Biomedical subjects

M J McGarvey

Publications and source records attributed to M J McGarvey.

At least 19 recordsLinked to original sources

PubMed2004

Negative correlation between intrahepatic expression of hepatitis C antigens and apoptosis despite high-level expression of Fas and HLA antigens.

The role of virus-related apoptosis in hepatic injury in chronic HCV is unclear. It is unknown whether HCV induces apoptosis directly or whether cellular injury is immunologically mediated. We studied the relationship between infected hepatocytes, apoptosis and necroinflammation. We established a Fluorescence Activated Cell Sorter (FACS) based intracellular staining technique for the HCV NS3 protein and examined intrahepatic viraemia, disease activity and apoptosis. We also stained infected cells for expression of human leucocyte antigen (HLA) class I and Fas antigens. We examined 34 liver biopsies (24 from patients with HCV) and found marked variation in the proportion of infected cells (2.5-42%). The number of infected cells correlated with serum viraemia but not histology. The number of infected cells was inversely related to the number of apoptotic cells (P < 0.001); infected cells expressed both HLA class I (14 cases) and Fas antigens (12 cases). The number of hepatocytes infected with hepatitis C is variable and does not influence histological activity. In infected patients, the majority of HCV-positive hepatocytes express target molecules for activated lymphocytes (Fas and HLA class I antigens) but they do not undergo apoptosis, suggesting that hepatitis C may inhibit apoptosis by modulating intracellular pro-apoptotic signals.

Adult↗

PubMed2001

Subcellular localisation of the X protein in HBV infected hepatocytes.

Hepatitis B virus X protein (HBx) is a multifunctional protein that exerts its effects primarily by acting as a transcriptional transactivator of viral and multiple host cell genes. HBx is thought to be essential for maintaining viral replication and has been implicated in the development of hepatocellular carcinoma in patients chronically infected with hepatitis B virus. Very little is known about its functional mechanisms and although interactions with several nuclear and cytoplasmic proteins have been demonstrated in vitro, there is no clear consensus as to where HBx localises in infected hepatocytes. In this study, the expression and intracellular distribution of HBx were examined in human liver biopsies using an anti-HBx rabbit polyclonal antiserum. HBx was detected in a high proportion (69%) of samples from patients with chronic HBV infection. Detection of HBx correlated with the absence of cirrhosis and the presence of serum e-antigen. HBx was detected predominantly in the cytoplasm; however, it was also found in the nuclei of up to 20% of positively stained hepatocytes, either exclusively nuclear or localised both in the nucleus and cytoplasm within the same cell. Furthermore, the intracellular distribution of HBx was analysed in transfected Huh-7 cells by confocal microscopy, using the monoclonal antibody 16F1. In these experiments, a substantial nuclear detection was confirmed in a significant proportion of HBx expressing cells. The data indicate a high functional significance of nuclear HBx, consistent with the concept that transactivation may involve interactions with nuclear proteins.

Adult↗

PubMed1999

Subcellular localisation of NS3 in HCV-infected hepatocytes.

Hepatitis C virus (HCV) NS3 is a multifunctional protein with both protease and helicase activities and has been shown to interact with host cell proteins. It is shown that NS3 is present in the hepatocytes from patients with chronic HCV infection by using anti-NS3 antisera. NS3 is detectable in approximately 4% of the hepatocytes from these patients. In most infected cells, NS3 is present in the cytoplasm; however, in a minority of HCV-infected cells, both the cytoplasm and the nucleus or the nucleus on its own are positive for NS3. The presence of NS3 in the nuclei of hepatocytes in chronically infected patients indicates that the protein may play a role other than in virus replication, such as in persistence of HCV infection.

Blotting, Western↗

PubMed1999

Characterization and mutational analysis of the helicase and NTPase activities of hepatitis C virus full-length NS3 protein.

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) possesses three activities which are likely to be essential for virus replication; a serine protease located in the N terminus and helicase and NTPase activities located in the C terminus. Sequence analysis of the helicase/NTPase domain has identified motifs indicative of the DEAD-box family of helicases. Here we present the characterization of the helicase and NTPase activities of full-length NS3, expressed as a His-tagged fusion protein in E. coli, and make comparisons with published data of NS3 helicase domain alone. The helicase and NTPase activities of full-length NS3 have been demonstrated and we have characterized the effects of amino acid substitutions on conserved motifs of NS3 helicase. Helicase and NTPase activities were dependent on Mg2+ and ATP and inhibited by monovalent cations. NS3 was able to hydrolyse all four NTPs and dNTPs to drive DNA duplex unwinding but with differing abilities. NTPase activity was stimulated by all polynucleotides tested, with poly(U) having the greatest effect. Mutational analysis of conserved motifs of NS3 helicase showed all conserved residues to be required for optimal activity. These results are in accord with a recently proposed model for NS3 helicase activity.

