Biomedical subjects

H Will

Publications and source records attributed to H Will.

At least 55 records · Page 3Linked to original sources

PubMed1998

Reactivation of hepatitis B in a long-term anti-HBs-positive patient with AIDS following lamivudine withdrawal.

BACKGROUND/AIMS: In HIV-infected patients, who have recovered completely from an acute hepatitis B infection and become anti-HBs positive, hepatitis B infection may be reactivated after progression to AIDS. CASE REPORT: We present the case of a homosexual male patient with AIDS who developed clinical and serological reactivation of hepatitis B with detectable HBV-DNA 18 years after complete recovery from acute hepatitis B infection. Prior to reactivation, antiretroviral triple therapy including lamivudine was changed to therapy without lamivudine. After reintroduction of lamivudine in the triple therapy, HBV-DNA became undetectable and the patient lost HBsAg and again developed anti-HBs antibodies. CONCLUSION: The hepatitis B in this patient can be explained best by reactivation of persistent HBV infection, possibly because of transient decline in antibodies against HBs-antigen due to a reduction in CD4+ lymphocyte numbers and B cell dysfunction. This observation points to the clinical relevance of HBV persistence in serum and blood cells of anti-HBs-positive subjects for many years after recovery from acute hepatitis B infection. The possible role of lamivudine withdrawal which immediately preceded HBV breakthrough in our patient is noteworthy. Regular monitoring of markers of HBV infection, including HBV-DNA, in patients with AIDS appears justified after discontinuation of lamivudine.

Acquired Immunodeficiency Syndrome↗

PubMed1998

The antinuclear autoantibodies Sp100 and gp210 persist after orthotopic liver transplantation in patients with primary biliary cirrhosis.

BACKGROUND/AIMS: Primary biliary cirrhosis is an autoimmune liver disease which is characterized by the presence of autoantibodies directed against mitochondrial components which belong to the pyruvate dehydrogenase enzyme complex. Apart from antibodies against mitochondrial components, primary biliary cirrhosis patients often show antibodies against nuclear components, of which anti-Sp100 and anti-gp210 are considered to be disease specific. We investigated the incidence and course of antibodies against nuclear components in primary biliary cirrhosis patients before and after liver transplantation. METHODS: Sera from 42 primary biliary cirrhosis patients were studied using indirect immunofluorescence to detect antibodies against mitochondrial components and antibodies against nuclear components, ELISA to detect anti-Sp100, and immunoblot analysis to detect anti-gp210 and antibodies against nuclear components subtypes. RESULTS: Ninety-three percent of primary biliary cirrhosis patients in our study were antimitochondrial antibody positive. Forty-three percent of the patients were antinuclear antibody positive. Of these, 35% had antibodies against Sp100 and 36% were positive for anti-gp210. After transplantation, antimitochondrial antibody titers as well as antinuclear antibody titers decreased in all patients. Autoantibodies in low titer persisted for up to 13 years. The pattern of nuclear autoantigens recognized by patient sera was unchanged after liver transplantation. However, the antinuclear antibody pattern was very different between the individual patients. Anti-Sp100 and anti-gp210 were not detected in sera of patients with autoimmune hepatitis, hepatitis C infection, inflammatory bowel disease, connective tissue diseases, or primary sclerosing cholangitis. The serum alkaline phosphatase level was not different in antinuclear antibody negative or positive patients before or after transplantation. CONCLUSIONS: We conclude that the persistence of antibodies against mitochondrial components, and anti-Sp100 and anti-gp210 in primary biliary cirrhosis patients after liver transplantation is disease specific, but that this does not reflect recurrent disease activity in the graft.

Adult↗

PubMed1998

Localization of the human membrane-type 2 matrix metalloproteinase gene (MMP15) to 16q12.1 near DNA elements that are part of centromeric and non-centromeric heterochromatin of 11 human chromosomes.

We have localized a second gene for membrane-type matrix metalloproteinases, MT2-MMP, to chromosome 16q12 by in situ hybridization. FISH experiments using a genomic PAC clone containing the MT2-MMP gene resulted in an unusual hybridization pattern detecting centromeric and non-centromeric heterochromatin regions or its flanking sequences in 11 human chromosomes in addition to the MT2-MMP locus on chromosome 16q12. The detailed analysis of this hybridization pattern using molecular cytogenetic methods together with the specific hybridization of the MT2-MMP cDNA allowed a refined mapping of the gene to 16q12.1, directly adjacent to the 16q heterochromatin. Our findings may give some insights into the evolution of the MMP gene family.

