Lamivudine Resistance and Precore Variants in Iranian Patients With Chronic Hepatitis B: Correlation With Virological and Clinical Features.

BACKGROUND: Long-term lamivudine therapy, despite its initial effectiveness against hepatitis B virus (HBV), is associated with the emergence of drug resistance mutations in polymerase protein. OBJECTIVES: The aim of the present study was to determine the prevalence of precore and lamivudine drug resistance mutations in lamivudine treated patients with chronic B hepatitis. PATIENTS AND METHODS: Sequential sera were obtained from 88 chronic HBV carriers who received lamivudine for more than 24 months. Polymerase and precore regions were directly sequenced for these groups: I (before treatment), II, and III (12 and 24 months after treatment, respectively). RESULTS: All patients (100%) were contained genotype D, subtype ayw2. One (1.1%), 12 (13.6%), and 22 (25%) members of groups I, II, and III had the replacement of either isoleucine or valine instead of methionine in tyrosine-methionine-aspartate-aspartate (YMDD) motif, respectively. The frequency of mutations from 0 time point to 12 and 24 months showed that there was an increasing trend between sequential samples (P < 0.001). In group I, 31 (35.2%); II, 36 (41.0%) and III, 41 (46.6%) members had the precore stop codon mutations. The frequency of mutations from 0 time point to 12 and 24 months showed that there was an ascending trend between sequential samples. Indeed, frequency of precore stop codon was significantly increased with the passage of time (P < 0.001). CONCLUSIONS: Presence of drug resistance mutations among the patients was significant. Precore mutations were common amongst Iranian HBV chronic carriers under lamivudine therapy and these mutations were accompanied by clinical relapse.

Negative Association of Plasma Levels of Vitamin D and miR-378 With Viral Load in Patients With Chronic Hepatitis B Infection.

BACKGROUND: Chronic Hepatitis B (CHB) is accompanied by inflammation of liver because of infection with Hepatitis B Virus (HBV). Previous studies revealed an inverse association between vitamin D and HBV DNA levels. OBJECTIVES: The current study aimed to investigate the levels of 25 (OH) D3 (the steady form of vitamin D), miR-378 and HBV DNA in the patients with CHB. PATIENTS AND METHODS: One hundred and seventy three patients with HBeAg negative CHB were recruited for the study. Plasma levels of HBVDNA and 25 (OH) D3 were quantified. The expression level of miR-378 in plasma was measured by a relative quantitative Real Time Polymerase Chain Reaction (qRT-PCR) assay. RESULTS: In the pathway regression analysis, the plasma level of 25 (OH) D3 showed a significant inverse correlation with plasma levels of HBV DNA (-0.198, P = 0.008) and direct correlation with miR-378 (0.188, P = 0.013). Similarly plasma level of miR-378 had inverse association with HBV DNA level (-0.177, P = 0.020). CONCLUSIONS: These results suggest that vitamin D could involve in a miRNA- mediated regulatory pathway in control of HBV replication. Further studies are recommended to understand the effects of miR-378 and anti-infective action of vitamin D on Hepatitis B Virus.

The Increased Level of Serum p53 in Hepatitis B-Associated Liver Cirrhosis.

BACKGROUND: The ability of tumour suppressor protein p53 (P53) to regulate cell cycle processes can be modulated by hepatitis B virus (HBV). While preliminary evidences indicates the involvement of protein-x of HBV (HBx) in altering p53 DNA binding, no further data have been accumulated for the significance of serum p53 in chronic hepatitis B virus infected patients. METHODS: 72 non-cirrhotic and 19 cirrhotic patients infected by HBV were enrolled for the analysis in this study. Enzyme linked immunosorbent assay (ELISA) was performed to study the concentrations of serum p53 protein. The tertiary structures of HBx and P53 were docked by Z-dock and Hex servers for in-silico protein-protein interaction analysis. RESULTS: There was a significant association between the serum p53 and cirrhosis (OR=1.81 95% CI: 1.017-3.2, P=0.044). Cirrhotic patients had higher level of serum p53 compare with chronic infection of HBV (1.98±1.22 vs. 1.29±0.72 U/ml, P=0.05). No evidence of correlation was seen between the different variables such as age, gender, log viral load, serum alkaline phosphatase (ALP) and alanine aminotransferase (ALT) with serum p53. Tertiary model shows that the amino acid residues from Arg110 to Lys132 of the N-terminal of P53 which is critical for ubiquitination, are bonded to a region in N- terminal of HBx amino acid residues from Arg19 to Ser33. CONCLUSION: There is an increase in serum p53 in HBV-related cirrhosis patients. In this case, HBx might be responsible for such higher concentration of p53 through HBx-p53 protein-protein interaction, as is shown by molecular modeling approach.

