Immunostaining of PD-1/PD-Ls in liver tissues of patients with hepatitis and hepatocellular carcinoma

AIM: To investigate the expression of programmed death (PD)-1,PD ligand 1 (PD-L1) and PD-L2 in liver tissues in the context of chronic hepatitis and hepatocellular carcinoma (HCC).METHODS: Liver biopsies and HCC specimens from patients were collected and histologically examined.The expression of PD-1,PD-L1,and PD-L2 in biopsy specimens of chronic hepatitis and HCC specimens was evaluated by immunohistochemical staining.The association between the expression level of PD-1,PD-L1,and PD-L2 and clinical and pathological variables was analyzed statistically.RESULTS: Expression of PD-1 was found in liverinfiltrating lymphocytes.In contrast,PD-L1 and PD-L2 were expressed in non-parenchyma liver cells and tumor cells.The expression of PD-L1 was significantly correlated with hepatitis B virus infection (1.42 ± 1.165 vs 0.50 ± 0.756,P = 0.047) and with the stage of HCC (7.50 ± 2.121 vs 1.75 ± 1.500 vs 3.00 ± 0.001,P = 0.018).PD-1 and PD-Ls were significantly up-regulated in HCC specimens (1.40 ± 1.536 vs 5.71 ± 4.051,P = 0.000;1.05 ± 1.099 vs 4.29 ± 3.885,P = 0.004;1.80 ± 1.473 vs 3.81 ± 3.400,P = 0.020).CONCLUSION: PD-L1 may contribute to negative regulation of the immune response in chronic hepatitis B.PD-1 and PD-Ls may play a role in immune evasion of tumors.

Interference of Hepatitis B Virus with Cellular Signaling

The presence of hepatitis B virus (HBV) proteins leads to changes in the cellular gene expression. As a consequence, the cellular signaling processes are influenced by the actions of HBV proteins. It has been shown that HBV nucleocapsid protein and the amino-terminal part of polymerase termed as terminal protein (TP) could inhibit interferon signaling. Further, the global gene expression profiles differ in hepatoma cells with and without HBV gene expression and replication. The expression of interferon (IFN) stimulated genes (ISGs) was differently regulated in cells with HBV replication and could be modulated by antiviral treatments. The HBV TP has been found to modulate the ISG expression and enhance the HBV replication. The modulation of the cellular signaling processes by HBV may have significant implications for pathogenesis.

Control of hepatitis B virus replication by interferons and Toll-like receptor signaling pathways

Hepatitis B virus(HBV) infection is one of the major causes of liver diseases, affecting more than 350 million people worldwide. The interferon(IFN)-mediated innate immune responses could restrict HBV replication at the different steps of viral life cycle. Indeed, IFN-α has been successfully used for treatment of patients with chronic hepatitis B. However, the role of the innate immune response in HBV replication and the mechanism of the anti-HBV effect of IFN-α are not completely explored. In this review, we summarized the currently available knowledge about the IFN-mediated anti-HBV effect in the HBV life cycle and the possible effectors downstream the IFN signaling pathway. The antiviral effect of Toll-like receptors(TLRs) in HBV replication is briefly discussed. The strategies exploited by HBV to evade the IFN- and TLR-mediated antiviral actions are summarized.

Genetic variation of hepatitis B virus and its significance forpathogenesis

Hepatitis B virus(HBV)has a worldwide distribution and is endemic in many populations.Due to its unique life cycle which requires an error-prone reverse transcriptase for replication,it constantly evolves,resulting in tremendous genetic variation in the form of genotypes,sub-genotypes,and mutations.In recent years,there has been considerable research on the relationship between HBV genetic variation and HBV-related pathogenesis,which has profound implications in the natural history of HBV infection,viral detection,immune prevention,drug treatment and prognosis.In this review,we attempted to provide a brief account of the influence of HBV genotype on the pathogenesis of HBV infection and summarize our current knowledge on the effects of HBV mutations in different regions on HBV-associated pathogenesis,with an emphasis on mutations in the pre S/S proteins in immune evasion,occult HBV infection and hepatocellular carcinoma(HCC),mutations in polymerase in relation to drug resistance,mutations in HBV core and e antigen in immune evasion,chronicalization of infection and hepatitis B-related acute-on-chronic liver failure,and finally mutations in HBV x proteins in HCC.

