Contributions of Nonhuman Primates to Research on Aging.

Aging is the biological process of declining physiologic function associated with increasing mortality rate during advancing age. Humans and higher nonhuman primates exhibit unusually longer average life spans as compared with mammals of similar body mass. Furthermore, the population of humans worldwide is growing older as a result of improvements in public health, social services, and health care systems. Comparative studies among a wide range of organisms that include nonhuman primates contribute greatly to our understanding about the basic mechanisms of aging. Based on their genetic and physiologic relatedness to humans, nonhuman primates are especially important for better understanding processes of aging unique to primates, as well as for testing intervention strategies to improve healthy aging and to treat diseases and disabilities in older people. Rhesus and cynomolgus macaques are the predominant monkeys used in studies on aging, but research with lower nonhuman primate species is increasing. One of the priority topics of research about aging in nonhuman primates involves neurologic changes associated with cognitive decline and neurodegenerative diseases. Additional areas of research include osteoporosis, reproductive decline, caloric restriction, and their mimetics, as well as immune senescence and chronic inflammation that affect vaccine efficacy and resistance to infections and cancer. The purpose of this review is to highlight the findings from nonhuman primate research that contribute to our understanding about aging and health span in humans.

HIV pathogenesis: the host.

Human immunodeficiency virus (HIV) pathogenesis has proven to be quite complex and dynamic with most of the critical events (e.g., transmission, CD4(+) T-cell destruction) occurring in mucosal tissues. In addition, although the resulting disease can progress over years, it is clear that many critical events happen within the first few weeks of infection when most patients are unaware that they are infected. These events occur predominantly in tissues other than the peripheral blood, particularly the gastrointestinal tract, where massive depletion of CD4(+) T cells occurs long before adverse consequences of HIV infection are otherwise apparent. Profound insights into these early events have been gained through the use of nonhuman primate models, which offer the opportunity to examine the early stages of infection with the simian immunodeficiency virus (SIV), a close relative of HIV that induces an indistinguishable clinical picture from AIDS in Asian primate species, but importantly, fails to cause disease in its natural African hosts, such as sooty mangabeys and African green monkeys. This article draws from data derived from both human and nonhuman primate studies.

IL-17-producing innate lymphoid cells are restricted to mucosal tissues and are depleted in SIV-infected macaques.

Innate lymphoid cells (ILCs) are an emerging subset of lymphocytes involved in surveillance against virally infected cells. Here, we show CD3(-)CD8(high) lymphocytes in macaque blood include major subsets of ILCs including natural killer (NK) cells expressing CD16, NKp46, and NKG2A, but also populations of ILCs in mucosal tissues having different properties. One ILC subset secreted interleukin (IL)-17 (ILC17), but these were restricted to mucosal tissues. Some mucosal ILC17 cells expressed classical NK-cell markers, but little NKG2A or NKG2D. Some ILC17 cells secreted IL-22 and tumor necrosis factor-α, but few produced interferon (IFN)-γ or contained granzyme B. IL-17 production by ILCs was induced by IL-6, transforming growth factor-ß, and IL-23. Further, simian immunodeficiency virus (SIV) infection resulted in a significant loss of ILC17 cells, especially in the jejunum, which persisted throughout SIV infection. These findings indicate that ILC17 cells may be involved in innate mucosal immune responses, and their loss may contribute to loss of intestinal mucosal integrity and disease progression in human immunodeficiency virus (HIV)/SIV infection.

The non-human primate model of tuberculosis.

Non-human primates (NHPs) are used to model human disease owing to their remarkably similar genomes, physiology, and immune systems. Recently, there has been an increased interest in modeling tuberculosis (TB) in NHPs. Macaques are susceptible to infection with different strains of Mycobacterium tuberculosis (Mtb), producing the full spectrum of disease conditions, including latent infection, chronic progressive infection, and acute TB, depending on the route and dose of infection. Clearly, NHPs are an excellent model of human TB. While the initial aim of the NHP model was to allow preclinical testing of candidate vaccines and drugs, it is now also being used to study pathogenesis and immune correlates of protection. Recent advances in this field are discussed in this review. Key questions such as the effect of hypoxia on the biology of Mtb and the basis of reactivation of latent TB can now be investigated through the use of this model.