Acid Anhydride Hydrolases↗

PubMed1998

Respiratory syncytial virus RNA in cells from the peripheral blood during acute infection.

It is not known whether respiratory syncytial virus spreads beyond the respiratory tract. With the use of reverse transcriptase polymerase chain reaction, we found viral and transcribed RNA in cells from the arterial blood of four children with bronchiolitis but none in serum or cerebrospinal fluid. Respiratory syncytial virus might therefore spread outside the respiratory tract.

Bronchiolitis, Viral↗

PubMed1997

The precore sequence of hepatitis B virus is required for nuclear localization of the core protein.

The cellular localization of the precore/core and core proteins was studied by immunofluorescence following transfection of 143 thymidine kinase-negative (TK ) and Hep-G2 cells with expression constructs containing wild-type (hepatitis B e antigen [HBeAg]-positive) and precore mutant (HBeAg-negative) sequences. Precore/core constructs with the wild-type phenotype result in strong nuclear staining, while, in contrast, constructs expressing core antigen alone have strong cytoplasmic staining. These differences in the pattern of immunofluorescence staining may be caused by expression of the precore/core protein, some of which may be translocated into the nucleus, following removal of the signal peptide. In vitro translation experiments showed that the main protein products obtained in the presence of microsomal membranes were the precore/core protein and a truncated product representing the same protein without its signal peptide. Core protein expression from the precore mutant constructs was very much reduced, indicating that translational re-initiation was not very efficient. The significance of the precore/core protein being present in the nucleus is not clear, but suggests that it may be important in the replicative cycle of the virus. Finally, HBeAg produced by some of the constructs could not be detected because amino acid substitutions affected antibody-binding epitopes.

Amino Acid Sequence↗

PubMed1997

Hepatitis G virus infection: clinical characteristics and response to interferon.

A new member of the Flaviviridae family has recently been cloned and completely sequenced. The new virus, tentatively named hepatitis G virus (HGV) and known to be closely related to GB virus C (GBV-C), is transmitted by blood and blood products, intravenous drug use and other behaviour associated with a high risk of parenteral exposure to blood. The association of the virus with hepatitis is demonstrated by the presence of raised liver transaminase (alanine aminotransferase, ALT) levels in patients infected with HGV in the absence of other identifiable causes of hepatitis. No patient sera from groups exposed to blood and blood products were found to be positive when tested for the presence of GBV-A or GBV-B sequences, two other recently described flaviviruses. Forty-five per cent of the HGV-infected patients investigated had normal ALT suggesting the existence of a normal carrier state. Persistent infection of up to 13 years duration was observed. Co-infection with hepatitis B or hepatitis C viruses (HBV and HCV) was commonly seen presumably because of shared risk factors. None of five patients with fulminant hepatic failure was positive for HGV infection. The virus is sensitive to interferon-alpha, but sustained responses were not seen with the treatment regimens used for HBV and HCV. Viral titres increased during immunosuppression following liver transplantation and the higher levels of viraemia were in one case accompanied by elavated ALT. Whether HGV (GBV-C) replicates in the liver in some or all cases remains to be established. Preliminary data suggest that it is present within peripheral blood lymphocytes.

Antiviral Agents↗

PubMed1996

The expression of hepatitis B virus polymerase in hepatocytes during chronic HBV infection.

A recombinant protein corresponding to part of the amino-terminal domain of hepatitis B virus (HBV) polymerase was expressed in Escherichia coli. Antisera raised against this protein stained hepatocytes, from human liver biopsies, predominantly in the nucleus but some cytoplasmic staining was also observed. No staining was observed in hepatocytes from uninfected patients. Liver biopsies, taken from patients who were infected with human immunodeficiency virus (HIV) as well as HBV showed more intense staining with these antisera than that seen in patients who were infected with HBV alone. The staining pattern suggests that either the whole HBV polymerase protein, or a portion encoding the amino-terminal domain, is translocated to the nucleus. This event may be an important early step in replication of the HBV genome.

AIDS-Related Opportunistic Infections↗

PubMed1995

The GB hepatitis viruses.

The genomes of three new flavi-like viruses, GBV-A, GBV-B and GBV-C have been identified. Nucleic acid molecules corresponding to the genomes of GBV-A and GBV-B were isolated from tamarins with hepatitis which had been infected with the GB agent. RNA sequences corresponding to GBV-C have been shown to be present in sera from humans with non-A-E hepatitis. Sequence comparisons show that these three viruses are more closely related to each other and to hepatitis C virus (HCV) than to any other known viruses. Together with HCV they appear to form a discrete cluster of related viruses within the larger genus of flaviviridae. The pathological significance of these viruses and their association with hepatitis is currently emerging.

