Lisa Bobroski

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.

OBJECTIVES: The majority of HIV-1-infected individuals manifest a plethora of central nervous system (CNS) diseases unrelated to opportunistic infections, including AIDS dementia complex, encephalitis, and various other disorders of the CNS. The present study sought to evaluate the cellular reservoirs and expression patterns of HIV-1 in brain tissue to gain further understanding of HIV-1 neuropathogenesis. DESIGN: CNS tissue, obtained post-mortem from 22 patients with AIDS and four HIV-1-seronegative controls, was analyzed. METHODS: CNS samples were evaluated using a combination of in situ DNA polymerase chain reaction (PCR), reverse transcriptase (RT)-initiated in situ PCR, and immunohistochemistry. By utilizing this triple-staining methodology, HIV-1 proviral DNA and HIV-1-specific mRNA can be identified at the single cell level. RESULTS: HIV-1 was detected in all 22 AIDS brain specimens and in none of the four brains from HIV-1-seronegative individuals. The most commonly infected cells in AIDS brains were microglia cells and macrophages, but variable levels of HIV-1 infections were demonstrated in many of the major histological cell types within the CNS, including neurons, microvascular endothelial cells (MVEC) and astrocytes. The presence of HIV-1-infected cells was not uniform with infected cells unevenly distributed throughout the brain parenchyma. The degree of HIV-1 mRNA expression varied from 39-65% of the cells in the CNS harboring HIV-1 provirus. Choroid plexus and MVEC exhibited relatively high levels of productive infection. CONCLUSION: These findings demonstrate that several cell types in the CNS, in addition to microglia or macrophages, may become infected with HIV-1 in vivo with variable levels of HIV-1 mRNA expression. The diverse cellular reservoirs for HIV-1 in the CNS may be critically linked to the molecular mechanisms involved in HIV-1 neuropathogenesis. In addition, in vivo infection of MVEC, and possibly cells in the choroid plexus, may directly contribute to penetration of the blood-brain barrier by HIV-1.

In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent in situ polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) to be prevalent in the plaque areas of post mortem brain tissue from patients with MS. In addition, we have obtained evidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demonstration of the presence of activated macrophages expressing high levels of iNOS and evidence of peroxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease.

Human herpesvirus (HHV) type 8 has been detected in both classical and AIDS~related Kaposi's sarcoma, body-cavity lymphomas, and other types of tumors. HHV-8 has also been detected in DNA from peripheral blood mononuclear cells (PBMC) of some Kaposi's sarcoma patients and more readily in B cell fractions derived from panned cell subpopulations. Two patients were followed using several methods: in situ hybridization, solution-based polymerase chain reaction (PCR), and in situ peR. HHV-8 was intermittently detected in plasma, and detection correlated with detection in PBMC. In situ PCR demonstrated HHV-8 sequences in both peripheral blood B lymphocytes and, to a lesser extent, T lymphocytes. HHV-8 may undergo periods of viremia while at other times it is undetectable and infects circulating B cells and some T cells.

Interleukin (IL)-2-induced microvascular lung injury is an experimental paradigm commonly used to investigate the pathogenesis of the adult respiratory distress syndrome. Since tumor necrosis factor-alpha (TNF-alpha) is known to induce such an injury in vivo and since TNF-alpha is involved in other models of lung injury, we postulated that it might also mediate pulmonary toxicity after IL-2 administration. The present study tested this hypothesis by evaluating the effect of TNF-alpha inhibition on IL-2-induced lung injury in the rat. Recombinant human IL-2 (10(6) U IV per rat, n = 6) elevated lung water, myeloperoxidase activity, and protein accumulation in bronchoalveolar lavage fluid and induced tissue hypoxia. Also, IL-2 enhanced lung tissue TNF-alpha mRNA and peptide (1543 +/- 496 pg/g lung wet weight) localized to alveolar macrophages by in situ hybridization. In marked contrast, IL-2 failed to affect serum TNF-alpha, which remained at undetectable levels. Pretreatment with anti-TNF-alpha monoclonal antibody (25 mg/kg IV, n = 7) or the TNF-alpha synthesis inhibitor rolipram (200 micrograms/kg IV, n = 7) attenuated lung injury and reverted tissue hypoxia. Furthermore, TNF-alpha inhibition prevented the upregulation of lung tissue IL-1 beta, IL-6, cytokine-induced neutrophil chemoattractant, and E-selectin (ELAM-1) but not intercellular adhesion molecule-1 mRNAs in response to IL-2. These data imply that locally produced TNF-alpha mediates IL-2-induced lung inflammation and tissue injury and point to the potential utilization of TNF-alpha inhibitors in treating the pulmonary toxicity of IL-2 immunotherapy.