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T J Harrison

Publications and source records attributed to T J Harrison.

At least 145 records · Page 8Linked to original sources
PubMed1997

Macular pattern retinal dystrophy, adult-onset diabetes, and deafness: a family study of A3243G mitochondrial heteroplasmy.

PURPOSE: To correlate mitochondrial DNA (mtDNA) mutation with phenotypic expression in three members of a Finnish family with macroreticular pattern dystrophy, non-insulin-dependent diabetes mellitus, and deafness. METHODS: A multiplex polymerase chain reaction/allele-specific oligonucleotide method was used to screen 10 mtDNA point mutations known to cause mitochondrial DNA disorders, often characterized by myopathy, retinopathy, or both. Quantitative analysis of mutant mitochondrial DNA was performed in three tissue types in each of three family members by determining the percentage of mutant mtDNA in blood, buccal cells, and hair follicles. RESULTS: A heteroplasmic A3243G mtDNA point mutation was found in each of the three family members studied. Heteroplasmy refers to the coexistence of normal and mutant mitochondria in the same cell. The average percentage of mutant heteroplasmy ranged from 11% to 25%. The severity of disease symptoms did not appear to correlate with the average degree of mutant heteroplasmy in the three tissues analyzed. CONCLUSIONS: Molecular confirmation in this family emphasizes the importance of mitochondrial DNA mutation analysis in patients with macular pattern retinal dystrophy and other mitochondrial associated nonocular disease, such as non-insulin-dependent diabetes mellitus and deafness. The detection of a disease-associated mitochondrial DNA mutation warrants genetic counseling, appropriate patient follow-up, and possibly the molecular testing of other at-risk family members.

Age of Onset↗
PubMed1997

Production of hepatitis B virus covalently closed circular DNA in transfected cells is independent of surface antigen synthesis.

Covalently closed circular DNA (cccDNA) is the first hepatitis B virus (HBV) DNA replicative intermediate formed from the genomic DNA and serves as the template for synthesis of viral mRNA and pregenomic RNA. It also appears to be produced by intracellular recycling of relaxed circular DNA intermediates. Here, we report that none of the forms of HBV surface antigen affect this intracellular recycling of HBV DNA. Elimination of the initiation codons for the large and middle surface proteins did not affect the detection of surface antigen in culture supernatants. In contrast, detection of surface antigen was eliminated by the removal of, or the introduction of two stop codons downstream of, the initiation codon of the small (major) surface antigen. None of the mutations affected the production, in transfected HepG2 cells, of HBV DNA replicative intermediates, including cccDNA.

Cell Line↗
PubMed1996

Expression of a human cytomegalovirus gp58 antigenic domain fused to the hepatitis B virus nucleocapsid protein.

Hepatitis B virus core antigen (HBcAg) has been used as a carrier for expression and presentation of a variety of heterologous viral epitopes in particulate form. The aim of this study was to produce hybrid antigens comprising HBcAg and an immunogenic epitope of human cytomegalovirus (HCMV). A direct comparison was made of amino and carboxyl terminal fusions in order to investigate the influence of position of the foreign epitope on hybrid core particle formation, antigenicity and immunogenicity. HCMV DNA encoding a neutralising epitope of the surface glycoprotein gp58 was either inserted at the amino terminus or fused to the truncated carboxyl terminus of HBcAg and expressed in Escherichia coli. The carboxyl terminal fusion (HBc3-144-HCMV) was expressed at high levels and assembled into core like particles resembling native HBcAg. Protein with a similar fusion at the amino terminus (HCMV-HBc1-183) could not be purified or characterised immunologically, although it formed core like particles. HBc3-144-HCMV displayed HBc antigenicity but HCMV antigenicity could not be detected by radioimmunoassay or western blotting using anti-HCMV monoclonal antibody 7-17 or an anti-HCMV human polyclonal antiserum. Following immunisation of rabbits with HBc3-144-HCMV, a high titre of anti-HBc specific antibody was produced along with lower titres of HCMV/gp58 specific antibody.

Animals↗
PubMed1996

Genotypes of hepatitis C virus in Nigeria.