Animals↗

PubMed1993

Expression of the terminal protein of hepatitis B virus is associated with failure to respond to interferon therapy.

The terminal protein domain of the hepatitis B viral polymerase can inhibit the cellular response to interferon. To clarify the clinical relevance of this inhibitory effect, we examined the expression of terminal protein in liver biopsy specimens from patients with chronic hepatitis B infection. We found that expression of terminal protein is associated with a failure of hepatocytes to respond to interferon, as assessed by expression of the interferon-inducible protein beta 2-microglobulin. Patients whose liver specimens contained large numbers of cells expressing terminal protein tended not to respond to treatment. During interferon therapy the percentage of cells expressing terminal protein remained constant, but treatment significantly reduced the number of cells that expressed the hepatitis B nucleocapsid protein. Hence expression of terminal protein in a cell may prevent elimination of the virus by interferon therapy.

Adult↗

PubMed1991

A recombinant vaccinia virus expressing hepatitis A virus structural polypeptides: characterization and demonstration of protective immunogenicity.

A recombinant vaccinia virus containing most of the P1 region of hepatitis A virus (HAV) was constructed. Cell lysates of cultures infected with the virus contained HAV proteins detectable by radioimmunoassay. Western blot analysis revealed the presence of a single protein of Mr 60K to 62K, bearing epitopes from structural polypeptides VP4, -3 and -2, and the N terminus of VP1. The size of the protein suggests that at least some of the vaccinia virus thymidine kinase is also expressed. Inoculation of tamarin monkeys with the recombinant virus resulted in the development of a specific anti-HAV immune response which was protective against challenge with a virulent strain of HAV. Recombinant viruses expressing the above region of HAV or the proteins expressed by such viruses may be useful in the development of a vaccine suitable for use in man.

Animals↗

PubMed1990

Histological and immunohistochemical study of hepatitis B virus in human immunodeficiency virus infection.

Because the risk factors for human immunodeficiency virus (HIV) infection and hepatitis B (HBV) are similar and therefore coinfection is not uncommon, a detailed histological and immunohistochemical study of chronic hepatitis B infection in a group of 20 HIV positive Caucasian males (who did not have AIDS) and 30 HIV negative controls were undertaken. Using both the conventional histological classification and the Knodell histological activity index it was shown that HIV negative patients were more likely to have active disease and also more scarring than HIV positive patients. Hepatitis B surface antigen (HBsAg) expression was not significantly different between the two groups but expression of hepatitis Be antigen (HBeAg) and HBV-DNA polymerase was greater in those who were HIV positive. HIV positive patients are therefore more likely to have immunohistochemical markers of active viral replication, although histologically, liver disease is less severe. These findings have important implications for assessing the biopsy specimens in this group of patients and for treatment strategies aimed at improving their immune function.

Adult↗

PubMed1989

Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection.

Some patients with chronic hepatitis B virus (HBV) infection are HB e antigen (HBeAg) negative, have circulating HBV particles, and often have especially severe chronic hepatitis. To test the hypothesis that the absence of HBeAg production may be due to a change in the nucleotide sequence of the pre-core region of the genome, 18 Greek and 3 non-Greek patients positive for HB surface antigen underwent direct sequencing of HBV-DNA amplified from sera. In 7 out of 8 HBeAg negative patients, two mutations (guanosine to adenosine) were found in the terminal two codons of the pre-core region, giving the sequence TAGGACATG. The remaining patient had the first mutation only. The sequence TGGGGCATG was found in 4 of 5 of the HBeAg positive patients. The first mutation results in a translational stop codon that is predicted to result in failure to produce HBeAg. The rest of the pre-core region in the HBeAg negative patients was otherwise homologous to that of the HBeAg positive patients and to known sequences.

Amino Acid Sequence↗

PubMed1989

Studies of GB hepatitis agent in tamarins.

Three tamarins (Saguinus labiatus), two of which had previously been infected with hepatitis A virus and parenteral non-A, non-B hepatitis, were inoculated intravenously with the agent of GB hepatitis. All three animals developed alanine aminotransferase abnormalities 2 weeks after inoculation. Peak alanine aminotransferase levels were recorded 4 weeks postinoculation. These declined thereafter but continued to fluctuate at abnormal levels for 32 weeks. Liver biopsies showed liver cell swelling and inflammation with focal necrosis. Portal tracts and areas around central veins were heavily infiltrated with mononuclear cells. A fourth animal (no previous exposure to hepatitis viruses) inoculated with GB was killed on Day 15 postinoculation. Serum and extracts of liver and feces from this day were used as inocula for three other animals. Only the serum and liver extract transmitted GB hepatitis. The fecal specimen did not transmit and a fecal extract taken at a later date from another animal was also noninfectious. GB hepatitis virus is distinct from the viruses causing Type A and blood-borne non-A, non-B-hepatitis. Although the virus is present in serum and has previously been transmitted per os, it is not shed in feces.

