[Relationship between PD-1 expression on peripheral T lymphcytes and HBeAg seroconversion after entecavir treatment in chronic hepatitis B patients].

To observe longitudinally the expression of Programmed death 1(PD-1) on peripheral blood T cells in chronic hepatitis B patients underwent antiviral treatment with entecavir (ETV) and to explore the relationship between PD-1 expression and HBeAg seroconversion. Twenty HBeAg positive patients underwent antiviral treatment with ETV were followed up for 51 weeks. 14 patients remained HBeAg positive and 6 patients achieved HBeAg seroconversion. Peripheral blood was collected at six time points: T0: baseline, T1: 2-4week; T2: 5-10week; T3: 11-20week; T4: 21-30week: T5: 31-51week. PD-1 expressions on T cells were assessed by flow cytometry. Serum HBV DNA loads were determined by real-time fluorescent quantitative polymerase chain reaction (PCR) and serum ALT levels were examined at the same time. At baseline, serum HBV DNA load of patients without HBeAg seroconversion and with HBeAg seroconversion were (7.54+/-0.67) log10 copies/ml and (7.30+/-0.79) log10 copies/ml (P more than 0.05), the ALT levels were (187.26+/-184.15) U/L and (272.17+/-215.07) U/L (P more than 0.05), PD-1 exprissions on CD4+ T cells were 6.04%+/-3.71% and 6.77%+/-2.88% (P more than 0.05), PD-1 exprissions on CD8+ T cells were 6.39%+/-3.33% and 8.88%+/-2.84% (P more than 0.05). After ETV treatment, serum HBV DNA loads and ALT levels both decreased gradually, which was positively correlated with PD-1 expressions on CD4+ and CD8+ T cells (r=0.212, P = 0.05; r=0.377, P less than 0.01; r=0.279, P less than 0.05; r=0.347, P less than 0.01). During the same monitoring period, the HBV DNA loads, ALT levels and PD-1 expressions on T cells of the patients with HBeAg seroconversion decreased significantly as compared with the patients without HBeAg seroconversion. Besides, the decrease of HBV DNA loads during period deltaT0-T1 and deltaT0-T2 and PD-1 expressions on CD8+ T cells during period deltaT0-T2 and deltaT0-T3 were significantly different between these two kinds of patients (49.9% vs 37.3%, P less than 0.05; 56.7% vs 47.4%, P less than 0.05; 70.1% vs -4.2%, P less than 0.05; 66.9% vs 24.5%, P less than 0.05). The rapid decrease of PD-1 expression on peripheral CD8+ T cells after antiviral treatment with ETV is positvely correlated with the decrease of serum HBV DNA loads and may be used as a predictive index for HBeAg seroconversion in HBeAg positive patients.

[Establishment of long-term culture system for human B cells derived from peripheral blood mononuclear cells activated via soluble CD40 ligand].

OBJECTIVE: To develop a method for long-term culture of human B cells from peripheral blood mononuclear cells (PBMCs) by means of human soluble CD40 ligand (sCD40L), and to investigate the proliferation and antigen-presenting function of the CD40-activated B cells. METHOD: Peripheral blood sample of 30 ml was collected from a healthy person. PBMCs were isolated and cultured in the presence of sCD40L. Flow cytometry was used to examine the proliferation, DNA cycle, and cell surface markers: CD86, CD80, major histocompatibility complex (MHC) class II, and MHC Class I of the B cells. The cells cultured for 45 days were divided into 2 groups: Group 1 incubated with the fluorescein isothiocyanate (FITC)-conjugated hepatitis-B core antigen (HBcAg-FITC) and Group 2 used as control group. Eighteen hours later cytometry and fluorescence microscopy were used to detect the peptide pulsing. RESULTS: The B cells could be cultured up to 50 days in the sCD40L culture system. The ratio of B cells in the PBMCs was 8.21% at the beginning, and increased to 70.67% by day 47, and the B cell absolute count was 6.56 x 10(5) at the beginning and increased to 8.61 x 10(6) at day 50, about 10 times higher. sCD40L promoted a strong up-regulation of cell surface markers. The expression rates of CD80, CD86, MHC-II, and MHC-I of the sCD40L-activated B cells were 83.97%, 84.73%, 99.59%, and 98.70% respectively. Flow cytometry showed that 98.10% of the B cells co-incubated with HBcAg-FITC were loaded with HBcAg. Fluorescence microscopy showed that the HBcAg was in the cytoplasm of the B cells. CONCLUSION: A human sCD40L culture system has been developed with the ability to culture human peripheral blood B cells, thus realizing the long-term proliferation and activation of human peripheral blood B cells that function as antigen-presenting cells.

Inhibitory effect of silymarin on CCl4-induced liver fibrosis by reducing Ly6Chi monocytes infiltration.

