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Biomedical subjects

M Roggendorf

Publications and source records attributed to M Roggendorf.

At least 91 records · Page 5Linked to original sources
PubMed1997

Determination of peripheral blood mononuclear cell responses to mitogens and woodchuck hepatitis virus core antigen in woodchucks by 5-bromo-2'-deoxyuridine or 2[3H]adenine incorporation.

Characterization of cellular immune response to antigens of woodchuck hepatitis virus (WHV) can contribute to the understanding of acute resolving and chronic outcome of hepadnavirus infection. Studies were limited because peripheral blood mononuclear cells (PBMCs) of woodchucks failed to incorporate [3H]thymidine sufficiently. Therefore, we established a non-radioactive proliferation assay for woodchuck PBMCs using 5-bromo-2'-deoxyuridine (BrdU) as thymidine analogue. Mitogen- and WHV core protein-(WHcAg) induced PBMC proliferation was detected by BrdU incorporation and compared to an assay using 2[3H]adenine. After stimulation with concanavalin A (ConA) and phytohaemagglutinin (PHA) we observed significant PBMC proliferation with both assays. Mitogen-induced nucleoside uptake of PBMCs into cellular DNA was confirmed by detection of 1',2'[3H]BrdU and 2[3H]adenine in extracted DNA. PBMCs obtained during the acute phase of WHV infection could be stimulated by WHcAg, whereas no WHcAg-induced proliferation of PBMCs was found in WHV-negative animals. PBMCs of chronic WHV carriers showed only a weak response to WHcAg. The established assays will be useful in determining the kinetics of cellular immune responses to different WHV antigens in the course of WHV infection and may provide an insight into mechanisms responsible for chronic outcome of hepadnavirus infection.

Adenine↗
PubMed1997

Characterization of antibody response to hepatitis C virus protein E2 and significance of hypervariable region 1-specific antibodies in viral neutralization.

Antibodies directed against hypervariable region 1 (HVR1) within the viral glycoprotein E2 of hepatitis C virus (HCV) are postulated to neutralize virus. An in vitro infection/binding assay of human fibroblast cells was established in order to study neutralization of HCV. Occurrence of mutations in the nucleotide sequence of HVR1 as compared to the inoculum after infection of human fibroblasts suggested replication of HCV in these cells. The significance of HVR1-specific antibodies in sera of patients who were infected in a single-source outbreak by an HCV contaminated anti-D immunoglobulin (IgG) preparation was studied. Using immunoprecipitation and ELISA, HVR1-specific antibodies could be detected in most of the sera obtained early (< or = 1 year p.i.) and late (up to 14 years p.i.) in single patients. Further characterization of the HVR1-specific antibodies in patient sera by attachment studies of HCV to the human fibroblasts suggested that HVR1-specific antibodies in sera obtained early p.i. can neutralize virus of the anti-D IgG preparation.

Amino Acid Sequence↗
PubMed1997

Different susceptibilities of genetic variants of hepatitis C virus (HCV) to interferon (IFN).

Genetic variants of HCV may have different degrees of resistance to IFN and may therefore influence the outcome of IFN therapy. However, selection of HCV variants by IFN has not been investigated in detail. In this paper, heteroduplex analysis was used to monitor major changes of HCV populations in 4 chronically infected patients under IFN therapy. We found that a major variant of the HCV 5' non-coding region (5' NCR) emerged in a responder. In other patients although no new variant of the 5' NCR was identified, significant changes occurred within the core and E1 region of the HCV genome. Disappearance and emergence of HCV variants may reflect their different susceptibilities to IFN. Our results indicate that responses of HCV populations to IFN are complex and need to be characterized by analysis of multiple HCV genome regions.

Antiviral Agents↗
PubMed1997

Therapeutic vaccination of woodchucks against chronic woodchuck hepatitis virus infection.

BACKGROUND/AIMS: Therapeutic vaccination is a new approach to treat patients with chronic hepatitis B virus infection. We have used the woodchuck model to examine the efficacy and safety of this approach. METHODS: Seven woodchucks chronically infected with woodchuck hepatitis virus were immunized with surface antigen from this virus, purified from plasma, in conjunction with a peptide named FIS (encompassing amino acids 106-118: FISEAIIHVLHSR from sperm whale myoglobin), which is recognized by T helper lymphocytes. As controls, two woodchucks chronically infected with woodchuck hepatitis virus were immunized: one with FIS only and the other with surface antigen only. RESULTS: Co-immunization with surface antigen and FIS, but not with FIS or surface antigen alone, induced anti-surface antibodies in 7/7 immunized woodchucks. In the two woodchucks in which the highest titer of anti-surface antibody was elicited, severe liver damage was observed: one died of fulminant hepatitis and the other became seriously ill with hepatic injury and had to be sacrificed. CONCLUSIONS: Co-immunization of chronically infected woodchucks with surface antigen and a peptide recognized by T helper cells produces a good anti-surface antibody response. However, this strategy needs to be optimized before its implementation in humans. Although our experiments are not strictly comparable to vaccination of chronically hepatitis B virus-infected patients with recombinant or plasma-derived vaccines, we believe that precautions should be taken to avoid the risk of severe liver injury when immunizing hepatitis B virus carriers.

