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Stephen Locarnini

Publications and source records attributed to Stephen Locarnini.

At least 19 recordsLinked to original sources
PubMed2007

Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.

UNLABELLED: Toll-like receptors (TLRs) play a key role in the innate immune response. The aim of this study was to examine the expression of TLR2 and TLR4 in chronic hepatitis B (CHB). The TLR2 and TLR4 expression on hepatocytes and Kupffer cells from fresh liver biopsies was measured from 21 patients with untreated hepatitis B e antigen (HBeAg)-positive and HBeAg-negative CHB. Parallel studies were also undertaken on monocytes from their peripheral blood. Expression of TLR2 on hepatocytes, Kupffer cells, and peripheral monocytes was significantly reduced in patients with HBeAg-positive CHB in comparison with HBeAg-negative CHB and controls, whereas it was significantly increased in HBeAg-negative CHB compared with controls. The level of TLR4 expression did not differ significantly between the groups. These results were confirmed in vitro using hepatic cell lines transduced with recombinant HBV baculovirus expressing wild-type HBV (HBeAg-positive), precore stop codon (G1896A) mutant HBV (HBeAg-negative). The functional relevance of these findings was established by the demonstration of significantly reduced cytokine production (TNF-alpha) and phospho-p38 kinase expression in the presence of the HBeAg. In the absence of HBeAg, HBV replication was associated with up-regulation of the TLR2 pathway leading to increased TNF-alpha production. CONCLUSION: This study demonstrates a potentially important interaction between HBeAg, HBV, and the innate immune response.

Adult↗
PubMed2006

Characteristics of drug resistant HBV in an international collaborative study of HIV-HBV-infected individuals on extended lamivudine therapy.

BACKGROUND: Little is known about the prevalence and pattern of hepatitis B virus (HBV) mutations in HIV/HBV co-infected individuals on long-term lamivudine (3TC) therapy. METHODS: HBV polymerase/envelope/basal core promoter/pre-core sequences from 81 HIV-HBV co-infected persons who received at least 6 months 3TC were compared to HBV reference sequences. Host and viral characteristics associated with HBV mutations were determined. RESULTS: HBV viraemia was detected in 53 persons (65%) and was associated with lower CD4 cell count nadir and higher HIV RNA at the time of testing but not with 3TC duration. Known 3TC-resistant mutations occurred in 50% and 94% of viremic patients with < 2 years and > 4 years 3TC, respectively. The CD4 cell count at testing was significantly higher in those with 3TC-resistant mutations. The triple polymerase mutant (rtL173V, rtL180M, rtM204V), which behaves as a vaccine escape mutant in vitro, occurred in 17% of viraemic patients. Polymerase mutations that may confer resistance to other anti-HBV agents were also detected. CONCLUSIONS: In HIV-HBV co-infected patients, greater immunocompromise is associated with continued HBV viraemia while on 3TC, and development of 3TC-resistant mutations are inevitable with prolonged 3TC use. These mutant viruses may limit future therapeutic options due to cross-resistance or may produce HBV vaccine escape mutants. Thus, timing and selection of antiretroviral therapy is critical in this population.

Adult↗
PubMed2006

Reduced expression of toll-like receptor 2 on peripheral monocytes in patients with chronic hepatitis B.

Persistent infection with hepatitis B virus (HBV) likely depends on viral inhibition of host defenses. We report that chronic hepatitis B e antigen-positive HBV infection is associated with a significant reduction in peripheral blood monocyte expression of Toll-like receptor 2, a key component of innate immunity, thereby providing a mechanism by which wild-type HBV may establish persistent infection.

Adolescent↗
PubMed2006

Comparison of real-time PCR assays for monitoring serum hepatitis B virus DNA levels during antiviral therapy.

The performance characteristics of the RealART and Molecular Beacons assays were compared with those of the Digene Hybrid Capture II assay (ultrasensitive). The results of the RealART and Digene Hybrid assays were related (r = 0.94; P < 0.001) and diverged by 2 orders of magnitude. The RealART assay can be used to effectively monitor serum hepatitis B virus DNA levels.

