Ana Gila

Mutations in E2-PePHD, NS5A-PKRBD, NS5A-ISDR, and NS5A-V3 of Hepatitis C Virus Genotype 1 and Their Relationships to Pegylated Interferon-Ribavirin Treatment Responses

ABSTRACT Mutations in several subgenomic regions of hepatitis C virus (HCV) have been implicated in influencing the response to interferon (IFN) therapy. Sequences within HCV NS5A (PKR binding domain [PKRBD], IFN sensitivity-determining region [ISDR], and variable region 3 [V3]) were analyzed for the pretreatment serum samples of 60 HCV genotype 1-infected patients treated with pegylated IFN plus ribavirin (1b, n = 47; 1a, n = 13) but with different treatment outcomes, those with sustained virologic responses (SVR; n = 36) or nonresponders (NR; n = 24). Additionally, the sequence of the PKR/eIF-2α phosphorylation homology domain (E2-PePHD) region was determined for 23 patients (11 SVR and 12 NR). The presence of >4 mutations in the PKRBD region was associated with SVR (P = 0.001) and early virologic responses (EVR; 12 weeks) (P = 0.037) but not rapid virologic responses (4 weeks). In the ISDR, the difference was almost statistically significant (68% of SVR patients with mutations versus 45% without mutations; P = 0.07). The V3 region had a very high genetic variability, but this was not related to SVR. Finally, the E2-PePHD (n = 23) region was well conserved. The presence of >4 mutations in the PKRBD region (odds ratio [OR] = 9.9; P = 0.006) and an age of ≤40 years (OR = 3.2; P = 0.056) were selected in a multivariate analysis as predictive factors of SVR. NS5A sequences from serum samples taken after 1 month of treatment and posttreatment were examined for 3 SVR and 15 NR patients to select treatment-resistant viral subpopulations, and it was found that in the V3 and flanking regions, the mutations increased significantly in posttreatment sera (P = 0.05). The genetic variability in the PKRBD (>4 mutations) is a predictive factor of SVR and EVR in HCV genotype 1 patients treated with pegylated IFN and ribavirin.

Importance of Host Genetic Factors HLA and IL28B as Predictors of Response to Pegylated Interferon and Ribavirin

OBJECTIVES:Viral factors are considered the best predictors of response to treatment for chronic hepatitis C (CHC), but genetic factors are known to have an important role in this respect. This paper investigates the relationships among the host genetic factors HLA and IL28B, viral factors, and the outcome of combination therapy.METHODS:A multicenter retrospective cohort of 428 previously untreated CHC patients was treated with pegylated interferon/ribavirin (pegIFN/RBV) for 48 weeks. In all, 378 (88%) of these patients were genotype 1 or 4, and 50 (12%) were genotype 2 or 3.RESULTS:Multivariate logistic regression showed the rs12979860 CC genotype (adjusted odds ratio (aOR)=4.3, 95% confidence interval (95% CI): 2.6–7), the HLA-DQB1*0301 allele (aOR=2.08, 95% CI: 1.2–3.5) and age, viral genotype, and viral load levels to be significantly associated with sustained virological response (SVR). When the variable rs12979860 was eliminated, the area under the receiver operating characteristic (ROC) curve (AUC) decreased significantly (0.76 vs. 0.69; P=0.03). AUC values derived from viral factors were lower than those corresponding to host genetic factors (0.67 vs. 0.72, respectively; P=0.04). The HLA-DQB1*0301 and A*0201 alleles were associated with rs12979860 CC genotype and SVR (P<0.0001).CONCLUSIONS:The HLA-DQB1*0301 allele and IL28B genotype are factors that are associated independently with SVR. There is a synergism between the HLA-DQB1*0301 and HLA-A*0201 alleles with polymorphism rs12979860 CC, which increases the SVR rate. IL28B genotype is the best predictor of SVR.

Quasispecies as predictive factor of rapid, early and sustained virological responses in chronic hepatitis C, genotype 1, treated with peginterferon-ribavirin

BACKGROUND The relationship between the complexity of the hypervariable region 1 quasispecies of HCV and responsiveness to therapy is not completely clear. OBJECTIVE To investigate the importance of quasispecies as a predictive factor of rapid (RVR), early (EVR) and sustained (SVR) virologic response. METHODS Prospective analysis of 82 patients with chronic hepatitis C, genotype 1, treated with peginterferon alfa-2a and ribavirin. Quasispecies (SSCP), HCV-RNA and viral load were determined at baseline and 2, 4, 8 and 12 weeks after beginning treatment. RESULTS Less fibrosis and lower serum GGT activity were the only predictive factors for EVR. SVR predictive factors were age < or =40 years, viral load < or =600,000IU/mL, and quasispecies < or =5 bands. By logistic regression analysis, the independent factors determining SVR were age (P=0.011), viral load (P=0.027), and quasispecies (P=0.010). Quasispecies and viral load were not predictive factors of RVR. During treatment, quasispecies decreased rapidly in the SVR group. In non-EVR patients, quasispecies were slightly lower up to 8 weeks and then increased. CONCLUSIONS Quasispecies are an important predictive factor for SVR, but are no better predictors than viral load. Quasispecies are not predictive factors for RVR or EVR.

