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Macaques with self-injurious behavior (SIB) have been used as a model of human SIB and have previously been shown to respond to treatments targeting enhancement of central serotonin signaling, whether by supplementation with tryptophan, or by inhibiting synaptic reuptake. Decreased serotonin signaling in the brain has also been implicated in many human psychopathologies including major depression disorder. A disturbance in tryptophan metabolism that moves away from the production of serotonin and toward the production of kynurenine has been proposed as a major etiological factor of depression. We hypothesized that in macaques with SIB, central tryptophan metabolism would be shifted toward kynurenine production, leading to lower central serotonin (5-hydroxytryptamine). We analyzed tryptophan metabolites in the cerebral spinal fluid (CSF) of macaques with and without SIB to determine whether and where tryptophan metabolism is altered in affected animals as compared with behaviorally normal controls. We found that macaques with SIB had lower CSF concentrations of serotonin than did behaviorally normal macaques, and that these deficits were inversely correlated with the severity of abnormal behavior. However, our results suggest that this decrease is not due to shifting of the tryptophan metabolic pathway toward kynurenine, as concentrations of kynurenine were also low. Concentrations of IL6 were elevated, suggesting central inflammation. Determining the mechanism by which serotonin function is altered in self-injurious macaques could shed light on novel therapies for SIB and other disorders of serotonin signaling.

Simian immunodeficiency virus (SIV) infection of macaques recapitulates many aspects of HIV pathogenesis and is similarly affected by both genetic and environmental factors. Psychosocial stress is associated with immune system dysregulation and worse clinical outcomes in people with HIV. This study assessed the impact of single housing, as a model of psychosocial stress, on innate immune responses of pigtailed macaques ( Macaca nemestrina ) during acute SIV infection.A retrospective analysis of acute SIV infection of 2- to si6-year-old male pigtailed macaques was performed to compare the innate immune responses of socially ( n = 41) and singly ( n = 35) housed animals. Measures included absolute monocyte count and subsets, and in a subset ( n ≤ 18) platelet counts and activation data.SIV infection resulted in the expected innate immune parameter changes with a modulating effect from housing condition. Monocyte number increased after infection for both groups, driven by classical monocytes (CD14 + CD16 - ), with a greater increase in socially housed animals (227%, p.001, by day 14 compared with preinoculation time points). Platelet numbers recovered more quickly in the socially housed animals. Platelet activation (P-selectin) increased by 65% ( p = .004) and major histocompatibility complex class I surface expression by 40% ( p = .009) from preinoculation only in socially housed animals, whereas no change in these measures occurred in singly housed animals.Chronic psychosocial stress produced by single housing may play an immunomodulatory role in the innate immune response to acute retroviral infection. Dysregulated innate immunity could be one of the pathways by which psychosocial stress contributes to immune suppression and increased disease severity in people with HIV.

Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage (CVL). CVL constituents have utility as biomarkers and play protective roles in wound healing and against HIV‐1 infection. However, several components of cervicovaginal fluids are less well understood, such as extracellular RNAs and their carriers, for example, extracellular vesicles (EVs). EVs comprise a wide array of double‐leaflet membrane extracellular particles and range in diameter from 30 nm to over one micron. The aim of this study was to determine whether differentially regulated CVL microRNAs (miRNAs) might influence retrovirus replication. To this end, we characterized EVs and miRNAs of primate CVL during the menstrual cycle and simian immunodeficiency virus (SIV) infection of macaques. EVs were enriched by stepped ultracentrifugation, and miRNA profiles were assessed with a medium‐throughput stem‐loop/hydrolysis probe qPCR platform. Whereas hormone cycling was abnormal in infected subjects, EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV‐depleted CVL supernatant. Only a small number of CVL miRNAs changed during the menstrual cycle or SIV infection, for example, miR‐186‐5p, which was depleted in retroviral infection. This miRNA inhibited HIV replication in infected macrophages in vitro. In silico target prediction and pathway enrichment analyses shed light on the probable functions of miR‐186‐5p in hindering HIV infections via immunoregulation, T‐cell regulation, disruption of viral pathways, etc. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract., By comparing the microRNA and extracellular vesicle profiles of cervicovaginal lavage of both uninfected and infected macaques, we observed an enrichment of microRNAs in extracellular vesicle‐depleted supernatant and discovered a significant reduction in miR‐186‐5p after simian immunodeficiency virus infection. Furthermore, once transfected into HIV‐infected human macrophages, synthetic miR‐186‐5p mimics inhibited HIV replication, demonstrating its antiretroviral potential.

Evidence suggesting that HIV pathogenesis differs by sex, a variable known to influence the extent and breadth of immune responses in health and disease continues to accumulate. Host factors that promote or inhibit HIV replication may do so in a way that varies by sex. Prior studies using cultured human macrophages demonstrated that osteopontin (OPN)/secreted phosphoprotein-1 (SPP1) stimulates HIV replication. To test whether OPN has the same positive impact on virus replication at the level of tissues, we used a humanized mice model of low-level chronic HIV infection and in which OPN RNA and protein expression was inhibited with targeted aptamers. Interestingly, 4 months after infection when there were no significant differences in HIV viral load in plasma between groups however in contrast, in the spleen, lung, and liver the tissue burden of HIV RNA, as well as the proportion of leukocytes in female and male mice differed depending on whether OPN was expressed or not. The findings collectively demonstrate the potential for complex interactions between host factors like OPN and sex to influence different facets of HIV tissue-level pathogenesis.

