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Biomedical subjects

K Blight

Publications and source records attributed to K Blight.

15 recordsLinked to original sources
PubMed1997

Formation of native hepatitis C virus glycoprotein complexes.

The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.

Animals↗
PubMed1996

Absence of double-stranded replicative forms of HCV RNA in liver tissue from chronically infected patients.

The mechanism of hepatitis C virus (HCV) replication is unknown, although the classification of HCV in the Flaviviridae has led to the postulation that HCV may adopt a replication strategy similar to that of the flaviviruses. To determine if HCV double-stranded replicative forms, consistent with this strategy, were present in total liver RNA extracted from HCV-infected individuals, HCV-specific RNA was detected by reverse transcription followed by polymerase chain reaction (RT-PCR). Initially, a strand-specific RT-PCR resulting from chemical modification of the 3' end of the RNA was established using in vitro transcribed HCV RNA. This procedure allowed the specific detection of positive and negative HCV RNA strands in HCV-infected liver tissue. The species of HCV RNA was then examined in RNA extracted from liver tissue from naturally infected individuals; total liver RNA was either: (i) fractionated with 2M LiCl (designed to precipitate single-stranded and partially double-stranded RNA); or (ii) digested with RNase A in high salt conditions (designed to digest single-stranded RNA only). Amplification of positive sense HCV RNA from the LiCl-insoluble fraction, but not from the LiCl-soluble fraction nor in the RNase A-digested sample, was consistent with the interpretation that single-strand, but not double-stranded HCV RNA, was contained in the liver samples. Thus, it is unclear if a double-stranded RNA species is formed during the HCV replication cycle.

Electrophoresis, Agar Gel↗
PubMed1994

Detection and distribution of hepatitis C-specific antigens in naturally infected liver.

Hepatitis C virus antigen expression was examined using peptide antibodies in liver tissue taken at biopsy from four chronic carriers of hepatitis C virus. Hepatitis C virus antigens E2/NS1, NS3, NS4 and NS5 were widespread in unfixed frozen liver sections and were present as distinct granules or foci within the cytoplasm of hepatocytes and in infiltrating lymphocytes in portal tracts. Fixation of frozen sections with 1% formalin improved the histological appearance of the tissue section without reducing the sensitivity of antigen detection. However, in tissue sections fixed in acetone, chloroform, carbon tetrachloride or methyl carnoys, detection of all hepatocyte-specific hepatitis C virus antigens was significantly reduced. Dual immunostaining of liver sections for lymphocyte cluster of differentiation markers and hepatitis C virus antigens determined that a high proportion of cluster of differentiation 20-positive B cells and cluster of differentiation 4-and cluster of differentiation 8-positive T cells, predominant in lymphoid aggregates, were positive for hepatitis C virus antigens.

Antigens, Differentiation↗
PubMed1993

Localisation of hepatitis C virus proteins in infected liver tissue by immunofluorescence.

Hepatitis C virus (HCV) antigen expression was examined by immunohistochemical staining in liver tissue taken at biopsy from 8 anti-HCV positive patients. Frozen liver sections were stained by indirect immunofluorescence for capsid, E2/NS1, NS3, NS4 and NS5 using polyclonal antibodies raised to synthetic peptides from these regions. The antigens E2 and NS3 were localised in scattered hepatocytes and also in cells within and around areas of inflammation. A weaker signal was observed for NS4 and NS5 and no signal was seen for capsid antigen. No staining was seen in liver tissue from 9 individuals, including 3 hepatitis B virus-positive and 2 hepatitis delta virus/positive patients, who were negative for serological markers of HCV. The specificity of the staining reaction was also confirmed by the lack of staining in HCV-positive liver samples, after the antisera was pre-adsorbed against the specific peptide. Collectively, the data suggests that HCV may not only be hepatotropic but also lymphotropic, and this may be an important factor in the pathogenesis of HCV infection.

Antigens, Viral↗
PubMed1993

Immunohistochemical detection of the NS4 antigen of hepatitis C virus and its relation to histopathology.

The immunohistochemical localization of the hepatitis C virus (HCV) nonstructural antigen 4 (NS4) was investigated in formalin-fixed human liver biopsy samples taken from 10 patients who were anti-HCV positive. NS4 was detected within the cytoplasm of hepatocytes in all HCV-positive patients studied, but not in the mononuclear cell infiltrates, bile duct epithelium, or endothelial cells. A high proportion of hepatocytes appeared positive, but the staining intensity was variable. After a coded histological evaluation of the liver tissue, the pattern of liver injury was shown to have no significant correlation with antigen-positive hepatocytes, and no direct relationship was observed between the distribution of antigen-positive hepatocytes and areas of hepatocyte necrosis. The staining pattern was considered to be specific because liver samples from patients chronically infected with hepatitis B virus or from uninfected individuals were negative. Furthermore, no staining was noted when either preimmune rabbit serum or anti-NS4 adsorbed against the specific synthetic peptide was substituted for the primary antibody.

Adult↗
PubMed1992

Detection of hepatitis C virus RNA by in situ hybridization.

Persistent infection with hepatitis C virus (HCV) is associated with chronic hepatitis and cirrhosis which may eventually develop into primary hepatocellular carcinoma. The mechanism of pathogenesis is ill-defined and nothing is known of the distribution, frequency or type of infected cell in the liver of HCV-infected individuals. In this study we have examined liver tissue taken at autopsy from 2 anti-HCV-positive patients by in situ hybridization for the presence of HCV RNA. Viral RNA was detected by autoradiography after hybridization with 125I-labelled riboprobes, representing approximately 35% of the HCV genome. Only a few positive cells were identified in the HCV-infected liver samples, but not in a normal liver sample. Hybridization with an unrelated probe was negative in all samples. The HCV RNA-positive cells were detected with anti-sense but not sense RNA probes, suggesting that they contained a high ratio of genomic:antigenomic RNA. The appearance and distribution of the HCV RNA-positive cells suggested that they were not hepatocytes and were more likely to be lymphocytes or macrophages.