Clinical feature of intrahepatic B-lymphocytes in chronic hepatitis B.

Humoral immunity constitutes major defense mechanism against viral infections. However, the association of hepatic injury and B-cells population in chronic hepatitis B virus (HBV) carriers has not been studied well. In this study, fifty seven hepatitis B surface antigen (HBsAg) positive and HBeAg negative patients were studied to determine the expression of CD20, a cell surface marker expressed on B-cells, in liver biopsy sections using immunohistochemistry. The patients' clinical data at the time of liver biopsy were acquired from their medical records. There was a significant association between log HBV DNA and both ALT (r = 0.36, P = 0.006) and histologic activity index (HAI) total score (r = 0.3, P = 0.02), respectively. The CD20 was expressed in all 57 liver biopsy samples with a submembranous and membranous staining pattern and its expression was significantly associated with HAI total score (r = 0.32, P = 0.01) and stage of fibrosis (r = 0.31, P = 0.02). The susceptible B lymphocytes to hepatitis B virus might be implicated in the development of immune mediated inflammation of HBV-induced hepatic injury. The present data also support that the liver is potentially one of the secondary lymphoid organs.

Surface protein mutations in chronic hepatitis B patients who received hepatitis B vaccine therapy.

OBJECTIVE S: The aim of this study was to determine the correlation between vaccine therapy and appearance of mutations in hepatitis B surface antigen (HBsAg)-positive chronic hepatitis B virus (HBV) patients. MATERIALS AND METHODS: 16 patients received the HBV vaccine and another 16 individuals from the control group did not. The surface gene was amplified and directly sequenced from samples prior to vaccination and six months after the third dose. RESULTS: Only one patient lost HBsAg. 48 and 44 amino acid mutations were found before and after vaccine therapy in the vaccine group respectively, 51 of which (55.4%) occurred in immune epitopes: 5 were in B cell, 21 in T helper (Th), and 25 in cytotoxic T-lymphocyte (CTL) epitopes. In the control group, 35 and 41 amino acid substitutions were found before and after therapy, respectively. 32 (42%) of 76 amino acid changes occurred within immune epitopes. There were no differences in age, gender, and duration of chronicity in both patient and control groups in terms of the frequency and the patterns of mutations. CONCLUSION: In chronic carriers who already had HBsAg variants selected by the host-immune response, any immune stimulation by the vaccine had no effect on the chronic state of these patients or selected any remarkable escape mutants. Newer strategies should be considered based on third generation or the use of DNA vaccines or new adjuvants.

The Possible Role of TLR2 in Chronic Hepatitis B Patients with Precore Mutation.

Recognition mechanisms of innate immune response help to improve immunotherapeutic strategies in HBeAg-negative chronic hepatitis B (CHB). Toll-like receptor 2 (TLR2) is an important component of innate immunity. In this study, the frequency of precore mutations of the hepatitis B virus (HBV) and serum TLR2 were evaluated in CHB patients. Fifty-one patients with chronic hepatitis B, negative for HBeAg and detectable HBV DNA, were examined for the presence of mutations in pre-core region of HBV genome by direct sequencing. Serum TLR2 was measured by enzyme-linked immunosorbent assay. Interactions of truncated HBeAg and TLR2 proteins were evaluated with molecular docking software. The G1896A pre-core mutation were detected in 29 (57%) which was significantly associated with higher concentration of serum TLR2 in comparison with patients without this mutation (4.8 ± 2.9 versus 3.4 ± 2.2 ng/mL, P = 0.03). There was also a significant correlation between serum ALT and TLR-2 (r = 0.46; P = 0.01). Docking results illustrated residues within the N-terminus of truncated HBeAg and TLR2, which might facilitate the interaction of these proteins. These findings showed the dominance of G1896A pre-core mutation of HBV variants in this community which was correlated with serum TLR2. Moreover TLR2 is critical for induction of inflammatory cytokines and therefore ALT elevation.

