The aim of the present study was to investigate the overall clinical expression characteristics of the cluster of differentiation (CD) 28 family receptors [CD28, inducible T-cell co-stimulator, programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 and B-and T-lymphocyte attenuator] on T cells in patients with chronic hepatitis B (CHB), analyze the correlations among these receptors and the clinical parameters, and to investigate the effects of PD-1 blockade on the receptor expression profiles, T-cell function and other biological effects. The expression characteristics of the CD28 family of receptors, the effects of PD-1 blockade on the receptor expression profiles and the levels of interferon (IFN)-gamma were investigated in the T cells of patients with CHB. In addition, the transcription factor, T-box 21 (T-bet) and GATA binding protein 3 (GATA-3) mRNA expression levels were investigated in the peripheral blood mononuclear cells (PBMCs) of patients with CHB. The expression levels of the CD28 family receptors in the T cells of patients with CHB demonstrated distinct characteristics, for example levels of PD-1 and CTLA-4 on CD4 T cells and ICOS, PD-1, and BTLA on CD8 T cells were increased in cells from patients with CHB compared with those from the healthy individuals. A significant positive correlation was demonstrated among the serum HBV DNA titers and the levels of PD-1 on CD8(+) T cells with the highest expression of PD-1 corresponding to viral levels >106 IU/ml. A significant positive correlation was observed between the serum HBV DNA titers and the expression levels of BTLA on CD8(+) T cells with the highest expression of BTLA corresponding to viral levels >10(6) IU/ml. PD-1 blockade altered the expression profiles of CD28 family receptors in the T cells of patients with CHB, partly enhanced T cell function and increased the ratio of T-bet/GATA-3 mRNA in PBMCs. Thus, CD28 family receptors are potential clinical indicators for the rapid monitoring of changes in T cell function during CHB treatment. Furthermore, PD-1 blockade has a therapeutic potential that may be enhanced by modulating the expression of co-stimulatory and -inhibitory receptors of the CD28 family.

Objective:To clarify the differences of host immune responses at different stages of HBV infection. Methods:We constructed three HLA-A*0201/HBV tetramers with immunodominant epitopes of core18-27,polymerase 575-583 and envelope 335-343,and analyzed antigen specific CD8+ T cells and the expression of CD127 in peripheral blood mononuclear cells ( PBMCs) from patients infected with HBV using these HLA-A*0201/HBV tetramers. Results: The frequencies and expansion ability of antigen specific CD8+ T cells in most self-limited HBV infected individuals were higher than that in chronically HBV infected patients. In low copy period the frequencies of antigen specific CD8+ T cells were similar to those in immune clearance phase at a high viral load and liver damage and in immune clearance phase, which had no significant correlation with virus quantitation and ALT level. In chronic infection the ability of antigen specific CD8+ T cells proliferation was inversely proportional to the viral titer. In most self-limited HBV infected individuals the IFN-γsecretion functions of antigen specific CD8+ T cells were higher than in chronic infection,but in immune tolerance phase these cells lost the ability. HBsAg level was different at different stages after HBV infection:it was highest in immune tolerance phase,but in immune clearance phase,activity period and low copy period the correlation with HBV DNA replication gradually declined. The frequency of CD8+ CD127+ T cells in chronic HBV infection was lower than the control group and self-limited infection group,especially in immune tolerance with HBeAg+ and immune clearance phase. Conclusion: The frequencies of antigen specific CD8+ T cells are not the main determinant of immune-mediated protection in chronic HBV infection,memory antigen specific CD8+ T cells are not clear or missing,which provides the possibility for therapeutic vaccines and immunization therapy.

Immune-mediated inflammatory injury is an important feature of the disease aggravation of hepatitis B virus-related acute-on-chronic liver failure (ACLF). Toll-like receptors (TLRs) have been shown previously to play a pivotal role in the activation of innate immunity. The purpose of this study was to characterize the TLR4 expression in peripheral blood mononuclear cells (PBMCs) of ACLF patients and its possible role in the disease aggravation. Twelve healthy subjects, 15 chronic HBV-infected (CHB) patients and 15 ACLF patients were enrolled in this study. The TLR4 expression in PBMCs and T cells of all subjects was examined by real-time PCR and flow cytometry. The correlation of TLR4 expression on T cells with the markers of disease aggravation was evaluated in ACLF patients. The ability of TLR4 ligands stimulation to induce inflammatory cytokine production in ACLF patients was analyzed by flow cytometry. The results showed that TLR4 mRNA level was upregulated in PBMCs of ACLF patients compared to that in the healthy subjects and the CHB patients. Specifically, the expression of TLR4 on CD4(+) and CD8(+) T cells of PBMCs was significantly increased in ACLF patients. The TLR4 levels on CD4(+) and CD8(+) T cells were positively correlated with serum total bilirubin (TBIL), direct bilirubin (DBIL), international normalized ratio (INR) levels and white blood cells (WBCs), and negatively correlated with serum albumin (ALB) levels in the HBV-infected patients, indicating TLR4 pathway may play a role in the disease aggravation of ACLF. In vitro TLR4 ligand stimulation on PBMCs of ACLF patients induced a strong TNF-α production by CD4(+) T cells, which was also positively correlated with the serum markers for liver injury severity. It was concluded that TLR4 expression is upregulated on T cells in PBMCs, which is associated with the aggravation of ACLF.

