Frank H. Michaels

Nitric oxide (NO) has been implicated as a pathogenic mediator in a variety of central nervous system (CNS) disease states, including the animal model of multiple sclerosis (MS) and experimental allergic encephalomyelitis. We have examined post-mortem brain tissues collected from patients previously diagnosed with MS, as well as tissues collected from the brains of patients dying without neuropathies. Both Northern blot analysis and reverse transcriptase (RT)-driven in situ PCR (RT-in situ PCR) studies demonstrated that inducible NO synthase (iNOS) mRNA was present in the brain tissues from MS patients but was absent in equivalent tissues from normal controls. We have also performed experiments identifying the cell type responsible for iNOS expression by RT-in situ PCR in combination with immunohistochemistry. Concomitantly, we analyzed the tissues for the presence of the NO reaction product nitrotyrosine to demonstrate the presence of a protein nitrosylation adduct. We report here that iNOS mRNA was detectable in the brains of 100% of the CNS tissues from seven MS patients examined but in none of the three normal brains. RT-in situ PCR experiments also demonstrated the presence of iNOS mRNA in the cytoplasm of cells that also expressed the ligand recognized by the Ricinus communis agglutinin 1 (RCA-1), a monocyte/macrophage lineage marker. Additionally, specific labeling of cells was observed when brain tissues from MS patients were exposed to antisera reactive with nitrotyrosine residues but was significantly less plentiful in brain tissue from patients without CNS disease. These results demonstrate that iNOS, one of the enzymes responsible for the production of NO, is expressed at significant levels in the brains of patients with MS and may contribute to the pathology associated with the disease.

Human immunodeficiency virus (HIV) frequently causes neurological dysfunction and is abundantly expressed in the central nervous system (CNS) of acquired immunodeficiency syndrome (AIDS) patients with HIV encephalitis or myelopathy. The virus is found mostly in cells of the monocyte-macrophage lineage within the CNS, but the possibility of infection of other glial cells has been raised. Therefore, the effects of different HIV-1 and HIV-2 strains were studied in primary cultures of adult human brain containing microglial cells, the resident CNS macrophages, and astrocytes. These cultures could be productively infected with macrophage-adapted HIV-1 isolates but not with T lymphocyte-adapted HIV-1 isolates or two HIV-2 isolates. As determined with a triple-label procedure, primary astrocytes did not express HIV gag antigens and remained normal throughout the 3-week course of infection. In contrast, virus replicated in neighboring microglial cells, often leading to their cell fusion and death. The death of microglial cells, which normally serve immune functions in the CNS, may be a key factor in the pathogenesis of AIDS encephalitis or myelopathy.

Kaposi's sarcoma (KS) arises more frequently in homosexual and bisexual men than in other groups of HIV-1 infected individuals. Clinico-epidemiologic data indicate that homosexuals often are infected with multiple microbial agents and/or subjected to other antigenic stimuli, preceding or accompanying HIV-1 infection. Signs of immune activation, in fact, frequently have been detected in these individuals, and the onset of KS can precede any sign of immunodeficiency. These data have suggested that products from activated immune cells may affect the development of AIDS-KS. Here we report that conditioned media from activated or dysregulated T cells contain a variety of cytokines that promote the growth of spindle cells derived from KS lesions of AIDS patients (AIDS-KS cells) and induce normal vascular cells, potential cell progenitors of the AIDS-KS cells, to acquire features of the KS cell phenotype ("spindle" cell morphology and growth responsiveness to the mitogenic effect of extracellular HIV-1 Tat protein). The same conditioned media or cytokines promote HIV-1 gene expression and rescue defective HIV-1 proviruses, interrupting HIV-1 latency and increasing Tat production. The cellular and viral effects of cytokines are increased in an additive or synergistic manner by picomolar concentrations of extracellular Tat. These data suggest that cytokines produced by activated immune cells cooperate with HIV-1 infection in AIDS-KS pathogenesis.

The human immunodeficiency virus (HIV) genetic determinant(s) responsible for tropism in human T cells or macrophages are not well defined. We studied the role of the HIV type 2 (HIV-2) nef and vpr genes in viral tropism. HIV-2 mutants, lacking either vpr or nef genes, or both vpr and nef, were obtained by site-specific mutagenesis of a biologically active HIV-2 proviral clone (HIV-2sbl/isy), which is infectious in both human T cells and macrophages. Viral progeny carrying mutations of nef, vpr, or of both nef and vpr genes replicated more efficiently than the parental virus in primary human peripheral blood cells and in the human Hut 78 T-cell line. In contrast, the HIV-2 nef- mutant infected human macrophages as efficiently as the parental virus, whereas viruses lacking the vpr gene either alone or in conjunction with the lack of the nef gene did not replicate in macrophages. Thus, some lack of nef in HIV-2 enhances viral replication in T cells and does not interfere with viral replication in primary macrophages, whereas vpr is essential for replication of HIV-2 in human macrophages. Because the parental HIV-2sbl/isy cloned virus also infects rhesus macaques, the use in animal studies of these HIV-2 mutants with differences in cell tropism and rates of replication will be highly useful in understanding the mechanism of viral infectivity and possibly pathogenicity in vivo.