Chromosome Mapping↗

PubMed1998

Neonatal fulminant hepatitis B: structural and functional analysis of complete hepatitis B virus genomes from mother and infant.

Transmission of hepatitis B virus (HBV) from anti-hepatitis B e (anti-HBe)-positive carrier mothers to their infants may result in neonatal fulminant hepatitis B (FHB). We investigated whether HBV variants with a particular DNA sequence and functional phenotype, responsible for FHB, are selected during transmission. Full-length HBV genomes from a mother-infant pair were completely sequenced and transfected into human hepatoma cells. The dominant neonatal and maternal HBV populations were nearly identical (homology 99.8%) and showed a precore stop codon mutation, T-1762 and A-1764 substitutions in the core promoter region, and pre-S2 start codon mutations. Cells transfected with variants from mother and child, compared with wild-type virus, synthesized and released a similar number or fewer HBV DNA-containing particles. In conclusion, no particular HBV strain emerged during neonatal FHB. In this case, a de novo infection with variants showing a defect in HBe antigen and pre-S2 protein synthesis but not a high replication competence probably contributed to the fulminant disease course.

Adult↗

PubMed1998

Wild-type levels of pregenomic RNA and replication but reduced pre-C RNA and e-antigen synthesis of hepatitis B virus with C(1653) --> T, A(1762) --> T and G(1764) --> A mutations in the core promoter.

Hepatitis B virus (HBV) isolates with A-1762 to T and G-1764 to A mutations in the core promoter have been associated with active hepatitis, severe liver disease following liver transplantation, hepatocellular carcinoma and acute fulminant courses--in the latter case combined with a C-1653 to T mutation. In this study, a mutant core promoter region containing the T-1653, T-1762 and A-1764 mutations was placed into the context of a wild-type HBV genome and analysed by transfection. The mutations reduced the level of pre-C mRNA (by 55%) and e-antigen secretion. In contrast, no significant effects on the levels of pregenome/C and pre-S/S mRNAs, intracellular core, polymerase, and pre-S /S2 proteins and secreted S-antigen were observed. The amount of progeny virus DNA in the cells and in the culture medium was increased marginally, if at all.

Base Sequence↗

PubMed1998

Amplification of full-length hepatitis B virus genomes from samples from patients with low levels of viremia: frequency and functional consequences of PCR-introduced mutations.

To facilitate the investigation of hepatitis B virus (HBV) sequence variation, we recently established a method for functional analysis of PCR-amplified full-length HBV genomes. This study aimed at estimating the number of mutations introduced during amplification of genomes from samples from patients with low levels of viremia and their influence on replication and antigen expression. Wild-type HBV DNA template molecules in concentrations like those present in samples from patients with very low levels of viremia were amplified, sequenced (30 kb total), and functionally tested. We found that Taq polymerase and a Taq-Pwo polymerase mixture introduced an average of 5.7 and 3.1 mutations per genome, respectively, corresponding to polymerase error rates of 12.1 x 10(-5) and 6.0 x 1(0-5). One of 8 genomes (12%) amplified with Taq polymerase, but 7 of 17 genomes amplified with Taq-Pwo polymerases (41%), remained replication competent. All replication-competent genomes expressed HBs and HBe antigens and had an average of only 0.9 mutations per genome. In contrast, replication-defective genomes had an average of 5.4 mutations, which frequently also disturbed viral antigen expression. From these data we conclude that many of the replication-competent HBV genomes from a clinical specimen will retain their replication and antigen expression phenotypes even after extensive amplification with Taq-Pwo polymerases. Because replication competence is highly sensitive to random mutations, it is the best marker for the identification of HBV genomes with few or no PCR-introduced mutations.

Cloning, Molecular↗

PubMed1998

Enhancement of hepatitis B virus infection by noninfectious subviral particles.

The biological function of the huge excess of subviral particles over virions in hepatitis B virus infections is unknown. Using the duck hepatitis B virus as a model, we unexpectedly found that subviral particles strongly enhance intracellular viral replication and gene expression. This effect is dependent on the multiplicity of infection, the ratio of virions over subviral particles, and the time point of addition of subviral particles. Most importantly, we show that the pre-S protein of the subviral particles triggers enhancement and requires the presence of the binding regions for putative cell-encoded virus receptor proteins. These data suggest that enhancement is due either to the recently described transactivation function of the pre-S protein or to signalling pathways which become activated upon binding of subviral particles to cellular receptors. The findings are of clinical importance, since they imply that infectivity of sera containing hepadnaviruses depends not only on the amount of infectious virions but also decisively on the number of particles devoid of nucleic acids. A similarly dramatic enhancing effect of noninfectious particles in other virus infections is well conceivable.

