Detection of double mutant X antigen/antibody in sera of patients with hepatitis B virus infection and its implication / 中华传染病杂志

Objective To explore the correlation between serum hepatitis B virus (HBV) X antigen/antibody (HBxAg-wild/HBxAb-wild,and HBxAg-mutant/HBxAb-mutant) and the disease progression in patients with chronic HBV infection.Methods A direct enzyme immunosorbent asssay (ELISA) was performed to detect HBxAb using recombinant antigen,and a double antibody sandwich ELISA assay to detect HBxAg using monoclonal antibody and specific rabbit polyclonal antibody.HBxAg-wild/HBxAb-wild and HBxAg-mutant/HBxAb-mutant were tested in sera from cases at different stages of chronic HBV infection.A chi-square test was employed to examine statistical significance.Results The positive rates of HBxAg-wild and HBxAg-mutant in the chronic asymptomatic HBV carriers,chronic hepatitis,hepatitis B-related cirrhosis and liver cancer were 6.2％ (2/32),10.7％ (3/28),28.6％ (6/21),43.6％ (17/39) and 3.1％ (1/32),10.7％ (3/28),33.3％ (7/21),48.7％ (19/39),respectively.The positive rates of HBxAb-wild and HBxAb-mutant in the above mentioned groups were 6.2％ (2/32),21.4％ (6/28),38.1％ (8/21),53.8％ (21/39)and 6.2％ (2/32),25.0％ (7/28),42.9％ (9/21),61.5％ (24/39) respectively.The positive rates of HBxAg-wild and HBxAg-mutant were not significantly different among the above groups (x2 =0.871,0.780,0.565 and 0.317,respectively; all P＞0.05) ; The positive rates of HBxAb-wild and HBxAb-mutant were also similar among all the groups (x2 =0.780,0.709,0.580 and 0.210,respectively; all P＞0.05).The positive rates of HBxAg-wild,HBxAb-wild,HBxAg-mutant,HBxAb-mutant in patients with low viral loads (HBV DNA＜1 × 104 copy/mL) were 36.5％ (23/63),44.4％ (28/63),42.9％ (27/63) and 54.0％ (34/63),respectively,those in patients with high viral loads (HBVDNA≥1×104 copy/mL) were 8.8％ (5/57),15.8％ (9/57),5.3％ (3/57) and 14.0％ (8/57),respectively.The positive rates of HBxAg and HBxAb were significantly higher in cases with low viral loads than those with high viral loads (x2 =12.869,11.522,22.556 and 20.976,respectively; all P＜0.05).The positive rates of HBxAg-wild,HBxAb-wild,HBxAg-mutant,HBxAb-mutant in the HBeAg positive group were 21.7％ (18/83),30.1％ (25/83),22.9％ (19/83) and 32.5％ (27/83),respectively,while those in the HBeAg negative group were 27.0％ (10/37),32.4％ (12/37),29.7％ (11/37) and 40.5％ (15/37),respectively.No significant difference of HBxAg/HBxAb positive rates between HBeAg positive group and HBeAg negative group was noticed (x2 =0.408,0.064,0.638 and 0.722,respectively; all P＞0.05).Conclusions The antigenicity and specificity of HBV X protein remains similar after the occurrence of A1762T/G1764A double mutant in X gene.It is also found that the positive rates of HBxAg and HBxAb increase with disease progression.HBxAg/HBxAb might be promoting factors for tumorigenesis in chronic HBV infection.HBxAg and HBxAb might have negative influence on HBV replication.

