Expression of HBsAg and HBcAg in liver tissue: correlation with disease activity.

The patterns of Hepatitis B surface antigen (HBsAg) and Hepatitis B core antigen (HBcAg) expression were studied in liver biopsies taken from 41 patients with chronic HBV disease. Immunohistochemical methods were used on deparaffinized sections for the identification of HBsAg and HBcAg in liver tissue. Twenty-one of the 41 cases (51.2%) were classified as inactive liver disease and 20 (48.8%) as active liver disease. In liver biopsies with inactive disease, HBsAg demonstrated varying types of cytoplasmic expression in a rather high number of hepatocytes distributed mainly in clusters, while HBcAg was rarely expressed in liver nuclei. On the other hand, in liver biopsies with active disease HBsAg was characterized by a diffuse cytoplasmic expression in a few discrete hepatocytes, while HBcAg was expressed in the nuclei of the hepatocytes in 70% of the cases and in half of the positive cases it was also detected in the cytoplasm. In conclusion, HBsAg expression in a few scattered hepatocytes correlates with active liver disease and positive HBcAg, while varying HBsAg cytoplasmic expression in a rather high number of clustered hepatocytes is related to chronic inactive liver disease and negative expression of HBcAg.     

Immunocytochemical investigation of hepatitis B virus-associated antigens in cases of liver cirrhosis and HBsAg antigenemia and their relationship to development of hepatocellular carcinoma

Summary Using light and ultrastructural immunoperoxidase techniques, we examined the distribution of hepatitis B virus (HBV)-associated antigens and the subcellular localization of hepatitis B surface antigen (HBsAg) in liver biopsies of HBsAg—positive patients with cirrhosis. The localization patterns of HBsAg in hepatocytes were membranous, cytoplasmic, festoon and inclusion body types. Cytoplasmic and festoon types were seen more often than the membranous type in pseudolobules, and hepatitis B core antigen (HBcAg)—positive cells with cytoplasmic type were distributed in the periphery of pseudolobules with active inflammation. Immunoelectron microscopy in the cytoplasmic or festoon type of HBsAg showed immunoreaction in the cisternae and on virus-like particles in the cisternae in patients with hepatitis B e antigen (HBeAg) antigenemia. Simultaneous staining of HBsAg and HBcAg revealed that hepatocytes with cytoplasmic or festoon type of HBsAg contained HBcAg—immunoreactivity. The inclusion body type of HBsAg was characteristic of liver cirrhosis with hepatocellular carcinoma (HCC); the subcellular localization of HBsAg was seen in clusters of the endoplasmic reticulum around the nucleus, and HBsAg—immunoreactivity was observed on many virus-like particles in most of the cisternae in those with HBeAg antigenemia. These findings suggest that the synthesis of HBsAg is active in patients with liver cirrhosis and that the formation of HBV is also active in those with HBeAg antigenemia and that HBV may be retained more in cirrhotic livers with hepatocellular carcinoma after proliferation than in those without it.     

Specificity of T lymphocyte cytotoxicity to autologous hepatocytes in chronic hepatitis B virus infection: evidence that T cells are directed against HBV core antigen expressed on hepatocytes

Peripheral blood T lymphocytes from 21 patients with chronic HBV infection were incubated with autologous hepatocytes in a microcytotoxicity assay. Cytotoxicity was significantly increased in 13 cases, and in 12 of these the cytotoxic effect of the T lymphocytes was inhibited by preincubating the liver cells with IgG containing antibodies to the hepatitis B core antigen (HBcAg). Normal human IgG and IgG containing antibodies to the hepatitis B surface antigen (HBsAG) were without effect. Control experiments using autologous fibroblasts as target cells showed low levels of T cell cytotoxicity and no blocking effect of anti-core antibody. All patients in whom it was possible to demonstrate HBcAg in liver tissue had significantly increased T cell cytotoxicity to autologous hepatocytes. These studies suggest that T cell cytotoxicity in patients with chronic HBV infection is directed against determinants resembling the hepatitis B core antigen on the plasma membrane of hepatocytes.     

Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen

There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.     

Detection of core antibody in hepatitis B infection.

Hepatitis B core antigen (HBcAg) is found on the decoated Dane particle and on a morphologically similar particle detected mainly in the nucleus of hepatocytes of patients with hepatitis B. HBcAg prepared from the liver of a chimpanzee infected with hepatitis B virus was used to test human serum for core antibody (anti-HBc) by complement fixation. Anti-HBc was found in serum collected from patients with hepatitis B in both the acute and convalescent stages, from carriers of hepatitis B surface antigen (HBsAg) and from patients with chronic liver or renal disease who were carriers of HBsAg. It was not found in patients with hepatitis A or infectious mononucleosis, or in healthy persons who were not carriers of HBsAg.     