DNA, Viral↗
PubMed2005

Epidemiological and virological characteristics of 2 subgroups of hepatitis B virus genotype C.

BACKGROUND: We aimed to investigate the characteristics of hepatitis B virus (HBV) genotype C subgroups in Hong Kong and their relationship with HBV genotype C in other parts of Asia. METHODS: Full-genome nucleotide sequences of 49 HBV genotype C isolates from Chinese patients with chronic hepatitis B were compared with the sequences of 69 HBV genotype C isolates and 12 non-genotype C isolates in the GenBank database. Phylogenetic analysis was performed to define the subgroups of HBV genotype C on the basis of >4% heterogeneity of the entire HBV genome. RESULTS: HBV in 80% of patients in Hong Kong belonged to a subgroup predominantly found in Southeast Asia (Vietnam, Thailand, Myanmar, and southern China) designated as HBV genotype "Cs," and HBV in the remaining 20% of patients belonged to another subgroup, predominantly found in the Far East (Korea, Japan, and northern China), designated as HBV genotype "Ce." Overall, the mean+/-SD nucleotide sequence difference between HBV genotype Cs and HBV genotype Ce was 4.2%+/-0.3%. When HBV genotype Cs and HBV genotype Ce were compared among patients in Hong Kong, HBV genotype Cs was associated with a higher tendency to develop basal core promoter mutations (80% vs. 50%; P=.14), a higher prevalence of C at nucleotide 1858 (95% vs. 0%; P<.001), and a lower prevalence of precore stop codon mutations (5% vs. 50%; P=.002). CONCLUSIONS: HBV genotype C can be differentiated into 2 subgroups--namely, genotype Ce and genotype Cs--that have different epidemiological distributions and virological characteristics.

Adult↗
PubMed2005

Sexual transmission of hepatitis B infection despite the presence of hepatitis B virus immunity in recipients of allogeneic bone marrow transplantation.

BACKGROUND: After hematopoietic cell transplantation (HCT), hepatitis due to hepatitis B virus (HBV) rarely occurred beyond the initial 12 months after transplantation. OBJECTIVES: We investigated the cause of "late" hepatitis due to HBV infection in two recipients after allogeneic HCT. STUDY DESIGN: Two male patients with acute myeloid leukemia and light chain myeloma, respectively, developed HBV-related hepatitis more than 2 years after HCT. All serum samples collected from the recipients, donors and their respective spouses were tested for HBV DNA by nested PCR, and if positive further quantified by Digene Hybrid Capture assay II. The HBV genotype was determined by PCR and sequencing. RESULTS: Genotypic analysis suggested that the cause of "late" hepatitis was due to acute HBV infection transmitted from their respective spouse. CONCLUSION: Our findings suggested that sexual precautions should be taken in these patients after HCT. Alternatively, or even additionally, active vaccination should be delivered to these patients once they have lost their HBV immunity.

Adult↗
PubMed2005

Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy.

BACKGROUND & AIMS: This study aimed to determine whether intrahepatic hepatitis B virus (HBV) covalently closed circular (ccc) DNA and total HBV DNA levels at the end of therapy would predict sustained response to therapy. METHODS: Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients receiving either lamivudine monotherapy or combination of peginterferon and lamivudine had liver biopsy at the end of 1 year therapy and were followed for 52 more weeks after cessation of therapy. Serum HBV DNA, intrahepatic HBV ccc DNA, and total HBV DNA levels were determined. RESULTS: Forty-seven patients, including 34 males and 13 females, were studied. Twenty-seven patients received combination therapy, and 20 patients received lamivudine monotherapy. Twenty-nine patients had end-of-treatment virologic response, and 15 patients had sustained response 52 weeks after therapy. At the end of treatment, log serum HBV DNA levels correlated well with log intrahepatic HBV cccDNA and log intrahepatic total HBV DNA levels. Log intrahepatic cccDNA and log intrahepatic total DNA levels were significantly lower among patients with sustained virologic response. The adjusted odds ratio for log cccDNA was 5.3 (95% CI: 1.5-18.2, P = .009) and, for log intrahepatic HBV DNA, was 4.4 (95% CI: 1.3-14.7, P = .015) to predict sustained virologic response. Using log cccDNA at -0.80 copies/genome equivalent as cutoff, the sensitivity, specificity, and positive and negative predictive values and accuracy of predicting sustained virologic response were 73%, 78%, 56%, 86%, and 77% respectively. CONCLUSIONS: Intrahepatic HBV cccDNA and intrahepatic total HBV DNA levels at the end of therapy are superior to serum HBV DNA as surrogates of sustained virologic response.

