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Objective To investigate the effects of HBcAg-specific CD8+T cells on inhibiting HBV replication in vitro,and to search the cytokine of noncytolytic mechanisms in viral clearance. Methods By the method of coculture of HepG2.2.15 cell (target cells) with HLA-A2 matched HBcAg-specific CD8+T cell clone (effector cells) at E:T ratios of 1:50,and monitoring HBV production (HBsAg,HBeAg,and HBV-DNA)in coculture supernatants at 24h,48h and 72h,the percentage of decrease in HBV replication level was observed. Furthermore,blocking experiment with neutralizing mAbs to IFN-? was performed to evaluate the effect of this cytokine. Results CD8+T clone produced high levels of IFN-?following coculture with 2.2.15 cells. HBsAg,HBeAg and HBV-DNA in coculture supernatants were significantly reduced,and the greatest effect was observed at 72h by 54.55%,50.36% and 74.55%,respectively. The reduction of HBV DNA was decreased followed by using neutralizing mAbs to IFN-?. The maximum activity of cytotoxicity of target cells was at 24h by 15.66%. Conclusion ①HBV-specific CD8+T cells inhibit HBV replication by cytolytic and noncytolytic mechanisms.②The effect of noncytolytic mechanisms is mainly mediated by IFN-?.
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Objective To investigate cytokine levels in serum and culture medium of peripheral blood mononuclear cells(PBMC) from patients with hepatitis B virus(HBV)infection.Methods PBMCs isolated from fresh heparinized blood were cultured and stimulated with rHBcAg.After 72h at 37℃ 5% CO2 in air,the culture supernatant was collected.Levels of interferon-gamma(IFN-?) and interleukin(IL)-4 in blood serum in spontaneous and supernatant were measured by enzyme-linked immunosorbent assay(ELISA).Results Serum IFN-? levels in patients with acute self-limited hepatitis B(AH) and chronic hapetiti B(CHB) were significantly higher than those in normal control(NC)(P
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Objective To generate HLA - A * 0201 and A * 2403 restricted HBcAg - specific cytotoxic T lymphocyte (CTL) clones. Methods Peripheral blood mononuclear cells (PBMCs) were derived from a HLA - A * 0201/2403 - positive patient with chron- ic hepatitis B . PBMCs were stimulated respectively using two synthetic peptides( HBc18 ～ 27 and HBc 117 ～ 125) , and epitope -specific CTL clones were generated by limiting dilution technique with PHA alone or combined with synthetic peptides. The cell clones were then characterized by IF staining,FCM and LDH release. Results After 2 weeks of in vitro stimulating PBMCs, specific CTL lines were established. 29 T clones were generated from those CTL lines using HBc18 ～27 stimulating. Among the 29 clones ,28 clones belonged to CD8~+ T cells and all displayed cytolytic activity. 12 CD8 +T clones were generated from those CTL lines using HBc117 ～ 125 stimulating. Specific cytotoxic activity was observed in 9 of those clones. The other three displayed less cytolytic activity. In the process of cloning, PHA was used alone or combined with synthetic peptides,and the achievement showed a rate of 15.62% and 14.58% . Conclusion HBc18 ～ 27 and HBc117 ～ 125 are capable of activating CD8~+ T cells in PBMCs of HLA - A * 0201 /2403 patient with chronic hepatitis B. In the process of CD8~+T cloning, synthetic peptides could not increase the rate of successful cloning.
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Objectives: To express HBeAg in prokaryotic and eukaryotic cells and compare the two types of HBeAg in the anti-HBeAg testing. Methods: HBeAg was expressed both in E.coli cells and in silk worm cells, purified by Sephacryl S-200.HBeAg protein concentration and antigenic titer were determined respectively by ultraviolet-spectroscopy and EIA. Results: HBeAg produced by E.coli cells: Activation ratio was 10 000/mg, HBeAg/HBcAg = 50; The specificity in testing anti-HbeAg was 96%;HBeAg produced by silk worm cells: Activation ratio was 160 000/mg, HBeAg/HBcAg = 5 000, The specificity in testing anti-HbeAg was 100%. Conclusions: HBeAg produced by eukaryotic cells contained much lower proportion of HbcAg and higher activation ratio, which therefore bring about a possibility to improve the quality of the kit for testing Anti-HBe.
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We have observed and studied the immune response, ultrastructure and phagocytosis of peritoneal macrophages (M?) of mice in protein deficiency by means of indirect fluorescent antibody test (IFAT), immunoenzymatic staining technique (IEST),fluorescence isothiocyanate antibody (FITC-Ab) quantitative assay, M? phagocytosis test, scanning electron microscopy (SEM) and im-munoelectron microscopy (IEM).The results showed that the body weight of mice was continuously declined after fed protein deficient diet. In the same time fluorescence reaction and enzyme stain on the M? surface was retarded. The amount of FITC-Ab on the M? membrane was decreased. The villi on the M? surface were shortened, the positive rates and positive degree of cells were lowered,the reaction of cell membrane and nuclear membrane was retarded in SEM and IEM.The phagocytic function of M? was inhibited.The results showed that in protein deficiency, the immune reaction, structure and function of peritoneal M? of mice were markedly affected.