It has been well established that silymarin has hepatoprotective and anti-fibrotic effects. But the mechanisms are poorly understood. In recent years, the role of Ly6Chi monocytes in liver fibrosis has been well demonstrated. Thus, in present study we aimed to investigate whether silymarin can relieve liver fibrosis by reducing Ly6Chi monocytes infiltration. The mouse model of liver fibrosis was established by injected with carbon tetrachloride (CCl4) via intraperitoneal repeatedly. Mice in silymarin group received silymarin treatment by gavage. Silymarin significantly reduced liver inflammation and fibrosis of the mice induced by CCl4 injection, as revealed by liver histological and pathological analysis. Mice administrated by silymarin exhibited less infiltration of Ly6Chi monocytes. But there was no difference on other tested leukocyte subsets between CCl4 group and silymarin group. Meanwhile, further study found that silymarin significantly reduced CCl4-induced increased expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1 and monocyte chemoattractant protein 1 (MCP-1), which was in line with the decreased numbers of intrahepatic Ly6Chi monocytes. In conclusion, our study showed that the anti-inflammatory and anti-fibrotic effects of silymarin could be contributed to the prevention of Ly6Chi monocytes infiltration into the injured livers, which will give us a better understanding on the cellular mechanism of hepatoprotective and anti-fibrotic effect for silymarin.

Association of serum gamma-glutamyl transferase with treatment outcome in chronic hepatitis B patients.

AIM: To investigate the association of serum gamma-glutamyl transferase (GGT) levels with chronic hepatitis B infection and hepatitis B e antigen (HBeAg) seroconversion. METHODS: A retrospective study was performed on clinical data collected from patients who had been positive for hepatitis B surface antigen for > 6 mo and who were antiviral-treatment naïve (n = 215) attending the Hepatitis Clinic at Nanjing Drum Tower Hospital between August 2010 and December 2013. Healthy individuals without liver disease (n = 83) were included as controls. Patients were categorized into four groups based on disease status as recommended by the European Association for the Study of the Liver: immune tolerance (IT; n = 47), HBeAg-positive hepatitis (EPH; n = 93), HBeAg-negative hepatitis (ENH; n = 20), and inactive carrier (IC; n = 55). Prediction of complete response (CR) based on serum GGT was also examined in EPH patients (n = 33) treated for 48 wk with nucleos(t)ide analogue (NA) therapy, including lamivudine plus adefovir combination therapy (n = 20) or entecavir monotherapy (n = 13). CR was defined as a serum hepatitis B virus DNA level < 500 copies/mL and HBeAg seroconversion by 48 wk of treatment. RESULTS: Serum GGT levels were significantly increased in EPH and ENH patients relative to the IT, IC, and healthy control groups (P < 0.01 for all). However, no significant difference in serum GGT levels was found between the EPH and ENH groups. Baseline serum GGT levels were significantly higher in patients who achieved CR (7/33; 21.2%) compared to patients in the non-CR group (26/33; 78.8%; P = 0.011). In addition, the decline in serum GGT was greater in CR patients compared to non-CR patients after 24 wk and 48 wk of treatment (P = 0.012 and P = 0.008, respectively). The receiver operating characteristic curve yielded a sensitivity of 85.71% and a specificity of 61.54% at a threshold value of 0.89 times the upper limit of normal for baseline serum GGT in the prediction of CR following NA therapy. CONCLUSION: Serum GGT is significantly elevated in EPH and ENH patients and is a potential biomarker for the prediction of HBeAg seroconversion following NA therapy.

[Effectiveness and safety of bicyclol combined with thymosin in treatment of chronic viral hepatitis B].

OBJECTIVE: To analyze the efficacy and safety of bicyclol combined with thymosin in treatment of chronic viral hepatitis B (CHB). METHODS: A total of 135 patients with CHB were randomized into experimental group and control group. The patients in the experimental group received bicyclol orally 75 mg daily and thymosin 20 mg intramuscular injection once every 2 days for 24 weeks and those in control group received bicyclol orally 75 mg daily alone for 24 weeks. The levels of serum aminotransferase (ALT/AST), HBV-DNA, HBeAg /antiHBe were observed. RESULTS: Compared with pre-treatment levels, the serum aminotransferase levels decreased significantly in both groups, but there were no statistically significant differences between them. HBeAg negative conversion rate was significantly higher in the experimental group than in the control group (35.3 percent vs.19.4 percent, P less than 0.05). HBV DNA negative conversion rate was significantly higher in the experimental group than in the control group (36.7 percent vs. 20.9 percent, P less than 0.05). No obvious adverse events which were probably related to the drugs were observed in this study. CONCLUSION: The combination of bicyclol with thymosin had better effect in treatment of chronic hepatitis B ias compared with bicyvlol alone.