Correlation between fluorescent antibody detection of hepatitis B core antigen in liver biopsies and the presence of e antigen in serum.

HBc Ag was detected by fluorescent antibody in liver biopsies taken from chronic hepatitis B surface antigen (HBs Ag) carriers having e antigen in their sera. HBc Ag could not be detected in liver biopsies from similar individuals having e antibody in their sera.     

Serum HBsAg, HBeAg, anti-HBe, and hepatitis B viral DNA in asymptomatic carriers in Taiwan

Plasma samples from 89 asymptomatic hepatitis B surface antigen (HBsAg) positive volunteer blood donors were titrated for HBsAg by radioimmunoassay using the parallel line method. HBsAg titers ranged widely from 0.01 to 325 micrograms/ml. The titers correlated well with hepatitis B viral DNA (HBV DNA) and hepatitis B e antigen (HBeAg) in the serum. The HBsAg titers in 55 HBV DNA positive carriers were 90.7 +/- 61.7 micrograms/ml (Mean +/- SD) vs. 6.3 +/- 12.2 micrograms/ml in the 34 carriers without HBV DNA in the serum. The titers were 93.9 +/- 59.1 micrograms/ml in 56 carriers with HBeAg, 6.4 +/- 10.1 micrograms/ml in 24 anti-HBe-positive carriers, and 4.9 +/- 4.6 micrograms/ml in 9 HBeAg/anti-HBe-negative carriers. 50 (89.3%) of the 56 HBeAg-positive carriers had HBV DNA, in contrast to four (16.7%) of 24 anti-HBe-positive carriers. The study indicated that high-titered HBsAg carriers were much more likely to be infectious, and confirmed that HBeAg is a practical marker of infectivity. Absence of HBeAg, however, did not exclude infectivity in asymptomatic HBsAg carriers, inasmuch as one-sixth of the carriers had HBV DNA.     

Expression of hepatitis B virus-specific markers in asymptomatic hepatitis B surface antigen carriers.

A study was undertaken to assess the state of hepatitis B virus infection in a group of asymptomatic hepatitis B surface antigen (HBsAg) carriers. This study confirmed that the presence of hepatitis B e antigen (HBeAg) in serum was closely associated with serum HBsAg-specific deoxyribonucleic acid polymerase activity, hepatitis B core antigen (HBcAg) in serum and liver cell nuclei, and a histological picture of chronic hepatitis. No HBsAg-specific deoxyribonucleic acid polymerase activity or HBcAg was detected in highly concentrated anti-HBe-positive sera. In addition, liver biopsy specimens from carriers with anti-HBe were negative for HbcAg by immunofluorescence, and the liver histology was either normal or revealed only fatty changes. These data indicate that the anti-HBe-positive sera contained either no Dane particles or, if present, at least a 500-fold-lower concentration of Dane particles than that found in HBeAg-positive sera.     

Hepatitis B virus core antigen (HBc Ag) accumulation in an HBV nonproducer clone of HepG2-transfected cells is associated with cytopathic effect

Two clones of the hepatoblastoma HepG2 cell line transfected with complete hepatitis B virus deoxyribonucleic acid (HBV DNA) were studied. The kinetics and cytopathic effect of HBV Ag production in these two clones (one of which was an HBV producer) were compared to those of the parent HepG2 cell line. The presence of hepatitis B surface antigen (HBs Ag) and hepatitis B e antigen (HBe Ag) was determined by commercial enzyme-linked immunosorbent assay (ELISA). A hepatitis B core antigen (HBc Ag)-specific ELISA assay was developed, using monoclonal anti-HBc to detect HBc Ag. Amounts of HBs, HBe, and HBC Ags were partially quantified in both intracellular and extracellular compartments. The HBV producer clone excreted high levels of HBc, HBe, and HBs Ags from the beginning of the growth phase, and no cytopathic effect was observed. The HBV nonproducer clone produced high levels of HBs and HBe Ags, but there was no detectable HBc Ag in the supernatant; instead, HBc Ag accumulated in the intracellular compartment. In this clone, significant cell death was observed 4 days after cell confluency, corresponding with notable HBc Ag release into the supernatant. These results suggest a cytopathic effect associated with HBc Ag accumulation in the HBV nonproducer clone, but no cytopathic effect in the HBV producer clone. This suggests that virological factors as well as the host's immune response may be considered in explaining liver injury occurring in hepatitis B.     

