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.

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.

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.

Resistance of neonatal monkeys to live attenuated vaccine strains of simian immunodeficiency virus.

A major safety concern of using live-attenuated vaccine strategies against AIDS is the potential exposure of neonates or fetuses to vaccine virus from the mother. Here we report that high viral loads and disease were observed in only 2 of 18 neonatal monkeys infected with gene-deleted vaccine strains of simian immunodeficiency virus. Pathogenicity was restricted to neonates born to unvaccinated mothers, that is, lacking maternal immunity, and that received extremely high doses of vaccine virus orally. No in utero transmission of vaccine virus was observed in four neonates born to mothers vaccinated during the second trimester. Our results suggest that the live attenuated vaccine approach should remain a viable option for preventing HIV infection and disease in high-risk human populations.

Progesterone implants enhance SIV vaginal transmission and early virus load.

Simian immunodeficiency virus (SIV) can cross the intact vaginal epithelium to establish a systemic infection in macaques (mac). Using this SIVmac model, we found that subcutaneous progesterone implants, which could mimic hormonally based contraceptives, thinned the vaginal epithelium and enhanced SIV vaginal transmission 7.7-fold over that observed in macaques treated with placebo implants and exposed to SIV in the follicular phase of the menstrual cycle. Progesterone treatment also increased the number of SIV DNA-positive cells in the vaginal lamina propria as detected by in situ polymerase chain reaction analysis. Moreover, plasma viral RNA was elevated for the first three months in macaques with progesterone implants, and three of the progesterone-treated macaques developed relatively rapid disease courses. This study shows that SIV genital infection and disease course are enhanced by subcutaneous implants containing progesterone when compared with the rate of vaginal transmission in the follicular phase.

HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected macrophages.

Infection of macrophage lineage cells is a feature of primate lentivirus replication, and several properties of primate lentiviruses seem to have evolved to promote the infection of macrophages. Here we demonstrate that the accessory gene product Nef induces the production of two CC-chemokines, macrophage inflammatory proteins 1alpha and 1beta, by HIV-1-infected macrophages. Adenovirus-mediated expression of Nef in primary macrophages was sufficient for chemokine induction. Supernatants from Nef-expressing macrophages induced both the chemotaxis and activation of resting T lymphocytes, permitting productive HIV-1 infection. These results indicate a role for Nef in lymphocyte recruitment and activation at sites of virus replication.

Inhibition of intracellular transport of B cell antigen receptor complexes by Kaposi's sarcoma-associated herpesvirus K1.

The B cell antigen receptor (BCR) is a large complex that consists of a disulfide-linked tetramer of two transmembrane heavy (mu) chains and two light (lambda or kappa) chains in association with a heterodimer of Igalpha and Igbeta. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a transforming protein called K1, which has structural and functional similarity to Igalpha and Igbeta. We demonstrate that K1 downregulates the expression of BCR complexes on the surface. The NH(2)-terminal region of K1 specifically interacts with the mu chains of BCR complexes, and this interaction retains BCR complexes in the endoplasmic reticulum, preventing their intracellular transport to the cell surface. Thus, KSHV K1 resembles Igalpha and Igbeta in its ability to induce signaling and to interact with mu chains of the BCR. However, unlike Igalpha and Igbeta, which interact with mu chains to direct BCR complexes to the cell surface, K1 interacts with mu chains to block the intracellular transport of BCR complexes to the cell surface. These results demonstrate a unique feature of the K1 transforming protein, which may confer virus-infected cells with a long-term survival advantage.

Perivascular macrophages are the primary cell type productively infected by simian immunodeficiency virus in the brains of macaques: implications for the neuropathogenesis of AIDS.

The macrophage is well established as a target of HIV and simian immunodeficiency virus (SIV) infection and a major contributor to the neuropathogenesis of AIDS. However, the identification of distinct subpopulations of monocyte/macrophages that carry virus to the brain and that sustain infection within the central nervous system (CNS) has not been examined. We demonstrate that the perivascular macrophage and not the parenchymal microglia is the primary cell productively infected by SIV. We further demonstrate that although productive viral infection of the CNS occurs early, thereafter it is not easily detectable until terminal AIDS. The biology of perivascular macrophages, including their rate of turnover and replacement by peripheral blood monocytes, may explain the timing of neuroinvasion, disappearance, and reappearance of virus in the CNS, and questions the ability of the brain to function as a reservoir for productive infection by HIV/SIV.

Early regeneration of thymic progenitors in rhesus macaques infected with simian immunodeficiency virus.

The thymus plays a critical role in the maturation and production of T lymphocytes and is a target of infection by human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV). Using the SIV/macaque model of AIDS, we examined the early effects of SIV on the thymus. We found that thymic infection by SIV resulted in increased apoptosis 7-14 d after infection, followed by depletion of thymocyte progenitors by day 21. A marked rebound in thymocyte progenitors occurred by day 50 and was accompanied by increased levels of cell proliferation in the thymus. Our results demonstrate a marked increase in thymic progenitor activity very early in the course of SIV infection, long before marked declines in peripheral CD4(+) T cell counts.

Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection.

Human and simian immunodeficiency virus (HIV and SIV) replicate optimally in activated memory CD4(+) T cells, a cell type that is abundant in the intestine. SIV infection of rhesus monkeys resulted in profound and selective depletion of CD4+ T cells in the intestine within days of infection, before any such changes in peripheral lymphoid tissues. The loss of CD4+ T cells in the intestine occurred coincident with productive infection of large numbers of mononuclear cells at this site. The intestine appears to be a major target for SIV replication and the major site of CD4+ T cell loss in early SIV infection.

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.

Dilated cardiomyopathy associated with simian AIDS in nonhuman primates.

BACKGROUND: Cardiomyopathy is being recognized with increasing frequency in patients with AIDS, yet the relationship between HIV infection and cardiac contractile dysfunction remains obscure. The purpose of the present study was to determine if infection with simian immunodeficiency virus (SIV) in nonhuman primates is associated with cardiac dysfunction and myocardial injury. METHODS AND RESULTS: Left ventricular size and function were determined by 2D echocardiography in 16 rhesus macaques before and at weekly intervals following infection with cloned pathogenic SIV(mac) 239 or the highly attenuated SIV(mac) 239 nef deletion mutant. A second group of 15 rhesus macaques chronically infected with pathogenic (n=6) or nonpathogenic (n=9) virus were studied at >2 years following infection. Cardiac tissues from 24 rhesus macaques chronically infected (>2 years) with pathogenic SIV were reviewed for evidence of cardiac pathology. Acute infection (<6 weeks) with either pathogenic or nonpathogenic SIV caused neither contractile dysfunction nor cardiac pathology. However, LV ejection fraction was significantly (P<0.05) depressed (43+/-7%) in rhesus macaques chronically infected with pathogenic SIV compared with rhesus macaques chronically infected with nonpathogenic SIV (61+/-3%). Furthermore, two thirds of rhesus macaques that succumbed to simian AIDS had myocardial pathology including lymphocytic myocarditis (n=9) and coronary arteriopathy (n=6), with complete vessel occlusion (n=4) and associated myocardial infarction and necrosis. CONCLUSIONS: This unique model is valuable in understanding the pathogenesis of cardiac injury associated with retroviral infection in a relevant nonhuman primate model of AIDS.

Early brain injury in the SIV-macaque model of AIDS.

OBJECTIVE: To specify the type and severity of cellular damage in the central nervous system soon after infection and at later stages of disease in the SIV-macaque model of AIDS. DESIGN AND METHODS: Adjacent samples of frontal cortical gray matter were taken from three groups of macaques: uninfected controls (n = 4), acute (14 days post-infection; n = 4), and chronic (mean 2 years post-infection; n = 7). In vitro high resolution magnetic resonance spectroscopy of snap frozen intact tissue and quantitative neuropathology measurements of synaptophysin, calbindin, and glial fibrillary acidic protein (GFAP) in formalin-fixed tissue were performed. RESULTS: Losses in n-acetylaspartate and calbindin (indicating neuronal injury and/or death) and decreases in synaptophysin immunoreactivity (indicating synaptodendritic injury) were detected along with increases in GFAP (indicating reactive gliosis). Cellular injury worsened progressively with increased time after infection. CONCLUSIONS: These results are the first direct evidence that neuronal injury occurs soon after infection. The exacerbation of injury with time suggests a connection between the early response of the central nervous system and dementia, which occurs late in the course of infection. This connection may have broad implications for the study of and the development of therapies for damage of the central nervous system by HIV.

Characterization of the cutaneous exanthem in macaques infected with a Nef gene variant of SIVmac239.

The molecularly cloned viruses known as SIVmac239/R17Y and SIVmac239/YEnef cause extensive lymphocyte activation and induce an acute disease syndrome in macaque monkeys. One manifestation of this syndrome is a severe diffuse cutaneous maculopapular exanthem that is similar to the exanthem associated with HIV-1 infection. To examine the pathogenesis of this exanthem, biopsies obtained throughout the course of clinically evident rash were examined for the presence of virus by in situ hybridization and immunohistochemistry, and the cellular infiltrate was characterized with respect to cellular immunophenotype and chemokine receptor expression. The onset of rash was associated with abundant simian immunodeficiency virus nucleic acid and protein within perivascular dermal infiltrates and occasionally within intraepithelial cells. Analysis of cellular infiltrates showed that biopsies, obtained on the day of rash onset, were composed of equal numbers of CD4+ and CD8+ lymphocytes and abundant alphaEbeta7 positive cells surrounding vessels with upregulated endothelial E-selectin. Moreover, by examining virus expression in sequential skin biopsies from the same animal, the clearance of virus and the resolution of rash were associated with an increase in the percentage of cells expressing CD8, the chemokine receptor CXCR3, and GMP-17, a marker of cytotoxic granules. These results suggest that activated cytotoxic T cells are trafficking to sites of inflammation in the skin and directly or indirectly affect levels of viral replication at these sites.

