Tumour necrosis factor-alpha, interleukin-10, interferon-gamma and vitamin D receptor gene polymorphisms in patients with chronic hepatitis delta.

No data exist to assess certain polymorphisms that have a potential effect on the immune response in patients with chronic hepatitis delta (CHD). The aim of this study was to investigate polymorphisms in 6 polymorphic sites: IL-10 -1082 (rs1800896), IL-10 -627 (rs1800872), IFN-γ +874 (rs62559044), TNF-α -308 (rs1800629), vitamin D receptor (VDR) FokI (rs2228570) and VDR TaqI (rs731236). The genotypes of 67 patients with CHD and 119 patients with chronic hepatitis B (CHB) were compared. In addition, 56 individuals with resolved hepatitis B virus (HBV) infection were used as a control group for patients with CHB. Polymorphisms in TNF-α, IL-10, and VDR genes were analysed using polymerase chain reaction/restriction fragment length polymorphism methods. The IFN-γ gene polymorphism was detected by allele-specific polymerase chain reaction (PCR). Patients with CDH were more likely to have advanced liver disease compared with patients with CHB (P < 0.0001). IL-10 -1082 and VDR TaqI polymorphisms showed significant differences between patients with CHD and CHB. The high secretory IL-10 -1082 genotype GG was less frequent in CHD compared with patients with CHB and resolved HBV (17.7%, 37.4% and 47.1%, respectively (P < 0.05 for CHD vs CHB and resolved HBV). The frequency of the high secretory VDR TaqI TT genotype was 86.6% in patients with CHD, 62.7% in patients with CHB and 62.5% in resolved HBV individuals (CHD vs CHB: P < 0.05). None of the polymorphisms analysed had an effect on HBV persistence. IL-10 -1082 and VDR TaqI polymorphisms may contribute to the more severe liver disease associated with CHD compared with CHB.

Natural history and treatment of chronic delta hepatitis.

Chronic delta hepatitis (CDH) represents a severe form of chronic viral hepatitis, induced by the hepatitis delta virus (HDV) in conjunction with the hepatitis B virus (HBV). Delta hepatitis may lead to disease in humans through co-infection. The former leads to acute hepatitis which clinically can range from mild hepatitis to fulminant hepatitis and death. Severe or fulminant hepatitis is more often observed with HBV-HDV co-infection compared to HBV mono-infection. Chronic infection after acute hepatitis B + D co-infection is infrequent and similar to the rate in mono-infected patients. CDH develops in 70-90% of patients with superinfection. CDH runs a more progressive course than chronic hepatitis B and may lead to cirrhosis within 2 years in 10-15% of patients. However, as with any immune-mediated disease, different patterns of progression, ranging from mild to severe progressive disease, are observed. Active replication of both HBV and HDV may be associated with a more progressive disease pattern. Further, different HDV and HBV genotypes may contribute to various disease outcomes. CDH may be frequently associated with hepatocellular carcinoma development although recent studies provided conflicting results. The only established therapy for CDH is treatment with interferons for a duration of at least 1 year. On treatment, 6 month HDV RNA assessment may give clues as to whether to stop treatment at 1 year or continue beyond 1 year. New approaches to treatment of CDH are an urgent need of which the use of prenylation inhibitors appears the most promising.

Treatment of chronic delta hepatitis with lamivudine vs lamivudine + interferon vs interferon.

Chronic delta hepatitis is the most severe form of chronic viral hepatitis for which interferon (IFN) is the only available treatment. In 39 patients (25 were treatment-naïve, 14 had previously used IFN), efficacy of 1-year treatment with IFN (9 MU, t.i.w.) or lamivudine (LAM; 100 mg, q.d.) alone was compared with IFN and LAM combination (2 months of LAM to be followed by combination treatment). IFN monotherapy was given only to treatment-naïve patients. In both treatment-naïve and previous IFN users, end of treatment virological and biochemical responses were similar with IFN-LAM combination and superior to LAM monotherapy (P < 0.05). Improvement in liver histology occurred more often with IFN +/- LAM than with LAM alone (P < 0.05). In treatment-naïve patients, combination treatment was not superior to IFN monotherapy. After treatment discontinuation, virological and biochemical response rates decreased in LAM and IFN combination and IFN monotherapy. On treatment virological response at month 6 of treatment predicted sustained virological response. The results of this study suggest that addition of LAM to IFN for the treatment of delta hepatitis is of no additional value and that both treatment modalities are superior to LAM monotherapy.

