| Co-Investigator(Kenkyū-buntansha) |
SAITO Sakura NIH,Dept. of Viral Disease and Vaccine Control, Chief Researcher, ウィルス製剤部, 主任研究員 (90175349)
MATSUURA Yoshiharu NIH,Dept. of Virology II,Laboratory Chief, ウィルス第二部, 室長 (50157252)
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| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1996: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1994: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
I.IFN treatments are applied to many diseases like cancers, chronic viral infections and some intractable diseases. In the case of chronic hepatitis C virus (HCV) infection, 70% patients are insensitive to the treatments in spite of long term and high dose of IFN.Kohase et. al., previously reported that IL-1, EGF,PDGF inhibited production of IFN and it's antiviral activity in human diploid fibroblast cells (FS-4). In this study, we examined whether cytokines have suppressive effects on induction of antiviral state in primary monkey hepatic parenchymal cells. Moreover, Hep G2 cells constitutively expressing HCV proteins were tested for their sensitivities to antiviral activities of IFNs. The level of antiviral state was quantitatively assayd by the method of virus (VSV) yield reduction. Results : 1) IL-1, EGF,HGF,but not TNF,IL-6 PDGF,TGF-beta suppressed antiviral activities of human IFN-alpha、beta、gamma in primary monkey hepatic parenchymal cells. 2) High doses of human IFN-gamma showe
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d cytotoxic activity on primary monkey hepatic parenchymal cells and the levels of the cell damage, measured by LDH-release assay, were suppressed by IL-1, EGF,HGF.In addition, IFN-gamma induced apoptosis in primary mouse hepatic parenchymal cells was suppressed by IL-1, EGF and HGF,and not by IL-6, TNF,PDGF,TGF-beta, IFN-alpha/beta. 3) Three different Hep G2 cell lines stably expressing various regions of the HCV gemome (core-E1-E2, DELTA NS2-NS3-NS4-DELTA NS5A and entire ORF) had equal sensitivity to IFNs as that of control Hep G2 cells. ^<**>Primary monkey hepatic cells were isolated from the cynomolgus monkeys used for the safety tests of virus vaccine
II.The double stranded (ds) RNA dependent protein kinase (PKR) hasimportant roles not only in the antiviral activity induced with interferon but also in the control mechanism of cell growth. Although it is reported to distribute over a wide range of animal cells, any laboratories have not been able to detect its activity in hyman diploidfibroblast cells by the in vitro assay system. We have succeeded to demonstrate its phosphorylation activity of a subunit of eIF-2 added as a substrate exogenously. Furthermore, we studied its protein immunologically using a polyclonal antiserum prepared against N-terminal half peptide of PKR.The PKR protein was detected in the extract of FS-4 cells by Western immunoblotting, but its increase by IFN was very little. The immunoprecipitation by this antiserum and the following electrophoresis made us possible to distinguish clearly between phosphorylated and unphosphorylated PKR.Taking advantage of this method, we have found revealed that almost all of PKR existed as phospho-rylated form in FS-4 cells. These results suggested that the auto-phosphorylation activity of PKR in the extract of FS-4 cells could not be detected in vitro because that it had been phosphorylated already in cells. Less
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