Biomedical subjects
Publications and source records attributed to F Deinhardt.
A cDNA fragment corresponding to the nonstructural gene region of Hepatitis C virus was cloned and sequenced. cDNA was obtained by reverse transcription of viral RNA extracted from serum of a German patient with chronic post transfusion hepatitis. "Nested" PCR resulted in a cDNA fragment of 345 nt. The sequence showed a homology of 96% to the American prototype HCV.
The prevalence of antibodies to the hepatitis C virus (HCV) was determined in 498 hemodialysis patients from three german dialysis units, 121 staff members and 42 family members using an enzyme-linked immunosorbent assay (ELISA) of the second generation which detects antibodies to a structural (C22) and to non-structural (C33c, C100, 5-1-1) recombinant antigens to HCV. Using the second generation ELISA 115 patients (23.1%) were anti-HCV positive versus 77 (15.5%) when sera were tested by an ELISA of the first generation containing only a non-structural antigen (C100). In 34 of these 40 discordant sera antibodies against at least one viral protein was found by a recombinant immunoblot assay. Of 5 sera containing antibodies to only one viral protein (C22) 3 were HCV RNA positive by polymerase chain reaction. Epidemiological evaluation of the patients revealed that the prevalence of anti-HCV was correlated to the duration of dialysis but not to the number of blood transfusions. Of 121 staff members 2 (1.6%) and 2 of 42 family members (4.7%) were positive indicating a low risk of the patients' contacts of acquiring HCV infection.
Before hepatitis A virus (HAV) was identified, spread of hepatitis A was prevented by public health measures. The first specific, preventive measure for hepatitis A was passive protection with standard, pooled human immune globulins. Human immune globulin contained sufficient HAV neutralizing antibodies for short-term, prophylactic passive protection and for control of the spread of local outbreaks. After many unsuccessful attempts, HAV was propagated in cell cultures and the development of vaccines for active immunization began. Formalin-inactivated, whole HAV induced protective immunity, and such formalin-inactivated hepatitis A vaccines are now being evaluated in large-scale clinical trials. HAV attenuated by serial propagation in cell culture has been used for several, live, attenuated hepatitis A vaccines and results of clinical trials are reassuring. Future approaches to protection against hepatitis A are likely to include vaccination with: hybrid viruses; hepatitis A antigen-expressing, genetically-engineered bacteria; purified hepatitis A antigens produced by molecular biological techniques and incorporated into slow or pulse-releasing systems; synthetic peptides or idiotypes.
Hepatitis A vaccine, strain HM175, was investigated for immunogenicity and tolerability in a prospective multicentre trial. The following vaccination schedules and antigen contents were evaluated: days 0 and 14 with 720 ELISA units (El.U) of antigen, days 0 and 28 with 720 El.U and days 0 and 28 with 360 El.U. In all study groups, the seroconversion rates following two vaccinations were between 95 and 100%. Higher geometric mean concentrations of antibody to hepatitis A virus (anti-HAV) were reached by the vaccine containing 720 El.U of HAV antigen. The vaccine was equally well tolerated in all groups. In addition, an abbreviated schedule, in which 720 El.U of HAV antigen was given on days 0 and 14, resulted in 100% seroconversion by day 28 and a level of anti-HAV that was substantially higher than that observed after passive immunization. This implies that such a vaccine could replace immune globulin administration if time permits.
A total of 114 healthy young adults were immunized with hepatitis A vaccine using different vaccination schedules. Individuals received either a single dose (group 1), two doses given simultaneously (group 2), two doses at days 0 and 14 (group 3) or at days 0 and 28 (group 4), or three doses at days 0, 7 and 21 (group 5). Two weeks after a single dose, seroconversion rates between 77 and 85% were achieved (groups 1, 3, 4). All individuals immunized with two doses within two weeks (groups 2, 3, 5) had antibodies to hepatitis A vaccine (anti-HAV positive) by week 3; these participants also showed clearly higher mean anti-HAV values (geometric mean titres, GMTs) at this time than those individuals vaccinated only once. GMTs at week 8 were 560 IU/l in group 5, 236, 339 and 428 IU/l in groups 2-4 and 102 IU/l in group 1. Of participants with anti-HAV at week 8, 82 were again tested 4 months later; all were still seropositive. Ten individuals were tested during the first three weeks at 3-4 day intervals for anti-HAV immunoglobulin M (IgM); specific IgM responses were not detectable before day 10 but were present in eight of 10 vaccinees by day 14.
The "high lymphoma-prone" baboon stock (Papio hamadryas) of the Sukhumi Primate Center colony is characterized by a high prevalence of antibodies to the STLV-I/HTLV-I type of retrovirus and a high manifestation of human ATL-type (adult T-cell leukemia/lymphoma) malignancies (Yakovleva et al., this symposium). This is in contrast to other primate colonies and wild monkeys, which have low seroprevalence and very few if any ATL-type T-cell malignancies. To characterize the type of T-cell lymphoma retrovirus involved in the Sukhumi disease, a PCR (polymerase chain reaction) DNA analysis of peripheral blood lymphocytes (PBL) and of various tissues of healthy "at-risk", or ill baboons was performed. Proviral STLV/HTLV sequences were detected in all monkeys with symptoms of T-cell malignancy and/or antibodies to STLV-I/HTLV-I. For precise identification and characterization of the Sukhumi T-cell lymphoma virus, parts of the virus genome were mapped and sequenced from PCR derived fragments. A 420 nucleotide fragment of the env (gp 46) gene (analysed from 3 different DNA's) revealed 16.2% nucleotide divergence to the Japanese strain of HTLV-I and 14.8% to the Japanese strain of STLV-I including one deletion of a triplet. On the level of amino acid (a.a) sequence this revealed an exchange of 6 a.a. to STLV-I (4.3%), but only of 4 a.a. to HTLV-I (2.8%). The analysis of 120 nucleotides of the tax sequence (identical in 6 different DNAs) resulted in 5% nucleotide divergence to the HTLV-I (2.4% on the a.a. level) and 10% (7.3% a.a.) to the STLV-1. These results indicate that the Sukhumi T-cell lymphoma virus is a representative of the T-cell leukemia/lymphoma virus family, apparently more closely related to HTLV-I than to STLV-I genome. Furthermore, the infected monkeys from Sukhumi develop at a high rate a T-cell malignancy not observed among other baboons carrying STLV.
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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.