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Biomedical subjects

M Flint

Publications and source records attributed to M Flint.

At least 19 recordsLinked to original sources
PubMed2005

Outcome of revision hip arthroplasty in patients with a previous total hip replacement for developmental dysplasia of the hip.

Our aim was to determine if the height of the cup, lateralisation or the abduction angle correlated with functional outcome or survivorship in revision total hip replacement in patients with a previous diagnosis of developmental dysplasia of the hip. A retrospective investigation of 51 patients (63 hips) who had undergone revision total hip replacement was performed. The mean duration of follow-up was 119 months. Forty-one patients (52 hips) were available for both determination of functional outcome and survivorship analysis. Ten patients (11 hips) were only available for survivorship analysis. The height of the cup was found to have a statistically significant correlation with functional outcome and a high hip centre correlated with a worse outcome score. Patients with a hip centre of less than 3.5 cm above the anatomical level had a statistically better survivorship of the cup than those with centres higher than this. Restoration of the height of the centre of the hip to as near the anatomical position as possible improved functional outcome and survivorship of the cup.

Adult↗
PubMed2004

Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner.

We recently reported that retroviral pseudotypes bearing the hepatitis C virus (HCV) strain H and Con1 glycoproteins, genotype 1a and 1b, respectively, require CD81 as a coreceptor for virus-cell entry and infection. Soluble truncated E2 cloned from a number of diverse HCV genotypes fail to interact with CD81, suggesting that viruses of diverse origin may utilize different receptors and display altered cell tropism. We have used the pseudotyping system to study the tropism of viruses bearing diverse HCV glycoproteins. Viruses bearing these glycoproteins showed a 150-fold range in infectivity for hepatoma cells and failed to infect lymphoid cells. The level of glycoprotein incorporation into particles varied considerably between strains, generally reflecting the E2 expression level within transfected cells. However, differences in glycoprotein incorporation were not associated with virus infectivity, suggesting that infectivity is not limited by the absolute level of glycoprotein. All HCV pseudotypes failed to infect HepG2 cells and yet infected the same cells after transduction to express human CD81, confirming the critical role of CD81 in HCV infection. Interestingly, these HCV pseudotypes differed in their ability to infect HepG2 cells expressing a panel of CD81 variants, suggesting subtle differences in the interaction of CD81 residues with diverse viral glycoproteins. Our current model of HCV infection suggests that CD81, together with additional unknown liver specific receptor(s), mediate the virus-cell entry process.

Amino Acid Sequence↗
PubMed2001

The B-cell receptor of a hepatitis C virus (HCV)-associated non-Hodgkin lymphoma binds the viral E2 envelope protein, implicating HCV in lymphomagenesis.

Hepatitis C virus (HCV) infection is associated with extrahepatic B-cell lymphoproliferative disorders. To determine whether a viral antigen drives this B-cell expansion, the B-cell receptors were cloned from HCV-associated lymphomas and were expressed as soluble immunoglobulins. The rescued immunoglobulins were then tested for their ability to bind the HCV-E2 envelope glycoprotein, an antigen that was previously implicated in the pathogenesis of HCV-associated B-cell diseases. One of 2 lymphoma immunoglobulin test cases bound the E2 protein in a manner identical to a bona fide human anti-E2 antibody. Moreover, it bound E2 from multiple viral genotypes, suggesting reactivity with a conserved E2 epitope. These findings support the hypothesis that some HCV-associated lymphomas originate from B cells that were initially activated by the HCV-E2 protein and might explain the association between HCV infection and some B-cell lymphoproliferative disorders.

Amino Acid Sequence↗
PubMed2001

In search of hepatitis C virus receptor(s).

