Objective:To explore the early predictors for clinical cure by sequential combined interferon therapy in nucleos(t)ide analogues (NAs) experienced patients with chronic hepatitis B(CHB).Methods:CHB patients received NAs treatment≥one year with hepatitis B surface antigen (HBsAg) ≤1 500 IU/mL, hepatitis B e antigen (HBeAg) negative and hepatitis B virus (HBV) DNA <100 IU/mL in the Third Affiliated Hospital of Sun Yat-sen University from June 2016 to September 2019 were included. According to the different treatment regimens, the patients were divided into interferon alone for 48 weeks group (group A), interferon combined with NAs for 12 weeks and continued NAs treatment for 48 weeks group (group B), interferon combined with NAs for 48 weeks group (group C). Basic data such as age and gender of patients were collected. HBsAg, hepatitis B surface antibody (anti-HBs) and alanine aminotransferase (ALT) were monitored at week 4, 8, 12, 24, 36 and 48. The decline of HBsAg from baseline, and the rates of clinical cure at 48 weeks were analyzed. The independent sample t test, chi-square test and rank sum test were used for statistical analysis. Logistic regression analysis was used to achieve the early prediction index of clinical cure at week 48. Results:A total of 1 020 CHB patients were followed up regularly for at least five time points. The rates of clinical cure at week 48 in group A, B and C were 34.6%(157/454), 32.7%(69/211) and 33.5%(119/355), respectively, with no statistical significance ( χ2=0.25, P=0.883). Patients were divided into the cured group (345 cases) and the uncured group (675 cases) according to the clinical outcomes at week 48. The age ((38±13) years old vs (43±12) years old), baseline HBsAg (131.00(359.80) IU/mL vs 437.60(531.50) IU/mL) and the proportion of male patients (81.7%(282/345) vs 89.5%(604/675)) of patients in the cured group were all lower than those of patients in the uncured group. The differences were all statistically significant ( t=6.32, Z=12.67, χ2=11.99, respectively, all P<0.050). There were 212 patients in the cured group who achieved clinical cure within 24 weeks of treatment. The rate of clinical cure at 48 weeks in patients whose HBsAg at week 4 decreased from baseline was higher than that in patients with increased HBsAg (41.6%(149/358) vs 28.2%(108/383)). The difference was statistically significant ( χ2=14.13, P<0.001). The rate of clinical cure at week 48 in patients with HBsAg at week 12 decreased ≤34.03% of baseline was only 6.9%(13/188). Multivariate logistic regression analysis showed that age (odds ratio ( OR)=0.962, 95% confidence interval ( CI) 0.936 to 0.989, P=0.006), HBsAg level at week 24 ( OR=0.950, 95% CI 0.934 to 0.966, P<0.001) and anti-HBs level at week 24 ( OR=1.012, 95% CI 1.005 to 1.019, P=0.001) were early predictors for clinical cure at week 48 of treatment in NAs experienced CHB patients. Conclusions:Clinical cure of NAs experienced CHB patients received sequential combined interferon therapy mostly occurs in the early stage (within 24 weeks). Age, HBsAg level at week 24, and anti-HBs level at week 24 are early predictors for clinical cure of 48-week sequential combined interferon treatment.

Objective: To investigate the key factor(s) of hepatitis B virus X protein (HBx) promoting B7-H6 gene activation. Methods: The DNA fragments of the B7-H6 promoter were amplified from the human genomic DNA using polymerase chain reaction(PCR). Products of PCR were digested by KpnI and HindⅢ, and inserted into luciferase reporter vector (pGL3-Basic). The correctness of the recombinant plasmid pGL3-B7-H6 was confirmed by sequencing. Human hepatoma cell line HepG2 were co-transfected with pGL3-B7-H6 and the eukaryotic expression vectors (pCMV-HBs、pCMV-HBc and pCMV-HBx), and the relative luciferase activity was detected.The different dose of HBx expression plasmids were transfected into the HepG2 cells, and the expression of B7-H6 protein were determined by Western blot. Results: A period of 2.2 Kb of B7-H6 promoter region were successfully cloned into pGL3-Basic, which was confirmed by DNA sequencing. The relative luciferase activity was significantly higher in the cells transfected with pGL3-B7-H6 than that in the cells transfected with the empty vector pGL3-Basic (5.24±1.25 vs. 1.12±0.31, P=0.005). The relative luciferase activity in HepG2 cells co-transfected with pGL3-B7-H6 and pCMV-HBx was remarkably increased compared with the control group (17.60±3.36 vs. 6.73±1.36, P=0.001). Western blot further demonstrated that HBx protein can significantly enhance B7-H6 protein expression. Conclusions Hepatitis B Virus X proteins enhanced B7-H6 promoter activity and promoted B7-H6 protein expression.