Detection of Hepatitis B Antigen in Circulating Immune Complexes in Acute and Chronic Hepatitis

By using polyethylene glycol precipitation at low concentration (PEG test) and the radiolabeled C1q binding test, immune complexes were detected sera from acute (23/28) and chronic (28/32) hepatitis patients, hemodialyzed patients with chronic hepatitis B surface (HBs) antigenemia (7/19), and asymptomatic HBs antigen carriers (2/11). After treatment of PEG precipitates with acidic pH, heating, or proteolytic enzyme (protease), electroimmunodiffusion or radioimmunoassay revealed the presence of HBs antigen or antibody in dissociated immune complexes in sera from several acute and chronic hepatitis patients. Electron microscopy showed immune complexes of HB virus in 9 of 12 PEG precipitates obtained from PEG-test-positive sera; these 9 precipitates were from patients with acute or chronic hepatitis and the other three from chronic HBs Ag carriers. Free HB virus particles were observed after protease digestion of PEG precipitates. Neither immune complexes nor virus particles were seen in precipitates from PEG-test-negative but HBs-Ag-positive sera from chronic carriers.     

Immunological and biophysical properties of hepatitis B antigen labeled by the chloramine-T and by the lactoperoxidase methods.

Optimal conditions were sought for the radiolabeling of microgram quantities of hepatitis B surface antigen (HBs Ag) employing the chloramine-T or lactoperoxidase iodination procedures. Preparations of HBsAg labeled by these procedures are referred to as chloramine-T preparations and lactoperoxidase preparations, respectively. Labeled HBsAg having specific activities between 10-20 muCi/mug were found to display the greatest degree of sensitivity for unlabeled HBsAg and for anti-HBs using a double-antibody radioimmunoassay (RIA-DA). Increasing the specific activity above this level redulted in a decreased affinity of labeled 1251-HBs Ag for anti-HBs, indicating that soluble antigenic alterations had developed. At equivalent specific activities, chloramine-T preparations competed less effectively for unlabeled HBs Ag than lactoperoxidase preparations, and anti-HBs endpoint titers were slightly reduced, especially among preparations of high specific activity (greater than or equal to 65 muCi/mug). Chloramine-T preparations of HBs Ag (sp. act. 15--30 muCi/mug) showed essentially no antigenic deterioration over a 2-month period at minus 196 degrees C or minus 70 degrees C. Utilization of optimally labeled 1251-HBs Ag has increased the sensitivity of the RIA-DA for unlabeled HBs Ag 30-fold to a level below 1 ng/ml and enhanced antiamine-T method revealed that only the most acidic population was labeled (pH 3.75+/-0.5). In contrast, six antigenic components with distinct pI values ranging from 3.7 to 5.2 were detected by RIA-DA in both unlabeled HBs ag and in the chloramine-T preparation. This indicated that the chloramine-T method did not radically change the relative number or charge of each of the pI populations present in purified preparations of HBs Ag. Analysis of HBs Ag iodinated by the lactoperoxidase procedure revealed the presence of three of four populations of particles with pI values ranging from 3.9 to 4.5, suggesting that this procedure labels HBs Ag more uniformly.     