Amino Acid Sequence↗
PubMed1997

Transmission of GBV-C/HGV from drug-addicted mothers to their babies.

BACKGROUND/AIMS: In 1995, a new flavivirus, GBV-C/HGV was identified. Little information is available on the clinical manifestations and epidemiology of GBV-C/HGV infection. We investigated the risk of mother-to-infant transmission in a group of GBV-C/HGV RNA positive women and followed up the GBV-C/HGV infected babies. METHODS: Twenty-eight anti-HCV positive women, of whom 25 have been intravenous drug users, and their children were included in the study. RNA was extracted from serum, reverse transcribed and amplified with primers from the NS5 region of GBV-C/HGV and 5'-UTR of HCV in a nested polymerase chain reaction. Amplified DNA fragments were gel purified and sequenced; the sequences obtained were subjected to a phylogenetic analysis. RESULTS: Transmission of GBV-C/HGV occurred in 10 (56%) of 18 infants born to GBV-C/HGV positive mothers; all these women were drug abusers. Only one (5%) of 19 babies whose mothers were HCV RNA positive by polymerase chain reaction, was infected with HCV during the follow up. High sequence homology in the NS5 region of GBV-C/HGV isolates in 10 mother-child pairs suggested mother-to-infant transmission. All 10 babies remained GBV-C/HGV RNA positive during follow up (2-12 months). None of the GBV-C/HGV infected infants became icteric or demonstrated any clinical or biochemical signs of liver disease. CONCLUSIONS: Mother-to-infant transmission of GBV-C/HGV may be high, at least in HCV-infected, drug-addicted women. In GBV-C/HGV RNA positive infants the rate of GBV-C/HGV persistent infection is high, but the infection is not accompanied by any symptoms of liver disease.

Base Sequence↗
PubMed1997

GBV-C/HGV infection in renal dialysis and transplant patients.

BACKGROUND: Recently a new human virus (GBV-C/HGV) was identified. With the use of the polymerase chain reaction (PCR) the possibility of a high prevalence of the GBV-C/HGV infection in haemodialysis patients was demonstrated. The aim of the present study was to use a combination of the PCR and a new diagnostic test for antibodies to the viral envelope protein E2 to assess the prevalence of the GBV-C/HGV infection in German patients with renal failure. METHODS: RT-PCR and ELISA were used to detect GBV-C/HGV RNA and antiviral antibodies (anti-E2) in the sera of 31 patients on a maintenance haemodialysis (HD)), 25 patients on a perioneal dyalisis (CAPD), and 92 renal transplant patients (RT). RESULTS: A statistical trend was noted for a higher rate of the GBV-C/HGV RNA in the whole group of patients with renal failure (11.5%) than in the control group of organ donors (5.5%); The difference between GBV-C/HGV RNA prevalence in RT patients (15.2%) and in organ donors (5.5%) was found to be significant. Anti-E2, which are considered as an indicator of a past GBV-C/HGV infection, were detected in 12.9% of HD patients, in 20.0% of CAPD patients, in 10.9% of RT patients, in 11.1% of organ donors, and in 10.9% of blood donors. These differences were not significant. Time on haemodialysis was significantly longer in GBV-C/HGV infected patients compared to uninfected patients and all patients with the GBV-C/HGV RNA have a history of blood transfusions. CONCLUSIONS: Patients with renal failure treated with dialysis or subjected to renal transplantation are at increased risk of aquiring the GBV-C/HGV infection. Higher rates of the GBV-C/HGV RNA and a similar prevalence of anti-E2 in patients with renal failure as compared to donors suggest that the rate of GBV-C/HGV infection resolution in immunosuppressed patients with renal failure might be lower than in immunocompetent patients.

Adult↗
PubMed1997

Cloning and characterization of a complete open reading frame of the hepatitis C virus genome in only two cDNA fragments.

