A, Capobianco R, Caudell D, Cline M, Doi T, et al. International harmonization of nomenclature and diagnostic criteria (INHAND): non-proliferative and proliferative lesions of the non-human primate (M. fascicularis). J Toxicol Pathol 2021;34(3 Suppl. l):1se182s. https://doi.org/10.1293/tox.34.1S [From NLM]. 27. Lowenstine LJ, McManamon R, Terio KA. Comparative pathology of aging great apes: bonobos, chimpanzees, gorillas, and orangutans. Vet Pathol 2016;53(2):250e76. https://doi.org/10.1177/ 0300985815612154 [From NLM]. 28. Donnelly KB. Cardiac valvular pathology: comparative pathology and animal models of acquired cardiac valvular diseases. Toxicol Pathol 2008;36(2):204e17. https://doi.org/10.1177/ 0192623307312707. 29. Marcato PS, Benazzi C, Bettini G, Masi M, Della Salda L, Sarli G, Vecchi G, Poli A. Blood and serous cysts in the atrioventricular valves of the bovine heart. Vet Pathol 1996;33(1):14e21. https:// doi.org/10.1177/030098589603300102 [From NLM]. 30. Blankenship B, Skaggs H. Findings in historical control Harlan RCCHanTM: WIST rats from 4-, 13-, 26-week studies. Toxicol Pathol 2012;41. https://doi.org/10.1177/0192623312460925. 31. Bodié K, Decker JH. Incidental histopathological findings in hearts of control beagle dogs in toxicity studies. Toxicol Pathol 2014;42(6):997e1003. https://doi.org/10.1177/0192623313508480 [From NLM]. 32. Porter BF, Frost P, Hubbard GB. Polyarteritis nodosa in a cynomolgus macaque (Macaca fascicularis). Vet Pathol 2003;40(5):570e3. https://doi.org/10.1354/vp.40-5-570. 33. Drevon-Gaillot E, Perron-Lepage MF, Clement C, Burnett R. A review of background findings in cynomolgus monkeys (Macaca fascicularis) from three different geographical origins. Exp Toxicol Pathol 2006;58(2e3):77e88. https://doi.org/10.1016/ j.etp.2006.07.003. 34. Albassam MA, Lillie LE, Smith GS. Asymptomatic polyarteritis in a cynomolgus monkey. Lab Anim Sci 1993;43(6):628e9. 35. Vogel P, Fritz D. Cardiomyopathy associated with angiomatous pheochromocytoma in a rhesus macaque (Macaca mulatta). Vet Pathol 2003;40(4):468e73. https://doi.org/10.1354/vp.40-4-468. 36. Konishi S, Kotera T, Koga M, Ueda M. Spontaneous hypertrophic cardiomyopathy in a cynomolgus macaque (Macaca fascicularis). J Toxicol Pathol 2018;31:49e54. 37. Rajiah P, Fulton NL, Bolen M. Magnetic resonance imaging of the papillary muscles of the left ventricle: normal anatomy, variants, and abnormalities. Insights into Imaging 2019;10(1):83. https://doi.org/ 10.1186/s13244-019-0761-3. 38. Sung KT, Yun CH, Hou CJ, Hung CL. Solitary accessory and papillary muscle hypertrophy manifested as dynamic mid-wall obstruction and symptomatic heart failure: diagnostic feasibility by multi-modality imaging. BMC Cardiovasc Disord 2014;14(34). https://doi.org/10.1186/1471-2261-14-34 [From NLM]. 39. Kobashi A, Suwa M, Ito T, Otake Y, Hirota Y, Kawamura K. Solitary papillary muscle hypertrophy as a possible form of hypertrophic cardiomyopathy. Jpn Circ J 1998;62(11):811e6. https://doi.org/ 10.1253/jcj.62.811 [From NLM].

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40. Chandra M, Mansfield KG. Spontaneous pericardial mesothelioma in a rhesus monkey. J Med Primatol 1999;28(3):142e4. https://doi.org/ 10.1111/j.1600-0684.1999.tb00261.x. 41. Alves DA, Bell TM, Benton C, Rushing EJ, Stevens EL. Giant thoracic schwannoma in a rhesus macaque (Macaca mulatta). J Am Assoc Lab Anim Sci 2010;49(6):868e72. 42. Remick AK, Van Wettere AJ, Williams CV. Neoplasia in prosimians: case series from a captive prosimian population and literature review. Vet Pathol 2009;46(4):746e72. https://doi.org/10.1354/vp.08-VP0154-R-FL. 43. Brown RJ, Kessler MJ, Kupper JL. Myocardial fibrosarcoma in rhesus monkey. Lab Anim Sci 1977;27(4):524e5. 44. Martin Jr CB, Misenhimer HR, Ramsey EM. Ovarian tumors in rhesus monkeys (Macaca mulatta): report of three cases. Lab Anim Care 1970;20(4 Pt 1):686e92. 45. Woodruff JM, Johnson DK. Hepatic hemangioendothelioma in a rhesus monkey. Pathol Vet 1968;5(4):327e32. https://doi.org/ 10.1177/030098586800500403. 46. Lombardini ED, Virmani R, Blanchard TW, Lafond JF, Menard S, Dore M. Epithelioid hemangioendothelioma in the right auricle of an adult, male Rhesus macaque (Macaca mulatta). J Med Primatol 2010;39(5):315e7. https://doi.org/10.1111/j.16000684.2010.00411.x. 47. Gozalo A, Chavera A, Dagle GE, Montoya E, Weller RE. Primary renal hemangiosarcoma in a moustached tamarin. J Med Primatol 1993;22(7e8):431e2. 48. Mejia AF, Gierbolini L, Jacob B, Westmoreland SV. Pediatric hepatic hemangiosarcoma in a rhesus macaque (Macaca mulatta). J Med Primatol 2009;38(2):121e4. https://doi.org/10.1111/j.16000684.2008.00309.x. 49. Myers Jr DD, Dysko RC, Chrisp CE, Decoster JL. Subcutaneous hemangiosarcomas in a rhesus macaque (Macaca mulatta). J Med Primatol 2001;30(2):127e30. https://doi.org/10.1034/j.16000684.2001.300209.x. 50. King CS, Streett JW, Brownstein DG. Cavernous lymphangioma in a squirrel monkey. Lab Anim Sci 1993;43(3):252e4. 51. Gozalo AS, Zerfas PM, Starost MF, Elkins WR, Clarke CL. Splenic angioleiomyoma in an owl monkey (Aotus nancymae). J Med Primatol 2010;39(6):385e8. https://doi.org/10.1111/j.16000684.2010.00425.x. 52. Lilbert J, Burnett R. Main vascular changes seen in the saline controls of continuous infusion studies in the cynomolgus monkey over an eight-year period. Toxicol Pathol 2003;31(3):273e80. https://doi.org/ 10.1080/01926230390204306. 53. Kast A. Pulmonary hair embolism in monkeys. Exp Toxicol Pathol 1994;46(3):183e8. https://doi.org/10.1016/S0940-2993(11)80079-5. 54. Engedal H, Jensen H, Saetersdal TS. Ultrastructure of abnormal membrane inclusions in nuclei of human myocardial cells. Br Heart J 1977;39(2):145. https://doi.org/10.1136/hrt.39.2.145. 55. Hendrickx AG, Cukierski M, Prahalada S, Janos G, Rowland J. Evaluation of bendectin embryotoxicity in nonhuman primates: I. Ventricular septal defects in prenatal macaques and baboon.

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Teratology 1985;32(2):179e89. https://doi.org/10.1002/ tera.1420320205. Hummler H, Korte R, Hendrickx AG. Induction of malformations in the cynomolgus monkey with 13-cis retinoic acid. Teratology 1990;42(3):263e72. https://doi.org/10.1002/tera.1420420310. Korte, R.; Hummler, H.; Hendrickx, A. G. Importance of early exposure to 13-cis retinoic acid to induce teratogenicity in the cynomolgus monkey. Teratology 1993, 47 (1), 37-45. DOI: 10.1002/ tera.1420470108. Howell LL, Schama KF, Ellis JE, Grimley PJ, Kitchens AJ, Byrd LD. Fetal development in rhesus monkeys exposed prenatally to cocaine. Neurotoxicol Teratol 2001;23(2):133e40. https://doi.org/10.1016/ s0892-0362(01)00121-0. Sasseville VG, Mansfield KG, Mankowski JL, Tremblay C, Terio KA, Matz-Rensing K, Gruber-Dujardin E, Delaney MA, Schmidt LD, Liu D, et al. Meeting report: spontaneous lesions and diseases in wild, captive-bred, and zoo-housed nonhuman primates and in nonhuman primate species used in drug safety studies. Vet Pathol 2012;49(6):1057e69. https://doi.org/10.1177/ 0300985812461655. Chapter 8 Shelton KA, Clarkson TB, Kaplan JR. nonhuman primate models of atherosclerosis. Elsevier Academic Press; 2012. https:// doi.org/10.1016/B978-0-12-381366-4.00008-0. Kaplan JR, Chen HY, Manuck SB. The relationship between social status and atherosclerosis in male and female monkeys as revealed by meta-analysis. Am J Primatol 2009;71(9):732e41. https://doi.org/ 10.1002/ajp.20707. Menna P, Salvatorelli E, Minotti G. Cardiotoxicity of antitumor drugs. Chem Res Toxicol 2008;21(5):978e89. https://doi.org/ 10.1021/tx800002r [From NLM]. Stoltz JH, Stern JO, Huang Q, Seidler RW, Pack FD, Knight BL. A twenty-eight-day mechanistic time course study in the rhesus monkey with hepatitis C virus protease inhibitor BILN 2061. Toxicol Pathol 2011;39(3):496e501. https://doi.org/10.1177/ 0192623311398276. Waheed MA, Oud L. Acute pulmonary edema associated with propofol: an unusual complication. West J Emerg Med 2014;15(7):845e8. https://doi.org/10.5811/westjem.2014.7.22942 [From NLM]. Kam PC, Cardone D. Propofol infusion syndrome. Anaesthesia 2007;62(7):690e701. https://doi.org/10.1111/j.13652044.2007.05055.x [From NLM]. Engelhardt JA, Fant P, Guionaud S, Henry SP, Leach MW, Louden C, Scicchitano MS, Weaver JL, Zabka TS, Frazier KS, et al. Scientific and regulatory policy committee points-to-consider paper*: drug-induced vascular injury associated with nonsmall molecule therapeutics in preclinical development: part 2. antisense oligonucleotides. Toxicol Pathol 2015;43(7):935e44. https://doi.org/ 10.1177/0192623315570341. Frazier KS. Antisense oligonucleotide therapies: the promise and the challenges from a toxicologic pathologist’s perspective. Toxicol Pathol 2015;43(1):78e89. https://doi.org/10.1177/ 0192623314551840.

Chapter 20

The endocrine system of the non-human primate Jennifer A. Chilton1, Ingrid Sjo¨gren2, Anne-Marie Mølck2 and Inger Thorup2 1

Charles River Laboratories, Reno, NV, United States; 2Novo Nordisk A/S, Måløv, Denmark

1. Introduction The tasks carried out by the endocrine system are farreaching and intimately involved in the homeostasis of immunity and organ function. Typically, the endocrine organs include the pituitary gland, thyroid gland, adrenal gland, endocrine pancreas (Islets of Langerhans), hypothalamus, pineal gland, and parathyroid gland; however, many additional organs have endocrine function including the skin, kidney, reproductive organs, heart, intestine, stomach, and bone. These latter organs are beyond the scope of this chapter and lesions seen in these locations in the non-human primate (NHP) may be found in other sections of this book. Evaluation of the endocrine organs within the context of preclinical or nonclinical studies primarily relies on the gross and microscopic morphology of the gland and assessment of the organ’s weight. Ancillary means of evaluation, such as serum, plasma, fecal, or urine hormone assays or immunohistochemistry (IHC) for hormonal peptides, are available, but are not commonly used in standard preclinical evaluations of NHP endocrine organs1; however, this may change in coming years, as noninvasive methods of monitoring endocrine organs via urine and fecal hormone assays, and blood-derived assays, are becoming more commonly used and validated.2 Nonetheless, lesions in the endocrine organs can affect parameters that are routinely evaluated in preclinical studies, including clinical pathology and flow cytometry, and lesions in one organ may impact another; therefore, knowledge of spontaneous findings is a valuable tool in the assessment of the endocrine organs.

2. Anatomy, histology, and embryology of the endocrine system The embryology of the endocrine organs involves all three layers of the primordial embryo: mesoderm, ectoderm, and endoderm. Migration of cells from these tissues eventually forms the subcompartments of many of the adult endocrine organs noted anatomically and histologically. Much of the embryology of the NHP endocrine system parallels that of other mammals, with alterations primarily in the timing of events and much is extrapolated from humans to NHPs and vice versa.

2.1 Adrenal gland The adrenal gland of the embryo is a complex, functional organ in primates. It consists primarily of the fetal cortex, sometimes referred to as the provisional cortex, which originates from neural crest and mesenchymal cells early in gestation and serves to provide essential hormones that support the developing fetal organs, including lung, brain, and gonads, and also regulates the fetal-placental unit.3,4 The key hormone, dehydroepiandrosterone (DHEA), and its sulfonated product, DHEAS, are produced by the fetal cortex and work synergistically at the developing organs. The cells of the adrenal medulla migrate from the neural crest and are recognized late in gestation or in the early postnatal period. The fetal adrenal gland is larger in relationship to the fetal kidney as compared to that of the adult organs (Fig. 20.1A and B).