The current review examines the role of brain macrophages, that is perivascular macrophages and microglia, as a potential viral reservoir in antiretroviral therapy (ART) treated, simian immunodeficiency virus (SIV)-infected macaques. The role, if any, of latent viral reservoirs of HIV and SIV in the central nervous system during ART suppression is an unresolved issue. HIV and SIV infect both CD4 lymphocytes and myeloid cells in blood and tissues during acute and chronic infection. HIV spread to the brain occurs during acute infection by the infiltration of activated CD4 lymphocytes and monocytes from blood and is established in both embryonically derived resident microglia and monocyte-derived perivascular macrophages. ART controls viral replication in peripheral blood and cerebrospinal fluid in HIV-infected individuals but does not directly eliminate infected cells in blood, tissues or brain. Latently infected resting CD4 lymphocytes in blood and lymphoid tissues are a well recognized viral reservoir that can rebound once ART is withdrawn. In contrast, central nervous system resident microglia and perivascular macrophages in brain have not been examined as potential reservoirs for HIV during suppressive ART. Macrophages in tissues are long-lived cells that are HIV and SIV infected in tissues such as gut, lung, spleen, lymph node and brain and contribute to ongoing inflammation in tissues. However, their potential role in viral persistence and latency or their potential to rebound in the absence ART has not been examined. It has been shown that measurement of HIV latency by HIV DNA PCR in CD4 lymphocytes overestimates the size of the latent reservoirs of HIV that contribute to rebound that is cells containing the genomes of replicative viruses. Thus, the quantitative viral outgrowth assay has been used as a reliable measure of the number of latent cells that harbor infectious viral DNA and, may constitute a functional latent reservoir. Using quantitative viral outgrowth assays specifically designed to quantitate latently infected CD4 lymphocytes and myeloid cells in an SIV macaque model, we demonstrated that macrophages in brain harbor SIV genomes that reactivate and produce infectious virus in this assay, demonstrating that these cells have the potential to be a reservoir.

In November 2015, an 83-d-old juvenile male common marmoset (Callithrix jacchus) in good body condition was found dead in his family cage with no previous premonitory signs. Necropsy revealed a gas-distended abdomen, feces-distended large bowel, and a full-thickness distal colonic perforation resulting in fecal peritonitis. The distal colon ended in a blind pouch at 7 mm prior to the expected anal opening, consistent with atresia ani. Here we present this case, briefly discuss the human and veterinary literature regarding correction of anorectal malformations, and highlight the importance of identifying such devastating congenital defects in breeding colonies while limiting the disruption and handling of seemingly healthy, young NHP raised in a complex social setting.

Extracellular vesicles (EVs) have great potential as novel drug carriers for the treatment of various diseases. These lipid bilayer vesicles are naturally abundant in mammalian tissues and circulation, can be loaded with therapeutic small molecule drugs, (si)RNA, proteins and CRISPR/Cas9, and may be engineered for retention by specific tissues. However, many questions remain on the optimal dosing, administration route, and pharmacokinetics of EVs. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available from larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had markedly longer circulation times in plasma than previously reported in mice, and were detectable in cerebrospinal fluid (CSF) after 30-60 minutes. Already after one minute following IV administration, we observed EV uptake by PBMCs, most notably B-cells. EVs were detected in liver and spleen within one hour of IV administration. None of the IN doses resulted in readily detectable EV levels in plasma, CSF, or organs, suggesting that IN delivery of EVs in large animals including humans may require reconsideration or pretreatment approaches. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.

To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 post-inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

To catalyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, including development of novel interventive and preventive strategies, the progression of disease was characterized in a robust coronavirus disease 2019 (COVID-19) animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at 2, 4, 7, 14, and 28 days after inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal time points, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 after inoculation, corresponding with widespread necrosis and inflammation. At day 7, when the presence of infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 after inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow-resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term MkL, had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an MkL-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in an MHC II-dependent manner both in vitro and in vivo. These data indicated that MkL had key immune regulatory roles dictated in part by the tissue environment.

Background Although social distancing is a key public health response during viral pandemics, psychosocial stressors, such as social isolation, have been implicated in adverse health outcomes in general [1] and in the context of infectious disease, such as human immunodeficiency virus (HIV) [2, 3]. A comprehensive understanding of the direct pathophysiologic effects of psychosocial stress on viral pathogenesis is needed to provide strategic and comprehensive care to patients with viral infection. Methods To determine the effect of psychosocial stress on HIV pathogenesis during acute viral infection without sociobehavioral confounders inherent in human cohorts, we compared commonly measured parameters of HIV progression between singly (n = 35) and socially (n = 41) housed simian immunodeficiency virus (SIV)-infected pigtailed macaques (Macaca nemestrina). Results Singly housed macaques had a higher viral load in the plasma and cerebrospinal fluid and demonstrated greater CD4 T-cell declines and more CD4 and CD8 T-cell activation compared with socially housed macaques throughout acute SIV infection. Conclusions These data demonstrate that psychosocial stress directly impacts the pathogenesis of acute SIV infection and imply that it may act as an integral variable in the progression of HIV infection and potentially of other viral infections.

Background Osteopontin (OPN) as a secreted signaling protein, is dramatically induced in response to cellular injury and neurodegeneration. Microglial inflammatory responses in the brain are tightly associated with the neuropathologic hallmarks of neurodegenerative disease, but understanding of the molecular mechanisms remains in several contexts, poorly understood. Methods Positron emission tomography (PET) neuroimaging using radioligands to detect increased expression of the translocator protein (TSPO) receptor in the brain, is a non-invasive tool used to track neuroinflammation in living mammals. Results In humanized, chronically HIV-infected mice in which OPN expression was knocked down with functional aptamers, uptake of TSPO radioligand, DPA-713 was markedly upregulated in the hippocampus, cortex, olfactory bulb, cerebellum and significantly increased in other key brain regions analyzed compared to controls. TSPO+ microglia were detected by immunolabeling of post-mortem brain tissue, thus validating the neuroimaging findings. Unexpectedly, two types of neurons also selectively stained positively for TSPO. The reactive cells were the specialized neurons of the cerebellum, Purkinje cells, and a subset of tyrosine hydroxylase positive neurons of the substantia nigra. Two-way ANOVA of immunoreactivity revealed that a well-validated marker of microglial activation in brain tissue, ionized calcium-binding adaptor molecule-1 (Iba-1) was significantly increased, in an interaction that depended on HIV replication. Interestingly, similar analyses of TSPO immunoreactivity showed a significant interaction with OPN expression. Conclusions Collectively, these findings using a model of chronic HIV-infection revealed for the first time two key findings: 1) two different pathways of neuroinflammation are activated, and 2) osteopontin acts as a molecular brake regulating in the brain, the inflammatory response to HIV infection.

