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Biomedical subjects

M Roggendorf

Publications and source records attributed to M Roggendorf.

At least 19 recordsLinked to original sources
PubMed2000

Detection of germ-cell tumor cells in the peripheral blood by nested reverse transcription-polymerase chain reaction for alpha-fetoprotein-messenger RNA and beta human chorionic gonadotropin-messenger RNA.

By establishing sensitive nested reverse transcription-PCRs for the detection of mRNA of alpha-fetoprotein (AFP) and beta human chorionic gonadotropin (betahCG), we investigated the presence of circulating tumor cells in the peripheral blood of 119 patients with germ-cell tumor. A total of 336 blood samples obtained before and during therapy were examined with regard to clinical applicability. The overall ratio of positive PCR results was 26.5% and was independent of the serum concentration of AFP and hCG/betahCG. No correlation of the positivity for AFP-mRNA to serum AFP level was found. In contrast, positive results in betahCG-PCR were twice as frequent in patients with elevated serum hCG/betahCG levels as in those with normal serum hCG/betahCG levels (P = 0.012). To develop a valid correlation to tumor stage, tumor histology, and serum level of tumor markers, a subgroup of 36 patients was evaluated before definite therapy. The subgroup revealed an overall ratio of 33.3% positive PCR results. The serum level of both of the markers did not correlate with the detection of corresponding mRNA in peripheral blood samples. However, positive betahCG-PCR results were found exclusively in patients with elevated serum hCG/betahCG (6 of 18 versus 0 of 18; P = 0.019). Patients with stage IIC/III germ-cell tumor demonstrated nearly twice the frequency of positive PCR results as patients with stage I tumor [7 (41.2%) of 17 versus 4 (23.5%) of 17] in this subgroup. With regard to histology, positive PCR results were found mostly in embryonal carcinoma.

Adolescent↗
PubMed2000

Expression and purification of woodchuck tumour necrosis factor alpha.

The production of recombinant woodchuck cytokines is an essential prerequisite to study the immune response to hepadnavirus infection in the woodchuck model. Woodchuck tumour necrosis factor-alpha (TNF-alpha) was expressed in mammalian cells and in Escherichia coli. A test system for the biological activity of woodchuck TNF-alpha was established on basis of its cytotoxic effect to the murine fibrosarcoma cell line L929. Recombinant TNF-alpha was purified and used for the production of neutralizing antisera.

Animals↗
PubMed2000

Molecular cloning and characterization of major histocompatibility complex class I cDNAs from woodchuck (Marmota monax).

The full-length cDNAs of woodchuck major histocompatibility complex (MHC) class I (MhcMamo-I or Mamo-I) genes were cloned by using cellular mRNA isolated from the peripheral blood mononuclear cells and liver tissues of woodchucks. DNA sequence analysis of Mamo-I cDNAs revealed that the coding regions of Mamo-I genes were about 1,080 bp long, encoding 359 amino acid residues. The deduced amino acid sequences of Mamo-I showed structural features like leader, alpha1, alpha2, alpha3, transmembrane and cytoplasmic domains, similar to their homologues in human and other mammals. Analysis of five full-length clones from unrelated woodchucks indicated a polymorphism within the alpha1 and alpha2 domains of Mamo-I heavy chain and a high conservation within the alpha3 and the transmembrane/cytoplasmic domains. Amino acid residues of the alpha2 and alpha3 domains that are supposed to be involved in the binding of MHC class I to CD8 molecule, were largely conserved in Mamo-I genes. Phylogenetic comparison of MHC class I genes of woodchuck and other mammals indicated a close evolutionary relationship between woodchuck and squirrel MHC class I. We tentatively named this region the locus A of Mamo-I genes (Mamo-A). Sequence analysis of 101 clones of alpha1 and alpha2 regions derived from 14 woodchucks revealed that at least 14 different alleles within Mamo-A exist. Among these 14 alleles identified so far, Mamo-A*01 and Mamo-A*09 were of the highest frequency of about 21.5% and 14.5%, respectively. Our results indicate that Mamo-I genes are of a similar molecular structure to those of human and other mammals.

Alleles↗
PubMed2000

Reduced risk of persisting cytomegalovirus pp65 antigenemia and cytomegalovirus interstitial pneumonia following allogeneic PBSCT.

