Early Transcriptional Changes within Liver, Adrenal Gland, and Lymphoid Tissues Significantly Contribute to Ebola Virus Pathogenesis in Cynomolgus Macaques.

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.

Maternal Zika Virus (ZIKV) Infection following Vaginal Inoculation with ZIKV-Infected Semen in Timed-Pregnant Olive Baboons.

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 106 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.

Attenuation of Simian Varicella Virus Infection by Enhanced Green Fluorescent Protein in Rhesus Macaques.

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.

In vitro responses of multiple cytokines to purified protein derivative in healthy and naturally Mycobacterium tuberculosis-infected rhesus monkeys (Macaca mulatta).

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.

Transcriptional regulation of follicle-stimulating hormone ß-subunit in marmoset by an alternate distal promoter.

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 ß-subunit gene (FSHß) is a rate-limiting step in the production of mature FSH, this study aimed to investigate the regulation of marmoset FSHß gene expression in comparison to human. In silico analysis of the FSHß promoter sequences identified a TATA box element upstream of the conventional TATA box element in marmoset but not in human sequence. FSHß 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 FSHß transcription in marmoset compared to humans.

Pregnancy-driven cardiovascular maternal miR-29 plasticity in obesity.

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.

Rotaviruses reach late endosomes and require the cation-dependent mannose-6-phosphate receptor and the activity of cathepsin proteases to enter the cell.

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.

Polioencephalomalacia secondary to hypernatremia in squirrel monkeys (Saimiri sciureus).

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.

Acute-phase responses in healthy and diseased rhesus macaques (Macaca mulatta).

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.

Inflammatory fibroid polyp in the duodenum of a common marmoset (Callithrix jacchus).

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.

Lipid metabolism and other metabolic changes in vervet monkeys experimentally infected with Trypanosoma brucei rhodesiense.

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.

Renoprotective effect of erythropoietin against ischaemia-reperfusion injury in a non-human primate model.

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.

Dramatic transcriptomic differences in Macaca mulatta and Macaca fascicularis with Plasmodium knowlesi infections.

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.

Immune Profile of the Normal Maternal-Fetal Interface in Rhesus Macaques and Its Alteration Following Zika Virus Infection.

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 αß 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.

Prenatal disorders and congenital Zika syndrome in squirrel monkeys.

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.

Functional inactivation of pulmonary MAIT cells following 5-OP-RU treatment of non-human primates.

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.

Evolution of a TRIM5-CypA splice isoform in old world monkeys.

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.

Fecal glucocorticoids as indicators of metabolic stress in female Sykes' monkeys (Cercopithecus mitis albogularis).

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.

Immunohistochemical study of the ubiquitin-nuclear factor-kB pathway in the endometrium of the baboon (Papio anubis) with and without endometriosis.

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.

Clinicopathologic characterization of naturally occurring diabetes mellitus in vervet monkeys.

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.