Hepatitis B virus infection:defective surface antigen expression and pathogenesis

Hepatitis B virus(HBV) infection is a global public health concern. HBV causes chronic infection in patients and can lead to liver cirrhosis, hepatocellular carcinoma, and other severe liver diseases. Thus, understanding HBV-related pathogenesis is of particular importance for prevention and clinical intervention. HBV surface antigens are indispensable for HBV virion formation and are useful viral markers for diagnosis and clinical assessment. During chronic HBV infection, HBV genomes may acquire and accumulate mutations and deletions, leading to the expression of defective HBV surface antigens. These defective HBV surface antigens have been found to play important roles in the progression of HBV-associated liver diseases. In this review, we focus our discussion on the nature of defective HBV surface antigen mutations and their contribution to the pathogenesis of fulminant hepatitis B. The relationship between defective surface antigens and occult HBV infection are also discussed.

Models for predicting hepatitis B e antigen seroconversion in response to interferon-α in chronic hepatitis B patients

AIM:To develop models to predict hepatitis B e antigen(HBe Ag)seroconversion in response to interferon(IFN)-αtreatment in chronic hepatitis B patients.METHODS:We enrolled 147 treatment-nave HBe Agpositive chronic hepatitis B patients in China and analyzed variables after initiating IFN-α1b treatment.Patients were tested for serum alanine aminotransferase(ALT),hepatitis B virus-DNA,hepatitis B surface antigen(HBs Ag),antibody to hepatitis B surface antigen,HBe Ag,antibody to hepatitis B e antigen(anti-HBe),and antibody to hepatitis B core antigen(anti-HBc)at baseline and 12 wk,24 wk,and 52 wk after initiating treatment.We performed univariate analysis to identify response predictors among the variables.Multivariate models to predict treatment response were constructed at baseline,12 wk,and 24 wk.RESULTS:At baseline,the 3 factors correlating most with HBe Ag seroconversion were serum ALT level>4×the upper limit of normal(ULN),HBe Ag≤500 S/CO,and anti-HBc>11.4 S/CO.At 12 wk,the 3 factors most associated with HBe Ag seroconversion were HBe Ag level≤250 S/CO,decline in HBe Ag>1 log10 S/CO,and anti-HBc>11.8 S/CO.At 24 wk,the 3 factors most associated with HBe Ag seroconversion were HBe Ag level≤5 S/CO,anti-HBc>11.4 S/CO,and decline in HBe Ag>2 log10 S/CO.Each variable was assigned a score of1,a score of 0 was given if patients did not have any of the 3 variables.The 3 factors most strongly correlating with HBe Ag seroconversion at each time point were used to build models to predict the outcome after IFN-αtreatment.When the score was 3,the response rates at the 3 time points were 57.7%,83.3%,and 84.0%,respectively.When the score was 0,the response rates were 2.9%,0.0%,and 2.1%,respectively.CONCLUSION:Models with good negative and positive predictive values were developed to calculate the probability of response to IFN-αtherapy.