Researchers have worked with animals as models for decades to expand our knowledge of basic biological processes and to systematically study the physiology of disease. In general, the public has an expectation that work with animals has a purpose and will ultimately reap benefits. The likelihood of such an outcome is directly dependent on the quality of the science being conducted with those animals. However, not all frameworks for consideration of the ethics around animal research overtly consider scientific quality. In the following review, we explore the complex relationship between scientific quality and animal research ethics. We advocate for the development of a detailed “Harm-Yield Analysis” for the evaluation of biomedical animal research that emphasizes scientific quality along with societal benefit in the ethical justification of the research. We reflect on the lost opportunity to establish best practices in animal research early in the career of scientists by introducing in the curriculum and encouraging the use of a paradigm of the iterative consideration of the ethics of animal research alongside other aspects of experimental design.

While social distancing is a key public health response during viral pandemics, social stress, which can be induced by social isolation, has been implicated in adverse health outcomes in general1 and in the context of infectious disease, such as HIV2,3. A comprehensive understanding of the direct pathophysiologic effects of social stress on viral pathogenesis is needed to provide strategic and comprehensive care to patients with viral infection. To determine the effect of social stress on HIV pathogenesis during acute viral infection without sociobehavioral confounders inherent in human cohorts, we compared commonly measured parameters of HIV progression between singly and socially housed SIV-infected pigtailed macaques (Macaca nemestrina). Singly housed macaques had a higher viral load in the plasma and cerebrospinal fluid and demonstrated greater CD4 T cell declines and greater CD4 and CD8 T cell activation compared to socially housed macaques throughout acute infection. These data demonstrate that social stress directly impacts the pathogenesis of acute HIV infection and imply that social stress may act as an integral variable in the progression of HIV infection and potentially of other viral infections.

Simian immunodeficiency virus (SIV) infection of pigtailed macaques is a highly representative and well-characterized animal model for HIV neuropathogenesis studies that provides an excellent opportunity to study and develop prognostic markers of HIV-associated neurocognitive disorders (HAND) for HIV-infected individuals. SIV studies can be performed in a controlled setting that enhances reproducibility and offers high-translational value. Similar to observations in HIV-infected patients receiving antiretroviral therapy (ART), ongoing neurodegeneration and inflammation are present in SIV-infected pigtailed macaques treated with suppressive ART. By developing quantitative viral outgrowth assays that measure both CD4+ T cells and macrophages harboring replication competent SIV as well as a highly sensitive mouse-based viral outgrowth assay, we have positioned the SIV/pigtailed macaque model to advance our understanding of latent cellular reservoirs, including potential CNS reservoirs, to promote HIV cure. In addition to contributing to our understanding of the pathogenesis of HAND, the SIV/pigtailed macaque model also provides an excellent opportunity to test innovative approaches to eliminate the latent HIV reservoir in the brain.

An 8-year-old, male Rhesus macaque (Macaca mulatta), previously used for dengue virus (DENV) vaccine research with viral challenge, was presented with adult-onset, chronic, cyclic thrombocytopenia. Platelet number, morphology, and function were evaluated by automated hematology, peripheral blood smears, electron microscopy, flow cytometry, and impedance aggregometry. Bone marrow was evaluated by cytology. Both serum anti-dengue nonstructural protein 1 (NS1) antibodies and anti-platelet antibodies were detected by ELISA. Platelet characterization showed a lack of aggregation to all agonists (ADP, ASP, and collagen), increased activation with increased expression of surface marker (HLA-ABC), and an absence of surface receptor GPIX during clinical episodes of petechiae and ecchymoses, even in the presence of normal platelet counts. Bone marrow aspirates identified potential mild megakaryocytic hypoplasia. All platelet functions and morphologic attributes were within normal limits during clinically normal phases. Presence of anti-dengue NS1 serum antibodies confirmed a positive DENV titer 8 years postvaccination. Based on the history and clinical findings, a primary differential diagnosis for this chronic, cyclic platelet pathology was autoimmune platelet destruction with potential bone marrow involvement.

This study provides further evidence that the latent reservoir is comprised of both CD4+ T cells and myeloid cells. The data presented here suggest that CD4+ T cells and macrophages found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. Additionally, we have shown that monocytes in blood contain latent virus and, though not considered a reservoir themselves due to their short life span, could contribute to the size of the latent reservoir upon entering the tissue and differentiating into long-lived macrophages. These new insights into the size and location of the SIV reservoir using a model that is heavily studied in the HIV field could have great implications for HIV-infected individuals and should be taken into consideration with the development of future HIV cure strategies., Human immunodeficiency virus (HIV) eradication or long-term suppression in the absence of antiretroviral therapy (ART) requires an understanding of all viral reservoirs that could contribute to viral rebound after ART interruption. CD4 T cells (CD4s) are recognized as the predominant reservoir in HIV type 1 (HIV-1)-infected individuals. However, macrophages are also infected by HIV-1 and simian immunodeficiency virus (SIV) during acute infection and may persist throughout ART, contributing to the size of the latent reservoir. We sought to determine whether tissue macrophages contribute to the SIVmac251 reservoir in suppressed macaques. Using cell-specific quantitative viral outgrowth assays (CD4-QVOA and MΦ-QVOA), we measured functional latent reservoirs in CD4s and macrophages in ART-suppressed SIVmac251-infected macaques. Spleen, lung, and brain in all suppressed animals contained latently infected macrophages, undetectable or low-level SIV RNA, and detectable SIV DNA. Silent viral genomes with potential for reactivation and viral spread were also identified in blood monocytes, although these cells might not be considered reservoirs due to their short life span. Additionally, virus produced in the MΦ-QVOA was capable of infecting healthy activated CD4s. Our results strongly suggest that functional latent reservoirs in CD4s and macrophages can contribute to viral rebound and reestablishment of productive infection after ART interruption. These findings should be considered in the design and implementation of future HIV cure strategies.