In order to evaluate the risk of cytomegalovirus (CMV) associated disease after allogeneic stem cell transplantation (SCT), 158 consecutive patients at risk for infection were analyzed. BMT was performed in 101 patients and peripheral blood stem cell transplantation (PBSCT) in 57 patients. CMV antigenemia was found in 57 cases (56%) after BMT and 27 cases (47%) after PBSCT, respectively. CMV antigenemia resistant to a 14-day course of GCV was found in 26 patients (26%) after BMT but in only four patients (7%) after PBSCT (P < 0.01). Eighteen patients (11%) developed CMV disease, 14 post BMT and four post PBSCT. Lethal CMV-related interstitial pneumonia (CMV-IP) occurred in 13 cases of whom 12 patients were bone marrow recipients (P = 0.04). The subgroup of seronegative patients with a CMV seropositive donor had a significantly lower risk of developing CMV antigenemia, GCV-resistant CMV antigenemia (P < 0.01) and CMV-related disease (P = 0.01). In conclusion, the incidence of persistent CMV antigenemia and CMV-IP was significantly reduced when allogeneic transplantation was performed with peripheral blood stem cells instead of bone marrow. These findings suggest that our previous in vitro data on improved immune reconstitution after allogeneic PBSCT as compared to allogeneic BMT have clinical relevance.

Adolescent↗
PubMed2000

Sequence heterogeneity of TT virus and closely related viruses.

TT virus (TTV) is a recently discovered infectious agent originally obtained from transfusion-related hepatitis. However, the causative link between the TTV infection and liver disease remains uncertain. Recent studies demonstrated that genome sequences of different TTV strains are significantly divergent. To assess genetic heterogeneity of the TTV genome in more detail, a sequence analysis of PCR fragments (271 bp) amplified from open reading frame 1 (ORF1) was performed. PCR fragments were amplified from 5 to 40% of serum specimens obtained from patients with different forms of hepatitis who reside in different countries (e.g., China, Egypt, Vietnam, and the United States) and from normal human specimens obtained from U.S. residents. A total of 170 PCR fragments were sequenced and compared to sequences derived from the corresponding TTV genome region deposited in GenBank. Genotypes 2 and 3 were found to be significantly more genetically related than any other TTV genotype. Moreover, three sequences were shown to be almost equally related to both genotypes 2 and 3. These observations suggest a merger of genotypes 2 and 3 into one genotype, 2/3. Additionally, five new groups of TTV sequences were identified. One group represents a new genotype, whereas the other four groups were shown to be more evolutionary distant from all known TTV sequences. The evolutionary distances between these four groups were also shown to be greater than between TTV genotypes. The phylogenetic analysis suggested that these four new genetic groups represent closely related yet different viral species. Thus, TTV exists as a "swarm" of at least five closely related but different viruses. These observations suggest a high degree of genetic complexity within the TTV population. The finding of the additional TTV-related species should be taken into consideration when the association between TTV infections and human diseases of unknown etiology is studied.

Base Sequence↗
PubMed2000

Changes in the epidemiology of hepatitis C infection in Germany: shift in the predominance of hepatitis C subtypes.

Hepatitis C virus (HCV) subtype distribution was studied in 395 chronically infected patients from Germany. HCV genotype 1 was most frequent (80.5%). One hundred forty-three individuals (36.2%) were infected with subtype 1a and 175 (44.3%) were suffering from subtype 1b infection, respectively. HCV subtype 3a was found in 53 (13.42%) persons. Subtypes 2a, 2b, and 2c have been detected in 5 (1.27%), 10 (2.53%), and 4 (1.01%) individuals. Genotypes 4 and 5a accounted for HCV infections in 4 (1.01%) and 1 (0.25%) subjects. There was a notable variation in the distribution of the prevalent subtypes 1a and 1b in different age groups. Subtype 1a was detected in 53.3% and 68.0% of patients aged 1-10 and 11-20 years, whereas subtype 1b in the same groups was present only in 33.3% and 28.0% of patients, respectively. In contrast, in individuals older than 50 years subtype 1b was most frequent. Thus, subtype 1b has been gradually substituted for subtype 1a during the last 20 years. Logistic regression analysis with adjustment for sex and different modes of HCV acquisition demonstrated that age of the infected subjects was a direct explanatory variable for subtype 1a and 1b distribution. Therefore, the observed shift in HCV subtype prevalence could not be attributed to changes in the epidemiological relevance of different known risk factors of HCV transmission, as had been assumed in previous studies. The altered subtype pattern reported here may have a profound influence on the future epidemiology of HCV infection.

Adolescent↗
PubMed1999

Inquiries on intraspousal transmission of hepatitis C virus: benefits and limitations of genome sequencing and phylogenetic analysis.

During recent years, courts had to decide in some notable cases whether or not defendants infected the plaintiffs with HIV. Lawsuits on the transmission of other viral pathogens up to now have hardly become known. We report here on the use of genome sequencing and phylogenetic analysis to investigate possible intraspousal transmission of hepatitis C virus (HCV). The high degree of genetic relatedness observed in phylogenetic analysis among the HCV strains isolated from the couple demonstrates that the man and the woman are infected with the same isolate of the virus. After other plausible routes of infection have been virtually excluded by anamnestic and conventional epidemiological evaluation we could infer that the man most probably has infected his girlfriend with HCV. This conclusion was further supported by the finding that both are also infected with closely related isolates of GB virus C (GBV-C). Thus, the results from molecular biological investigations and epidemiological evaluation are complementary pieces of evidence in inquiries on possible intraspousal transmission of HCV.

