Nitric oxide (NO) has been implicated as a pathogenic mediator in a variety of central nervous system (CNS) We have reported (1) that Borna disease virus, rabies virus, and herpes simplex virus induce the increased expression of inducible nitric oxide synthase (iNOS) mRNA in the brains of intrathecally infected mice and rats. We and others (2) also found that the induction of experimental allergic encephalomyelitis resulted in a similar increase in iNOS mRNA expression, suggesting that a similar phenomenon may occur in the brains of patients with multiple sclerosis (MS). We investigated this hypothesis by detecting iNOS mRNA in extracts of brains from patients who died with MS and from extracts of brain tissues from patients who died from nonneurological diseases. We have also colocalized the iNOS expressing cells by using immunocytochemical techniques combined with reverse transcriptase (RT) driven-in situ PCR and demonstrated the presence of iNOS protein by the same double labeling procedures. Finally, as a surrogate marker for nitric oxide (NO) presence, we have immunocytochemically localized nitroty-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.rosine adducts in histological sections of brain from MS patients. MATERIALS AND METHODSSources and Selection of Specimens. The samples used in these studies were collected from patients with an antemortem diagnosis of MS based on clinical presentation and reconfirmed by histopathology after death. All MS samples were found to contain pathognomonic MS plaques; control brains were examined and found to be pathologically unremarkable. In most cases, autopsy samples were of frontal lobes, which provided mixed white and gray matter.In two of the MS cases, special efforts were made to preserve the tissue morphology. Small portions of the brain specimens were frozen in liquid nitrogen immediately after sectioning and then stored at -70°C. This procedure preserved the architecture of the brain significantly better than direct freezing of fresh brain specimens at -80°C, which results in ice crystal formation in the tissue and distorts its architecture.Cell Culture and Cytokine Activation. A549 human pulmonary epithelial cells were purchased from the American Type Culture Collection. Cells were grown in Ham's F-12 medium supplemented with 10% (vol/vol) fetal calf serum, penicillin (50 units/ml), streptomycin (50 ,ug/ml), and 5 ,tM L-glutamine. Cells were seeded into 6-well tissue culture clusters (Falcon) for Northern blot analysis or seeded into 8-well Lab-Tek culture slides (Nunc) for RT-in situ PCR. For those cultures stimulated for the expression of iNOS, interleukin 1(3 (IL-1,B at 100 units/ml), tumor necrosis factor a (TNF-a at 10 ng/ml), and interferon y (IFN-,y at 500 units/ml) were added for 8 h.For use in Northern blot analysis, the cells were scraped from the culture substrate, washed three ti...

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.

We have engineered tomato plants (Lycopersicon esculentum Mill var. UC82b) to express a gene for the glycoprotein (G-protein), which coats the outer surface of the rabies virus. The recombinant constructs contained the G-protein gene from the ERA strain of rabies virus, including the signal peptide, under the control of the 35S promoter of cauliflower mosaic virus. Plants were transformed by Agrobacterium tumefaciens-mediated transformation of cotyledons and tissue culture on selective media. PCR confirmed the presence of the G-protein gene in plants surviving selection. Northern blot analysis indicated that RNA of the appropriate molecular weight was produced in both leaves and fruit of the transgenic plants. The recombinant G-protein was immunoprecipitated and detected by Western blot from leaves and fruit using different antisera. The G-protein expressed in tomato appeared as two distinct bands with apparent molecular mass of 62 and 60 kDa as compared to the 66 kDa observed for G-protein from virus grown in BHK cells. Electron microscopy of leaf tissue using immunogold-labeling and antisera specific for rabies G-protein showed localization of the G-protein to the Golgi bodies, vesicles, plasmalemma and cell walls of vascular parenchyma cells. In light of our previous demonstration that orally administered rabies G-protein from the same ERA strain elicits protective immunity in animals, these transgenic plants should provide a valuable tool for the development of edible oral vaccines.

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.

KS Y-1 cells in the in vivo mouse model can be used to study the effects of therapeutic compounds in advanced KS.

A model for hepatitis B virus-associated chronic liver disease has been made using cloned hepatitis B virus DNA as a transgene in a severe combined immunodeficient host. These mice consistently support virus gene expression and replication. After adoptive transfer of unprimed, syngeneic splenocytes, these mice cleared virus from liver and serum, and developed chronic liver disease. This model will permit identification of the host and virus contributions to chronic liver disease in the absence of tolerance.

The human immunodeficiency virus type 1 (HIV-1) vif gene (viral infectivity gene) plays an important role in viral replication in vitro. We demonstrated that the Vif protein is membrane associated in HIV-1-infected cells and have investigated the role in viral replication of the equivalent gene in HIV-2. We constructed an HIV-2 vif minus mutant and studied its virulence and cellular tropism in vitro. Parallel experiments were also performed with an HIV-1 vif mutant to ascertain whether the two distantly related HIV-2 and HIV-1 genes might exert the same effect on viral replication. The results indicated that both HIV-1 and HIV-2 vif minus cell-free infection was not impaired when the SupT-1 cell line was used. However, differential degrees of impairment in viral replication were observed when other cell lines were used (Molt-3, U-937). Nevertheless, when viral production could not be detected, rescue experiments by coculture with the permissive cell line SupT-1 were generally positive, indicating that the viruses were still present in the inoculated cells. In contrast, when primary human cells (peripheral blood mononuclear cells and purified macrophages) were infected with HIV-1 and HIV-2 vif minus viruses no productive infection was observed and generally no virus was rescued by cocultivation. Thus, like in HIV-1, the vif gene of HIV-2 is crucial for viral infectivity in primary cells and might represent an attractive target for therapy.

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