Douglas R. Pauley

Neuroinvasion by simian immunodeficiency virus coincides with increased numbers of perivascular macrophages/microglia and intrathecal immune activation

During peak viremia and initial antibody response, rhesus macaques infected with pathogenic and nonpathogenic isolates of SIV show distinct differences in viral load and tissue distribution. Animals infected with pathogenic isolates of SIV invariably have virus in the CSF and brain parenchyma by two weeks postinoculation, whereas animals infected with nonpathogenic isolates do not. Mechanisms underlying neuroinvasion by SIV and HIV are unknown, but recruitment of latently infected mononuclear cells from the peripheral circulation (Trojan horse theory) is frequently proposed. Circulating monocytes, from which perivascular macrophage/microglia are derived, are a likely vehicle for cell-associated transport of virus across the blood-brain barrier. This transport and the kinetics of perivascular macrophage/microglial turnover in the CNS likely depend on endothelial and leukocyte adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), which has previously been shown to be upregulated on cerebrovascular endothelium in SIV encephalitis. To investigate the role of peripheral monocyte recruitment into the perivascular macrophage/microglial cell pool at the time of initial viral neuroinvasion, we examined the temporal relationships among perivascular macrophage/microglia density, endothelial VCAM-1 expression and localization of viral nucleic acid in the CNS of macaques acutely infected with pathogenic and nonpathogenic molecular clones of SIV. The concentration of CSF quinolinic acid, a marker of intrathecal immune and macrophage activation, was examined concurrently. We found that significant increases in the density of perivascular macrophages/microglia coincided with viral neuroinvasion and marked elevations in CSF quinolinic acid. Furthermore, combined in situ hybridization and immunohistochemistry demonstrated that infected perivascular cells were macrophages/microglia. These findings provide evidence suggesting that neuroinvasion occurs through an influx of infected monocytes which take up residence in the CNS as perivascular macrophages/microglia. VCAM-1 expression, however, was not clearly correlated with these events, thus its contribution to initial viral neuroinvasion is unclear.

Association of Simian Virus 40 with a Central Nervous System Lesion Distinct from Progressive Multifocal Leukoencephalopathy in Macaques with AIDS

The primate polyomavirus SV40 is known to cause interstitial nephritis in primary infections and progressive multifocal leukoencephalopathy (PML) upon reactivation of a latent infection in SIV-infected macaques. We now describe a second central nervous system manifestation of SV40: a meningoencephalitis affecting cerebral gray matter, without demyelination, distinct from PML. Meningoencephalitis appears also to be a primary manifestation of SV40 infection and can be seen in conjunction with SV40-induced interstitial nephritis and pneumonitis. The difference in the lesions of meningoencephalitis and PML does not appear to be due to cellular tropism, as both oligodendrocytes and astrocytes are infected in PML and meningoencephalitis, as determined by in situ hybridization or immunohistochemistry for SV40 coupled with immunohistochemistry for cellular determinants. This is further supported by examination of SV40 nucleic acid sequences from the ori-enhancer and large-T-antigen regions, which reveals no tissue-or lesion-specific variation in SV40 sequences.

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.

Ephrin/Eph receptor expression in brain of adult nonhuman primates: Implications for neuroadaptation

In developing brain, Eph receptors and their ephrin ligands (Ephs/ephrins) are implicated in facilitating topographic guidance of a number of pathways, including the nigrostriatal and mesolimbic dopamine (DA) pathways. In adult rodent brain, these molecules are implicated in neuronal plasticity associated with learning and memory. Cocaine significantly alters the expression of select members of this family of axonal guidance molecules, implicating Ephs, ephrins in drug-induced neuroadaptation. The potential contribution of Ephs, ephrins to cocaine-induced reorganization of striatal circuitry brain in primates [Saka, E., Goodrich, C., Harlan, P., Madras, B.K., Graybiel, A.M., 2004. Repetitive behaviors in monkeys are linked to specific striatal activation patterns. J. Neurosci. 24, 7557-7565] is unknown because there are no documented reports of Eph/ephrin expression or function in adult primate brain. We now report that brains of adult old and new world monkeys express mRNA encoding EphA4 receptor and ephrin-B2 ligand, implicated in topographic guidance of dopamine and striatal neurons during development. Their encoded proteins distributed highly selectively in regions of adult monkey brain. EphA4 mRNA levels were prominent in the DA-rich caudate/putamen, nucleus accumbens and globus pallidus, as well as the medial and orbitofrontal cortices, hippocampus, amygdala, thalamus and cerebellum. Immunocytochemical localization of EphA4 protein revealed discrete expression in caudate/putamen, globus pallidus, substantia nigra, cerebellar Purkinje cells, pyramidal cells of frontal cortices (layers II, III and V) and the subgranular zone of the hippocampus. Evidence for EphA4 expression in dopamine neurons emerged from colocalization with tyrosine-hydroxylase-positive terminals in striatum and substantia nigra and ventral tegmental area cell bodies. The association of axonal guidance molecules with drug-induced reorganization of adult primate brain circuitry warrants investigation.

