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Biophysical and biochemical characterization of hepatitis B antigen.

Hepatitis B surface antigens specified by the genome of the hepatitis B virus are shared by various particulate forms which circulate in the sera of chronic HBs Ag carriers. As purified from sera, HBs Ag consists of at least seven polypeptides, two of which appear to be glycoproteins. Most or all of these polypeptides contain both group-specific (a) and subtype-specific (d or y) determinants aspart of their structure. One particulate form, the Dane particle, is present as a minor component in most sera and may actually represent the virus of type B hepatitis.

Animals

[Change of the prevalent subtype of hepatitis B antigen in acute hepatitis B infections (author's transl)].

Subtyping of hepatitis B antigen (HBs Ag) in patients with acute hepatitis B revealed subtype ay in 75 percent while subtype ad was much more common in chronic hepatitis B infections: 81 percent of HBs Ag positive blood donors and 76 percent of patients with HBs Ag positive chronic aggressive hepatitis revealed subtype ad. In contrast to blood donors and chronic hepatitis patients, a change of the prevalent subtype was noted in acute hepatitis patients between 1970 and 1974. Before May 1972, subtype ad was found in 67 percent of patients, whereas later subtype ay dominated in 93 percent. An unequal distribution of subtypes between acute and chronic forms of hepatitis B infections has been explained by differences in the virulence of virus strains. Our results suggest that the higher prevalence of subtype ad in longterm carriers may be due to infection during an earlier time when subtype ad was also common in acute infections. The change of the prevalent subtype in acute infections may be attributed to differences in contagiosity rather than differences in virulence of virus strains.

Acute Disease

The incidence of hepatitis-B-antigen and anti-hepatitis-B among blood donors in Ndola, Zambia.

Hepatitis-B antigen (HBsAG) was detected in the sera of 29 or 250 (11.6%) blood donors in Ndola, Zambia, most of whom were prisoners or students. The incidence among prisoners was 15.1%, among students only 1.6%. Thirteen sera were subtyped, nine carried the y and four the d antigenic determinant. Although there was a marked difference in the incidence of HBs antigen between prisoners and students, when donors were tested for antibody to HBsAg (anti-HBs) this positive. Overall, anti-HBs was detected in 132 of 250 blood donors (52.8%). The sensitivity of radioimmunoassay, counter-immunoelectrophoresis and a newly developed passive haemagglutination (PHA) test, employing tanned formalinised antibody coated turkey erythrocytes, was compared. Although radioimmunoassay provided the most sensitive method, the PHA test compared extremely favourably and was considered to be particularly suitable for use in the tropics.

Adolescent

The detection of hepatitis B antigen in hepatic parenchyma by the fluorescent antibody technic.

Tissue sections from 42 specimens of liver were examined by indirect immunofluorescence microscopy for the presence of hepatitis B antigen (HB Ag). In all cases the serologic status of HB Ag was known. Fourteen of the specimens were also examined by electron microscopy. In four biopsies from three patients positive cytoplasmic fluorescence was detected using antisera prepared in animals and 20-nm. nuclear particles were found by electron microscopy. These patients were all seropositive for HB Ag, all had chronic aggressive hepatitis or active cirrhosis, and all were receiving immunosuppressive therapy at the time of examination. Nuclear fluorescent staining was demonstrated when one of these biopsies was re-examined using a human antiserum.

Cell Nucleus

Differential distribution of hepatitis B surface antigen and hepatitis B core antigen in the liver of hepatitis B patients.

One hundred liver biopsies from 100 patients with clinical presumptive diagnosis of hepatitis were examined by immunofluorescence for the presence of hepatitis B surface antigen (HBSAg) and hepatitis B core antigen (HBcAg). Of the 60 HBsAg-positive livers, 51 were diagnosed as chronic hepatitis on histological grounds, 6 as acute hepatitis, and 3 as "near-normal liver." From the 60 tissue-positive cases, 3 subjects were HBsAg seronegative. HBcAg was detected in 44 livers, all of which also had HBcAg in the localized in the cytoplasm and the membranes of the hepatocytes, and HBcAg in the nuclei and in 4 cases also in the cytoplasm. Predominant HBsAg expression in the cytoplasm was observed in near-normal liver, chronic persistent hepatitis, and cirrhosis with little activity. This correlated with the amount of ground glass hepatocytes in the biopsies. HBcAg and membrane-localized HBsAg were minimal in those conditions. HBcAg was most prevalent in patients with chronic aggressive hepatitis and active cirrhosis treated with immunosuppressive drugs, whereas the amounts of HBsAg and HBcAg in nontreated patients of those two groups and in acute hepatitis with signs of transition to chronicity were almost equal. HBsAg expression in liver cell membranes was most prominent in active forms of chronic hepatitis (chronic aggressive hepatitis and in active cirrhosis) and in acute hepatitis with signs of transition to chronicity. This observation correlated in the presence of HBcAg in the biopsies of those patients. In acute hepatitis both HBsAg and HBcAg were detected rarely and no membrane expression of HBsAg was observed. The over-all results show a significant relationship between the different degrees of accumulation of HBsAg and HBcAg in the liver and the various histological types of hepatitis and further suggest an interplay of both hepatitis B virus and host immune response in the development and pathogenesis of hepatitis B.

