Andrew V. Albright

Pathogenesis of human immunodeficiency virus-induced neurological disease

Infection of the central nervous system by the type 1 human immunodeficiency virus (HIV-1) commonly results in a number of neurological impairments known, in their most severe form, as HIV-associated dementia (HAD). The persistence of HIV encephalitis (HIVE), the pathological correlate of HAD, in spite of highly active antiretroviral therapy (HAART) underscores the importance of continued research focused on the neurobiology of HIV. To elucidate direct and indirect mechanisms of HIV neuropathogenesis, current investigation is focused on neuroinvasion, HIV-1-mediated mechanisms of neuronal damage and apoptosis, and compartmentalized evolution of virus in the brain. The aim of this review is to provide a selective overview of the most recent research on the neurobiology of HIV, adding only a brief introduction regarding established principles.

Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages.

ABSTRACT Human immunodeficiency virus (HIV) replication in the major natural target cells, CD4+ T lymphocytes and macrophages, is parallel in many aspects of the virus life cycle. However, it differs as to viral assembly and budding, which take place on plasma membranes in T cells and on endosomal membranes in macrophages. It has been postulated that cell type-specific host factors may aid in directing viral assembly to distinct destinations. In this study we defined annexin 2 (Anx2) as a novel HIV Gag binding partner in macrophages. Anx2-Gag binding was confined to productively infected macrophages and was not detected in quiescently infected monocyte-derived macrophages (MDM) in which an HIV replication block was mapped to the late stages of the viral life cycle (A. V. Albright, R. M. Vos, and F. Gonzalez-Scarano, Virology 325:328-339, 2004). We demonstrate that the Anx2-Gag interaction likely occurs at the limiting membranes of late endosomes/multivesicular bodies and that Anx2 depletion is associated with a significant decline in the infectivity of released virions; this coincided with incomplete Gag processing and inefficient incorporation of CD63. Cumulatively, our data suggest that Anx2 is essential for the proper assembly of HIV in MDM.

High sensitivity detection of JC-virus DNA in postmortem brain tissue by in situ PCR.

APJ is a recently described seven-transmembrane (7TM) receptor that is abundantly expressed in the central nervous system (CNS). This suggests an important role for APJ in neural development and/or function, but neither its cellular distribution nor its function have been defined. APJ can also serve as a co-receptor with CD4 for fusion and infection by some strains of human immunodeficiency virus (HIV-1) in vitro, suggesting a role in HIV neuropathogenesis if it were expressed on CD4-positive CNS cells. To address this, we examined APJ expression in cultured neurons, astrocytes, oligodendrocytes, microglia and monocyte-derived macrophages utilizing both immunocytochemical staining with a polyclonal anti-APJ antibody and RT - PCR. We also analyzed the ability of a recently identified APJ peptide ligand, apelin, to induce calcium elevations in cultured neural cells. APJ was expressed at a high level in neurons and oligodendrocytes, and at lower levels in astrocytes. In contrast, APJ was not expressed in either primary microglia or monocyte-derived macrophages. Several forms of the APJ peptide ligand induced calcium elevations in neurons. Thus, APJ is selectively expressed in certain CNS cell types and mediates intracellular signals in neurons, suggesting that APJ may normally play a role in signaling in the CNS. However, the absence of APJ expression in microglia and macrophages, the prinicpal CD4-positive cell types in the brain, indicates that APJ is unlikely to mediate HIV-1 infection in the CNS.

The Effect of Human Herpesvirus-6 (HHV-6) on Cultured Human Neural Cells: Oligodendrocytes and Microglia

Human herpesvirus-6 (HHV-6) is a betaherpesvirus that has been frequently associated with pediatric encephalitis. In 1995 Challoner et al reported that HHV-6 variant B (HHV-6B) was linked to multiple sclerosis (MS) due to the presence of viral DNA and antigen in the oligodendrocytes surrounding MS plaques. These findings led us to examine HHV-6Bs in vitro tropism for primary neural cells. HIV-6B mediated cell-to-cell fusion in cultured adult oligodendroglia. Infection of oligodendrocytes was further confirmed by transmission electron microscopy (EM), which showed the presence of intracellular HHV-6 particles, and by PCR for HHV-6 DNA. However, the release of infectious virus was low or undetectable in multiple experiments. Microglia were also susceptible to infection by HHV-6B, as demonstrated by an antigen capture assay. We did not detect infection of a differentiated neuronal cell line (NT2D). Our findings suggest that HHV-6B infection of oligodendrocytes and/or microglia could potentially play a role in neuropathogenesis.

CXCR3 expression in human central nervous system diseases

The CXCR3 chemokine receptor, expressed on activated T lymphocytes, is seen within the central nervous system (CNS) in inflammatory conditions where a T‐cell response is prominent. However, the distribution of CXCR3 in parenchymal CNS cells is unknown. Using a monoclonal antibody against CXCR3 and post‐mortem tissue of patients with and without CNS pathology, we have determined its expression pattern. CXCR3 was found in subpopulations of cells morphologically consistent with astrocytes, particularly reactive astrocytes, and in cerebellar Purkinje cells. It was also detected in arterial endothelial and smooth muscle cells, particularly in areas associated with atherosclerotic plaques. CXCR3‐positive astrocytes were particularly prominent in the CNS of HIV‐positive patients, in patients with Multiple Sclerosis (MS), in ischaemic infarcts and in astrocytic neoplasms. Immunofluorescence studies of mixed adult primary glial cultures and fetal glial cultures also showed expression of CXCR3 in astrocytes. CXCR3 mRNA was detected in Purkinje cells by in situ hybridization with a CXCR3‐specific probe. Thus, the predominant expression of CXCR3 in reactive astrocytes may indicate that it plays a role in the development of reactive gliosis in a variety of infectious, inflammatory, vascular and neoplastic processes in the CNS. The relationship between CXCR3 expression in astrocytes to its expression in Purkinje cells, endothelial cells and smooth muscle cells is yet to be determined.