Adult↗
PubMed1999

Selection of genetic variants of the 5' noncoding region of hepatitis C virus occurs only in patients responding to interferon alpha therapy.

Interferon alpha (IFN alpha) can suppress the replication of hepatitis C virus (HCV) in chronically infected patients. However, HCV persists in a significant number of patients despite the normalization of alanine transaminase (ALT) during IFN alpha therapy. In this study, HCV variants in patients under IFN alpha therapy were characterized to examine their role in viral persistence during the therapy. Sixteen patients selected for this study were infected with HCV genotype 1b and remained HCV RNA positive for at least 1 month after onset of therapy. Nine patients responded to the therapy in terms of normalization of ALT (responders), whereas seven patients did not show a significant decrease of ALT level (nonresponders). To examine HCV populations in these patients, the HCV 5' noncoding region (5' NCR) was analyzed by polymerase chain reaction amplification and sequencing. Newly emerging variants of the HCV 5' NCR replaced predominant variants present prior to IFN alpha therapy in six of nine responders. Most predominant HCV variants during IFN alpha therapy carried a nucleotide substitution G to A at nt 231 within the 5' NCR. An analysis of the HCV quasispecies population in one responder revealed that a preexisting variant became predominant under IFN alpha therapy. These results emphasized the importance of the genetic heterogeneity of the HCV genome for viral resistance to IFN alpha. Five of seven HCV isolates from nonresponders were identical to those found in responders with regard to the nucleotide sequence of the 5' NCR. However, no selection of variants of the HCV 5' NCR occurred in nonresponders during the course of therapy. We conclude that IFN alpha treatment leads to the selection of variants of the HCV 5' NCR only in responders and may act differently in nonresponders. Our results suggest that the HCV 5' NCR may be a target of anti-HCV actions of IFN alpha.

5' Untranslated Regions↗
PubMed1999

Immunodominant B-cell domains of hepatitis C virus envelope proteins E1 and E2 identified during early and late time points of infection.

BACKGROUND/AIMS: We characterized immunoreactive B-cell domains of hepatitis C virus (HCV) envelope proteins E1 and E2 by a peptide ELISA using sera of patients who were infected by the same isolate of HCV (HCV-AD78). METHODS: Fifty-four overlapping peptides which corresponded to the sequence of E1 and E2 of isolate HCV-AD78 were used to detect specific antibodies. Three groups of HCV-AD78 related sera were analyzed. Two groups were from sera obtained at early time points of infection (months 4-15) from patients who later resolved infection (group A), or who later developed chronic disease (group B). Group C sera were from later time points of chronic disease. As a control, sera of chronic HCV patients who did not have HCV-AD78 infection were also analyzed (group D). RESULTS: In group A, 25 of the 54 peptides produced OD405 above the cut-off, whereas 17 peptides produced such values in group B. Only 10 and 3 peptides yielded such values in groups C and D, respectively. The overall prevalence of antibodies against peptides was high in the early phase of infection (means of 28.7+/-14.8% and 25.9+/-14.5% in groups A and B, respectively). At later time points of chronic infection (group C), the overall prevalence was lower (mean 18.6+/-15.4%). Group D sera produced the lowest overall prevalence (mean 13.2+/-14.1%). Three peptides, covering aa271-290, aa481-500 and aa551-570, were recognized significantly more frequently (p<0.05) by group A sera than group B sera. CONCLUSIONS: We conclude that more linear epitopes of the HCV envelope are recognized with a high prevalence of antibodies, as was suggested previously. However, most B-cell domains of the HCV envelope induce a similarly high antibody response in patients who resolve infection or develop chronic disease.

Adult↗
PubMed1999

Detection of TT virus DNA in specimens other than blood.

BACKGROUND: Recently, a new infective agent of humans, TT virus (TTV), was identified. Very high prevalence rates of TTV in different population groups, including apparently healthy individuals, were reported for several countries. OBJECTIVES, STUDY DESIGN: In order to investigate whether or not non-parenteral transmission routes can contribute to TTV spread, we have tested saliva, urine, and stool samples from eight TT viraemic individuals for the presence of TTV DNA by polymerase chain reaction. RESULTS: TTT DNA was detected in saliva of five subjects and stools of four patients. None of the urine samples contained TTV DNA. Viral titres in saliva were close to those found in serum. In feces, TTV DNA could only be detected in low concentrations. CONCLUSIONS: Our findings on the presence of TTV DNA in saliva and stool suggest that TTV might be transmitted non-parenterally.

