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Biomedical subjects

M Roggendorf

Publications and source records attributed to M Roggendorf.

At least 109 records · Page 6Linked to original sources
PubMed1995

Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment.

It has been postulated that antibodies specific to the hypervariable region 1 (HVR1) within the putative envelop protein E2 of hepatitis C virus (HCV) can neutralize virus. We studied such antibodies in sera of patients who were infected in a single-source outbreak by a contaminated anti-D immunoglobulin preparation (HCV-AD78). The nucleotide sequences of cDNAs encoding HVR1 of HCV-AD78 were determined. The four major variants (HVR1.A, B, C, and D) were expressed as fusion proteins in Escherichia coli. Sixty-seven percent of sera contained antibodies to HVR1.A. Sera unrelated to infection of the outbreak also recognized HVR1.A but to a lesser extent (15%), suggesting that not all HVR1-specific antibodies are absolutely isolate-specific. Antibodies directed against individual variants of HVR1 were found in sera obtained early postinfection (p.i.) (< or = 1 year) but also in sera obtained several years later. An in vitro binding assay of HCV to tissue culture cells was employed to further characterize these sera. Five of seven sera that were obtained early p.i. prevented binding of HCV to cells. Preincubation of such sera with HVR1-specific fusion proteins restored binding of HCV to cells in four of five sera. These findings suggest that the majority of neutralizing antibodies are directed against HVR1.

Amino Acid Sequence↗
PubMed1995

Acquired pure red cell aplasia: a study of six cases.

Persistent infection by parvovirus B19 associated with pure red cell aplasia (PRCA) has been documented in immunocompromised patients. Bone marrow failure is associated with conditions in which immune surveillance is impaired, and in these instances occult parvovirus infection may be suspected. In this study we have assessed by serological and molecular methods whether parvovirus B19 infection may be a more frequent cause of PRCA than hitherto suspected and whether it may be present in the absence of a typical bone marrow picture. Six patients with PRCA--two with isolated PRCA and no apparent underlying disease, two with a lymphoproliferative disease, one with thymoma, and one with chronic myelomonocytic leukemia--have been studied. Four of the six patients had overt PCRA and were clearly immunocompromised. Parvovirus B19 was not detected in any of the six patients by PCR analysis and serology investigating the presence of IgM or IgG antibodies. Although parvovirus B19 infection needs to be ruled out in PRCA it represents only one, and probably not the most frequent, etiological factor of PRCA.

Adult↗
PubMed1995

Seroprevalence of hantavirus antibodies in Germany as determined by a new recombinant enzyme immunoassay.

In order to elucidate the epidemiological importance of hemorrhagic fever with renal syndrome in Germany, the prevalence of antibodies against hantaviruses was determined in 13,358 sera from residents of various geographic regions, 1,284 sera from occupational risk groups and 287 sera from chronic hemodialysis patients. Serological investigations were performed using a highly specific transferable solid phase enzyme immunoassay based on the recombinant nucleocapsid proteins of a Hantaan and a Puumala serotype strain. The overall antibody prevalence was found to be 1.68%. In the serum panels from western and southern Germany, it was determined to be 1.83% on average in contrast to only 0.8% in the panel from eastern Germany. An endemic focus revealing an antibody prevalence of 3.12% was detected in a low-mountain area called Suebian Alb, which is located in the federal state of Baden-Württemberg. Occupational risk groups and a group of chronic hemodialysis patients showed a significantly elevated antibody prevalence ranging from 3.3% to 10%. The Puumala serotype was found to be the prevailing virus, but the percentage of sera predominantly recognizing the Hantaan nucleocapsid protein increased towards the south and the east and was significantly elevated in dialysis patients.

Antibodies, Viral↗
PubMed1995

Analysis of hepatitis C virus quasispecies populations by temperature gradient gel electrophoresis.

Hepatitis C virus (HCV) forms complex quasispecies populations which consist of a large number of closely related genetic variants. This genetic heterogeneity may cause antigenic variation or drug resistance. We used heteroduplex analysis by temperature gradient gel electrophoresis (TGGE) to characterize genetic variants of HCV. The high resolution of TGGE was proven by comparison of DNA sequence data of different cDNA clones from the HCV 5'NCR with their corresponding migration pattern in TGGE. Using this method we were able to identify virus variants of the HCV 5'NCR even if they only differed from each other by a single base. HCV populations from three patients with chronic hepatitis C were found to consist of genetic variants, although the degree of the heterogeneity varied. In addition, we compared the genetic heterogeneity of the core and E2 regions of the HCV genome in one patient. Our results demonstrate that TGGE is a useful tool for characterization of the genetic heterogeneity of virus populations in vivo.

