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Biomedical subjects

M Roggendorf

Publications and source records attributed to M Roggendorf.

At least 37 records · Page 2Linked to original sources
PubMed2001

Large nontransplanted hepatocellular carcinoma in woodchucks: treatment with adenovirus-mediated delivery of interleukin 12/B7.1 genes.

BACKGROUND: Cytokine-based gene therapy strategies efficiently stimulate immune responses against many established transplanted tumors, leading to rejection of the tumor. In this study, we investigated the therapeutic potential of cancer immunotherapy in a clinically more relevant model, woodchucks with primary hepatocellular carcinomas induced by woodchuck hepatitis virus. METHODS: Large (2-5 cm), established intrahepatic tumors were given an injection once with 1 x 10(9) plaque-forming units of AdIL-12/B7.1, an adenovirus vector carrying genes for murine interleukin 12 and B7.1, or of AdEGFP, the control virus, and regression of the tumors was then monitored. Five animals were used in total. RESULTS: In four tumor-bearing animals, the antitumor response was assessed by autopsy and histologic analysis within 1-2 weeks after treatment. In all animals treated with AdIL-12/B7.1 therapy versus AdEGFP therapy, we observed substantial tumor regression (P =.006; two-sided unpaired Student's t test) accompanied by a massive infiltration of T lymphocytes. These tumors also contained increased levels of CD4(+) and CD8(+) T cells and interferon gamma (IFN gamma). In continuously growing tumor nodules given an injection of the control virus or in nontumoral liver, no such effects (i.e., tumor regression and increased levels of CD4(+) and CD8(+) T cells and IFN gamma) were detected. In the fifth animal, monitored for long-term antitumor efficacy by magnetic resonance imaging (MRI) after intratumoral vector administration by MRI guidance, the tumor was almost completely eliminated (> or = 95%) 7 weeks after treatment. CONCLUSION: Adenovirus vector-based immunotherapy appears to be an effective treatment of large nontransplanted (orthotopic) tumors that acquire malignant characteristics in a stepwise process, reflecting the real-world scenario of hepatocellular carcinoma in humans.

Adenoviridae↗
PubMed2001

Thymidine utilization abnormality in proliferating lymphocytes and hepatocytes of the woodchuck.

Effective incorporation of tritiated thymidine ([(3)H]TdR) into proliferating lymphocytes is important because [(3)H]TdR is a standard label to study proliferate T-cell responses. We analyzed the thymidine utilization of woodchuck peripheral blood lymphocytes (PBL) since the [(3)H]TdR incorporation assay was not applicable to measure proliferative immune responses in the woodchuck, a current major virus/host model for human hepatitis B virus infection. Incorporation of [(3)H]TdR into DNA as well as the activity of the salvage pathway enzyme thymidine kinase (TK) of proliferating woodchuck PBL was low compared to human lymphocytes. Furthermore, [(3)H]TdR incorporation of proliferating woodchuck PBL remained residual regardless of the use of methotrexate, an inhibitor of the competitive deoxythymidine monophosphate de novo synthesis pathway. Using a human probe, specific for the proliferation-associated TK1, we proved the genomic presence and transcription of TK1 sequences in various species. TK1 sequences were detected in the genome of human, mouse, woodchuck, and chicken specimens. In contrast to proliferating human PBL and 3T3 mouse fibroblasts, no TK1 transcript was found in proliferating woodchuck PBL and hepatic cells. Transfection experiments with vectors containing the murine or human TK1 and selection assays demonstrated the ability of woodchuck cells to transcribe TK1 and to express functional TK1 proteins. Our study characterizes the unique failure of sufficient [(3)H]TdR incorporation into proliferating woodchuck cells and demonstrates tritiated adenine and serine as alternative labels to monitor PBL proliferation in the woodchuck.

3T3 Cells↗
PubMed2001

Targeting murine immune responses to selected T cell- or antibody-defined determinants of the hepatitis B surface antigen by plasmid DNA vaccines encoding chimeric antigen.