A pilot survey of hepatitis C virus (HCV) infection in Nigeria was carried out on healthy adult blood donors and children of preschool age. Sixteen of 200 (8%) donors were positive for antibodies using a second generation enzyme-linked immunosorbent assay (ELISA) but all of the children were negative. Supplementary testing of the ELISA-positives using a recombinant immunoblot assay (RIBA-2) confirmed the presence of antibody in four and two others were indeterminate. Four of the anti-HCV-positive sera and one found positive by ELISA but which was negative by RIBA-2 were found to be positive for HCV RNA using reverse transcriptase-polymerase chain reaction (RT-PCR) and primers specific for the 5' untranslated region (5'UTR) of the HCV genome. The NS5 and core regions also were amplified and the PCR products from all three regions were sequenced. Sequences from the 5'UTR could be divided into two groups: one group comprised three isolates with greater than 95% sequence identity with published sequences of genotype 1 and the other comprised two isolates with greater than 93% sequence identity with genotype 4. Analysis of three sequences amplified from the NS5 region confirmed this assignment to genotypes 1 and 4. Pairwise comparisons of the NS5 region sequences with representatives of 1a, 1b, 1c (for the first group) and 4a-4h (for the second group) show the first group to include subtypes classifiable as 1a and a novel sequence and the second group to include a novel sequence within genotype 4. Sequence analysis of the core region was consistent with this interpretation. These data confirm the presence of at least two major HCV genotypes in Nigeria (genotypes 1 and 4) and we report two novel sequences which have been designated provisionally as genotypes 1d and 4i.

Adult↗
PubMed1996

Selection of mutations in the hepatitis B virus polymerase during therapy of transplant recipients with lamivudine.

We describe mutations in the hepatitis B virus (HBV) polymerase gene in viruses which reactivated in two patients during therapy with -2'-deoxy-3'-thiacytidine, or lamivudine (3TC), and following orthotopic liver transplantation for chronic hepatitis B. Virus resistance to 3TC is associated with mutations which lead to amino acid substitutions in the highly conserved tyr-met-asp-asp (YMDD) motif, part of the active site of the polymerase, and which parallel those seen in resistant human immunodeficiency virus (HIV). Substitutions of valine and isoleucine for methionine were found in the two cases. The significance of single secondary mutations, which differ between viruses from the two patients, remains to be determined. Thus, viral resistance to lamivudine of hepatitis B virus mimics that of HIV and can occur in the setting of immunosuppression after liver transplantations.

Adult↗
PubMed1996

Current issues in the diagnosis of hepatitis B and C virus infections.

Diagnostic tests for hepatitis B virus infection are well established, although development of format and components continues. Variants of HBV with amino acid changes in the major antigenic determinant of the surface protein (HBsAg), and which may escape neutralisation by anti-HBs, have been described in many countries. The increasing reliance on monoclonal antibodies in the formulation of new assays for HBsAg raises the question of whether these surface variants may escape detection. The prevalence of variants which are unable to synthesise the e antigen (precor mutants), especially in certain geographical areas, means that the absence of HBeAg in carriers, with or without anti-HBe, does not necessarily indicate clearance of viraemia. The discovery of hepatitis C virus was followed rapidly by evidence of considerable sequence variation among different isolates. At least six major genotypes of HCV are recognised worldwide. Nonetheless, current assays seem reliable for detection of antibodies to this diverse virus. Assays for antigen are not available and diagnosis of viraemia requires sensitive detection of the viral genome, for example using reverse transcription and the polymerase chain reaction (RT-PCR). Evaluation of the efficacy of anti-viral therapy requires quantitative assays, adding a further degree of complexity. Other tests, such as the branch DNA (bDNA) assays are available, but lack sensitivity. Whether different genotypes of HCV vary in their pathogenicity and response to therapy remains contentious and convenient methods for determination of the genotype (or equivalent serotyping assays) are required to resolve this issue.

Journal Article↗
PubMed1996

HCV-associated hepatocellular carcinoma without cirrhosis.

BACKGROUND/AIMS: Hepatocellular carcinoma is an aggressive malignancy and carries a poor prognosis. Hepatitis B and C virus infection, cirrhosis and aflatoxin B1 exposure are considered major risk factors. The role of hepatitis C virus in the causation of hepatocellular carcinoma has been debated. It is a positive, single-stranded RNA virus without a DNA intermediate in its replicative cycle, so that integration of hepatitis C virus nucleic acid sequences into the host genome seems unlikely. The most plausible explanation of hepatitis C virus-associated hepatocellular carcinoma so far is that the virus causes necroinflammatory hepatic disease with vigorous regeneration, fibrosis, and eventually cirrhosis. The aim of this study was to examine the relationship of hepatitis C, cirrhosis and hepatocellular carcinoma. METHODS: Sixty-six consecutive patients with hepatocellular carcinoma undergoing resection or transplantation at the Royal Free Hospital were reviewed. A combination of serological data and polymerase chain reaction assay was used to assign hepatitis C virus and hepatitis B virus infection. RESULTS: We found four HCV-RNA positive patients with hepatocellular carcinoma without cirrhosis. All four cases were positive for HCV-RNA and negative for all markers of hepatitis B virus infection. CONCLUSIONS: These four cases show that hepatocellular carcinoma may develop in patients with hepatitis C virus without pre-existing cirrhosis. However, the precise role of hepatitis C virus in hepatocarcinogenesis, the carcinogenic potential of the different genotypes and whether this role is influenced by other risk factors still have to be clarified.

