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Plasmodium knowlesi, a model malaria parasite, is responsible for a significant portion of zoonotic malaria cases in Southeast Asia and must be controlled to avoid disease severity and fatalities. However, little is known about the host-parasite interactions and molecular mechanisms in play during the course of P. knowlesi malaria infections, which also may be relevant across Plasmodium species. Here we contrast P. knowlesi sporozoite-initiated infections in Macaca mulatta and Macaca fascicularis using whole blood RNA-sequencing and transcriptomic analysis. These macaque hosts are evolutionarily close, yet malaria-naïve M. mulatta will succumb to blood-stage infection without treatment, whereas malaria-naïve M. fascicularis controls parasitemia without treatment. This comparative analysis reveals transcriptomic differences as early as the liver phase of infection, in the form of signaling pathways that are activated in M. fascicularis, but not M. mulatta. Additionally, while most immune responses are initially similar during the acute stage of the blood infection, significant differences arise subsequently. The observed differences point to prolonged inflammation and anti-inflammatory effects of IL10 in M. mulatta, while M. fascicularis undergoes a transcriptional makeover towards cell proliferation, consistent with its recovery. Together, these findings suggest that timely detection of P. knowlesi in M. fascicularis, coupled with control of inflammation while initiating the replenishment of key cell populations, helps contain the infection. Overall, this study points to specific genes and pathways that could be investigated as a basis for new drug targets that support recovery from acute malaria., (© 2021. The Author(s).)

Targeting MAIT cells holds promise for the treatment of different diseases and infections. We previously showed that treatment of Mycobacterium tuberculosis infected mice with 5-OP-RU, a major antigen for MAIT cells, expands MAIT cells and enhances bacterial control. Here we treated M. tuberculosis infected rhesus macaques with 5-OP-RU intratracheally but found no clinical or microbiological benefit. In fact, after 5-OP-RU treatment MAIT cells did not expand, but rather upregulated PD-1 and lost the ability to produce multiple cytokines, a phenotype resembling T cell exhaustion. Furthermore, we show that vaccination of uninfected macaques with 5-OP-RU+CpG instillation into the lungs also drives MAIT cell dysfunction, and PD-1 blockade during vaccination partly prevents the loss of MAIT cell function without facilitating their expansion. Thus, in rhesus macaques MAIT cells are prone to the loss of effector functions rather than expansion after TCR stimulation in vivo, representing a significant barrier to therapeutically targeting these cells., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

The maternal decidua is an immunologically complex environment that balances maintenance of immune tolerance to fetal paternal antigens with protection of the fetus against vertical transmission of maternal pathogens. To better understand host immune determinants of congenital infection at the maternal-fetal tissue interface, we performed a comparative analysis of innate and adaptive immune cell subsets in the peripheral blood and decidua of healthy rhesus macaque pregnancies across all trimesters of gestation and determined changes after Zika virus (ZIKV) infection. Using one 28-color and one 18-color polychromatic flow cytometry panel we simultaneously analyzed the frequency, phenotype, activation status and trafficking properties of αβ T, γδ T, iNKT, regulatory T (Treg), NK cells, B lymphocytes, monocytes, macrophages, and dendritic cells (DC). Decidual leukocytes showed a striking enrichment of activated effector memory and tissue-resident memory CD4+ and CD8+ T lymphocytes, CD4+ Tregs, CD56+ NK cells, CD14+CD16+ monocytes, CD206+ tissue-resident macrophages, and a paucity of B lymphocytes when compared to peripheral blood. t-distributed stochastic neighbor embedding (tSNE) revealed unique populations of decidual NK, T, DC and monocyte/macrophage subsets. Principal component analysis showed distinct spatial localization of decidual and circulating leukocytes contributed by NK and CD8+ T lymphocytes, and separation of decidua based on gestational age contributed by memory CD4+ and CD8+ T lymphocytes. Decidua from 10 ZIKV-infected dams obtained 16-56 days post infection at third (n=9) or second (n=1) trimester showed a significant reduction in frequency of activated, CXCR3+, and/or Granzyme B+ memory CD4+ and CD8+ T lymphocytes and γδ T compared to normal decidua. These data suggest that ZIKV induces local immunosuppression with reduced immune recruitment and impaired cytotoxicity. Our study adds to the immune characterization of the maternal-fetal interface in a translational nonhuman primate model of congenital infection and provides novel insight in to putative mechanisms of vertical transmission., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moström, Scheef, Sprehe, Szeltner, Tran, Hennebold, Roberts, Maness, Fahlberg and Kaur.)

During the Zika virus (ZIKV) outbreak in Brazil (2015-2016), the clinical manifestations associated with its infection were complex and included miscarriage and congenital malformations, not previously described. In this study, we evaluated the prenatal conditions of pregnant female squirrel monkeys (Saimiri collinsi) infected during different gestational thirds (GTs) and assessed all clinical aspects, diagnostic imaging, viremia and the immune response. In our study, 75% of the infected animals in the 1st GT group had significant clinical manifestations, such as miscarriage and prolonged viremia associated with a late immune response. Consequently, their neonates showed fetal neuropathology, such as cerebral hemorrhage, lissencephaly or malformations of the brain grooves, ventriculomegaly, and craniofacial malformations. Thus, our study demonstrated the relevance of pregnant squirrel monkeys as a model for the study of ZIKV infection in neonates due to the broad clinical manifestations presented, including the typical congenital Zika syndrome manifestations described in humans.

We present a unique case of metastatic cholangiocarcinoma with concurrent abdominal cestodiasis in an African green monkey (Chlorocebus pygerythrus) that presented with respiratory insufficiency and abdominal discomfort. There were multiple white-grey masses in the liver and colonic serosa alongside intra-abdominal parasitic cysts. Histopathologically, the liver masses were composed of poorly-differentiated epithelial cells that formed densely cellular solid areas and trabeculae. The neoplastic cells were strongly immunopositive for CK7 but negative for Hep-Par1 antigen, which confirmed a diagnosis of cholangiocarcinoma. Interestingly, there was strong and diffuse neoexpression in the tumour of the cell cycle regulator 14-3-3σ, which is not constitutively expressed in normal liver. There was aberrantly strong expression of E-cadherin, a key cell-cell adhesion protein, in neoplastic cells with evidence of cytoplasmic internalization. This is the first immunohistochemical analysis of 14-3-3σ and E-cadherin in a liver neoplasm in an animal species and the use of these markers requires further investigation in animal liver neoplasms., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ebola virus (EBOV) continues to pose a significant threat to human health, as evidenced by the 2013-2016 epidemic in West Africa and the ongoing outbreak in the Democratic Republic of the Congo. EBOV causes hemorrhagic fever, organ damage, and shock culminating in death, with case fatality rates as high as 90%. This high lethality combined with the paucity of licensed medical countermeasures makes EBOV a critical human pathogen. Although EBOV infection results in significant damage to the liver and the adrenal glands, little is known about the molecular signatures of injury in these organs. Moreover, while changes in peripheral blood cells are becoming increasingly understood, the host responses within organs and lymphoid tissues remain poorly characterized. To address this knowledge gap, we tracked longitudinal transcriptional changes in tissues collected from EBOV-Makona-infected cynomolgus macaques. Following infection, both liver and adrenal glands exhibited significant and early downregulation of genes involved in metabolism, coagulation, hormone synthesis, and angiogenesis; upregulated genes were associated with inflammation. Analysis of lymphoid tissues showed early upregulation of genes that play a role in innate immunity and inflammation and downregulation of genes associated with cell cycle and adaptive immunity. Moreover, transient activation of innate immune responses and downregulation of humoral immune responses in lymphoid tissues were confirmed with flow cytometry. Together, these data suggest that the liver, adrenal gland, and lymphatic organs are important sites of EBOV infection and that dysregulating the function of these vital organs contributes to the development of Ebola virus disease. IMPORTANCE Ebola virus (EBOV) remains a high-priority pathogen since it continues to cause outbreaks with high case fatality rates. Although it is well established that EBOV results in severe organ damage, our understanding of tissue injury in the liver, adrenal glands, and lymphoid tissues remains limited. We begin to address this knowledge gap by conducting longitudinal gene expression studies in these tissues, which were collected from EBOV-infected cynomolgus macaques. We report robust and early gene expression changes within these tissues, indicating they are primary sites of EBOV infection. Furthermore, genes involved in metabolism, coagulation, and adaptive immunity were downregulated, while inflammation-related genes were upregulated. These results indicate significant tissue damage consistent with the development of hemorrhagic fever and lymphopenia. Our study provides novel insight into EBOV-host interactions and elucidates how host responses within the liver, adrenal glands, and lymphoid tissues contribute to EBOV pathogenesis., (Copyright © 2020 American Society for Microbiology.)