By exchanging sequences from the middle-surface (MS) and small-surface (S) Ag of hepatitis B virus (HBV) with corresponding sequences of the MS Ag of woodchuck hepatitis virus, we constructed chimeric MS variants. Using these constructs as DNA vaccines in mice, we selectively primed highly specific (non-cross-reactive) Ab responses to pre-S2 of the HBV MS Ag and the "a" determinant of the HBV S Ag, as well as L(d)- or K(b)-restricted CTL responses to HBV S epitopes. In transgenic mice that constitutively express large amounts of HBV surface Ag in the liver we could successfully suppress serum antigenemia (but not Ag production in the liver) by adoptive transfer of anti-pre-S2 or anti-"a" immunity but not CTL immunity. DNA vaccines greatly facilitate construction of chimeric fusion Ags that efficiently prime specific, high-affinity Ab and CTL responses. Such vaccines, in which sequences of an Ag of interest are exchanged between different but related viruses, are interesting tools for focusing humoral or cellular immunity on selected antigenic determinants and elucidating their biological role.

Animals↗
PubMed2001

Topical treatment of acute adenoviral keratoconjunctivitis with 0.2% cidofovir and 1% cyclosporine: a controlled clinical pilot study.

OBJECTIVE: To evaluate the efficacy of 0.2% cidofovir eyedrops and 1% cyclosporine eyedrops administered 4 times daily (qid) to treat acute adenoviral keratoconjunctivitis. METHODS: A randomized, controlled, double-masked study was conducted on 39 patients with acute adenoviral keratoconjunctivitis of recent onset. Patients were divided into 4 treatment groups: (1) cidofovir qid, (2) cyclosporine qid, (3) cidofovir + cyclosporine qid, and (4) sodium chloride qid (control). The diagnosis was confirmed using adenoviral polymerase chain reaction from conjunctival swabs. Duration of treatment was 21 days. MAIN OUTCOME MEASURES: Severity of conjunctival hyperemia, conjunctival chemosis, superficial punctate keratitis during treatment, and presence and severity of corneal subepithelial infiltrates were evaluated using a clinical score. Duration until subjective improvement of symptoms was recorded. RESULTS: Subjective improvement of local symptoms was accelerated in the cyclosporine group. All other clinically relevant variables showed no statistically significant difference among the 4 treatment groups. Particularly, we did not find a difference in the frequency of corneal subepithelial infiltrates at the end of treatment. CONCLUSIONS: Use of cidofovir, cyclosporine, or both did not accelerate the improvement of clinical symptoms of acute adenoviral keratoconjunctivitis compared with the natural course of the infection as demonstrated by this pilot study. This might be because of the wide spectrum of the clinical course of the infection, low sensitivity to cidofovir, too low of a concentration of cidofovir, or early cessation of viral replication in the course of the infection. The effect of a higher concentration of topical cidofovir with and without cyclosporine requires investigation in a larger group of patients.

Acute Disease↗
PubMed2001

Differences in HCV antibody patterns in haemodialysis patients infected with the same virus isolate.

Eight cases of de novo hepatitis C virus (HCV) infection in a haemodialysis unit in Rio de Janeiro, Brazil, were retrospectively studied. HCV viraemia was demonstrated by RT nested PCR in seven of the seroconverters. Genotyping showed that six patients were infected with a genotype 1b strain and one with a genotype 1a strain. A phylogenetic analysis of nucleotide sequences of the HCV core region revealed that five of the six 1b isolates form a separate cluster when compared with other 38 HCV 1b core sequences randomly chosen from the GenBank. The revealed sequence similarities indicated the nosocomial spread of a single HCV strain within the unit. To investigate whether the patients infected with the same viral isolate display similar patterns of antibody response to individual proteins, serial serum samples were examined. A line immunoassay for qualitative and semi-quantitative determination of specific antibodies against recombinant and synthetic HCV antigens was employed. Despite infection with the same virus strain, the patients sera demonstrated different patterns of reactivity against individual structural and nonstructural HCV proteins immediately after seroconversion. For each patient, however, antibody responses remained mostly stable throughout the follow-up of 8 to 24 months.

Genotype↗
PubMed2001

Performance characteristics of a transcription-mediated nucleic acid amplification assay for qualitative detection of hepatitis C virus RNA.