Spontaneous Pathology of the Laboratory Non-human Primate. https://doi.org/10.1016/B978-0-12-813088-9.00001-X Copyright © 2023 Elsevier Inc. All rights reserved.

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FIGURE 20.1 (A) Left adrenal gland and kidney from an adult macaque and (B) late-term fetal macaque: The fetal adrenal gland (FG) is relatively large compared to the fetal kidney, a relationship that reverses in the adult macaque’s adrenal gland (AG) versus its kidney.

The fetal cortex consists of a distinct population of cells that are large and polygonal with central nuclei. Because there is significant hematopoietic activity in the fetal adrenal gland, it is not uncommon to see islands of hematopoietic cells present (Fig. 20.2). The fetal cortex will eventually regress, mostly within the first 2 weeks postpartum in macaques, and be replaced by cells of the adult cortex that migrated from the mesothelium of the embryo, and the adrenal gland after birth will include the familiar zona glomerulosa and zona fasciculata. Formation of the zona reticularis varies between species and is not present in adult male marmosets (Fig. 20.3A and B).5 Otherwise, the process of transition from the fetal to the adult adrenal confirmation is similar between macaques, marmosets, and humans, with the timing of transitional events being the main difference.6 In general, the cells of the cortical zones produce mineralocorticoids, glucocorticoids, and androgens while the medullary cells primarily produce catecholamines. However, the adrenal gland function may be more complicated;

FIGURE 20.2 It is not uncommon to see islands of hematopoietic cells (arrow) present in the NHP fetal adrenal cortex, due to the hematopoietic activity of the gland prior to birth. Note the distinct population of cells that are large and polygonal with central nuclei that make up the fetal adrenal cortex.

some researchers suggest that the adrenal gland in the NHP has additional functions subtly associated with homeostasis that are autologous and under the influence of a seasonal circadian physiology rather than direct pituitaryhypothalamic regulation.7 The most common configuration of the adrenal cortex has an approximately 1-2-1 relationship of the width of the zona glomerulosa-zona fasciculata-zona reticularis, respectively. Although there may be variation of size of the zones of the cortex, usually due to specific endocrine demands, on occasion, the zona reticularis may be excessively wide. This finding in the adrenal is characterized by bland cell population and is without hypertrophic or hyperplastic features; however, the width of the zona reticularis is quite noticeable (Fig. 20.4).

2.2 Thyroid and parathyroid glands The thyroid gland develops from an epithelial proliferation in the floor of the primitive pharynx, referred to as foramen cecum. The thyroid anlage descends, connected by the thyroglossal duct, and forms a bilobed diverticulum in front of the primitive gut. In macaques, follicle formation and colloid production in the fetus is first recognized by day 50 of pregnancy and small colloid-filled follicles and connecting tissue are present from around day 75 of pregnancy. From day 75 to 150, the content of iodine increases significantly with enlargement of the follicles and colloid accumulation. Despite the functionality of the fetal thyroid gland, T4 and T3 are transported from the dam to the fetus throughout pregnancy.8 The ultimobranchial body (or duct) arises from the fifth pharyngeal pouch, which is the last one to develop and is usually considered part of the fourth pharyngeal pouch. The ultimobranchial body/duct fuses with the thyroid anlage, differentiates and proliferates to form C-cells within the thyroid gland. C-cells were originally considered to be of neural crest cells origin; however, recent data from lineage-tracing experiments of neural crest cells using transgenic mice and specific staining for

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FIGURE 20.3 (A) Male marmoset adrenal cortex versus (B) male macaque adrenal cortex: Note that the male marmoset adult adrenal gland lacks the zona reticularis that is present in adult macaque adrenal gland. M, medulla; ZF, zona fasciculata; ZG, zona glomerulosa; ZR, zona reticularis.

FIGURE 20.4 Rarely, there may a noticeably wide zona reticularis (ZR) as compared to the other zones of the adrenal cortex of the macaque that should not be interpreted as hypertrophy or hyperplasia, as there are no cellular alterations to support this diagnosis.

endodermal epithelium markers strongly suggest an endodermal origin in mammals.9 The adult NHP thyroid gland is situated in the front and lateral sides of the neck. It consists of two elongated lateral lobes connected by a narrow isthmus ventrally placed. However, the isthmus may be absentda variation between as well as within species.10 Cells of the thyroid gland follicular epithelium are central to the production of thyroid hormones via a complicated process from cellular excretion into the follicle, reabsorption, structural alteration, and secretion into the bloodstream. The thyroid gland of the NHP is composed of tightly packed follicles that are generally spheroid with the largest follicles situated at the periphery of the gland (Fig. 20.5A). The follicles are lined by a single layer of low to tall cuboidal cells lying on a thin

basal membrane and are separated by sparse connective tissue containing abundant capillaries, lymphatics, and nerves (Fig. 20.5B). The secretory product of the follicular epithelium, colloid, is a semi-fluid gellike substance and consists primarily of thyroglobulin, an iodinated glycoprotein. During life, colloid contacts the follicular epithelium. Resorption of the colloid by the epithelium results in clear vesicles at the colloid-epithelial interface in histologic sections that should be differentiated from artifactual vacuoles that occasionally are present within the colloid. The height of the cuboidal epithelium and the size of the follicles vary with the activity of the gland, i.e., high activity results in smaller follicles with less colloid and taller epithelium and vice versa for low activity (Fig. 20.6A and B).10 When demand for thyroid product is exceptionally high, there may be markedly reduced follicular lumens throughout the gland with diffusely cubodial to collumnar epithelium or hypertrophic epithelium (Fig. 20.7). The morphology of the thyroid gland of marmosets corresponds in principle to that of other monkey species; however, the follicles vary greatly in size, from an average mean of 61e83 mm in diameter to large, apparently less active follicles with flat but vital epithelium and diameters up to 700 mm (Fig. 20.8). The existence of these large follicles is a characteristic of the thyroid gland of adult marmosets.11,12 C-cells, also called parafollicular cells or clear cells, comprise the second population of cells in the thyroid gland. They are polygonal or elongated, pale, and larger than the follicular cells with larger and paler nuclei. These cells may be difficult to discern on H&E stained tissue sections. To ease identification, IHC for presence of calcitonin, the main secretion product of C-cells, may be

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FIGURE 20.5 (A) The NHP thyroid gland is composed of tightly packed follicles with the largest follicles (arrows) situated at the periphery of the gland. (B) The thyroid follicles are lined by a single layer of follicular cells lying on a thin basal membrane and are separated by sparse connective tissue containing abundant capillaries, lymphatics, and nerves. The secretory product, colloid, fills the follicular lumens.

performed (Fig. 20.9). In cynomolgus monkeys, C-cells either surround part of follicles or they are placed between follicles where they predominantly are seen in clusters of 2e10 cells, but rarely larger cluster can be seen. In rhesus monkeys, clusters of 2e4 cells are described.13,14 In marmosets a similar pattern is seen in adult animals, with C-cells located within the follicular basal membrane.15 Based on IHC detection of calcitonin in cynomolgus monkey thyroid glands, C-cells are primarily distributed distally to the internal parathyroid in the middle third of the lateral lobes in a dorsal-medial position, whereas no C-cells are present in the poles or isthmus of the gland (Diagram 20.1). The same distribution pattern is seen for marmosets and rhesus monkeys.13,15,16 Concurrent with thyroid gland development and migration, the external parathyroid gland arises from the third pharyngeal pouch and the internal parathyroid gland arises

FIGURE 20.6 The height of the follicular epithelium and the size of the follicles in the thyroid gland vary with the activity of the gland. When viewed on equal magnification, the low activity of the gland is reflected by (A) follicles that are large with abundant luminal colloid and low cuboidal to flattened follicular epithelium, while high glandular activity results in (B) smaller thyroid follicles with less colloid and taller epithelium.

from the fourth pharyngeal pouch, respectively, and align with the adjacent thymus. Both thymus and external parathyroid glands together lose connection with the third pharyngeal wall and move caudo-medially, and eventually the external parathyroid gland comes to rest between the thyroid poles. The internal parathyroid glands lose the connection with the pharyngeal wall and attach themselves to the migrating thyroid gland. Eventually, the external parathyroid glands take up residence at the tracheal surface of thyroid at a more cranial position than the thyroidattached internal parathyroid glands.17 For the adult external parathyroid gland, proximity to the thyroid gland may vary on occasion, with most embedded in the surface of the thyroid gland, and others completely separated within adjacent adipose and connective tissue subtending the capsule. The external parathyroid gland may have multiple islands of tissue present (Fig. 20.10A and B). The distribution of the parathyroid glands within the thyroid gland is primarily in the middle and cranial third of the gland (Diagram 20.1).

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FIGURE 20.9 C-cells within the interstitium of the NHP thyroid gland may be difficult to discern on hematoxylin and eosin (H&E) stained sections but may be elucidated by immunohistochemical staining (IHC) for calcitonin (arrow). Image courtesy of NovoNordisk.

FIGURE 20.7 When the systemic demand for thyroid gland hormone is high there may be diffuse changes in the thyroid gland with reduced follicular lumens and increased height (hypertrophy) of the follicular epithelium.

FIGURE 20.8 The thyroid gland of the marmoset has follicles that vary greatly in size, from an average mean of 61e83 mm in diameter to large, less active follicles with flat but vital epithelium and diameters up to 700 mm (asterisk). Image courtesy of Heather Simmons.

The parathyroid gland consists of two cell types: chief (principal) cells and oxyphil cells. The chief cells which constitute the majority of the gland cell population possess faintly stained granular cytoplasm and are usually arranged in a characteristic cord or sheet pattern, although there may be pattern variability among individuals. The oxyphilic cells are fewer in number, are larger than the chief cells, usually have an eosinophilic appearance, and occur singly or in small clusters between the chief cells. Oxyphilic cells can be quite difficult to discern on H&E stained sections of NHP parathyroid glands.18 Staining with Periodic Acid Schiff (PAS) helps distinguish the chief cells (with PAS positive cytoplasmic granules) from the oxyphils (PAS negative). The number of oxyphil cells tends to increase with age.18,19 Both the chief and the oxyphil cells may vary in H&E staining intensity, which probably reflects differences in functional state or is related to submicroscopic cellular components, such as mitochondria number.18,20 The gland has a connective tissue capsule of its own. The connective tissue stroma is minimal but contains many blood capillaries. The parathyroid glands are responsible for maintaining physiological levels of calcium and phosphorus in the body. The major product of the parathyroid gland is parathyroid hormone (PTH) produced by the chief cells. Together with other hormones (calcitonin, vitamin D, and fibroblast growth factor 23 (FGF23)), it plays an important role for regulating calcium and phosphorous homeostasis in the body.21 PTH is released in response to decreased ionized calcium in the circulation, exerting a direct effect

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DIAGRAM 20.1 C-cell distribution and percentage of C-cell content versus length of lobe in the thyroid gland of cynomolgus monkeys (n ¼ 9) with location of the parathyroid glands within the thyroid gland, based on serial transverse sections: The distribution of the parathyroid glands within the thyroid gland is primarily in the middle and cranial third of the gland while C-cells are primarily distributed distally to the internal parathyroid in the middle third of the lateral lobes of the thyroid gland in a dorsal-medial position. No C-cells are present in the poles or isthmus of the thyroid gland. White bars ¼ thyroid lobe, dotted bars ¼ Ccells, gray bars ¼ internal parathyroid gland, black bars ¼ external parathyroid gland. Diagram courtesy of NovoNordisk.

2.3 Pituitary gland and hypothalamus

FIGURE 20.10 (A) The parathyroid glands (asterisks) are commonly imbedded within the thyroid gland (internal parathyroid) or in close apposition to the thyroid follicular surface (external parathyroid) (B) occasionally the external parathyroid gland is located in the adjacent subcapsular adipose tissue. Note: There are multiple lobules of external parathyroid gland present (arrows).

on target cells in bone and kidney, and indirectly targeting the intestine regulating release and retention of calcium.22 The chief cells also synthesize Chromogranin A which is an autocrine inhibitor of PTH secretion.18

The pituitary gland originates from two areas of the embryo. An outpouching of the embryonic ectoderm, the stomodeum, migrates dorsally to form Rathke’s Pouch attached to the craniopharyngeal duct. Eventually the duct regresses, and Rathke’s Pouch forms into the early adenohypophysis, or anterior pituitary gland. While the ectoderm is migrating to form the adenohypophysis, the neuroectoderm of the diencephalon (primitive hypothalamus) simultaneously migrates ventrally to form the neurohypophysis, or posterior pituitary gland, and is attached by the infundibulum to the floor of the diencephalon. The division of the early fetal brain and origin of the hypothalamus itself is debated; however, once it gains nuclei it is recognized as a formed region of the brain.23,24 Rathke’s Cleft forms at the junction of Rathke’s Pouch and the neurohypophysis. Rathke’s Pouch subdivides into the pars distalis, pars intermedia, and the highly vascular pars tuberalis. The 5 cell types of the pars distalis begin to populate the gland, with somatotrophs, gonadotrophs, and corticotrophs appearing first, followed by thyrotrophs and lactotrophs. Over the course of development, axons from the hypothalamic nuclei extend down the infundibulum (Fig. 20.11) to the vasculature of the posterior pituitary gland and to the vasculature of the infundibulum.25 The vasculature of the infundibulum and posterior pituitary gland is fenestrated, characterized by gaps between the vascular endothelial cell foot processes spanned by a thin diaphragmatic membrane; therefore the combined structure is considered a circumventricular organ where communication and transfer of materials between blood and tissue parenchyma is highly intimate and specific. For instance, this type of vasculature plays an important role in fluid balance, energy metabolism, and immunomodulation of the tissues.26 The pituitary gland rests within a depression of the ventral calvarium called the sella turcica.