Aged↗
PubMed1996

Genetic variation in hepatitis B virus.

In wild type isolates of hepatitis B virus (HBV), the sequence of the genome may vary in up to 10% of nucleotide positions despite conservation of coding and function. Changes as small as of a single base, however, may disrupt expression of viral proteins. For example, precore mutants fail to synthesize HBeAg and point mutations in the region encoding the a determinant of HBsAg may have a profound effect on antigenicity. Investigation of HBV sequences in virus from patients with unusual serological profiles continues to reveal novel variants.

Genome, Viral↗
PubMed1995

Precore and core mutations in HBV from individuals in India with chronic infection.

Ten hepatitis B surface antigen seropositive carriers (5 asymptomatic and 5 with chronic liver disease) were tested for HBeAg/anti-HBe and for HBV-DNA using the polymerase chain reaction. Five were DNA-positive, 2 with HBeAg and 3 with anti-HBe. Nucleotide sequences were determined for these 5 cases. Hepatitis B virus DNA from one cirrhotic carrier with anti-HBe had a mutation in the precore region (nucleotide position 1862) which may affect signal peptide cleavage and HBeAg synthesis. In the other 2 anti-HBe- and DNA-positive cases, a cirrhotic carrier and an asymptomatic case, there was a mutation at nucleotide position 1896 leading to a termination codon in the precore region. In all 5 patients, except for one or two missense mutations, there was no significant variation in the core region.

Amino Acid Sequence↗
PubMed1995

Amplification and sequence analysis of the precore and core region of the HBV genome from sera of Spanish patients with HBV2-like infections.

Hepatitis B virus (HBV) infections are occasionally associated with unusual serological profiles which may be attributed to the lack of immune responsiveness of the patients or to variants of the virus. The term HBV2 was introduced to describe potential variants of HBV which do not elicit a detectable immune response to the nucleocapsid (anti-HBc) in apparently immune-competent patients. The entire nucleotide sequences of the nucleocapsid genes of viruses from two Spanish patients with HBV2-like infections are reported here. In-frame deletions and other mutations in these sequences may account, at least in part, for the unusual serological profiles of the patients. Antibody responses to viral proteins containing deletions may not be detected by commercially available assays.

Amino Acid Sequence↗
PubMed1995

Molecular epidemiology of hepatitis B virus vaccine variants in Singapore.

Perinatal infection with variants of hepatitis B virus occurs despite combined immunoprophylaxis with hepatitis B immunoglobulin and currently licensed plasma-derived and recombinant yeast hepatitis B vaccines. Several variants have been detected during a large study of infants born to carrier mothers in Singapore. The most frequent variant was a virus in which a single amino acid substitution Gly to Arg occurred at amino acid position 145 of the outer protein coat of the virus. Similar mutations have been described in Italy, Japan, the USA and a number of other countries. The emergence of antibody escape mutants is a cause for concern for the detection of virus and possibly for future immunization programmes.

Amino Acids↗
PubMed1995

Diversity of genotypes of hepatitis C virus in southern India.

A second generation assay for antibody to hepatitis C virus (anti-HCV) was used to screen 78 southern Indian individuals with a high risk of infection. RT-PCR targeted at the 5' end untranslated region (5'UTR) of the HCV genome was used to evaluate evidence of viraemia in 32 anti-HCV positive sera. The PCR products amplified from the 5'UTR of the HCV genome from 24 patients were sequenced, revealing the existence of two distinct groups of sequences: 21 corresponded to HCV type 1 while the other three sequences had 95% to 99% identity to HCV type 3. Two of these three isolates had more than 90% nucleotide identity in the NS5 region to established 3b sequences whereas the other had less than 74% nucleotide identity to any of the published genotype 3 (3a, 3b, 3c, 3d, 3e and 3f) sequences. However, a search of the EMBL nucleotide database revealed 91% identity to the unpublished sequence of an isolate of HCV from Indonesia. We provide evidence that these two isolates may represent a novel subtype within genotype 3. Our data also suggest that HCV genotype 1 predominates over HCV genotype 3 in southern India.