Since the genomic sequence of HCV was determined, significant progress has been made towards understanding the functions of the HCV-encoded proteins, despite the lack of an efficient in-vitro replication system or convenient small-animal model. The identity of the receptor for HCV remains elusive, however. Low-density lipoprotein receptor, CD81, and GAGs may all act as receptors for HCV, either sequentially or by different viral quasispecies. Recent work using pseudotypic VSV bearing E1 or E2 chimeric molecules showed that entry of the E1 pseudotype can be inhibited by recombinant LDLr, whereas the E2 pseudotype is more sensitive to inhibition by recombinant CD81 or heparin. These results suggest that E1 and E2 may be responsible for interactions with different cellular molecules. It is also conceivable that additional, yet unidentified, cellular proteins are involved in viral binding and entry. Intriguingly, the reports of HCV-RNA associated with PBMC suggest that HCV infection may not be restricted to hepatocytes. Thus, separate reservoirs of virus may exist, and HCV may use different receptors to access these different cell types.

Animals↗
PubMed2001

Psychosocial and health-related characteristics of religious well-being.

This investigation examined the relationship between religions well-being and eight psychosocial and health-related characteristics. This study assessed the hypothesis that religious well-being is related to overall health. Participants were 462 college students at two separate colleges in the Pacific Northwest. Analysis showed those subjects scoring higher on the measure of religious well-being scored lower on indices like loneliness and hopelessness and higher on self-esteem. Alcohol and drug use also differed significantly between the high and the low, religious well-being groups.

Adolescent↗
PubMed2000

Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein.

The E2 protein of hepatitis C virus (HCV) is believed to be a virion surface glycoprotein that is a candidate for inclusion in an antiviral vaccine. A truncated soluble version of E2 has recently been shown to interact with CD81, suggesting that this protein may be a component of the receptor for HCV. When expressed in eukaryotic cells, a significant proportion of E2 forms misfolded aggregates. To analyze the specificity of interaction between E2 and CD81, the aggregated and monomeric forms of a truncated E2 glycoprotein (E2(661)) were separated by high-pressure liquid chromatography and analyzed for CD81 binding. Nonaggregated forms of E2 preferentially bound CD81 and a number of conformation-dependent monoclonal antibodies (MAbs). Furthermore, intracellular forms of E2(661) were found to bind CD81 with greater affinity than the extracellular forms. Intracellular and secreted forms of E2(661) were also found to differ in reactivity with MAbs and human sera, consistent with differences in antigenicity. Together, these data indicate that proper folding of E2 is important for its interaction with CD81 and that modifications of glycans can modulate this interaction. Identification of the biologically active forms of E2 will assist in the future design of vaccines to protect against HCV infection.

Amino Acid Sequence↗
PubMed2000

Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2.

Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).

Amino Acid Sequence↗
PubMed1999

Production of interleukin-12 as a self-processing 2A polypeptide.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-linked subunits (p40 and p35) encoded by separate genes. We used the apparent autocleavage property of a 2A peptide from the foot-and-mouth disease virus (FMDV) to express bovine (Bo) IL-12 as a self-processing polypeptide (p402Ap35). We demonstrate that 2A will mediate the cleavage of p402Ap35 into two separate subunits in a manner similar to that observed during the processing of the FMDV polypeptide. Furthermore, this 2A polypeptide encoded a functional heterodimer, which elicited activities associated with IL-12 in other species. We propose that this strategy of self-processing polypeptides may be used in many applications where the coordinated and stoichiometric expression of complex proteins is required.

Animals↗
PubMed1999

Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81.