Influence of B cells in liver fibrosis associated with hepatitis B virus harboring basal core promoter mutations.

The development of the liver disease in chronic hepatitis B with common viral variants can be determined through the interaction between the virus and the host immune response. B cells constitute half of the intrahepatic lymphocyte population with an impact on fibrosis. A proliferation-inducing ligand (APRIL) has been shown to have a co-stimulatory activity on B cells. For this study HBV DNA was amplified and then sequenced to show the presence of the basal core promoter (BCP) mutations in the serum from 57 patients with chronic hepatitis B. The range of IgD-positive B cells was detected by immunohistochemistry in liver biopsies; and patients serum was assayed for APRIL levels by enzyme immunoassay. Twenty-seven patients (47.4%) harbored the A1762T-G1764A BCP mutations. Coefficients of logistic regression showed that the effect of increasing IgD-positive B cells in rising odds of the liver disease is the same in the patients with BCP mutation A1762T-G1764A and in the patients without mutation, nevertheless the effect of APRIL is not similar in these two groups of patients. Logistic regression in patients with BCP A1762T-G1764A mutations demonstrated that increasing one score of APRIL decreased the odds of fibrosis stage about 8%. These results suggest that in infection with viral variants of hepatitis B virus, the population of IgD-positive B cells may play a decisive role in later stages of the liver disease which is reduced by APRIL in chronic hepatitis patients with BCP mutations.

Plasma myeloperoxidase activity and apolipoprotein A-1 expression in chronic hepatitis B patients.

BACKGROUND: Hepatitis B virus initiates a complicated cascade process leading to chronic hepatitis B, cirrhosis, and hepatocellular carcinoma. In inflammatory situations, myeloperoxidase is released in plasma and binds to apolipoprotein A-1 in high-density lipoproteins. This study aims to evaluate the level of plasma myeloperoxidase as well as the pattern of plasma proteins in patients with chronic hepatitis B. METHODS: Included in this study were 30 male subjects: 19 chronic hepatitis B patients, 6 HBV-related cirrhotic patients, and 5 healthy controls. Plasma myeloperoxidase was measured using enzyme-linked immunosorbent assay. Proteomic analysis of plasma proteins was performed by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. One way ANOVA was used for data analysis. RESULTS: Mean plasma myeloperoxidase levels were higher in patients with liver cirrhosis (65.5±12.5; P=0.007) and chronic hepatitis B (53.7±10.6; P=0.18) when compared with healthy subjects (45±7.6). Moreover, a positive correlation was found between plasma myeloperoxidase levels and hepatic fibrosis stage (r=0.53, P=0.002; r=0.63, P=0.000). Proteomic analysis showed an altered plasma protein pattern in progressive hepatitis B and down-regulation of the major apolipoprotein A-1 along with the appearance of a variety of spots noted to be apolipoprotein A-1isoforms with different molecular masses. CONCLUSION: In this study, progressive liver injury due to HBV infection correlated with higher plasma myeloperoxidase and an altered plasma apolipoprotein A-1pattern.

The inverse association of serum HBV DNA level with HDL and adiponectin in chronic hepatitis B infection.

