Correlation of hepatocyte expression of hepatitis B viral antigens with histological activity and viral titer in chronic hepatitis B virus infection: an immunohistochemical study

Background and Aim: The localization of hepatitis B virus (HBV) core antigen to the nucleus or cytoplasm of hepatocytes has biological implications for viral packaging and persistence. This study examined the relationship between the localization of hepatitis B virus antigens, histological activity, and viral titer in patients with chronic HBV infection. Methods: Liver biopsies from 110 patients with chronic HBV infection were studied. Ishak's scoring system was used for the histological analysis. The localization of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) and the percentage of hepatocytes stained positive by immunohistochemistry were correlated with viral titer, histological activity, and fibrosis indices using Spearman rank correlation. Results: In 88 hepatitis B e‐antigen (HBeAg)‐positive individuals, the nuclear localization of HBcAg correlated significantly with DNA titer (r = 0.435, P = 0.001) and negatively with fibrosis (r = ?0.297, P = 0.005). The cytoplasmic localization correlated significantly with histological activity (r = 0.211, P = 0.049). In 22 HBeAg‐negative individuals, the nuclear localization of HBcAg correlated significantly with histological activity (r = 0.625, P = 0.002), DNA titer (r = 0.651, P = 0.009), and fibrosis (r = 0.447, P = 0.042). The cytoplasmic localization correlated significantly with DNA titer (r = 0.524, P = 0.045) and fibrosis (r = 0.528, P = 0.012). There was no correlation of HBsAg expression with DNA titer, histological activity index, or fibrosis in both groups. HBeAg‐positive patients presented at a younger age. Conclusion: In HBeAg‐positive individuals, nuclear core antigen correlated with DNA titer, and cytoplasmic localization with histological activity, whereas in HBeAg‐negative individuals, nuclear localization correlated with DNA titer, histological activity, and fibrosis, and cytoplasmic localization correlated with DNA titer and fibrosis, but not with histological activity. These observations suggest biological differences between HBeAg‐positive and ‐negative disease.     

乙型肝炎病毒前S1抗原对判断病毒复制和乙型肝炎预后的价值

目的探讨乙型肝炎病毒前S1抗原与HBVM和HBV DNA的相关性,评价前S1抗原与病毒复制和在判断乙型肝炎预后中的作用。方法采用ELISA法检测前S1抗原;采用时间分辨免疫荧光法检测HBVM;采用酶学速率法检测肝功能;采用荧光定量PCR方法检测HBV DNA。结果在HBeAg阳性的141例患者中,HBV DNA和前S1抗原的检出率分别为97.2%和92.9%,抗-HBe阳性的183例中,分别为39.9%和37.2%;在HB-sAg阳性/抗-HBc阳性的59例中分别为62.7%和54.2%;慢性乙型肝炎轻度159例,中度132例,重度96例,其血清前S1抗原阳性率分别为56.6%、73.5%和61.5%,三组之间无显著性相差。结论检测前S1抗原可以较好地反映HBV的存在和复制,其在乙型肝炎发病中的临床意义还有待探讨。     

Subcellular localization of hepatitis B core antigen in relation to hepatocyte regeneration in chronic hepatitis B

To test whether the dominant cytoplasmic expression of hepatitis B core antigen (HBcAg) in active chronic hepatitis B is secondary to liver damage and regeneration, the relationship between subcellular localization of HBcAg, liver inflammatory activity, and hepatocyte regeneration in chronic hepatitis B was studied. Correlation of the clinical and laboratory data with the topographical distribution of HBcAg was studied in 30 patients. The subcellular localization of HBcAg in relation to hepatocyte cell cycles was studied by double immunostaining of HBcAg and proliferating cell nuclear antigen. Patients with predominant cytoplasmic HBcAg had significantly higher levels of biochemical and histological activities and proliferating cell nuclear antigen expression than patients with predominant nuclear HBcAg. The levels of proliferating cell nuclear antigen expression correlated positively with biochemical and histological activities and degrees of cytoplasmic HBcAg expression but negatively with degrees of nuclear HBcAg expression. Proliferating cell nuclear antigen expression was shown in 49% of hepatocytes with cytoplasmic HBcAg but in only 2% of hepatocytes with nuclear HBcAg. These findings suggested that, following liver damage, the regeneration of surviving hepatocytes might cause the shift of intracellular HBcAg from nucleus to cytoplasm. As a result, the extent of nuclear HBcAg expression reduces with concomitant increase in cytoplasmic HBcAg expression.     