Impact of Short-Term Combined Antiretroviral Therapy on Brain Virus Burden in Simian Immunodeficiency Virus-Infected and CD8+ Lymphocyte-Depleted Rhesus Macaques

Antiretroviral drugs suppress virus burden in the cerebrospinal fluid of HIV-infected individuals; however, the direct effect of antiretrovirals on virus replication in brain parenchyma is poorly understood. We investigated the effect of short-term combined antiretroviral therapy (CART) on brain virus burden in rhesus monkeys using the CD8-depletion model of accelerated simian immunodeficiency virus (SIV) encephalitis. Four monkeys received CART (consisting of the nonpenetrating agents PMPA and RCV) for four weeks, beginning 28 days after SIV inoculation. Lower virus burdens were measured by real-time RT-PCR in four of four regions of brain from monkeys that received CART as compared with four SIV-infected, untreated controls; however, the difference was only significant for the frontal cortex (P < 0.05). In contrast, significantly lower virus burdens were measured in plasma and four of five lymphoid compartments from animals that received CART. Surprisingly, despite normalization of neuronal function in treated animals, the numbers of activated macrophages/microglia and the magnitude of TNF-alpha mRNA expression in brain were similar between treated animals and controls. These results suggest that short-term therapy with antiretrovirals that fail to penetrate the blood-cerebrospinal fluid barrier can reduce brain virus burden provided systemic virus burden is suppressed; however, longer treatment may be required to completely resolve encephalitic lesions and microglial activation, which may reflect the longer half-life of the principal target cells of HIV/SIV in the brain (macrophages) versus lymphoid tissues (T lymphocytes).

Alterations in Expression of Monocyte Chemotactic Protein-1 in the Simian Immunodeficiency Virus Model of Disseminated Mycobacterium avium Complex

Mycobacterium avium complex (MAC) infection is the most common disseminated bacterial infection in untreated patients with acquired immunodeficiency syndrome (AIDS). We investigated the potential role of monocyte chemotactic protein-1 (MCP-1) in the pathogenesis of disseminated MAC, using the simian immunodeficiency virus (SIV)/macaque model of AIDS. Macaques were inoculated with SIV, followed by challenge with a pathogenic AIDS isolate of M. avium 14 days later. After challenge with M. avium, marked increases in serum MCP-1 levels were detected in SIV-infected macaques, a finding that was duplicated in coinoculated bronchoalveolar macrophages. MCP-1 levels were significantly higher in SIV-infected macaques than in non-SIV-infected controls (327.1 vs. 151.5 pg/mL, respectively; P=.04), suggesting that up-regulation of MCP-1 contributes to the development of progressive mycobacterial disease. Similarly, morphometric analysis revealed increased expression of MCP-1 in hepatic microgranulomas from SIV-infected macaques. We conclude that the pronounced increases in MCP-1 levels demonstrated in tissue and serum samples after M. avium inoculation may play a role in the development of disseminated mycobacterial disease.

Tissue-specific reduction in DC-SIGN expression correlates with progression of pathogenic simian immunodeficiency virus infection

Studies were undertaken to determine whether previously described reductions in splenic DC-SIGN expression in simian acquired immune deficiency syndrome (AIDS) are limited to pathogenic simian immunodeficiency virus (SIV) infection. DC-SIGN expression was evaluated by immunohistochemistry in lymphoid tissues from AIDS-susceptible Asian macaque monkeys as compared with AIDS-resistant sooty mangabey monkeys in the presence and absence of SIV infection. The phenotype of DC-SIGN+ cells in susceptible and resistant species was identical and most consistent with macrophage identity. Significantly lower levels of DC-SIGN expression were identified in spleen, mesenteric lymph node, and bone marrow of macaques with AIDS (P<0.05). Reduced levels of splenic DC-SIGN correlated significantly with CD4T cell depletion in long-term pathogenic infection of macaques (P<0.01), whereas SIV-infected mangabeys retained high levels of DC-SIGN expression in spleen despite persistent infection. Reduced expression of DC-SIGN in spleen specifically characterizes pathogenic forms of SIV infection, correlates with disease progression, and may contribute to SIV pathogenesis.

Expression of peripherin in the brain of macaques (Macaca mulatta and Macaca fascicularis) occurs in astrocytes rather than neurones and is associated with encephalitis

Peripherin is a member of the type III intermediate filament family, expressed in neurones of the peripheral nervous system of many species and in a discrete subpopulation of neurones of the central nervous system (CNS) during early development in rodents. Previous studies on rats have shown that peripherin immunoreactivity increased significantly in cell bodies of spinal motor neurones following axonal injury. Our study examined the expression of peripherin in the cerebrum of normal macaques (Macaca mulatta and Macaca fascicularis) and those with encephalitis of viral (simian immunodeficiency virus and simian virus 40) or autoimmune (experimental allergic encephalomyelitis) aetiology. Immunohistochemistry, immunoelectronmicroscopy, immunofluorescence and confocal microscopy were performed on tissue sections using antibodies against cell‐specific markers and peripherin. Peripherin‐positive cells were absent in the cerebrum of normal macaques of all ages examined, whereas animals with encephalitis had peripherin‐positive cells associated with inflammatory infiltrates. Further evaluation revealed that these peripherin‐positive cells were not neurones, but were predominantly astrocytes expressing glial fibrillary acidic protein. Our study suggests that peripherin is not neurone‐specific in the CNS of macaques; peripherin is expressed in astrocytes of animals with encephalitis.

EVALUATION OF ANTI-HUMAN ANTIBODIES FOR IMMUNOHISTOCHEMISTRY ON ARCHIVAL NONHUMAN PRIMATE TISSUES

Abstract: A panel of commercially available antibodies which recognize specific antigens on human tissues was developed for use in immunohistochemistry on tissues from eight species of nonhuman primates. Antibodies were selected for potential usefulness in diagnostic pathology, and for effectiveness in formalin‐fixed, paraffin‐embedded tissues. Tissues from four species of macaques and four New World monkeys were evaluated. Using these antibodies we were able to identify 17/21 antigens examined in all eight species, and 21/21 antigens in the four species of macaques. Detailed immunohistochemistry protocols are presented, along with a systematic approach to developing a protocol for a new antibody.