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FIGURE 20.11 The infundibulum (I) provides the neural pathway from the hypothalamus to the pituitary gland (pars distalis (PD), pars nervosa (PN), pars tuberalis (PT), and pars intermedia (PI)). The infundibulum contains fenestrated vasculature and is considered a circumventricular organ.

The adult pituitary gland is divided into subcompartments consisting of the neurohypophysis (including the posterior pituitary gland containing pars nervosa and infundibulum) and adenohypophysis (including the anterior pituitary gland containing the pars distalis and pars tuberalis). Historically, the function of the pars tuberalis was unclear; however, more recent investigations have elucidated endocrine activity for this portion of the pituitary gland. There is high expression of melatonin receptors present in the pars tuberalis, and these have been linked to seasonal reproductive activity in mammals.27 A third subcompartment, the pars intermedia (intermediate lobe or neurointermediate lobe) lies between the pars nervosa and the hypophyseal cleft and is also derived from Rathke’s pouch. While most references include the pars intermedia with the adenohypophysis, some include it with the posterior pituitary structures and the neurohypophysis. In standard hematoxylin and eosin (H&E) sections, the pars distalis, pars intermedia, and pars nervosa are commonly

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visualized (Fig. 20.12); however, there is great variability in orientation of the gland at time of processing, leading to highly variable amounts of each subcompartment available for microscopic evaluation between animals assigned to toxicological studies. The pars nervosa and infundibulum that attach to the ventral brain are generally considered a unified extension of the hypothalamus. Cell bodies within the hypothalamus extend nonmyelinated fibers down the infundibulum to the pars nervosa where they secrete antidiuretic hormone (ADH) and oxytocin into the recipient vasculature for systemic release. The endpoints of these axons form bulbous structures that serve as hormone storage sites and are often visible microscopically as Herring bodies within the pars nervosa. Additionally, the neural tissue is supported by glial cells visible as dark nuclei within the pars nervosa of H&E stained sections (Fig. 20.13). The pars distalis and pars intermedia are primarily regulated by the hypothalamus. The pars distalis receives inhibiting and releasing hormones via a capillary bed located in the ventral hypothalamus that drains into the hypothalamic-hypophyseal portal veins of the pars tuberalis, which, in turn, drains into the gland capillaries for cellular distribution. The pars intermedia cells are under tonic inhibition from hormone secretion by the neural axons of the hypothalamus through the neurohypophysis infundibulum.28 Histologically, the pars distalis has cell populations that are distinct on H&E stained slides. Cells that retain greater amounts of hematoxylin have blue cytoplasm and are called basophils, those retaining eosin in the cytoplasm are eosinophils, and lastly, those that resist both chromogens are chromophobes (Fig. 20.14). The relative abundance of any one of these stained cell populations is highly variable between individual animals based on the metabolic and endocrine status at the time of gland collection. Each of

FIGURE 20.12 The standard histologic section of the NHP pituitary gland includes the pars distalis (PD), pars intermedia (PI), and pars nervosa (PN) within the plane of section.

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blue cytoplasm on H&E-stained sections and form follicles filled with pale secretion.

2.4 Endocrine pancreas

FIGURE 20.13 The pars nervosa of the NHP: Cell bodies within the hypothalamus extend nonmyelinated axons (A) down the infundibulum to the pars nervosa where they form bulbous end structures often visible as Herring Bodies (HB). Additionally, the neural tissue is supported by pituicytes (P-glial cells) visible as dark nuclei within the pars nervosa and contains fenestrated capillaries (C).

Similar to other organs that originate from the endodermderived gut tube of the embryo, the pancreas arises from two buds of the tube, one between what will eventually develop into stomach and intestine and the other between the embryonic liver and biliary structures (pancreatic buds). Signals from surrounding tissues are essential for the orientation of the endocrine pancreas from the pancreatic epithelial cells in the fetus, directing the differentiation and morphogenesis into endocrine, exocrine, or ductal structures. Likewise, the lineage of the endocrine pancreas cells, eventually resulting in specific hormone producing cells, is directed by signals from surrounding tissues, both within the developing pancreas and externally. Eventually, these cells will express insulin (beta cells), glucagon (alpha cells), ghrelin (epsilon cells), pancreatic polypeptide (PP cells), and somatostatin (delta cells) and will be congregated in the Islets of Langerhans. Expression of these cellular products may be identified by IHC (Fig. 20.15AeC). The pancreatic islets of both human and NHPs are distinguished from rodents by the increased incidence of mixed endocrine cell populations within the structure.29 There can be a large variation in the size and distribution of normal Islets of Langerhans within the pancreas of macaques, some of which may be due to sectioning variation (Fig. 20.16).

2.5 Pineal gland (epiphysis cerebri) FIGURE 20.14 The pars distalis has cell populations that are visually distinct as basophils (B) that retain hematoxylin stain, eosinophils (E) with retained eosin staining, and chromophobes (C) that resist either stain. Each of these cell staining characteristics is associated with specific endocrine functions or secretions (see body of the text for more information). The relative abundance of any one of these stained cell populations is highly variable between individual animals based on the metabolic and endocrine status at the time of gland collection.

these stained cells represents one or more of the 5 secretory cell types in the anterior pituitary gland. Basophils represent gonadotrophs (secrete LH and FSH), corticotrophs (secrete ACTH and MSH), and thyrotrophs (secrete TSH). Eosinophils represent somatotrophs (secrete GH) and lactotrophs (secrete prolactin). Chromophobes have a less clear function. They may represent degranulated eosinophils or basophils, or may be a “flexible resting” cell population that has capacity to change into hormonesecreting cells.28 The cells of the pars intermedia are melanotrophs (secrete a-MSH), and are also known as “MSH-producing corticotropes.” They generally have light

The pineal gland originates as an invagination of the neural tube neuroepithelium in the region of the diencephalon. Eventually, the invagination moves ventrally, forming the lobed structure recognized as the pineal gland (Fig. 20.17). The pineal gland is designated as a photo-neuro-endocrine transducing organ within the brain, with functions associated with circadian rhythm and regulation of internalexternal integration of biochemical and physiological needs.30 Much of the information on the fetal development of the pineal gland has been extrapolated to mammals from lower vertebrates, such as zebra fish, and has as of yet not been confirmed in NHPs, but it is considered likely that the various species share common sequences, if not common stimuli, for the development of the gland. Additionally, some cell types present in the adult pineal gland are likely immigrants, such as the two types of glial cells-microglia that serve as phagocytes and astrocytic glial support cells, both of which may be visualized more clearly with the aid of IHC or special stains. Microglial cells express variable amounts of ionized calcium-binding adapter molecule 1 (Iba-1), also known as allograft inflammatory factor 1

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FIGURE 20.15 The identity of islet cell populations within the NHP pancreas may be elucidated by IHC for cellular endocrine products: (A) Double labeling for glucagon producing alpha cells (brown chromogen) and insulin producing beta cells (red chromogen) in islet cells of the macaque. (B) IHC for somatostatin producing delta cells (red chromagen). (C) IHC for pancreatic polypeptide producing PP cells (red chromogen). Images courtesy of NovoNordisk.

(AIF1), and astrocytes express glial fibrillary acidic protein (GFAP) (Fig. 20.18A and B). The microglia are unique to higher vertebrates suggesting a more recent phylogenic addition to the pineal gland.31 The pineal gland is well known as a producer of melatonin and the regulator of sleep/wake cycles. In the NHP, release of melatonin promotes sleep.32 The pineal gland has a complicated neural connection with the suprachiasmatic nuclei of the hypothalamus but has no neurons of its own. Axon fibers from the hypothalamus may be noticed in some sections of the pineal gland (Fig. 20.19). In addition, the pineal gland is included as a circumventricular organ by many authors, due to its fenestrated vasculature. FIGURE 20.16 There may be a large variation in the size and distribution of normal islets of Langerhans within a section of the pancreas from an NHP. Some variation may be artifact due to variation of plane of section through a roughly circular to oval-shaped, or 3-dimensional, islet of Langerhans.

3. The hypothalamic endocrine axes The hypothalamic endocrine axes are complicated pathways. The following paragraphs are provided as a

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FIGURE 20.17 The normal macaque pineal gland: The pineal gland is characterized by ribbons of melatonin-secreting pinealocytes. The pineal gland appears sporadically in standard histologic sections of the NHP brain as a bulbous structure within the third ventricle attached to the ventral surface of the brain by a stalk.

FIGURE 20.18 Supports cells within the normal pineal bland may be identified using immunohistochemical methods (IHC): (A) Astrocytes (brown chromagen) express glial fibrillary acidic protein (GFAP) detected by IHC and are concentrated near vasculature of the pineal gland. (B) Microglial cells express ionized calcium-binding adapter molecule 1 (Iba-1) and are scattered throughout the normal NHP pineal gland.

superficial summary and the reader is advised to search out more specific information on the World Wide Web or advanced endocrine publications.

FIGURE 20.19 The pineal gland has a complicated neural connection with the suprachiasmatic neuclei of the hypothalamus. Axon fibers from the hypothalamus may be noticed in the pineal gland (arrows).

Hormones and neural input from the hypothalamus to the pituitary gland are initiators or inhibitors that affect multiple endocrine axes in the periphery, including the adrenal axis, gonadal axis, and thyroid axis. In turn, feedback from target organs in the periphery regulates the function at the hypothalamus and pituitary gland. For ease of concept, each axis will be considered separately here; however, they are not completely isolated entities, some utilizing identical cell populations. Dysfunction in one endocrine circuit at the level of the hypothalamus or pituitary gland may eventually result in dysfunction of others. For example: hyperadrenocorticism may be associated with decreased concentration of circulating thyroid hormones.33 The Hypothalamic Pituitary Adrenal Axis (HPAA) is partially responsible for homeostatic regulation of body fluid, energy storage, digestion, and immune function, in conjunction with peripheral organs including kidneys, fat, stomach, intestine, lymph nodes, thymus, and spleen. From the periventricular nucleus of the hypothalamus, corticotropin releasing hormone (CRH) is produced in response to low levels of serum cortisol or in response to neural input (stress response). CRH targets the anterior pituitary gland corticotrope cells (basophils), stimulating the production and release of adrenocorticotropic hormone (ACTH). ACTH targets the adrenal cortical cells, primarily the zona fasciculata and zona reticularis, stimulating the production and release of cortisol. As the cortisol level rises there is feedback inhibition at the hypothalamus and the pituitary gland, reducing secretion of CRH and ACTH, respectively, and maintaining homeostasis. However, the system is not as simple as it might seem. Hormones from other endocrine systems, including angiotensin II converted from angiotensin I in the lung, and vasopressin produced by the hypothalamus that targets the renal collecting ducts have the

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potential of stimulating CRH and ACTH production and release.3 The HPAA is subject to the general adaptation syndrome in which a stressor, real or imagined, results in physiological or neural stimulation of the hypothalamus and subsequent downstream release of cortisol in addition to adrenaline and noradrenaline (epinephrine and norepinephrine) from the adrenal medulla.34 The end result in NHPs may be alterations in glandular morphology and organ weights that may impact preclinical study interpretations.35 Regulation of thyroid function occurs through a feedback inhibition loop connecting to both the hypothalamus and pituitary glanddthe Hypothalamic-Pituitary-Thyroid Gland Axis (HPTA). When circulating thyroid hormones drop, the hypothalamus responds by producing and secreting thyrotropin-releasing hormone (TRH) that acts at the pituitary gland to increase thyroid-stimulating hormone (TSH) secretion, which in turn, targets thyroid follicular cells and stimulates production, secretion, and transformation of thyroid hormone precursors into functional thyroid hormones. As circulating thyroid hormones increase, they feed back to both the hypothalamus and pituitary gland to inhibit production and release of TSH. This balance between peripheral demand and production of hormones by the HPTA is remarkably stable throughout the time of wakefulness in NHPs, and also over the years with age. Therefore, any alterations in TSH from baseline could be considered indicative of thyroid or HPTA dysfunction.1 Although there are now validated means of measuring TSH and thyroid hormones in NHPs, they are not standard components of preclinical toxicological studies, rather, changes in morphology of the thyroid gland, pituitary gland, and hypothalamus are currently considered adequate for general evaluation of test article effects on the HPTA organ components. The Hypothalamic Gonadal Axis (HGA) is central to the sexual maturation and regulation of reproductive function of both male and female NHPs and this endocrine circuit is, perhaps, the most commonly monitored by hormone assays when questions of dysfunction arise. Dysfunction within the HGA is related to lesions that may occur either in the hypothalamus, pituitary gland, or gonads. The reader is referred to the sections of this book specifically addressing male and female reproductive organs for more detailed information on effects of HGA disruption and organ-specific lesions.

successful embryological and fetal development. Severe congenital lesions that hinder gland function are usually lethal at these stages and therefore, are not routinely identified in NHPs in the postneonatal stages. However, some nonlethal congenital abnormalities of endocrine organs are not uncommon in NHPs and are summarized here.