Base Sequence↗
PubMed1995

The woodchuck: an animal model for hepatitis B virus infection in man.

Since the discovery of woodchuck hepatitis virus (WHV) in 1978, the virus and its host, the American woodchuck, have been studied and used as the most suitable model for human hepatitis B virus infection. WHV is closely related to the human virus, having strong similarities in morphology, genome structure and gene products, replication, epidemiology, the course of infection and in the development of illness and hepatocellular carcinoma. Because of this high homology, the woodchuck model is used for many studies for the development of new vaccines, therapeutic vaccination and antiviral agents. In addition, the woodchuck system is used for investigation of molecular mechanisms of the viral life cycle, the mechanisms of carcinogenesis and cell infection.

Animals↗
PubMed1994

[Current developments in the diagnosis of hepatitis C].

Since the discovery of hepatitis C virus five years ago eight complete isolates and a large number of partial isolates have been sequenced. By comparing sequences, six HCV types can be differentiated which show more than 35% divergency in the NS5 proteins. The course of hepatitis C and the response rate after interferon therapy may be dependent on the HCV type. Serological tests for the diagnosis of acute and chronic hepatitis C have been improved, so that more than 90% of patients seroconvert at the peak of transaminases during acute infection; however in single cases, seroconversion can last up to nine months after onset of disease. Antibodies which can be detected in the acute and chronic phase of hepatitis C are directed against structural and nonstructural proteins. Most recently, also antibodies enveloping proteins E1 and E2 have been identified. These antibodies obviously do not seem to neutralize the virus. In patients with acute hepatitis C and complete recovery antibodies may persist up to ten years after onset of disease. At present there is no marker for past infection or immunity to HCV. Chronicity of hepatitis C and infectivity of patients can only be shown by detection of viral RNA using RT-PCR. Indications to perform PCR are patients prior to and after interferon therapy, hemodialysis patients, patients undergoing immunosuppression, new-born babies of mothers with chronic hepatitis C and patients with acute hepatitis C who are negative for antibodies.

Algorithms↗
PubMed1994

Sequence variability in the env-coding region of hepatitis C virus isolated from patients infected during a single source outbreak.

The variability of the hepatitis C virus genome was investigated in a group of German patients who developed chronic hepatitis C after parenteral administration of contaminated immunoglobulin to prevent Rh sensitization after pregnancy. The nucleotide and deduced amino acid sequence alterations of the E1 and the first hypervariable region of the E2 gene of the hepatitis C virus (HCV) genome from sera of two randomly selected patients were studied by comparison of HCV sequences obtained from the original inoculum (anti Rh immunoglobulin) and from patient sera collected in 1979 and 1989. All isolates were classified as subtype 1b but showed nucleotide insertions of up to 12 nucleotides at the cleavage site of E1/E2. Microheterogeneity of HCV genomes was found in the immunoglobulin supporting the quasispecies model of HCV distribution. Remarkable nucleotide exchanges over the 10 year period in the E1 region (0.9-5.2 x 10(-3) base substitutions per genome site per year) and especially in the first hypervariable region of the E2 gene (about 1.5 x 10(-2)) occurred. The HCV genome undergoes a selection of variants, though it is not known if this derives from mutation or selection of pre-existing rare variants.

Amino Acid Sequence↗
PubMed1994

Demonstration of HCV-RNA and HBV-DNA in the serum of HBsAg negative patients with hepatocellular carcinoma.

Forty patients with chronic liver disease and HCC were analyzed for infection with hepatitis C (HCV) and hepatitis B (HBV) viruses. All patients were negative for HBsAg, 16 were alcoholics, 6 had previous blood transfusions and 18 had sporadic chronic hepatitis. Antibodies to HCV were determined by EIA 2nd generation. HBV-DNA was detected by PCR using primers of the precore region. Analysis of HCV-RNA was done with nested PCR amplifying the 5' non-coding region of the HCV genome, using primers complementary to nucleotides 1-20 and 305-320 and nested primers complementary to nucleotides 21-31 and 271-286 of the HCV-J1. Anti-HCV were positive in 35/40 patients (87.5%). HCV-RNA was detected by PCR in 34 patients (85%) all of them positive for the anti-HCV. HCV-RNA was detected in 70.5% of the alcohol abusers, in 100% of patients with history of transfusion(s) and 94.1% of patients with cryptogenic chronic liver disease. HBV-DNA was detected in only 2 patients. In conclusion, there is a high rate of HCV and a low rate of HBV viremia detected by PCR in Spanish patients with HCC HBsAg negative. No patient without anti-HCV presents HCV-RNA. Our results suggest that persistent HCV replication may play a role in hepatic carcinogenesis, as HBV-DNA could be found in only 5% of our HCC patients.