Adult↗
PubMed2005

Molecular virology and the development of resistant mutants: implications for therapy.

Two of the key events in the viral life cycle of the hepatitis B virus (HBV) involve (1) generation from genomic DNA of the covalently closed circular DNA transcriptional template and (2) reverse transcription of the viral pregenomic RNA to form the HBV DNA genome. Because the virus employs reverse transcription to copy its genome, mutant viral genomes are found frequently. Particular selection pressures, both endogenous (host immune clearance) and exogenous (vaccines and antivirals), readily select out these escape mutants. The introduction of nucleoside/nucleotide analogue therapy has seen the emergence of drug resistance as the major factor limiting drug efficacy. The development of drug resistance is not unexpected if viral replication continues in the setting of ongoing treatment, especially monotherapy. Thus, prevention of resistance requires the adoption of strategies that effectively control virus replication.

Antiviral Agents↗
PubMed2005

Selection of hepatitis B virus polymerase mutations in HIV-coinfected patients treated with tenofovir.

BACKGROUND: Tenofovir (TDF) is an adenosine nucleotide analogue that has been approved for the treatment of HIV-1 infection. It also shows activity against hepatitis B virus (HBV) in patients with or without lamivudine (LAM)-associated mutations. Development of clinical or virological HBV breakthrough during TDF therapy has not been reported so far. The aim of this study was to analyse the HBV polymerase (pol) from HIV/HBV-coinfected patients with detectable serum levels of HBV DNA during treatment with TDF for longer than 6 months. METHODS: The HBV pol was sequenced from 43 patient's serum before and during TDF therapy. Phenotypic analyses were performed using HBV replication-competent plasmids carrying unique mutations selected under TDF therapy. RESULTS: Mean exposure to LAM was 35.3 +/- 27.5 months and to TDF 11.2 +/- 6.7 months. Genotypic analyses from 21 of the patients revealed LAM-associated mutations, and a further two patients developed a novel mutation, rtA194T, along with LAM-resistance-associated mutations. Phenotypic analyses revealed that constructs harbouring rtA194T combined with rtL180M and rtM204V displayed an over 10-fold increase in the IC50 for TDF compared with the wild type. CONCLUSION: The selection of HBV mutations in HBV/HIV-coinfected patients failing TDF therapy is an unlikely event within the first 12 months of treatment. However, HBV from two of the 43 patients treated with TDF for more than 12 months was found to contain one novel mutation located distal to the catalytic site of the HBV pol. In vitro, rtA194T conferred a reduced susceptibility to TDF in the presence of LAM-associated mutations.

Adenine↗
PubMed2004

Hepatitis B virus: pathogenesis, viral intermediates, and viral replication.

Although HBV has the potential to generate an almost limitless spectrum of quasispecies during chronic infection, the viability of the majority of these quasispecies is almost certainly impaired due to constraints imposed by the remarkably compact organization of the HBV genome. On the other hand, single mutations may affect more than one gene and result in complex and unpredictable effects on viral phenotype. Better understanding of the constraints imposed by gene overlap and of genotype-phenotype relationships should help in the development of improved antiviral strategies and management approaches. Although the probability of developing viral resistance is directly proportional to the intensity of selection pressure and the diversity of quasispecies, potent inhibition of HBV replication should be able to prevent development of drug resistance because mutagenesis is replication dependent. If viral replication can be suppressed for a sufficient length of time, viral load should decline to a point where the continued production of quasispecies with the potential to resist new drug treatments no longer occurs. Clinical application of this concept will require optimization of combination therapies analogous to highly active antiretroviral therapy (HAART) for HIV infection. Total cure of hepatitis B will require elimination of the intranuclear pool of viral minichromosomes, which will probably only be achieved by normal cell turnover, reactivation of host immunity, or elucidation of the antiviral mechanisms operating during cytokine clearance in acute hepatitis B (see Fig. 1).