A pilot study of 2 years of interferon treatment in patients with chronic delta hepatitis.

High dose interferon treatment for 1 year is the only established treatment for chronic hepatitis D, but it is associated with a high relapse rate after treatment discontinuation. In this study, patients were treated with 10 MU interferon alpha 2b, thrice weekly for 2 years. Twenty-three patients were recruited and 15 completed the 2-year treatment and 6 months follow-up periods. Treatment response was assessed biochemically [normal alanine aminotransferase (ALT)], virologically (undetectable hepatitis D virus RNA) and histologically (at least 2 point decrease in the Knodell score) at the end of treatment (EOT) and at the end of follow-up. Out of 15 patients who finished the 2-year treatment period, seven patients (47%) had a biochemical response but only two (13%) had a normal ALT after follow-up. ALT decreased from the baseline value of 143.1 +/- 121.7 (mean +/- SD) to 39.7 +/- 20.6 (P < 0.01) at EOT. Virological response was observed in six patients at EOT and in two patients at follow-up. Two patients lost hepatitis B surface antigen. Of the 12 patients with paired liver biopsies, a histological improvement was observed in eight patients. Interferon treatment leads to a complete or partial response in a substantial number of patients but 2 years of treatment does not appear to increase sustained response rates over 1 year treatment.

Inhibition of hepatitis B virus replication by shRNAs in stably HBV expressed HEPG2 2.2.15 cell lines.

In this study, the effect of RNAi on HBV replication was observed in a cell culture model, HepG2 2.2.15 cell line, which supports human HBV ayw replication and expression. Aim of the study was to investigate effects of shRNAs (small hairpin RNAs) targeting hepatitis B virus mRNAs on the viral replication in HepG2 2.2.15 cells. We selected three target HBV mRNA regions with different putative secondary structures to test whether the secondary structure of RNA may affect the inhibition efficacy on the target HBV RNA. Three HBV-specific siRNAs (small interfering RNA) were designed targeting X (1689-1708), Core (2229-2248) and S (765-784 nt) transcripts. HepG2 2.2.15 cells were transfected with shRNA expressing plasmids, P765, P2229 and P1689 targeting S, core and X region, respectively or a mock plasmid targeting lacZ gene. The culture media was collected throughout six days after transfection and analyzed by real-time PCR. Viral DNA production was suppressed for 7 days. The HBV DNA levels were decreased by 73, 72 and 79% with P765, P2229 and P1689 vectors, respectively. In conclusion, the shRNAs designed for X, core and S regions, specifically and significantly suppressed HBV DNA. siRNAs potentially may be used in treatment of hepatitis B.

Cytotoxic CD4 T cells in viral hepatitis.

CD4+ T cells are thought to contribute to antiviral immune responses by secretion of cytokines thereby providing help to CD8+ T and B cells. However, perforin-positive cytotoxic CD4+ T cells have been described in human immunodeficiency virus-positive patients suggesting a role not only of CD8+ but also of CD4+ T cells for killing virus-infected cells. We investigated 76 patients with viral hepatitis [15 hepatitis B virus (HBV), 22 HBV/hepatitis D virus and 17 hepatitis C virus (HCV)] for cytotoxic CD4+ T cells. The frequency of perforin-positive CD4+ T cells in viral hepatitis was highly variable ranging from < 1% to more than 25%. Perforin-positive CD4+ T cells displayed the phenotype of terminally differentiated effector cells (CD28-, CD27-). The highest frequencies of CD4+ cytotoxic T lymphocytes (CTLs) were found in patients with delta hepatitis (P = 0.04 vs HBV and HCV patients), and the presence of CD4+ CTLs was associated with elevated aspartate aminotransferase levels (P = 0.01) and decreased platelet counts (P = 0.03). Perforin-positive CD4+ T cells decreased in two individuals during spontaneous clearance of acute hepatitis C. Significant associations were found between the frequency of perforin-expressing CD4+ cells and age (P = 0.04), perforin-positive CD8+ cells (P < 0.001) and perforin-positive CD4-/CD8- lymphoid cells (P = 0.002). Differentiated CD27- effector CD4+ CTLs can be detected in patients with viral hepatitis. In particular in patients with more advanced liver disease, the accumulation of perforin-positive T cells with age could be one correlate for the more severe course of viral hepatitis in elderly individuals.