The synthesis of long cDNA molecules encoding the complete genome of RNA viruses has recently been demonstrated; this major improvement has numerous practical applications such as construction of infectious cDNA clones or study of sequence variability at the level of a single RNA molecule. Using hepatitis C virus (HCV) as a model, we established an RT-PCR technique for amplification of cDNA fragments with a length of about 5 kb. The RT reaction was carried out with a Moloney murine leukaemia virus reverse transcriptase lacking detectable RNase H activity. For PCR reactions an enzyme mix containing Taq and Pwo DNA polymerases was used. Hot start and addition of 5% DMSO were also important to efficiently achieve long PCR products. About 10(6) HCV genome equivalents/ml in serum were needed in order to amplify the HCV genome in only two cDNA fragments covering about 98% of the complete genome. Analysis of the HCV quasi-species is also possible by this method as shown by sequencing of the hypervariable region 1 (HVR1) after cloning of cDNAs. The integrity of the long cDNA clones was proven by (1) restriction analyses, (2) partial sequencing and (3) expression of respective gene products. In vitro transcribed cDNAs were translated in rabbit reticulocyte lysate. Structural and nonstructural HCV proteins were identified by immunoprecipitation using patient serum. These results suggest that the two cDNA clones encode a complete and functional open reading frame of HCV.

Amino Acid Sequence↗
PubMed1997

Genetic heterogeneity of hepatitis G virus isolates from different parts of the world.

Comparative sequence analysis of a 354 nt fragment of the NS5 region of hepatitis G virus (HGV) isolates was performed to assess two levels of HGV genomic variability: (1) heterogeneity of HGV within an infected individual, and (2) heterogeneity of different HGV isolates. Comparison of nucleotide sequences of DNA clones from two virus isolates demonstrated that in each infected individual HGV is represented by a population of virions with closely related but heterogeneous genomes (quasi-species). Phylogenetic analysis of nucleotide sequences of 42 isolates collected from 14 countries revealed less significant genome variability of HGV as compared to hepatitis C virus. Sequences of all HGV isolates fell into one group of distribution of evolutionary distances. On a phylogenetic tree all HGV sequences segregated into numerous branches. All sequences of isolates from Africa, South and South-East Asia, however, were clustered together and were separated from those of other isolates collected in Europe, North America and Central Asia.

Base Sequence↗
PubMed1997

GB virus C infection in patients who underwent liver transplantation.

To elucidate the impact of an infection with the recently discovered GB virus C (GBV-C) on the clinical course after orthotopic liver transplantation (OLT), we studied eight patients who were GBV-C RNA positive after transplantation. Five individuals had been viraemic before transplantation, three became GBV-C RNA positive thereafter. A control group comprised eight patients without pre- or post-transplant GBV-C infection. GBV-C RNA was detected by reverse-transcription followed by nested polymerase-chain-reaction (PCR) with primers corresponding to the NS5 genome region. Nested PCR products were sequenced directly. The five patients infected with GBV-C before transplantation remained GBV-C RNA positive throughout the time of observation. Pre- and post-transplant GBV-C RNA titres were almost identical. Phylogenetic analysis revealed a very close relationship between the pre- and post-transplant viral nucleotide sequences indicating persistent GBV-C infection. No signs of hepatitis could be detected after transplantation in all GBV-C infected patients. However, four out of eight GBV-C RNA positive patients had a clinical course complicated by severe cholestasis, which was not observed in the control group. Although GBV-C infection does not lead to an increase in the rate of post-transplant hepatitis, it might be associated with severe unexplained cholestatic courses after OLT.

Adult↗
PubMed1997

Characterization of T-cell response to woodchuck hepatitis virus core protein and protection of woodchucks from infection by immunization with peptides containing a T-cell epitope.

Specific activation of T cells appears to be a prerequisite for viral clearance during hepatitis B virus (HBV) infection. The T-cell response to HBV core protein is essential in determining an acute or chronic outcome of HBV infection, but how this immune response contributes to the course of infection remains unclear. This is due to results obtained from humans, which are restricted to phenomenological observations occurring during the clinical onset after HBV infection. Thus, a useful animal model is needed. Characterization of the T-cell response to the core protein (WHcAg) of woodchuck hepatitis virus (WHV) in woodchucks contributes to the understanding of these mechanisms. Therefore, we investigated the response of woodchuck peripheral blood mononuclear cells (PBMCs) to WHcAg and WHcAg-derived peptides, using our 5-bromo-2'-deoxyuridine assay. We demonstrated WHcAg-specific proliferation of PBMCs and nylon wool-nonadherent cells from acutely WHV-infected woodchucks. Using a cross-reacting anti-human T-cell (CD3) antiserum, we identified nonadherent cells as woodchuck T cells. T-cell epitope mapping with overlapping peptides, covering the entire WHcAg, revealed T-cell responses of acutely WHV-infected woodchucks to peptide1-20, peptide100-119, and peptide112-131. Detailed epitope analysis in the WHcAg region from amino acids 97 to 140 showed that T cells especially recognized peptide97-110. Establishment of polyclonal T-cell lines with WHcAg or peptide97-110 revealed reciprocal stimulation by peptide97-110 or WHcAg, respectively. We vaccinated woodchucks with peptide97-110 or WHcAg to prove the importance of this immunodominant T-cell epitope. All woodchucks immunized with peptide97-110 or WHcAg were protected. Our results show that the cellular immune response to WHcAg or to one T-cell epitope protects woodchucks from WHV infection.