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo

瞄准：为了调查 APOBEC3G 的效果，调停了对在房间文化和复制的肝炎 B (HBV ) 的抗病毒的活动能干的 HBV 基于向量的老鼠模型。方法：哺乳动物的肝细胞瘤房间 Huh7 和 HepG2 是有编码 APOBEC3G 和复制的驾驶 CMV 的表示向量的各种各样的数量的 cotransfected 能干 1.3 褶层在长度上 HBV。在 transfected 房间的媒介的 HBsAg 和 HBeAg 的层次被 ELISA 决定。在 transfected 房间的 HBcAg 的表示被西方的污点检测。从细胞内部的核心粒子的 HBV DNA 和 RNA 被北、南部的污点分析检验。估计 APOBEC3G 在活体内的活动，一个 HBV 基于向量的模型在哪个 APOBEC3G 和 HBV 向量经由大量的尾巴静脉注射被共同交付被使用。在重量的单位的 HBsAg 和 HBV DNA 的层次一象在老鼠的肝的 HBV 联系核心的 RNA 一样的 of 老鼠被 ELISA 和量的 PCR 分析分别地决定。结果：在细胞内部的联系核心的 HBV DNA 的层次和 HBsAg 和 HBeAg 的细胞外的生产有剂量依赖者减少。细胞内部的联系核心的病毒的 RNA 的层次也减少了，但是在 transfected 房间的 HBcAg 的表示没几乎显示出变化。与在试管内结果一致，在重量的单位的 HBsAg 的层次一 of 老鼠戏剧性地被减少。在浆液 HBV DNA 和肝 HBV RNA 的层次的超过 1.5 log10 减少与控制组相比在对待 APOBEC3G 的组被观察。结论：这些调查结果显示 APOBEC3G 能压制 HBV 复制和抗原表示在活体内和在试管内，在 HBV 感染的处理答应进展。

Development of HBsAg-Binding Aptamers that bind HepG2.2.15 cells via HBV surface antigen

肝炎 B 病毒表面抗原(HBsAg ) ，肝炎 B 病毒(HBV ) 的膜上的特定的抗原感染了房间，为治疗学的药提供一个完美的目标。有与 HBsAg 的高亲密关系和特性的试剂的发展具有到 HBV 感染的早阶段的诊断和治疗的大意义。此处，我们报导能明确地绑在 HBsAg 蛋白质和 HBsAg 积极的 hepatocytes 的 RNA aptamers 的选择。一高亲密关系 aptamer， HBs-A22，被孤立从一起始锝 ?.1 脳 1 的 115 mer 图书馆用 SELEX 过程的 015 个随机顺序的 RNA 分子。选择 aptamer HBs-A22 明确地跳了到表示 HBsAg 的 hepatoma 房间线 HepG2.2.15 但是没绑在 HBsAg 缺乏的 HepG2 房间。这是能绑在 HBV 特定的抗原的首先报导的 RNA aptamer。这最新孤立的 aptamer 能被修改交付成像，目标为感染 HBV 的房间的诊断、治疗学的代理人。关键词 Aptamer - 由指数的丰富(SELEX ) 的 ligands 的系统的进化 - 肝炎 B 病毒(HBV )- HBsAg - Hepatocytes 基础项目：中国(2008ZX10002-011 ) 的国家大研究节目；中国(30700701 ) 的国家自然科学基础；国家高技术研究和中国(2006AA02Z128 ) 的发展节目。

Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients

AIM: To establish a cell model harboring replicative clinical hepatitis B virus (HBV) isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B (CHB) patients. METHODS: The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction (PCR). All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap Ⅰ?digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel. RESULTS: A total of 25 independent HBV isolateswere obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent. CONCLUSION: The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.

Inhibitory effect of miR-125b on hepatitis C virus core protein-induced TLR2/My D88 signaling in THP-1 cells