Animals↗

PubMed1998

[The serologic detection of virus-induced infections in the domestic goose (Anser anser dom.)].

The most important virus-induced diseases associated with heavy losses in the domestic goose are Derzsy's disease which is caused by a goose parvovirus and duck plague (duck viral enteritis) which is caused by an avian herpesvirus. Both diseases still occur but can be prevented by timely vaccinations. Antibodies against Influenza A viruses of the subtypes H1, H5, and H7 as well as against avian paramyxoviruses of the serogroups 4, 6, and 8, respectively, were not detected in any of the examined sera. However, antibodies against paramyxovirus type 1 were detected in sera of one source. Haemagglutination inhibition or neutralizing antibodies against avian adenoviruses (EDS76 virus and goose adenovirus of the serotypes 1, 2, and 3) were quite often detected. Based on the present knowledge their pathogenic potential is minor. Neutralizing antibodies against a reovirus originating from Muscovy ducks and against a chicken reovirus (strain U Con S 1133) were quite frequently detected. In 35 of 564 examined geese sera hepatitis B virus was found.

Animals↗

PubMed1998

Detailed characterization of the binding site of the lipoprotein lipase-specific monoclonal antibody 5D2.

Monoclonal antibody (MAb) 5D2 recognizes lipoprotein lipases (LPL) from different species but not related lipases. This MAb is a unique reagent, used world-wide, because it differentiates between monomeric inactive and dimeric active LPL, inhibits human LPL enzyme activity, and binds to C-terminal LPL sequences involved in interactions with lipoproteins, lipoprotein receptors, and heparin. In this study we have analyzed the fine specificity of the MAb epitope recognition in order to better understand its functional properties and species-specific LPL immune reactivity. In peptide scan assays, MAb 5D2 reacted with all, except two, 13 amino acid-long peptides located between positions 380 and 410. Peptides from the amino terminal end of this region reacted more strongly than those from the carboxyl terminal end. Furthermore, only a peptide from the amino terminal end competed effectively with the binding of MAb 5D2 to native LPL bound to microtiter plates or nitrocellulose. A systematic peptide mutagenesis study indicated that 8 amino acids of the reactive region, mainly located in the amino terminal end, are critical for binding and probably directly interact with MAb 5D2. The experimentally determined antigenicities of species-specific LPL peptides and of the corresponding denatured full-length LPL proteins on immunoblots were consistent with these findings. According to a proposed 3D-model for LPL, only the amino terminal end of the antigenic region is easily surface-accessible. These data combined with 3D-modelling of monoclonal antibody (MAb)-lipoprotein lipase (LPL) protein interaction provide new insight into the known biological effects of MAb 5D2 on LPL and the antigenic determinants that are recognized.

Amino Acid Sequence↗

PubMed1997

Evidence for covalent modification of the nuclear dot-associated proteins PML and Sp100 by PIC1/SUMO-1.

PML and Sp100 proteins are associated with nuclear domains, known as nuclear dots (NDs). They were discovered in the context of leukemic transformation and as an autoantigen in primary biliary cirrhosis, respectively. Both proteins are expressed in the form of many COOH-terminally spliced variants, and their expression is enhanced by interferons (IFN). The recent finding that PIC1/SUMO-1, a small ubiquitin-like protein, is covalently linked to the RanGAP1 protein of the nuclear pore complex and also binds PML in yeast cells led us to determine whether PML is covalently modified by PIC1/SUMO-1 and whether the same is true for Sp100. We found an immune reaction of PML and Sp100 proteins with a PIC1/SUMO-1-specific monoclonal antibody by immunoblotting when using cell extracts prepared from stably transfected cells inducibly expressing one isoform of each protein as well as from nontransfected cells. In contrast, both proteins did not react when synthesized in vitro. Immunofluorescence staining showed that PIC1/SUMO-1 colocalized with Sp100 and PML in NDs except in mitotic cells, in which PML and Sp100 are dissociated. Cell fractionation and immunoblotting demonstrated that PIC1/SUMO-1 immunoreactive Sp100 in IFN-treated and untreated cells was exclusively nuclear, whereas nonmodified Sp100 was also found in the cytoplasm. Taken together, these data strongly suggest covalent modification of specific nuclear isoforms of Sp100 and PML by PIC1/SUMO-1. This modification may play a regulatory role in ND structure, composition, and function.