4. Congenital lesions of the endocrine system

FIGURE 20.20 Adreno-hepatic fusion as characterized by intermingled hepatocytes and adrenal gland cells, usually within the liver but liver cells within the autonomous adrenal gland also occur (pictured). The liver tissue may be identified by the presence of hepatic architecture including bile ducts (arrows), sinusoids or canaliculi and the two tissues are comingled without capsule present.

Congenital lesions of the endocrine organs in NHPs are generally rare, primarily due to their intricate ties to

4.1 Adrenal gland 4.1.1 Adrenohepatic fusion and liver embedded adrenal gland Adrenohepatic fusion is characterized by intermingled hepatocytes and adrenal cortical cells within the liver and has been noted in a low number of cynomolgus monkeys.19 Less commonly, there may be similar retention of liver tissue within the autonomous adrenal gland. The cellular morphology of hepatic tissue in the adrenal gland may be difficult to differentiate from retained fetal adrenal gland cortex (see Retained fetal adrenal cortex), but in general, the liver tissue will have other hepatic elements such as bile ducts, canaliculi, or sinusoids (Fig. 20.20). Additionally, the right adrenal gland may be embedded within the liver lobe in macaques, and may be differentiated from adrenohepatic fusion by the presence of a capsule around the adrenal gland tissue with complete segregation of liver and adrenal tissue (Fig. 20.21).19,36,37

4.1.2 Ectopic adrenal gland Foci of microscopic adrenal gland tissue in extraglandular locations is common in NHPs. Referred to as adrenal gland ectopia or accessory adrenal gland, it usually forms nested adrenal gland tissue adjacent to or within an abdominal organ. The kidney and reproductive organs (particularly the epididymis) are common locations for ectopic adrenal

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FIGURE 20.21 The right adrenal gland (AG) may be adhered to or embedded in the liver lobe (L) in macaques and may be differentiated from adreno-hepatic fusion by the presence of a connective tissue septum (arrows) between the two organs, or a complete adrenal capsule (C) present around the adrenal gland tissue.

gland foci due to embryological proximity of the organs (Fig. 20.22A and B).37 Accessory or ectopic adrenal gland is usually characterized by well-formed adrenal gland tissue surrounded by a capsule, and is very commmonly noted adjacent to, but independent of, the primary adrenal glands (Fig. 20.23). In some cases, no capsule is noted. The author has noted ectopia of the entire adrenal gland within the thoracic cavity on gross examination, either as an isolated organ or conjoined to an ectopic thoracic kidney, consistent with a failure of embryological organ migration.

4.1.3 Ectopic bone in the adrenal gland Ectopic tissue within the adrenal gland is less common than adrenal gland ectopia, but foci of bone with bone marrow are reported on occasion in macaques (Fig. 20.24). These foci appear well organized, having a formed marrow cavity within bone, and are most common at the corticomedullary junction of the gland.

4.1.4 Retained fetal adrenal cortex Retention of the fetal adrenal gland into adulthood of a monkey has only a single reported case in the literature38; however, it is not uncommonly encountered incidentally in the adrenal glands of adolescents and some adults. While it has been reported as a very rare finding in animals from China, animals from Cambodia may have a much higher incidence of this finding. The fetal cortical cells generally form a layer between the cortex and the medulla, and occasionally large regions of these cells may be present. These cells, or nests of cells, consist of large, fetal cortical cells with abundant eosinophilic to finely granular cytoplasm,

large central nuclei, and single prominent nucleoli (Fig. 20.25A and B). The retained fetal adrenal cortex is noted at the corticomedullary junction, presumably due to the fetal migration and involution pattern from outer to inner cortex, and may have associated foci of necrosis, mineralization, microhemorrhage, or hemosiderin deposits. More rarely, there may be large aggregates of foamy or vacuolated cells at the corticomedullary junction with pale brown-hued cytoplasm that is indicative of steroidogenic activity. The cells have large nuclei, single prominent nucleoli, indistinct cell borders and form dense fascicles or aggregates interspersed between bands of fibrovascular tissue. They form in association with the vascular bed between the adult cortex and medulla (Fig. 20.26A and B). Small clusters or individual cells of similar morphology are not uncommonly noted near the adrenal gland corticomedullary junction in adjult macaques. Many of these features have been described for the human fetal adrenal cortex during involution,39 and this is considered a rare variation of retained fetal adrenal cortex by the author; however, this has not been yet been confirmed by ancillary means and remains a speculative diagnosis.

4.2 Thyroid and parathyroid glands 4.2.1 Congenital thyroid gland cysts Several forms of congenital cysts may occur in the thyroid gland or parathyroid gland of NHPs. These cysts are primarily derived from a partially retained branchial apparatus, either the branchial pouch or the branchial cleft. Ultimobranchial cystic ducts characterized by dilated and occasionally multilobulated structures surrounded by flat, cuboidal, or less commonly squamous epithelium, are occasionally noted in both the thyroid and parathyroid glands of NHPs. They are most often positioned near or within the internal parathyroid gland due to the common origin from the fourth and fifth pharyngeal pouch complex. Ultimobranchial cystic ducts are remnants of the embryological ultimobranchial body, and as such, contain calcitoninpositive C-cells on immunohistochemical evaluation. The embryological thyroglossal duct also may persist into adulthood, forming a persistent thyroglossal duct or thyroglossal duct cyst. The ductal cyst usually contains a mucinous substance and is lined by simple cuboidal to columnar epithelium which may be ciliated. In general, the various forms of cysts that derive from embryological remnants (Fig. 20.27A and B) may be difficult to differentiate on H&E-stained tissue sections, therefore, the terminology recommended for reporting purposes would be simply “cyst, congenital.”

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FIGURE 20.22 (A) Ectopic adrenal gland characterized by well-formed adrenal gland tissue, usually with a capsule, that may be commonly noted adjacent to the kidneys or reproductive organs. (B) Ectopic adrenal tissue may occur embedded within other organs (arrow) and may be without capsule, in this case, embedded within the kidney.

FIGURE 20.23 Accessory adrenal gland is characterized by well-formed adrenal gland tissue surrounded by a capsule (arrows), and very commonly attached to, but independent of, the primary adrenal gland.

FIGURE 20.24 Adrenal gland of a macaque: A focus of well-formed ectopic bone with bone marrow surrounded by fiborus tissue is present at the corticomedullary junction.

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FIGURE 20.25 (A) The adult NHP adrenal gland has a retained fetal adrenal cortex that appears as an additional layer of eosinophilic cells between the adult cortex and medulla (asterisk). (B) The retained fetal cortex consists of large cells with abundant eosinophilic to finely granular cytoplasm, large central nuclei, and single prominent nucleoli. Note the focus of mineral (arrow), a common finding associated with the retained fetal adrenal cortex and commonly noted in adrenal glands of macaques.

4.2.2 Ectopic thymus in the thyroid gland or parathyroid gland Ectopic thymus within the thyroid and/or parathyroid glands is exceptionally common in NHPs. Ectopic thymus tissue is usually located at the periphery of the gland as a single, variably sized nodule that may contain both medulla and cortex or cortex only (Fig. 20.28); however, it may also appear embedded within the glands. The high prevalence of ectopic thymus is linked to the common origin with that of parathyroid glands.40 The presence of very small foci of ectopic thymus within the thyroid or parathyroid glands should be carefully differentiated from mononuclear cell infiltratesdanother common finding in NHPs.

4.2.3 Ectopic thyroid or parathyroid gland Grossly, the thyroid gland may easily be visualized as a discrete organ; however, additional microscopic foci of thyroid gland, or less commonly parathyroid gland, may be found nearly anywhere in the cervical to thoracic tissues

FIGURE 20.26 (A) Very rarely, there may be large aggregates of foamy or vacuolated cells at the corticomedullary junction (arrows) that may represent a variation of retained fetal adrenal cortex. (B) The cells are highly vacuolated with pale brown-hued cytoplasm, large nuclei, single prominent nucleoli and indistinct cell borders that form dense fascicles or aggregates interspersed between bands of fibrovascular tissue. (M-medulla).

and most commonly is noted in sections of epicardium of the heart, or superficial connective or adipose tissue in trachea, esophagus, or pharynx (Fig. 20.29).

4.2.4 Ectopic salivary gland in the thyroid gland Ectopic salivary gland may be noted in the thyroid or parathyroid gland of NHPs, usually as a single small focus of glandular or ductal tissue in the subcapsular adipose tissue (Fig. 20.30).19

4.2.5 Thyroid gland hypoplasia or agenesis On occasion, macaques have unilateral thyroid and/or parathyroid gland hypoplasia or agenesis (also referred to as hemiagenesis) noted grossly or microscopically; however, no biological consequences have been recorded so far for this congenital anomaly, perhaps a result of contralateral gland lobe compensatory function, but thyroid function in macaques on study are rarely surveyed and only clinical observation and general clinical pathology parameters are used to determine

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FIGURE 20.29 Microscopic foci of thyroid gland (arrow) that are extraneous to the main organ may be found nearly anywhere in the cervical to thoracic tissues and most commonly are noted in sections of epicardium, trachea, esophagus, or pharynx.

FIGURE 20.27 Congenital cysts of the thyroid gland may have variable presentations: (A) Columnar ciliated cells, with or without cilia, are often visible lining congenital cysts of the thyroid gland and attributed most commonly to remnant ultimobranchial tissue. (B) Congenital cysts lined by thyrogenic epithelium are most commonly attributed to thyroglossal duct remnants and may have regions of dysplastic, hyperplastic or hypertrophic thyrogenic epithelial cells, some containing globular, eosinophilic cytoplasmic vacuoles (V) may be present in the thyroglossal cyst. FIGURE 20.30 Occasionally, foci of salivary gland tissue (arrow) are present in the subcapsular adipose tissue of the thyroid gland in macaques.

FIGURE 20.28 Ectopic thymus tissue is usually located at the periphery of the gland as a single small nodule that may contain both medulla and cortex or cortex only.

normalcy. While most cases encountered by the author had normal morphology or mild follicular hyperplasia of the contralateral intact thyroid gland, in the case of unilateral hypoplasia presented, the contralateral thyroid gland remaining had follicular epithelial hypertrophy with basilar, globular to glassy, eosinophilic cytoplasmic vacuoles; small follicular lumens; increased resorption vacuoles; and apically displaced nuclei (Fig. 20.31A and B) consistent with increased activity of the remaining gland. The glassy vacuoles with apical nuclear displacement have been previously described for NHP thyroid glands with hyperplasia of the endoplasmic reticulum as confirmed by electron microscopy41; however, no further investigation was performed in this case. There has been discussion in publication over whether these conditions in

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humans may predispose to thyroid gland abnormalities later in life, including adenocarcinoma, adenoma, multinodular goiter, and chronic thyroiditis.42,43 In one study, when the authors followed 40 human patients with thyroid hemiagenesis they found that there was risk of additional thyroid pathology and that most patients had increased thyrotropin (TSH) and free triiodothyronine (FT3).43 No such published evaluations have been performed on NHPs.

4.2.6 Congenital goiter Congenital goiter has been diagnosed in three newborn rhesus macaques from the same parents. These infants had normal triiodothyronine levels, high TSH levels, and low thyroxine levels. Further investigation indicated a postorganification defect leading to thyroid hyperplasia.44 In very rare cases, enlarged hyperplastic thyroid glands have been identified at necropsy in otherwise normal individuals (see Hyperplastic lesions of the thyroid gland).

4.3 Pituitary gland 4.3.1 Congenital cysts of the pituitary gland

FIGURE 20.31 Thyroid gland hypoplasia in a macaque: (A) There is unilateral hypoplasia of the thyroid gland with a few follicular structures (F), several degenerative cystic follicles (CF), a small ductal remnant (D), and ectopic thymus (T) present in section. The contralateral thyroid (CLT) gland is present in section. (B) Higher magnification of the contralateral thyroid gland in Fig. 20.31A: The follicles are lined by cuboidal to columnar cells with abundant, basilar, globular to glassy, eosinophilic cytoplasmic vacuoles and apically displaced nuclei. Note: There are abundant resorption vacuoles (arrows) along the epithelial-luminal interface indicative of a highly active gland.

FIGURE 20.32 (A) Rathke’s pouch cysts or clefts are lined by cuboidal or columnar pseudostratified epithelium and may contain accumulation of mucin, blood, or sloughed cells. (B) The epithelium lining the Rathke’s pouch cyst is often ciliated (arrow).

Degenerative cysts of the pituitary gland of non-human primates are quite common in the pars distalis or pars intermedia (see Degenerative lesions of the pituitary gland). Other cystic structures are formed from the remnants of Rathke’s pouch (Rathke’s cyst or cleft). Rathke’s pouch cysts or clefts are usually lined by cuboidal or columnar pseudostratified epithelium that is variably ciliated, and may be vacant or contain accumulation of mucin, blood, or sloughed cells (Fig. 20.32A and B). More rarely, there may be cysts lined by goblet cells, consistent with their origin from the craniopharyngeal pouch (Fig. 20.33).