Polymerase chain reaction analysis of human herpesvirus-6 sequences in the sera and cerebrospinal fluid of patients with multiple sclerosis

Several studies have suggested a possible association of human herpesvirus-6 (HHV-6) with multiple sclerosis (MS), a demyelinating disease with a variable course and progression. To determine whether HHV-6 could be detected in the sera of CSF of patients with different subtypes of MS, we performed nested polymerase chain reaction (PCR) on samples obtained from MS patients as well as samples from normal adults or individuals with other neurological diseases. Ninety-six serum samples from 24 patients with MS, including 13 individuals with relapsing remitting MS, one individual with primary progressive MS, seven individuals with secondary progressive MS and three individuals with an unspecified type were analyzed. Multiple serum samples were examined from individuals over varying periods of time and included samples obtained during exacerbations, remissions, and at different stages of progressive disease. HHV-6 DNA was detected only in one out of 15 serum samples that were collected over a number of years from one individual with secondary progressive MS. No HHV-6 DNA was detected in CSF from six patients with MS or 14 patients with other neurologic disease. These results indicate that the presence of HHV-6 DNA in the serum or CSF of patients with MS is not a common phenomenon, at least within the limits of the sensitivity of our assay.

Interactions between HIV-1 gp120, chemokines, and cultured adult microglial cells

HIV dementia (HIVD), a disease that is apparently mediated by neurotoxins and viral proteins secreted by HIV infected microglia, is characterized neuropathologically by an increased number of activated microglia in the brains of affected individuals. Consequently, the rational design of potential therapeutic strategies should take into account the mechanisms that lead to microglial activation and to their increased prominence in the adult brain. In this regard, one leading hypothesis proposes that microglia are recruited to specific sites in the central nervous system (CNS) as a result of interactions between microglial chemokine receptors and chemokines, or even the viral glycoprotein gp120, which binds chemokine receptors in the process of cellular entry. Adult microglia express the functional chemokine receptors CCR5 and CXCR4 molecules that mediate chemotaxis in these and other cell types. We determined that purified adult microglial cultures contain a heterogeneous population with respect to their ability to respond to the α- and β-chemokines, SDF1α, and MIP-1β. A mean of 14.6% of the microglia assayed responded to both α- and β-chemokines (CCR5+CXCR4+ phenotype); 45.4% of microglia were phenotyped as CCR5+CXCR4−; 12.9% of the microglia were CXCR4+CCR5−; and 27.0% of microglia did not respond to either chemokine. No increase in intracellular calcium levels was seen in the vast majority of microglia exposed to the soluble HIV envelope protein, gp120, or to HIV envelope (gp120/gp41) expressed on MLV virus pseudotypes. However, exposure of microglia to soluble fractalkine or to other chemokines resulted in an intracellular calcium flux. Our results raise the possibility of microglial heterogeneity with respect to their response to chemokines, and indicate that any effects due to gp120 are likely to be considerably less robust than the response of microglia to the natural ligands of their chemokine receptors, for example SDF1α and MIP-1β.

Efavirenz is a potent nonnucleoside reverse transcriptase inhibitor of HIV type 1 replication in microglia in vitro

The objective of this study was to determine whether reverse transcriptase inhibitors (RTIs) could decrease viral replication in microglia. Human microglia obtained from individuals undergoing temporal lobectomy were cultured and infected with HIV-1 isolates from the central nervous system (CNS) as previously described (Strizki JM, et al. J Virol 1996;70:7654-7662). These microglial cultures were treated with one of three nucleoside RTIs (NRTIs) or with efavirenz, a nonnucleoside RTI (NNRTI), at various time points before and during HIV-1 infection. The drug levels sufficient to provide > 90% inhibition of microglial HIV replication (IC90) were determined by comparison of p24(gag) release in the cultures among treated and untreated microglia. Infectious virus released from the infected cultures was also measured with U373-MAGI-CCR5 cells. Efavirenz, an NNRTI, blocked HIV-1(DS-br) infection of microglia with an IC(90) of 0.7-7 nM. This value is similar to the efavirenz IC(90) values for inhibition of laboratory and clinical isolates in lymphocytes, is 2-3 logs lower than the IC90 values of AZT and d4T, and is 1-2 logs lower than that of ddC in microglia. Efavirenz also inhibited infection with other neurotropic isolates, and with viruses isolated from other compartments that also replicated well in microglia. Thus, efavirenz is a potent inhibitor of HIV-1 infection in microglia. Furthermore, efavirenz IC(90) drug levels are present in the cerebrospinal fluid (CSF) of patients taking this once daily NNRTI.

HIV-1 infection of a CD4-negative primary cell type: The oligodendrocyte

HIV-1 infection of CD4-negative cells has been described both in vitro and in vivo. Two closely related glycolipids, galactosylceramide (GalCer) and 3′-sulfo-galactosylceramide (GalS), have been proposed as potential alternative cellular receptors for HIV-1. Human adult cultured oligodendrocytes are CD4-negative/GalCer-positive cells that are infected by HIV-1 in vitro, resulting in the presence of stable HIV-1 DNA sequences and the production of virus [Albright et al., Virology, (1996) in press]. The in vivo relevance and the effects of HIV infection on oligodendrocyte biology need to be studied further.