Hepatocyte hepatitis B surface antigen expression in chronic hepatitis B virus carriers in Singapore: Correlation with viral replication and liver pathology

The hepatocyte hepatitis B surface antigen (HBsAg) expression in 149 liver biopsies from 124 chronic hepatitis B virus (HBV) carriers was correlated with serum HBV DNA status and histologic activity. Hepatocyte HBsAg was stained by the peroxidase-antiperoxidase method and serum HBV DNA was determined by dot blot hybridization. Sixty-five biopsies (44%) showed minimal changes (MC), 82 biopsies (55%) showed chronic liver disease (CLD) and 2 biopsies (1%) showed hepatocellular carcinoma. Hepatocyte HBsAg was found in 144 biopsies (97%). It was present in the cytoplasm of 141 specimens (95%) and/or plasma membrane of 48 specimens (32%). Approximately half (45%) of the cytoplasmic HBsAg-positive biopsies showed discrete distribution, while the other half (55%) were grouped. Fifty-five per cent (77 of 141) of cytoplasmic HbsAg-positive biopsies had CLD, while 44% (62 of 141) showed MC. There was no relationship between the presence of cytoplasmic HBsAg or its topographic distribution with disease activity. Membrane HBsAg distribution was similar for both groups of patients (MC vs CLD: 25 of 65 (38%) vs 23 of 82 (28%); P = NS). Serum HBV DNA was detected in 98 patients (66%) and was seen mostly in association with CLD (CLD vs MC: 61% vs 39%, P less than 0.001). It was also detected more often in the sera of patients with membrane HBsAg than in those with cytoplasmic HBsAg staining (41 of 48 (85%) vs 97 of 141 (67%); P less than 0.02). However, discrete distribution of cytoplasmic HBsAg was associated with positive serum HBV DNA when compared with grouped distribution (52 of 63 (83%) vs 43 of 78 vs (55%); P less than 0.005).     

乙型肝炎病毒丹氏颗粒抗原在慢性乙型肝炎病毒检测中应用的研究

目的分析乙肝病毒五项检测方法和乙肝病毒丹氏颗粒抗原检测试剂在检测慢性乙型肝炎感染中的一致性,探讨丹氏颗粒抗原检测试剂在检测慢性乙肝感染的经济性和可靠性。方法ELISA方法检测134例慢性乙肝患者的血清标志物和丹氏颗粒抗原,荧光定量PCR方法检测HBV DNA。比较各组丹氏颗粒抗原水平及HBVDNA定量水平和乙肝五项的相互关系。结果慢性乙肝患者HBsAg、HBeAg、HBcAb阳性组、HBsAg、HBcAb阳性组、HBsAg、HBeAb、HBcAb阳性组和HBsAb、HBeAb、HBcAb阳性组丹氏颗粒抗原阳性率分别为100.0(、100.0(、94.3(和0。结论丹氏颗粒抗原试剂在检测慢性乙肝感染中与乙肝五项指标及HBV DNA定量之间具有一致性。丹氏颗粒抗原是一个能较好的反应乙肝病毒复制的指标,对乙肝的诊断、预后及抗病毒疗效观察有较好的实用价值。     

High-level expression of hepatitis B viral antigens in fibrosing cholestatic hepatitis.

The expression of hepatitis B viral antigens was quantified in liver tissue from four transplant recipients with fibrosing cholestatic hepatitis (FCH) and compared with five other transplant recipients who did not develop this syndrome and 30 patients with chronic hepatitis B virus (HBV) infection. As measured by radioimmunoassays, the liver tissue from patients with FCH had significantly greater amounts of both hepatitis B surface antigen (HBsAg) and nucleocapsid antigens than to transplant patients without this syndrome (P less than 0.01) or patients with chronic HBV infection (P less than 0.001). Intrahepatic expression of pre-S1/pre-S2 in FCH was also extensive with a distribution parallel to that of HBsAg. High-level expression of intrahepatic HBsAg and hepatitis B core antigen in the explanted liver was associated with subsequent development of FCH in the liver graft, suggesting that viral/host factors may also be important. This pattern of intrahepatic hepatitis viral antigen expression, by analogy with Chisari's transgenic mice model and Roingeard's HBV-transfected HepG2 cell model, may be the cause of direct hepatocytopathic injury in this condition.     