Alanine Transaminase↗

PubMed1989

Subclasses of antibodies to hepatitis B core antigen in chronic HBV infection: changes during treatment with alpha interferons and predictors of response.

Response to interferon therapy in chronic hepatitis B virus (HBV) carriers is preceded by the appearance of IgM class anti-HBc (antibody to hepatitis B core antigen). The temporal relationship and magnitude of the IgM anti-HBc response is variable suggesting that the antibody is not directly involved in hepatocyte lysis, but is merely a marker of a changed state of immunity to the nucleocapsid proteins, induced by interferon. IgG 1, 2, 3, and 4 class anti-HBc did not change during therapy, but IgG 3 anti-HBc was significantly lower in responders than non-responders. IgG anti-HBc of all subclasses was absent in two Chinese HBV carriers. Lower than normal titres of anti-HBc (p less than 0.001) were detected in human immunodeficiency virus antibody positive (anti-HIV) HBV carriers. These data indicate the presence of altered immunity to the nucleocapsid antigens in these two types of chronic HBV carrier that are known to respond poorly to antiviral therapy.

Adolescent↗

PubMed1986

The phosphorylation of ribosomal protein S6 by protein kinases from cells infected with pseudorabies virus.

We examined the ability of protein kinase activities from BHK (baby-hamster kidney) cells infected with pseudorabies virus to catalyse the phosphorylation of ribosomal protein S6 in vitro. When the cytosol from infected cells was fractionated on DEAE-cellulose, 40S ribosomal protein kinase activity was found associated with the two isoforms of the cyclic AMP-dependent protein kinase, protein kinase C and a protein kinase (ViPK, virus-induced protein kinase) only detected in infected cells. The phosphorylation of ribosomal protein by ViPK was of particular interest because the appearance of the protein kinase and the increase in the phosphorylation of protein S6 in infected cells shared a similar time course. At moderate concentrations of KCl the major ribosomal substrate for ViPK was ribosomal protein S7, a protein not found to be phosphorylated in vivo. However, at 600 mM-KCl, or in the presence of 5-10 mM-spermine at 60-150 mM-KCl, the phosphorylation of ribosomal protein S7 was suppressed and ribosomal protein S6 became the major substrate. The maximum stoichiometry of phosphorylation obtained under the latter conditions was 1-2 mol of phosphate/mol of S6, and only mono- and di-phosphorylated forms of S6 were detected on two-dimensional gel electrophoresis. As the infection of BHK cells by pseudorabies virus results in the appearance of phosphorylated species of S6 containing up to 5 mol of phosphate/mol of S6 protein, it appears unlikely that ViPK alone can be responsible for the multiple phosphorylation seen in vivo. Nevertheless, tryptic phosphopeptide analysis did indicate that in vitro ViPK catalysed the phosphorylation of at least one of the sites on ribosomal protein S6 phosphorylated in vivo, so that a contributory role for the enzyme in the phosphorylation in vivo cannot be excluded.

Cells, Cultured↗

PubMed1984

Identification and expression in Escherichia coli of merozoite stage-specific genes of the human malarial parasite Plasmodium falciparum.

The key steps in the development of a malaria vaccine through gene cloning are the identification of the proteins involved in host protective immunity and the cloning, identification, and expression of the genes coding for these proteins. Recent data have indicated that certain proteins synthesized at the late schizont-merozoite stage of Plasmodium falciparum play a major role in malaria immunity. This paper reports the identification, in a cDNA library, of recombinant clones corresponding to genes expressed specifically during the late schizont-merozoite stage of P. falciparum development. The 132 cDNA clones thus identified out of 10,000 were found to correspond to only 12 different genes, probably representing most of the major schizont-merozoite specific genes. The stage-specific cDNAs can be efficiently expressed in Escherichia coli cells. The protein products of some of these clones are recognized by monoclonal antibodies specific for late schizont-merozoite proteins. We conclude that only a small set of genes is specifically induced in the schizont-merozoite stage and that the stage-specific cDNA clones we have isolated are very likely to include the genes coding for the immunologically relevant proteins of P. falciparum.

Animals↗

PubMed1983

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells.

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.

Adenosine Triphosphate↗