Objective To retrospectively investigate the relapse rates of different duration of extended consolidation after withdrawal of nucleos(t) ide analogues (NAs) treatment in patients with chronic hepatitis B (CHB) who met NAs cessation criteria.Methods 102 CHB patients discontinued treatment according to NAs cessation criteria or extended duration of consolidation therapy after meeting the cessation criteria.30 patients meeting the cessation criteria were Group A.72 patients extending consolidation therapy after meeting the cessation criteria were Group B.Based on different duration of extended consolidation therapy,72 patients were divided into 3 groups.Patients with a duration of extended 12 months after meeting NAs cessation criteria were Group B1.Patients with a duration of extended 24 months were Group B2.Patients with duration of extended 36 months were Group B3.After cessation of NAs treatment,the cumulative relapse of different group was calculated by the Kaplan-Meier method.The cumulative relapses between the selected groups were analyzed with Log-rank test.Results The cumulative relapse rates after 6,12,18,24,36 and 48 months after cessation of NAs treatment were 52.3％,70.0％,74.3％,76.7％,82.4％ and 88.4％ in Group A;24.0％,38.8％,40.6％,43.3％,43.3％ and 43.3％ in Group B;respectively.The relapse rate of Group B was much lower than that of Group A.The cumulative relapse rates after 6,12,18,24,36 and 48 months after cessation of NAs treatment were 35.0％,48.2％,51.9％,57.9％,57.9％ and 57.9％ in Group B1;18.1％,30.7％,30.7％,30.7％,30.7％ in Group B2;7.1％,21.4％,21.4％,21.4％ in group B3;respectively.The relapse rate of Group B1 was the highest,the following was of Group B2,and of Group B3 was the lowest one.The total amount of relapse in Group A,B1,B2 and B3 was 26,21,5 and 3 respectively.Conclusions A longer duration of extended consolidation therapy after meeting NAs cessation criteria may contribute to the lower relapse rates.

OBJECTIVETo determine the immune repertoires of peripheral CD4+T cell receptor (TCR) Vb CDR3 in primary biliary cirrhosis (PBC) and analyze TCR diversity and preferred usage at sequence-level resolution.

METHODSARM-PCR and high-throughput sequencing were used to obtain millions of TCR Vb CDR3 sequences from peripheral CD4+T cells isolated from 7 patients with PBC and healthy volunteers. All sequencing data were analyzed, together with corresponding clinical information, by bioinformatic software. The Mann-Whitney U test was used for statistical analysis.

RESULTSThe PBC patients showed a lower level of diversity among the peripheral CD4+TCR Vb CDR3 than the healthy volunteers, and patients with higher level progression of the disease showed a greater lack of diversity. In addition, 4 specific preferred-usage amino acid sequences were discovered for the PBC patients: ASSFTGGPVEQY, ASSLISSGNNEQF, ATSRDTLAGGPGDTQY, and SASLEGNTEAF; these sequences were also found in higher frequencies in patients with later stages of PBC.

CONCLUSIONSDecreased TCR Vb CDR3 diversities and specific preferred usage of TCR CDR3 sequences in peripheral CD4+T lymphocytes in PBC suggest that clonal expansion of a large number of CD4+T cells may be an important factor for PBC progression. These data provide a better understanding about the general characteristics of CD4+T cells in PBC patients and related to pathogenesis of the disease, and may provide useful insights into potential targets for immunotherapy.