Zika virus (ZIKV) infection is now firmly linked to congenital Zika syndrome (CZS), including fetal microcephaly. While Aedes species of mosquito are the primary vector for ZIKV, sexual transmission of ZIKV is a significant route of infection. ZIKV has been documented in human, mouse, and nonhuman primate (NHP) semen. It is critical to establish NHP models of the vertical transfer of ZIKV that recapitulate human pathogenesis. We hypothesized that vaginal deposition of ZIKV-infected baboon semen would lead to maternal infection and vertical transfer in the olive baboon ( Papio anubis ). Epidemiological studies suggest an increased rate of CZS in the Americas compared to the original link to CZS in French Polynesia; therefore, we also compared the French Polynesian (FP) ZIKV isolate to the Puerto Rican (PR) isolate. Timed-pregnant baboons ( n  = 6) were inoculated via vaginal deposition of baboon semen containing 10 6 focus-forming units (FFU) of ZIKV ( n  = 3 for FP isolate H/PF/2013; n  = 3 for PR isolate PRVABC59) at midgestation (86 to 95 days of gestation [dG]; term, 183 dG) on day 0 (all dams) and then at 7-day intervals through 3 weeks. Maternal blood, saliva, and cervicovaginal wash (CVW) samples were obtained. Animals were euthanized at 28 days ( n  = 5) or 39 days ( n  = 1) after the initial inoculation, and maternal/fetal tissues were collected. Viremia was achieved in 3/3 FP ZIKV-infected dams and 2/3 PR ZIKV-infected dams. ZIKV RNA was detected in CVW samples of 5/6 dams. ZIKV RNA was detected in lymph nodes but not the ovaries, uterus, cervix, or vagina in FP isolate-infected dams. ZIKV RNA was detected in lymph nodes (3/3), uterus (2/3), and vagina (2/3) in PR isolate-infected dams. Placenta, amniotic fluid, and fetal tissues were ZIKV RNA negative in the FP isolate-infected dams, whereas 2/3 PR isolate-infected dam placentas were ZIKV RNA positive. We conclude that ZIKV-infected semen is a means of ZIKV transmission during pregnancy in primates. The PR isolate appeared more capable of widespread dissemination to tissues, including reproductive tissues and placenta, than the FP isolate. IMPORTANCE Zika virus remains a worldwide health threat, with outbreaks still occurring in the Americas. While mosquitos are the primary vector for the spread of the virus, sexual transmission of Zika virus is also a significant means of infection, especially in terms of passage from an infected to an uninfected partner. While sexual transmission has been documented in humans, and male-to-female transmission has been reported in mice, ours is the first study in nonhuman primates to demonstrate infection via vaginal deposition of Zika virus-infected semen. The latter is important since a recent publication indicated that human semen inhibited, in a laboratory setting, Zika virus infection of reproductive tissues. We also found that compared to the French Polynesian isolate, the Puerto Rican Zika virus isolate led to greater spread throughout the body, particularly in reproductive tissues. The American isolates of Zika virus appear to have acquired mutations that increase their efficacy., (Copyright © 2020 Gurung et al.)

Background: As the widely used biomarker of whole-blood stimulation assays for tuberculosis diagnosis, the release of IFN-γ might be affected by multiple factors, such as immunosuppression and some infectious agents. Here, we evaluated additional cytokines as diagnostic biomarkers., Methods: Forty-three cytokines were measured by Luminex xMAP technologies in 30 healthy and 10 naturally Mycobacterium tuberculosis (MTB)-infected rhesus monkeys pre- and post-stimulation by purified protein derivative (PPD)., Results: After stimulation, production of 23 and 38 cytokines was markedly increased in healthy and MTB-infected macaques, respectively. A comparison of the stimulation index (SI) between MTB infections and healthy macaques showed that the SIs of 32 cytokines in MTB-infected macaques were significantly higher than those in healthy macaques. Pooling the results, eight cytokines were suggested as ideal biomarkers for a whole-blood stimulation assay for MTB diagnosis., Conclusion: PPD could induce multiple cytokine responses in either healthy or MTB-infected monkeys, and eight cytokines had reliable predictive capacity as diagnostic biomarkers of MTB infection., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

An experimentally naïve, 9-y-old, intact male cynomolgus macaque was reported for bleeding from an unidentified site. Sedated physical examination indicated mild gingival separation from the lingual aspect of the upper right canine tooth as the source of the hemorrhage. Physical exam revealed a firm mass adhered to the left zygomatic arch, 2 subcutaneous nodules on the chest, and a large mass in the cranial abdomen. Radiographs revealed a large soft-tissue mass in the cranial abdomen and multifocal nodules in the caudal lung fields. On ultrasonography, the liver was grossly enlarged and contained a cavi- tated mass. Hematology and serum chemistry results demonstrated severe regenerative anemia with normal clotting times and adequate platelet count. For humane reasons, euthanasia was elected. On gross examination, the liver was markedly enlarged by an expansile mass primarily affecting the median lobe, with multiple, smaller nodules throughout the remaining lobes. Multifocal round, firm nodules were observed on the surface of all lung lobes and throughout the omentum. Histologic examination of the hepatic, cutaneous, osseous, and pulmonary lesions demonstrated well-defined, endothelium-lined vascular channels arranged in cords with abundant hemorrhage; endothelial-cell immunomarkers confirmed these results. On the basis of these findings, hepatic hemangiosarcoma, with metastases to the lungs, omentum, subcutis, and bone, was diagnosed. This case study is the first report of spontaneous hepatic hemangiosarcoma in a cynomolgus macaque and the first case with metastasis to bone in a NHP.

