[Hurdles in the fight against viral hepatitis].
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Biomedical subjects
Publications and source records attributed to F Deinhardt.
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Frozen serial serum specimens obtained from past studies on the natural history and prevention of Type B hepatitis in children were retested by radioimmunoassay for the following markers of hepatitis B infection: hepatitis B surface antigen (HBsAg) and antibody (anti-HBs), hepatitis B e antigen (HBeAg) and antibody (anti-HBe), and antibody to hepatitis B core antigen (anti-HBc). The interval between exposure and evidence of viremia (HBsAg) was as short as six days. HBsAg and HBeAg persisted for two to five months and occasionally for more than one year after recovery. After the disappearance of their respective antigens, anti-HBc and anti-HBs persisted for more than seven years and anti-HBe for one to two years. Treatment with hepatitis B immune globulin after exposure induced complete or partial protection or prolongation of the incubation period. Administration of heat-inactivated hepatitis B virus, MS-2 strain, to 29 children induced an inapparent infection in three, characterized by a transient appearance of HBsAg and HBeAg, and the persistence of anti-HBc, anti-HBe and anti-HBs for more than two years.
Hepatitis B virus surface antigen (HBsAg) could be studied until recently only by isolating it from the blood of carriers, thus making incorporation of radioactive precursors into this protein(s) impossible. The isolation of a cell line producing HBsAg [Alexander et al, 1978] has eliminated this obstacle. The cell line was therefore used for labeling HBsAg either with 35S-methionine or with 35S-cystine. HBsAg was purified by pelleting the component and by isopycnic centrifugation in CsCl gradients. HBsAg-positive fractions (as determined by solid-phase radioimmunoassay) were isolated from the gradients and analyzed in sodium dodecyl sulfate-containing polyacrylamide gels. It was found that although HBsAg contains substantial amounts of 35S-cystine, very little 35S-methionine was incorporated into this protein. In contrast, both labels were found in other structures having a buoyant density of about 1.3 gm/cm3 in CsCl. It was concluded that HBsAg is very low in methionine, and therefore this amino acid should not be used for labeling HBsAg in cells or in a cell-free system. Analysis of 35S-cystine-labeled HBsAg-positive material (buoyant density about 1.2 gm/cm3 in CsCl) revealed five proteins with molecular weights in the range of 48,000-82,000.
Hepatitis A virus (HAV) was isolated directly from human feces and propagated serially in an HBsAg producing human hepatoma cell line. No cytopathic effect was observed in the tissue culture and no detectable amounts of HAV were present in the tissue culture supernatant fluid. However, increasing amounts of hepatitis A antigen (HAAg) were detected by radioimmunoassay in the cell extracts obtained by freezing and thawing of cells. Specificity of the HAAg determination was shown by neutralization with convalescent sera of marmosets experimentally infected with the MS-1 strain of hepatitis A and by the absence of this neutralization with preinoculation sera. HAAg was first detected after four weeks in the cell extract of infected cultures after inoculation of 10(2)--10(4) tissue culture infectious doses of HAV from second passage.
In order to evaluate the potential infectivity of blood of hepatitis B patients, the Dane particle associated DNA polymerase was determined, which is a reliable marker for the presence of complete viral particles. Enzyme activities were compared with hepatitis B e antigen (HBeAg) titers determined by radioimmunoassay. Detectable DNA polymerase activity was only present in HBeAg positive blood, preferentially in samples with high antigen titers (1 : 1000 and above). These samples therefore have to be considered as highly infectious. However, blood with low HBeAg levels and free of detectable polymerase activity can still be infectious, since the polymerase reaction is rather insensitive compared to the radioimmunological HBeAg determination.
In 1968, studies of infectious hepatitis in volunteers were reported. Immunologic procedures for serologic study of the hepatitis A virus were not available at that time, and only the clinical and biochemical parameters of the disease were reported. Serial serum specimens from the participants in the study were retained; these specimens had been taken before inoculation and up to more than 100 days after inoculation. When a radioimmune assay for antibody to hepatitis A virus was developed, the series of sera was analyzed retrospectively. Forty-four male volunteers were involved in a series of three studies. Twenty (46%) of the volunteers were found to be initially immune to hepatitis A virus. Eighteen susceptible volunteers (with no preexisting antibody) were challenged with infectious virus. Eight of these volunteers developed clinical hepatitis and seroconverted; one seroconverted without evidence of clinical disease; and nine neither seroconverted nor had evidence of clinical disease. The radioimmune assay provided a method for diagnosis of immune status and of the acute disease caused by hepatitis A virus.
Using a solid phase radioimmunoassay, antibody to hepatitis A virus (anti-HAV) was determined in 3890 sera from populations in seven European countries. Prevalence of anti-HAV was lowest in Scandinavian countries and highest in Greece and France. Antibodies were found in 77 (13%) of 602 blood donors in Sweden, in 29 (17%) of 175 blood donors and women taking birth control pills in Norway, in 273 (39%) of 700 blood donors in Switzerland, in 262 (52%) of 505 blood donors in Holland, in 365 (55%) of 661 accident patients in West Germany, in 452 (75%) of 600 blood donors in France and in 530 (82%) of 647 persons in Greece. Prevalence of anti-HAV increased with age in all populations tested, indicating nearly total exposure to HAV in persons over 19 years of age in Greece and in persons over 39 years of age in West Germany, Holland and France. Antibody was found more frequently in rural than in urban populations in Greece and Switzerland. Calculation of the age-specific incidence of HAV infections suggests a remarkable decline in the exposure rate in the last few decades.