Base Sequence↗
PubMed1994

Southern hybridisation analysis of HBV DNA in peripheral blood leucocytes and of different cell types: changes during the natural history and with interferon-alpha therapy in patients with hepatitis B virus infection.

Hepatitis B virus (HBV) DNA was evaluated in peripheral blood mononuclear cells (PBMC) from 50 individuals utilising Southern hybridisation analysis. HBV DNA sequences were detected in PBMC from 16/29 (55 percent) of chronic hepatitis B virus (HBV) carriers with serum HBeAg and HBV DNA, compared with 1/8 (13%) of carriers with anti-HBe and HBV DNA negative (P = NS). Two of 7 patients with previous HBV infection and chronic liver disease had detectable HBV DNA in PBMC. Of the 19 patients with HBV DNA in PBMC, 18 had high molecular weight species. In addition, five of these had free, monomeric HBV DNA and six patients had low molecular weight bands. For nine of the above patients, total peripheral blood leucocytes were separated into PBMC and polymorphonuclear cells. Four had HBV DNA in PBMC only, two only in polymorphonuclear cells and three in both types of cell. Eleven patients with chronic HBV infection were studied at monthly intervals for 6 months. Six were untreated and five received IFN-alpha. Three patients who responded to IFN-alpha had HBV DNA present in PBMC before therapy, and two became negative. Two of 3 untreated patients had intermittent HBV DNA in PBMC and the other remained persistently negative. Of the patients positive on more than one occasion, the pattern of HBV DNA was similar. Peripheral blood leucocytes often contain multimers of free HBV DNA, more commonly in patients with serum HBeAg and HBV DNA and may occur even in the absence of serum HBsAg. These findings have implications for recurrence of disease after hepatic transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Southern↗
PubMed1994

Exclusion in liver by polymerase chain reaction of hepatitis B and C viruses in acute liver failure attributed to sporadic non-A, non-B hepatitis.

Hepatitis B and C viruses have been implicated in a few cases of acute liver failure attributed to sporadic (community acquired) non-A, non-B hepatitis, but reports are conflicting. We determined whether hepatitis B virus and hepatitis C virus were detectable in prospectively stored hepatectomies from seven British patients grafted for acute liver failure attributed to sporadic non-A, non-B hepatitis. For hepatitis B virus, we used nested polymerase chain reaction with primers to the core and surface regions. For hepatitis C virus, we used one round of reverse transcription polymerase chain reaction with primers to the 5' untranslated region and Southern hybridization using an internal oligonucleotide probe as well as nested PCR for the E1 region. Positive controls were native livers from two patients with unequivocal fulminant hepatitis B and from four patients with cirrhosis attributed to hepatitis C virus. Our negative findings suggest that, in the UK, acute liver failure attributed to sporadic non-A, non-B hepatitis most likely is caused by agent/s other than hepatitis B and C viruses.

Blotting, Southern↗
PubMed1994

Analysis of integrated hepatitis B virus DNA and flanking cellular sequences in the hepatocellular carcinoma cell line HCC36.

A hepatocellular carcinoma cell line, HCC36, was established from an adult HBV carrier in Taiwan. From Southern blot analysis, there were at least four sites of integration of HBV DNA, and no viral replicative intermediates were detected. A genomic library was constructed from HCC36 DNA, and two phage clones, designated lambda 36A and lambda 36B, were shown to contain HBV DNA and flanking cellular sequences. In lambda 36A, HBV DNA sequences were quite conserved, and 7.4% base variation was detected. The viral sequences in lambda 36A and lambda 36B differed in only four bases, in addition to the microdeletion and -insertion observed in lambda 36B. The flanking cellular sequences identified in lambda 36A were human Alu sequences and in lambda 36B satellite sequences.

Base Sequence↗
PubMed1994

Dot blot hybridization assay for the detection of duck hepatitis B virus DNA among healthy Indian country ducks.

A DNA molecular hybridization technique employing Duck Hepatitis B Virus (DHBV) DNA of 3.0 kilobase pairs as a probe was used to screen for the presence of DHBV DNA in blood samples, collected from 90 apparently healthy Indian country ducks. Six out of 90 ducks showed positivity for DHBV DNA in serum (5.4%) and only 4 out of 6 DHBV DNA positive ducks answered in Counter Immuno Electrophoresis (CIEP) using specific antibody against DHBV surface antigen raised in Guinea pig. The results indicate the pilot observation that (a) DHBV carrier status exists to a tune of 5.4% among apparently healthy Indian country ducks also and (b) DHBV probe can be employed as a sensitive and reliable assay for DHBV DNA detection in DHBV infected ducks.

Animals↗