Chronic lymphocytic enteritis (CLE) is a frequent disease in common marmosets. However, no diagnostic test for early detection of CLE is available. Mast cells have an important role in gastrointestinal disease. The purpose of this study was to measure fecal concentrations of N-methylhistamine (NMH), a breakdown product of histamine metabolism, in common marmosets. A previously established NMH gas chromatography-mass spectrometry assay for canine feces and urine was used, and partial validation was performed. The reference intervals ( n = 30) established for fecal NMH concentrations in common marmoset were 118.2 ng/g or less for a single fecal sample, 121.7 ng/g or less for the 3-d mean, and less than or equal to 167.5 ng/g for the 3-d maximum. Considerable day-to-day variation was observed in fecal NMH concentrations; the mean %CV was 42.2% (minimum, 7.1%; maximum, 141.4%). Fecal NMH concentrations were measured in 14 marmosets for which necropsy reports were available; 7 of the 8 marmosets with CLE and the 1 animal with lymphoma and ulcerative enteritis had increased fecal NMH concentrations. Increased fecal NMH concentrations may serve as a potential marker for CLE; however, further studies exploring the role of mast cells in marmosets with CLE are needed.

Genetic and environmental factors such as high-fat diet are involved in the development of type 2 diabetes mellitus (T2DM). Cynomolgus monkey shares similar genetic makeup, tissue structures, physiology and metabolic function to human. This study aimed to establish T2DM model in cynomolgus monkey and compare expression profiles of hepatic genes and their associated pathways in normal cynomolgus monkeys and those with T2DM. We employed RNA-seq technique and identified 1451 differentially expressed genes (DEGs) with a false discovery rate (FDR) of 0.1% between normal and T2DM animals. KEGG pathway analysis revealed that DEGs were associated with 12 KEGG pathways (P < 0.05). Two of these pathways were associated with metabolism and five were related to immunity. Unexpected, we found ECM-receptor interaction pathway. In conclusion, our data suggest that three major pathways may be implicated in the development of T2DM, including steroid biosynthesis, immune response and ECM. Further characterization of these pathways may provide new targets for the prevention and therapy of T2DM., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Simian varicella virus (SVV), the primate counterpart of varicella-zoster virus, causes varicella (chickenpox), establishes latency in ganglia, and reactivates to produce zoster. We previously demonstrated that a recombinant SVV expressing enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and in infected monkeys. Here, we generated two additional recombinant SVVs to visualize infected cells in vitro and in vivo One harbors eGFP fused to the N terminus of open reading frame 9 (ORF9) (rSVV.eGFP-2a-ORF9), and another harbors eGFP fused to the C terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (i.t.) or i.t. plus intravenous (i.v.) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) fluid at 10 days postinoculation (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP-expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis. IMPORTANCE SVV infection in nonhuman primates has served as an extremely useful animal model to study varicella-zoster virus (VZV) pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to that with SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on in vitro results should be handled with care, since eGFP expression could result in attenuation of the virus., (Copyright © 2018 American Society for Microbiology.)

Follicle-stimulating hormone (FSH) is essential for mammalian folliculogenesis and spermatogenesis. Common marmoset (Callithrix jacchus) is a New World primate which exhibits an unusual FSH profile across the ovarian cycle with a mid-follicular FSH peak that is not observed in Catarrhini primates like humans. Since transcription of FSH β-subunit gene (FSHβ) is a rate-limiting step in the production of mature FSH, this study aimed to investigate the regulation of marmoset FSHβ gene expression in comparison to human. In silico analysis of the FSHβ promoter sequences identified a TATA box element upstream of the conventional TATA box element in marmoset but not in human sequence. FSHβ mRNA transcript longer than the conventional transcript was detected in marmoset pituitary implying presence of a distal transcription start site. In luciferase reporter assays, the marmoset putative distal promoter had higher activity than the corresponding human region even in absence of the conventional proximal promoter. Indeed higher affinity binding of TATA box-binding protein to the putative distal TATA box element was obtained in electrophoretic mobility shift assay. This suggests existence of a differential regulation of FSHβ transcription in marmoset compared to humans., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Obesity in pregnancy (MO) is a risk factor for maternal and/or fetal cardiovascular system disorders. This study evaluated maternal CVS expression of microRNA-29 family and its target molecules in MO to test the hypotheses: CVS miR-29 concentrations are increased in pregnancy and decreased in MO., Methods: Non-pregnant (n=4), pregnant obese (POb, n=4), and pregnant non-obese (PnOb, n=4) baboons (Papio spp.) were studied. Maternal left ventricle (LV), left atrium (LA), and aortic arch (AA) were collected at the end of gestation. Expression of MiR-29 and elastin (ELN) mRNA were quantified., Results: LA miR-29 (a, c) expression was highest in PnOb. In the LV, miR-29b expression trended lower (P=.059) for PnOb animals. ELN mRNA expression correlated positively with miR-29b expression in AA (r=.76, P=.03)., Conclusion: Maternal obesity diminishes miR-29 adaptation to pregnancy. Pharmacologic, tissue-specific targeting of miRNA-29 may represent a strategy for prevention and treatment of MO complications., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Context: Endometriosis is a common gynecological disease affecting one in 10 women of reproductive age and is a major cause of pelvic pain and impaired fertility. Endometrial stromal cells of women with endometriosis exhibit a reduced response to in vitro decidualization. NOTCH1 is critical for decidualization of both mouse and human uterine stromal cells., Objective: This study aimed to determine whether decidualization failure in women with endometriosis is a consequence of impaired Notch signaling., Setting and Design: We investigated expression levels of Notch signaling components in the endometrium of women and baboons with or without endometriosis. We identified NOTCH1-regulated genes during decidualization of human uterine fibroblast (HuF) cells by microarray and quantified their expression levels in in vitro-decidualized endometrial stromal cells isolated from women with or without endometriosis., Results: Notch signaling receptors NOTCH1 and NOTCH4, ligands JAGGED2 and DLL4, as well as direct target genes HES5 and HEY1 were decreased in the eutopic endometrium of women and baboons with endometriosis. Notch signaling was decreased in stromal cells isolated from women with endometriosis, which was associated with impaired in vitro decidualization. Genes that were down-regulated by NOTCH1 silencing in decidualized HuF cells were also decreased in decidualized endometrial stromal cells of women with endometriosis. FOXO1 acts as a downstream target of Notch signaling and endometriosis is associated with decreased expression of NOTCH1-regulated, FOXO1-responsive genes during decidualization., Conclusions: Decreased Notch signaling is associated with endometriosis and contributes to impaired decidualization through the down-regulation of FOXO1.

Epithelioid hemangiosarcoma is a rare malignant endothelial neoplasia with a unique, predominantly epithelioid morphology. A 4-y-old rhesus monkey from our laboratory had multiple neoplastic nodules in a digit, limb skin, hindlimb muscle, and visceral organs including lung, heart, and brain. The nodules were composed of pleomorphic, polygonal, epithelioid, neoplastic cells that were arranged in sheets, nests, and cords and supported by variably dense fibrovascular connective tissue. The morphologic features of this tumor were predominantly epithelioid. However, some regions contained cystic spaces, clefts, and channel-like structures, all of which were lined with morphologically distinct neoplastic endothelial cells. These neoplastic cells, with or without epithelioid morphology, were positive immunohistochemically for CD31, factor VIII-related antigen, and vimentin. The presence of multiple metastatic nodules, high mitotic rate, and extensive Ki67-positive staining were consistent with malignancy. This report is the first description of epithelioid hemangiosarcoma in a rhesus monkey.