Prevalence of Williams e1 antigen in comparison with e2 antigen in hepatitis B antigen carriers and patients in hemodialysis unit

The prevalence of both e1 and e2 antigens in 1,158 sera of asymptomatic HBsAg carriers, carriers in hemodialysis units, and HBsAg-negative blood donors was examined. The detection rate of e1 antigen was as high as 80% in asymptomatic carriers, 95% in hemodialysis patients, and even 13.1% in HBsAg-negative donors. All of the e1 antigen-positive specimens in such HBsAg-negative sera were found to have both or either anti-HBs and anti-HBc, suggesting the past history of Hepatitis B virus (HBV) infection of the donors. In the HBsAg-positive serum, the detection rate of e2 antigen (17%) was lower than that of e1 (80%), and all sera having e2 antigen were positive for e1 antigen. The titers of HBsAg, HBcAg, and anti-HBc in e2 antigen-positive sera were higher than that of sera detecting only e1 antigen. The appearance of e1 antigen and e2 antigen in the course of post-transfusion hepatitis B was studied with five cases. Retrospective study showed that three of them each received one unit of HBsAg-positive blood, and the other two received HBsAg-negative blood but with high-titered anti-HBc. In four cases out of five, in which e2 antigen was detected during the course of infection, the initial detection of e2 antigen occurred at or just before the elevation of liver enzyme levels. On the other hand, e1 antigen was detected relatively early after transfusion, and the time of onset. Moreover, the detection period of e1 antigen persisted longer, even after the disappearance of HBsAg antigenemia. These two separate studies suggest that not only e2 antigen but also e1 antigen are associated with the infection of HBV, but they are distinct from each other; the e2 antigen may have the properties of a signal of the viral activity in the patient as suggested by many others, but e1 antigen does not seem to bear such diagnostic values.     

Subtypes of HBsAG in Morocco: general distribution in HBsAG carriers in Casablanca in terms of their clinical state. Relations with the markers of the HBe system

HBs Ag subtypes ad and ay were determined by counter-electrophoresis (CEP) among 301 persons from Casablanca found HBs Ag positive by CEP: 147 were asymptomatic HBs Ag carriers and 154 were patients with acute or chronic hepatitis. w and r determinants were investigated among 82 from them only. HBe antigen and antibody (AB) were investigated by gel double immunodiffusion among 294 persons. Into the whole population, the prevalence of ad and ay subtypes was respectively 20 and 80%. This distribution, intermediate between those reported in West Europe and West Africa, was in agreement with data previously reported by others in the Maghreb, but differing from these ones in the distribution of w and r determinants. ayr subtype was found dominant (39%) and adw rare (2.4%), the prevalences of adr and ayw being intermediate (25-30%). Among asymptomatic HBs carriers and patients only adr and ayr distribution differed: in the formers, adr was dominant and ayr rare and, in contrast, ayr was frequent and adr rare among patients. Elsewhere, some findings were in agreement with previously reported data by others, such as the frequent association of ad to chronic liver disease or long-term HBs Ag carriage, ay being rather associated to short-term carriage, the frequency of HBe Ab in ad subtype and the presence of ay among almost all HBs positive haemodialysed patients.     

Serial liver biopsies in blood donors with persistent HBs antigenaemia.

Initial liver biopsies from asymptomatic HBs antigen positive blood donors showed a range of histological abnormalities ranging from minor parenchymal lesions to cirrhosis. Twenty of these have now been followed up for periods of up to four years and during that time have had at least two liver biopsies. Throughout the period of study all the donors have remained carriers of HBs Ag, and there was no significant variation in the titers of antigen or the electron microscopic appearances of the serum in individual donors. The histological appearances of subsequent liver biopsies were not always the same as those seen initially, and while there appeared to be some improvement in three cases, there were nine in which the histological appearances were worse.     

