Observation of PD-1+CXCR5+CD4+T lymphocyte and sPD-1 levels in HBeAg positive chronic hepatitis B virus carriers treated with entecavir / 中华肝脏病杂志

Objective: To dynamically observe the clinical efficacy of entecavir and the changes of PD-1+CXCR5+CD4+T lymphocytes and sPD-1 levels in peripheral blood of HBeAg-positive chronic hepatitis B virus carriers treated with entecavir, and further explore its clinical significance. Methods: There were 31 cases of chronic hepatitis B virus carriers in the treatment group (A), 32 cases of chronic hepatitis B virus carriers in the treatment group (B), and 15 cases of chronic hepatitis B virus carriers in the non-treatment group (C).Three groups peripheral blood samples and clinical data at 0, 24 and 48 weeks were collected and compared. PD-1+CXCR5+CD4+T lymphocytes were detected by flow cytometry, and the level of sPD-1 was detected by enzyme-linked immunosorbent assay. ANOVA and Spearman correlation analysis were performed on the measurement data among the three groups. Results: At week 0, the serum levels of HBsAg, HBeAg and HBV DNA were significantly higher in groups A and C than group B. PD-1+CXCR5+CD4+T lymphocytes in peripheral blood were significantly higher in group B (4.70%±1.58%) than group A (3.25%±1.01%) and group C (2.77%±0.67%) (F=16.65, P<0.05). There was no significant difference between group A and group C (P>0.05). Peripheral blood sPD-1 in group B [(1 866.62±1 472.70) pg/ml] was significantly higher than group A [(824.86±538.66) pg/ml] and group C [(618.19±602.62) pg/ml] (F=10.95, P<0.05). There was no significant difference between group A and group C (P>0.05). At 48 weeks, the serum HBsAg did not decrease significantly in groups A and C than baseline (P>0.05), but were significantly higher than group B (P<0.05). Serum HBeAg levels were decreased significantly in groups A and B than baseline (P<0.05). <0.05), but group A was significantly higher than group B (P<0.05), and there was no significant difference between group A and group C (P>0.05). Serum HBV DNA level was significantly lower in groups A and B than group C (P<0.05), and there was no significant difference between group A and group B (P>0.05). Peripheral blood PD-1+CXCR5+CD4+T lymphocytes were significantly lower in Group A (1.56%±0.73%) and group B (1.32%±0.43%) than group C (2.64%±0.85%) (P<0.05). Peripheral blood sPD-1 were significantly lower in group A [(289.05±215.86) pg/ml] and group B [(236.01±173.92) pg/ml] than group C [(650.34±598.46) pg/ml] (P<0.05). There was no significant difference between group A and group B. Correlation analysis results: In group A at 48 weeks, the decreased level of PD-1+CXCR5+CD4+T lymphocyte ratio had no correlation with the decreased level of HBsAg and HBV DNA, but was positively correlated with the decreased level of HBeAg (r=0.376, P<0.05). The decreased level of sPD-1 had no correlation with the changes of HBsAg, but was positively correlated with the decreased levels of HBeAg and HBV DNA (r=0.598 and 0.384, P<0.05). In group B at 48 weeks, the decreased levels of PD-1+CXCR5+CD4+T lymphocytes and sPD-1 were positively correlated with the decreased levels of HBsAg, HBeAg, and HBV DNA (P<0.05). Conclusion: Hepatitis B virus replication and expressions in HBeAg-positive chronic hepatitis B virus carriers were significantly inhibited after 48 weeks of antiviral treatment, which is related not only to entecavir treatment, but also to the immunological mechanism involved in sPD-1. Moreover, the inhibition of HBeAg expression is associated with a decrease in the number and/or activity of PD-1+CXCR5+CD4+T lymphocytes.