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FIGURE 20.33 Congenital cysts of the pituitary gland originating from the craniopharyngeal pouch may have a significant goblet cell population (arrow) lining the structure.

4.4 Endocrine pancreas 4.4.1 Giant islets of Langerhans Greatly enlarged, coalescing islets of Langerhans have very rarely been noted in cynomolgus monkeys and other NHPs, and likely represent a congenital alteration of these structures (Fig. 20.34).

FIGURE 20.35 Light yellow to gold pigmented granules (arrow) within the cell cytoplasm are most commonly associated with lipofuscin accumulation, a common pigment associated with aging and lysosomal digestion or degradation of lipid-rich components.

beginning in young adulthood. The lipofuscin may begin as a light yellow to gold (Fig. 20.35) and progress with age to darker, brown pigmented granules within the cortical cell cytoplasm. It is considered a form of age-related lysosomal degradation of lipid-rich cellular materials.

5.1.2 Adrenal gland necrosis Within the adrenal glands of NHPs there may be degenerative foci that many pathologists diagnose as necrosis, with or without hemorrhage, usually at the corticomedullary junction in NHPs (Fig. 20.36). Whether this is a degenerative finding or an extension of cortical involution is not clear.

FIGURE 20.34 Large, coalescing islets of Langerhans (asterisks) are rarely reported in NHPs and are considered a likely congenital malformation.

5. Degenerative lesions of the endocrine system 5.1 Adrenal gland 5.1.1 Adrenal gland pigment Spontaneous degenerative changes of the adrenal gland are usually minor in NHPs and occur with high frequency near the corticomedullary junction. Lipofuscin within adrenal cortical cells may be noted in NHPs, primarily macaques,

FIGURE 20.36 Foci of necrosis and/or hemorrhage (arrow) at the corticomedullary junction are occasionally noted in NHPs.

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5.2 Thyroid and parathyroid glands 5.2.1 Degenerative cysts of the thyroid and parathyroid glands Degenerative changes in the thyroid and parathyroid glands of NHPs may be quite variable and include cysts, atrophy, and follicular accumulation of cell debris or inspissated thyroglobulin. These changes may occur separately or together and in some cases, may have associated inflammation. The degenerative type of follicular cyst is very common in NHPs and usually appears in low numbers within the thyroid and parathyroid glands (Fig. 20.37A and B). The thyroid cysts may resemble normal follicles, but are larger and often displace or compress the adjacent thyroid follicles. Thyroid and parathyroid degenerative cysts are primarily lined by bland flattened or low cuboidal epithelium. The luminal colloid may appear pale or basophilic and may contain desquamated follicular cells and macrophages. The pathologic significance of these cysts is unclear.19 It may be difficult to distinguish a pronounced dilated follicle from a congenital ultimobranchial duct cyst; however, congenital cysts are usually differentiated by a squamous or ciliated epithelial lining (see Congenital cyst of thyroid gland). Cysts and dilated follicles are more

FIGURE 20.37 (A) Thyroid cysts (asterisk) often displace or compress the adjacent thyroid follicles. (B) Thyroid and parathyroid degenerative cysts (asterisk) are primarily lined by bland flattened or low cuboidal epithelium. The luminal material may contain inspisated material, sloughed epithelial cells or appear pale eosinophilic or basophilic.

common in males compared to females according to some authors.37

5.2.2 Thyroid gland follicular atrophy and degeneration Focal to extensive follicular atrophy is frequently seen in the thyroid of cynomolgus monkeys. Microscopically follicular cell atrophy is characterized by loss and collapse of follicles and tiny follicles that are lined by flattened or low cuboidal epithelial cells (Fig. 20.38). Desquamation of follicular epithelial cells in to the lumen or macrophage infiltration may appear in either degenerative cysts or atrophic follicles (Fig. 20.39A).19 These luminal cells are predominantly positive for CD68 antigen when evaluated by IHC (Fig. 20.39B), consistent with macrophages. Atrophy of the parathyroid gland cells is much rarer but has been noted in macaques at a low level. It may occur secondary to injury, chronic hypercalcemia, or may be idiopathic.

5.2.3 Follicular epithelial vacuolation Vacuolar degeneration of the thyroid follicular epithelium is an uncommon finding but has been recorded as spontaneous in cynomolgus monkeys without associated degenerative changes or loss of function. The reported gross appearance of the thyroid glands is usually unremarkable and the organ weight is within the normal range. Microscopically, the follicular epithelial cells contain large vacuoles and apically displaced nuclei (Fig. 20.40A) which are negative when stained with PAS staining method and negative when evaluated by antithyroglobulin IHC. Although referred to as “vacuolar degeneration” the process elucidated by electron microscopy is often an enlarged, hyperplastic endoplasmic reticulum.41 Similar findings have occurred in macaques from the author’s facility, along with other forms of vacuolar degeneration in which there were numerous clear microvacuoles present (Fig. 20.40B).

FIGURE 20.38 Microscopically, thyroid follicular atrophy is characterized by regional to diffuse loss and collapse of follicles with remaining follicles lined by flattened or low cuboidal epithelial cells and containing little or no colloid, or inspissated colloid.

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FIGURE 20.39 (A) Desquamation of follicular epithelial cells into the lumen and/or macrophage infiltration (arrow) may appear in degenerative cysts. (B) These luminal cells are primarily positive for CD68 antigen when evaluated by IHC (arrow) consistent with macrophages.

5.2.4 Thyroid gland and parathyroid gland fibrosis

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FIGURE 20.40 (A) Vacuolar degeneration in which there are large vacuoles and apically displaced nuclei of the follicular epithelium (arrow). This finding has been diagnosed as hypertrophy of the endoplasmic reticulum when evaluated by electron microscopy.41 (B) Diffuse cytoplasmic microvacuolation is occasionally noted in the thyroid follicular epithelium of NHPs.

5.3 Pituitary gland

Increased fibrous tissue in the capsule or interstitium of the parathyroid gland or interstitium of the thyroid gland is occasionally noted in NHPs, particularly with increased age, or may be noted in organized fashion within the parathyroid gland at any age (Fig. 20.41). Fibrosis can be seen following inflammation, necrosis, or hemorrhage, and the diagnosis is used when the amount of connective tissue is considered greater than commonly observed in the gland.

Histologically visible, spontaneous degenerative changes in the pituitary gland of macaques and marmosets have not been greatly reported. Most degenerative changes in the pituitary gland of NHPs are primarily described through biochemical alterations of hormone production noted as a consequence of aging or social stress.46

5.2.5 Fat infiltration of the thyroid gland or parathyroid gland

The most commonly noted degenerative changes in young NHPs are mineralization and increased fibrous tissue (Fig. 20.43), however with age there may be significantly increased fibrous tissue that sections lobules of the pars distalis or pars intermedia.

The normal NHP thyroid and parathyroid glands are relatively low in adipose tissue; however, rarely there are noted cases with significant fatty infiltration. Whether these cases represent a true degenerative process is unknown, but they resemble thyrolipomatosis described in humans with the characteristic feature of diffuse mature adipose cell infiltration among the nonneoplastic thyroid follicles and parathyroid cell cords or pseudofollicles.40,45 (Fig. 20.42A and B).

5.3.1 Pituitary gland fibrosis and mineralization

5.3.2 Vacuolar degeneration of the pituitary gland Although cytoplasmic vacuolation of the pars distalis or pars intermedia (Fig. 20.44A and B) is sometimes referred

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FIGURE 20.41 Increased fibrous tissue in the capsule or interstitium of the parathyroid gland is occasionally noted in NHPs. Fibrosis is diagnosed when the amount of connective tissue is considered greater than commonly observed in the gland.

FIGURE 20.42 (A-B) Rarely there are thyroid and parathyroid glands with significant fatty infiltration noted in NHPs. The characteristic feature is of diffuse mature adipose cell infiltration between the normal glandular tissue.

FIGURE 20.43 Increased fibrous tissue (arrows) within the pituitary gland of NHPs is a rare finding among young animals but may be significantly increased in aged non-human primates.

FIGURE 20.44 (A) Cytoplasmic vacuolation of the pars distalis or (B) pars intermedia is sometimes referred to as “vacuolar degeneration”; however, the underlying etiology is usually of hypertrophic or hyperplastic nature.

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to as “vacuolar degeneration,” this finding is usually due to hypertrophic or hyperplastic conditions (see Hyperplastic lesions of the pituitary gland).

5.3.3 Degenerative cysts of the pars distalis or pars intermedia Degenerative cysts may occasionally be noted in the pars distalis or pars intermedia of the pituitary gland of NHPs. These cysts are characterized by enlarged glandular spaces filled with amphophilic hyaline material or inspissated material occasionally admixed with small amounts of cell debris. They are often lined by flattened or low cuboidal epithelium that may be attenuated and are generally increased in number in aged animals.

5.4 Endocrine pancreas 5.4.1 Islet of Langerhans atrophy Atrophy of the islet cells as a spontaneous finding is an extremely rare phenomenon in macaques, characterized by loss of cytoplasmic volume, leaving clusters of nuclei that may also be decreased in size. The finding is more commonly reported in conjunction with amyloidosis of the islets of Langerhans, due primarily to diabetes mellitus which may be naturally occurring in aged animals or toxicologically induced. Fibrosis of islets may occur, usually as sequelae of previous injury or inflammation, or in conjunction with fibrosis or inflammation of the pancreas in general.

5.5 Pineal gland 5.5.1 Fibrosis and mineralization of the pineal gland Degenerative changes of the pineal gland are rare but include mineralization and increased fibrous tissue. Immunohistochemistry for GFAP highlights the tissue that is part of the astrocytosis and glial scar formation in these “fibrotic” foci (Fig. 20.45A and B).

6. Inflammatory and vascular lesions of the endocrine system 6.1 Mononuclear cell infiltrates of endocrine organs The most common inflammatory finding in the endocrine glands of NHPs is minimal to mild mononuclear and/or mixed cell infiltrates (Fig. 20.46). These infiltrates are nearly ubiquitous in NHP tissues and a common incidental finding in the endocrine organs. A search of the historical control database (HCD) from the editor’s facility for

FIGURE 20.45 (A) The most commonly noted degenerative changes in the NHP pineal gland are mineralization and increased fibrous tissue. (B) The “fibrous tissue” noted in many of these lesions is often glial scar tissue and can be better visualized following immunohistochemistry for GFAP. Note: Compare this image to the normal pineal gland GFAP in Fig. 20.18A.

mononuclear cell infiltrates in endocrine organs is presented in Table 20.1.

6.2 Adrenal gland 6.2.1 Adrenalitis Spontaneous inflammatory lesions of the adrenal glands of NHPs are exceedingly rare. Chronic adrenal inflammation as a spontaneous finding has been noted at the editor’s facility and characterized by lymphohistiocytic infiltrates concentrated within the zona glomerulosa and zona fasciculata of the cortex, accompanied by hemorrhages and alternating cortical hyperplasia and atrophy that distorted the normal, three-layer architecture of the gland (Fig. 20.47A and B). The features of the inflammation were similar to those described with autoimmune adrenalitis.47 This animal had no clinical observations and no changes in clinical chemistry or hematology to indicate either an underlying cause or systemic ramifications of the adrenal

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at the margins of these cavernous foci (Fig. 20.49A and B) bu the significance is unknown.

6.3 Thyroid and parathyroid glands 6.3.1 Thyroiditis

FIGURE 20.46 Macaque pituitary gland: minimal to mild mononuclear cell infiltrates (arrow) are a common incidental finding in the endocrine organs of NHPs, here noted at the junction of the pituitary gland pars intermedia and pars nervosa.

inflammation; however, the lesion was noted with histopathology incidentally and no additional endocrine evaluation was performed.