白细胞介素10(interleukin 10,IL-10)是一种多效细胞因子,在免疫调控中发挥了重要作用,其在病毒持续性感染中的作用也日益受到关注.有研究显示HBeAg阳性患者血清高水平的IL-10和IL-12与早期自发的HBeAg血清转换相关[1],也有研究结果显示IL-10与病毒感染的持续与清除相关[2].IL-10在肝脏疾病中还显示出抗炎和抗纤维化作用[3].HepG2.2.15细胞是HBV稳定持续表达的肝癌细胞系,是可以在一定程度上代表HBV慢性感染的细胞模型.在前期研究中,我们发现该细胞模型能分泌表达IL-10蛋白[4],研究肝细胞IL-10的表达在HBV感染中所发挥的作用也许能为HBV感染后肝细胞在免疫应答中发挥的作用提供新的观点.因此,本研究旨在构建IL-10的真核表达载体,使其在肝细胞系HepG2及小鼠模型中表达,为后续的功能及其他相关研究奠定基础.

To investigate the action of isothiafludine (NZ-4), a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus (HBV) in vitro and in vivo. HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA (pgRNA) and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 (25, 50 or 100 mg·kg−1·d−1) for 15 d. NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC50 value of 1.33 μmol/L, whereas the compound inhibited the cell viability with an IC50 value of 50.4 μmol/L. Furthermore, NZ-4 was active against the replication of various drug-resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 (5, 10, 20 μmol/L) concentration-dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks. NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process, thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat HBV infection.

Background & Aims The natural course of chronic hepatitis B virus (HBV) infection is characterized by different immune responses, ranging from immune tolerant (IT) to immune activated (IA) stages. In our study, we investigated the natural killer (NK) cells activity in patients at different immunological stages of chronic HBV infection. Methods Blood samples obtained from 57 HBeAg positive patients with chronic hepatitis B (CHB), including 15 patients in the immune tolerant (IT) stage, 42 patients in the immune activated (IA) stage, and 18 healthy individuals (HI). The analyses included flow cytometry to detect NK cells, the determination of cytokine levels as well as of surface receptor expression and cytotoxicity. Results NK cells in peripheral blood were significantly lower in patients in the IA stage of CHB compared to HI (p<0.05). Patients in the IA stage of CHB had lower levels of NK cells activating receptor NKp30 and NKG2D expression, cytokine interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production, as compared to patients in the IT stage and HI, respectively (p<0.05). Cytotoxicity of NK cells was lower in patients in the IA stage of CHB compared to patients in the IT stage and HI, respectively (p<0.05). The level of IFN-γ but not level of TNF-α and cytotoxicity of NK cells was inversely correlated with serum HBV load in patients with CHB. Peripheral NK cells activity did not correlate with ALT level. Conclusion NK cells activity was lower in CHB patients, especially in those in the IA stage.

Long-term compliance with regular surveillance is important for the prevention and timely management of chronic hepatitis B (CHB). However, there are no researches focusing on the compliance of hepatitis B virus infected patients in regular surveillance so far. The purpose of our study was to investigate the outpatient compliance with long-term regular surveillance in China. Data of 3257 CHB outpatients was pooled and analyzed to assess the outpatient’s compliance with the long-term regular surveillance plan. In all outpatients, the non-follow-up and the follow-up group accounted for 73.2% and 26.8%, respectively. Among the follow-up outpatient’s, only 48.9% received ongoing-follow-up and 51.1% were finally lost to follow-up; the median length of visiting duration was 25 months; and the predictive 1-, 2-, 3-, 4- and 5-year ongoing follow-up rate was 72.7%, 52.5%, 42.4%, 33.8%, and 26.3%, respectively. In conclusion, our survey proved that the regular long-term surveillance on Chinese chronic HBV carrier is difficult to be fully implemented. A large proportion of outpatients do not receive routine follow-up and are at risk of treatment delay due to various social reasons.