The persistence of antibody to hepatitis A antigen (anti-HAV) of the immunoglobulin M (IgM) class was evaluated in 88 sera of 51 acute hepatitis A patients. IgM was separated from IgG by a 2-h reorienting sucrose gradient ultracentrifugation, and the titer of anti-HAV was determined in the IgG- and IgM-containing fractions by solid-hase radioimmunoassay. IgM anti-HAV was the predominating antibody at onset of jaundice and persisted in these patients for at least 60 days, but not longer than 115 days. The demonstration of IgM anti-HAV is therefore a valuable tool for the diagnosis of recent hepatitis A infection.
The parameters for the use of reorienting gradients in vertical rotors for rapid separation of immunoglobulin M from immunoglobulin G on a preparative scale for the rapid diagnosis of infectious diseases are described.
Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.
Minced salivary glands from seven white-lipped marmosets (Saguinus fuscicollis and Saguinus nigricollis) and one cotton-topped marmoset (Saguinus oedipus) were cocultivated with marmoset cell cultures. A viral agent, designated SSG, was isolated from two Saguinus fuscicollis. Slowly progressing foci of rounded, vacuolated, refractile cells were first observed at 40-43 days incubation. Electron microscopy revealed intranuclear herpesvirus nucleocapsids and intracytoplasmic and extracellular enveloped particles. Infected cells stained with hematoxylin and eosin contained eosinophilic intranuclear and cytoplasmic inclusion bodies. SSG could be passaged in cell cultures only using viable whole cells; infectious cell-free virus was not detected in either culture supernatants or cell lysates. SSG replicated in marmoset fibroblastic but not in marmoset epithelioid or human fibroblastic cell cultures. Plasma antibodies to SSG were detected by indirect immunofluorescence assays in 16 of 56 (28.6%) adult wild-caught marmosets but were absent in 40 colony-born, hand-reared marmosets. Antigenic cross-reactivity of SSG with a rhesus monkey (Macaca mulatta) cytomegalovirus (bidirectional) and with a human cytomegalovirus (unidirectional) was also demonstrated by indirect immunofluorescence assays. SSG was identified as a herpesvirus by morphology and was classified as a cytomegalovirus by its site of isolation, biologic properties in vitro, and antigenic characteristics.
TPA, a highly active tumor-promoting agent, is an effective mitogen for primate peripheral blood lymphocytes. Optimal stimulation of human lymphocytes was obtained 4 days after the addition of TPA at a concentration of 7.5 ng/ml. Lymphocyte fractionation experiments demonstrated that both T and B cells incorporated 3H-thymidine significantly in response to TPA. Lymphocyte blastogenesis was not due to the reactivation of latent herpesviruses by the tumor promoter, since similar responses to TPA were obtained with virus-genome positive or negative cells. Increased levels of DNA synthesis were observed when TPA was added to marmoset, baboon, rhesus monkey, or chimpanzee peripheral blood lymphocytes. Canine peripheral blood lymphocytes and spleen cells from guinea pigs, rats, and mice were not stimulated by TPA. These observations suggest that TPA-induced lymphocyte blastogenesis may be useful for studies of lymphocyte activation and of the molecular mechanisms of action of tumor-promoting phorbol esters.
Sera collected from patients with nasopharyngeal carcinoma, patients with other head and neck neoplasms, patients with infectious mononucleosis (IM), and healthy donors were titrated for antibodies (IgM, IgG, IgA) against Epstein-Barr-Virus (EBV) specific antigens (VCA, EA, EBNA) by indirect and by antikomplementary immunfluorescence. NPC-patients develop significantly high anti-EBV titers of IgG and IgA. In contrast patients with neoplasms (mainly carcinomas) arising in sites of the head and neck other than the nasopharynx revealed a lower incidence of high titers. Our results emphasize the remarkable predominance of IgA-antibodies to VCA and EA in NPC-patients and shows that Europeans to not differ in that respect from Asian patients. The significance and implications of these findings are discussed.
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Hepatitis-B-surface antigen (HBsAg) was not detected by sensitive radioimmunoassays in the tear fluids of 6 HBsAg carriers with low and medium titers of HBsAg (less than 1:10,000) in the serum. However, HBsAg could be demonstrated in low concentrations in tear fluids of 5 of 6 HBsAg carriers with high serum titers (greater than 1:10,000). The concentration of HBsAg in the tear fluid was at least 100 times lower than in the sera of these 5 persons. Correspondingly HBsAg could be found in only 1 of the rinsing and in none of the storage solutions of the contact lenses of 7 persons with high titers of HBsAg in the serum (greater than 1:32,000). HBsAg was not adsorbed to smooth HEMA-lenses. Because of the low concentration of HBsAg in tear fluids and the dilution effect (about 5 x 10(-10)) the transmission of hepatitis B by multiple use of contact lenses by several persons during adaption is lighly unlikely. In addition, a special cleaning solution (Liprofin) can destroy nearly completely the antigenicity of HBsAg at 60 degrees C.
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Circulating lymphocytes from three species of marmoset monkeys were transformed in vitro with Herpesvirus ateles (HVA) either by co-cultivation with lethally X-irradiated HVA-producing lymphoblastoid cells or by infection with cell-free virus: 42 transformed lymphocyte cultures were obtained from 72 transformation attempts. Attempts to transform squirrel monkey lymphocytes were unsuccessful in 29 attempts. Association of HVA with each transformed culture was demonstrated by staining of antigen-positive cells in indirect fluorescent antibody (FA) tests and recovery of HVA after co-cultivation of the transformed cells with permissive monolayer cells. Cells of most transformed cultures possessed T lymphocyte properties: i.e. formation of E rosettes, reactivity with a specific anti-marmoset T lymphocyte serum and lack of surface Ig.
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.