The detection of hepatitis C virus (HCV) RNA by nucleic acid amplification techniques is the method of choice to differentiate between ongoing and past infection, and can be used to monitor the course of HCV infection. In this study, we evaluated the performance characteristics of a newly developed transcription-mediated amplification (TMA)-based assay, the VERSANT HCV RNA qualitative assay, which was designed to qualitatively detect HCV RNA. Samples tested by the TMA assay included 100 HCV antibody negative sera; serial dilutions of an HCV genotype 1a panel; the WHO HCV RNA standard 76/790; an HCV genotyping panel; and 150 clinical specimens, including sera from patients who had received alpha interferon (IFN) treatment or liver transplants. TMA test results were compared with the Cobas Amplicor HCV polymerase chain reaction (PCR) assay. The analytical specificity of the HCV TMA assay was > 98%. No carry-over contaminations were observed. The assay demonstrated an analytical sensitivity of 100% at 41 HCV RNA copies/mL (genotype 1a panel) and 5 IU/mL (WHO standard), respectively. HCV genotypes and subtypes did not affect the results. Qualitative RNA detection by diagnostic Amplicor PCR and TMA was in agreement in > 97% of all 150 clinical samples tested. In our study, the TMA-based assay proved to be a specific and sensitive method for qualitative HCV RNA detection. The test may turn out to be an attractive alternative to already established techniques for HCV RNA amplification in routine clinical laboratories.

Enzyme-Linked Immunosorbent Assay↗
PubMed2001

Adenoviral infection after allogeneic stem cell transplantation (SCT): report on 130 patients from a single SCT unit involved in a prospective multi center surveillance study.

The incidence of adenovirus (AV) infections following SCT was determined in a prospective multicenter trial. Over 1 year, 130 consecutive patients undergoing allogeneic SCT at Essen University Hospital were included and followed for 6 months. Source of stem cells was blood in 68 cases. Fifty-eight patients had HLA-identical sibling donors. Throat swabs, urine and stool samples were screened weekly for AV antigen and DNA by ELISA and nested PCR, respectively. In 35 cases adenovirus infection was detected. There was no seasonal variation. Throat swabs were positive in 24, urine in 12, and stool in 11 cases, resulting in a cumulative risk of infection of 29%. The incidences of AV infection of the respiratory, gastrointestinal and urinary tract were 19%, 10%, and 9%, respectively, and infections were diagnosed after a median (range) interval of 44 (-2-179), 37 (-2-168), and 53 (17-153) days after transplantation. On multivariate analysis, presence of AV antibody in the donor and acute graft-versus-host disease grade IV were found to be independent risk factors for AV infection. Eleven patients had AV isolated from more than one site and five patients had probable AV disease. We were not able to identify patients in whom AV infection was the leading cause of death. The majority of patients infected with AV suffered from severe acute graft-versus-host disease often accompanied by other opportunistic infections, such as aspergillosis or CMV reactivation. Nineteen out of 36 patients who died during the observation period had AV infection. In summary, AV infection after allogeneic SCT was observed in a substantial number of patients. In addition to well-known risk factors for viral infection after SCT we were able to demonstrate that a positive AV antibody test in the donor is an important risk factor for AV infection. Further studies are needed, however, before final conclusions on the clinical sequelae of AV infection can be made and the role of preventive and therapeutic strategies toward AV infection after allogeneic SCT can be defined.

Actuarial Analysis↗
PubMed2001

Coadministration of gamma interferon with DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen enhances the specific immune response and protects against WHV infection.

DNA vaccinations are able to induce strong cellular immune responses in mice and confer protection against infectious agents. However, DNA vaccination of large animals appears to be less effective and requires repeated injections of large amounts of plasmid DNA. Enhancement of the efficiency of DNA vaccines may be achieved by coapplication of cytokine-expressing plasmids. Here we investigated, with woodchucks, whether coadministration of an expression plasmid for woodchuck gamma interferon (IFN-gamma), pWIFN-gamma, can improve DNA vaccination with woodchuck hepatitis virus core antigen (WHcAg). Animals were immunized with pWHcIm (a plasmid expressing WHcAg) alone or with a combination of pWHcIm and pWIFN-gamma using a gene gun. Six weeks postimmunization, all animals were challenged with 10(5) genome equivalents of woodchuck hepatitis virus (WHV). The antibody and lymphoproliferative immune responses to WHV proteins were determined after immunization and after challenge. Vaccination with pWHcIm and pWIFN-gamma led to a pronounced lymphoproliferative response to WHcAg and protected woodchucks against subsequent virus challenge. Two of three animals vaccinated with pWHcIm alone did not show a detectable lymphoproliferative response to WHcAg. A low-level WHV infection occurred in these woodchucks after challenge, as WHV DNA was detectable in the serum by PCR. None of the pWHcIm-vaccinated animals showed an anti-WHcAg antibody response after DNA vaccination or an anamnestic response after virus challenge. Our results indicate that coadministration of the WIFN-gamma gene with pWHcIm enhanced the specific cellular immune response and improved the protective efficacy of WHV-specific DNA vaccines.

