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G Wildner

Publications and source records attributed to G Wildner.

35 records · Page 2Linked to original sources
PubMed1992

Hepatitis B virus polymerase gene: expression of the long open reading frame using the baculovirus expression system.

A recombinant baculovirus was constructed containing a copy of the hepatitis B virus (HBV) genome which was inserted to produce an in-frame fusion of the precore (pre-C) coding region with the first 11 amino acids of the polyhedrin gene. The recombinant baculovirus expressed the 25K pre-C protein and two novel proteins, of approximately 93K and 72K. Both the 93K and 72K proteins are recognized by an anti-polymerase monoclonal antibody. Northern blot analysis of the mRNA produced during infection of Spodoptera frugiperda cells by the HBV recombinant baculovirus detected only one HBV mRNA species, suggesting that the three HBV-specific proteins expressed are translated from the same mRNA. No larger fusion proteins cross-reacting with either anti-core or polymerase antibodies were detected. These findings suggest that the two proteins encoded within the HBV polymerase gene are not produced via a core-polymerase fusion intermediate but by internal binding of ribosomes. These results are the first clear demonstration of efficient expression of two bona fide unprocessed polymerase proteins in a 1:1 ratio from an unspliced pre-C mRNA-like transcript. With the successful expression of the polymerase gene in insect cells it is now possible to produce large amounts of these proteins, allowing a more detailed structural and functional analysis of these proteins.

Baculoviridae↗
PubMed1992

Lymphocytes, macrophages and HLA-DR expression in vitreal and epiretinal membranes of proliferative vitreoretinopathy. An immunohistochemical study.

Proliferative vitreoretinopathy (PVR) as a severe complication of retinal detachment and also the main cause of failure in its treatments is not yet completely understood. In the present study, we examined 16 PVR epiretinal membrane specimens removed during vitrectomy from 16 patients with complicated rhegmatogenous retinal detachment by immunostaining of cryosections. We looked for the presence of lymphocytes and detected helper/inducer T lymphocytes (CD4+), suppressor/cytotoxic T lymphocytes (CD8+), B lymphocytes (CD22+), macrophages (CD68+), and HLA-DR expression. The results showed that 6 out of 16 specimens reacted positively with monoclonal antibody against the helper/inducer T cell subset (38%), 5 out of 16 reacted positively with a suppressor/cytotoxic T cell-specific monoclonal antibody (31%), 7 out of 16 reacted with B cell-specific monoclonal antibody (44%), and 14 out of 16 were positive for macrophages and HLA-DR (88%). Nevertheless, we think that the invasion of macrophages and lymphocytes into the membranes is not the cause for the pathogenesis, but a secondary event that might further complicate the course of the disease.

Antibodies, Monoclonal↗
PubMed1991

Mechanism, kinetics, and role of duck hepatitis B virus e-antigen expression in vivo.

No duck hepatitis B virus (DHBV) pre-C transcript has been identified so far, and neither the interrelationship of e-antigen (DHBeAg) with the expression of other viral antigens or virus replication nor its function is known. In this study we identified in infected livers a minor transcript from which the precursor protein of DHBeAg could be synthesized. Mutation of the first AUG on this transcript abolished expression of DHBeAg. DHBV genomes containing this mutation were infectious in Pekin ducks, the kinetics of pre-S envelope protein expression and virus secretion were not significantly different from wild-type, and the mutant genomes did not revert to wild-type to a detectable level after several passages. In contrast to pre-S protein, the level of DHBeAg in the serum was independent of the level of viremia, accumulated gradually to a high and constant level after a lag phase, and was also easily detectable in a mixed infection containing less than 0.1% of wild-type in a pre-C mutant virus containing inoculum. These data indicate that precore protein is synthesized from a minor pre-C mRNA with translation initiation at the pre-C AUG codon, and leads to high levels of DHBeAg rather late in infection. High levels of DHBeAg can even be produced efficiently by a very small subpopulation of wild-type virus in a mixed infection with predominantly pre-C mutant virus. Lack of DHBeAg appears to have no effect on DHBV viability and kinetics of virus secretion into the bloodstream when ducklings are infected with the pre-C AUG mutant virus a few days after birth.

Animals↗
PubMed1991

Characterization of infectious and defective cloned avian hepadnavirus genomes.

