TAKAKI Akinobu Okayama University, University Hospital of Medicine and Dentistry, Assistant, 医学部・歯学部附属病院, 助手 (80359885)
SHIRATORI Yasushi Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Professor, 大学院・医歯薬学総合研究科, 教授 (70196624)
|Budget Amount *help
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2005: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2004: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
Background and Aims : It has been reported that dendritic cells (DC) of chronic hepatitis C patients showed reduced allo-stimulatory function. However, the exact mechanisms of this phenomenon are not well defined. To investigate the component of HCV polyprotein that has the most important effect on DC cell function, we examined the modulatory effects of 7 HCV protein genes (g-core, g-NS2, g-NS3, g-NS4A, g-NS4B, g-NS5A, and g-NS5B) and 5 recombinant proteins (p-core, p-NS3, p-NS4, p-NS5A, and p-NS5B) on the DC surface marker expression and its functions.
Methods : Peripheral blood mononuclear cells (PBMC) were separated from healthy volunteers using a Ficoll gradient, and CD14+ cells were enriched using CD14 microbeads. On day 5, the cells were harvested and pulsed with 7 HCV protein plasmids by the lipofection method or incubated with 5 recombinant HCV proteins. After culturing for 48 hrs, the cells were assayed for cell surface marker expression by flow cytometric analysis. For cytokin
e analysis and proliferative T cell response analysis, the immature DC and LPS matured DC were cultured with auto CD4+ T cells and PPD 0.1 microgram / well. After 2 more days of culture, the supernatants were measured for cytokine concentration. After a further 3 days of culture, 3H-thymidine uptake was measured. The results were all shown as the ratio of HCV protein data to data of the control vector or buffer control.
Results : The NS4A and NS4B genes and NS4 protein reduced the expression of CD86. The NS4B and NS5A genes and NS4 protein induced relatively low T cell responses. Cytokines of the culture supernatant showed that the concentration of Th1 cytokines of the supernatants were low in g-NS4 transduced and p-NS4 - added DC and CD4 T cell cocultures. However, these effects of HCV proteins on DC function were all restored by LPS maturation.
Conclusion : HCV NS4 protein may reduce the CD86 expression levels and the function of DC and hamper the Th1 immune responses. However, this effect can be restored when the DCs were well matured. Less