Hepatic Expression of Adiponectin Receptors Increases with Non-alcoholic Fatty Liver Disease Progression in Morbid Obesity in Correlation with Glutathione Peroxidase 1

BackgroundThe prevalence of non-alcoholic fatty liver disease (NAFLD) in obesity is very high. The role of adiponectin receptors in NAFLD progression remains still unclear. We speculate that changes in the hepatic expression levels of the two adiponectin receptors may be associated with the expression of oxidative stress-related genes.MethodsWe studied 60 morbidly obese patients with NAFLD, who underwent liver biopsy at the time of bariatric surgery. We measured the hepatic messenger-RNA concentration of adiponectin receptors (ADIPOR1 and ADIPOR2), glutathione peroxidase 1 (GPx1), glutathione reductase (GRd) and inducible oxide nitric synthase. Additionally, biochemical parameters and oxidative stress markers were determined in blood samples. According to the Kleiner score, the patients were divided into two groups: group 1 (25 patients without steatohepatitis) and group 2 (25 patients with probable steatohepatitis and ten patients with steatohepatitis).ResultsThe messenger-RNA concentration of all genes analysed in the study was higher among the patients in group 2. However, no differences in blood oxidative stress markers were observed. Strong correlations were found among the expression levels of ADIPOR1, ADIPOR2 and GPx1. The multivariate analysis showed that the only independent variable associated with NAFLD progression was the increase in GPx1 expression levels.ConclusionsNAFLD progression in morbid obesity is associated with increase in hepatic adiponectin receptor and oxidative stress-related genes. The linear correlations suggest that ADIPOR1, ADIPOR2 and GPx1 share key molecular factors in the regulation of the genetic expressions.

Importance of IL-10 and IL-6 during chronic hepatitis C genotype-1 treatment and their relation with IL28B.

UNLABELLED This paper investigates serum levels of interleukin 10 (IL-10) and interleukin 6 (IL-6) in patients with chronic hepatitis C genotype 1 (CHC-GT1), the relation of each with clinical and virological characteristics, how they affect the response to combined therapy and their relation with the IL28B polymorphisms rs12979860. Serum level expression and the polymorphism of IL-10, IL-6 and IL28B were determined in 138 CHC-GT1 patients, treated with pegylated interferon/ribavirin (pegIFN-α/RBV) for 48 weeks, in the following samples: baseline, week-12 (during treatment) and week-72 (post-treatment). 77 patients (56%) presented Sustained Virological Response (SVR) and 61 (44%) were non-SVR. Multivariate logistic regression showed that age ≤ 40 years (aOR=3.7, 95%CI=1.5-8.9, P=0.004), low activity of gamma glutamyl transferase (GGT) (aOR=0.9, 95%CI=0.98-0.99, P=0.028), CC genotype of IL28B polymorphism (aOR=2.7, 95%CI=1.0-7.2, P=0.044) and low IL-6 (aOR=0.5, 95%CI=0.3-1.0, P=0.038) were predictor factors of virological response. In all patients, following treatment, IL-6 decreased at week-12 (P=0.004) from baseline and had returned to basal values at week-72. Serum IL-10 concentration was significantly decreased at week-72 only in SVR patients (P ≤ 0.001). When patients were stratified by IL28B polymorphisms rs12979860 CC vs non-CC patients, a statistically significant decrease in IL-10 at week-72 in both groups was observed (P=0.003 and P ≤ 0.001, respectively). None of the polymorphisms of IL-10 or IL-6 studied were associated with SVR. CONCLUSIONS CC genotype of IL28B and low IL-6 serum concentration are factors associated independently with SVR. Moreover, decreased IL-10 at week-72 is associated with SVR in both CC and non-CC patients, and both factors are important to determine the effectiveness of treatment.

