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W K Roth

Publications and source records attributed to W K Roth.

At least 73 records · Page 4Linked to original sources
PubMed1995

Evaluation of a reverse hybridization assay for genotyping of hepatitis C virus.

BACKGROUND/AIMS: Several strains of the hepatitis C virus exist; distinct genotypes and subtypes can be identified by sequence comparison of the viral genomes. Recent evidence that the genotype/subtype of hepatitis C virus may influence the clinical course of chronic hepatitis C and the response to interferon-alpha therapy for this disease suggests that methods to identify the genotype may become clinically useful. In the present study we evaluated a recently introduced reverse hybridization assay. METHODS: HCV-RNA was isolated from serum samples from 61 consecutive patients attending our out-patient clinic and subsequently sequenced in the 5'-noncoding and the nonstructural-5 region by the dideoxynucleotide chain termination method. HCV-genotyping was performed by phylogenetic analysis of nonstructural-5 sequences. The amplification product for the reverse hybridization assay was obtained by "nested" polymerase chain reaction using biotinylated primers corresponding to the 5'-noncoding region. The assay is based on hybridization of the resulting polymerase chain reaction product with oligonucleotide probes immobilized as parallel lines on membrane strips. RESULTS: According to the phylogenetic analysis of the nonstructural-5 region the prevalence of hepatitis C virus subtypes was as follows: 1a 18%, 1b 51%, 2a 3%, 2b 3%, 2c 7% and 3a 18%. The reverse hybridization assay correctly identified each hepatitis C virus genotype (1, 2, and 3). However, differentiation of hepatitis C virus subtypes was insufficient. 1/11 HCV-1a isolates was incorrectly classified by the reverse hybridization assay as HCV-1b and vice versa 3/31 HCV-1b isolates as HCV-1a. Classification of hepatitis C virus subtypes 2a, 2b and 3a was correct, but 4/4 HCV-2c isolates were misinterpreted by the assay as HCV-2a. CONCLUSIONS: The reverse hybridization assay can differentiate between hepatitis C virus genotypes 1, 2, and 3, but is not completely reliable for hepatitis C virus subtyping.

Base Sequence↗
PubMed1995

[Viral hepatitis C].

Soon after the isolation of the hepatitis C virus (HCV) genome in 1988 it became evident that HCV is the most important cause of non-A, non-B-hepatitis. In recent years the structure of this (+)-stranded RNA-virus, the different genotypes of HCV and the replication in hepatic and extrahepatic sites have been investigated. HCV has a remarkable degree of genetic heterogeneity, mutates rapidly, leading to the simultaneous coexistence of different genoms in the same individual (quasispecies) and most likely to the generation of neutralization escape mutants. The cytotoxicity of the hepatitis C virus appears to be mainly immune-mediated. This review article summarizes basic, diagnostic and clinical aspects of acute and chronic HCV-infection, association with other diseases and complications such as liver cirrhosis and hepatocellular carcinoma. Interferon-alpha has been shown useful in normalizing serum aminotransferases and decreasing liver inflammatory lesions in about half of the patients with chronic hepatitis C. However, relapses after the cessation of interferon-alpha are frequent, leading to a sustained response in less than 30% of treated patients. Several clinical and biochemical parameters for response to interferon-alpha have been proposed. In patients with orthotopic liver transplantation due to progressive chronic hepatitis C and decompensated liver cirrhosis, reinfection of the donor organ frequently occurs. However, in transplanted patients under immunosuppression the course of hepatitis C reinfection is usually mild. Due to screening programs of blood and blood products the incidence of posttransfusion-acquired hepatitis C has declined. However, further efforts in understanding the transmission of community-acquired hepatitis C are necessary. The development of a hepatitis C vaccine will be difficult due to the high degree of viral genetic heterogeneity.

Carcinoma, Hepatocellular↗
PubMed1994

Clinical evaluation of a new polymerase chain reaction assay (Amplicor HCV) for detection of hepatitis C virus.

Direct detection of hepatitis C virus (HCV) by reverse transcription (RT) and polymerase chain reaction (PCR) has clinical impact on diagnosis and the assessment of anti-viral therapy. However, recent results of a quality control study on the detection of HCV-RNA by RT-PCR revealed inappropriate sensitivity and specificity in the majority of participating laboratories. In this study we evaluated the first standardized RT-PCR-assay (Amplicor HCV) for routine detection of hepatitis C virus in serum samples from patients with hepatitis C (n = 111), patients with resolved acute hepatitis C (n = 7) and controls (n = 101). The Amplicor HCV assay was convenient to handle, detected all genotypes of the hepatitis C virus commonly present in Europe (type 1, 2 and 3 according to Simmonds et al.) and had a lower detection limit of 10(2)-10(3) copies as assessed by quantifiable HCV-specific RNA templates. In the clinical evaluation the Amplicor HCV system reached a sensitivity of 100% and a specificity of 97%.