Animals↗
PubMed2001

Replication of naturally occurring woodchuck hepatitis virus deletion mutants in primary hepatocyte cultures and after transmission to naive woodchucks.

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.

Animals↗
PubMed2001

Prevalence of TTV DNA and GBV-C RNA in patients with systemic sclerosis, rheumatoid arthritis, and osteoarthritis does not differ from that in healthy blood donors.

OBJECTIVE: To determine the prevalence of GB virus-C (GBV-C) RNA and TT virus (TTV) DNA in patients with systemic sclerosis (SSc), rheumatoid arthritis (RA), and osteoarthritis (OA) as well as to compare the autoantibody pattern in patients with SSc with and without evidence of viral infection. PATIENTS AND METHODS: The study included 168 patients (84 SSc, 41 RA, and 43 OA) diagnosed according to the American College of Rheumatology criteria and 122 volunteer blood donors. The presence of GBV-C RNA and TTV DNA in serum was assessed by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and semi-nested PCR, respectively. Autoantibodies in patients with SSc were determined by enzyme linked immunosorbent assay (ELISA) and Hep-2 immunofluorescence. RESULTS: TTV-DNA was detected in 10/84 (12%) patients with SSc, 9/41 (22%) patients with RA, 3/43 (7%) patients with OA, and 16/122 (13%) blood donors. GBV-C RNA was present in 4/84 (5%) patients with SSc, 2/43 (5%) patients with OA, and 5/122 (4%) blood donors. No patient with RA was positive for GBV-C RNA. One patient with SSc and one patient with OA showed a double infection with GBV-C and TTV. 74/84 (88%) patients with SSc were positive for at least one autoantibody species tested: 18/84 (21%) showed anticentromeric autoantibodies, 55/84 (66%) a speckled (36/84 (43%) fine, 19/84 (23%) coarse), and 20/84 (24%) a homogeneous nuclear Hep-2 pattern, and 21/84 (25%) had antinucleolar autoantibodies. Anti-Scl-70 antibodies were found in 31/84 (37%) and anti-RNP antibodies in 5/84 (6%) patients with SSc. No differences in the autoantibody pattern in patients with SSc with or without viral infection could be detected. CONCLUSION: The prevalence of GBV-C RNA and TTV DNA in serum samples from patients with SSc, RA, and OA was low and comparable with that in blood donors. A continuing infection with TTV and or GBV-C was not associated with a significant change in the autoantibody pattern in patients with SSc. These data provide no evidence for an association between GBV-C and/or TTV infections and SSc and/or arthritis (RA and OA).

Adult↗
PubMed2001

Evaluation of new approaches to prophylactic and therapeutic vaccinations against hepatitis B viruses in the woodchuck model.

The woodchuck model is most suitable for vaccine studies of prophylaxis and therapeutic treatment of hepatitis B virus infection. Recently, methodological advances allowed the examination of antigen-specific T cell responses in woodchucks during woodchuck hepatitis virus (WHV) infection and vaccinations. Similar to hepatitis B virus infection in humans, multispecific T-cell responses to WHV occur during acute self-limiting infection in woodchucks. Immunizations with WHV core antigen (WHcAg) or DNA vaccines expressing WHcAg demonstrated that priming of specific T-cell responses leads to the control of WHV infection. B-cell responses but no T-cell responses to WHV surface antigens (WHsAg) were induced in chronically WHV-infected woodchucks by the therapeutic immunizations with WHsAg. Breaking T-cell tolerance appears to be critical for immunotherapeutic approaches to chronic hepatitis B.