DNA Virus Infections↗
PubMed1999

Immunization of woodchucks with plasmids expressing woodchuck hepatitis virus (WHV) core antigen and surface antigen suppresses WHV infection.

DNA vaccination can induce humoral and cellular immune response to viral antigens and confer protection to virus infection. In woodchucks, we tested the protective efficacy of immune response to woodchuck hepatitis core antigen (WHcAg) and surface antigen (WHsAg) of woodchuck hepatitis virus (WHV) elicited by DNA-based vaccination. Plasmids pWHcIm and pWHsIm containing WHV c- or pre-s2/s genes expressed WHcAg and WHsAg in transient transfection assays. Pilot experiments in mice revealed that a single intramuscular injection of 100 microgram of plasmid pWHcIm DNA induced an anti-WHcAg titer over 1:300 that was enhanced by boost injections. However, two injections of 100 microgram of pWHcIm did not induce detectable anti-WHcAg in woodchucks. With an increase in the dose to 1 mg of pWHcIm per injection, transient anti-WHcAg response and WHcAg-specific proliferation of peripheral mononuclear blood cells (PMBCs) appeared in woodchucks after repeated immunizations. Four woodchucks vaccinated with pWHcIm were challenged with 10(4) or 10(5) of the WHV 50% infective dose. They remained negative for markers of WHV replication (WHV DNA and WHsAg) in peripheral blood and developed anti-WHs in week 5 after challenge. In contrast, woodchucks not immunized or immunized with the control vector pcDNA3 developed acute WHV infection. Two woodchucks immunized with 1 mg of pWHsIm developed WHsAg-specific proliferative response of PBMCs but no measurable anti-WHsAg response. A rapid anti-WHsAg response developed during week 2 after virus challenge. Neither woodchuck developed any signs of WHV infection. These data indicate that DNA-based vaccination with WHcAg and WHsAg can elicit immunity to WHV infection.

Amino Acid Sequence↗
PubMed1999

High prevalence of TT virus infection in Brazilian blood donors.

A recent report has described the molecular cloning and characterization of a novel, single-stranded DNA virus, named TT virus (TTV), which was present in the sera of Japanese patients with posttransfusion hepatitis of unknown etiology [Okamoto et al. (1998) Hepatology Research 10:1-16]. Using a nested polymerase chain reaction assay, sera from Brazilian patients with acute non A-C hepatitis and blood donors were examined for the presence of TTV DNA sequences. Thirty-seven of 52 (71%) patients with acute non A-C hepatitis and 45 of 72 (62%) blood donors were found to have TTV sequences in their sera. Such a high proportion in blood donors indicated that TTV infection is common in the general Brazilian population. Partial nucleotide sequences (326 bases in open reading frame 1) from seven isolates were determined. By phylogenetic analysis, four TTV strains were classified into the genomic subgroup G1a described previously. The three others belonged to subgroup G1b. Sequence homologies between strains belonging to a same subgroup were 92.9-99.1%, whereas homologies of 85.9-90.2% were calculated between isolates from different subgroups.

Adolescent↗
PubMed1998

Antibodies directed to envelope proteins of hepatitis C virus outside of hypervariable region 1.

The relatively high variability of the hepatitis C virus (HCV) envelope proteins E1 and E2 suggests that parts of these proteins other than the hypervariable region 1 (HVR1) might be involved in the induction of virus neutralizing antibodies. To test this hypothesis, two HCV proteins, pE1 and pE2 delta, were generated by in vitro translation. They represent amino acids 174-337 of E1 and 411-688 of E2, respectively, of isolate HCV-AD78; the protein pE2 delta contained no HVR1. As a control, protein pG.HVR1, which represents amino acids 384-410 of HVR1 of isolate HCV-AD78, was expressed separately. These three proteins were used in an immunoprecipitation assay to detect the presence of antiviral antibodies in sera of patients infected with the same isolate of HCV (HCV-AD78). Sera were obtained 4-8 months postinfection from patients who later resolved an acute infection or developed chronic liver disease. A high prevalence of antibodies (up to 85.7%) against pE1 and pE2 delta could be detected in both groups of patients, suggesting that these forms of the HCV envelope proteins contain B-cell epitopes. The antibody responses against proteins pE1 and pE2 delta did not differ significantly between patients with resolving or chronic infection, whereas antibodies against protein pG.HVR1 were associated with resolution of infection. Rabbit antisera raised against pE1 and pE2 delta were tested for their ability to neutralize the binding of HCV to susceptible cells in tissue cultures. The results suggested that although a few B-cell epitopes outside of HVR1 can induce virus neutralizing antibodies, these antibodies are probably not associated with the resolution of infection.

Animals↗