This study suggests that CD4+ T cells found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. In addition, this study demonstrates that macrophages in tissues are another cellular reservoir for SIV and may contribute to viral rebound after treatment interruption. This new insight into the size and location of the SIV reservoir could have great implications for HIV-infected individuals and should be taken into consideration for the development of future HIV cure strategies., Understanding the cellular and anatomical sites of latent virus that contribute to human immunodeficiency virus (HIV) rebound is essential for eradication. In HIV-positive patients, CD4+ T lymphocytes comprise a well-defined functional latent reservoir, defined as cells containing transcriptionally silent genomes able to produce infectious virus once reactivated. However, the persistence of infectious latent virus in CD4+ T cells in compartments other than blood and lymph nodes is unclear. Macrophages (Mϕ) are infected by HIV/simian immunodeficiency virus (SIV) and are likely to carry latent viral genomes during antiretroviral therapy (ART), contributing to the reservoir. Currently, the gold standard assay used to measure reservoirs containing replication-competent virus is the quantitative viral outgrowth assay (QVOA). Using an SIV-macaque model, the CD4+ T cell and Mϕ functional latent reservoirs were measured in various tissues using cell-specific QVOAs. Our results showed that blood, spleen, and lung in the majority of suppressed animals contain latently infected Mϕs. Surprisingly, the numbers of CD4+ T cells, monocytes, and Mϕs carrying infectious genomes in blood and spleen were at comparable frequencies (∼1 infected cell per million). We also demonstrate that ex vivo viruses produced in the Mϕ QVOA are capable of infecting activated CD4+ T cells. These results strongly suggest that latently infected tissue Mϕs can reestablish productive infection upon treatment interruption. This study provides the first comparison of CD4+ T cell and Mϕ functional reservoirs in a macaque model. It is the first confirmation of the persistence of latent genomes in monocytes in blood and Mϕs in the spleen and lung of SIV-infected ART-suppressed macaques. Our results demonstrate that transcriptionally silent genomes in Mϕs can contribute to viral rebound after ART interruption and should be considered in future HIV cure strategies. IMPORTANCE This study suggests that CD4+ T cells found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. In addition, this study demonstrates that macrophages in tissues are another cellular reservoir for SIV and may contribute to viral rebound after treatment interruption. This new insight into the size and location of the SIV reservoir could have great implications for HIV-infected individuals and should be taken into consideration for the development of future HIV cure strategies.

Objective Resting CD4 T cells have been recognized as the major cell reservoir of latent HIV-1 during antiretroviral therapy (ART). Using an simian immunodeficiency virus (SIV)/macaque model for AIDS and HIV-related neurocognitive disorders we assessed the contribution of the brain to viral latency and reactivation. Design Pigtailed macaques were dual inoculated with SIVDeltaB670 and SIV17E-Fr and treated with an efficacious central nervous system-penetrant ART. After 500 days of viral suppression animals were treated with two cycles of latency reversing agents and increases in viral transcripts were examined. Methods Longitudinal plasma and cerebrospinal fluid (CSF) viral loads were analyzed by quantitative and digital droplet PCR. After necropsy, viral transcripts in organs were analyzed by PCR, in-situ hybridization, and phylogenetic genotyping based on env V1 loop sequences. Markers for neuronal damage and CSF activation were measured by ELISA. Results Increases in activation markers and plasma and CSF viral loads were observed in one animal treated with latency reversing agents, despite ongoing ART. SIV transcripts were identified in occipital cortex macrophages by in-situ hybridization and CD68 staining. The most abundant SIV genotype in CSF was unique and expanded independent from viruses found in the periphery. Conclusion The central nervous system harbors latent SIV genomes after long-term viral suppression by ART, indicating that the brain represents a potential viral reservoir and should be seriously considered during AIDS cure strategies.

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due to marked systemic toxicities. Herein we demonstrate that DON inhibits glutamine metabolism and provides antitumor efficacy in a murine model of glioblastoma, although toxicity was observed. To enhance DON's therapeutic index, we utilized a prodrug strategy to increase its brain delivery and limit systemic exposure. Unexpectedly, simple alkyl ester-based prodrugs were ineffective due to chemical instability cyclizing to form a unique diazo-imine. However, masking both DON's amine and carboxylate functionalities imparted sufficient chemical stability for biological testing. While these dual moiety prodrugs exhibited rapid metabolism in mouse plasma, several provided excellent stability in monkey and human plasma. The most stable compound (5c, methyl-POM-DON-isopropyl-ester) was evaluated in monkeys, where it achieved 10-fold enhanced cerebrospinal fluid to plasma ratio versus DON. This strategy may provide a path to DON utilization in glioblastoma multiforme patients.

In November 2015, an 83-d-old juvenile male common marmoset (Callithrix jacchus) in good body condition was found dead in his family cage with no previous premonitory signs. Necropsy revealed a gas-distended abdomen, feces-distended large bowel, and a full-thickness distal colonic perforation resulting in fecal peritonitis. The distal colon ended in a blind pouch at 7 mm prior to the expected anal opening, consistent with atresia ani. Here we present this case, briefly discuss the human and veterinary literature regarding correction of anorectal malformations, and highlight the importance of identifying such devastating congenital defects in breeding colonies while limiting the disruption and handling of seemingly healthy, young NHP raised in a complex social setting.