Objective To compare the antiviral treatment situation between the elderly and young chronic hepatitis C (CHC) patients,and to discuss the influence factors of antiviral outcome in patients with chronic hepatitis C.Methods We retrospectively investigated 48 cases of chronic hepatitis C patients.They were divided into elderly patients (≥60 years,n =16) and young patients (＜60 years,n =32).After interferon plus ribavirin combination antiviral therapy,we observed virological response,biochemical response and adverse effects incidence in the two groups,and analyzed the antiviral outcome of possible factors.Results The rates of rapid virological response (RVR),early virological response (EVR) and sustained virological response (SVR) were 93.75％ (15/16),100.00％ (16/16),50.00％ (8/16)respectively in elderly patients.The rates of RVR,EVR and SVR were 90.63％ (29/32),100.00％ (32/32),78.13％ (25/32) respectively in young patients.There were significant differences in SVR rate between the two groups (P ＜ 0.05).Compared with the younger patients,the incidence of fatigue,neutropenia and anemia increased in elderly patients [93.75％ (15/16) vs 59.38％ (19/32),87.50％ (14/16) vs 53.13％ (17/32),56.25％ (9/16) vs 21.88％ (7/32)] (P ＜0.05).There were no significant differences in biochemical response and other adverse effects incidence between the two groups (P ＞ 0.05).The SVR rates and peg-interferon plus ribavirin antiviral treatment in young patients were higher than elderly patients and ordinary interferon plus ribavirin antiviral treatment patients respectively [78.12％ (25/32) vs 50.00％(8/16),80.00％ (28/35)vs 38.46％ (5/13)] (P ＜ 0.05).Conclusions The SVR rate of elderly CHC patients decreases significantly and the adverse effects incidence is high.The age and treatment options are related to antiviral outcome in patients with chronic hepatitis C.

慢性乙型肝炎患者临床结局受多种因素的影响,主要包括宿主因素和病毒学因素两大方面,其中病毒学因素包括HBVDNA载量,HBV基因型、HBV基因变异及HBV蛋白表达等。本文就影响慢性乙型肝炎病毒感染者临床结局的病毒学因素研究进展进行了介绍。

Objective To explore the factors in duck hepatitis B virus infection model by different kinds of ducklings and observe the antiviral effect in models.Methods Collecting duck sera,qualitative detecting viral DNA by PCR method and detecting the changes of viral DNA load by the quantitative PCR method in duck sera.Observing the antiviral effects in duck DHBV infection models after antiviral drug treatment.Results Natural infection rate of duck DHBV is different,Cherry Valley duck for 8.75%,two batches in Hubei sheldrake were 17.80% and 10.68% respectively.Both intravenous and intraperitoneal injection can cause ducklings infected with DHBV,infection rates of two ways were 80% and 65% respectiviely.Ducks infected with DHBV,the viral load is maintained at 106~108 copies / mL and persist for more than 20 days.Changing trends of antiviral effect in different DHBV models are coincident after treating by the antiviral drugs.Conclusion Duck species and artificial routes of infection can affect DHBV infection rate,in vivo the viremia of duck is stably persistent after DHBV infection.Therefore,DHBV infection model is an available model in antiviral drug screening.

HBV (hepatitis B virus) vaccine is an effective way to control HBV infection, so viruses must be in variance to escape the clearance of host's immunity. The mutations induced by HBV vaccine majored in 'a' determination. In order to study mutations those how to influence the biologic characteristic of S gene, especially in antigenicity and immunogenicity of HBsAg, We constructed series variant clones of HBV S gene 'a' determination according to data of epidemiology. After verified by sequence, we constructed a series variant clone of HBV S gene with "site-mutation method of PCR (gene SOEing PCR, gene splice of overlap extending PCR)". After cellular expression, the antigen expressed by different mutant clones were all recognized by corresponding monoclonal antibody and not done by common HBsAb with immunofluorescence assay. In a conclusion, the mutations in " a " determination can alter the biologic characteristic of S gene, especially in the antigenicity of HBsAg, in a extend. The series mutant clones of "a" determination, which we constructed, may be provide the basic theory for how to develop new HBsAg detected kit, vaccine or HBV immunoglobulin(HBIG). [Xiaofei Li, ZujiangYu, DongliangYang, Jun Zhang, Youhua Hao, Baoju Wang, Kan Quancheng. Cloning of HBV S Gene with Artificial Mutation Sites in 'a' Determinant and Analyzing the Protein Antigenicity Expressed. Life Sci J 2012;9(3):143-147]. (ISSN: 1097-8135). http://www.lifesciencesite.com. 19