Antiviral Agents↗
PubMed1992

Expression of platelet-derived growth factor and its receptor in AIDS-related Kaposi sarcoma in vivo suggests paracrine and autocrine mechanisms of tumor maintenance.

As previously described, proliferation of Kaposi sarcoma (KS)-derived cells in vitro is dependent on the presence of platelet-derived growth factor (PDGF). To test the hypothesis that PDGF may also be a major growth factor for KS cells in vivo, we performed in situ hybridization and immunohistochemical staining for PDGF and PDGF receptors in tissue sections of AIDS-related KS. The data suggest that KS consists of two types of tumor cells. (i) The main population are spindle-shaped cells with elongated nuclei (KS-s cells). They reveal a strong expression of PDGF beta receptors but do not express the PDGF-A and PDGF-B isoforms. (ii) A minor population of KS cells express PDGF beta receptor as well as PDGF-A and PDGF-B (KS-p cells). These cells are often grouped in whorls and surrounding vascular slits. They reveal spherical nuclei with evenly distributed chromatin and inconspicuous nucleoli. PDGF alpha receptor is not expressed in either form of KS cells. The results suggest that the isoforms of PDGF and the PDGF beta receptor are differentially expressed in two different cell types in KS and that PDGF isoforms may contribute to the pathogenesis of KS.

Acquired Immunodeficiency Syndrome↗
PubMed1992

'Run-off' polymerization with digoxigenin labelled nucleotides creates highly sensitive and strand specific DNA hybridization probes: synthesis and application.

In this paper the in vitro synthesis and application of non-radioactively labelled strand specific DNA probes is described. The probe is labelled by incorporation of nucleotides with the hapten digoxigenin into single-stranded DNA during a 'run-off' reaction catalyzed by Thermus aquaticus (Taq) DNA-polymerase. The 'run-off' reaction requires a linearized plasmid template and one primer binding site at a defined distance from the restriction site. Single-stranded DNA can be synthesized during repeated cycles of denaturation, annealing, and extension. The conditions for the incorporation of digoxigenin-11-dUTP (dig-11-dUTP) during polymerization were optimized to generate strand specific DNA hybridization probes up to a length of 5000 nt. The strand specificity is demonstrated by a dot-blot, with in vitro-transcribed target RNA of c-sis. The sensitivity of the probe was tested in a Northern blot, and found to be identical to a probe radiolabelled by nick-translation (specific activity 6.5 x 10(8) cpm micrograms-1). The resolution of the signals and speed of development was even superior compared to the radiolabelled probe.

Actins↗
PubMed1992

Kaposi's sarcoma: a review of gene expression and ultrastructure of KS spindle cells in vivo.

The ultrastructural features and the gene expression pattern of Kaposi's sarcoma (KS) spindle cells in vivo suggest that KS is a tumor of the mixed cell type. The expression pattern of cytokines and cytokine receptors in the tumor lesion, together with the results obtained from in vitro characterization of KS-derived cells, provide evidence that paracrine mechanisms of growth factor action are important for the maintenance of KS. The reports on virus infection of KS cells suggest an indirect role of virus infection in the induction of KS, most likely mediated by immunostimulation and subsequent production of cytokines.

Biomarkers↗
PubMed1991

HIV-associated Kaposi's sarcoma: new developments in epidemiology and molecular pathology.

New epidemiological data give evidence for an unknown etiological agent of Kaposi's sarcoma (KS). Experimental support is provided by research on cultivated KS cells. These results contradict a direct involvement of HIV-1 in the pathogenesis of KS. Research on cultivated KS cells confirmed the hypothesis that KS spindle cells originate from endothelial cells and gave new insight into the pathogenesis of tumor cell growth. KS spindle cells secrete an autocrine acting growth promoting activity. Nevertheless, they seem to depend on several growth factors like PDGF and IL-6 provided by surrounding endothelial cells and macrophages, respectively. The results support the hypothesis of a tumor relying on paracrine acting factors more than on autocrine acting factors.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Fibroblast growth factor 5 proto-oncogene is expressed in normal human fibroblasts and induced by serum growth factors.