S100ß as a novel and accessible indicator for the presence of monocyte-driven encephalitis in AIDS.

AIMS: The pathogenesis of human/simian immunodeficiency virus encephalitis (HIVE/SIVE) remains incompletely understood, but is associated with alterations in the blood-brain barrier. At present, it is not possible to easily determine if an individual has HIVE/SIVE before post mortem examination. METHODS: We have examined serum levels of the astroglial protein S100ß in SIV-infected macaques and show that it can be used to determine which animals have SIVE. We also checked for correlations with inflammatory markers such as CCL2/MCP-1, IL-6 and C-reactive protein. RESULTS: We found that increased S100ß protein in serum correlated with decreased expression of the tight junction protein zonula occludens-1 on brain microvessels. Furthermore, the decrease in zonula occludens-1 expression was spatially related to SIVE lesions and perivascular deposition of plasma fibrinogen. There was no correlation between encephalitis and plasma levels of IL-6, MCP-1/CCL2 or C-reactive protein. CONCLUSIONS: Together, these data indicate that SIVE lesions are associated with vascular leakage that can be determined by S100ß protein in the periphery. The ability to simply monitor the presence of SIVE will greatly facilitate studies of the neuropathogenesis of AIDS.

Monitoring alpha4beta7 integrin expression on circulating CD4+ T cells as a surrogate marker for tracking intestinal CD4+ T-cell loss in SIV infection.

Intestinal CD4+ T cells are rapidly and profoundly depleted in human immunodeficiency virus (HIV)-infected patients and simian immunodeficiency virus (SIV)-infected macaques. However, monitoring intestinal cells in humans is difficult, and identifying surrogate markers in the blood, which correlate with loss or restoration of intestinal CD4+ T cells could be helpful in monitoring the success of therapeutic strategies and vaccine candidates. Recent studies indicate HIV utilizes the intestinal homing molecule alpha4beta7 for attachment and signaling of CD4+ T cells, suggesting this molecule may have a central role in HIV pathogenesis. Here, we compared beta7(HIGH) integrin expression on CD4+ T cells in blood with loss of CD4+ T cells in the intestine of macaques throughout SIV infection. The loss of beta7(HIGH) CD4+ T cells in blood closely paralleled the loss of intestinal CD4+ T cells, and proved to be a more reliable marker of intestinal CD4+ T-cell loss than monitoring CCR5+ memory CD4+ T cells. These data are consistent with a recent hypothesis that alpha4beta7 has a role in the selective depletion of intestinal CD4+ T cells, and indicate that monitoring beta7(HIGH) expression on CD4+ T cells in the blood may be a useful surrogate for estimating intestinal CD4+ T cell loss and restoration in HIV-infected patients.

Expression of serotonin transporters by peripheral blood mononuclear cells of rhesus monkeys (Macaca mulatta).

It has been well established that serotonin (5-hydroxytryptamine, 5-HT) plays a key role in neuro-endocrine-immune networks, mostly through its receptors and/or transporters. Although the presence of 5-HT receptor mRNAs in peripheral blood mononuclear cells (PBMCs) of rhesus monkeys has been reported, there is little information about serotonin transporter (SERT) expression by these cells. To examine SERT expression at the transcription and translation level, one-step RT-PCR, confocal microscopy and flow cytometry were used to detect SERT mRNA and protein expression by rhesus monkey PBMCs. It was found that SERT mRNA could be detected by RT-PCR from all of the rhesus macaque PBMC RNA samples and the nucleotide sequence of the amplicons was identical to the published SERT mRNA sequence. Low level SERT immunoreactivity was also demonstrated on the surface of rhesus PBMCs by confocal microscopy. Almost all lymphocytes and most monocytes were positive for SERT by flow cytometry. In the 2 rhesus macaques examined by multicolor flow cytometry, SERT(bright) cells were more than 84%, 94%, and 96% among CD20+, CD3+, and CD3+CD4+ lymphocytes respectively. These data demonstrate expression of SERT by rhesus macaque PBMCs, and indicate that rhesus macaques would be suitable models to test the in vivo immune regulatory effects of 5-HT or drugs targeting SERT.