Platelet decline is a feature of many acute viral infections, including cytomegalovirus (CMV) infection in humans and mice. Platelet sequestration in association with other cells, including endothelium and circulating leukocytes, can contribute to this decline and influence the immune response to and pathogenesis of viral infection. We sought to determine if platelet-endothelial associations (PEAs) contribute to platelet decline during acute murine CMV (mCMV) infection, and if these associations affect viral load and production. Male BALB/c mice were infected with mCMV (Smith strain), euthanized at timepoints throughout acute infection and compared to uninfected controls. An increase in PEA formation was confirmed in the salivary gland at all post-inoculation timepoints using immunohistochemistry for CD41+ platelets co-localizing with CD34+ vessels. Platelet depletion did not change amount of viral DNA or timecourse of infection, as measured by qPCR. However, platelet depletion reduced viral titer of mCMV in the salivary glands while undepleted controls demonstrated robust replication in the tissue by plaque assay. Thus, platelet associations with endothelium may enhance the ability of mCMV to replicate within the salivary gland. Further work is needed to determine the mechanisms behind this effect and if pharmacologic inhibition of PEAs may reduce CMV production in acutely infected patients.

Musculoskeletal disease is one of the most prevalent categories of pathology in the captive common marmoset. In this chapter, we will review the basic physiology of the musculoskeletal system, highlight the peculiarities of this system in marmosets, and discuss common methods for the diagnosis of musculoskeletal disease, including radiographic and biomarker assays. We will then specifically review the etiology, diagnosis, and treatment of common diseases of the musculoskeletal system in marmosets, including metabolic bone disease, and marmoset wasting syndrome, and discuss how marmosets have been used as translational animal models for osteoporosis, arthritis, and other musculoskeletal diseases.

Lentiviruses infect myeloid cells, leading to acute infection followed by persistent/latent infections not cleared by the host immune system. HIV and SIV are lentiviruses that infect CD4+ lymphocytes in addition to myeloid cells in blood and tissues. HIV infection of myeloid cells in brain, lung, and heart causes tissue-specific diseases that are mostly observed during severe immunosuppression, when the number of circulating CD4+ T cells declines to exceeding low levels. Antiretroviral therapy (ART) controls viral replication but does not successfully eliminate latent virus, which leads to viral rebound once ART is interrupted. HIV latency in CD4+ lymphocytes is the main focus of research and concern when HIV eradication efforts are considered. However, myeloid cells in tissues are long-lived and have not been routinely examined as a potential reservoir. Based on a quantitative viral outgrowth assay (QVOA) designed to evaluate latently infected CD4+ lymphocytes, a similar protocol was developed for the assessment of latently infected myeloid cells in blood and tissues. Using an SIV ART model, it was demonstrated that myeloid cells in blood and brain harbor latent SIV that can be reactivated and produce infectious virus in vitro, demonstrating that myeloid cells have the potential to be an additional latent reservoir of HIV that should be considered during HIV eradication strategies.

Introduction: The goal of this study was to characterize extracellular vesicles (EVs) and miRNA profiles of primate cervicovaginal lavage (CVL) during the menstrual cycle and simian immunodeficiency virus (SIV) infection, and to determine if CVL-associated miRNAs might influence replication of retroviruses. Methods: CVL and peripheral blood were collected for five weeks from two uninfected and four SIV-infected macaques. EVs were enriched by stepped ultracentrifugation and characterized by population-level and single vesicle analyses. miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform and validated by targeted qPCR assays. Influence of miR-186-5p (miR-186) on HIV protein and RNA production was monitored in monocyte-derived macrophages. Results: Although menstrual hormone cycling was abnormal in infected subjects, EV concentration increased with progesterone in uninfected subjects. miRNAs were present predominantly in the EV-depleted supernatant fraction of CVL. Few changes in miRNA levels correlated with the menstrual cycle or SIV infection. miR-186, depleted in retroviral infection, was investigated for a possible role in controlling retroviral infection. miR-186 inhibited HIV production when transfected into monocyte-derived macrophages. Conclusions: We report profiles of a targeted set of miRNAs in CVL fractions. The menstrual cycle may affect quantity of EVs recovered from CVL, but has only minor effects on abundance of the miRNAs we examined. miR-186 decline appears to be associated with SIV infection, and miR-186 inhibits HIV replication in macrophages in vitro. Further studies are required to characterize the role of EVs and small RNAs as biomarkers of disease in the reproductive tract.

Background It is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART. Methods and findings Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection. Conclusions We report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission., Timothy Henrich and colleagues study the effect of very early antiretroviral treatment on the reservoir of HIV-infected cells in two patients., Author summary Why was this study done? Early initiation of ART following infection may limit the total body burden of HIV. It is not known if starting ART extremely early after HIV infection will lead to ART-free remission or cure. We studied 2 individuals who started ART an estimated 10 and 12 days after HIV infection with very low peak viral load measurement; extensive testing of blood and tissue for HIV persistence was performed. One participant stopped ART in order to test if and when HIV would rebound. What did the researchers find? No HIV could be definitively detected for up to 2 years in the participant who initiated ART approximately 10 days after HIV infection. Intermittent, very low levels of HIV were detected in blood but not tissue in the participant who initiated ART an estimated 12 days following infection. The participant with no detectable HIV following ART experienced viral rebound 225 days after stopping ART. What do these findings mean? HIV relapsed despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission.

Sensitive assays are needed for the detection of residual viral reservoirs in HIV-1-infected subjects on suppressive combination antiretroviral therapy regimens to determine whether eradication strategies are effective. Mouse viral outgrowth assays have recently been developed and have the potential to be more sensitive than traditional in vitro quantitative viral outgrowth assays. In this article we describe these assays and review several studies that have used them to measure the latent reservoir.