A truncated soluble form of the hepatitis C virus E2 glycoprotein, E2661, binds specifically to the surface of cells expressing human CD81 (hCD81) but not other members of the tetraspanin family (CD9, CD63, and CD151). No differences were noted between the level of E2661 binding to hCD81 expressed on the surface of rat RBL or KM3 cells compared to Daudi and Molt-4 cells, suggesting that additional human-cell-specific factors are not required for the primary interaction of E2 with the cell surface. E2 did not interact with African green monkey (AGM) CD81 on the surface of COS cells, which differs from the hCD81 sequence at four residues within the second extracellular region (EC2) (amino acids [aa] 163, 186, 188, and 196), suggesting that one or more of these residues defines the site of interaction with E2. Various recombinant forms of CD81 EC2 show differences in the ability to bind E2, suggesting that CD81 conformation is important for E2 recognition. Regions of E2 involved in the CD81 interaction were analyzed, and our data suggest that the binding site is of a conformational nature involving aa 480 to 493 and 544 to 551 within the E2 glycoprotein. Finally, we demonstrate that ligation of CD81 by E2661 induced aggregation of lymphoid cells and inhibited B-cell proliferation, demonstrating that E2 interaction with CD81 can modulate cell function.

Animals↗
PubMed1999

Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein.

Hepatitis C virus (HCV) glycoproteins E1 and E2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (ER). C-terminal truncation of E2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. We demonstrate cell surface expression of a chimeric glycoprotein consisting of E2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza A virus hemagglutinin (HA), termed E2661-HATMCT. The E2661-HATMCT chimeric glycoprotein was able to bind a number of conformation-dependent monoclonal antibodies and a recombinant soluble form of CD81, suggesting that it was folded in a manner comparable to "native" E2. Furthermore, cell surface-expressed E2661-HATMCT demonstrated pH-dependent changes in antigen conformation, consistent with an acid-mediated fusion mechanism. However, E2661-HATMCT was unable to induce cell fusion of CD81-positive HEK cells after neutral- or low-pH treatment. We propose that a stretch of conserved, hydrophobic amino acids within the E1 glycoprotein, displaying similarities to flavivirus and paramyxovirus fusion peptides, may constitute the HCV fusion peptide. We demonstrate that influenza virus can incorporate E2661-HATMCT into particles and discuss experiments to address the relevance of the E2-CD81 interaction for HCV attachment and entry.

Animals↗
PubMed1998

Heterotypic recognition of recombinant FMDV proteins by bovine T-cells: the polymerase (P3Dpol) as an immunodominant T-cell immunogen.

In this study we have examined the recognition of VP0, VP1, VP2, VP3 and P3Dpol by PBMC and CD4+ T-cells from infected, vaccinated-challenged, and multiply-vaccinated (O1, A24, C1 or ASIA1) cattle using recombinant proteins of an O1 serotype virus. The structural protein VP1 was recognised in an homotypic context whereas VP2, VP3, VP4 and P3Dpol were also recognised by T-cells from animals exposed to heterotypic viruses. Only the non-structural protein P3Dpol was consistently recognised by T-cells from the majority of animals examined and heterotypic recognition correlated with the presence of serologically detectable P3Dpol in purified virus. Thus, P3Dpol is a major cross-reactive immunodeterminant of FMDV, eliciting heterotypic T-cell responses and, therefore, with possible potential for inclusion in a subunit vaccine.

Animals↗
PubMed1998

Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in ELISA.

A profiling ELISA was developed to detect antibody to the non-structural (NS) proteins Lb, 2C, 3A, 3D, and the polyprotein 3ABC, of foot-and-mouth disease virus (FMDV). The assay was used to examine panels of sera from naive cattle, and from experimentally infected or vaccinated animals. All sera from cattle experimentally infected with any of the seven serotypes of FMDV were positive for antibody to 2C, 3A, 3D and 3ABC, and the majority were positive for Lb. The three categories of sera could be differentiated on the basis of the presence or absence of antibody to the structural and/or NS proteins of FMDV. The assay is simple, rapid and reproducible and can be used to identify previous infection in animals which are seropositive for antibody to the structural proteins of the virus. Validating the assay with field sera demonstrated that antibody to 3ABC, and usually one or more of the other non-structural proteins, was detected only in animals reported to have shown clinical signs of FMD. Vaccinated cattle which had received less than five vaccinations, were frequently positive for antibody to 3D but were negative for antibody to 3ABC. Occasional animals which had received more than ten vaccinations had NS protein antibody profiles which were similar to those seen following infection.