人原发性肝细胞肝癌中丙型肝炎病毒C_（33c）抗原及HBxAg的分布及意义

作者应用抗ＨＣＶＮＳ３区Ｃ３３ｃ抗原２Ｂ６株单克隆抗体和抗ＨＢｘＡｇ多克隆抗体,采用ＡＢＣ法对１０２例人原发性肝细胞肝癌（ＰＨＣ）组织进行了ＨＣＶ及ＨＢＶ抗原定位研究。ＨＣＶＣ３。抗原及ＨＢｘＡｇ在ＰＨＣ中的阳性检出率分别是８１．４％及７４．５％,Ｃ３３ｃ抗原或ＨＢｘＡｇ阳性占所检病例９４．１％,相同病例二者同时阳性为６１．８％。１０２例ＰＨＣ中５０例有癌旁肝组织,其Ｃ３３ｃ抗原和ＨＢｘＡｇ的阳性检出率分别是６２％和９２％。ＨＣＶＣ３３ｃ抗原定位于肝癌细胞的胞浆内,胞核未见阳性信号。Ｃ３３ｃ抗原阳性细胞在ＰＨＣ中呈散在、局灶分布为主,在癌旁肝组织呈弥漫分布为主。本文结果提示ＨＣＶ感染在ＰＨＣ的发生中可能起重要作用。     

乙型肝炎病毒急性感染小鼠模型的建立

采用高压水注射方法,通过尾静脉将具有复制能力的HBV质粒导入BABL/cJ小鼠体内,应用real-time PCR、ELISA、RIA、Southern Blot、Northern Blot,以及免疫组化等方法,检测小鼠病毒血症、血清和肝组织中HBV 抗原表达动态变化、肝组织中HBV转录和复制情况,以及小鼠免疫应答状况。结果HBV基因可以在小鼠体内表 达和复制,并诱导小鼠产生特异性免疫应答,其应答模式及HBV清除过程与人类的HBV急性感染类似。实验显 示高压注射具有复制能力的HBV质粒可以在小鼠体内建立HBV急性感染模型,这种模型可以用于HBV病毒 学、免疫学以及抗病毒药物筛选等方向的研究。     

Hepatitis B in a hospital for the mentally subnormal in southern England.

The prevalence of hepatitis B viral (HBV) infection was assessed in 340 patients and 268 staff in a hospital for the mentally subnormal in Wessex. Hepatitis B surface antigen (HBsAg), antibody to hepatitis B core antigen, antibody to HBsAg, e antigen (eAg), and antibody to eAg were used as markers of such infection. Forty patients and 10 staff had evidence of recent or current infection, while 149 patients and 50 staff had evidence of past infection. HBV markers were more common in mongols, epileptics, patients with cerebral palsy, and those of lower mental grades and reached a peak after 5-15 years of hospitalisation. eAg was detected in 12 out of 26 patients with HBsAg but in none of the four staff with HBsAg. Abnormal liver function values were found in 24 (60%) of the patients with recent or current HBV infection but in only 30 (19%) of those without HBV markers. Among the staff the prevalence of HBV markers correlated with the duration of employment and degree of contact with patients. Of those with recent or current infection, 4 (40%) had abnormal liver function values compared with 25 (12%) of those without HBV markers. Despite the high prevalence of markers clinically overt hepatitis B was rare.     

西伯利亚旱獭中类人乙肝病毒的研究

对111份西伯利亚旱獭血清标本进行血清学,形态和组织病理学检测,HBsAg阳性22份,阳性率19.8％;抗-HBs1份,阳性率0.9％;HBV-DNA核酸杂交,阳性斑点16份,阳性率14.4％。IEM观察在3份标本中发现以22—24nm球形颗粒为主,其中有42—45mm的Dane样颗粒。20.7％的肝脏标本有组织病理改变,其中19份为急性肝类,4份为慢性肝炎。超薄切片肝细胞核内有20nm左右的HBeAg颗粒,结果进一步证实我国西伯利亚旱獭中有嗜肝病毒感染。     

A Mouse Model for Studying the Clearance of Hepatitis B Virus In Vivo Using a Luciferase Reporter