Objective To investigate short-term efficacy and security of transplantation of human umbilical cord mesenchymal stem cells (HUCMSCs)in the course of treatment of decompensated cirrhosis.Methods Ninety-six Hepatitis B patients with decompensated cirrhosis were enrolled,among which 50 were sbject to transplantation of HUCMSCs in addition to routine therapy (the treatment group) and the rest patients were underwent routine therapy(the control group).We observed the efficacy and security at 2,4,6 and 12 week of the treatment course.Results The symptoms of atigue,anorexia,bloating and edema were greatly relieved in both groups after 4 weeks,and sustained remission after 4-12 weeks.The overall survival rate was 96％ after 12 weeks of the treatment group,2 patients were died after 8 and 12 weeks of the HUCMSCs transplantation due to hepatic encephalopathy repecticely.Compared to the baseline,ALT,AST,TBil and ALB(albumin) were improved after 2,4,6 and 12 weeks in both groups(P ＜ 0.05),and the averages of ALB(albumin) of treatment group were higher than the control group.PT(prothrombin time) and PTA(prothrombin time activity) were improved after 4,6,12 weeks in the treatment group (P ＜ 0.05),however,there were no differences in the control group during the 4,6 and 12 weeks(P ＞0.05).During the course of HUCMSCs tranplantation,the security was satisfied and no special side effects were observed.Conclusion The transplantation of HUCMSCs for therapy of Hepatitis B patients with decompensated cirrhosis is a safety and effective therapy,and can improved ALB,PT,PTA,liver function and clinical symptoms within a short period,which is an alternative method of treatment recommended.

Objective To investigate expression and significance of serum interleukin-32 of patients with chronic hepatitis B (CHB) treated with different anti-viral drugs.Methods One hundred and eighty CHB patients were randomly divided into two groups.The first group included 92 CHB patients,who were orally treated with lamivudine (LAM) 100 mg+ ADV 10 mg,once daily.The second group included 88 CHB patients who were subcutaneously treated with interferon (IFN),180 μg,once a week.The treatment period was 48 weeks.Serum IL-32 expression of patients was tested by ELISA,and the difference in IL-32 expression between two groups was compared by using statistical methods.Then the correlations between IL-32 and alanine aminotranferase (ALT),aspartate aminotranferase (AST),total bilirubin (TBIL),HBV DNA were analyzed,respectively.Results Serum IL-32 expression of all patients gradually decreased after anti-viral treatment,but the difference in IL-32 expression between two groups was not statistically significant until weeks 48.Serum IL-32 expression was positively related with serum ALT,AST and HBV DNA load,respectively,and was not related with serum TBIL.Conclusion Serum IL-32 expression of CHB patients gradually decreased after anti-viral treatment.IL-32 might be involved in the pathogenesis of CHB.

Objective To investigate the expression of chemokine receptors CCR4,CCR6,CXCR3 on Th17 cell,and analyze the correlation between chemokine receptors and serum biochemical parameters of liver function test and hepatitis B virus (HBV) DNA load in peripheral blood of chronic hepatitis B (CHB) patients.Methods Thirty patients with CHB (CHB group) and 15 healthy persons (control group) were enrolled in the study.CCR4,CCR6,CXCR3 expression levels on Th17 cell were assayed by flow cytometry.The correlation between chemokine receptors and alanine aminotransferase (ALT),total bilirubin (TBil),and HBV DNA load were analyzed using Pearson' s correlation analysis,respectively.Results CCR4,CCR6,CXCR3 expression levels on CD4 + Th17 cell in CHB group were higher than that of control group.Moreover,the difference between them was statistically significant (P ＜ 0.05).CCR4,CCR6 expression levels on CD8 + Th17 cell in CHB group were higher than that of control group (P ＜ 0.05).Although CXCR3 expression level on CD8 + Th17 cell in CHB group was higher than that of control group,there was no significant difference between them (P ＞ 0.05).CCR4,CCR6 expression levels were positively correlated with serum ALT,HBV DNA load,respectively (P ＜ 0.05),but were not correlated with TBil (P ＞ 0.05).Conclusion Expression levels of chemokine receptor on Th17 cell were increased in patients with CHB and were positively associated with degree of liver inflammation.Therefore,CCR4,CCR6 expression on Th17 cell might be involved in liver injury resulted from Th17 cell.