Correlation of hepatitis B virus X antigen expression with liver cell apoptosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the roles of hepatitis B virus (HBV) X antigen (HBxAg) in development of HBV-related liver diseases and carcinogenesis.</p><p><b>METHODS</b>Liver tissues were collected from patients with HBV infection (HBV carriers, n = 14; chronic hepatitis B (CHB), n = 24), HBV-related liver cirrhosis (LC, n = 20), or hepatocellular carcinoma (HCC, n = 20). Immunohistochemistry was used to detect the expression of HBxAg and the host apoptosis-related genes Fas and Fas ligand (Fas-L). The correlations of HBxAg with HBV DNA level in serum, inflammation grade, and fibrosis stage were statistically analyzed. Liver inflammation grade and fibrosis stage were in accordance with Knodell standard. x2test and Fisher's exact test were adopted in count data, x2split method was adopted in pariwise comparisons between multiple samples, Rank-sum test was adopted in ranked data, Spearman rank correlation analysis was adopted in correlation analysis.</p><p><b>RESULTS</b>The rates of HBxAg-positivity were similar between the patients with HBV infection (71.1%), LC (60.0%), and HCC (65.0%) (x2= 0.754, P = 0.686). The rates of Fas- and Fas-L-positivity in liver cells were also similar between the three groups (Fas: 28.9% vs. 20.0% vs. 5.0%, x2= 4.667, P = 0.101; Fas-L: 36.8% vs. 50.0% vs. 60.0%, x2= 2.988, P = 0.225). However, the positive rate of Fas in lymphocytes of liver tissue was significantly higher in the HCC patients than in the HBV-infected patients (90.0% vs. 68.4%, Z = -4.360, P = 0.00001). The expressions of HBxAg and Fas-L corresponded to regions of severe inflammation in tissues from LC patients and some HCC patients. Furthermore, the expression of HBxAg was positively correlated with Fas (r = 0.304, P = 0.02) and Fas-L (r = 0.368, P = 0.004) in the HBV-infected patients and LC patients, and the expression of Fas was positively correlated with that of Fas-L (r = 0.448, P = 0.0004). Patients with high and medium loads of HBV DNA showed significantly higher rates of HBxAg-positivity than those with low loads (88.9% and 69.2% vs. 26.7%, P less than 0.05).</p><p><b>CONCLUSION</b>In the early stage of chronic HBV infection, HBxAg may induce liver cell apoptosis by up-regulating Fas expression, and in the later stage, HBxAg may induce immune escape by up-regulating Fas-L expression in liver cells. Together, HBxAg and high HBV DNA load may promote chronic HBV infection and progression to hepatocarcinogenesis.</p>

Integrated pharmacokinetic study of multiple effective components of tea polyphenols and its correlation with anti-free radical pharmacodynamics in rats / 药学学报

LC-MS/MS method was used to simultaneously determine anti-oxidative active catechins EGCG, ECG, EGC and EC in plasma of rats treated with tea polyphenols (TP). The integrated plasma concentration (C') of TP was calculated by means of self-defined weighing coefficient based on percent AUC of individual components, thereby assessing integrated pharmacokinetic (PK) parameters of TP via log C'-T curve. The anti-free radical effects of TP were estimated using inhibitory rate of drug-containing serum collected at different times from rats against in vitro lipid peroxidation of mouse liver homogenate. The obtained E-T curves were used to calculate anti-free radical pharmacodynamic (PD) parameters of TP. E-logC and E-log C' plots and linear regression were carried out in order to obtain the correlation coefficient (R2). The results indicated that the log C'-T curves of TP, which could be best described by three-compartment model, corresponded to elimination rule of iv administration of drugs. The integrated PK parameters showed that TP was distributed in body rapidly and widely, and eliminated from deep compartment slowly. From comparison of R2 values and consistence of C'-T course and E-T course, it was evident that TP integrated PK behaviors correlated much better with its PD behaviors than individual active components, and thus demonstrated that integrated PK parameters could characterize to maximal extent holistic disposition of Chinese herbal drugs and reflect residence properties of holistic effective substances in biological body.

The cloning, expression, purification and immunological identification of wild-type and mutant hepatitis B virus X gene in pGEX-6P-2 system / 病毒学报

To settle the foundation for the future research on the influence of wild and mutant (A1762T/ G1764A) HBV X gene on the progress of chronic HBV infection and hepatic tumorigenicity, wild and mutant (A1762T/G1764A) HBxAgs expression system was constructed. The wild and mutant (A1762T/ G1764A) HBV X genes were amplified with polymerase chain reaction (PCR) from HBV genome were inserted into pGEX-6P-2 and confirmed by sequencing respectively. Prokaryotic expression vectors pGEX-6P-2-hbvx(w) and pGEX-6P-2-hbvx(m) (A1762T/G1764A) were constructed and transformed to Trans1-blue; wild and mutant HBxAgs were expressed through IPTG induction respectively; after refolding of inclusion body, the wild and mutant HBxAgs were purified with GSTrap FF; and analysised by SDS-PAGE, Western blot and ELISA. SDS-PAGE analysis showed that the expression system was able to express target protein efficiently; the concentrations of purified wild HBxAg and mutant HBxAg were 4.88 mg/mL and 5.07 mg/mL respectively; Western blot analysis certified both the wild HBxAg and the mutant HBxAg could be recognized by the same monoclonal antibody against HBxAg; the two expressed fusion antigens coated in microtiter plate were able to react with the sera of HBV infected patients but not with the sera from healthy donors in ELISA. Results demonstrated that we successfully established a system for expression of hepatitis B x antigen and lay the foundation for further research on the role and molecular mechanisms of the mutant HBxAg in the progress of chronic HBV infection and hepatic tumorigenicity.