Hepatitis B virus(HBV) infection remains a global problem, despite the effectiveness of the Hepatitis B vaccine in preventing infection. The resolution of Hepatitis B virus infection has been believed to be attributable to virus-specific immunity. In vivo direct evaluation of anti-HBV immunity in the liver is currently not possible. We have developed a new assay system that detects HBV clearance in the liver after the hydrodynamic transfer of a reporter gene and over-length, linear HBV DNA into hepatocytes, followed by bioluminescence imaging of the reporter gene (Fluc). We employed bioluminescence detection of luciferase expression in HBV-infected hepatocytes to measure the Hepatitis B core antigen (HBcAg)-specific immune responses directed against these infected hepatocytes. Only HBcAg-immunized, but not mock-treated, animals decreased the amounts of luciferase expression, HBsAg and viral DNA from the liver at day 28 after hydrodynamic infection with over-length HBV DNA, indicating that control of luciferase expression correlates with viral clearance from infected hepatocytes.     

Hepatitis B Virus-Specific miRNAs and Argonaute2 Play a Role in the Viral Life Cycle

Disease-specific serum miRNA profiles may serve as biomarkers and might reveal potential new avenues for therapy. An HBV-specific serum miRNA profile associated with HBV surface antigen (HBsAg) particles has recently been reported, and AGO2 and miRNAs have been shown to be stably associated with HBsAg in serum. We identified HBV-associated serum miRNAs using the Toray 3D array system in 10 healthy controls and 10 patients with chronic hepatitis B virus (HBV) infection. 19 selected miRNAs were then measured by quantitative RT-PCR in 248 chronic HBV patients and 22 healthy controls. MiRNA expression in serum versus liver tissue was also compared using biopsy samples. To examine the role of AGO2 during the HBV life cycle, we analyzed intracellular co-localization of AGO2 and HBV core (HBcAg) and surface (HBsAg) antigens using immunocytochemistry and proximity ligation assays in stably transfected HepG2 cells. The effect of AGO2 ablation on viral replication was assessed using siRNA. Several miRNAs, including miR-122, miR-22, and miR-99a, were up-regulated at least 1.5 fold (P<2E-08) in serum of HBV-infected patients. AGO2 and HBcAg were found to physically interact and co-localize in the ER and other subcellular compartments. HBs was also found to co-localize with AGO2 and was detected in multiple subcellular compartments. Conversely, HBx localized non-specifically in the nucleus and cytoplasm, and no interaction between AGO2 and HBx was detected. SiRNA ablation of AGO2 suppressed production of HBV DNA and HBs antigen in the supernatant.

Conclusion

These results suggest that AGO2 and HBV-specific miRNAs might play a role in the HBV life cycle.     

用rAAV8-1.3HBV制备两种品系小鼠乙型肝炎病毒感染模型的比较研究

我们先前用rAAV8-1.3HBV静脉注射C57BL/6小鼠成功地制备了慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染模型。为了探讨不同品系的小鼠对rAAV8-1.3HBV静脉注射是否具有不同反应,本研究比较了C57BL/6和BALB/c小鼠静脉注射重组病毒后外周血中HBV抗原和抗体水平、病毒载量和肝脏组织HBcAg表达情况,以及不同剂量重组病毒注射与这些指标的关系。将低(4×109 Viral genome,vg)、中(4×1010vg)和高(4×1011vg)三种剂量的rAAV8-1.3HBV通过尾静脉注射至C57BL/6和BALB/c小鼠,分别利用ELISA和荧光定量PCR方法检测血清中的HBV抗原、抗体水平以及HBV DNA,利用免疫组化检测肝脏组织HBcAg的表达。结果发现,对于C57BL/6小鼠,三种不同剂量rAAV8-1.3HBV注射均可造成100%小鼠出现HBV持续感染;血清HBsAg、HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,其表达水平随重组病毒注射剂量的增加而升高,高剂量注射时可造成超过40%的肝细胞感染HBV,血清中HBV DNA可达105 IU/mL以上;未检测到针对HBV的抗体。对于BALB/c小鼠,三种不同剂量rAAV8-1.3HBV注射也可造成100%小鼠出现HBV持续感染;血清HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,但是血清HBsAg在重组病毒注射2周之后显著下降甚至消失;在中剂量注射组的BALB/c小鼠血清中检测到低水平的Anti-HBs;血清HBeAg和肝组织HBcAg的表达水平随重组病毒注射剂量的增加而增高,并且各剂量组表达水平均高于C57BL/6小鼠,高剂量注射时可造成超过50%的肝细胞感染HBV。本研究表明,低至4×109 vg剂量的rAAV8-1.3HBV注射即可造成C57BL/6和BALB/c两种品系小鼠出现HBV持续感染,并且HBV复制水平随重组病毒注射剂量增加而增高;BALB/c小鼠对HBV的免疫反应强于C57BL/6小鼠,可以产生针对HBsAg的体液免疫反应而使血清HBsAg转阴,但无法清除携带HBV的肝细胞。     