Increased frequency of HLA DR13 in hepatitis C virus carriers with persistently normal ALT levels

The aim of this study was to clarify the relationship between human leukocyte antigen DR allele distribution and the degree of liver cell injury of hepatitis C virus (HCV) carriers in Japan. The subjects, 68 HCV carriers, were divided into two groups according to the laboratory data and liver histology. Those in the asymptomatic carrier group (n = 19) had normal ALT levels persistently for 8–153 months (mean 25.7 months) and were diagnosed histologically as normal liver, nonspecific reactive hepatitis or chronic persistent hepatitis. Those in the chronic active hepatitis group (n = 49) had elevated ALT levels and were diagnosed histologically with chronic active hepatitis. The human leukocyte antigen DR alleles of all subjects were defined using the polymerase chain reaction restriction fragment length polymorphism method. The expression of human leukocyte antigen class I antigen and intercellular adhesion molecule 1 on the hepatocyte membrane were also examined in 14 patients from each group using an indirect immunohistochemical method. The frequency of DR13 (42.1%) in the asymptomatic carrier group was significantly higher (Pc < 0.003) than that of the chronic active hepatitis group (4.1%). There were no significant differences for the other DR alleles. The frequencies of expression of human leukocyte antigen class I antigen and intercellular adhesion molecule 1 on the hepatocyte membrane of the asymptomatic carrier group were significantly less than those of the chronic hepatitis group (64% vs. 100% P < 0.05, 29%; vs. 71% P < 0.05, respectively), although there was no significant difference in the serum HCV-RNA titer between the two groups (10^6.4±1.1 vs. 10^6.5±0.7 copies/mL). These results demonstrate that the cellular immune response of the asymptomatic carrier group is less activated than the response of the chronic active hepatitis group and that HLA DR13 may be closely associated with this low activity of hepatitis among HCV carriers. © 1996 Wiley-Liss, Inc.     

HBsAg-induced interferon-gamma secretion in T cells from asymptomatic HBsAg carriers and HB-immune donors in vitro.

Peripheral T lymphocytes obtained from asymptomatic carriers of hepatitis B surface antigen (HBsAg) and donors immune to hepatitis B (HB) through natural infection or vaccination were induced by the envelope protein (HBsAg) of the hepatitis B virus (HBV) into secretion of interferon-gamma (IFN-gamma) in vitro. The kinetics of the IFN-gamma response varied between individuals, but was constantly found to be biphasic, with an early peak attained after 12 hr-4 days and a late peak after 5-8 days of antigen stimulation. The early release of IFN-gamma activity was antigen-specifically induced, as it was in T cells from HB-immune and asymptomatic carriers of HBsAg but not HB-susceptible controls. The second peak of HBsAg-induced IFN-gamma secretion was induced in all three donor groups and the kinetics of IFN-gamma release were similar to that of the mitogen phytohemagglutinin(PHA)-induced IFN-gamma production in similarly prepared T-cell cultures. Thus the late burst of IFN-gamma activity seems to result from a mitogenic property contained within the envelope material of HBV. The mitogenic response was three- to fivefold higher for 4/7 asymptomatic carriers of HBsAg compared to HB-immune donors and HB-susceptible controls, indicating that some patients with chronic asymptomatic carriership of HBsAg exhibit enhanced mitogenic responses to HBsAg.     

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.     

Age-specific prevalence and significance of hepatitis B e antigen and antibody in chronic hepatitis B virus infection in Taiwan: a comparison among asymptomatic carriers, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma

The age-specific prevalence of hepatitis B e antigen (HBeAg) and its antibody (anti-HBe) were studied by radioimmunoassay, and compared in a large series of patients with chronic hepatitis B virus (HBV) infection, including 268 asymptomatic carriers, 389 chronic hepatitis, 114 liver cirrhosis, and 278 hepatocellular carcinoma (HCC). The prevalence of HBeAg/anti-HBe in asymptomatic carriers and patients with chronic hepatitis correlated closely with age as HBeAg prevalence decreased and anti-HBe prevalence increased with increasing age (P less than 0.0005), and is probably due to high infection rate at young age in Taiwan. The prevalence of HBeAg in patients with both cirrhosis and HCC are much significantly lower and had no correlation with age. Two peaks of age-specific prevalence of HBeAg and anti-HBe were observed in patients with HCC, implicating two patterns of HBV infection in these patients. The difference in the prevalence of HBeAg and anti-HBe might indicate that asymptomatic carriers, chronic hepatitis, liver cirrhosis, and HCC are sequential sequelae of HBV infection.     