Acute Disease

Antibodies to hepatitis B core antigen in hepatitis B surface antigen-positive and -negative chronic hepatitis.

Antibody to hepatitis B core antigen (anti-HBc) is a sensitive indicator of hepatitis B virus (HBV) infection. However, anti-HBc has been found in only a few patients with chronic hepatitis. Therefore, we tested for anti-HBc in 124 sera from 67 patients with histologically proven chronic hepatitis by the indirect fluorescent antibody technique. All patients, except for one with chronic hepatitis who was seropositive for hepatitis B surface antigen (HBs Ag), had anti-HBc that persisted throughout the follow-up period (three months to three years). Of 33 HBs Ag-seronegative patients, anti-HBc was detected in seven patients and persisted for six months to two years. These findings suggest that in this study 21% of patients with chronic hepatitis with undetectable amounts of HBs Ag in the serum had evidence of recent or continued HBV replication.

Adolescent

Incidence and nature of cytoplasmic hepatitis B antigen in hepatocytes.

Hepatitis B antigen (HB Ag) in the hepatocytic cytoplasm is detected by immunofluorescence after reaction with fluoresceinated antiserum to HB Ag or by electron microscopy as numerous 20- to 30-nm. tubular and circular structures in dilated cisternae of excess endoplasmic reticulum. On light microscopy, these hepatocytes can be recognized because their cytoplasm has a ground-glass appearance and stains with Gomori's aldehyde fuchsin. Aldehyde fuchsin-positive ground-glass hepatocytes were detected in all 14 asymptomatic carriers of HB Ag and in 16 of 60 HB Ag-seropositive patients with chronic hepatitis, but not in HB Ag-seropositive acute viral hepatitis or in various other HB Ag-seronegative liver diseases. These cells are helpful in identifying on light microscopy HB Ag carriers and a portion of patients with HB Ag-positive chronic hepatitis. Nuclear HB Ag did not stain with aldehyde fuchsin. Nucleic acids were not detected in the ground-glass cytoplasm by special stains at the light or electron microscopic level. We suggest that the tubular and circular structures in the hepatocytic cytoplasm are coat material of the hepatitis B virus or virally coded host cell reaction product rather than the complete hepatitis B virus.

Acute Disease

Demonstration of hepatitis B e antigen in hepatitis B core particles obtained from the nucleus of hepatocytes infected with hepatitis B virus.

Liver tissue infected with hepatitis B virus was homogenized and nuclei were separated by centrifugation. Hepatitis B core particles were obtained from the nucleus by the digestion with pronase followed by ultracentrifugation in a sucrose density gradient. Hepatitis B core particles were then treated with sodium dodecyl sulphate and 2 mercaptoethanol and tested for hepatitis B e antigen (HBeAg) by the haemagglutination method. The antigenicity of HBeAg was clearly demonstrated in hepatitis B core particles so treated, although untreated core particles did not reveal any detectable HBeAg activity. The localization of HBeAg in hepatitis B core particles was further supported by the results of a fluorescent antibody technique. When a frozen section of the liver infected with hepatitis B virus was stained with the specific rabbit antibody against HBeAg labelled with fluorescent isothiocyanate, only nuclei of hepatocytes were stained, in a similar distribution to hepatitis B core antigen visualized by fluorescent antibody against hepatitis B core antigen in a nearby section.

Cell Nucleus

Hepatitis B antigen - an incomplete history.

Hepatitis B virus still cannot be grown in an in vitro system; therefore, research into hepatitis B antigen (HB Ag) is limited to laboratory methods such as serology, electron microscopy, and biochemistry. These have established the presence of two distinct antigenic components of HB Ag, that associated with the small forms and the outer covering of the Dane particle (HBs Ag), and that of the Dane particle core (HBc Ag). Current findings make it almost certain that the Dane particle represents the hepatitis B virus (HBV), the smaller forms of the antigen representing excess viral lipoprotein. The group of individuals positive for the antigen are considered and the immunopathology of the disease is discussed. Present understanding of the antigen has made it possible to consider the use of HBs Ag as a means of vaccination. However, further information on the immune mechanisms associated with HB Ag are required before this can be accepted as a general means of protection. Finally, looking at currently available techniques, it would appear that passive hemagglutination and radioimmunoassay are both highly specific and sensitive methods of screening for HB Ag.

Adult

Hepatitis B antigen: advancement in knowledge quickens. (A review).