Antibodies against HBV, HCV and HAV detected by using microparticle enzyme immunoassay in hepatitis outpatients

The presence of the hepatitis B surface antigen (HBsAg), of the antibodies against HBc, HCV and HAV was determined in outpatients in the period September 2005 - December 2006. The serum samples were analyzed by using Enzyme Immunoassay microparticles (Abbott AxSYM System). At least one test was positive in 238 patients (15.4%) of the total of 1547 patients. Of the 238 positive subjects, in 130 positive subjects (54.6%) the existence of HBV infection could be ascertained based on the presence of HBsAg or of the antibodies against HBc or of their association; 83 patients (34.9%) presented antibodies against HCV and in other 12 patients the antibodies against HCV were associated with HBsAg or with antibodies against HBc, suggesting the coexistence of HCV and HBV infection. The antibodies against HCV and the associations between HCV and HBV were mostly detected in subjects with the diagnosis of cirrhosis, liver failure or chronic hepatitis. Of the 13 (5.46%) patients with antibodies against HAV, 6 patients presented the associations: in 2 cases antibodies anti-HAV with positive HBsAg, in 1 case antibodies anti-HAV and anti-HBc with positive HBsAg, in 2 cases antibodies anti-HAV and anti-HBc and in 1 case antibodies anti-HAV and anti-HCV.     

Chronic Hepatitis B Virus Infection: The Relation between Hepatitis B Antigen Expression,Telomere Length,Senescence, Inflammation and Fibrosis

BackgroundChronic Hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that HBV might accelerate hepatocyte ageing and investigated the effect of HBV on hepatocyte cell cycle state and biological age. We also investigated the relation between inflammation, fibrosis and cell cycle phase.MethodsLiver samples from patients with chronic HBV (n = 91), normal liver (n = 55) and regenerating liver (n = 15) were studied. Immunohistochemistry for cell cycle phase markers and HBV antigens was used to determine host cell cycle phase. Hepatocyte-specific telomere length was evaluated by quantitative fluorescent in-situ hybridization (Q-FISH) in conjunction with hepatocyte nuclear area and HBV antigen expression. The effects of induced cell cycle arrest and induced cellular senescence on HBV production were assessed in vitro.Results13.7% hepatocytes in chronic HBV had entered cell cycle, but expression of markers for S, G2 and M phase was low compared with regenerating liver. Hepatocyte p21 expression was increased (10.9%) in chronic HBV and correlated with liver fibrosis. Mean telomere length was reduced in chronic HBV compared to normal. However, within HBV-affected livers, hepatocytes expressing HBV antigens had longer telomeres. Telomere length declined and hepatocyte nuclear size increased as HBV core antigen (HBcAg) expression shifted from the nucleus to cytoplasm. Nuclear co-expression of HBcAg and p21 was not observed. Cell cycle arrest induced in vitro was associated with increased HBV production, in contrast to
in vitro induction of cellular senescence, which had no effect.ConclusionChronic HBV infection was associated with hepatocyte G1 cell cycle arrest and accelerated hepatocyte ageing, implying that HBV induced cellular senescence. However, HBV replication was confined to biologically younger hepatocytes. Changes in the cellular location of HBcAg may be related to the onset of cellular senescence.     

Prevalence of occult hepatitis B virus infection in kidney transplant recipients

In this cross-sectional study, 207 hepatitis B surface antigen (HBsAg)-negative kidney transplant recipients were evaluated based on demographic and epidemiological data and on the levels of serological markers of hepatitis B virus (HBV) and hepatitis C virus infection and liver enzymes. Patients with HBV or human immunodeficiency virus infection were excluded. Sera were analysed for the presence of HBV-DNA. HBV-DNA was detected in two patients (1%), indicating occult hepatitis B (OHB) infection (the HBV-DNA loads were 3.1 and 3.5 IU/mL in these patients). The results of the liver function tests were normal and no serological markers indicative of HBV infection were detected. The prevalence of OHB infection was low among kidney transplant recipients, most likely due to the low HBsAg endemicity in the general population of the study area.     