Simian immunodeficiency virus disrupts extended lengths of the blood--brain barrier.

It is known that there is disruption of the blood-brain barrier during terminal AIDS encephalitis in both human immunodeficiency virus (HIV)-infected humans and simian immunodeficiency virus (SIV)-infected rhesus macaques. Much, although by no means all, of the neuropathological findings of HIV and SIV infection involves accumulation of monocytes/macrophages that have likely crossed the blood-brain barrier (BBB). There is no convincing, rigorous, demonstration of HIV (or SIV) infecting endothelial cells in vivo. However, this is not to say that HIV infection would not have any effects on the physiology of microvascular brain endothelial cells. Because of the elaborate nature of cerebral microvessels, previous studies of cerebral endothelial cells have been constrained by sectioning artifacts. Examination of freshly isolated cerebral microvessels allows investigation of extended lengths of vessels (>150 mum) without sectioning artifacts. These studies determine the changes in the expression of the tight junction protein zo-1 protein on the endothelial cells of cerebral capillaries at terminal acquired immune deficiency syndrome, demonstrating that there is a decreased expression of zo-1 protein over extended lengths of microvessels.

Activation of the blood-brain barrier by SIV (simian immunodeficiency virus) requires cell-associated virus and is not restricted to endothelial cell activation.

The primary cell infected during acute HIV neuropathogenesis is the monocyte-derived macrophage. We have demonstrated that there is activation of the BBB (blood-brain barrier) during acute viral infection and at terminal AIDS. However, it has never been determined if there is a requirement for the virus to be carried through the BBB or how these Trojan horses would be induced to cross the BBB. We added SIVmac251-infected (SIV is simian immunodeficiency virus) mononuclear cells (and their supernatants) to the luminal or abluminal compartment of our BBB model. There was activation of both sides of the BBB model, only if viral-infected cells were added to the luminal compartment, as opposed to the addition of cell-free supernatants. This suggests that cell-associated virus is essential for the activation of the BBB. This, in turn, would be expected to lead to further infiltration of cells capable of viral infection. VCAM-1 (vascular cell adhesion molecule 1) staining revealed, for the first time, that there is an absolute requirement for virally infected cells, and not just the presence of virus for the activation of the BBB.

Quantitative evaluation of Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected rhesus monkeys.

The association of the microsporidia Enterocytozoon bieneusi with chronic diarrhea and wasting in individuals with acquired immunodeficiency syndrome (AIDS) has been demonstrated. The disease caused by E. bieneusi has been linked to decreased levels of circulating CD4+ T lymphocytes. In this study, we investigated the relationship between the extent of excretion of E. bieneusi in feces of simian immunodeficiency virus (SIV)-infected juvenile macaques and the CD4+ T lymphocyte counts in the peripheral blood. Twelve juvenile rhesus monkeys (Macaca mulatta) were intravenously inoculated with the pathogenic molecular clone SIVmac239. Numbers of CD4+ T lymphocytes were assessed by three-color flow cytometry. The presence of E. bieneusi DNA in feces was assessed by nested PCR. In addition, selected samples of feces were examined by competitive quantitative PCR to assess the level of E. bieneusi infection. Low (n = 5) to undetectable (n = 7) quantities of E. bieneusi were present in feces of the twelve animals in prior to inoculation with SIV. After SIV inoculation the number of animals shedding E. bieneusi increased (n = 10) as did the quantity of E. bieneusi shedding in the feces. Of the twelve juvenile animals, five animals died within 8 months post-SIV inoculation with symptoms of AIDS. Four of the five deceased animals showed shedding of E. bieneusi DNA in feces (> or =100 spores/g) for at least three consecutive months. Increased number of E. bieneusi in feces was accompanied by decreased counts of circulating CD4+ T lymphocytes and increased SIV plasma viral load.