Lamivudine vs lamivudine and interferon combination treatment of HBeAg(-) chronic hepatitis B.

To determine whether combination treatment of HBeAg(-) chronic hepatitis B is beneficial we studied 78 patients with HBeAg(-), HBV DNA-positive chronic hepatitis B who were randomized to lamivudine, 100 mg, qd, for 12 months or lamivudine-interferon (9 MU, t.i.w.) in combination. In the combination arm, 2 months of lamivudine treatment preceded 10 months of combination treatment. Biochemical, virologic and histologic responses were assessed at the end of treatment, after six and a median 27 months of drug-free follow-up (short- and long-term follow-up, respectively). Virologic response was defined as undetectable HBV DNA with a hybridization assay and biochemical response as normal alanine aminotransferase (ALT). Change in HBV DNA was also assessed by real-time polymerase chain reaction (PCR). Presence of YMDD mutants at the end of treatment was investigated with a line probe assay. Both treatment regimes led to a median 2 log decline in HBV DNA levels. Virologic end of treatment responses were 90 and 92% with mono- and combination treatment, respectively. Corresponding virologic responses at short- and long-term follow-up were 59 and 54%, and 27 and 25%, respectively. Patients having a baseline HBV DNA value > or =200 pg/mL were more likely to relapse within 6 months off therapy than those patients with a baseline HBV DNA level <200 pg/mL (P = 0.041). YMDD mutants were observed in 53% of patients receiving lamivudine compared with 24% of patients receiving the combination regime (P = 0.017). In conclusion, efficacy of combination treatment is similar to lamivudine monotherapy. However, combination treatment decreases the development of YMDD mutant strains compared with lamivudine monotherapy.

Molecular epidemiology of hepatitis B, C and D viruses in Turkish patients.

Different genotypes of the hepatitis viruses may influence the clinical outcome of the disease. The distribution of genotypes may vary according to geographical regions. The aim of this study was to evaluate hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV) genotypes in Turkish patients with chronic hepatitis in a large cohort of patients. Genotyping was performed in 41, 59 and 365 patients with chronic hepatitis B, D and C, respectively, and 36 hemodialysis patients with chronic hepatitis C. Genotypes were determined by direct sequencing in hepatitis B and by polymerase chain reaction-restriction fragment length polymorphism in hepatitis C and D patients. In addition, HBV subtyping by multiplex PCR and subtype specific ELISA were performed in 83 and 71 HBsAg (+) blood donors, respectively. All hepatitis B (100%) and hepatitis D (100%) patients had genotype D and type I, respectively. HBsAg subtyping by two methods yielded that 99% of the patients were subtype ayw. S gene amino acid sequence in the 41 patients included for HBV genotyping revealed the ayw2 subtype. Genotype distribution of 365 patients with chronic C hepatitis were as follows: 306 (84%) patients genotype 1b, 43 (11%) patients genotype 1a, 10 (3%) patients genotype 2, 3 (1%) patients genotype 3, 3 (1%) patients genotype 4. Among 36 patients receiving hemodialysis, 28 (78%) patients had genotype 1b and 8 (22%) patients had genotype 1a. The study indicates that Turkish patients with chronic viral hepatitis show very little genotypic heterogeneity. Subtype ayw and the genotype D of HBV DNA, and the type I of HDV RNA represent almost 100% of related infections. The genotype 1b of HCV RNA was found to be significantly dominant in Turkish patients.

YSDD: a novel mutation in HBV DNA polymerase confers clinical resistance to lamivudine.