T细胞功能耗竭是HBV持续性感染的一个重要特征,由T细胞表面表达的抑制性受体、抑制性细胞及细胞因子组成的抑制性微环境在介导特异性T细胞功能耗竭中扮演着重要作用.改善或阻断这些抑制性环境相关因子有可能恢复和提高慢性乙型肝炎(CHB)患者的耗竭的病毒特异性的T细胞功能,并最终清除患者体内的病毒. 关键词：肝炎病毒,乙型;T淋巴细胞;T细胞功能耗竭

Objective:To investigate the constituent ratio and its variation trend of different clinical diagnostic subtypes for patients with chronic HBsAg infection in their first outpatient visit.Methods:Through the database the clinical informations of patients with chronic HBsAg infection in the initial diagnosis during 2007~2009 were obtained.The constituent ratio and its variation trend of different diagnostic subtypes for patients with 9 chronic HBsAg infection were statistically analyzed.Results:There were 2 677 outpatients with initial diagnosis of chronic HBsAg infection during 3 years,the constituent ratio of the different diagnostic classification are ranked by HBeAg positive chronic hepatitis B in 32%,inactive HBsAg carriers in 24.1%,HBeAg negative chronic hepatitis B in 23.9%,chronic HBV carriers in 12.1%,HBeAg negative compensatory cirrhosis in 4.4%,hepatitis virus related hepatocellular carcinoma in 1.4%,HBeAg positive decompensated cirrhosis in 0.9%,hepatitis B complicated with other diseases in 0.6%,decompensated liver cirrhosis in 0.4%.During the 3 years,the type constituent ratio and sorting remained relatively stable,with HBeAg positive chronic hepatitis B patients constituent ratio present downtrend of year after year,while HBeAg negative chronic hepatitis B constituted an increasing tendency year by year;the mean age of patients and HBV natural history maintained consistent in each type.Conclusions:Chronic hepatitis B and chronic HBV carriers are the main body in outpatients.The constituent ratio of patients with HBeAg positive chronic hepatitis B has downtrend of year after year,this may be affected by the hepatitis B vaccination project in our country.

Objective To investigate the expression of innate immune-associated molecules in Huh7,HepG2,HepG2.2.15 and L02 hepatocyte lines and the influence of their expression by hepatitis B viral transfection in intro.Methods The total mRNA of the four cultured cells and three transfected cells by HBV replication clone(pAAV-HBV1.2)were extracted and reverse transcripted to cDNA.The expression of the innate immune-associated molecules such as TLRs,NLRs and RLRs were detected by PCR.Results The results showed that all of the four cell lines not only expressed the TLRs such as TLR1,TLR3,TLR4 and TLR9,but also expressed the NLRs such as NOD1,and RLRs such as RIG1.The expression of MDA5 and A20 decreased in transfected HepG2 and Huh7 cells.Conclusion Hepatocytes can express a series of innate immune-associated molecules such as TLRs and RLRs.There are distinct expression in different hepatocyte lines.HBV could influence the expression of the innate immune-associated molecules in hepatocyte lines.

Objective To construct eukaryotic expression vectors of vacuolar protein sorting 4B(VPS4B) gene with site-directed mutagenesis.Methods VPS4B gene was amplified by RT-PCR from Huh7 cells and cloned into the eukaryotic vector pXF3H.Site-directed mutagenesis method based on overlap extension PCR was used to construct two dominant negative(DN) mutants,K180Q and E235Q.The point mutation was verified by DNA sequencing.The mammalian hepatoma cell HepG2 was transfected with VPS4B and the 2 mutants.The expression of the fused proteins was determined by Western blot.Results VPS4B gene was cloned and the eukaryotic vector pXF3H-VPS4B was constructed.The mutants were obtained by overlap extension PCR.DNA sequencing showed that AAG at 538-540 bp site,which encoded the 180-amino acid,was changed to CAG,and in another mutant site,GAA at 703-705 bp site,which encoded the 235-amino acid,was changed to CAA.No mutation in other base pair sites of the recombinant plasmids.The fusion protein HA-VPS4B was detectable by Western blot in HepG2 cells transfected with the 2 mutant vectors.Conclusion The eukaryotic expression vectors for VPS4B and its two DN mutants K180Q and E235Q were successfully implemented.