For many years viral hepatitis and its causative agent have been an enigma to investigators. In the last decade there has been such an increase of information on the subject that it challenges one's ability to keep abreast of the advancement in knowledge. The purpose of this paper is to bring into focus some of the latest developments with regard to viral hepatitis B and its causative agent.

Acute Disease

The nature and incidence of cryoproteins in hepatitis B antigen (HbsAg) positive patients.

Hepatitis B (HbsAg) surface antigen has been detected in the serum of patients with a variety of diseases and immune complexes of this antigen and antibody have been implicated in tissue damage to various organs. Previously we have demonstrated that serum cryoproteins occur in a variety of immune complex disorders and represent pathogenic complexes of antigen and specific antibody. Sera from patients with acute HbsAg positive hepatitis, chronic hepatitis B antigenemia, acute and chronic HbsAg negative hepatitis, as well as a variety HbsAg negative miscellaneous liver diseases and normals were studied for the presence and nature of cryoproteins. Cryoproteins were detected in a large number of patients with acute and chronic HbsAg positive hepatitis and chronic HbsAg carriers. The quantity of these cold insoluble precipitates was highest in acute hepatitis. Cryoproteins were detected with much less frequency in HbsAg negative patients and were not found in normals. The precipitates in HbsAg patients contained either HbsAg, anti-HBsAg or both, along with immunoglobulins and occasionally complement and rheumatoid factor. The cryoproteins in these patients had biological properties attributable to immune complexes and several of the patients had clinical manifestations of acute or chronic serum sickness. Cryoproteins from HbsAg negative patients did not contain HbsAg or antibody to HbsAg and did not have biologic properties of immune complexes. In HbsAg positive patients HbsAg and antibody to HbsAg were concentrated in the cryoprecipitate. The preliminary studies suggest that investigation on cryoproteins in hepatitis may be of clinical and immunopathogenic value.

Antigen-Antibody Complex

Host components in hepatitis B antigen.

Purified [125I]-labelled 20 to 25 nm hepatitis B antigen particles were found to give low affinity immunoprecipitation reactions with antisera to several normal serum components, which were immunologically distinct from the reaction due to the classical hepatitis B antigen surface determinant. These additional antigenic determinants were acid-stable and tightly bound to the particles; they could not be released by treatment with Tween 80 or ether, but were removed by protease digestion with the preservation of particle integrity. It was not possible to distinguish whether they were due to the presence of trace amounts of partly denatured serum components, or to a weak cross-reaction with antigens present in normal serum. The implications of this finding for hepatitis B antigen and antibody detection in sensititive assays are discussed. No evidence was found for native antigenic material, present in normal serum or normal liver cells, being integral to the structure of these particles.

Bromelains

Some epidemiological and clinical aspects of hepatitis B antigen and its subtypes.

The close association between hepatitis B antigen (HBAg) and the infectious agent of hepatitis B is clear. Many investigations have shown HBAg to be a useful tool for epidemiological studies of hepatitis B. The relation between HBAg and the postulated hepatitis B virus (HBV) is as yet not clear. In light of recent results a possible candidate could be the so-called Dane particle, which has HBAg reactivity on the surface, but possesses an antigenically distinct core. The core has been shown to have associated DNA polymerase activity. The particles which carry HBAg reactivity have surfaces which are antigenically complex. One common specificity a and 2 pairs of mutually exclusive determinants have been recognized namely d and y and w and r but further possible specificities are under investigation. Four different phenotypes have been described, adw, adr, ayw and ayr. Present evidence indicates that adw, adr and ayw are the phenotypic expression of 3 different transmissible strains of HBV. Studies on the epidemiology of these subtypes have shown 3 different geographic patterns. In the USA and Northern Europe both Dw (adw) and YW (ayw) are common, but in the Eastern Mediterranean and Middle East Yw is practically the only type found. In the Far East DR (adr) is the dominating subtype. Investigations have been done to determine whether there are any clinical differences in hepatitis caused by the different tubtypes. No certain differences have been shown.

Antibodies, Viral

[Clinical importance of hepatitis B antigen (author's transl)].

The clinical importance of the hepatitis B antigen (HBsAg) is based mainly on the differentiation between different courses of inflammatory liver diseases and/or nonvirus induced diseases of the liver. Epidemiologic studies have shown that beside a parenteral inoculation there is a possibility of a non-parenteral inoculation. Furthermore, there is evidence that the sporadic hepatitis is more commonly hepatitis B. Epidemiologic investigations revealed that clinically healthy HBsAg carriers, the "carrier" status in immunodeficiency syndromes and the natural circulation of the hepatitis B virus are of major interest. The demonstration of HBsAg enables us to characterize some diseases which show associations to hepatitis B virus, but where the pathogenic role of HBsAg/Ab immunocomplexes is questionable. Furthermore, it was possible by the detection of HBsAg to establish newer therapeutic and preventive interventions of virus B hepatitis.

Adult