Serial phlebotomy is a common sampling practice for repeated-measures studies in biomedical research. In NHP, the effect of serial blood collection on RBC parameters has been characterized, but the effects on platelet parameters and other aspects of the hemogram have not been well studied. We sought to characterize the circulating platelet phenotype throughout the course of 7 serial phlebotomies spanning 30 d in pigtailed macaques (Macaca nemestrina). Phlebotomy was performed on 23 animals at days 0, 2, 4, 7, 10, 21, and 30 to quantify the circulating platelet count and markers of both hemostatic and immune platelet activation. Platelet immune activation was characterized by increases in surface MHC class I and II expression and increases in circulating platelet-leukocyte aggregates. These changes occurred in the absence of increases in the prohemostatic markers P-selectin and CD40L and without evidence of adverse clinical effects. Mild increases in platelet count, mean platelet volume, and immune activation occurred early in the study. After day 21, mean platelet volume and other hematologic parameters returned to baseline while changes in platelet count and immune activation were greater than during the first 10 d of the study. These data demonstrate that serial phlebotomy in NHP has delayed effects on platelet parameters, which may be a source of clinically silent, immunologic and physiologic variability within repeated measures studies. The impact of these effects on research aims should be considered when designing protocols requiring serial phlebotomy in NHP.

A retrospective neuropathologic review of 30 SIV-infected pigtailed macaques receiving combination antiretroviral therapy (cART) was conducted. Seventeen animals with lymphocyte-dominant inflammation in the brain and/or meninges that clearly was morphologically distinct from prototypic SIV encephalitis and human immunodeficiency virus encephalitis were identified. Central nervous system (CNS) infiltrates in cART-treated macaques primarily comprised CD20+ B cells and CD3+ T cells with fewer CD68+ macrophages. Inflammation was associated with low levels of SIV RNA in the brain as shown by in situ hybridization, and generally was observed in animals with episodes of cerebrospinal fluid (CSF) viral rebound or sustained plasma and CSF viremia during treatment. Although the lymphocytic CNS inflammation in these macaques shared morphologic characteristics with uncommon immune-mediated neurologic disorders reported in treated HIV patients, including CNS immune reconstitution inflammatory syndrome and neurosymptomatic CSF escape, the high prevalence of CNS lesions in macaques suggests that persistent adaptive immune responses in the CNS also may develop in neuroasymptomatic or mildly impaired HIV patients yet remain unrecognized given the lack of access to CNS tissue for histopathologic evaluation. Continued investigation into the mechanisms and outcomes of CNS inflammation in cART-treated, SIV-infected macaques will advance our understanding of the consequences of residual CNS HIV replication in patients on cART, including the possible contribution of adaptive immune responses to HIV-associated neurocognitive disorders.

Peripheral neuropathy is the most frequent neurological complication of HIV infection, affecting more than one-third of infected patients, including patients treated with antiretroviral therapy. Although emerging noninvasive techniques for corneal nerve assessments are increasingly being used to diagnose and monitor peripheral neuropathies, corneal nerve alterations have not been characterized in HIV. Here, to determine whether SIV infection leads to corneal nerve fiber loss, we immunostained corneas for the nerve fiber marker βIII tubulin. We developed and applied both manual and automated methods to measure nerves in the corneal subbasal plexus. These counting methods independently indicated significantly lower subbasal corneal nerve fiber density among SIV-infected animals that rapidly progressed to AIDS compared with slow progressors. Concomitant with decreased corneal nerve fiber density, rapid progressors had increased levels of SIV RNA and CD68-positive macrophages and expression of glial fibrillary acidic protein by glial satellite cells in the trigeminal ganglia, the location of the neuronal cell bodies of corneal sensory nerve fibers. In addition, corneal nerve fiber density was directly correlated with epidermal nerve fiber length. These findings indicate that corneal nerve assessment has great potential to diagnose and monitor HIV-induced peripheral neuropathy and to set the stage for introducing noninvasive techniques to measure corneal nerve fiber density in HIV clinical settings.

Background Thrombocytopenia is a known consequence of HIV infection, and decreased production of platelets has been previously implicated in the pathogenesis of platelet decline during asymptomatic infection. Thrombopoietin (THPO) drives platelet production by stimulating the maturation of bone marrow megakaryocytes and can be transcriptionally downregulated by cytokines that are increased during infection such as transforming growth factor β (TGFβ) and platelet factor 4 (pf4). Design To determine whether transcriptional downregulation of THPO contributed to decreased platelet production during asymptomatic infection in the simian immunodeficiency virus (SIV)/macaque model of HIV, we compared hepatic THPO mRNA levels to platelet number and megakaryocyte density. To identify potential inhibitory factors that decrease THPO transcription during asymptomatic infection, we measured TGFβ and pf4 plasma levels. To determine whether combined antiretroviral therapy (cART) could correct platelet decline by altering cytokine levels, we measured TGFβ and pf4 in cART-treated SIV-infected macaques and compared these values to cART-untreated SIV-infected macaques. Results Hepatic THPO transcription was downregulated during asymptomatic SIV infection concurrent with platelet decline. Hepatic THPO mRNA levels correlated with bone marrow megakaryocyte density. In contrast, plasma TGFβ levels were inversely correlated with hepatic THPO transcription and bone marrow megakaryocyte density. With cART treatment, plasma TGFβ levels and platelet count returned to values similar to those in uninfected macaques. Conclusions TGFβ-mediated downregulation of hepatic THPO may lead to decline in platelet number during asymptomatic SIV infection, and cART may prevent platelet decline by normalizing plasma TGFβ levels.