6.2.2 Adrenal gland hemorrhage There may be minor hemorrhage, primarily at the corticomedullary junction, in NHPs that may result in accumulation of hematoidin or hemosiderin pigment in the adrenal glands, which is usually present in the cytoplasm of macrophages rather than cortical cells (Fig. 20.48A).37 On rare occasions, the hemorrhage is more significant, and may be in conjunction with other alterations of the gland such as nodular hyperplasia (Fig. 20.48B). Large cavernous foci of hemorrhage and fluid accumulation between the adrenal cortex and medulla are most rare but have been recorded in control animals assigned to toxicological studies. These large accumulations have acute to peracute features suggesting they formed close to the time of euthanasia. In some cases, retained fetal adrenal gland tissue has been identified

Chronic inflammation in the thyroid gland has occasionally been reported in young macaques, primarily females,37 and in marmosets.48 Inflammation should be differentiated from the benign-appearing incidental mononuclear cell infiltrates or the very small foci of ectopic thymus that are more commonly noted in macaques. The macroscopic appearance of chronic inflammation may be unremarkable or there may be irregularity to the glandular profile and organ weights may or may not be increased. The microscopic changes are characterized by extensive lymphoplasmacytic cellular infiltrates that may include nodular aggregates, some with lymphoid follicular structures including germinal centers (Fig. 20.50). When the inflammation is extensive, it may be accompanied by follicular atrophy or tissue effacement, fibrosis, or alterations of follicular epithelial cells, such as cellular hypertrophy or hyperplasia. Some follicles may be degenerative and contain desquamated epithelial cells or mononuclear cells. There may be compensatory pituitary gland changes associated with chronic thyroiditis, including adenohypophyseal cell hypertrophy or hyperplasia.37,49 Spontaneous inflammation of the parathyroid gland alone is very rare as it is usually in conjunction with thyroid gland inflammation. When noted only in the parathyroid gland, the lesion may be diffuse and chronic, with infiltration by lymphocytes, plasma cells, and macrophages, or may be chronic-active with inclusion of neutrophils. There may be associated tissue damage and or vascular changes with the inflammation. In severe cases the parathyroid tissue may be effaced by inflammatory cells and fibrosis.21

TABLE 20.1 Mononuclear cell infiltrates in the endocrine organs of cynomolgus macaques (Macaca fascicularis) utilized as study control animals. Cambodia

China

Mauritius

Cynomolgus macaques

Male

Female

Male

Female

Male

Female

Thyroid gland

1 (55) 1.82%

3 (55) 5.45%

32 (958) 3.34%

29 (925) 3.14%

12 (275) 4.36%

10 (272) 3.68%

Adrenal gland

1 (55) 1.82%

0 (55) 0.0%

12 (957) 1.25%

19 (925) 2.05%

2 (276) 0.72%

6 (272) 2.21%

Parathyroid gland

0 (52) 0.0%

1 (53) 1.89%

5 (846) 0.59%

3 (811) 0.37%

5 (236) 2.12%

5 (233) 2.15%

Pituitary gland

1 (54) 1.85%

0 (55) 0.0%

19 (955) 1.99%

13 (922) 1.41%

11 (275) 4.0%

10 (271) 3.69%

A Data search limited to animals examined as study terminal, interim or recovery control cohorts and excluding unscheduled decedents: Islets of Langerhans and pineal gland were omitted from table as no specifics for these compartments/organs were noted in the HCD. Bold type indicates the total number of animals with the finding of mononuclear cell infiltrates in the respective organ. Numbers in parentheses indicate the number of animals examined. Percent values in parentheses were calculated as (total number of animals with the finding/total number of organs examined * 100). Data harvested on March 29, 2022, from Charles River, Inc.

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FIGURE 20.48 (A) Deposits of hemosiderin may be noted within the adrenal glands of NHPs, particularly at the corticomedullary junction due to previous hemorrhage. (B) Hemorrhage at the corticomedullary junction is usually minimal in NHPs; however, on rare occasion, it may be quite prominent and in conjunction with other findings, such as nodular hyperplasia (asterisk).

be present, such as melanophages or lymphocytes (Fig. 20.51B)

FIGURE 20.47 A spontaneous case of adrenalitis in a macaque: (A) Alternating cortical hyperplasia and atrophy distorts the normal, threelayer architecture of the gland. (B) Lymphohistiocytic infiltrates are concentrated within the zona glomerulosa and zona fasciculata of the cortex, accompanied by hemorrhage.

6.5 Endocrine pancreas

6.4 Pituitary gland and pineal gland

6.5.1 Inflammation of the islets of Langerhans

Inflammation of the pituitary gland, including lymphocytes, histiocytes, or neutrophils, has been reported only in experimentally induced autoimmune hypophysitis50 and in cases of cytomegalovirus infection, and is not reported for the pineal gland as a spontaneous finding.51 Rarely, the pineal gland will experience minor microgliosis (Fig. 20.51A) and on occasion other cell populations may

Spontaneous inflammation of the pancreas is occasionally noted in NHPs and can be quite extensive, but not usually isolated to the islets of Langerhans unless associated with islet amyloidosis. Nevertheless, ubiquitous inflammation has consequential effects that may result in endocrine pancreas insufficiency (hypoinsulinemia) due to effacement of the islets of Langerhans.

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FIGURE 20.49 (A) Large cavernous foci of acute to subacute hemorrhage and fluid accumulation between the adrenal cortex and medulla are rare but have been recorded in control animals assigned to toxicological studies. (B) In some cases, retained fetal adrenal gland tissue (arrow) has been identified at the margins of these cavernous foci. FIGURE 20.51 (A) Rarely, the pineal gland will experience minor microgliosis. (B) Other cell populations may be present with microglial clusters of the pineal gland, such as melanophages or lymphocytes.

6.5.2 Vascular ectasia of the islets of Langerhans Vascular ectasia as a focal or multifocal finding is not an uncommon conformation within the islets of Langerhans of NHPs (Fig. 20.52).

7. Hyperplastic lesions of the endocrine system 7.1 Adrenal gland FIGURE 20.50 The microscopic changes of thyroiditis in NHPs may include extensive lymphoplasmacytic cellular infiltrates that may include nodular aggregates, some with lymphoid follicular structures including germinal centers.

Hypertrophy and hyperplasia of the adrenal cortex of NHPs is perhaps one of the most common findings in preclinical studies and is often noted together. Expansion of the zona

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FIGURE 20.52 Vascular ectasia as a focal or multifocal finding is not an uncommon conformation within the islets of Langerhans of NHPs.

fasciculata is most commonly noted (Fig. 20.53AeC); however, rarely there may be expansion of the zona reticularis that lacks hypertrophic or hyperplastic features and may be a morphologic variation of the gland in macaques. Physiological cortical hypertrophy or hyperplasia is not uncommon in NHPs due to metabolic demand or stress during a study and may be focal or multifocal, but most commonly is diffuse, possibly resulting in significantly increased organ weights.35 Cellular enlargement without significant compression of adjacent tissue is the hallmark of cortical hypertrophy. Focal or nodular hyperplasia is characterized by a region of enlarged cortical cells, some with admixed multinucleated cells, usually from the zona fasciculata or zona reticularis that may contain mitoses and that compresses the adjacent cortical tissue. Some consider hypertrophy and hyperplasia of the adrenal cortex in NHPs to be a continuum, with the first eventually converting to the second when the inciting stimuli remain constant or escalate in intensity.

7.2 Thyroid and parathyroid glands Hypertrophy and/or hyperplasia may affect any of the cell populations within the thyroid gland, and in some cases, both hypertrophy and hyperplasia are present together; therefore, separation of the two features may be difficult.18 Most commonly the follicular lining cells are affected and the combination of hypertrophy and hyperplasia are not uncommonly noted in the thyroid glands of NHPs at a low severity level. Hypertrophy of the follicular epithelium may be noted as a normal physiological response to increased demand for the thyroid hormone. Follicular epithelial hyperplasia may be diffuse or localized, and when exuberant, may be noted grossly as nodules of the thyroid gland or generalized gland enlargement (Fig. 20.54A). Typically, follicular hyperplasia is characterized by irregularly sized follicles with cuboidal to columnar epithelium. There may be excessive resorption vacuoles at the follicular-luminal

FIGURE 20.53 (A) Focal or nodular cortical hyperplasia (asterisk) is characterized by a region of enlarged cortical cells, some with admixed multinucleated cells, usually from the zona fasciculata or zona reticularis that may contain mitoses and that compresses the adjacent cortical tissue. (B) Higher magnification of Fig. 20.53A: The enlarged cells of the hyperplastic focus (H) with multinucleation as compared to the smaller cells of the normal adjacent cortex (CX). (C) Diffuse cortical hypertrophy and/ or hyperplasia of the zona fasciculata is not uncommon in NHPs due to metabolic demand or stress and may significantly increase organ weights.

interface. Epithelial cells may contain cytoplasmic eosinophilic droplets (Fig. 20.54C) or crystals and there is usually folding of the epithelium on collagenous stalks (Fig. 20.54B).52 In contrast, follicular cell hypertrophy is represented by enlarged lining cells often with small sized follicles and may be a feature of highly active glands

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FIGURE 20.55 (A) Follicular cell hypertrophy is characterized by small follicles lined by enlarged follicular cells and is a feature of highly active thyroid glands. (B) The hypertrophic follicular epithelium is typically cuboidal to columnar and follicles typically have smaller lumens as compared to the quiescent gland.

C-cell hyperplasia also occurs in NHPs, most commonly noted in marmosets but also rarely in macaques, and is characterized by either cellular clusters or diffuse

FIGURE 20.54 (A) The thyroid glands from sex, age and weight matched cynomolgus monkeys: Diffuse follicular hyperplasia has resulted in bilateral enlargement of the thyroid glands (left-arrow) as compared to the normal thyroid glands (right). (B) Follicular hyperplasia is characterized by irregularly sized follicles with cuboidal to columnar epithelium. There may be excessive resorption vacuoles at the follicular-luminal interface and folding of the epithelium on collagenous stalks. (C) Epithelial cells in hyperplastic foci may contain cytoplasmic eosinophilic droplets.

(Fig. 20.55A and B). There may be both hypertrophy and hyperplasia of follicular epithelium present in glands with degenerative changes as well (Fig. 20.56).

FIGURE 20.56 A common presentation in NHPs is of thyroid gland hypertrophy and hyperplasia in conjunction with follicular atrophy and degeneration.

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increases in the cell population (Fig. 20.57).53 Hyperplasia of the parathyroid gland of NHPs as a spontaneous finding has not been reported.

FIGURE 20.57 C-cell hyperplasia also occurs in NHPs, most commonly noted in marmosets but also rarely in macaques, and is characterized by either cellular clusters or diffuse increases in the cell population.

7.3 Pituitary gland 7.3.1 Hypertrophy and hyperplasia of the pars distalis NHPs may develop discrete foci of pituitary hypertrophy and/or hyperplasia that must be differentiated from microadenomas; however, these conditions are generally very rare as spontaneous findings in routine toxicological studies that utilize young animals. Hyperplasia of the pituitary adenohypophysis of cynomolgus monkeys has been reported in conjunction with chronic thyroiditis49 and associated with an alcohol consumption study, but may occur spontaneously in some cases (Fig. 20.58A and B).54 Vacuolar changes of the pituitary gland pars distalis and pars intermedia are occasionally noted in NHPs as well, some in conjunction with hypertrophy and/or hyperplasia. Chronic stimulation of the pituitary gland, due to failure of the feedback mechanism, eventually results in cystic dilation of the endoplasmic reticulum, noted histologically as pale, eosinophilic cytoplasmic vacuoles (Fig. 20.44A and B).55 As with many findings in the pituitary gland, weight of evidence should be applied in order to separate possible test article affects from those occurring spontaneously.

7.4 Endocrine pancreas Islet hyperplasia is reported in a number of NHPs; however, cynomolgus and rhesus monkeys are currently not among those noted. Marmosets have been noted to have spontaneous islet hyperplasia under conditions of obesity or diabetes.56

FIGURE 20.58 (A) Hyperplasia of the pars distalis may be focal or diffuse and is characterized by an enlargement that blends at the margins with the normal gland. (B) Hyperplastic cells usually have irregular architecture composed of enlarged cells. Mitoses are very rarely noted in these foci. Note: There are distinct, large, pale vacuoles present in many of the hypertrophic cells (arrows) usually attributed to cystic dilation of the endoplasmic reticulum.

8. Neoplastic lesions of the endocrine system 8.1 Adrenal gland 8.1.1 Pheochromocytoma Pheochromocytoma is the most commonly reported tumor of the adrenal gland of both macaques and marmosets.57e59 As with most neoplasia, these tumors are more common in aged animals as compared to the young monkeys commonly assigned to preclinical studies, and are primarily identified by their morphological features. Originating from medullary chromaffin cells, pheochromocytomas are neuroendocrine tumors characterized by polygonal cells arranged in cords or packets supported by a fine, fibrovascular stroma (Fig. 20.59). Neoplastic cells usually have granular cytoplasm and express chromogranin A with variable expression of met-enkephalin, b-endorphin, neuron-specific enolase, S-100, or synaptophysin. Other features variably noted in the literature include anisocytosis

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Spontaneous Pathology of the Laboratory Non-human Primate

These tumors generally compress or distort the adjacent tissue (Fig. 20.60A) and are composed of large cells with abundant, clear, or finely vesicular cytoplasm, single to multiple hyperchromatic nuclei, and rare mitoses that may palisade around blood vessels (Fig. 20.60B)

8.1.3 Adrenal hemangioma Adrenal gland hemangioma usually presents as noninvasive, expansile, well-demarcated, and often encapsulated mass with hemorrhagic gelatinous contents (Fig 20.61). With increased size of the neoplasm, the adjacent adrenal gland tissue becomes compressed and atrophic.