OBJECTIVE:This report aims to investigate the Toll-like receptor (TLR) signaling pathways and induced antiviral activity in hepatocytes.METHODS:We isolated primary hepatocytes from wild-type C57BL/6 mice and examined the expression of TLR by realtime RT-PCR. Hepatocytes were stimulated with TLR 1-9 agonists and the supernatants were harvested. The secretion of cytokines were tested by ELISA. The antiviral effectors in supernatants were assayed via virus protection assay (in EMCV system) and the control of HBV replication were assessed via Southern blotting (in HBV system).RESULTS:We demonstrated that hepatocytes expressed TLR1-9. In accordance with these TLR expression profiles, hepatocytes responded to all TLR ligands by producing inflammatory cytokines (TNF-α or IL-6), to TLR -1,-3,-7 and -9 ligands by producing type I IFN (IFN-α or IFN-β). Only TLR 3 and TLR 7 agonists could stimulate the production of high amounts of antiviral mediators by hepatocytes in virus protection assay. By contrast, supernatants from TLR1, -3 and -4 directly stimulated hepatocytes and TLR 3, -7 and -9 transfected hepatocytes were able to potently suppress HBV replication.CONCLUSION:Primary hepatocytes display a unique TLR signaling pathway and can control HBV replication after stimulation by TLR agonists in mice. It may be helpful for the development of TLR-based therapeutic approaches against hepatotropic virus.

This study investigated the expression profiles of IL-10 gene in three human hepatoma cell lines including Huh7, HepG2, and HepG2 transfected with a plasmid containing hepatitis B virus (HBV) named HepG2.2.15. RT-PCR analysis demonstrated that IL-10 message RNA was absent in HepG2 and Huh7 cells, whereas it was present in HepG2.2.15 cells, which was consistent with ELISA result. Furthermore, except for lamivudine other antiviral treatments did not significantly decrease the HBV DNA level in HepG2.2.15 cells, while they had different effects on the expression of IL-10 protein, although stimulation by LPS had no significant effect. In addition, except for poly(I:C), the other treatments decreased the expression of IL-10 protein to different degrees, but had no significant effects on the expression of NF-κB and MyD88. Meanwhile, all treatments we used had effect on the expression of STAT1. In conclusion, IL-10 was expressed in HepG2.2.15 cells and STAT1 pathway might be involved in the regulation of IL-10 expression in HepG2.2.15 cells, but it was not the sole pathway, the exact mechanism warrants further study.

Objective:To construct a prokaryotic expression vector of mouse Tim-3 molecule and prepare the polyclonal antibody against mouse TIM-3.Methods:The extracellular TIM-3 gene was cloned from the spleen cells of BALB/c mice by RT-PCR and inserted into the vector pRSET-B in order to construct prokaryotic expression vector pRSET-B-TIM-3.Mouse TIM-3 protein was expressed by IPTG induction and purified by Ni-NTA column.Polyclonal antibody against TIM-3 protein was generated by immunizing rabbit with the routine method.The specificity and sensitivity of this antibody was confirmed by ELISA,Western blot,immunohistochemistry assay and FACS.Results:The fragment of 510 bp extracellular DNA sequence was obtained from the spleen cells of BALB/c mice by RT-PCR,and the prokaryotic expression vector pRSET-B-TIM-3 was constructed successfully.Induced by IPTG,the recombinant TIM-3 protein could be expressed effectively.After immunization of rabbit with the recombinant TIM-3 protein,the polyclonal antibody against mouse TIM-3 protein with a high affinity and specificity was obtained.Conclusion:The TIM-3 gene is cloned and the prokaryotic expression vector pRSET-BTIM-3 is constructed successfully.The polyclonal antibody with high affinity and specificity is generated successfully.It can be used to study the mouse TIM-3 effectively.

Objective To investigate the inhibitory effect of vacuolar protein sorting 4B(VPS4B) dominant negative mutant K180Q on hepatitis B virus(HBV) replication in vitro.Methods Huh7 cells were co-transfected with the replication competent 1.3-fold over-length HBV plasmid pHBV1.3,and various amounts of wild-type VPS4B in eukaryotic expression plasmid pXF3H-VPS4B or its dominant negative mutant K180Q in eukaryotic expression plasmid pXF3H-K180Q.HBsAg and HBeAg in the media of the transfected cells were quantitatively determined by time resolved fluorescence immunoassay.DNA related to intracellular HBV core capsid was examined by Southern blot,and mRNA related to HBV was determined by real-time PCR.Results Wild-type VPS4B inhibited the secretion of HBsAg and HBeAg but showed a weak inhibition on the levels of intracellular core-related HBV DNA and mRNA.Comparatively,dominant negative mutant VPS4B-K180Q significantly reduced the secretion of HBsAg and HBeAg at very low concentration,and displayed a does-dependent inhibitory effect on viral DNA and mRNA.Conclusion VPS4B and its dominant negative mutant may possess the effect against HBV in vitro,and the dominant negative mutant appears to be far more potent inhibitory effect than wild-type VPS4B.