The emergence of drug-resistant virus in hepatitis B virus (HBV) patients treated with lamivudine is well documented. In this study, we determined the mutations occurring in the tyrosine-methionine-aspartate-aspartate (YMDD) amino acid motif of the HBV DNA polymerase gene, as well as upstream and downstream of this region, in patients with breakthrough virus during lamivudine therapy. Thirty-one Turkish patients (20 patients HBeAg positive, 11 patients HBeAg negative and anti-HBe positive) with chronic HBV infection who completed at least 104 weeks of lamivudine treatment were investigated. All patients received lamivudine, (150 mg/day), for 104 weeks, with or without 4 months of interferon (IFN) combination. HBV-specific sequences were amplified by polymerase chain reaction (PCR) from sera of patients with breakthrough virus, and the PCR products were directly analysed by sequencing. Breakthrough virus was detected in seven of the 31 patients (22.6%) between 9 and 18 months of therapy. Of the seven patients, six were HBeAg positive at baseline, and four had a double mutation consisting of rtM204V and rtL180M, while two had an rtM204I change. In one patient, two base substitutions at rt204 (ATG --> AGT; T to G and G to T) lead to a methionine to serine change (YMDD --> YSDD). This novel DNA pol mutation was detected at month 18 of lamivudine treatment. In addition, this new variant had the rtL180M mutation and a 12 base pair deletion in the pre-S1 region between nucleotides 43-54. The YSDD mutation was still present 6 months after lamivudine discontinuation. In vitro transfection studies also confirmed that the YSDD strain is resistant to lamivudine. In conclusion, the results indicate that, in addition to a Met --> Val and Met --> Ile change in YMDD, a Met --> Ser change at rt204 (YMDD --> YSDD) associated with the rtL180M change can also emerge during lamivudine treatment, which confers lamivudine resistance in vivo and in vitro, leading to virological breakthrough and ALT increases.

Nucleotide divergences in the core promoter and precore region of genotype D hepatitis B virus in patients with persistently elevated or normal ALT levels.

BACKGROUND: Mutation in the hepatitis B virus precore codon 28, creating a translational stop codon and double 1762-1764 T/A mutations in the core promoter region, controlling the transcription of the precore RNA and the core RNA have been suggested to correlate with the HBeAg status of patients with HBV infection. OBJECTIVES: The aim of the study was to further investigate the association of nucleotide divergences in both core promoter and precore regions with liver cell injury (reflected by ALT levels) in patients with chronic HBV infection. STUDY DESIGN: The sequences of the core promoter and the precore region of HBV isolated from 67 patients, all having genotype D and subtype ayw were analyzed. The patients were divided into two groups and four subgroups according to their HBeAg and Anti-HBe status, and ALT profile. RESULTS: It was found that the nucleotide divergences in the core promoter but not in the precore region were higher in patients having persistently elevated serum ALT than in serum ALT normal patients in both HBeAg positive and Anti-HBe positive groups (P<0.05). The number of T/A and A1896 stop codon mutations did not yield a statistically significant difference between ALT normal and elevated groups. It was also found that 1762-1764 T/A and precore A 1896 mutation existed in five and six out of 29 HBeAg positive patients, respectively. In 38 anti-HBe positive patients, 1762-1764 T/A and precore A1896 mutation were detected in three and 16 patients respectively, and coexisted in 10 patients. CONCLUSIONS: Precore A 1896 stop codon mutation seems to play an essential role in the loss of HBeAg in Turkish patients. Serum viremia levels of HBV in patients having precore stop codon and/or T/A mutation were not significantly different from the other patients carrying wild type strains. Nucleotide variability in the core promoter region may be one of the factors linked to hepatitis B disease activity.

Circulating IL-2 and IL-10 in chronic active hepatitis C with respect to the response to IFN treatment.