Magnetic resonance spectroscopy: an in vivo tool for monitoring cerebral injury in SIV-infected macaques.

The purpose of our study was to demonstrate the feasibility of using in vivo proton Magnetic Resonance Spectroscopy (MRS) to monitor the brain manifestations of SIV infection in the macaque model of AIDS. Previous spectroscopy work on macaque brain tissue and in vivo work in humans is reviewed to provide the motivation and context for this study. We collected 34 MRS data sets on 14 uninfected rhesus macaques. From this data, we demonstrate that we are capable of detecting changes similar to those observed in human MRS studies for most metabolites using less than 10 animals. The juvenile macaques utilized in this study demonstrate age-related changes in the levels of N-acetyl aspartate (NAA), a neuronal marker. The quantity and distribution of neurochemicals in the macaque are found to be slightly, but significantly, different than in the human.

Developmental expression patterns of CCR5 and CXCR4 in the rhesus macaque brain.

Emerging data indicate that chemokine receptors on neurons and glia in the central nervous system (CNS) play a role in normal CNS development, intercellular communication, and the neuropathogenesis of AIDS. To further understand chemokine receptors in the brain and explore their potential role in HIV neuropathogenesis, particularly in pediatrics, we examined the regional and cellular distribution of CCR5 and CXCR4 in normal fetal, neonatal, and adult rhesus macaques. CCR5 and CXCR4 were detected by immunohistochemistry and immunofluorescence within the cytoplasm of subpopulations of neurons in the neocortex, hippocampus, basal nuclei, thalamus, brain stem, and cerebellum and by flow cytometry on the surface of neurons and glia. Interestingly, expression of CCR5 and CXCR4 increased significantly (p<0.05) from birth to 9 months of age. We further characterize this dynamic developmental pattern of CCR5 and CXCR4 expression in resident cells of the CNS.

Simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in gastrointestinal tissues of chronically SIV-infected rhesus monkeys.

Although systemic virus-specific cytotoxic T lymphocyte (CTL) responses are of critical importance in controlling virus replication in individuals infected with human immunodeficiency virus 1 (HIV-1), little is known about this immune response in the gastrointestinal (GI) tract. This study investigated the GI tract CTL response in a nonhuman primate model for HIV-1 infection, simian immunodeficiency virus (SIV)-infected rhesus monkeys. Lymphocytes from duodenal pinch biopsy specimens were obtained from 9 chronically SIVmac-infected rhesus monkeys and GI tract lymphocytes were harvested from the jejunum and ileum of 4 euthanized SIVmac-infected rhesus monkeys. Lymphocytes were also assessed in GI mucosal tissues by in situ staining in histologic specimens. SIVmac Gag-specific CTLs were assessed in the monkeys using the tetramer technology. These GI mucosal tissues of chronically SIVmac-infected rhesus monkeys contained levels of CTLs comparable to those found in the peripheral blood and lymph nodes. The present studies suggest that the CD8(+) CTL response in GI mucosal sites is comparable to that seen systemically in SIVmac-infected rhesus monkeys.

The mucosal immune system: primary target for HIV infection and AIDS.