8.1.4 Other tumors of the adrenal gland Various tumors have been reported in the adrenal glands of NHPs including a medullary fibroma in a rhesus monkey,36 and adrenal ganglioneuroma in a marmoset.33

FIGURE 20.59 Pheochromocytoma of a aged NHP: (A) there is an expansile mass of neoplastic cells efacing the adrenal medulla and compressing the adrenal cortex. (B) Originating from medullary chromaffin cells, pheochromocytomas are neuroendocrine tumors characterized by polygonal cells arranged in cords or packets supported by a fine, fibrovascular stroma.

and anisokaryosis, giant or irregular hyperchromatic nuclei, multinucleation, and cytoplasmic invagination. Use of special stains (such as Churukian-Schenk or Sevier-Munger method) or immunohistochemistry may elucidate the cytoplasmic granules that are characteristic of these tumors. Pheochromocytomas may be malignant, invading the adrenal cortex and occasionally form metastases in other organs. In some reported cases, anecdotal evidence of catecholamine-induced lesions of heart (catecholamine cardiomyopathy) and blood vessels (arteriosclerosis) in NHPs with pheochromocytomas have been observed.58,60

8.1.2 Adrenal cortical adenoma Adrenal cortical adenomas have been reported in rhesus macaques, and are rarely noted in cynomolgus macaques.58

FIGURE 20.60 An adrenal cortical adenoma in a macaque: (A) The tumor is expansile, causing distortion and compression of the adjacent cortex and medulla. (B) The neoplastic cells form palasades with fine fibrovascualr stroma between. The neoplasitic cells are large with abundant, clear or finely vesicular cytoplasm, single to multiple hyperchromatic nuclei, and rare mitoses.

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FIGURE 20.61 An adrenal gland hemangioma in a macaque: The central region of the adrenal gland has been effaced by a dark red, lobular, gelatinous mass and the remaining cortex is a thin rim at the periphery. Image courtesy of Rebekah Keesler.

8.2 Thyroid and parathyroid glands 8.2.1 C-cell neoplasia Neoplasia of the thyroid gland cells (C-cells and follicular cells) is rarely reported, and parathyroid gland tumors have no recorded cases in NHPs. One case of invasive C-cell carcinoma in macaques has been described as consisting of “islands and clusters of Ccells separated by thin connective tissue septa” and malignancy determined by metastasis to the mediastinal lymph node.61 The benign counterpart to the C-cell tumor, the adenoma, has not yet been reported in NHPs commonly used in nonclinical studies. Although C-cell hyperplasia is a common finding in marmosets, neoplastic lesions have not been described. Thus, the significance of this hyperplastic finding in relation to progression to tumor has not been demonstrated.

8.2.2 Thyroid follicular adenoma and follicular adenocarcinoma Thyroid follicular adenomas and carcinomas are more commonly encountered and reported in thyroid glands of marmosets and macaques as compared to C-cell tumors.48,61 The majority of these tumors were noted microscopically in young animals. When grossly visible, the tumor results in an enlarged, thyroid gland with firm, tan nodules. Carcinomas are separated from adenomas based on the morphology of the neoplastic cells, lack of encapsulation or multifocal nature, or the presence of invasion or metastasis (Fig. 20.62A and B). Both tumors may be composed of cuboidal to columnar neoplastic cells, but carcinomas typically exhibit greater cellular pleomorphism. Carcinomas also may have significant nuclear atypia.

Nuclear molding or multinucleation may be common and mitoses variable. Follicular growth patterns of the tumors are most commonly reported in literature; however, a combination of solid and follicular or papillary growth patterns occurs (Fig. 20.63A-C). Rarely, the carcinomas may contain cells with eosinophilic, granular cytoplasm resembling Hurthle cells (oxyphilic/oncocytic cells).

8.3 Pituitary gland 8.3.1 Pituitary adenoma Cynomolgus macaques have been the primary species associated with pituitary tumors, primarily pituitary adenomas. Rarely, rhesus monkeys are also reported with pituitary adenomas.62 Malignant pituitary tumors in these species have not been reported. As with other endocrine organ tumors, functionality is rarely evaluated for pituitary tumors when identified and, for animals assigned to nonclinical studies, the tumors are usually found incidentally at standard tissue evaluation. Literature review, however, indicates that these tumors as noted in older animals may be functional in many animals.62e64 In one study, classification of the adenomas was accomplished using IHC. Majority of the tumors across species were positive for prolactin (lactotrophs), and in some cases, had serum elevations of prolactin.64 This agrees with other published accounts of prolactin secreting pituitary adenomas in macaques, some with associated galactorrhea.63 Other tumors in macaques were reported as chromophobe adenoma or nonsecreting adenoma of the pars intermedia.64 The example of a typical pituitary adenoma (Fig. 20.64) provided occurred in a young animal (w2.5 years old). This micro-tumor was characterized by a well-demarcated,

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Other tumors of the pituitary gland of NHPs include a single report of a pituicytoma in a rhesus macaque.66

FIGURE 20.62 Patterns of thyroid follicular adenocarcinoma (ACA) in NHPs: (A) Papillary ACA (asterisk) in the thyroid gland of an aged animal forms a large nodule, with the tumor filling an expanded follicular lumen. (B) A young (˂4 year old) NHP with multifocal follicular ACA (asterisks).

unencapsulated, expansile mass composed of neoplastic cells arranged in sheets and ribbons. Neoplastic cells were polygonal with scant cytoplasm, round stippled nuclei, and indistinct nucleoli. Occasional pseudorosettes were present. Less commonly there may be atypical adenomas in NHPs. The case presented was a 2.5-year-old female with a welldemarcated nodular enlargement of one lobe of the pars distalis that compressed the adjacent pars intermedia (Fig. 20.65A) The neoplastic population consisted of primarily small eosinophilic cells and large basophilic cells with perinuclear, cytoplasmic, hyaline to granular, eosinophilic inclusions (Fig. 20.65B) Inclusions of this type have been described in somatotrophic pituitary tumors as aggregates of keratin-intermediate filaments and referred to as “fibrous bodies.”65

FIGURE 20.63 Patterns of thyroid follicular adenocarcinoma (ACA): (A) Papillary ACA generally forms ribbons or trabecula that may form solid masses (B) Higher magnification of Fig. 20.62A: Thyroid ACA may have fronds of fibrous tissue lined by neoplastic cells and may have concretions of mineral. (C) Higher magnification of Fig. 20.62B: Thyroid Follicular ACA retains much of the follicular structure but is lined by neoplastic cells.

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FIGURE 20.64 A typical pituitary microadenoma in a macaque: Within the pars distalis, there is a well-demarcated, unencapsulated, expansile mass composed of neoplastic cells arranged in sheets and ribbons and containing tumor cells arranged radially around small vessels (pseudorosettes). Neoplastic cells are polygonal with scant cytoplasm, round stippled nuclei, and indistinct nucleoli. Image courtesy of Rebekah Keesler.

8.4 Endocrine pancreas 8.4.1 Pancreatic neuroendocrine tumors (PanNETs) Pancreatic neuroendocrine tumors (PanNETs) are rare in NHPs, but several cases have been reported in the literature under various names, such as islet cell adenoma, or islet cell tumor.59,67,68 Malignant tumors of islet cells are reported in various primates, such as colobus monkeys69 and recently in a rhesus macaque described as an acinarneuroendocrine carcinoma.67 Carcinomas of the endocrine pancreas have not yet been described in marmosets or cynomolgus monkeys in the literature. Islet cell adenomas are usually solitary nodules, characterized by ribbons or pseudoglands of cuboidal to columnar cells with small, usually uniform nuclei, inconspicuous nucleoli, and rare mitoses. They have a fibrous capsule that can vary in thickness (Fig. 20.66A and B). One insulin secreting tumor (insulinoma) has been documented in literature70; however, all other PanNETs reported either had no indication of functionality or were not evaluated for function.

9. Other findings in endocrine organs of non-human primates 9.1 Adrenal gland multinucleated cells It is not uncommon to encounter multinucleated cells or syncytial cells in the adrenal cortex of macaques. These

FIGURE 20.65 An atypical pititary adenoma in a macaque: (A) There is nodular enlargement of the pars distalis (circle) with compression of the pars intermedia (PI). (B) The neoplastic population contains primarily small eosinophilic cells and large basophilic cells with perinuclear, cytoplasmic, hyalin to granular, eosinophilic inclusions (I) referred to as “fibrous bodies”.

formations are characterized by multiple nuclei within an amorphous cellular structure containing foamy cytoplasm. There may be few or numerous nuclei included (Fig. 20.67A and B).

9.2 Euthanasia artifact Euthanasia artifact has been documented in multiple organs of NHPs, including lung and liver,71 but it is not uncommonly noted in the adrenal gland as well. It usually presents as smudgy vascular profiles with dissociation and degeneration of adjacent cellular components (Fig. 20.68). Occasionally, there may be acute extravasation of blood or fluid with pooling in the affected region. There is no inflammation associated with the tissue changes.

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FIGURE 20.66 An Islet cell adenoma in a macaque: (A) There is a solitary nodule of neoplastic cells encased by a fibrous capsule within the pancreas of a cynomolgus monkey. (B) The neoplastic cells form ribbons of cuboidal to columnar cells with small uniform nuclei, inconspicuous nucleoli, and rare mitoses.

FIGURE 20.67 (A) Multinucleated cells in the adrenal cortex of macaques (arrows) are relatively common. (B) Large syncytial cells characterized by multiple nuclei within an amorphous foamy cytoplasm. There may be few or numerous nuclei included.

9.3 Fat metaplasia of the adrenal gland Fatty metaplasia of the adrenal gland zona reticulata is exceptionally rare in macaques (Fig. 20.69A and B). Fat metaplasia has been described in humans of the zona fasciculata or the zona reticularis, often in conjunction with bone marrow metaplasia.72 The authors speculated that the pathogenesis was related to localized cellular necrosis in combination with local endocrine stimulation. Although this finding is most often associated with additional pathology when reported in humans, no such association has been made with NHPs.

10. Toxicologically induced lesions of the endocrine system 10.1 Immune-mediated hypersensitivity reactions One of the most common, indirect effects associated with biologic test articles administered to macaques is immune-

FIGURE 20.68 Euthanasia artifact of the adrenal gland presents as smudgy vascular profiles with dissociation and degeneration of adjacent cellular components (arrow). There may be fluid or blood accumulation in these regions.

mediated hypersensitivity reactions, particularly, immunecomplex disease (ICD) secondary to the development of antidrug antibodies (ADAs). The fine- to medium-sized vasculature that is common within the endocrine system is prone to ICD injury, leading to vascular congestion, perivascular hemorrhage, increased leukocyte trafficking

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FIGURE 20.69 Fat metaplasia of the adrenal gland: (A) Within the adrenal gland of a cynomolgus monkey, there are fat-laden cells in the zona reticularis near the corticomedullary junction (arrows). (B) The cells are large with multiple cytoplasmic lipid vacuoles.

within small caliber vessels, and vasculitis (Fig. 20.70A and B) Peracute infusion reactions may be due to ICD or may occur via immediate-type immune reactions or cytokine release syndrome and usually have little or no inflammation if tissue is examined within a short period following onset; however, there may be significant hemorrhage, edema, and necrosis associated with vascular beds (Fig. 20.71).

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FIGURE 20.70 (A) Immune complex disease due to antidrug antibody formation is exceptionally common in NHPs following the administration of biologic agents and may result in vascular congestion, perivascular hemorrhage, increased leukocyte trafficking, and vasculitis in the adrenal gland or other endocrine organs. (B) At higher magnification of Fig. 20.70A, the increased leukocyte and blood content of the dilated microvasculature is easily identified in the adrenal gland.

nodes draining the administration site, and in the adrenal gland of NHPs (Fig. 20.72)

10.2 Postvaccination bacille Calmette-Gue´rin (BCG) granulomas Bacille Calmette-Guérin (BCG), a live, attenuated Mycobacterium bovis vaccine, has been utilized for immunization against tuberculosis. BCG is also occasionally utilized to sensitize NHPs in the development of a delayed-type hypersensitivity animal model. Reports of disseminated BCG infections postvaccination in humans, particularly immunocompromised individuals, are not uncommon.73e75 A similar presentation has been noted in NHPs vaccinated with BCG a the editor’s facility. These infections produce multiorgan characteristic epithelioid granulomas with Langhans giant cells and may have necrosis or suppuration as a component. They have been typically noted in lymph

FIGURE 20.71 The adrenal gland from an animal that died from peracute infusion reaction: The typical findings of multifocal microhemorrhages throughout the tissue have little or no inflammation present.

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article to cholesterol-rich cells of the adrenal cortex.78 Over the years, a number of toxins, particularly environmental contaminants and plant-based toxins, have been noted to cause adrenal gland toxicity in NHPs. Examples of these adrenotoxic agents include some dioxins (increased adrenal gland weight),79 oleander plant leaves (Nerium oleanderdcortical necrosis/hemorrhage),80 and ricin toxin from castor beans (Ricinus communisdcortical necrosis).81

10.4 General toxicity of the thyroid and parathyroid glands FIGURE 20.72 Following vaccination with bacille Calmette Guérin (BCG), there may be multiorgan granuloma formation in the NHP, of which the adrenal gland is a common location. The granulomas have typical epithelioid macrophages with Langhans giant cells and may have necrosis or suppuration as a component.

