Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

Expression of nuclear factor kappa B in hepatitis C virus core gene transfected cholangiocarcinoma cells

OBJECTIVE: To establish an experimental model for exploring the role of hepatitis C virus (HCV) in the development of cholangiocarcinoma. METHODS: Recombinant plasmids of HCV-C gene were constructed by molecular cloning techniques and identified by PCR and restriction enzyme mapping.The plasmids were then transfected into QBC939 cells (a cholangiocarcinoma cell line) by Lipofection. After selection with G418, resistant colonies were obtained and analyzed by immunocytochemistry and Western blotting. The morphology was observed by trans mission electron microscopy (TEM). The expression of NF-(k)B was detected by immunocytochemistry. RESULTS: Recombinant plasmid was shown by PCR and restriction enzyme mapping to carry the target gene. Moreover, it could efficiently express HCV-C protein in QBC939 cells. HCV-like particles were found in the cytoplasm by TEM, which were spherical with a diameter of 50-80 nm and possessed an outer membrane. Moreover, NF-(k)B activation could be shown in HCV core-transfected cells. CONCLUSION: Expression of the HCV-C gene in cholangiocarcinoma cells was achieved. Transfected tumor cells (QBC939-HCVc) could be used as a model to study the effect of HCV on the development of cholangiocarcinoma.