In the United States, breast cancer is the most common malignancy among women, with an estimated lifetime incidence of approximately 12% in American women. Invasive ductal carcinoma is the most common form of breast cancer in women, accounting for approximately 60% of all breast carcinomas. Prognostic markers are used to assess aggressiveness, invasiveness, and extent of spread of a neoplasm and thus may be correlated with patient survival. Immunohistochemistry is currently widely used for this purpose, with a variety of prognostication markers available. Classic markers for breast cancer in women include estrogen and progesterone receptor steroid hormone proteins and human epidermal growth factor receptor 2. Many additional markers have been used in diagnosis and prognostication, including p53, p63, and E-cadherin and cell proliferation markers such as Ki67. Despite an estimated lifetime incidence of approximately 6.1%, naturally occurring mammary neoplasms in nonhuman primates are uncommonly reported, with only sporadic references over the past 75 y. The majority of reported tumors occur in rhesus macaques, although this prevalence has been suggested to be a consequence of their high frequency of usage in biomedical research. Here we present 2 cases of mammary carcinoma in adult female intact rhesus macaques, with cytology, histopathology, and extensive immunohistochemical analysis. According to current classifications for human breast tumors, both tumors were classified as invasive ductal carcinoma. The prognostic value of immunohistochemical markers in human breast cancer and in reported cases in nonhuman primates is discussed.

Five acute-phase reactants-serum amyloid A (SAA), C-reactive protein (CRP), haptoglobin, albumin, and iron-were measured using commercially available assays in 110 healthy rhesus macaques (Macaca mulatta), and reference intervals were established for future use in health monitoring of this species. Reference intervals established were as follows: SAA, 29.5-87.7 mg/L; CRP, 0-17.5 mg/L; haptoglobin, 354.3-2,414.7 mg/ L; albumin, 36.1-53.0 g/L; and iron, 13.3-40.2 micromol/L. Furthermore, changes in the acute-phase reactants were studied in two additional groups of animals: eight rhesus macaques suffering from acute traumatic injuries and nine rhesus macaques experimentally infected with Mycobacterium tuberculosis reflecting a chronic active inflammation. In animals with inflammation, SAA and haptoglobin concentrations were moderately increased, while CRP increased more than 200-fold. In addition, marked decreases in albumin and iron concentrations were observed. These results show that SAA, CRP, and haptoglobin are positive acute-phase proteins, whereas albumin and iron are negative acute-phase reactants in rhesus macaques.

Squirrel monkeys (Saimiri spp) are one of the most consistently used New World primates in biomedical research and are increasingly being used in neuroscience research, including models of drug abuse and addiction. Spontaneous neurologic disease in the squirrel monkey is uncommonly reported but includes various infectious diseases as well as cerebral amyloidosis. Hypernatremia is an extremely serious condition of hyperosmolarity that occurs as a result of water loss, adipsia, or excess sodium intake. Neurologic effects of hypernatremia reflect the cellular dehydration produced by the shift of water from the intracellular fluid space into the hypertonic extracellular fluid space. Severe hypernatremia may result in cerebrocortical laminar necrosis (polioencephalomalacia) in human patients as well as in a number of domestic species, including pigs, poultry, and ruminants. We report the clinical, histopathologic, and immunohistochemical findings of polioencephalomalacia in 13 squirrel monkeys. Polioencephalomalacia in these animals was associated with hypernatremia that was confirmed by serum levels of sodium greater than 180 mmol/L (reference range, 134.0-154.0 mmol/L [mEq/L]). All animals had concurrent diseases or experimental manipulation that predisposed to adipsia. Immunohistochemical investigation using antibodies to neuronal nuclei (NeuN), CNPase, Iba-1, and CD31 revealed necrosis of predominantly cerebral cortical layers 3, 4, and 5 characterized by neuronal degeneration and loss, oligodendrocytic loss, microglial proliferation, and vascular reactivity. The squirrel monkey is exquisitely sensitive to hyperosmolar metabolic disruption and it is associated with laminar cortical necrosis.

Unlabelled: Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention., Importance: Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.

Oncocytic adrenocortical tumours are rare in man and have never been described in non-human primates. An oncocytic adrenocortical carcinoma was identified in an 18-year-old female putty-nosed monkey (Cercopithecus nictitans) with hyperadrenocorticism and invasive aspergillosis. Microscopically, the tumour consisted of large cells with abundant eosinophilic, granular cytoplasm containing numerous mitochondria as identified by electron microscopy. Tumour cells had large nuclei with occasional intranuclear cytoplasmic pseudoinclusions. Immunohistochemically, tumour cells expressed vimentin, synaptophysin and neuron-specific enolase, while they were negative for cytokeratin, chromogranin-A, melan-A and S100., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A 25-year-old, female rhesus macaque (Macaca mulatta) presented with a history of weight loss despite a normal appetite and supportive care. The animal was humanely destroyed due to poor prognosis. Post-mortem examination revealed a focally extensive, firm, white annular constriction at the ileocaecal junction and an incidental finding of a pale white nodule approximately 0.8 cm in diameter in the left renal pelvis. Based on the microscopical findings, ileocaecal adenocarcinoma and renal pelvis transitional cell carcinoma (TCC) were diagnosed. The use of cytokeratin (CK)-7 and -20 and uroplakin III as potential renal TCC markers was evaluated. The neoplastic cells were labelled intensely with antibodies to uroplakin III, but not to CK-7 or -20. Spontaneous intestinal adenocarcinoma has been documented in the rhesus macaque, but concurrent renal pelvis TCC is highly unusual., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Interleukin-21 (IL-21) is produced primarily by CD4+ T cells and regulates immunity against human/simian immunodeficiency virus (HIV/SIV) infection. Activated CD8+ cells and their secreted interferon-gamma (IFN-γ) are crucial for the control of acute HIV/SIV infection. However, whether IL-21 can regulate IFN-γ production by CD8+ cells remains controversial. Rhesus macaques (RMs, n = 8) were infected with SHIV and the levels of plasma IL-21, IFN-γ and the frequency of peripheral blood activated T cells were measured longitudinally. Following infection with SHIV, the levels of plasma IL-21 and IFN-γ increased, peaked at 17 days postinfection and declined later. Furthermore, IL-21 induced IL-21 receptor (IL-21R) and IFN-γ, perforin, but not granmyze B, expression in CD8+ cells from four selected SHIV-infected RMs. The regulatory effect of IL-21 on CD8+ cell function appeared to be associated with increased levels of STAT3, but not STAT5, phosphorylation in CD8+ cells from SHIV-infected RMs. In parallel, treatment with soluble IL-21R/Fc, an inhibitor of IL-21-induced activation of JAK1/3 and STAT3, abrogated IL-21-induced STAT3 activation and IFN-γ production in CD8+ cells from SHIV-infected RMs in vitro. Our data indicated that IL-21 was a positive regulator of IFN-γ-secreting CD8+ cells and increased the STAT3 phosphorylation, regulating T-cell immunity against acute SHIV infection in RMs. Our findings may provide a new basis for the development of immunotherapies for the control of SHIV/HIV infection.

A 32-month-old male common marmoset had a firm and white-colored mass in the duodenal wall. The cut surface was smooth and grayish white in color. Histologically, the mass consisted of a proliferation of spindle cells with an oval to spindle-shaped nucleus and scant eosinophilic cytoplasm in a loose myxoid or fibrotic background. Most of the lesion displayed no specific growth pattern whereas some of the cells concentrated around the vessels and created an onion-bulb structure. Additionally, marked inflammatory cellular infiltration, mainly eosinophils, was observed throughout the lesion. Immunohistochemically, the spindle cells were positive for vimentin, α-smooth muscle actin, fascin, and cyclin D1, and negative for S-100, factor VIII-related antigen, and c-kit. These histological and immunohistochemical features did not meet any differential diagnoses such as gastrointestinal stromal tumor, inflammatory myofibroblastic tumor, solitary fibrous tumor/hemangiopericytoma, smooth muscle tumor, schwannoma, and hemangiosarcoma. Collectively, the authors diagnosed the mass as a lesion that corresponded to an inflammatory fibroid polyp (IFP) in humans. IFP is defined as a mesenchymal proliferation composed of spindle stromal cells, small blood vessels, and inflammatory cells, particularly eosinophils, and is currently classified as a nonneoplastic lesion. To the best of our knowledge, this is the first case of spontaneous IFP in nonhuman primates.