Objective HIV-associated neurocognitive deficits remain a challenge despite suppressive combined antiretroviral therapy. Given the association between HIV-induced central nervous system (CNS) disease and replication of HIV in immune-activated macrophages, CCR5 antagonists may attenuate CNS disease by modulating inflammatory signaling and by limiting viral replication. Design To establish whether initiating CCR5 inhibition during early infection altered CNS disease progression, outcomes were compared between simian immunodeficiency virus (SIV)-infected macaques treated with maraviroc (MVC) versus untreated SIV-infected macaques. Methods Six SIV-infected rhesus macaques were treated with MVC monotherapy for 5 months beginning 24 days postinoculation; 22 SIV-infected animals served as untreated controls. SIV RNA levels in plasma, cerobrospinal fluid, and brain, and CNS expression of TNFα and CCL2 were measured by qRT-PCR. Immunostaining for CD68 and amyloid precursor protein in the brain was measured by image analysis. Plasma sCD163 was measured by ELISA. Results SIV RNA and proviral DNA levels in brain were markedly lower with MVC treatment, demonstrating CCR5 inhibition reduces CNS replication of SIV and may reduce the CNS latent viral reservoir. MVC treatment also lowered monocyte and macrophage activation, represented by CNS CD68 immunostaining and plasma sCD163 levels, and reduced both TNFα and CCL2 RNA expression in brain. Treatment also reduced axonal amyloid precursor protein immunostaining to levels present in uninfected animals, consistent with neuroprotection. Conclusion CCR5 inhibitors may prevent neurologic disorders in HIV-infected individuals by reducing inflammation and by limiting viral replication in the brain. Furthermore, CCR5 inhibitors may reduce the latent viral reservoir in the CNS. Adding CCR5 inhibitors to combined antiretroviral regimens may offer multiple neuroprotective benefits.

Platelets are key participants in innate immune responses to pathogens. As a decrease in circulating platelet count is one of the initial hematologic indicators of human immunodeficiency virus (HIV) infection, we sought to determine whether decline in platelet number during acute infection results from decreased production, increased antibody-mediated destruction, or increased platelet activation in a simian immunodeficiency virus (SIV)/macaque model. During acute SIV infection, circulating platelets were activated with increased surface expression of P-selection, CD40L and major histocompatibility complex class I. Platelet production was maintained and platelet autoantibodies were not detected during acute infection. Concurrent with a decrease in platelet numbers and an increase in circulating monocytes, platelets were found sequestered in platelet-monocyte aggregates, thereby contributing to the decline in platelet counts. Because the majority of circulating CD16(+) monocytes formed complexes with platelets during acute SIV infection, a decreased platelet count may represent platelet participation in the innate immune response to HIV.

Thrombocytopenia is common in HIV and SIV infection, and is often associated with disease progression. HIV and SIV-associated thrombocytopenia arise through multiple mechanisms, including decreased platelet production, increased platelet destruction due to HIV-mimetic anti-platelet antibodies, and increased use of activated platelets. Activated platelets have the potential to contribute to the pathogenesis of HIV and SIV by interacting directly with inflammatory cells and endothelium and by releasing soluble immunomodulatory cytokines.

Background Sensitive assays are needed for detection of residual human immunodeficiency virus (HIV) in patients with undetectable plasma viral loads to determine whether eradication strategies are effective. The gold standard quantitative viral outgrowth assay (QVOA) underestimates the magnitude of the viral reservoir. We sought to determine whether xenograft of leukocytes from HIV type 1 (HIV)-infected patients with undetectable plasma viral loads into immunocompromised mice would result in viral amplification. Methods Peripheral blood mononuclear cells or purified CD4(+) T cells from HIV or simian immunodeficiency virus (SIV)-infected subjects with undetectable plasma viral loads were adoptively transferred into NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. The mice were monitored for viremia following depletion of human CD8(+) T cells to minimize antiviral activity. In some cases, humanized mice were also treated with activating anti-CD3 antibody. Results With this murine viral outgrowth assay (MVOA), we successfully amplified replication-competent HIV or SIV from all subjects tested, including 5 HIV-positive patients receiving suppressive antiretroviral therapy (ART) and 6 elite controllers or suppressors who were maintaining undetectable viral loads without ART, including an elite suppressor from whom we were unable to recover virus by QVOA. Conclusions Our results suggest that the MVOA has the potential to serve as a powerful tool to identify residual HIV in patients with undetectable viral loads.

HIV-induced damage to the CNS remains a major challenge for over 30 million people in the world despite the successes of combined antiretroviral therapy in limiting viral replication. Predicting development and progression of HIV-associated CNS disease is crucial because prevention and early intervention could be more effective than attempts to promote repair. The SIV/macaque model is the premier platform to study HIV neuropathogenesis, including discovery of predictive factors such as neuroprotective host genes and both blood and CSF biomarkers that precede and predict development of SIV CNS disease. This report details the role of macaque MHC class I genes, longitudinal alterations in biomarkers in the circulation, and expression of inflammatory and neuronal damage markers in CSF using samples from SIV-inoculated pigtailed macaques collected during acute, asymptomatic, and terminal stages of infection.

Self-injurious behavior (SIB) is a common comorbidity of psychiatric disorders but there is a dearth of information about neurological mechanisms underlying the behavior, and few animal models exist. SIB in humans is characterized by any intentional self-directed behavior that leads to wounds, whereas in macaques it is not always accompanied by wounds. We describe a cohort of rhesus macaques displaying SIB as adults, in which changes within the central nervous system were associated with the SIB. In these macaques, increases in central nervous system striatal dopamine (DA) receptor binding (BPND) measured by positron emission tomography (PET) [11C]raclopride imaging correlated with severity of wounding (rs=0.662, P=0.014). Furthermore, utilizing standardized cognitive function tests, we showed that impulsivity (stop signal reaction time, SSRT) and deficits in attentional set shifting (intra-/extradimensional shift) were correlated with increased severity of SIB (rs=0.563, P=0.045 and rs=0.692, P=0.009, respectively). We also tested the efficacy of guanfacine, an α2A adrenergic agonist that acts to improve postsynaptic transmission of neuronal impulses, in reducing SIB. A subset of these animals were enrolled in a randomized experimenter-blinded study that demonstrated guanfacine decreased the severity of wounding in treated animals compared with vehicle-only-treated controls (P=0.043), with residual beneficial effects seen for several weeks after cessation of therapy. Animals with the highest severity of SIB that received guanfacine also showed the most significant improvement (rs=−0.761, P=0.009). The elevated PET BPND was likely due to low intrasynaptic DA, which in turn may have been improved by guanfacine. With underlying physiology potentially representative of the human condition and the ability to affect outcome measures of disease using pharmacotherapy, this model represents a unique opportunity to further our understanding of the biology and treatment of SIB in both animals and humans.