BACKGROUND: The importance of circulating immunoregulatory cytokines in response to IFN treatment and the change of in vivo production of these cytokines during interferon (IFN) treatment are not well known. We aimed to determine whether pretreatment serum levels of IL-2 and IL-10 are predictive of the response to IFN treatment and to investigate if treatment response or nonresponse has any effect on the circulating levels of these cytokines. PATIENTS AND METHODS: 37 patients (18 responders and 19 non-responders) with chronic hepatitis C virus (HCV) infection who received IFN-alpha2b for 6 months were studied. Responders were defined by complete alanine aminotransferase (ALT) normalization and loss of HCV RNA as detected by bDNA assay while patients who had elevated ALT levels and positive HCV RNA after 6 months were considered as nonresponders. RESULTS: Genotype distribution, ALT and HCV RNA levels were similar in responders and nonresponders. A significant number of patients with chronic hepatitis C (20/37 = 54%) had elevated IL-2 levels while IL-10 levels were not different from controls. No difference in baseline cytokine levels was observed between responders and non-responders. In the posttreatment serum samples some patients lost their detectable IL-2 or IL-10; some patients developed detectable cytokine levels after treatment irrespective of the treatment response. CONCLUSION: These results suggest that active liver injury in chronic hepatitis C is associated with increased circulating Th1 cytokine IL-2 but not with Th2 cytokine IL-10 and that circulating levels of these cytokines do not predict the response to IFN treatment. There is no constant and regular change in circulating levels of these cytokines under IFN treatment with respect to treatment response.

Influence of viral load and alanine aminotransferase on viral genetic heterogeneity in patients with chronic hepatitis C virus infection.

BACKGROUND/AIM: Hepatitis C virus (HCV) populations in vivo consist of genetically different heterogeneous mixtures defined as 'quasispecies', which vary in the hypervariable region 1 (HVR1) mostly. To further address the role of quasispecies diversity in hepatitis C infection, this study aimed to evaluate the influence of ALT, viral load and genotypes on quasispecies heterogeneity in patients with HCV infection. METHODS: Thirty-six chronic hepatitis C patients with high levels of alanine aminotransferase (ALT) were studied. None of them received any antiviral therapy. HCV RNA serum levels, genotype and genetic heterogeneity were determined by branched-chain DNA assay, restriction fragment length patterns and RT-PCR single-strand conformational polymorphism analysis of HVR1, respectively. RESULTS: Twenty-eight patients had genotype 1b (28/36; 78%), 6 patients had genotype 1a (6/36; 17%), 1 patient was 2a (1/36; 3%) and genotype could not be determined in 1 patient. The patients were categorized into two groups according to the number of bands representing the dominant strains in the circulation: group A with 2 bands having 1 strain (14/36 patients; 39%) and group B with more than 2 bands indicating more than 1 strain (22/36 patients; 61%). The serum viremia and ALT levels for these groups were 11 +/- 8.8 and 5.3 +/- 4.6 mEq/ml (p < 0.05), and 79 +/- 20, and 127 +/- 80 IU/l (p < 0.05), respectively. CONCLUSION: The results of this study suggest that hepatitis C patients having 1 dominant strain in the circulation may show a relatively weaker immune response resulting in lower ALT and higher viremia levels, whereas patients with high degrees of virus quasispecies diversity have higher ALT levels and a more active immune response causing the selection of new genome variants and depressing viral replication partly.

Polymorphism of precore region of hepatitis B virus DNA among patients with chronic HBV infection in Turkey.

The prevalence of mutations in the precore and core promoter regions of hepatitis B virus DNA and the association with the hepatitis B e antigen-negative phenotype vary in different geographical areas. It is rather high especially in the Far East and Mediterranean countries. The mutations occurring in the precore and the minimal essential region of the core promoter of HBV-DNA were analyzed in the sera of 81 patients (HBeAg-positive, 47 patients; HBeAg-negative, 34 patients) with chronic hepatitis B virus infection by direct sequencing of amplified polymerase chain reaction products. All patients had thymine at nucleotide 1858. Seven of 47 HBeAg-positive patients (15%) and 29 of 34 HBeAg-negative patients (85%) had precore stop codon mutations (G to A change at nucleotide 1896). No nucleotide change was found in the minimal essential region of HBV core promoter in any patient studied. In conclusion, the hepatitis B e antigen-negative phenotype in Turkish patients with chronic hepatitis B is associated with mutations in the precore but not in the minimal essential region of the core promoter. These results representing a part of the eastern Mediterranean support the studies conducted for the other populations of the region.