BACKGROUND: The natural history of hepatitis B virus (HBV) is complex and influenced by the level of viral replication and host factors. The hepatoprotective role of high density lipoproteins (HDL) and adiponectin as host factors on HBV persistence is less well understood. METHODS: To investigate correlation between HBV DNA level with clinical parameters in patients with chronic hepatitis B, 92 male subjects with HBV infection without any risk factors for diabetes were enrolled in this study. Age and BMI of the study population were matched and HBV DNA, ALT, tumor necrosis factor alpha (TNF-α), adiponectin and lipid levels was measured. RESULTS: Serum HBV DNA correlated inversely with serum HDL level (r = -0.23; P = 0.014). The median of log copies/ml for HBV DNA (3.67) was considered as cut off point. Patients with HBV DNA level higher than cut off point had lower adiponectin (8.7 ± 5.3 vs 10.7 ± 4.9 µg/ml p = 0.05). Also, adiponectin had a negative correlation with TNF-α (r = -0.21, P = 0.04) and positive correlations with HDL (r = 0.18, P = 0.043). Multivariate regression models show that serum HDL level is an independent factor to predict serum HBV DNA. CONCLUSION: Our findings showed that higher HBV DNA levels are associated with lower HDL and adiponectin but induced TNF-alpha values.

The role of mutations in core protein of hepatitis B virus in liver fibrosis.

The core protein of hepatitis B virus encompasses B- and T-cell immunodominant epitopes and subdivided into two domains: the N-terminal and the functional C-terminal consisted phosphorylation sites. Mutations of the core gene may change the conformation of the core protein or cause alteration of important epitopes in the host immune response. In this study twenty nine men (mean age 40 +/- 9 years old) with chronic hepatitis B were recruited for direct sequencing of the core gene. Serum ALT and HBV DNA level were measured at the time of liver biopsy. The effects of core protein mutations on patients' characteristics and subsequently mutations in B cell, T helper and cytotoxic T lymphocyte (CTL) epitopes and also C-terminal domain of core protein on the activity of liver disease was evaluated. Liver fibrosis was significantly increased in patients with core protein mutation (1.0 +/- 0.8 vs 1.9 +/- 1.4 for mean stage of fibrosis P = 0.05). Mutations in CTL epitopes and in phosphorylation sites of C-terminal domain of core protein also were associated with higher liver fibrosis (P = 0.003 and P = 0.04; Fisher's exact test for both). Patients with mutation in C-terminal domain had higher serum ALT (62 +/- 17 vs 36 +/- 12 IU/l, p = 0.02). Patients with mutations in B cell and T helper epitopes did not show significant difference in the clinical features. Our data suggests that core protein mutations in CTL epitopes and C-terminal domain accompanied with higher stage of liver fibrosis may be due to alterations in the function of core protein.

Correlation between clinical severity score and the lower esophageal sphincter relaxation pressure in idiopathic achalasia.

OBJECTIVES: Achalasia is an esophageal motor disorder characterized by aperistalsis and incomplete relaxation of the lower esophageal sphincter (LES). The meaningful correlation between LES relaxation pressure and the severity of clinical symptoms is uncertain. The aim of this study was to elucidate the correlation between the clinical scoring and the LES relaxation pressure. METHODS: Over a 4-yr period from 1997 to 2001, all newly diagnosed patients with idiopathic achalasia were consecutively enrolled in a study. Diagnosis was established based on clinical, radiographic, endoscopic, and manometric criteria. The severity of five cardinal symptoms was scored on a scale of 0-3, and each patient received a total symptom score of 1-15. Manometry was subsequently performed, and the mean of five complete pull-through measurements was recorded as the resting LES relaxation pressure. RESULTS: A total of 115 patients (67 male and 48 female) with a mean age of 37.7 yr (range 12-90 yr) were included in the study. The mean total symptom score was 9.32 (range 3.00-14.00) and mean LES relaxation pressure before therapy was 56.29 mm Hg (range 8.00-107.80 mm Hg). Linear regression analysis showed a significant association between the total symptom score and LES relaxation pressure (p < 0.002, r = 0.290). Among the main symptoms, active and passive regurgitation showed significant correlation with LES relaxation pressure when compared to other individual symptoms using Pearson's correlation coefficient (p < 0.001 and 0.002, respectively). CONCLUSIONS: Our study showed that a clinical symptom score can be an appropriate predictor of the LES relaxation pressure in patients with idiopathic achalasia before therapy. Further studies are needed to evaluate similar correlations after therapeutic intervention.