Variation of Transaminases, HCV-RNA Levels and Th1/Th2 Cytokine Production during the Post-Partum Period in Pregnant Women with Chronic Hepatitis C

This study analyses the evolution of liver disease in women with chronic hepatitis C during the third trimester of pregnancy and the post-partum period, as a natural model of immune modulation and reconstitution. Of the 122 mothers recruited to this study, 89 were HCV-RNA+ve/HIV-ve and 33 were HCV-RNA-ve/HIV-ve/HCVantibody+ve and all were tested during the third trimester of pregnancy, at delivery and post-delivery. The HCV-RNA+ve mothers were categorized as either Type-A (66%), with an increase in ALT levels in the post-partum period (>40 U/L; P<0.001) or as Type-B (34%), with no variation in ALT values. The Type-A mothers also presented a significant decrease in serum HCV-RNA levels in the post-delivery period (P<0.001) and this event was concomitant with an increase in Th1 cytokine levels (INFγ, P = 0.04; IL12, P = 0.01 and IL2, P = 0.01). On the other hand, the Type-B mothers and the HCV-RNA-ve women presented no variations in either of these parameters. However, they did present higher Th1 cytokine levels in the partum period (INFγ and IL2, P<0.05) than both the Type-A and the HCV-RNA-ve women. Cytokine levels at the moment of delivery do not constitute a risk factor associated with HCV vertical transmission. It is concluded that differences in the ALT and HCV-RNA values observed in HCV-RNA+ve women in the postpartum period might be due to different ratios of Th1 cytokine production. In the Type-B women, the high partum levels of Th1 cytokines and the absence of post-partum variation in ALT and HCV-RNA levels may be related to permanent Th1 cytokine stimulation.

Effectiveness and safety of first-generation protease inhibitors in clinical practice: Hepatitis C virus patients with advanced fibrosis.

AIM To evaluates the effectiveness and safety of the first generation, NS3/4A protease inhibitors (PIs) in clinical practice against chronic C virus, especially in patients with advanced fibrosis. METHODS Prospective study and non-experimental analysis of a multicentre cohort of 38 Spanish hospitals that includes patients with chronic hepatitis C genotype 1, treatment-naïve (TN) or treatment-experienced (TE), who underwent triple therapy with the first generation NS3/4A protease inhibitors, boceprevir (BOC) and telaprevir (TVR), in combination with pegylated interferon and ribavirin. The patients were treatment in routine practice settings. Data on the study population and on adverse clinical and virologic effects were compiled during the treatment period and during follow up. RESULTS One thousand and fifty seven patients were included, 405 (38%) were treated with BOC and 652 (62%) with TVR. Of this total, 30% (n = 319) were TN and the remaining were TE: 28% (n = 298) relapsers, 12% (n = 123) partial responders (PR), 25% (n = 260) null-responders (NR) and for 5% (n = 57) with prior response unknown. The rate of sustained virologic response (SVR) by intention-to-treatment (ITT) was greater in those treated with TVR (65%) than in those treated with BOC (52%) (P < 0.0001), whereas by modified intention-to-treatment (mITT) no were found significant differences. By degree of fibrosis, 56% of patients were F4 and the highest SVR rates were recorded in the non-F4 patients, both TN and TE. In the analysis by groups, the TN patients treated with TVR by ITT showed a higher SVR (P = 0.005). However, by mITT there were no significant differences between BOC and TVR. In the multivariate analysis by mITT, the significant SVR factors were relapsers, IL28B CC and non-F4; the type of treatment (BOC or TVR) was not significant. The lowest SVR values were presented by the F4-NR patients, treated with BOC (46%) or with TVR (45%). 28% of the patients interrupted the treatment, mainly by non-viral response (51%): this outcome was more frequent in the TE than in the TN patients (57% vs 40%, P = 0.01). With respect to severe haematological disorders, neutropaenia was more likely to affect the patients treated with BOC (33% vs 20%, P ≤ 0.0001), and thrombocytopaenia and anaemia, the F4 patients (P = 0.000, P = 0.025, respectively). CONCLUSION In a real clinical practice setting with a high proportion of patients with advanced fibrosis, effectiveness of first-generation PIs was high except for NR patients, with similar SVR rates being achieved by BOC and TVR.

Plasma ribavirin trough concentrations during treatment of chronic hepatitis C in genotype-1 patients.