乙型肝炎病毒胎内传播的研究

为了探索HBV胎内感染的可能性及机率,随机配对选取了22例HBsAg携带者母亲的引产胎儿和22例HBsAg阴性母亲的引产胎儿,对其心血、肝脏进行了系统研究,简报如下: 1.对象 随机选择HBsAg阳性无症状带毒者的雷夫奴尔(Rivanol)引产胎儿22例,     

IL-12增强HBV融合抗原DNA疫苗在小鼠诱导的细胞免疫应答

乙型肝炎病毒(hepatitis B virus,HBV)极易形成慢性感染,主要机制在于感染者不能产生强有力的细胞免疫应答以清除病毒[1].慢性HBV感染者体内虽然存在HBV抗原特异性T淋巴细胞,但对HBV抗原的反应性较低.研究发现,增强这类T淋巴细胞的反应性,可以促进HBV的清除[2].     

A case of hepatitis B reactivation due to the hepatitis B virus escape mutant in a patient undergoing chemotherapy

A 62-year-old man had chronic hepatitis B virus (HBV) infection and was diagnosed with liver cirrhosis. At the time of diagnosis the patient’s virologic markers were positive for hepatitis B surface antigen (HBsAg), antibody to hepatitis B e antigen (anti-HBe) and antibody to hepatitis B core antigen (anti-HBc), while antibody to hepatitis B surface antigen (anti-HBs) and HBV DNA were negative. Later the patient received chemotherapy for malignancy. However, this was interrupted due to elevated liver enzymes. At the same time HBV DNA became positive. Lamivudine (LMV) therapy was administered immediately. However, the levels of serum aminotransferase and total bilirubin (TB) were still rising. Finally the patient died of fulminant hepatic failure. A sequence revealed HBV genotype C (HBsAg subtype adw) with immune escape mutations, F8L, S34L, F41S, G44V, F93C, V96G, L110I, C149Y and F161Y. The high morbidity and mortality of this complication is one of the major obstacles to completing the standard treatment for malignancy in HBV carriers. Therefore, the relative risk of antiviral prophylactic failure should be further assessed and the optimal strategy for antiviral prophylaxis in HBsAg-positive patients with oncologic and hematologic malignancies undergoing chemotherapy should be revised.     

Inhibition of hepatitis B virus in mice by RNA interference

Hepatitis B virus (HBV) infection substantially increases the risk of chronic liver disease and hepatocellular carcinoma in humans. RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we show that RNAi can be applied to inhibit production of HBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid. Cotransfection with plasmids expressing short hairpin RNAs (shRNAs) homologous to HBV mRNAs induced an RNAi response. Northern and Southern analyses of mouse liver RNA and DNA showed substantially reduced levels of HBV RNAs and replicated HBV genomes upon RNAi treatment. Secreted HBV surface antigen (HBsAg) was reduced by 94.2% in cell culture and 84.5% in mouse serum, whereas immunohistochemical detection of HBV core antigen (HBcAg) revealed >99% reduction in stained hepatocytes upon RNAi treatment. Thus, RNAi effectively inhibited replication initiation in cultured cells and mammalian liver, showing that such an approach could be useful in the treatment of viral diseases.     

Alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV

The incidence of hepatocellular carcinoma (HCC) is closely correlated with hepatitis B virus (HBV)-induced liver cirrhosis. Structural changes in the glycans of serum and tissue proteins are reliable indicators of liver damage. However, little is known about the alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV infection. This study compared the differential expression of liver glycopatterns in 7 sets of normal pericarcinomatous tissues (PCTs), cirrhotic, and tumor tissues from patients with liver cirrhosis and HCC induced by HBV using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were further utilized to validate and assess the expression and distribution of certain glycans in 9 sets of corresponding liver tissue sections. Eight lectins (e.g., Jacalin and AAL) revealed significant difference in cirrhotic tissues versus PCTs. Eleven lectins (e.g., EEL and SJA) showed significant alteration during cirrhotic and tumor progression. The expression of Galα1-3(Fucα1-2)Gal (EEL) and fucosyltransferase 1 was mainly increasing in the cytoplasm of hepatocytes during PCTs-cirrhotic-tumor tissues progression, while the expression of T antigen (ACA and PNA) was decreased sharply in cytoplasm of tumor hepatocytes. Understanding the precision alteration of liver glycopatterns related to the development of hepatitis, cirrhosis, and tumor induced by HBV infection may help elucidate the molecular mechanisms underlying the progression of chronic liver diseases and develop new antineoplastic therapeutic strategies.