Background Diastolic dysfunction is a highly prevalent cardiac abnormality in asymptomatic as well as ART ‐treated human immunodeficiency virus ( HIV ) patients. Although the mechanisms underlying depressed cardiac function remain obscure, diastolic dysfunction in SIV ‐infected rhesus macaques is highly correlated with myocardial viral load. As cardiomyocytes are not productively infected, damage may be an indirect process attributable to a combination of pro‐inflammatory mediators and viral proteins. Methods and Results Given the diverse roles of CCR5 in mediating recruitment of leukocytes to inflammatory sites and serving as a receptor for HIV entry into cells, we investigated the role of CCR5 in the SIV/macaque model of diastolic dysfunction. We found that in SIV‐infected macaques, CCR5 inhibition dramatically impacted myocardial viral load measured by qRT ‐PCR and prevented diastolic dysfunction measured by echocardiography. Complementary in vitro experiments using fluorescence microscopy showed that CCR5 ligands impaired contractile function of isolated cardiomyocytes, thus identifying CCR5 signaling as a novel mediator of impaired cardiac mechanical function. Conclusions Together, these findings incriminate SIV / HIV gp120‐ CCR 5 as well as chemokine‐ CCR 5 interactions in HIV ‐associated cardiac dysfunction. These findings also have important implications for the treatment of HIV ‐infected individuals: in addition to antiviral properties and reduced chemokine‐mediated recruitment and activation of inflammatory cells, CCR 5 inhibition may provide a cardioprotective benefit by preventing cardiomyocyte CCR 5 signaling.

The increasing use of the common marmoset (Callithrix jacchus) in research makes it important to diagnose spontaneous disease that may confound experimental studies. Bone disease and gastrointestinal disease are two major causes of morbidity and mortality in captive marmosets, but currently no effective antemortem tests are available to identify affected animals prior to the terminal stage of disease. In this study we propose that bone disease and gastrointestinal disease are associated disease entities in marmosets and aim to establish the efficacy of several economical antemortem tests in identifying and predicting disease. Tissues from marmosets were examined to define affected animals and unaffected controls. Complete blood count, serum chemistry values, body weight, quantitative radiographs, and tissue-specific biochemical markers were evaluated as candidate biomarkers for disease. Bone and gastrointestinal disease were associated, with marmosets being over seven times more likely to have either concurrent bone and gastrointestinal disease or neither disease as opposed to lesions in only one organ system. When used in tandem, serum albumin

Amyloidosis is a progressive and ultimately fatal disease in which amyloid, an insoluble fibrillar protein, is deposited inappropriately in multiple organs, eventually leading to organ dysfunction. Although this condition commonly affects macaques, there is currently no reliable method of early diagnosis. Changes in clinical pathology parameters have been associated with amyloidosis but occur in late stages of disease, are nonspecific, and resemble those seen in chronic, idiopathic enterocolitis. A review of animal records revealed that amyloidosis was almost always diagnosed postmortem, with prevalences of 15% and 25% in our rhesus and pig-tailed macaque colonies, respectively. As a noninvasive, high-throughput diagnostic approach to improve antemortem diagnosis of amyloidosis in macaques, we evaluated serum amyloid A (SAA), an acute-phase protein and the precursor to amyloid. Using necropsy records and ELISA analysis of banked serum, we found that SAA is significantly elevated in both rhesus and pig-tailed macaques with amyloid compared with those with chronic enterocolitis and healthy controls. At necropsy, 92% of rhesus and 83% of pig-tailed had amyloid deposition in either the intestines or liver. Minimally invasive biopsy techniques including endoscopy of the small intestine, mucosal biopsy of the colon, and ultrasound-guided trucut biopsy of the liver were used to differentiate macaques in our colonies with similar clinical presentations as either having amyloidosis or chronic, idiopathic enterocolitis. Our data suggest that SAA can serve as an effective noninvasive screening tool for amyloidosis and that minimally invasive biopsies can be used to confirm this diagnosis.

Fur mites are one of the most common ectoparasites of laboratory mice and traditionally are diagnosed through surveillance of individual colony animals. Although multiple diagnostic modalities exist, few recommendations suggest optimal testing methods, target colony populations, or sampling sites. We compared the fur pluck and sticky paper techniques for the diagnosis of Myocoptes musculinus in naturally infested immunocompetent mice and evaluated the effect of mouse age and sampling site on the efficacy of fur plucks. We found that the sticky paper technique was more likely to detect fur mites than were fur plucks. Housing mice individually increased the incidence of false-negative fur pluck tests, whereas sensitivity was equivalent for preweanling and adult mice. The ventral abdomen was the most likely single sampling location to detect evidence of any stage of Myocoptes musculinus, but fur mite eggs were overrepresented on the neck. We found that the surface temperature of the murine neck surface was warmer than was the rump and therefore may represent a unique microenvironment for fur mite egg development. Given our findings, we recommend that group-housed adult or preweanling mice should be selected for Myocoptes musculinus evaluation and that the ventral abdomen should be sampled. When possible, the postmortem sticky paper technique should be used rather than the antemortem fur pluck method.