Goals: To investigate the correlation between virological response and plasma ribavirin trough concentrations (RBV Ctrough) during the full period of chronic hepatitis C (CHC) treatment. Study: Multicenter prospective cohort study. Total 119 patients with CHC genotype-1 were treated with peginterferon alfa-2a (pegIFN) and RBV for 48 weeks. RBV quantification was carried out at week 4 (W4), W8, W12, W16, W24, W32, and W40 of treatment. Results: The mean RBV Ctrough value during treatment was 2.5±0.9 mg/L in total patients. At no time point of treatment were patients with RBV Ctrough average correlated with early and sustained virological response (SVR), but those with RBV Ctrough ≥5 mg/L (95th percentile) at any time point (22/119, 18%) were correlated with SVR (P=0.02). Such high RBV Ctrough values were found from the second to the fourth months of treatment in 73% of these patients (16/22), and this was independently associated with SVR (odds ratio=3.6, 95% confidence interval:1.02−13.2, P=0.04). Conclusion: Our data do not support RBV plasma monitoring as a tool to optimize treatment in patients with CHC genotype-1, but show that a high RBV plasma concentration could improve SVR rates.

Influence of HLA class I, HLA class II and KIRs on vertical transmission and chronicity of hepatitis C virus in children.

Background & aim There is evidence that maternal viral load of HCV during delivery influences the risk for Mother-to-child transmission (MTCT), but this does not explain all cases. We study the role of the immunogenetic profile (HLA, KIRs and KIR-ligand binding) of mothers and children in HCV-MTCT and in chronicity in the children. Methodology 79 HCV-RNA (+) mothers and their 98 children were included. 24 children were infected, becoming chronic in 8 cases and clearing in 16. HLA-class-I and II and KIRs were determined by Luminex. Results MTCT study: The presence of HLA-C1-ligand in mothers and/or their children reduces the risk of transmission (mothers: Pc = 0.011, children: P = 0.033), whereas the presence of HLA-C2C2-ligand in mothers increases it (Pc = 0.011). In children KIR2DL3-HLA-C1 is a protector factor (Pc = 0.011). Chronicity in children study: Maternal DQA1*01 allele (Pc = 0.027), KIR2DS1 (Pc = 0.011) or KIR3DS1 (Pc = 0.011) favours chronicity in the child. The presence of the DQB1*03 allele (Pc = 0.027) and KIR2DS3 (P = 0.056) in the child and homozygosity for KIR3DL1/3DL1 (Pc = 0.011) and for the HLA-Bw4/Bw4 ligand (P = 0.027) is associated with viral clearance, whereas the presence of HLA-Bw6 ligand (P = 0.027), the binding of KIR3DS1-HLA-Bw4 (P = 0.037) and heterozygosity for KIR3DL1/3DS1 (Pc = 0.011) favour viral chronicity. Mother/child allele matching: In the joint HLA analysis, matching was greater between mothers and children with chronic infection vs those who had cleared the virus (67%±4.1 vs 57%±1.2, P = 0.003). Conclusions The HLA-C1 ligand in the mother is related to MTCT, while several genetic factors of the mother or child are involved in the chronification or clearance of infection in the child. Matching allelic data is considered to be an indicator of HCV chronicity in the child and can be used as a potential prognostic test. This implies that NK cells may play a previously undocumented role in protecting against MTCT and that both NK cell immunity and adaptive T-cell responses may influence viral clearance in infected children.

The antigenic variability of HCV in viral HLA-Ag binding is related to the activation of the host immune response

Our previous data show that hepatitis C virus (HCV) genotype 1 patients expressing the HLA-DQB1 * 0301 allele have a combined response probability of 69%, while the remaining 31% do not respond, probably because the HCV immunodominant epitope (IE) against the DQB1 * 0301 allele is mutated. HCV IE (region sequenced in NS3 is a region encoding aa 1253–1272) from 37 patients (21 Sustained Virological Response, SVR; 16 non-SVR) HLA-DQB1 * 0301+, were analysed by pyrosequencing. In vitro cultures were also determined by CD4+ proliferation, using non-mutated IE (wild-type synthetic peptide) and synthetic mutated peptide. The pyrosequencing study revealed 34 different haplotypes. The SVR patients had fewer haplotypes (P = 0.07), mutations/haplotypes (P = 0.01) and polymorphic sites (P = 0.02) than non-SVR. Three polymorphic sites were associated with the non-SVR patients: haplotype 7 (L5P); haplotype 11 (L7P); and haplotype 15, (L15S) (P = 0.02). The in vitro study (n = 7) showed that in 4/7 patients (Group 1) the CD4+ proliferation obtained with wild-type synthetic peptide was higher than that obtained with the negative control and with the synthetic mutated peptide (P = 0.039). However, in the remaining 3/7 patients (Group 2) this pattern was not observed (P = 0.7). Our findings suggest that HLA-DQB1 * 0301+ patients with high antigenic variability in HCV IE (NS31253-1272) have a lower SVR rate, due to reduced CD4+ proliferation as a result of incorrect viral HLA-Ag binding.