Aged↗
PubMed1994

Typing of hepatitis C virus isolates by DNA enzyme immunoassay.

Recently, at least six types of hepatitis C viruses (HCV) have been identified. Different types of HCV appear to possess different pathogenic properties and a different sensitivity to interferon treatment. Typing of HCV isolates may therefore be an important diagnostic procedure. We report on a new method for identification of HCV types 1a, 1b, 2a, 2b and 3a which are most prevalent in Europe, North America and Japan. The assay is based on a combination of two well established techniques, the polymerase chain reaction (PCR) and DNA enzyme immunoassay (DEIA). In the first step of the method a cDNA of about 250 bp corresponding to the HCV core-region is amplified by nested PCR. The target cDNA is then hybridized to type-specific oligonucleotides fixed to a solid phase through an avidin-biotin bridge. The formed hybrids are detected by a standard ELISA using monoclonal antibodies reacting with double-stranded DNA. Typically, signal-to-noise (S/N) ratios between 18.2 and 48.6 could be observed when different HCV types/subtypes were analyzed by this method. The test was evaluated using cloned HCV cDNAs of known types and by sequence determination of some of the typed cDNAs. Typing of 115 isolates from Germany, Russia and Turkey revealed that subtype 1b (59-100%) and 1a (24-32%) are most prevalent in these countries.

Antibodies, Monoclonal↗
PubMed1994

[Significance of HCV infection for post-transfusion hepatitis].

Five years ago the hepatitis C virus has been first characterized, and diagnostic tools have been developed to demonstrate acute or chronic infection. At least six different genotypes have been identified. After a primary HCV infection reinfection with other genotypes seems to be possible. The serological tests currently available cannot differentiate between acute, past or chronic hepatitis C. Blood donors which show a reactive result by ELISA should be retested by a confirmatory assay and PCR for the demonstration of viral RNA to exclude or demonstrate persistence of HCV. Systematic screening of blood units for antibodies to HCV reduced the posttransfusion hepatitis up to 95%. However, there is still a risk for acquiring posttransfusion hepatitis C which is actually calculated to be 1:20,000 up to 1:50,000.

Blood Donors↗
PubMed1993

Variability of the envelope regions of HCV in European isolates and its significance for diagnostic tools.

Following the original description of HCV in 1989 a tremendous amount of sequence data is now available. Based on the 8 complete nucleotide sequences published so far at least 4 genotypes can be distinguished. Partial sequences of additional HCV isolates indicate the existence of further genotypes. A serological typing is not yet possible. For detection of virus, reverse transcription and amplification of the 5' non coding region is most commonly performed. This region of the genome is highly conserved among all isolates. In this study we used regions of the E1 and E2 gene in order to classify HCV isolates. The nucleotide sequences of regions in E1 and E2 gene of different European isolates from Germany, Croatia, Hungary, and Rumania were determined and compared to recently published RNA sequences of American and Japanese HCV isolates. The cDNA, obtained by reverse transcription of viral RNA extracted from sera was amplified by nested PCR, cloned and sequenced. Within 564 nucleotides (nt) of E1 we found 87-90% homology (and 89-92% homology at aa level) compared to sequences of Japanese origin and 73-74% homology (77-81% at aa level) compared to the prototype HCV sequence (ptHCV-I). In all characterized isolates the sequence of E2 (643 nucleotides) showed a homology of about 83% at the nucleotide level as compared to genotype II sequences, and a homology of about 70% to genotype I. Our results confirm the existence of two hypervariable regions in the E2 gene of genotype II sequences. Our results also indicate together with other reports from European HCV isolates that genotype II is predominant in Europe.

Amino Acid Sequence↗
PubMed1993

Detection of tick-borne encephalitis virus RNA in ticks (Ixodes ricinus) by the polymerase chain reaction.