AIM: To investigate the role of mi R-125 b in regulating monocyte immune responses induced by hepatitis C virus(HCV) core protein.METHODS: Monocytic THP-1 cells were treated with various concentrations of recombinant HCV core protein, and cytokines and mi R-125 b expression in these cells were analyzed. The requirement of Tolllike receptor 2(TLR2) or My D88 gene for HCV core protein-induced immune responses was determined by the transfection of THP-1 cells with gene knockdown vectors expressing either TLR2 si RNA or My D88 si RNA. The effect of mi R-125 b overexpression on TLR2/My D88 signaling was examined by transfecting THP-1 cells with mi R-125 b mimic RNA oligos.RESULTS: In response to HCV core protein stimulation, cytokine production was up-regulated and mi R-125 b expression was down-regulated in THP-1 cells. The modulatory effect of HCV core protein on cellular events was dose-dependent and required functional TLR2 or My D88 gene. Forced mi R-125 b expression abolished the HCV core protein-induced enhancement of tumor necrosis factor-α, interleukin(IL)-6, and IL-10 expression by 66%, 54%, and 66%, respectively(P < 0.001), by inhibiting My D88-mediated signaling, including phosphorylation of NF-k Bp65, ERK, and P38.CONCLUSION: The inverse correlation between mi R-125 b and cytokine expression after HCV core challenge suggests that mi R-125 b may negatively regulate HCVinduced immune responses by targeting TLR2/My D88 signaling in monocytes.

N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA.The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

Construction and Characterization of a Hepatitis B Virus Replicon

建立复制细胞肝炎 B 病毒(HBV ) 当模特儿并且在抗病毒的药评估决定它的应用程序，我们构造了表情包含了 HBV 染色体的 1.3 个拷贝，并且在 Huh7 房间在短暂 transfection 以后测量了病毒的复制的水平的 plasmid。我们然后观察了抗病毒的药管理的效果。HBV (ayw ) 基因碎片的 1.3 褶层被 PCR 和限制 endonuclease 消化克隆进 pCR2.1。recombinant plasmid 是进 Huh7 房间， HBsAg， HBeAg 和 HBV 的短暂 transfected 在 Huh7 房间的上层清液的 DNA 被 ELISA 和即时 PCR 分别地测量；细胞内部的 HBV replicative 中介和细胞内部的 HBV 抄本被南部的污点和北污点分别地检测。adefovir 的抗病毒的效果，新奇 anti-HBV 核苷酸类似物，在这个细胞的模型系统被评估。结果显示 HBV replicon 的 recombinant plasmid 成功地被构造；在 plasmid pHBV1.3 带的 HBV 染色体能高效地复制并且被表示在哈 7 个房间， adefovir 能在这个细胞的模型，和抑制禁止 HBV 复制是剂量依赖者。结论是 HBV replicon，能在 hepatoma 房间高效地开始病毒的复制，可以是在 HBV 复制和抗病毒的药的学习的一个有用工具。关键词肝炎 B 病毒 - 传染 replicon - 表示向量 CLC 数字 R373 基础条款：国家自然科学基础(No.30271170， No.30170889 ) 。

Silencing of UBP43 by shRNA Enhances the Antiviral Activity of Interferon against Hepatitis B Virus

Previous studies have shown that expression of the interferon-sensitive gene (ISG)15 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.

Effect of Hepatitis C Virus Core Protein on Interferon-Induced Antiviral Genes Expression and Its Mechanisms

Emerging data indicated that HCV subverts the antiviral activity of interferon(IFN);however,whether HCV core protein contributes to the process remains controversial.In the present study,we examined the effect of HCV core protein on interferon-induced antiviral gene expression and whether the effect is involved in the activation and negative regulation of the Jak/STAT signaling pathway.Our results showed that,following treatment with IFN-α,the transcription of PKR,MxA and 2'-5'OAS were down-regulated in HepG2 cells expressing the core protein.In the presence of HCV core protein,ISRE-dependent luciferase activity also decreased.Further study indicated that the core protein could inhibit the tyrosine phosphorylation of STAT1,whereas the level of STAT1 expression was unchanged.Accordingly,SOCS3,the negative regulator of the Jak/STAT pathway,was induced by HCV core protein.These results suggests that HCV core protein may interfere with the expression of some interferon-induced antiviral genes by inhibiting STAT1 phosphorylation and induction of SOCS3.