A 25-year-old female mandrill (Mandrillus sphinx) died after exhibiting weakness and recumbency with serosanguineous ascites. Gross findings included haemoperitoneum and multifocal to diffuse serosal thickening with petechiae and ecchymoses throughout the peritoneum. The uterus was covered entirely with large blood clots and was adherent to the ovaries and pelvic wall. Microscopical and immunohistochemical examination revealed extra- and intra-uterine growth of ectopic endometrial tissue with marked fibrosis. The ectopic endometrial tissues predominantly consisted of stromal cells expressing CD10 and progesterone receptor and variably-sized glands lined by the epithelium with occasional slight expression of oestrogen receptor α. A diagnosis of endometriosis was made. This is the first report of naturally occurring endometriosis in a mandrill., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Human African trypanosomiasis is associated with metabolic changes which have not been well characterized., Methods: Chlorocebus aethiops were experimentally infected with Trypanosoma brucei rhodesiense and late-stage disease induced at 28 days post-infection. Ear prick blood for glucose determination and blood samples were obtained at weekly intervals for 56 days. Analysis was carried out using dry chemistry analysis., Results: In early infection, there was a significant increase in creatine kinase, while during early and transitional stage of infection there was a significant decrease in glucose and high-density lipoprotein and an increase in triglyceride levels. In the late stage, there was a significant increase in both total cholesterol and LDL levels., Conclusions: Further investigations should focus on levels of total cholesterol during the follow-up period in curatively treated vervet monkeys. Apart from their importance in disease staging, the changes in lipids levels may also affect the pharmacokinetics of some trypanocides., (© 2011 John Wiley & Sons A/S.)

Endometriosis affects a large percentage of the rhesus macaques (Macaca mulatta) at our institution. When the disease is diagnosed in macaques on long-term research protocols, the treatment of choice in our facility is monthly administration of medroxyprogesterone acetate (MPA) to decrease estrogen release and subsequently diminish clinical signs associated with the disease. Because hormonal fluctuations associated with the normal menstrual cycle are known to affect parameters of glucoregulatory function in rhesus macaques, we evaluated the effect of MPA treatment on glucoregulatory function cross-sectionally in 6 animals and longitudinally in 4 animals with endometriosis. Our hypothesis was that monthly administration of MPA for the treatment of endometriosis would negatively affect glucoregulatory function in rhesus macaques. We found that adult female rhesus macaques on MPA therapy for 1.4 to 36.1 mo had lower insulin sensitivity than did age- and weight-matched healthy control animals. In addition, glucoregulatory function was reduced after MPA treatment as compared with pretreatment levels in a group of 4 macaques. These data suggest that glucoregulatory function should be considered when endometriosis treatment is planned for rhesus macaques.

A 3-y-old male rhesus macaque (Macaca mulatta) was noticed to be lethargic in the compound. Physical exam revealed cyanotic mucous membranes, dyspnea, bilateral harsh lung sounds, wheezing on expiration, and a firm mass possibly associated with the liver. Radiographs revealed bilateral soft tissue opacities in the thorax. Due to poor prognosis, the rhesus was euthanized, and a necropsy was performed. Both right and left lung lobes were consolidated and had multifocal white-tan masses. On cut section, the masses were firm, had areas of necrosis, hemorrhage, and often contained a tenacious exudate. Masses were identified in the liver and both kidneys. Given the morphologic features of the neoplasm, a diagnosis of squamous cell carcinoma was made. Immunohistochemistry staining for thyroid transcription factor, a nuclear transcription factor normally found in lung, thyroid, and tumors arising from either of those tissues, confirmed that the masses originated from the lung. Malignant primary lung tumors are divided into 8 main histologic subtypes: squamous cell carcinoma, small-cell carcinoma, large-cell carcinoma, adenocarcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, carcinoid tumor, and salivary gland tumors. Clinical signs associated with lung tumors include, but are not limited to, dyspnea, coughing, hemoptysis, lethargy, anorexia, and weight loss. Although squamous cell carcinoma will be low on the differential list for these clinical signs, we encourage clinicians and researchers to not rule it out solely based on incidence and age of the animal.

Background: The renoprotective effect of erythropoietin (Epo) against ischaemia-reperfusion injury (IR/I) was evaluated in a non-human primate model., Methods: Crab-eating macaques were divided into two groups: Control (n = 10), treated with saline, and EPO (n = 10), treated with Epo. Epo was injected intravenously at a dose of 12,000 units, 5 min before clamping the renal pedicle and 5 min before declamping. Renal IR/I was created by clamping the left renal artery for 90 min following right nephrectomy. Haemoglobin (Hb), haematocrit (Ht), creatinine (Cr), blood urea nitrogen (BUN), cystatin C and interleukin-6 (IL-6) were measured before (Pre) and after (Day 0) the operation, and on post-operative days: Day 1, Day 3, Day 5 and Day 7. Apoptotic cells were counted on Day 1., Results: There were no differences in Hb and Ht between the two groups. Cr, BUN, cystatin C and IL-6 levels in the EPO group were lower than those in the Control group at most of the observation points. The number of apoptotic cells in the Control was significantly higher than that of and EPO group., Conclusions: Epo significantly ameliorates renal IR/I in this non-human primate model. Our findings justify the clinical application of Epo, not only for acute renal failure, but also in transplantation.

Background: In humans and in baboons, protracted gastro-esophageal reflux (GER) transforms the squamous-lined esophagus into columnar-lined (that is Barrett's mucosa, BM). Alcian blue stain (AB) is used to evidence sialomucin-producing goblet cells in human BM., Aim: To assess the frequency and distribution of sialomucin-producing cells in BM in baboons., Materials and Methods: Sections from 137 consecutive baboon esophagi were alternatively stained with hematoxylin-eosin (H&E) and with AB (pH 2.5), without counterstain., Results: Out of 137 baboons, 131 (95.6%) had BM. Columnar and intramucosal glandular cells produced sialomucin in all 131 of these animals. Many BM cells were ballooned and filled with sialomucins, despite goblet cells not being found in H&E sections., Conclusion: In humans, protracted GER is a disease requiring medication that may lead to BM; AB stains mainly goblet cells and occasional columnar cells in BM. In baboons, in contrast, BM is a natural postnatal process of adaptation to GER, triggered by regurgitation and rumination. AB stains all columnar and intra-mucosal glandular cells. Sialomucin-overstuffed cells were more frequent and larger in baboons than in humans. The extra load of sialomucin in BM might be an integrated part of the postnatal life-long process of adaptation to regurgitation and rumination in baboons.