DNA, Viral↗
PubMed2004

Hepatitis B virus genotypes, core gene variability and ethnicity in the Pacific region.

BACKGROUND/AIMS: The world-wide distribution of hepatitis B virus (HBV) genotypes follows a geographic pattern under the influence of ethnic background. METHODS: Forty eight core genes from four pacific islands were compared with the following findings. RESULTS: First, island-specific variant substitutions were found for only two out of four islands. Second, 11 amino acid and 90 nucleotide changes specific for pacific genotypes C and D were defined. Third, the nucleotide diversity of genotype C (all but one were silent) was greater than that of genotype D. CONCLUSIONS: These results suggest an early appearance of genotype C in the pacific with few subsequent amino acid changes because of shared immunological responses across the region followed by random silent changes, some of which reflect isolation of individual island populations. Genotype D appeared later.

Databases, Genetic↗
PubMed2004

Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.

BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a unique episomal replicative intermediate responsible for persistent infection of hepatocytes. Technical constraints have hampered the direct study of cccDNA maintenance and clearance mechanisms in patients. The aim of this study was to develop a sensitive and specific assay for quantifying cccDNA in biopsy samples from chronic hepatitis B patients during different natural history phases and in patients undergoing antiviral therapy. METHODS: Intrahepatic cccDNA levels were quantified by a specific real-time PCR assay. Ninety-eight liver biopsy samples from patients in the major phases of the natural history of chronic hepatitis B and 32 pairs of samples from patients receiving adefovir dipivoxil (ADV) therapy were assessed. RESULTS: cccDNA was detected, at levels ranging over 3 orders of magnitude, in patients in different phases of the natural history of chronic hepatitis B. cccDNA levels were strongly correlated with levels of total intracellular HBV DNA and serum HBV DNA. Forty-eight weeks of ADV therapy resulted in a significant 0.8 log decrease in cccDNA copies/cell. Changes in cccDNA were correlated with a similar reduction in serum HBsAg titer but not with a decrease in the number of HBV antigen-positive cells during ADV treatment. CONCLUSIONS: cccDNA persists throughout the natural history of chronic hepatitis B, even in patients with serologic evidence of viral clearance. Long-term ADV therapy significantly decreased cccDNA levels by a primarily noncytolytic mechanism.

Adenine↗
PubMed2004

Molecular virology of hepatitis B virus.

Hepatitis B virus (HBV) has evolved a unique life cycle that results in the production of enormous viral loads during active replication without actually killing the infected cell directly. Because HBV uses reverse transcription to copy its DNA genome, mutant viral genomes emerge frequently. Particular selection pressures, both endogenous (host immune clearance) and exogenous (vaccines and antiviral drugs), readily select out these escape mutants. Which particular viral mutations or combination of mutations directly affect the clinical outcome of infection are not known. Further studies are clearly needed to identify the pathogenic basis and clinical sequelae arising from the selection of these mutants.

Adenine↗
PubMed2004

Comparison of sequence analysis and a novel discriminatory real-time PCR assay for detection and quantification of Lamivudine-resistant hepatitis B virus strains.

We report a rapid and accurate real-time PCR-based method to quantify wild-type and lamivudine-resistant hepatitis B virus by using a common forward primer paired with different reverse primers. Excellent concordance was demonstrated between sequencing and the discriminatory real-time assay; however, a mixture of quasispecies was more frequently detected by discriminatory real-time PCR.

Base Sequence↗