Animals↗
PubMed1997

Epitope mapping of antibodies directed against hypervariable region 1 in acute self-limiting and chronic infections due to hepatitis C virus.

Epitopes of hypervariable region 1 (HVR1) were mapped by enzyme-linked immunosorbent assay using follow-up sera of patients, all of whom were infected with the same isolate of hepatitis C virus (HCV). Our results suggest that (i) an early appearance (up to month 13 postinfection) of antibodies directed to the N terminus of HVR1 is associated with acute self-limiting infections of HCV and (ii) isolate-independent antibodies which are mainly directed to the C terminus of HVR1 seem to persist in chronically infected patients. The relevance of HVR1-specific antibodies for neutralization was evaluated by characterization of a rabbit serum.

Acute Disease↗
PubMed1997

Hepatitis C virus genotypes in different regions of the former Soviet Union (Russia, Belarus, Moldova, and Uzbekistan).

The prevalence of HCV genotypes in four republics of the former Soviet Union (Russia, Belarus, Moldova, and Uzbekistan) was investigated. Overall, 197 HCV isolates from 66 blood donors and 131 patients with chronic hepatitis were typed. Viral sequences from sera of infected subjects were amplified by nested RT-PCR using primers from the core region and typed by one or two techniques: (1) DNA enzyme immunoassay (DEIA) and (2) PCR with a set of type-specific primers. Only three major HCV genotypes were identified in this study population. HCV 1b was found to be the predominant virus type both among blood donors and chronic hepatitis patients, followed by 3a, 2a, and 1a (chronic hepatitis patients: 1b-82%; 3a-10%; 2a-4%, 1a-5% and 2c-1%; blood donors: 1b-77%; 3a-17%; and 2a-6%). No significant difference in genotype distribution was observed between different countries or between blood donors and chronic hepatitis patients within the same country. Results of the genotyping procedures were confirmed by direct sequencing of 216 nt PCR fragments corresponding to part of HCV core gene. Phylogenetic analysis of HCV 1b sequences from this study and from the Genbank demonstrated that the sequences from the former Soviet Union do not form evolutionary lineage(s) different from those of strains of the same subtype circulating in other geographical regions.

Adult↗
PubMed1996

Assessment of peripheral blood mononuclear cell proliferation by [2-3H]adenine uptake in the woodchuck model.

The levels of incorporated exogenous [3H]thymidine of peripheral blood mononuclear cells (PBMC) of the woodchuck were low after stimulation with mitogens concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM) when compared with other cell systems. The use of EDTA as an anticoagulant for blood sampling and AIM-V medium for culturing of PBMC improved the [3H]thymidine uptake of PBMC. A pronounced uptake is observed after use of [3H]adenine instead of [3H]thymidine for PBMC proliferation measurement. One likely explanation for the difference in [3H]adenine versus [3H]thymidine uptake is that the alternative pathway for thymidine monophosphate synthesis is important: the conversion of uridine to uridine monophosphate and, thereafter, to thymidine monophosphate. The optimal conditions for mitogen-induced proliferation of PBMC of the woodchuck were 2 micrograms/ml ConA and PHA at day 4 and 0.14 micrograms of PWM/ml at day 5. No consistent differences of [3H]adenine uptake were observed between PBMC from four woodchuck hepatitis virus-infected woodchucks and five uninfected animals.

Adenine↗
PubMed1996

Genotypes of hepatitis C virus isolates from different parts of the world.