Objective To investigate short-term efficacy and security of telbivudine as a sequential therapy in the HBeAg positive chronic hepatitis B (CHB) patients with pegylated IFNα-2a treatment failure.Methods 27 CHB patients with HBeAg positive and HBV DNA detectable after a 48-week pegylated IFNα-2a therapy were enrolled into this study,and were assigned to group A,with telbivudine as a sequential therapy.54 CHB patients with HBeAg positive were assigned to group B,with telbivudine as a naive treatment.To assessment the efficacy and security of telbivudine at week 48.Results At week 12,the rates of aminotransferases normalization,HBeAg seroconversion and HBV DNA undetectable (＜ 100 IU/ ml) among the two groups were 59.3％,14.8％,66.7％ vs 75.9％,5.6％,46.3％ respectively.At week 24,the rates among the two groups were 92.6％,25.9％,70.3％ vs 92.6％,7.4％,85.2％,respectively.At week 48,the rates among the two groups were 88.9％,29.6％,81.5％ vs 98.1％,28.0％,83.3％ respectively.All these were not statistically significant.But the rate of HBeAg seroconversion in group A is higher than that in group B.The virological breakthrough rate of group B at week 48 is 14.8％,no virological breakthrough was observed in group A.During the treatment,38.3％ of patients had creatine kinase(CK) elevation,but there was no case stopping treatment for severe adverse effect.Conclusion CHB patients with HBeAg positive after a 48-week pegylated IFNα-2a treatment failure can also achieve the same efficacy and security as the na(i)ve treatment,after receiving telbivudine as a sequential therapy.

Objective To investigate the efficacy of combined treatment of PEG IFNα-2a and recombinant hepatitis B vaccine in CHB patients with HBeAg positive.Methods 75 CHB patients with HBeAg positive were enrolled into this study.45 patients received the monotherapy of pegylated IFNα-2a (group A),and 30 patients were treated with PEG IFNα-2a combined with recombinant hepatitis B vaccine (group B).The two groups were compared clinical features,such as ALT,HBsAg levels and HBeAg seroconversion rates,HBV DNA suppression,at different time point(At 0,24,48,72 week).Results At week 0,levels of aminotransferases,HBsAg and HBV DNA were not statistically significant between the two groups(P ＞0.05).But the level of HBeAg in group B was much more than that in group A.This diversity show statistical significance(P ＜ 0.05).During week 24 to week 48,rates of aminotransferases normalization HBsAg seroconversion HBeAg seroconversion,and HBV DNA suppression were also not statistically significant between group A and B (P ＞ 0.05).At the 72 week of follow up,levels of aminotransferases,HBeAg seroconversion rate and HBsAg levels were not statistically significant among the two groups (P ＞0.05),but the negative conversion rate of HBV DNA drop in group B was much more than that in group A,the difference was statistically significant (P =0.032).Conclusion The combined treatment of PEG IFNα-2a and recombinant hepatitis B vaccine in CHB patients with HBeAg positive can improve the negative conversion rate of HBV DNA 72 weeks after the end of the 48 week of treatment,but wasn't associated with HBeAg seroconversion and HBsAg levels.

Objective To investigate the expression of IL-6 and IL-18 in serum and its correlation with aspartate aminotransferase (AST),alanine aminotransferase (ALT),total bilirubin (TBil),albumin (ALB),creatinine (Cr) and plasma ammonia in patients with hepatic encephalopathy.Methods Forty patients with liver cirrhosis complicated with hepatic encephalopathy were aligned to group A,where 18 patients with grade Ⅲ to grade Ⅳ were divided into group A1 and 22 patients with grade Ⅰ to grade Ⅱ were divided into group A2.Twenty liver cirrhosis patients were aligned to group B and 20 healthy persons were aligned to group C.The concentration of IL-6 and IL-18 in serum was detected by enzyme-linked immunosorbent assay (ELISA).The correlation between IL-6 or IL-18 and AST,ALT,TBil,ALB,Cr or plasma ammonia was analyzed by Pearson' s correlation analysis.Results Serum IL-6 and IL-18 levels in both A1 and A2 groups were significantly higher than those of group B and C.The difference was statistically significant (P ＜0.05).Both IL-6 and IL-18 levels were positively correlated with the ammonia,but there were no significant correlation with AST,ALT,TBil,ALB and Cr (P ＞ 0.05).Conclusion Serum IL-6 and IL-18 levels significantly increased in patients with hepatic encephalopathy.IL-6 and IL-18 may have a synergistic effect with ammonia,which were jointly involved in the pathogenesis of hepatic encephalopathy.