Study on the replication of Hepatitis B Virus in primary hepatocytes from heterogenous species / 中华肝脏病杂志

<p><b>OBJECTIVES</b>By studying the possibility and degree of the replication and expression of human hepatitis B virus (HBV) genes in normal liver cells from heterogenous species, such as primary duck hepatocytes (PDH) and primary rat hepatocytes (PRH), to investigate the species-specificity of HBV infection and replication.</p><p><b>METHODS</b>PDH and PRH isolated by in situ perfusion with low concentration collagenase were transfected with complete HBV genome by electroporation (transfection group, about 1.19 10(12) copies of linear HBV DNA/1 10(7) PRH/PDH). From 1 day to 15 days after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH/PDH were measured with IMX System, HBcAg was assayed with western blotting, Immunol dot blotting and Immunocytochemistry. Moreover, HBV S-mRNA and X-mRNA were tested with RT-PCR. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PRH/PDH electroporated only was used as control group.</p><p><b>RESULTS</b>HBsAg in the lysates of transfected PDH group were 15.24 (1day), 14.55 (3 days) and 5.13 ( 5 days; P/N values, positive>or=2.1), HBeAg all was negative (<2.1), and both were negative in the supernatants of transfected group. Viral antigen production in transfected rat hepatocytes: HBsAg in the lysates of transfected hepatocytes was positive, P/N values ranging from 2.17 to 93.41, The average P/N values was 14.74+/-31.82, and could be maintained for 15 days after transfection. Whereas, HBsAg in the supernatants of transfected group was only found positive on 1 day after transfection, which P/N value was 6.66. HBeAg and HBcAg in the lysates of PRH of transfection group were positive during the first 3 days following transfection, P/N values was around 2.8. The total amount of HBV DNA in the transfected PDH and PRH groups was strongly positive by dot blotting, whereas that of the control group was negative. Southern blot analysis of intracellular total HBV DNA indicated that there were relaxed circular (RC), covalently closed circular (ccc) and single-stranded (SS) HBV DNA replicative intermediates in the transfected PDH and PRH groups, there was no integrated HBV DNA in the cellular genome. Control groups were negative at all.</p><p><b>CONCLUSION</b>Expression of HBV genes and production can occur in hepatocytes from nonmammalian species or mammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.</p>

An in vitro model of hepatitis B virus gene replication and expression in primary rat hepatocytes transfected with circular viral DNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish an in vitro model of hepatitis B virus (HBV) replication and expression in primary rat hepatocytes (PRH) transfected with circular viral DNA for further study on the interaction of HBV with hepatocytes.</p><p><b>METHODS</b>Circular viral DNA containing complete HBV genome were transfected into PRH by electroporation (transfected group, about 4mug of circular viral DNA/1 10(7)cells). From day 1 to day 10 after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH were measured with IMX system. HBcAg was assayed with western blotting, immunol dot blotting and immunocytochemistry. Meanwhile, HBV S-mRNA and X-mRNA were tested with RT-PCR, and replicative intermediates of HBV DNA were analyzed by southern blotting and dot blotting. Moreover, Transmission electron microscopy was used if viral particles were produced in transfected rat hepatocytes. PRH electroporated only was used as control group.</p><p><b>RESULTS</b>(1) Viral antigen production in transfected rat hepatocytes: HBsAg in cell lysates was positive. P/N values ranged from 4.83 to 85.69, and could be maintained for 10 days after transfection. The average P/N values was 18.239 27.459. Whereas, HBsAg was negative in the supernatants of transfected group (P/N values, negative<2.1). HBeAg in the supernatants and lysates of transfected hepatocytes all was negative (P/N values<2.1) during 10 days following transfection. HBcAg was only found positive in transfected hepatocytes by immunol dot blotting. (2) Detection of viral transcripts: transcription of HBV DNA was investigated by preparing total RNA from rat hepatocytes 2 days after transfection and looking for S-mRNA and X-mRNA by RT-PCR. Results showed S-mRNA positive, X-mRNA negative. (3) HBV DNA replication analysis: intracellular total DNA was extracted 2 days after transfection and analysed by southern blotting. All replicative DNA intermediates, including relaxed circular (rcDNA), covalently closed circular (cccDNA), and single-stranded (ssDNA) linear HBV DNA forms, were indicated. Dot blotting showed intracellular HBV DNA positive in transfected group during 10 days after transfection. However, viral particles were not found in transfected hepatocytes during 3 days after transfection.</p><p><b>CONCLUSIONS</b>Circular HBV DNA transfected into primary adult rat hepatocytes could obtain continuous replication and stable expression of HBV surface antigen. This in vitro model has high reproducibility and stability, and is useful for directly studying the interaction of HBV with hepatocytes.</p>