Animals↗
PubMed1997

The cleavage activities of aphthovirus and cardiovirus 2A proteins.

The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/beta-glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high (approximately 85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1 D sequences increases the activity. In addition, we have demonstrated that the cardiovirus Theiler's murine encephalomyelitis virus(TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high efficiency--if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into the CAT/GUS artificial polyprotein system (in a single ORF). This recombinant [CAT-deltaTME2A-GUS] polyprotein was able to mediate cleavage with high (approximately 85%) efficiency--directly comparable to the activity observed when FMDV 2A was inserted. A similar insertion into [CAT-GUS] of the C-terminal 19 aa of the cardiovirus encephalomyocarditis virus (EMC) 2A, together with the N-terminal proline residue of protein 2B, produced a [CAT-delta EMC2A-GUS] polyprotein which also mediated cleavage at approximately 85%. Analysis of the products of expression of these artificial polyproteins in a prokaryotic translation system did not, apparently, reveal any GUS cleavage product.

Amino Acid Sequence↗
PubMed1994

Foot-and-mouth disease virus particles contain replicase protein 3D.

An antibody against the Escherichia coli-expressed RNA polymerase of foot-and-mouth disease virus (FMDV) reacts with the virus in ELISA and radioimmunoprecipitation experiments and with a protein of the disrupted virus particle in an immunoblot analysis. Treatment of the virus with trypsin, which cleaves capsid protein VP1 and a 56-kDa polypeptide present in trace amount in the particles, reduces the level of the reaction in ELISA and radioimmunoprecipitation and eliminates the immunoblot reaction. Electron microscopy showed that only approximately 20% of the virus particles reacted with the anti-polymerase antibody, whereas most reacted with an antibody against the immunodominant G-H loop of the virus. In the presence of ammonium ions, the expressed polymerase degrades the RNA of the virus into molecules sedimenting at approximately 12 S, indicating that it can act as a hydrolytic as well as a polymerizing enzyme. Moreover, the RNA in trypsin-treated virus particles is degraded when incubated at 37 degrees C, suggesting that the cleaved 56-kDa protein still possesses hydrolytic activity. In addition, the anti-polymerase antibody, which inhibits the polymerase activity of the E. coli-expressed protein, also partially inhibits the hydrolytic activity of the previously described endonuclease of the virus particle, suggesting that this enzyme is identical with the polymerase or forms part of it.

Antibodies, Viral↗
PubMed1986

Changes in the morphology and synthetic activity of cultured rat tail tendon.

Isolated single fascicles from tail tendons of young rats were freed of epitenon cells and cultured in vitro for up to 7 days. The tissue remained viable, as judged by the structural integrity of cell organelles and the ability to synthesize DNA and glycosaminoglycans (GAG). The rate of DNA synthesis peaked after 2 days in culture and decreased slowly thereafter. Concomitantly, an increase in cell number was noted at the periphery of the fascicle. GAG production also increased during culture, sulphated GAG being increased proportionately more than hyaluronic acid. Dermatan sulphate was the predominant sulphated GAG in freshly isolated fascicles, but in cultured tissue, the newly synthesized sulphated GAG was more sensitive to degradation by chondroitinase AC and had an increased electrophoretic mobility. fine structural changes were observed in cultured tissues such as the retraction of cell processes. rounding up of cell bodies and the appearance of gaps between collagen fibrils. Cultured tenocytes also frequently contained apparently phagocytized collagen fibrils which were not seen in freshly isolated fascicles, and this appearance was suggestive of collagen degradation occurring in vitro, although no change in the total hydroxyproline content was noted. The data show that when individual fascicles are cultured in vitro they undergo a process of matrix remodelling which has features in common with events occurring in vivo when tendons have been surgically manipulated.

Animals↗