Nuclear localization of hepatitis B core antigen and its relations to liver injury,hepatocyte proliferation,and viral load

GOALS: The aim of this study was to determine the factor(s) independently affecting the HBcAg expression pattern in HBV-infected livers. BACKGROUND: Subcellular localization of HBcAg have been found to be related to the activity of liver disease, hepatocyte proliferation rate and the level of HBV replication.STUDY A total of 98 patients with biopsy proven chronic hepatitis B were included. HBcAg and proliferative cell nuclear antigen (PCNA) were immunohistochemically detected. HBcAg expression and its relationship with histologic activity index, ALT levels, PCNA score and HBV DNA levels were investigated. RESULTS: Forty-three (44%) patients were positive for HBcAg staining, with most of them (37 patients) having nuclear localization. Forty-seven percent of patients with detectable HBV DNA had nuclear staining while none of the HBV DNA negative patients had nuclear staining ( < 0.0005). Patients with positive nuclear staining had lower HAI (<8) and a lower PCNA score (<353/1,000 cell) than those with negative staining (62% vs. 39% and 90% vs. 66%, respectively, P< 0.05). In multivariate analysis, both high HBV DNA level and low HAI ( P< 0.0005 and P< 0.05, respectively) but not PCNA score, were independently associated with nuclear staining of HBcAg. CONCLUSIONS: Our results suggest that viral load and the severity of liver damage but not the rate of hepatocyte proliferation independently affects the HBcAg expression pattern.     

Effect of nitric oxide on iron-mediated oxidative stress in primary rat hepatocyte culture

An iron-mediated oxidative stress caused by an increase of the intracellular pool of low molecular weight complex of iron (LMWC) can be observed with iron overloading or ethanol metabolism. The aim of this study was to determine whether nitric oxide (NO) behaved as a pro-oxidant or an antioxidant in such an iron-mediated oxidative stress in rat hepatocytes. The cells were set up in primary cultures and incubated with lipopolysaccharide (LPS) and γ-interferon (IFN) for 18 hours to induce NO synthase and to trigger NO production. Then 20 μmol/L iron or 50 mmol/L ethanol were added. Oxidative stress was evaluated by measuring lipoperoxidation using two markers: malondialdehyde (MDA) and conjugated dienes. Simultaneously, NO production was followed by the quantitation of nitrites in the culture medium, dinitrosyl iron complexes (DNICs) and mononitrosyl iron complexes (MNICs) in intact hepatocytes. DNIC and MNIC, evaluated by electron paramagnetic resonance (EPR), corresponded to NO bound to iron-containing molecules and to free NO, respectively. In cultures preincubated with LPS and IFN before iron or ethanol addition, a net decrease of lipid peroxidation induced by either NO, iron, or ethanol was noted. Moreover, an elevation of iron-bound NO and a decrease of free NO were observed in these cultures compared with the cultures incubated with only LPS and IFN. These data support the idea that there is a relationship between the changes of NO pool and the inhibition of oxidative stress. In addition, using N^G-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, NO was shown to be involved in the inhibition of oxidative stress induced by iron or ethanol. Addition of the chelator of LMWC iron, deferiprone, was followed by the inhibition of the increase of iron-bound NO and the reincrease of lipid peroxidation extent, which was as high as in cultures incubated only with LPS and IFN. Thus LMWC iron appeared to be involved also in the inhibition of oxidative stress induced by NO. All the results favor the conclusion that NO acts as an antioxidant in iron-mediated oxidative stress in rat hepatocytes. NO reacted with LMWC iron to form inactive iron complexes unable to induce oxidative stress in rat hepatocytes. Thus NO played a critical role in protecting the liver from oxidative stress.(Hepatology 1997 Jan;25(1):122-7)     

Effect of human immunodeficiency virus (HIV) infection on chronic hepatitis B hepatic viral antigen display

Immunofluorescent and immunoperoxidase monoclonal antibody-based techniques were used to demonstrate hepatitis B e antigen (HBeAg) and hepatitis B c antigen (HBcAg) display in the liver biopsy specimens of 45 chronic hepatitis B virus (HBV) carriers. Anti-human immunodeficiency virus (anti-HIV)-positive HBV carriers had many more HBe- and HBc-positive hepatocyte nuclei than anti-HIV-negative carriers (P less than 0.0003 and less than 0.02, respectively), and HBV-DNA levels were slightly, but not significantly, increased in the positive subjects. The number of HBe- and HBc-positive nuclei were positively correlated with serum HBV-DNA levels (P less than 0.05 comparing high serum HBV-DNA levels of greater than 2880 pg/ml and levels of 1-480 pg/ml), and were negatively correlated with disease activity (P less than 0.05 comparing those with severe chronic active hepatitis (CAH) and those with mild CAH and chronic persistent hepatitis (CPH]. These results indicate that male homosexual HBV carriers, positive for anti-HIV, may be immunosuppressed before there are clinical signs of immunodeficiency, and this allows an increased level of replication of at least one other virus (HBV).     