Fibroblast growth factor 5 (FGF-5) is a member of the fibroblast growth factor family with transforming potential. It has been found to be expressed in several human tumor cell lines, but nothing is known about expression of this growth factor in normal cells and its biological functions. Here we show that the FGF-5 gene is expressed in exponentially growing normal human fibroblasts. In quiescent fibroblasts, expression of FGF-5 is strongly induced by serum and several growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). This induction can be mediated by at least two different pathways involving protein kinase C or cAMP-dependent kinases. Since the effect is independent of de novo protein synthesis, FGF-5 represents the product of a primary response gene. In addition our data suggest that FGF-5 is mitogenic for human fibroblasts, indicating the existence of an FGF-5-mediated positive feedback in these cells which could amplify and prolong the cellular response to the initial stimulus.

Animals↗
PubMed1991

[Morphologic HIV demonstration in formalin embedded material--techniques, problems, results].

Formalin-fixed, paraffin-embedded material of archival specimens is suitable for a morphological HIV-detection: Infected cells with HIV in the proliferative phase can be demonstrated with reliable results on tissue sections by immunohistological technics using new antibodies. In situ nucleic acid hybridisation technics can also show HIV in the expression phase on paraffin-embedded material, but often fail in demonstrating latently HIV-infected cells. The DNA-Polymerase chain reaction can detect latent Provirus in morphologically defined areas of paraffin sections even in autopsy material, i.e. lymphnodes and even eyes of patients with HIV-Infection, but requires precaution and control with respect to contamination.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Low mitogenic response to EGF and TGF-alpha: a characteristic feature of cultured Kaposi's sarcoma derived cells.

We have investigated the mitogenicity of Epidermal Growth Factor (EGF) and Transforming Growth Factor alpha (TGF-alpha) for cultured Kaposi's sarcoma derived cells (KS cells). In contrast to control fibroblasts from the same patients, KS cells revealed only a weak mitogenic response to these growth factors. Neither EGF nor TGF-alpha were able to substitute for Platelet-derived growth factor (PDGF) when KS cells were grown in PDGF-depleted Platelet-Poor-Plasma serum (PPPS)-supplemented medium. The low mitogenicity of EGF and TGF-alpha for KS cells is not based upon a reduced expression of EGF receptor mRNA and protein in KS cells. However, the binding of EGF to KS cells is about 50% lower than that to fibroblasts. This reduced binding is not due to an occupation of the receptors by TGF-alpha since the expression level of this mitogen in different KS cell lines does not correlate with their capacity to bind EGF. In contrast, the low EGF binding seems to be an intrinsic feature of the EGF receptors of KS cells. In spite of the low mitogenicity of EGF for the tumor cells, the expression of c-myc, PDGF-A and Fibroblast growth factor 5 (FGF-5) mRNA is equally induced by purified EGF in KS cells and fibroblasts. This shows that at least the signal transduction pathways which lead to the expression of these genes are functional in KS cells.

Blotting, Northern↗
PubMed1990

"Run-off" synthesis and application of defined single-stranded DNA hybridization probes.

A simple and efficient method for synthesizing radioactively labeled single-stranded DNA hybridization probes with Thermus aquaticus (Taq) DNA polymerase is described. This is done in a "run-off" polymerization with repeated cycles of denaturation, annealing, and extension. It leads to high yields of a single-stranded DNA of defined length (up to 5000 nt), which is labeled to a high specific activity (1.3 x 10(8) cpm/micrograms DNA). These hybridization probes are equally sensitive as nick-translated DNA probes, but strand specific. This was tested by slot blot hybridization with in vitro-transcribed target RNAs and by Northern blotting. The use of single-stranded DNA hybridization probes combines the benefits of DNA stability and single-strand RNA probes.

Animals↗
PubMed1990

Cultured Kaposi's sarcoma-derived cells express functional PDGF A-type and B-type receptors.

We have investigated the role of platelet-derived growth factor (PDGF) for the growth of cultured Kaposi's sarcoma (KS)-derived cells. They were found to bind 125I-PDGF-AA and 125I-PDGF-BB in a manner which is consistent with the presence of both PDGF A-type and PDGF B-type receptors. Furthermore, KS cells responded mitogenically to both PDGF homodimers. This shows that KS cells have functional A- and B-type PDGF receptors, suggesting an important role of PDGF for the growth of KS cells in vitro. In addition we found that KS cells produce PDGF-A chain mRNA in response to PDGF stimulation. This suggests that a PDGF-induced autocrine stimulation involving PDGF-AA which could amplify the response to PDGF occurs in these cells.

Binding, Competitive↗
PubMed1990

Infection of human fibroblasts and osteoblast-like cells with HIV-1.