10.3 General toxicity of the adrenal gland The adrenal gland of the macaque is sensitive to toxins, much like that of the human primate, and acquires lesions from toxic substances in a similar manner. However, the response of the adrenal glands to toxic insult may be species specific due to variable sensitivities or alterations in metabolic pathways. For example, o,p’-DDD (Mitotane) is utilized for adrenal cortical carcinoma ablation in humans; however, the macaque is relatively resistant to the effects of this compound.76 Nonetheless, toxic injury to the adrenal gland may occur with test article administration, and may manifest in a variety of lesions, including inflammation, atrophy, hyperplasia, hypertrophy, necrosis, fibrosis, and vacuolar degeneration. These findings predominate in the adrenal cortex because many compounds are converted to toxic metabolites by the abundant cytochrome P450 enzymes of the cortex and many are soluble in the lipid-rich cortical cells. There are a number of published accounts of adrenotoxic substances, many of which have been recorded in monkeys as test subjects, and which outline associated mechanisms of action. For example, suramin, a polysulfonated naphthylurea, has been used for decades in the treatment of African sleeping sickness (trypanosomiasis) and was considered for treatment of HIV in the 1980s. Administration of suramin to macaques resulted in cortical architectural disruption with loss of the zona glomerulosa and zona fasciculata, along with inflammation and reduced function based on serum cortisol concentration.77 In one study, administration of an acyl-CoA: cholesterol acyltransferase inhibitor, to cynomolgus monkeys resulted in dose-related decreased cytoplasmic fine vacuolation and increased eosinophilia of the zona fasciculata and reticularis with associated reduction of serum cholesterol. The author attributed the toxic changes to targeting of the test

A variety of lesions may be noted in the thyroid glands of NHPs following administration of drugs or toxins. Perhaps the most common toxic changes in thyroid glands consist of hypertrophy, hyperplasia, or atrophy. These findings have complicated sources associated with the thyroid directly or the pituitary-thyroid axis. For example, administration of thiouracil, developed in the 1940s as a treatment for Grave’s Disease, leads to follicular cell hyperplasia in macaques82 while administration of synthetic thyroid hormones leads to follicular atrophy. Both results are due to enzyme inhibition: the first results in direct thyroid peroxidase inhibition in the follicular apical epithelium, decreased thyroid hormone biosynthesis, and compensatory hyperplasia; the second leads to downregulation of enzymes for TSH production due to feedback inhibition of the pituitary-thyroid axis. Administration of polychlorinated biphenyl (PCB) to NHPs caused alterations in colloid staining and follicular epithelial hypertrophy with nuclear enlargement, enlarged follicles, and desquamated follicular cells by an unknown mechanism.83 Other toxic changes in thyroid glands may be noted on occasion. In monkeys administered minocycline (a tetracycline antibiotic) for a month, nonfluorescent, black pigment developed in the follicular intraepithelial colloid droplets, but can also be seen in the colloid. Similar has been seen in rats and dogs of the study. Minocycline may inhibit iodide peroxidase leading to enzyme degradation into pigmented deposits.84 Similar black pigment had been noted in humans receiving the drug, and after investigation, it was determined to be lipofuscin with increased phagolysosomes presentda disorder of the lysosome/substrate induced by the drug.85 Similar to humans, NHPs are sensitive to radiation of the thyroid gland. Total body irradiation of rhesus macaques produced morphological alterations of the thyroid gland with decreased organ weight; however, the authors found no evidence of hypothyroidism.86 Toxic effects are not limited to follicular cells. For instance, diffuse C-cell hyperplasia has been induced in the rhesus monkey by daily intramuscular injections of vitamin D and adding a CaCl2 solution to their feed and drinking

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water. This led to an increased number of C-cells after 10 days.16

10.5 General toxicity of the pituitary gland and hypothalamus Exogenous compounds that cause injury to the pituitary gland or hypothalamus have far ranging downstream affects. Toxins affecting the hypothalamicdpituitaryd gonadal axis are plentiful in the literature and the reader is referred to the reproductive chapters of this book and the standard literature search engines for further information. The hypothalamic-pituitary-adrenal axis has been targeted for alteration for therapeutic reasons resulting in either inhibition or stimulation of the system. For example, yohimbine hydrochloride is a a2-adrenergic receptor antagonist that produces increased serum prolactin and cortisol and which has been used extensively in NHPs to upregulate the HPA in this respect.87,88

10.6 General toxicity of the endocrine pancreas Toxins specific to the endocrine pancreas have found use in experimental models of diabetes mellitus (DM). Streptozotocin (STZ), a selective b-cell toxicant derived from the bacterium Streptomyces achromogenes, is widely used to induce DM in NHPs. Gatifloxacin, a broad spectrum fluoroquinolone antibiotic, causes vacuolation of the beta cells due to dilatation of the smooth endoplasmatic reticulum while synthetic progestogens administered for long periods may cause hyalinization and fibrosis of the islets in monkeys.89

11. Conclusion An understanding of the spontaneous and toxicologically induces lesions of the endocrine organs is required for basic toxicologic studies, and separation of the incidental from the toxicologically induced findings is not always straightforward. With the current materials provided in this chapter, in conjunction with the rising utilization of ancillary tests and assays, the line between incidental and otherwise should be more clear, and the future of endocrinology as a core of NHP studies has a bright future.

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87. Gold MS, Donabedian RK, Redmond DE. Further evidence for alpha2 adrenergic receptor mediated inhibition of prolactin secretion: the effect of yohimbine. Psychoneuroendocrinology October 1978;3(3e4):253e60. 88. Coplan JD, Mathew SJ, Smith EL, et al. Effects of LY354740, a novel glutamatergic metabotropic agonist, on nonhuman primate

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Chapter 21

Clinical pathology of the non-human primate Angela L. Wilcox1, William Siska1 and Florence M. Poitout-Belissent2 1

Clinical Pathology, Charles River Laboratories, Reno, NV, United States; 2Clinical Pathology, Charles River Laboratories, Montreal ULC,

Senneville, QC, Canada

1. Introduction Non-human primates (NHPs) are genetically and physiologically similar to humans and are frequently used in preclinical studies, although their use is tightly regulated by animal welfare regulations and presents significant ethical dilemmas.1 Regulatory guidelines specify the use of two animal species for preclinical toxicity testing and NHPs are the most common nonrodent species used given their relevance to humans. Adherence to the 3R’s (reduce, replace, and refine) and generally high cost of conducting NHP studies result in small group sizes that present comprehensive challenges for study data interpretation, including clinical pathology.

2. Challenges and rationale for clinical pathology evaluations of non-human primates in biomedical research Clinical pathology testing in NHPs includes evaluation of hematology, coagulation, clinical chemistry, and urinalysis parameters as part of routine preclinical safety assessment and is an essential tool to identify potential target organ toxicity during pharmaceutical drug development. These evaluations are carried out noninvasively and typically antemortem. Additionally, these assessments are necessary to help determine dose relationships and adverse effects that may influence dose selection and design of future studies, the results of which may be extrapolated to humans for risk assessment and management. Evaluation of clinical pathology is important to identify the onset of toxic effects, which can be monitored during the course of the study and may serve as critical endpoints for monitoring in human patients. In 1996, The Joint Scientific Committee for International Harmonization of Clinical Pathology Testing

recommended clinical pathology evaluation for large animal studies within 7 days of the first day of dosing and at least one time point during the study as well as at study termination for subchronic and chronic studies.2 Years later, safety assessment studies have become increasingly more complex, and the timing of clinical pathology sample collection needs to be carefully planned in order to increase the likelihood of identifying pharmacodynamic and toxicologic effects and limit the impact of other aspects of the study design on clinical pathology data. For example, less frequent clinical pathology monitoring (e.g., biweekly, monthly) may be appropriate for a small molecule administered once daily via oral gavage over a long duration, while intravenous administration of a large molecule with rapid effects on hematopoietic cells would require more frequent monitoring and sample collections within 1e2 days of dose administration.3 Clinical pathology evaluation prior to dose administration (often referred to as pretreatment or prestudy) is recommended to provide baseline data and ensure animals are healthy for inclusion in the study. Recently, the Regulatory Affairs Committee of the American Society for Veterinary Clinical Pathology reviewed current guidelines provided by regulatory agencies including the Organization for Economic Co-operation and Development (OECD), United States Food and Drug Administration (FDA), European Medicines Agency (EMA), Ministry of Health, Labor and Welfare (MHLW), and Environmental Protection Agency (EPA) for inclusion of clinical pathology in routine toxicology studies. A “fit-for-purpose” approach emerged as an acceptable method of design depending on the class and type of test article.4 Additional clinical pathology endpoints such as hormones, cardiac troponin I, urinary fractional excretions of electrolytes, D-dimers, acute phase proteins (e.g., C-reactive protein), etc., may be indicated based on

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previous experience with the test article or known class effects and should be included as part of the “fit-for-purpose” approach in order to fulfill the study objectives. Clinical pathology laboratories that perform analysis of NHP samples in support of applications to regulatory agencies are governed by Good Laboratory Practices (GLP), a stringent set of requirements applicable to all facets of sample handling, processing, analysis, and reporting of clinical pathology data. In the authors’ experiences, data generated from each study are most commonly reported in separate tables containing individual animal and summary data, which also include the results of statistical analyses when applicable. Clinical pathology data should be correlated with various study endpoints including clinical observations, toxicokinetics, antidrug antibodies, immunophenotyping, organ weights, and histopathology to provide a more meaningful interpretation and enhance understanding of the toxiclogic profile of the test article. Clinical pathology data of NHPs are best interpreted with consideration of both concurrent control values and baseline data, when available, to reduce potential interpretive challenges associated with small group sizes, inter-animal variability, and study design factors.5 A number of factors can influence changes in clinical pathology parameters including age, species, sex, handling procedures, and stress; therefore, availability of control data generated under the same study conditions (e.g., time of day, handling, etc.) is ideal. However, when writing a clinical pathology report, the author must decide which of the data sets to report as the principal comparator for interpretation. Dose range finding and maximum tolerated dose studies are often small with only one animal per sex per group and may not include a control group. In such cases, comparison to baseline values is necessary. In studies in which two baseline time points are collected, the second time point (e.g., closest to initiation of dosing) is generally preferred for evaluation because the animals have had additional time to acclimate to handling and study procedures, therefore, the data are less likely to be influenced by stress, excitement, and other variables. Determination of test articleerelated effects is usually decided using a “weight-of-evidence” approach. Factors to consider include the magnitude and incidence of the change, potential study design influences (e.g., handling and blood sampling), inter- or intra-animal variability, presence of a dose relationship, and correlations to clinical observations, immunophenotyping, and anatomic pathology data. Statistical analysis and reference interval data can be helpful to support a test articleerelated interpretation when used with a “weight-of-evidence” approach but should not be used alone as the primary justification for describing a change as test article related. Further, the small number of animals on standard NHP studies (usually 3 to 5 per sex per group) limits the statistical power to identify subtle test articleerelated changes.

3. Clinical pathology sample collection and analysis Blood for clinical pathology is most often collected from the femoral vein in NHPs, but other venipuncture sites may include the saphenous, cephalic, and jugular veins.6 Blood is usually collected from alert animals that are manually restrained or, less frequently, restrained in primate chairs. Infrequently, blood samples may also be collected from animals under sedation or anesthesia. Care must be taken when interpreting clinical pathology data under these circumstances because differences have been reported when compared with data from alert animals.7 NHPs are routinely fasted (withholding of food, but with access to water) prior to blood sample collection in order to minimize the effects of feeding on glucose and lipid values. Appropriate acclimation of animals prior to study initiation helps them develop familiarity with handling and study procedures, and other study design factors and reduces stress (such as social housing) that can decrease the amount of intraanimal data variability, particularly for hematology parameters.8 The amount of blood sampling required depends on the type of toxicity study, with recommended sampling limits based on the weight of the animal. A 5 kg cynomolgus monkey has approximately 65 mL/kg or 325 mL of blood and a 5 kg rhesus macaque has approximately 56 mL/kg or 280 mL of blood.6 Guidelines for toxicity studies with single blood collection suggest 10% removal with a 2-week recovery with a maximum of up to 15% with a 4-week recovery period. Studies with multiple blood collections (e.g., toxicokinetic studies) often require removal of higher volumes over a 24-h period of time with maximal collection of up 20% followed by a 3-week recovery period.6 The type of blood collection tubes used for routine clinical pathology evaluation are selected based on the type of analysis being conducted. For clinical chemistry, empty tubes without anticoagulant or serum separator tubes are most commonly used to yield serum, although lithium heparin tubes for collection of plasma may also be used. Collection of samples for hematology and coagulation testing require the use of tubes containing anticoagulants. For hematology, tubes containing ethylenediamine tetraacetic acid (EDTA) are used for analysis of whole blood, while for coagulation, plasma is harvested using tubes containing sodium citrate. Gentle inversion of the tubes containing anticoagulant ensures adequate mixing of the blood and minimizes clotting. The use of a tube rocker for hematology samples is recommended until the samples are analyzed. Heparin is not recommended as an anticoagulant for hematology evaluation because it can alter cell morphology and cause platelet clumping. In order to attain the most accurate results for samples that cannot be analyzed within 8 h of collection, blood-filled tubes should be refrigerated and analyzed within 24 h. Blood smears

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should also be prepared from the EDTA-preserved sample using a microhematocrit tube. Samples for clinical chemistry should be allowed to clot at ambient temperature for 30 min before removal of the serum. Samples for coagulation should be centrifuged and the plasma removed for analysis. Samples for clinical chemistry and coagulation testing can be frozen for future analysis as most analytes are stable for weeks to months in frozen serum and plasma.

4. Hematology Hematologic evaluation includes assessment of erythrocyte, leukocyte, and platelet parameters. Leukocytes comprise the total white blood cell count and consist of neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Most hematology analyzers are capable of running a standard panel of parameters on