The infectivity in vivo, replication competence in vitro, and expression of viral genes of several molecularly cloned duck hepatitis B virus (DHBV) genomes were investigated. In addition, replication competence, core protein expression, and secretion of viral proteins were investigated for a grey heron hepatitis B virus genome. Except two, all DHBV isolates tested induced a systemic infection in Pekin ducks when injected as cloned viral DNA into the liver. After transfection of chicken hepatoma cells, both defective DHBV genomes expressed intracellular nucleocapsid and pre-S envelope proteins and secreted DHBs/pre-S particles into the medium. One of the defective DHBV genomes and HHBV produced within the cells replicative intermediates encapsidated in core particles and secreted virions, whereas the other defective DHBV genome did not and was unable to efficiently encapsidate the RNA pregenome. Comparative sequence analysis was performed to identify potential amino acid changes in viral proteins of both defective DHBV genomes. The data obtained demonstrate that most cloned avian hepadnaviruses are infectious or replication competent and suggest defects in envelope, polymerase or encapsidation function, respectively, in two cloned DHBV genomes.

Amino Acid Sequence↗
PubMed1990

Latency and reactivation of a precore mutant hepatitis B virus in a chronically infected patient.

In human patients infected with hepatitis B virus (HBV) seroconversion from HBe to anti-HBe often signals virus elimination. Occasionally, a second viremic phase is observed which may be due to superinfection with a variant or reactivation of a latent virus. To differentiate between these two possibilities we investigated the nucleotide sequences of virus populations from sera of a chronically infected patient who had two distinct viremic phases, one e-antigen positive and one anti-HBe antibody positive. By direct sequencing of amplified HBV C- and pre-S gene sequences we found that the viruses in the two populations differed by only two point mutations, one of which prevents expression of precore derived e-antigen. In the nonviremic phase viral DNA and antigen were found in the liver but nucleocapsid protein expression appeared drastically downregulated. These data demonstrate that HBV can enter a latent phase with low expression of core protein and selection for HBV variants which cannot synthesize e-antigen. This suggests that e-antigen expression can be a critical target for virus elimination and that loss of its expression can prolong chronicity and provoke latency of HBV infection.

Antigens, Viral↗
PubMed1990

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.

Amino Acid Sequence↗
PubMed1989

The use of fusion proteins to study HLA-B27-specific allorecognition.

Potential antigenic regions of the various external domains of the HLA-B27 antigen were expressed as fusion proteins in bacterial hosts and analyzed for their ability to induce humoral and cellular responses. Monoclonal antibodies directed against the proteins recognized monomorphic determinants of denatured HLA-antigens, but not B27-antigens expressed by intact lymphocytes. T-cell proliferation and IL-2 secretion were induced with a fusion protein representing regions of the first and second domains around amino acid residue 114. None of the fusion proteins stimulated cytotoxic T-lymphocytes (CTL) in an HLA-specific manner, although several included those amino acid sequences thought to be important for CTL recognition.

Animals↗
PubMed1989

Expression and immunogenicity of HLA-B27 in high-transfection recipient P815: a new method to induce monoclonal antibodies directed against HLA-B27.

The immunization of a (BALB/c x C57BL/6) FI mouse with murine transfectants expressing the HLA-B27 antigen resulted in a panel of polymorphic monoclonal antibodies with specificity for HLA-B27 and some additional HLA-antigens. Specificity of the antibodies was defined by cytofluorometric analysis on a panel of lymphoblastoid cell lines (LCL) derived from HLA typed individuals. Three of these antibodies are cytotoxic, and one of them inhibits B27-specific T cell cytotoxicity. Our results indicate that HLA-class I transfectants could be used to generate polymorphic antibodies, and that these antibodies may be helpful for HLA typing and for definition of epitopes recognized by T cells.

Animals↗
PubMed1985

Somatic mutation and clonal expansion of B cells in an antigen-driven immune response.

The variable (V) regions of three closely related monoclonal antibodies produced by hybridomas which had been isolated from a single mouse were sequenced at the level of the mRNA. The sequences and the restriction analysis of the immunoglobulin loci carried by the hybridoma cells indicate that the antibodies are derived from cells belonging to a single B cell clone. The sequence data imply a high frequency and stepwise occurrence of somatic point mutations in the expressed V region genes and substantial clonal expansion of B cells in the mouse. The mutations appear to be randomly introduced into heavy and light chain V region genes. Mutations are also seen in the complementarity determining regions which may thus have been involved in the selection of the cells producing the three antibodies.

Amino Acid Sequence↗
PubMed1980

Flow cytofluorometry, cytomorphology and histology in gastric carcinoma.

68 cases of gastric carcinoma and 31 tissue samples adjoining the carcinomas were investigated by impulse cytophotometry, cytomorphology and histology, respectively. Differentiated adenocarcinoma tended to aneuploidy, while undifferentiated carcinoma often showed euploidy. The adjoining mucosa was characterized by polymorphism of the mucosa superficial epithelial cells and the histograms showed slight abnormalities.

Adenocarcinoma↗