Anti-DNA,anti-HBc correlations with RIA-detected HBeAg and anti-HBe in chronic HBsAg carriers

The interrelations of 1) antibody to hepatitis B core antigen (HBcAg) — anti-HBc; 2) single-stranded DNA-binding antibodies (anti-DN A); and 3) the e-antigen/antibody system — hepatitis B e antigen (HBeAg) and antibody (anti-HBe), were studied in 150 hepatitis B surface antigen (HBsAg) carriers, in 43 of whom diagnostic liver biopsies had been performed. There was a good correlation between titers of anti-HBc and anti-DN A, regarded as indicators of viral and pathological activity, respectively, as well as between levels of these two antibodies and the presence of HBeAg or anti-HBE as detected by radio-immune assay (RIA). In general, HBeAg-positive carriers showed high anti-HBc and high anti-DNA titers, while the carriers positive for anti-HBe had low titers of both. These findings were in accord with the histopathological results. The three serologic parameters, anti-HBc, anti-DNA, and e-antigen/anti-body, should together prove useful for the evaluation of the clinical status of chronic HBsAg carriers.     

Molecular characterization of occult hepatitis B cases in Greek blood donors

The use of sensitive nucleic acid testing for hepatitis B virus in blood donors revealed a number of HBV DNA(+) cases among HBsAg(?) donors, a status known as occult HBV infection. The purpose of this study was the serological and molecular characterization of occult HBV infection in Greek blood donors. A prospective study was undertaken in order to identify occult HBV infection cases in blood donors. As part of the routine screening of blood donations in Greece, blood units were screened individually by a multiplex HIV‐1/HCV/HBV nucleic acid assay. Initially reactive samples were retested with discriminatory assays. HBV DNA(+)/HBsAg(?) samples were tested further for HBV serological markers and HBV DNA was quantified by real‐time PCR. Molecular characterization was performed by sequencing the envelope and polymerase genes of HBV. Preliminary screening revealed 21 occult cases with the following patterns: anti‐HBc only: 7 donors, anti‐HBc/anti‐HBs: 7 donors, anti‐HBc/anti‐HBe: 5 donors, anti‐HBc/anti‐HBs/anti‐HBe: 2 donors. In all cases, the HBV DNA load was <351 IU/ml. Sequencing was successful in 10 donors (classified within genotype D) revealing several amino acid substitutions related to diagnostic escape and antiviral resistance. HBsAg diagnostic failure and low viral replication in occult HBV infection carriers could possibly be attributed to multiple changes in envelope and polymerase regions, respectively. J. Med. Virol. 81:815–825, 2009. © 2009 Wiley‐Liss, Inc.     

Studies on natural cytotoxicity of human monocytes in viral hepatitis

Studies on natural cytotoxicity of peripheral blood monocytes were conducted in patients with acute viral hepatitis B, patients with chronic aggressive hepatitis etiopathogenically linked to viral hepatitis B, and in asymptomatic carriers of HBs antigen. In the majority of cases of viral hepatitis in the acute stage of the disease and in patients with chronic aggressive hepatitis a significant reduction in the examined function of monocytes was noted which became normalized during convalescence. Results obtained for HBs antigen carriers did not differ from those obtained for normal blood donors. The observations may indicate that restricted natural cytotoxicity of monocytes in the course of viral hepatitis B is related to liver injury. The disturbed monocyte natural cytotoxicity was becoming normal after in vitro incubation with thymic preparations (TFX, Thymex L).     