针对HBV X基因的siRNA抑制HepG2细胞HBV的复制和表达

目的构建能转录产生针对乙型肝炎病毒（HBV）X基因转录体的小干扰RNA（siRNA）转录载体pSIH—BV／X,研究其在体外对HBV复制和抗原表达的抑制作用。方法将构建成功的pSIHBV／X与HBV1．3倍体真核表达质粒pHBV1．3共转染HepG2细胞，转染后24、48、72h检测上清液中HBsAg、HBeAg的变化，并检测72h时HBV DNA的变化。结果成功构建了针对HBVX基因转录体的siRNA表达载体pSIHBV／X，并发现它能抑制HBsAg、HBeAg的分泌，抑制高峰在72h．抑制率分别是64％和61％，而无关对照序列的siRNA无此作用。荧光定量PCR证实HBV DNA的复制亦受到抑制，抑制率31％。结论针对HBVX基因转录体的siRNA在体外具有抑制HBV复制和抗原表达的作用。     

Early prognostic significance of hepatitis B surface antigen specific circulating immune complexes in acute type B viral hepatitis

A total of 120 patients with acute viral hepatitis B was investigated for the presence of circulating immune complexes, HBsAg-specific immune complexes, and the behavior of some other immune parameters (immunoglobulins, rosette inhibitory factor RIF, histamine induced inhibition of spontaneous rosette forming lymphocytes). Pathologically elevated levels of circulating immune complexes were seen in 89 per cent of patients, often related to extrahepatic signs in the prodromal phase. In 95.7 per cent of them, HBsAg-containing complexes were found. There were no differences in the behavior of immune complexes between patients with recovery and patients developing chronic hepatitis B. On the contrary, the levels of RIF and differences in the inhibitory effect of histamine on rosette forming test of patient's lymphocytes did allow to distinguish patients with recovery from patients with development of chronic hepatitis B early in the course.     

苦参素联合促肝细胞生长素治疗急性乙型临界性重型肝炎的疗效观察

目的观察苦参素联合促肝细胞生长素在治疗急性乙型临界性6重型肝炎并防止其向重型肝炎发展方面的临床疗效。方法选择30例病人治疗，同时随机设对照组30例。治疗组除应用苦参素外还与对照组一样应用促肝细胞生长素。并观察治疗前后肝功能指标的变化。结果两组在病程TBil、ALT、A/G、PTA和PT等指标方面经统计学处理有非常显著性差异（P〈0．01）。结论苦参素联合促肝细胞生长素在防治急性乙型临界性重型肝炎向急性重型肝炎发展上有良好的作用。     

Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection

To elucidate the biologic significance of hepatocyte hepatitis B core antigen (HBcAg) expression and its relation to the natural course of hepatitis B virus (HBV) infection, the patterns of HBcAg were correlated with HBV virus replication state and the disease activity in 598 needle liver biopsies performed on 569 hepatitis B surface antigen (HBsAg) carriers aged 1-81 years. A good correlation of liver HBcAg with serum HBeAg and HBV DNA status was demonstrated. HBcAg was present in the hepatocyte nuclei (nHBcAg) or cytoplasm (cHBcAg), or in both (mixed). Pure nHBcAg was seen mainly in children and young adults; 86% of the patients had non-aggressive disease, but rare cases of chronic active hepatitis (CAH) and HBeAg seroconversion were observed. In contrast, cHBcAg was predominantly associated with CAH (52%) and accompanied by a significantly higher HBeAg seroconversion rate (27%). The HBeAg-negative group, particularly the liver HBcAg-negative subgroup, had a lower frequency of CAH, but an increased incidence of non-aggressive disease as well as cirrhosis and/or hepatocellular carcinoma, indicating that HBeAg seroconversion to anti-HBe does not necessarily mean a favorable prognosis. The results suggest that expression of HBcAg correlates with the liver pathology and the three phases of chronic HBV infection: (1) the early immune tolerance phase is characterized by nHBcAg, mild disease and low HBeAg seroconversion rate; (2) the virus replication/elimination phase by cHBcAg or negative HBcAg, frequent CAH, and high HBeAg seroconversion rate; and (3) the inactive virus replication phase by negative HBcAg and a bipolar disease spectrum.