The focus of this proposal is to elucidate the molecular processes occurring in immune responses of the central nervous system (CNS). Interferon-gamma (IFN-gamma) is a powerful effector molecule with major implication in immunologically-mediated inflammatory diseases of the CNS. Since IFN-gamma induced gene products are the ultimate effectors of the biological activities achieved by IFN-gamma, the role of novel IFN-gamma inducible genes and their products in glial cells will be investigated. Recently a group of II CDNA library made in RAW 264.7 cells stimulated with IFN-gamma-rich medium.
Specific aims proposed are: 1) to analyze the differential expression of these IFN-gamma inducible genes in astrocytes and microglia, and investigate whether viruses or other cytokines in addition to IFN-gamma induce them, 2) study the induction mechanisms of one of these genes, crg-2, in astrocytes, 3) determine the role of CRG-2 protein in Ia induction by IFN-gamma or virus and 4) study the in vivo expression of crg-2 in measles infected Lewis rats and correlate its expression with measles virus-induced CNS immunopathology. To accomplish these goals, primary astrocytes, microglia and macrophages from rat will be used. Transcriptional regulation and the identification of cis and trans regulatory elements of crg-2 gene will be studied by northern analysis, nuclear run-on assays, mobility shift DNA-binding analysis and CAT assays. Anti CRG-2 antibodies and anti-sense oligonucleotides will be used to test whether Ia induction requires CRG-2. In vivo studies of measles infected brains will include immunocytochemistry, northern analysis and in-situ hybridization. The proposed studies in this application, we believe, will generate such needed information on certain CNS diseases by delineating molecular mechanisms involved in inflammatory pathways.
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