Hepatocyte localization of hepatitis B core and surface antigens in renal transplant recipients

Summary A prospective series of 45 liver biopsies taken from 22 renal transplant patients was investigated for the presence of hepatitis B antigen core (HBc) and surface (HBs) components by electron microscopy. At the time of each biopsy serum HBs Ag was sought by radioimmunoassay. Sections were taken for the detection of HBs Ag by immunofluorescence.In seropositive patients, intravesicular tubular structures resembling HBs Ag were found in 61% of biopsies while the intranuclear core HBc was present in 69%. No correlation could be made between the ultrastructural pattern of the viral components and the intensity of the histological liver damage. During the follow up, there was an accumulation of both HBs and HBc Ag even in a period as short as 1 year. The 9 liver specimens examined after three years of transplantation showed a marked accumulation of both antigens. Thus the expression of HB Ag at the hepatocellular level seems to correlate better with the duration of antigenaemia than with the histological pattern.Lastly, on matched semithin and ultrathin sections, the ground glass appearance of cytoplasm appeared to correlate with smooth endoplasmic reticulum distorsion, irrespective of the simultaneous presence or absence of intravesicular tubular structures. The sanded nuclei expressed a rare massive accumulation of core antigen.     

e抗体阳性的慢性乙型肝炎病人携带乙肝病毒前C区变异的研究

某些抗－ＨＢｅ（＋）的慢性乙型肝炎病人血清仍可检出ＨＢＶＤＮＡ。本工作对９例这类病人的病毒基因组，在聚合酶链反应后直接进行序列分析，发现８例其前Ｃ区第８３位核苷酸发生Ｇ→Ａ（Ａ８３）点突变，使编码色氨酸（ＴＧＧ）的第２８个密码变异为终止密码（ＴＡＧ）。其中２例Ａ８３变异发生在ＨＢｅＡｇ→抗－ＨＢｅ血清转换过程中。分析１例肝内ＨＢＶＤＮＡ，亦与其血清有相同的Ａ８３变异。以３例抗－ＨＢｅ（＋）的慢性无症状携带者为对照，均无Ａ８３变异。本文６例为慢性活动性肝炎，３例为慢性重症肝炎。变异病毒逃避了免疫清除，可能与病变持续活动甚至重症化有关。     

Anti-HBc titers in HBeAg and anti-HBe positive asymptomatic HBsAg carriers

A study was undertaken to establish markers for HBV replication in relation to HBeAg and anti-HBe. HBsAg carriers with serum HBeAg had DNA polymerase activity in the serum and HBcAg in the liver nuclei. Anti-HBe positive and anti-HBe/HBeAg negative sera lacked these markers. For anti-HBc the following geometrical mean titers were calculated: 1: 12,000 for HBeAg positive, 1:9, 100 for anti-HBe and anti-HBc positive, and 1:2,800 for anti-HBc positive anti-HBe/HBeAg negative asymptomatic HBsAg carriers. Follow up studies revealed mostly unchanged anti-HBc titers in all three groups over an observation period of ten to twenty months. Our data argue for a prolonged HBV replication in all HBsAg carrier subgroups compared to individuals with an uncomplicated acute virus-B-hepatitis. This study gives no final answer whether HBeAg negative HBsAg carriers have a continous HBV replication.     

Prevalence of hepatitis B e antigen and its antibody as detected by radioimmunoassays

A solid-phase radioimmunoassay (RIA) for the detection of hepatitis B e antigen (HBeAg) and antibody (anti-HBe) was developed. The RIA was approximately 1,000-fold more sensitive than rheophoresis for HBeAg, and approximately 6,000-fold more sensitive than rheophoresis for anti-HBe. Generally, less than one-fifth of hepatitis B antigen (HBsAg)-positive sera from blood donors were positive for either HBeAg or anti-HBe by rheophoresis; in contrast, more than 90% of the samples were positive by the RIA method. The ratio of HBeAg to anti-HBe among HBsAg carriers varied in different geographic localities. Also, the presence of HBeAg correlated directly with the titer of HBsAg and the presence of Dane core particles. Anti-HBe was associated with lower titers of serum HBsAg.