Hepatitis C virus (HCV) causes most cases of posttransfusion non-A, non-B hepatitis. HCV isolates were classified by their genetic relatedness into at least six genotypes and a series of subtypes. Methods for typing included amplification of certain genomic regions using universal or type/subtype specific primers, restriction fragment length polymorphism analysis, differential hybridization, nucleotide sequencing, and serologic genotyping. HCV genotypes and their subtypes coexist in various geographic locations but show different prevalences. The identification of genotypes/subtypes is useful for studies on the molecular epidemiology and pathogenesis of HCV infection.

DNA Primers↗
PubMed1996

Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA.

BACKGROUND/AIMS: In the past few years the detection of HCV-RNA by polymerase chain reaction has become a well-established diagnostic tool for patients with chronic hepatitis C. However, the lack of reproducible results between laboratories and the relatively high proportion of false-positive results, has indicated the need for a standardized and reliable polymerase chain reaction assay. In the present study we have analyzed the performance of a commercial, HCV-RNA polymerase chain reaction assay based on a single, combined reverse transcription and amplification reaction and on the use of Uracil-N-glycosilase to prevent carry-over contamination (Amplicor HCV, Roche Molecular Systems). METHODS: In this assay the amplification products are detected in microwell plates using biotinylated primer and the HRP avidin colorimetric system. Serum samples collected from 446 patients, including 181 with chronic active hepatitis C, 50 with autoimmune chronic hepatitis, 117 in hemodialysis, 30 asymptomatic carriers of anti-HCV and 68 with indeterminate serology (RIBA indeterminate results), as well as from 121 controls were tested with the commercial, single-step assay and with nested polymerase chain reaction. Both techniques use primers located within the 5' non-coding region of the HCV genome. RESULTS: In all cases a good concordance was observed between the commercial, single-step assay and nested polymerase chain reaction which, for patients with chronic active hepatitis, showed a sensitivity and specificity of 100% and 99.3% for the former and of 98.8% and 100% for the latter, when compared to clinical diagnosis taken as the gold standard. Most of the 11 discordant samples were seen in the group of RIBA-indeterminate cases and in patients with chronic active hepatitis C. Further analysis of these cases, based on repeat testing and clinical data showed that 64% and 36% of the discrepancies were due, respectively, to nested polymerase chain reaction and Amplicor inconsistent reactions. In hemodialyzed patients, patients with autoimmune hepatitis and asymptomatic carriers of anti-HCV, both assays produced results which were consistent with the clinical diagnosis. In the former group, polymerase chain reaction was able to identify the presence of active viral replication in some antibody negative samples. CONCLUSIONS: Taken together, these results indicate that the commercial, single-step polymerase chain reaction assay has the same clinical sensitivity and specificity as nested polymerase chain reaction and that, because of its simplified procedures and fast turn-around time, it may be a valuable test for routine diagnostic applications.

Hepacivirus↗
PubMed1996

Rational use of diagnostic tools in hepatitis C.

Six years after the introduction of serological tests for the detection of antibodies to HCV these diagnostic procedures have been improved significantly. The ELISA's of the third generation contain 4-5 fragments of structural and nonstructural proteins which have increased the sensitivity and specificity for antibody detection. Several confirmatory assays have been established to detect non-specific reactions. Viremia in acute and chronic HCV infection can be detected by RT-PCR. However, studies on the quality of PCR results with serum panels in a large number of diagnostic labs indicate the urgent need to standardize the RT-PCR techniques. Quantification of viral genomes during acute and chronic HCV infection seem to be of major importance to predict the response rat to IFN. Quantitative RT-PCR techniques using internal standards and the branched DNA technology have been established. Techniques for genotyping HCV isolates have been introduced which also seemed to be important for prediction of IFN response. Recently serotyping of HCV isolates has been evaluated and seems to correlate well with genotyping by RT-PCI and specific probes. The detection of quasispecies in the 5'NCR may, in the future, be an additional prospective test for the prognosis of IFN treatment.

Enzyme-Linked Immunosorbent Assay↗
PubMed1995

Transmission of hepatitis C virus to children and husbands by women infected with contaminated anti-D immunoglobulin.

In 1978-79 a single-source outbreak of hepatitis C occurred in 2533 women who had received virus-contaminated anti-D immunoglobulin. Children and husbands of 74 women with self-limited, and of 86 women with chronic, hepatitis C were followed up for over 10-15 years. In 3 of 231 investigated children (1.3%) serological evidence for HCV infection was found. However, none of the children developed an apparent or chronic hepatitis. Serum samples of the 94 husbands investigated showed no HCV antibodies or HCV RNA. We consider the risk of intrauterine or perinatal transmission of HCV, as well as that of transmission through close family contacts, to be low. No evidence was found for sexual transmission for women to men.

Antibodies, Anti-Idiotypic↗