Acute Disease↗
PubMed2001

Vaccination against hepatitis delta virus infection: studies in the woodchuck (Marmota monax) model.

Hepatitis delta virus (HDV) superinfection of hepatitis B virus carriers causes severe liver disease and results in a high rate of chronicity. So far, neither sufficient therapy nor vaccines to prevent HBV carriers from superinfection are available. A good model to test vaccine candidates is the woodchuck chronically infected with the woodchuck hepatitis virus (WHV); the woodchuck can be superinfected with HDV and shows a course of infection similar to that of patients. Different strategies have been investigated to establish a protective vaccine against HDV superinfection. Both proteins of HDV (HDAg p24 and p27), which differ only in the C-terminal amino acid sequence, have been used as vaccine candidates. Synthetic peptides derived from B cell epitopes of HDAg and HDAg p24 expressed in Escherichia coli, yeast, or baculovirus have been used to immunize woodchucks. The protein immunization induced a specific antibody response, however, no protection from HDV superinfection was achieved. Vaccinations with vaccinia virus expressing HDAg p24 or p27 and DNA immunization with vectors expressing p24 were also not able to induce a protective immune response, but seemed to modulate the course of HDV superinfection. Thus, new strategies to develop a vaccine to prevent HDV superinfection are needed.

Animals↗
PubMed2001

Induction of a cellular and humoral immune response against preprocalcitonin by genetic i: a potential new treatment for medullary thyroid carcinoma.

Currently, no effective therapy exists for patients suffering from progressive medullary thyroid carcinoma (MTC), a calcitonin (CT)-secreting C cell tumor. As CT, which arises from the precursor protein preprocalcitonin (PPCT), is expressed by almost all MTC cases, these molecules may represent target antigens for immunotherapy against MTC. In our study we investigated whether DNA immunization is able to induce cellular and humoral immune responses against human PPCT (hPPCT) in mice. Antigen-encoding expression plasmids were delivered intradermally by gene gun. One group of mice received DNA encoding hPPCT only. Two groups were coinjected with mouse cytokine genes. We observed in lymphocyte proliferative assays substantial proliferation against hPPCT in mice coinjected with the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, in contrast to mice vaccinated with hPPCT expression plasmid only. In addition, codelivery of the GM-CSF gene augmented the frequency of anti-hPPCT antibody seroconversions in sera of immunized animals, as shown by enzyme-linked immunosorbent assay. These results illustrate that cellular and humoral immune responses against hPPCT can be generated by DNA immunization and increased by coinjection of the GM-CSF gene. Our findings may have implications for the use of DNA immunization as a potential novel immunotherapeutic treatment for patients suffering from progressive MTC.

Animals↗
PubMed2000

Naturally occurring woodchuck hepatitis virus (WHV) deletion mutants in chronically WHV-infected woodchucks.

Deletion mutants of hepatitis B virus (HBV) are often found in chronically HBV-infected patients. It has not been possible to study the significance of such deletion mutants on liver diseases in a suitable animal model. In this study, we characterized naturally occurring deletion mutants of woodchuck hepatitis virus (WHV) in 11 chronically WHV-infected woodchucks. Deletions within the WHV preS region (nt 2992-338) had a length of 72 or 84 bp and were located in the amino terminal part of preS1. Internal deletions within the core gene (CID) had variable lengths (103 to 312 bp) and were identified within the center of this gene (nt 2021-2587). Four of seven CIDs were in-frame deletions, whereas the remaining three CIDs were out-of-frame deletions and led to the interruption of the reading frame. Sequence analysis of cloned PCR products of CIDs showed that heterogeneous WHV deletion mutants coexisted in single woodchucks. In addition, WHV genomes with double deletions in the preS1 and the core region could be found. We were unable to detect the expression of truncated core proteins in transfection experiments. The CID mutations led to a marked increase of the expression of the luciferase gene which was fused to the start codon of WHV polymerase, probably due to the shortening of the untranslated region or the removal of AUGs preceding the polymerase start codon. The characterization of naturally occurring WHV deletion mutants will allow us to study their biological and pathogenic properties in the woodchuck model in the future.

Animals↗