Despite intensive research, several questions remain regarding the pathogenesis of infection with HIV-1. Recently, it has been shown that simian immunodeficiency virus (SIV) selectively targets and destroys specific subsets of CD4+ T cells that are abundant in mucosal tissues but rare in peripheral lymphoid tissues. This finding could be highly relevant in explaining a major paradox in the infection and elimination of CD4+ T cells during HIV infection: the progressive decline in the number of CD4+ T cells in the blood, despite the paucity of HIV-infected cells in this tissue. This article discusses the hypothesis that infection with HIV and SIV, and the resulting disease, is governed by the state of cellular activation and the expression of chemokine receptors by specific subsets of CD4+ T cells residing in mucosal lymphoid tissues, rather than those found in the peripheral blood or lymph nodes.

Emergence and kinetics of simian immunodeficiency virus-specific CD8(+) T cells in the intestines of macaques during primary infection.

In this report, three Mamu-A*01(+) rhesus macaques were examined to compare the emergence of simian immunodeficiency virus (SIV)-specific CD8(+) T cells in the intestines and blood in early SIV infection using a major histocompatibility complex class I tetramer complexed with the Gag(181-189) peptide. Fourteen days after intravenous inoculation with SIVmac251, large numbers of SIV Gag(181-189)-specific CD8(+) T cells were detected in the intestinal mucosa (3.1 to 11.5% of CD3(+) CD8(+) lymphocytes) as well as in the blood (3.1 to 13.4%) of all three macaques. By 21 days postinoculation, levels of tetramer-binding cells had dropped in both the intestines and blood. At day 63, however, levels of SIV Gag(181-189)-specific CD8(+) T cells in the intestines had rebounded in all three macaques to levels that were higher (8.6 to 18.7%) than those at day 21. In contrast, percentages of tetramer-binding cells in the peripheral blood remained comparatively stable (2.5 to 4.5%) at this time point. In summary, SIV Gag(181-189)-specific CD8(+) T cells appeared in both the intestinal mucosa and peripheral blood at a comparable rate and magnitude in primary SIV infection. Given that the intestine is a major site of early viral replication as well as the site where most of the total body lymphocyte pool resides, these data indicate that it is also an early and important site of development of antiviral immune responses.

Central nervous system perivascular cells are immunoregulatory cells that connect the CNS with the peripheral immune system.

Perivascular cells are a heterogeneous population found in the central nervous system (CNS) and the peripheral nervous system (PNS). Several terms are used for these cells, including perivascular cells, perivascular macrophages, perivascular microglia, fluorescent granular perithelial cells (FGP), or Mato cells. Different terminology used may reflect subpopulations of perivascular cells within different anatomic regions and experimental paradigms, neuropathological conditions, and species studied. Different terminology also points to the lack of clear consensus of what cells are perivascular cells in different disease states and models, especially with breakdown of the blood-brain barrier (BBB). Despite this, there is consensus that perivascular cells, although a minor component of the CNS, are important immunoregulatory cells. Perivascular cells are bone marrow derived, continuously turn over in the CNS, and are found adjacent to CNS vessels. Thus, they are potential sensors of CNS and peripheral immune system perturbations; are activated in models of CNS inflammation, autoimmune disease, neuronal injury and death; and are implicated as phagocytic and pinocytotic cells in models of stroke and hypertension. Recent evidence from our laboratory implicate perivascular cells as primary targets of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection in the CNS of humans and macaques. This article reviews current knowledge of perivascular cells, including anatomic location and nomenclature and putative immunoregulatory roles, and discusses new data on the infection of these cells by SIV, their accumulation after SIV infection, and a possible role of the immune system in SIV encephalitis.

Rhesus macaque brain microvessel endothelial cells behave in a manner phenotypically distinct from umbilical vein endothelial cells.