Animals↗

PubMed1997

Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis.

We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association. In vitro binding studies indicate that the ayw and adr viral subtypes of HBx bind similar amounts of glutathione S-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.

Adult↗

PubMed1997

A new class of defective hepatitis B virus genomes with an internal poly(dA) sequence.

Sequence heterogeneity of hepatitis B virus (HBV) is increasingly recognized to play a role in virus-host interaction. We have used a recently established method for HBV full-length genome amplification to search for novel types of HBV variants and to investigate further the sequence heterogeneity of HBV genome populations. Using this method, a substantial fraction of HBV genomes much shorter than wildtype size was found in some sera and liver biopsies from infected patients. Cloning and sequencing of a number of these HBV genomes as well as hybridization studies revealed a new minor class of HBV genomes with an internal poly(dA) sequence approximately 60 to more than 100 nucleotides long in 4 of 10 patients. The 5'-ends of the internal poly(dA) sequences are located at positions corresponding to the authentic processing/polyadenylation sites of the RNA pregenome, whereas the positions of the 3'-ends are variable due to different sizes of adjacent deletions. These data suggest that the poly(A) tail of the pregenomic RNA is occasionally reverse transcribed by the HBV P-protein and during this process a deletion seems to be introduced into the DNA minus strand. We propose a mechanism by which this could be accomplished during DNA minus strand synthesis.

Base Sequence↗

PubMed1997

Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases.

Soluble proenzyme forms of the catalytic domains of membrane-type matrix metalloproteinases 1 and 2 (MT1-MMP and MT2-MMP) and a form of MT1-MMP containing the catalytic and hemopexin domains were expressed as soluble recombinant proteins. Purified, activated forms of the MT-MMP were shown to degrade fibronectin, tenascin, nidogen, aggrecan and perlecan. Only MT2-MMP showed activity against laminin. MT1-MMP retaining the hemopexin domain was able to specifically cleave native type-I and type-III collagens into the 3/4-1/4 fragments typical of the specific collagenases. The catalytic domain alone did not retain this activity. The MT-MMP did not degrade interleukin-1beta, but, similarly to many other MMP, could process a pro [tumor necrosis factor (TNF) alpha] fusion protein to release mature TNF. However, the latter was subsequently degraded into smaller fragments. These results demonstrate that, in addition to their ability to activate other MMP, such as progelatinase A/proMMP2 and procollagenase-3/proMMP13, MT-MMP degrade a number of extracellular matrix macromolecules. Their location at the surface of cells implies that they could play a significant role in the modulation of cell-matrix interactions.

Aggrecans↗

PubMed1997

Heterogeneity and common features of defective hepatitis B virus genomes derived from spliced pregenomic RNA.

Defective hepatitis B virus (HBV) genomes derived from packaging and reverse transcription of spliced RNA pregenomes were reported to be associated with progression to chronic infection. Since only two types with similarly spliced regions were characterized so far we reasoned that additional "spliced" genome variants may exist. Therefore, we isolated a large number of defective HBV genomes from sera of seven chronic carriers by full-length PCR. Forty-eight were found to contain deletions caused by splicing as identified by cloning, subgenomic PCR, and sequencing. In total, 11 types of spliced genomes derived from excision of 10 different introns were present in various combinations in each serum. This diversity resulted from alternative usage of five splice donor and four acceptor sites present in most but not all HBV genotypes. All spliced genomes shared sequence elements essential for replication as well as for transcription of the pre-C and pregenome/C mRNAs and the X mRNA. Moreover, all contained the coding regions for the X protein and for precore/core or precore/ core fusion proteins but lacked the pre-S/S gene promoters. These data demonstrate substantial and HBV genotype-dependent diversity of spliced genomes from which a variety of aberrant precore/core fusion proteins and normal X protein but no functional envelope and P proteins could be expressed. These genomes and the encoded proteins may play a role in the viral life cycle, persistence, and pathogenesis.

Cloning, Molecular↗

PubMed1997

Naturally occurring hepatitis B virus genomes bearing the hallmarks of retroviral G-->A hypermutation.