Primary human skin- and lung-derived fibroblast cell cultures and continuous human osteoblast-like and fibroblast-like cell lines were infected with different strains of HIV-1. Infection was measured at the single-cell level using the immunoperoxidase staining method to detect viral proteins. No cytopathic effects were observed in HIV-1-infected cell cultures. One continuous cell line (LC5), derived from embryonic lung, was readily infectable with HIV-1 and showed continuous production of infectious virus. Infection of LC5 cells could be blocked with anti-CD4 monoclonal antibodies. These findings indicate that fibroblasts of skin and lung, and osteogenic cells may be considered as potential target cells for HIV-1, thereby possibly contributing to the establishment of local HIV reservoirs.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Invasive migration of epidemic Kaposi's sarcoma cells in vitro.

Kaposi's sarcoma (KS) is a low grade malignant neoplasm which shows invasive growth and often occurs in immunosuppressed patients with the Acquired Immune Deficiency Syndrome (AIDS; epidemic KS). It is also found in elderly men where it is usually limited to the skin (classic KS). The present study investigated the chemotaxis and invasive migration of epidemic KS cells in vitro and compared them to cells grown from classic KS lesions and to fibroblasts. Epidemic KS cells demonstrated invasive migration through reconstituted basement membrane (Matrigel) as well as through interstitial connective tissue (collagen I) in early passages, whereas fibroblasts did not invade either barrier. Epidemic KS cells in late passages did not show any invasive migration. Following pretreatment with tumour necrosis factor alpha (TNF-alpha) there was no enhanced migration through the Matrigel and collagen I for epidemic KS cells, whereas classic KS cells showed an increased migration through the type I collagen barrier.

Basement Membrane↗
PubMed1989

Cells derived from sporadic and AIDS-related Kaposi's sarcoma reveal identical cytochemical and molecular properties in vitro.

We have established 40 cell cultures from biopsies of 5 patients with sporadic Kaposi's sarcoma (KS) and compared them with cell cultures derived from KS biopsies of AIDS patients. Immunocytochemical staining of sporadic KS cells revealed the same endothelial cell markers as those expressed on AIDS-KS cells, indicating that both types of KS might be of endothelial cell origin. In contrast to clinical features, in vitro growth properties showed no differences. KS cells of both types reveal a low degree of malignancy but can be distinguished from fibroblasts by a higher passage number in low serum concentrations and a more pronounced dependence on platelet-derived growth factor (PDGF). The DNA of 12 cell lines of both types of KS was negative for genomic equivalents of hepatitis B virus (HBV), cytomegalovirus (CMV) and, in the case of AIDS-KS, for human immunodeficiency virus (HIV). No rearrangements or amplifications of several oncogenes, often involved in human tumors, were detected. The expression of these oncogenes by sporadic and AIDS-KS cells, as analyzed by Northern blotting, was comparable to that of normal dermal fibroblasts from the same patients. Our results indicate that the 2 types of Kaposi's sarcoma possess identical cytochemical and molecular properties. They are probably weakly malignant neoplasms of endothelial cell origin, and paracrine growth stimulation appears to be important for their maintenance and progression.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Cytochemical and molecular properties of simian virus 40 transformed Kaposi's sarcoma-derived cells: evidence for the secretion of a member of the fibroblast growth factor family.

Contact-inhibited Kaposi's sarcoma-derived cells (KS cells) were transfected with Simian Virus 40 (SV40) DNA. Transformed cells (SV-KSC) were selected for their capacity to form foci on monolayers of the low-malignant KS cells. Isolated SV-KSC foci were found to contain integrated SV40 DNA sequences and to express SV40 large T-antigen. Several differentiation properties of KS cells are retained in the SV40 transformants, e.g., expression of vimentin and the endothelial cell marker BMA 120. In contrast to the maternal KS cells, SV-KSC are capable of growing in platelet-derived growth factor (PDGF)-depleted platelet-poor-plasma serum (PPPS) and in soft agar. However, they are not tumorigenic in nude mice. Expression of the oncogenes c-myc, c-N-ras, c-Ha-ras, and p53 is significantly elevated in SV-KSC, whereas c-fos and c-erb B expression is comparable to that of KS cells and fibroblasts. Conditioned medium from SV-KSC can substitute for PDGF when PDGF-dependent, nontransformed KS cells are grown in PPPS. Biochemical analysis of the SV-KSC supernatant and PDGF A and B mRNA expression analysis provide evidence that the mitogenic activity is not due to a PDGF-like growth factor. On the other hand, there is evidence to indicate that the SV-KSC mitogen is a member of the fibroblast growth factor family. SV-KSC represent an interesting model system for the study of different degrees of malignancy of cultured mesenchymal cells and especially provide an important source for the isolation of a potent growth factor for KS cells and other mesenchymal cells in vitro.

Cell Line, Transformed↗