Activation of endothelium is a critical step in leukocyte recruitment to the CNS and in development of neurological diseases, such as HIV-associated dementia. Due to limited availability of early disease course data, it is important to develop in vitro models of the blood-brain barrier (BBB) that can be used to address these early events. No such model of the BBB has been established for the macaque. Here, we characterize rhesus microvascular brain endothelial cells (MBEC), comparing them with rhesus umbilical vein endothelial cells (RUVEC), and discuss their suitability for future use in developing in vitro models of simian immunodeficiency virus (SIV) neuropathogenesis. We conclude that MBEC are distinct from RUVEC with respect to growth characteristics, culture requirements, morphology and expression of surface molecules important for leukocyte adhesion and immune activation.

Induction of disseminated Mycobacterium avium in simian AIDS is dependent upon simian immunodeficiency virus strain and defective granuloma formation.

Mycobacterium avium complex (MAC) is the most common disseminated bacterial disease in patients infected by the human immunodeficiency virus. Although murine models of disseminated MAC exist, they are primarily based on underlying genetic susceptibilities and cannot adequately address the complex interactions that occur between host, mycobacteria, and immunosuppressive lentivirus. To address this problem we have developed an experimental system to co-inoculate rhesus macaques with the simian immunodeficiency virus (SIV) and a clinical M. avium isolate that results in a disease virtually identical to that observed in human cases. Using this experimental system we have found that the development of disseminated MAC is dependent on viral strain. Animals co-infected with SIVmac251 and M. avium developed progressive disease, whereas control animals and animals inoculated with closely related viruses (SIVmac239 and SIVmac239MER) developed self-limiting infections. The ability of animals infected with SIVmac239 or SIVmac239MER to eliminate mycobacterial disease was independent of viral load and CD4 T-cell number but was correlated with the size and composition of microgranulomas. This work establishes a novel primate model of disseminated MAC in the context of immunosuppressive lentiviral infection and advances our understanding of why human immunodeficiency virus-infected patients are remarkably sensitive to the development of mycobacterial disease.

Enteropathogenic Escherichia coli and ulcerative colitis in cotton-top tamarins (Saguinus oedipus).

The cotton-top tamarin (CTT; Saguinus oedipus) is an endangered New World primate that develops a highly prevalent idiopathic colitis resembling human ulcerative colitis. This study found that enteropathogenic Escherichia coli (EPEC) caused acute colitis in CTTs, which was associated with ulcerative colitis. EPEC clinical isolates revealed localized adherence patterns by HEp-2 assay and were devoid of Shiga-toxin production. Sequencing of the eae gene (GenBank accession no. AF319597) revealed 99.2% identity to sequences of human isolates (GenBank AF116899) and corresponded to the epsilon intimin gene subtype. Detection of intimin sequences by polymerase chain reaction on primary fecal cultures indicated widespread EPEC infection in the CTT colony. Prospective analysis revealed that animals with fecal cultures positive for intimin sequences had a higher frequency of active colitis (75.0% vs. 27.2%; P<.005, chi(2) test) and higher histological scores of colonic inflammation (0.875 vs. 0.455, respectively; P<.05, Mann-Whitney rank sum test).

Macaques with rapid disease progression and simian immunodeficiency virus encephalitis have a unique cytokine profile in peripheral lymphoid tissues.

The influence of host cytokine response on viral load, disease progression, and neurologic lesions was investigated in the simian immunodeficiency virus (SIV)-infected macaque model of AIDS. Cytokine gene expression (interleukin-1beta [IL-1beta], IL-2, IL-6, IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and viral loads were evaluated by semiquantitative reverse transcription-PCR in lymph nodes of 5 control animals and 28 animals infected with SIVmac251 at the terminal stages of AIDS. Infected animals showed higher expression of IFN-gamma, IL-6, and IL-10 mRNAs compared with controls. Levels of all cytokines were comparable between animals with rapid (survival, <200 days) or slow/normal (survival, >200 days) disease progression. However, among rapid progressors, the eight animals with SIV encephalitis had a unique cytokine profile (increased IL-2, IL-6, and IFN-gamma) that was associated with higher viral loads. These observations provide evidence that host cytokine responses may influence SIV neuropathogenesis independent of disease progression.