Two hypermutated genomes of hepatitis B virus (HBV) were cloned from sera of chronic virus carriers. Twelve percent and 26% of guanosine residues were replaced by adenosine, with the transitions being erratically distributed along the genome. G-->A substitutions showed a strong dinucleotide preference, decreasing in the order GpA > GpG > > GpC > or = GpT. Such traits are typical of retroviral G-->A hypermutation which results from cDNA synthesis coinciding with fluctuations in the intracellular [dTTP]/[dCTP] ratio. The observations offer an explanation for the high prevalence of HBV variants bearing a tryptophan 28-->stop codon in the pre-core region of carriers with chronic active or fulminant hepatitis. The HBV hypermutants indicate that a small proportion of hepatocytes have distorted dNTP pools, which might have implications for the fidelity of hepatocyte DNA replication or repair.

Adenine↗

PubMed1997

Cellular localization, expression, and structure of the nuclear dot protein 52.

Nuclear dots containing PML and Sp100 proteins (NDs) play a role in the development of acute promyelocytic leukemia, are modified after infection with various viruses, and are autoimmunogenic in patients with primary biliary cirrhosis (PBC). PML and Sp100 gene expression is strongly enhanced by interferons (IFN). Based on immunostaining with a monoclonal antibody (mAb C8A2), a third protein, nuclear dot protein 52 (NDP52), was recently localized in NDs. Here we analyzed the cellular localization, expression, and structure of NDP52 in more detail. Our NDP52-specific sera revealed mainly cytoplasmic staining but no ND pattern, neither in untreated nor in IFN-treated cells. Cells transfected with NDP52 expression vectors showed exclusively cytoplasmic staining. In subcellular fractionation experiments, NDP52 was found in cytoplasmic and nuclear fractions. Unlike as described for Sp100 and PML, NDP52 mRNA and protein levels were only marginally enhanced by IFN gamma and not enhanced at all by IFN beta. NDP52 homodimerization but no heterodimerization with Sp100 or PML could be demonstrated. None of the 93 PBC sera tested contained autoantibodies against NDP52. Finally, mAb C8A2 reacted not only with NDP52 but also with a conformation-dependent epitope on the Sp100 protein. These data imply that NDP52 forms homodimers but no heterodimers with Sp100 and PML, lacks autoantigenicity in PBC, localizes mainly in the cytoplasm, and is associated with the nucleus, but not with NDs. Finally, unlike Sp100 and PML, NDP52 expression is neither markedly enhanced nor localization detectably altered by type I and II IFNs.

Animals↗

PubMed1997

Structural and functional analysis of the promoter of the hepatic lipase gene.

Hepatic lipase (HL) gene transcription is almost exclusively limited to hepatocytes. Here we have studied sequences and transcription factors regulating basal and hepatocyte-restricted HL promoter activity. Sequencing of a cloned 3.4-kb HL promoter fragment revealed three Alu repeat sequences and a consensus hepatocyte-enriched nuclear transcription factor 1 (HNF1) binding site located upstream of one major and one minor transcription initiation site. By transfection of cell lines of hepatic and non-hepatic origin and of primary hepatocyte cultures, sequences controlling basic HL promoter activity and negative elements located downstream and upstream thereof which extinguish or enhance this activity were defined. Some HL-promoter fragments with internal deletions were active only in primary hepatocyte cultures. Human HNF1 protein was shown to bind to the HL-specific HNF1 response element and the activity of a heterologous promoter was enhanced by HL-HNF1 in rat primary hepatocyte cultures but not in the context of the authentic 3.4-kb HL promoter sequences. In cell lines the presence of HNF4 but not of HNF1 and vHNF1 mRNA was found to correlate with HL gene expression although no perfect consensus HNF4 binding motif was detected in the promoter region tested. Taken together, these data indicate that hepatocyte-specific HL gene transcription is controlled by positive and negative transcription regulatory proteins which bind to sequence motifs within and outside of the proximal 3.4-kb promoter fragment studied. For the elucidation of the control of HL promoter activity in vivo the use of primary hepatocyte cultures is essential.

Animals↗

PubMed1997

Membrane-type-2 matrix metalloproteinase can initiate the processing of progelatinase A and is regulated by the tissue inhibitors of metalloproteinases.

Membrane-type-1 matrix metalloproteinase has been identified as an activator of the matrix metalloproteinase progelatinase A at cell surfaces. We report here that a soluble active form of membrane-type-2 matrix metalloproteinase can also process progelatinase A in a comparable fashion to the type-1 at rates which are dependent on the concentration of the proenzyme. Activation is inhibited by tissue inhibitors of metalloproteinases TIMP-2 and TIMP-3, but only partially by TIMP-1. These results suggest that cellular activation of progelatinase A may be initiated by different members of the membrane-type matrix metalloproteinase family depending on tissue distribution.

Amino Acid Sequence↗