Because of their mediating role in the stress response and potential effects on fitness, glucocorticoid (GC) hormones are increasingly used to assess the physiological costs of environmental and behavioral variation among wild vertebrates. Identifying the proximate causes of GC variation, however, is complicated by simultaneous exposure to multiple potentially stressful stimuli. Here, we use data from a partially provisioned social group of Sykes' monkeys to evaluate the effects of potential psychological and metabolic stressors on temporal and individual variation in fecal GC (fGC) excretion among 11 adult females. Despite high rates of agonism over provisioned foods fGCs declined during periods of high provisioning frequency when fruit availability was dominated by neem (Azadirachta indica), an item requiring great feeding effort. Provisioned foods did not prevent fGC increases when availability of the most preferred main fruit item, tamarind (Tamarindus indica), declined drastically. Although rank-related differences in access to provisioned foods and rates of agonism did not lead to an overall effect of rank on fGCs, low-ranking females excreted more fGCs than high-ranking females during a period of high provisioning intensity and low fruit availability. The emergence of this rank effect was associated with elevated feeding effort in all females, a greater access to provisioned items by high-ranking females, and a higher proportion of time spent moving in low-ranking females. Our findings suggest that metabolic stressors were the primary determinants of both temporal and individual variation in fGCs, indicating potential fitness benefits for high-ranking females when food availability is limited., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Diabetes mellitus (DM) is a group of chronic metabolic diseases characterized by persistent fasting hyperglycemia, and it can be of either polygenic or monogenic origin. Animal models have played an important role in elucidating the pathophysiology of the polygenic Type 1 and type 2 DM forms; however, useful animal models of the monogenic forms do not exist. The authors describe 4 cases of naturally occurring DM in vervet monkeys (Chlorocebus aethiops sabaeus), 1 of which has clinicopathologic findings consistent with type 2 DM, including persistent hyperglycemia, hypertriglyceridemia, islet amyloidosis, and reduced islet insulin immunostaining. In contrast, the 3 remaining animals have clinicopathologic similarities to a monogenic form of the disease, including a lack of islet amyloidosis and hypertriglyceridemia, as well as normal islet insulin immunostaining. In addition, pedigree analysis conducted on one of these animals is consistent with either an autosomal dominant or mitochondrial inheritance pattern, which supports a monogenic form of DM. The authors thus hypothesize that a naturally occurring monogenic form of diabetes may occur in vervet monkeys, making them a potential animal model for future studies.

Hyperglycemia, a prevalent condition in premature infants, is thought to be a consequence of incomplete suppression of endogenous glucose production and reduced insulin-stimulated glucose disposal in peripheral tissues. However, the molecular basis for these conditions remains unclear. To test the hypothesis that the insulin transduction pathway is underdeveloped with prematurity, fetal baboons were delivered, anesthetized, and euthanized at 125 d gestational age (GA), 140 d GA, or near term at 175 d GA. Vastus lateralis muscle and liver tissues were obtained, and protein content of insulin signaling molecules [insulin receptor (IR)-beta, IR substate-1, p85 subunit of phosphatidylinositol 3-kinase, Akt, and AS160] and glucose transporters (GLUT)-1 and GLUT4 was measured by Western blotting. Muscle from 125 d GA baboons had markedly reduced GLUT1 protein content (16% of 140 d GA and 9% of 175 d GA fetuses). GLUT4 and AS160 also were severely reduced in 125 d GA fetal muscle (43% of 175 d GA and 35% of 175 d GA, respectively). In contrast, the protein content of IR-beta, IR substate-1, and Akt was elevated by 1.7-, 5.2-, and 1.9-fold, respectively, in muscle from 125 d GA baboons when compared with 175 d GA fetuses. No differences were found in the content of insulin signaling proteins in liver. In conclusion, significant gestational differences exist in the protein content of several insulin signaling proteins in the muscle of fetal baboons. Reduced muscle content of key glucose transport-regulating proteins (GLUT1, GLUT4, AS160) could play a role in the pathogenesis of neonatal hyperglycemia and reduced insulin-stimulated glucose disposal.

The aim of the present study was to conduct a semiquantitative immunohistochemical investigation into the levels of intermediary proteins within the nuclear factor (NF)-kappaB pathway throughout the menstrual cycle in a non-human primate, namely the baboon (Papio anubis), with and without endometriosis. Formalin-fixed eutopic (n = 2-4) and ectopic (n = 6-7) endometrial tissues from baboons at the mid-luteal phase were embedded in paraffin and examined for NF-kappaB pathway components (i.e. IkappaB kinase (IKK) alpha, IKKbeta, phosphorylated (phospho-) IkappaBalpha and phospho-NF-kappaB p65 subunit), ubiquitin, 19S proteasome and the NF-kappaB activator tumour necrosis factor (TNF)-alpha. Similarly, endometrial tissues from baboons at the late follicular, mid-luteal and menses phase (n = 2-4) were investigated to determine the levels of these proteins throughout the menstrual cycle. Cytoplasmic stromal IKKalpha and glandular 19S proteasome immunostaining was elevated in the ectopic endometrium, whereas levels of ubiquitin, phospho-p65, IKKbeta, TNF-alpha and nuclear 19S proteasome were similar in the eutopic and ectopic endometrium. A significant decrease in phospho-IkappaBalpha nuclear immunostaining was observed within glandular cells of the ectopic endometrium. In the eutopic endometrium, IKKalpha, ubiquitin and 19S proteasome immunostaining was elevated in different phases of the menstrual cycle, whereas levels of phospho-p65, IKKbeta, phospho-IkappaBalpha and TNF-alpha remained unchanged. We have demonstrated that, in the baboon endometriosis model, levels of IKKalpha immunostaining are elevated, whereas those of phospho-IkappaBalpha are reduced, consistent with the hypothesis that excessive NF-kappaB activity plays a role in reducing ectopic endometrial apoptosis, which contributes to the pathophysiology of endometriosis. Further studies are required to confirm a causal association between elevated IKKalpha levels and reduced endometrial apoptosis.

Background: In previous studies, the length of the glandulo-metaplastic esophageal mucosa (GMEM) at the gastroesophageal junction was assessed in a selected group of baboons. In this study, the length of the GMEM was measured in the entire esophagus in a cohort of unselected adult baboons., Materials and Methods: In 15 female baboons, the entire esophagus was removed en bloc at autopsy, from the tongue to the angle of His. No part of the stomach was included. The length of GMEM was measured using a calibrated ocular microscale., Results: GMEM was found in 11 out of the 15 esophagi. The total length of GMEM recorded in the 11 cases was 115 mm (mean 10.5 mm, range 1-45 mm). The mean age for animals with GMEM was 15.5 years (range 7-32 years) and for animals without GMEM was 14.0 years (range 7-20 years); the difference was non-significant (p<0.6). No significant association was found between the length of the GMEM and the age of the animals (p<0.6)., Conclusion: This study substantiates the notion that GMEM in baboons is a postnatal physiological adaptative process of the esophageal mucosa to daily regurgitation with rumination of gastric juices of low pH. The GMEM apparently progresses upwards, along the esophageal mucosa. The baboon might be an excellent animal model to study the series of histological events that take place in the distal esophagus under the influence of protracted gastroesophageal reflux.

Background: Glands with glassy cells (GGCs) were recently found in 1.8% of patients showing Barrett's mucosa in esophageal biopsies. Similar GGCs were more recently detected in a baboon having glandulo-metaplastic esophageal mucosa (GMEM). The aim was to assess the frequency of baboons with GMEM having GGCs., Materials and Methods: GGCs were sought in 68 consecutive baboons having GMEM. Sections were stained with H&E, and with alcian blue (pH 2.5) to detect sialomucins in goblet cells (a marker of Barrett's mucosa in GMEM)., Results: Two out of the 68 baboons with Barrett's mucosa (2.9%) showed GGCs., Conclusion: In similarity to humans, the Barrett's mucosa in baboons may show GGCs. Although the significance of GGCs in baboons (and in humans) remains poorly understood, their presence might not be a fortuitous event but may be linked to the molecular events leading to the development of intestinal metaplasia in Barrett's mucosa, a known pre-neoplastic mucosal change in the distal esophagus in humans.