Neurobiology of simian and feline immunodeficiency virus infections.

Experimental and clinical evidence indicates that all lentiviruses of animals and humans are neurotropic and potentially neurovirulent. The prototypic animal lentiviruses, visna virus in sheep and caprine arthritis encephalitis virus in goats have been known for decades to induce neurologic disease. More recently, infection of the brain with the human immunodeficiency virus (HIV) has been linked to an associated encephalopathy and cognitive/motor complex. While the visna virus and caprine arthritis encephalitis virus are important models of neurologic disease they are not optimal for the study of HIV encephalitis because immune deficiency is only a minor component of the disease they induce. By contrast, the recently isolated lentiviruses from monkeys and cats, the simian and feline immunodeficiency viruses (SIV and FIV respectively), are profoundly immunosuppressive as well as neurotropic. SIV infection of the central nervous system of macaques now provides the best animal model for HIV infection of the human brain due to the close evolutionary relationship between monkeys and man, the genetic relatedness of their respective lentiviruses, and the similarities in the neuropathology. This chapter will compare and contrast the neurobiology of SIV and FIV with HIV.

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.

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.

Intrathecal synthesis of IgG in simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta).

We examined cerebrospinal fluid (CSF) and serum from 25 simian immunodeficiency (SIV)-infected rhesus macaques for the presence of SIV-specific immunoglobulin G (IgG) and for intrathecal synthesis of IgG. SIV-specific IgG was present in CSF from almost 50% of the macaques. In approximately half of these animals the SIV-specific IgG appeared to be derived from serum by leakage across a disrupted blood-brain barrier, whereas in the remaining animals there was evidence of intrathecal IgG synthesis. There were no significant associations between CSF SIV-specific IgG, intrathecal IgG synthesis and isolation of virus from CSF, clinical status, or neuropathological findings. However, SIV-specific IgG was absent from CSF in all four of the macaques with SIV encephalitis. The presence of SIV-specific IgG in CSF may have a modulating effect on the development of SIV-associated neurological disease.

Recombinant simian immunodeficiency virus expressing green fluorescent protein identifies infected cells in rhesus monkeys.

We engineered recombinant derivatives of simian immunodeficiency virus (SIV) to express enhanced green fluorescent protein (EGFP). Replacement of vpr sequences with EGFP resulted in a genome that did not produce detectable levels of replication-competent virus. Replication-competent virus and bright fluorescence of infected cells were obtained with two other constructs, one in which SIV nef sequences were replaced by EGFP and another in which EGFP was inserted into the SIV nef locus and HIV-1 nef sequences were expressed by downstream placement of an internal ribosomal entry site. These strains were infectious in rhesus monkeys and green fluorescing cells were detected in the tissues of infected monkeys by FACS analysis and by direct microscopic visualization. EGFP sequences were absent from recovered virus by 8 weeks following infection. We conclude that recombinant SIV that is engineered to express EGFP can be used to directly detect productively infected cells and aid in the immunophenotypic characterization of these cells within the first 2 weeks of infection of rhesus monkeys.

Enterocytozoon bieneusi as a cause of proliferative serositis in simian immunodeficiency virus-infected immunodeficient macaques (Macaca mulatta).

CONTEXT: Enterocytozoon bieneusi is the most frequent microsporidian parasite of human patients with acquired immunodeficiency syndrome and is a significant cause of diarrhea and wasting. Recently, this organism has also been recognized as a spontaneous infection of several species of captive macaques. As in humans, E bieneusi frequently causes enteropathy and cholangiohepatitis in immunodeficient simian immunodeficiency virus (SIV)-infected macaques. OBJECTIVE: To examine E bieneusi as an etiologic agent of nonsuppurative proliferative serositis in immunodeficient rhesus macaques (Macaca mulatta). DESIGN: Retrospective analysis of necropsy material obtained from immunodeficient SIV-infected rhesus macaques. RESULTS: Examination of SIV-infected rhesus macaques (n = 225) revealed E bieneusi proliferative serositis in 7 of 16 cases of peritonitis of unknown origin. The organism could be identified by in situ hybridization and polymerase chain reaction in sections of pleura and peritoneum obtained at necropsy. Serositis was always accompanied by moderate-to-severe infection of the alimentary tract, and morphologic evidence suggested dissemination through efferent lymphatics. Colabeling experiments revealed most infected cells to be cytokeratin positive and less frequently positive for the macrophage marker CD68. Sequencing of a 607-base pair segment of the small subunit ribosomal gene revealed 100% identity to sequences obtained from rhesus macaques (Genbank accession AF023245) and human patients (Genbank accession AF024657 and L16868). CONCLUSIONS: These findings indicate that E bieneusi disseminates in immunodeficient macaques and may be a cause of peritonitis in the immunocompromised host.