Autoradiography↗
PubMed1991

Characterization of murine thymic stromal-cell lines immortalized by temperature-sensitive simian virus 40 large T or adenovirus 5 E1a.

The heterogeneity of thymic stromal cells is probably related to their role in providing different microenvironments where T cells can develop. We have immortalized thymic stromal elements using recombinant retroviral constructs containing a temperature-sensitive simian virus 40 (SV40tsA58) large-T antigen gene or the adenovirus 5 E1a region linked to the gene coding for resistance to G418. Cell lines containing the thermolabile large T antigen encoded by SV40 proliferate at the permissive temperature of 33 degrees C and arrest growth when transferred to the nonpermissive temperature of 39 degrees C. At the nonpermissive temperature, ts-derived cell lines are shown to alter their phenotype but remain metabolically active, as indicated by the inducible expression of class I and class II MHC antigens. Here we describe the generation of a total of 84 thymic stromal-cell lines, many of which show distinct morphologic, phenotypic, and functional properties consistent with fibroblastoid, epithelial, or monocytoid origins. Several E1a and SV40tsA58-derived cell lines generated exhibit the epithelial characteristic of desmosome formation and, in addition, two of these lines (15.5 and 15.18) form multicellular complexes (rosettes) when incubated with unfractionated thymocytes from syngeneic mice. A single line (14.5) displays very strong nonspecific esterase activity, suggesting it may represent a macrophagelike cell type. We describe the generation of stromal cell lines with different properties, which is consistent with the heterogeneity found in the thymic microenvironment. In addition to documenting this diversity, these cell lines may be useful tools for studying T-cell development in vitro and give access to model systems in which stromal-thymocyte interactions can be examined.

Adenovirus Early Proteins↗
PubMed1988

Identification and localization of proteins encoded by two DIF-inducible genes of Dictyostelium.

We show that pDd56 and pDd63, two related DIF-inducible genes of Dictyostelium, respectively encode the ST310 and ST430 polypeptides identified by Morrissey, Devine, and Loomis (1984, Dev. Biol. 103, 414-424). We localize the two proteins by immunoelectron microscopy to the extracellular matrix surrounding the stalk cells and the stalk tube. Coupled with their predicted amino acid sequence and biochemical properties, this suggests that they are structural proteins of the stalk.

Dictyostelium↗
PubMed1988

Two DIF-inducible, prestalk-specific mRNAs of Dictyostelium encode extracellular matrix proteins of the slug.

The migratory slug of Dictyostelium discoideum is surrounded by, and continuously synthesizes, an extracellular protein-cellulose matrix known as the slime sheath which is deposited on the substratum as a trail marking the slug's progress. We show that the stalk-specific proteins, ST310 and ST430, are exclusively located in the slime sheath and trail and that fusion genes, containing upstream sequences from the cognate genes, direct correct mRNA accumulation during development and correct localization of the fusion protein. Immunoelectron microscopy shows the ST310 and ST430 proteins to be present throughout the entire thickness of the slime sheath and almost totally absent from the cells of the slug. The genes that encode the ST310 and ST430 polypeptides are inducible by DIF, a stalk-specific inducing agent, and the mRNAs are highly enriched in prestalk over prespore cells. The production of these extracellular proteins by prestalk cells suggests that, in a manner somewhat analogous to that of extracellular matrix proteins of higher eukaryotes, the anterior region of the slug may be responsible for the continuous deposition of a track, along which the slug cells migrate. In the mature culminant, the ST310, and possibly the ST430, protein form part of the stalk tube and stalk cell wall. Therefore, the results also show that there are proteins common to both slime trial and stalk tube, indicating a possible precursor-product relationship between these chemically similar integuments.

Dictyostelium↗
PubMed1987

Immunological analysis of mucin molecules expressed by normal and malignant mammary epithelial cells.

Many existing MAbs raised against the human milk fat globule or against carcinoma cells recognise epitopes on high-molecular-weight glycoproteins. In a comparative ELISA assay a number of these antibodies have been shown to react with an extract of the human milk fat globule. In comparative immunoblots of cultured normal milk cells and breast cell lines, all were found to bind to large molecules which show some variation in molecular weight depending on the cell source. The HMFG-2 antibody, which is widely used in cancer diagnosis, also recognises epitopes on lower-molecular-weight components. In T47D cells these may be as small as 80,000 Mr, and with electron microscopy this cell line can be shown to accumulate HMFG-2-reactive components in the Golgi apparatus. Using an HMFG-2 affinity column we have immunopurified HMFG-2-reactive material from the 2 breast cancer cell lines MCF-7 and T47D and shown that all of the above antibodies react in a solid-phase ELISA with the purified material. In addition to high-molecular-weight components, the immunopurified material was found to contain lower-molecular-weight components including a glycoprotein of 68,000 Mr that was not, however, recognised by the HMFG-2 antibody on a Western blot. We have used this immunopurified material to generate new MAbs. All of these recognise the high-molecular-weight bands seen with the other antibodies, but 2 of them also recognise a band at 68,000 Mr in blots of MCF-7 and T47D. The second-generation antibodies show a spectrum of reactivity on tissues similar to HMFG-2 and one reacts at least as strongly as HMFG-2 with methanol-acetone-fixed sections of breast cancers.

Antibodies, Monoclonal↗