A previously undescribed spontaneous cardiomyopathy was identified by routine light microscopic examination of the heart from four clinically healthy purpose-bred cynomolgus monkeys that ranged from four to nine years of age and included 2 males and 2 females. Special stains of Sirius red, Masson's trichrome, and Mallory's phosphotungstic acid hematoxylin (PTAH); and immunohistochemistry using anti-CD68, troponin-I, and desmin antibodies were used to facilitate lesion characterization and assess cardiomyocyte viability. Microscopically, the apical to mid-ventricular myocardium to subendocardium had foci of cardiomyocyte disarray with cytoplasmic pallor to stippling and karyomegaly, vacuolization of the perimyseal connective tissue, a meshwork of fibrous tissue that concentrated around medium-sized blood vessels and dissected between or less often replaced affected cardiomyocytes; and a minimal, predominantly macrophage infiltrate. The disrupted cardiomyocytes were immunoreactive to desmin and troponin-I antibodies and had a normal cross-striation pattern by PTAH, indicating the chronic cardiomyopathy was not associated with active cardiomyocyte damage. The consistent distribution and morphology of the cardiomyopathy suggested a common etiology and pathogenesis. The features were reminiscent of chronic catecholamine-induced experimental cardiomyopathy and stress cardiomyopathy in monkeys and humans, respectively. This report documents another spontaneous heart lesion in clinically healthy monkeys for consideration during interpretation of toxicology studies.

Purpose: To determine if bestrophin is present in the basal membrane of macular retinal pigment epithelium (RPE) and in drusen of rhesus monkeys with age-related drusenoid maculopathy., Methods: The macular region of three rhesus monkeys (Macaca mulatta), 23-24 years of age, with drusenoid maculopathy was dissected from eyes fixed with 4% paraformaldehyde. The macula was sectioned into rectangular pieces. The sclera was removed from each segment and the remainder separated into segments of neural retina with retinal epithelium or choroid with retinal epithelium. These segments were incubated with a goat polyclonal antibody to human bestrophin 1, reacted with gold-labeled rabbit antibody to goat IgG, silver-enhanced, and processed for transmission electron microscopy., Results: Bestrophin-labeled gold particles were found in quasi-linear arrays on the basal surface of the macular RPE and also within drusen where bestrophin was found in segments of membranous-like material. The array density of the bestrophin-linked gold particles on the basal membrane of the epithelium had a maximal value of about 5-100 bestrophin molecules/micron(2). Immuno-detection of bestrophin was most effective when examined in an RPE layer that remained attached to the neural retina, where the basal surface of the epithelium is more directly exposed to the antibodies., Conclusion: Bestrophin is present on the basal membrane of macular RPE of rhesus monkeys with age-related drusenoid maculopathy, and also found in the membranous-like structures of drusen. The latter finding provides insight into the pathogenesis of drusen by indicating that segments of the basal membrane of RPE contribute to the material that accumulates within drusen.

Background: The pig-tailed macaques are the only Old World monkeys known to be susceptible to human immunodeficiency virus type 1 (HIV-1) infection. We have previously reported that the TRIM5-Cyclophilin A (TRIMCyp) fusion in pig-tailed macaques (Macaca nemestrina) is dysfunctional in restricting HIV-1, which may explain why pig-tailed macaques are susceptible to HIV-1 infection. Similar results have also been reported by other groups. However, according to the current primate taxonomy, the previously reported M. nemestrina are further classified into three species, which all belong to the Macaca spp. This calls for the need to look into the previous studies in more details., Results: The local species Northern pig-tailed macaque (M. leonina) was analyzed for the correlation of TRIM5 structure and HIV-1 infection. Eleven M. leonina animals were analyzed, and all of them were found to possess TRIM5-CypA fusion at the TRIM5 locus. The transcripts encoding the dysfunctional TRIM5-CypA should result from the G-to-T mutation in the 3'-splicing site of intron 6. Polymorphism in the putative TRIMCyp recognition domain was observed. The peripheral blood mononuclear cells (PBMCs) of M. leonina were susceptible to HIV-1 infection. Consistent with the previous results, expression of the M. leonina TRIMCyp in HeLa-T4 cells rendered the cells resistant to HIV-2ROD but not to SIVmac239 infection., Conclusion: The susceptibility of M. leonina to HIV-1 infection is due to the dysfunctional TRIM5-CypA fusion in the TRIM5 locus. This finding should broaden our perspective in developing better HIV/AIDS non-human primate animal models.

Background: Chronic diarrhea can be challenging to manage in captive rhesus macaques (Macaca mulatta) leading to ongoing diagnostics, medications, monitoring, and potential euthanasia. Coconut has been used as a dietary supplement for people with inflammatory bowel disease, with anecdotal reports of decreased diarrhea following the dietary addition. A dietary trial in rhesus macaques was initiated to evaluate the hypothesis that dietary coconut decreases symptoms of chronic diarrhea in rhesus macaques., Methods: Ten rhesus macaques with chronic diarrhea were selected for the trial. Five of the subjects were fed coconut macaroons and five of the subjects were fed a sham cookie. Stool consistency was monitored daily for both groups., Results and Conclusions: Data of chi-squared analysis obtained from eight rhesus macaques with chronic diarrhea showed that the use of coconut macaroons as a dietary supplement did not have a statistically significant effect on their diarrhea.

Purpose: To examine patterns of retinal pigment epithelial autofluorescence and lipofuscin accumulation in relation to drusen and to explore the pathogenesis of drusen in rhesus monkeys., Methods: The macular areas of six rhesus monkeys, euthanized at 19 to 28 years of age, were studied by bright field and fluorescence light microscopy and transmission electron microscopy., Results: There was strong autofluorescence in the retinal epithelium that tended to diminish over drusen. Electron microscopy revealed that all retinal epithelial cells had large concentrations of lipofuscin bodies. The epithelial cells overlying drusen, however, tended to have less lipofuscin than epithelial cells not associated with drusen. Electron microscopy revealed that the epithelial cells overlying drusen were losing segments of cytoplasm containing lipofuscin bodies. Macrophage-like cells were consistently present in Bruch's membrane microns away from this lipofuscin-containing cytoplasmic material., Conclusions: Retinal epithelial cells overlying drusen have less lipofuscin than neighboring epithelial cells. The loss of lipofuscin seems due to a loss of cytoplasm containing lipofuscin that contributes to drusen formation. Macrophages in Bruch's membrane may be responsible for removing this lipofuscin debris. The results support in vivo studies showing reduced autofluorescence over drusen and support the "budding" of epithelial cytoplasm as a source of drusen material.

The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5alpha has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA). We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform. All pig-tailed macaques (M. nemestrina) screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101) of rhesus macaques (M. mulatta). The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares approximately 80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a remarkable example of convergent evolution. Based on the presence of the CypA insertion in separate macaque lineages, and its absence from sooty mangabeys, we estimate that the Macaca TRIM5-CypA variant appeared 5-10 million years ago in a common ancestor of the Asian macaques. Whether the formation of novel genes through alternative splicing has played a wider role in the evolution of the TRIM family remains to be investigated.