A polymerase chain reaction for the detection of tick-borne encephalitis virus (TBEV) RNA in ticks was developed. Two pairs of primers for nested PCR were selected from the 5'-NCR and the 5'-terminus of the C protein coding region, which are highly conserved among the TBEV isolates sequenced so far. The sensitivity of the nested PCR was tested by dilution experiments of a TBEV positive brain suspension. The specificity of the PCR products was confirmed by Southern blotting. In a pilot study, 60 homogenates of 7200 ticks (I. ricinus) were examined by PCR. Two homogenates were found positive. The PCR for TBEV RNA appears to be a valuable method to define endemic areas of TBE.

Animals↗
PubMed1993

Immunization with recombinant woodchuck hepatitis virus nucleocapsid antigen or hepatitis B virus nucleocapsid antigen protects woodchucks from woodchuck hepatitis virus infection.

Woodchucks were immunized with recombinant woodchuck hepatitis virus (WHV) nucleocapsid antigen (WHcAg) or hepatitis B virus (HBV) nucleocapsid antigen (HBcAg) and challenged with 10(6) WHV ID50. Six out of six woodchucks immunized with WHcAg and four out of six immunized with HBcAg were protected from WHV infection. Woodchucks immunized with WHcAg or HBcAg developed high serum antibody titres against WHcAg or HBcAg. Antibodies against WHc and HBc displayed little cross-reactivity (< 1%). This confirms and extends previous reports of protection against homologous challenge after immunization with HBcAg/WHcAg which are both internal viral antigens. As the dominant B-cell epitope(s) on particulate WHcAg and HBcAg appear not to be conserved it also demonstrates that antibodies against HBcAg/WHcAg are not important for this protection. Woodchucks immunized with WHcAg/HBcAg reacted with a fast serum antibody response against viral envelope proteins upon challenge with WHV, indicative of functional intrastructural/intermolecular T-cell help as one potential mechanism of protection after immunization with an internal viral antigen.

Animals↗
PubMed1993

Production of human cloned antibodies specific for hepatitis D virus-encoded small and large protein.

Cloned antibodies to specific epitopes of hepatitis D virus were produced by transformation with Epstein-Barr virus and subsequent cloning of peripheral blood B lymphocytes from a patient with chronic hepatitis D virus infection. Several stable cloned B cell lines, derived from two parent cultures, produced hepatitis D-virus-specific IgG antibodies. Some cloned IgG antibodies detected hepatitis D virus-associated antigen in hepatitis D virus-infected woodchuck liver tissue sections by indirect immunofluorescence staining and some reacted in an inhibition ELISA test detecting hepatitis D virus antibodies; most cloned IgG lines detected hepatitis D antigen both in immunofluorescence tests and in inhibition ELISA. Cloned antibodies to hepatitis D antigen detected by ELISA and/or immunofluorescence staining recognized the two major specific native and denatured polypeptides, p27 and p29, in Western blot analysis. Such cloned antibodies for hepatitis D virus are potentially useful for clinical diagnosis and research.

Antibodies, Viral↗
PubMed1993

Prevalence of antibodies to hepatitis C virus in patients on peritoneal dialysis--a multicenter study.

The prevalence of antibodies to the hepatitis C virus (HCV) was determined in 333 peritoneal dialysis (PD) patients from 10 German dialysis units, using an enzyme-linked immunosorbent assay of the second generation (ELISA 2nd gen) which detects antibodies to a structural (C22) and to non-structural (C33c, C100, 5-1-1) recombinant antigens of HCV. Sera from 18/333 (5.4%) patients were anti-HCV positive versus 11/295 (3.7%) when the sera were tested by an ELISA of the first generation (ELISA 1st gen) containing only a nonstructural antigen (C100). In the 18 sera positive by ELISA 2nd gen, antibodies against at least one viral protein were found by recombinant immunoblot assay (RIBA) in 15/333 (4.5%) patients. In the sera of 11/15 (73.3%) patients HCV RNA was detected by nested PCR. Epidemiological evaluation of the patients revealed that the prevalence of anti-HCV was correlated to the female sex (p = 0.005), presence of anti-HBc (p = 0.006), duration of total dialysis (hemodialysis HD and PD) (p = 0.012), duration of HD alone (p = 0.025) and previous renal transplantation (p < 0.001). Only a weak correlation was found to blood transfusions (p = 0.041) and elevation of serum ALT concentration (p = 0.055). But no correlation was found to diagnoses of renal failure (p = 0.129), duration of PD (p = 0.963) and past surgical procedures (p = 1.0). Four of nine peritoneal dialysates of anti-HCV positive patients were found positive for HCV RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme-Linked Immunosorbent Assay↗