Gastrointestinal disease and inflammation are common sequelae of human and simian immunodeficiency virus (SIV) infection. Nevertheless, the molecular mechanisms that lead to gastrointestinal dysfunction remain unclear. We investigated regulation of the interleukin (IL)-6-JAK-STAT3 pathway in jejunum and colon, collected at necropsy, from 10 SIV-infected macaques with diarrhea (group 1), 10 non-SIV-infected macaques with diarrhea (group 2), and 7 control uninfected macaques (group 3). All group 1 and 2 macaques had chronic diarrhea, wasting, and colitis, but group 1 animals had more frequent and severe lesions in the jejunum. A significant increase in IL-6 and SOCS-3 gene expression along with constitutive STAT3 activation was observed in the colon of all group 1 and 2 macaques and in the jejunum of only group 1 macaques compared to controls. Further, in colon, histopathology severity scores correlated significantly with IL-6 (groups 1 and 2) and SOCS-3 (group 2) gene expression. In jejunum, a similar correlation was observed only in group 1 animals. Phosphorylated STAT3 (p-STAT3) was localized to lymphocytes (CD3+) and macrophages (CD68+), with fewer CD3+ lymphocytes expressing p-STAT3 in group 1 macaques. Despite high SOCS-3 expression, STAT3 remained constitutively active, providing a possible explanation for persistent intestinal inflammation and immune activation that may favor viral replication and disease pro-gression.

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium, causes self-limited diarrhea in immunocompetent hosts and severe life-threatening diarrhea in AIDS patients. Highly active antiretroviral therapy has been used to effectively treat cryptosporiosis in some but not all AIDS patients. Therefore, there is an urgent need for innovative drugs to treat this disease. Cryptosporidium infection results in intestinal pathophysiological changes such as glucose malabsorption, increased chloride ion (Cl(-)) secretion, and epithelial barrier disruption, leading to disease pathogenesis. In order to develop tools to combat this opportunistic pathogen, it is vital to understand mediators involved in disease pathogenesis. Substance P (SP), a neuropeptide and pain transmitter, is located in the gastrointestinal tract. SP can cause Cl(-) secretion in human gastrointestinal explants. However, its role in cryptosporidiosis has not been fully studied. Jejunal samples from macaques before and after Cryptosporidium parvum infection were assayed for SP and SP receptor mRNA and protein levels by reverse transcription-PCR and by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The role of SP in pathophysiological alterations, such as Cl(-) secretion and glucose malabsorption, was studied using tissues derived from macaques infected with C. parvum by the Ussing chamber technique. SP and SP receptor mRNA and protein expression levels were increased in jejunal samples following C. parvum infection and were accompanied by increased basal ion secretion and glucose malabsorption. In vitro treatment of samples obtained from infected macaques with the SP receptor antagonist aprepitant (Emend; Merck, Whitehouse Station, NJ) completely reversed the increase in basal ion secretion and corrected the glucose malabsorption. Our findings raise the possibility of using SP receptor antagonists for the treatment of symptoms associated with cryptosporidiosis.

Pituitary adenomas were identified in 14 of 491 (2.9%) cynomolgus macaques evaluated from 1994 to 2004. Cases included male (8) and female (6) cynomolgus macaques ranging from 18 to 32 years of age. Seven of the pituitary adenomas caused gross enlargement of the pituitary gland that was visible on postmortem examination, whereas the remaining 7 were multifocal microadenomas identified on histologic examination. A total of 35 adenomas were identified in the 14 macaques, 6 of which were being treated for diabetes mellitus. Mean (+/- SD) pituitary weight was 0.31 +/- 0.42 g, compared with 0.07 +/- 0.02 g for 430 historical control animals (P < 0.0001). Immunohistochemical staining for follicle-stimulating hormone, luteinizing hormone, prolactin, human growth hormone, thyroid-stimulating hormone, and adrenocorticotropic hormone was applied to pituitary tissue from all cases. Immunostaining revealed 22 of 35 (62.9%) lactotroph adenomas, 5 of 35 (14.3%) plurihormonal cell adenomas, 3 of 35 (8.6%) corticotroph adenomas, 2 of 35 (5.7%) null cell adenomas, 1 of 35 (2.9%) somatotroph adenomas, 1 of 35 (2.9%) mixed corticotroph-somatotroph adenomas, 1 of 35 (2.9%) mixed lactotroph-corticotroph adenomas, 0 of 35 gonadotroph adenomas, and 0 of 35 thyrotroph adenomas. This study represents the first extensive retrospective case series performed to evaluate the histologic and immunohistochemical characteristics of pituitary adenomas in cynomolgus macaques. Our findings indicated that macaque pituitary adenomas frequently had mixed histologic appearance and hormone expression, and that, similar to human pituitary adenomas, prolactin-secreting neoplasms were the most prevalent type.

Macaques provide an important animal model for the study of hormonal agents and their effects on risk biomarkers for breast cancer. A common criticism of this model is that spontaneous breast cancer has rarely been described in these animals. In this report, we characterize 35 mammary gland lesions ranging from ductal hyperplasia to carcinoma in situ and invasive ductal carcinoma in cynomolgus and rhesus macaques. Based on a retrospective analysis, we estimated the lifetime incidence of mammary gland neoplasia in aged female macaques to be about 6%. Hyperplastic lesions (n = 19) occurred segmentally along ducts and included such features as columnar alteration, micropapillary atypia, and fibroadenomatous change. In situ carcinomas (n = 8) included solid, comedo, cribriform, and micropapillary elements, encompassing 4 of the major architectural patterns seen in human lesions. Invasive ductal carcinomas (n = 8) were generally solid, with prominent central necrosis and mineralization, often on a background of micropapillary ductal hyperplasia and in situ carcinoma. Cytologic changes of invasive lesions included increased mitoses, nuclear pleomorphism, extensive microinvasion, and stromal desmoplasia. Axillary lymph-node metastases were confirmed in 5 of the 8 invasive carcinomas. On immunohistochemistry, intraductal and invasive carcinomas had increased Ki67/MIB1 and HER2 expression and selective loss of estrogen and progesterone receptors. These findings suggest that breast cancer is an underreported lesion in macaques and highlight unique morphologic and molecular similarities in breast cancer between human and macaque species.

Background: Previous studies have characterized alopecia in captive rhesus macaques (Macaca mulatta) by a mixed partial to complete alopecia in a bilateral symmetric pattern., Methods: In this study, coat condition assessments were related to exogenous and endogenous factors in captive rhesus macaques under different housing conditions in order to identify disturbances in environmental factors controlling or influencing hair growth. Additionally, the degree of alopecia was investigated in relation to adrenal endocrine function as an indicator of social stress using faecal glucocorticoid measurements., Results: Hair loss was found to vary with season and sex, was most pronounced in adult females during the winter and spring months. Generally, infants were not affected, but alopecia developed during adolescence. However, the housing system, available enclosure space and variations in group size and composition also appeared to influence coat condition. Levels of immunoreactive cortisol metabolites (11-oxoetiocholanolone) in faeces were significantly negatively correlated with alopecia, suggesting a relationship between hypothalamic-pituitary-adrenal (HPA) axis activity and hair loss in captive rhesus macaques., Conclusions: Although the present study demonstrates the influence of the HPA axis on coat condition, it is not known if hair loss is caused by abnormal behaviour or hormonal imbalances of the HPA axis itself. Our data suggest that alopecia in rhesus macaques is a highly complex multicausal disorder.