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.

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.

Effects of ribavirin monotherapy on the viral population in patients with chronic hepatitis C genotype 1: direct sequencing and pyrosequencing of the HCV regions.

Ribavirin remains essential to chronic hepatitis C treatment. This paper investigates the influence of ribavirin priming to steady state before combined pegylated-interferon/ribavirin treatment on viral kinetics, ribavirin trough concentrations, genetic variability within HCV-core, -NS5B and -NS5A, and response to antiviral therapy. A prospective cohort study was made of 27 chronic hepatitis C genotype 1 naïve patients who received four weeks of ribavirin followed by pegIFN-α-2a/ribavirin for 48 weeks (Group A). The results obtained were compared with those for a control/historical group (Group B). In addition, direct sequencing and pyrosequencing were applied to determine ribavirin monotherapy-induced sequence changes. The rapid, early, and sustained virological response values obtained were 48%, 89%, and 52%, respectively, in Group A, and 52%, 90%, and 52% in Group B (P > 0.05). In the four-week combined treatment, the Group A patients showed a greater decrease in HCV-RNA (2.3 log10 IU/ml vs. 1.2 log10 IU/ml; P = 0.04), lower alanine aminotransferase levels (23.5 ± 1.33 U/L vs. 60.11 ± 18 U/L; P < 0.001) and higher mean ribavirin trough concentrations (3.28 ± 1.26 mg/L vs. 1.74 ± 0.7 mg/L; P = 0.001). No general increase in rates of nucleotide substitutions in the ribavirin monotherapy-treated patients was observed in NS5B, ISDR, or PKRbd, but there was a decrease in silent mutations in the HCV core (P = 0.04). This result was confirmed by pyrosequencing in the NS5A region. It is concluded that the ribavirin priming combined treatment with pegIFN-α-2a does not improve sustained virological response rates in HCV genotype 1 naïve infected patients. However, the greater reductions in viral load and alanine aminotransferase levels, together with the higher ribavirin trough concentration values obtained, could reflect the greater effectiveness of the treatment. Ribavirin does not have a mutagenic effect on the virus in patients with chronic hepatitis C.

Polymorphisms in histone deacetylases improve the predictive value of IL-28B for chronic hepatitis C therapy.

Histone deacetylases (HDACs) influence many cellular processes, including the modulation of signal transducer and activator of transcription activity (STAT) in response to interferon (IFN). To identify genetic markers that help optimize the IL-28B prediction of chronic hepatitis C (CHC) sustained virological response (SVR), we evaluated 27 single-nucleotide polymorphisms (SNPs) in HDAC1-11. Three SNPs, rs3778216, rs976552 and rs368328 in HDAC2, HDAC3 and HDAC5, respectively, were independently associated with SVR (P<0.05). The addition of these three HDAC's SNPs to the IL-28B predictive model (area under the curve (AUC)=0.630) rendered an important improvement of AUC-receiver operating characteristic value (AUC=0.747, P=0.021). Chi-squared Automatic Interaction Detector (CHAID) analysis denoted the significance of the rs3778216 C/C genotype in identifying a group of good responders despite carrying IL-28B T allele (79.2% of SVR), whereas HDAC5 G allele characterized a subgroup with poor response rate (25.5%). However, HDAC3 rs976552 did not display a relevant role for the hierarchical classification of patients. Variables related to SVR in hepatitis C virus genotype 1 (HCV-1) cohort were the same of those obtained for the overall population. Interestingly, in non-HCV-1 patients (n=56) the HDAC2 C/C genotype was the unique predictive variable related to SVR (AUC=0.733, P<0.007). Thus, these preliminary results suggest the potential usefulness of combined IL-28B and HDAC genotyping for the CHC patients' classification by likelihood of an SVR.

Histological and immunohistochemical assessment of liver biopsies in morbidly obese patients.

AIMS: To study liver lesions in morbidly obese patients who underwent liver biopsy at the time of bariatric surgery to define histological lesions, especially inflammatory infiltrate, diagnostic categories and the possible influence of gender in this respect. METHODS AND RESULTS: 110 biopsies (36 males-M- and 76 females -F-) were evaluated and categorised, according to the NAS (NAFLD -non alcoholic fatty liver disease- Activity Score) system and other criteria, as non-NAFLD (15.5%, F predominance), non-alcoholic steatohepatitis (NASH) (16.5%, M predominance), non-alcoholic hepatosteatosis (NAHS) (21%, F predominance) and, the most numerous group, NASH-borderline (NASH-BORD) (47%), with three subgroups, characterised by centrozonal lesions, portal area preferential involvement or affecting both areas. The predominant form of hepatocytesteatosis was mixed with a multivesicular component that was present in most cases with fibroinflammatory portal involvement. Nuclear glycogenosomes were found in greater number of biopsies in patients in the third and sixth decades. Portal inflammation was present in a large number of cases (M predominance); the application of immunohistochemical techniques (myeloperoxidase and CD68 antibodies) to evaluate lobular inflammation revealed "surgical hepatitis" in one third of the cases, and the presence of microgranulomas (CD68+) (M predominance), which were more abundant with increasing lesion severity. CONCLUSIONS: Portal inflammation and multivesicular hepatocytesteatosis are highly prevalent in morbidly obese patients. This study identifies a new subtype of NASH-BORD characterized by centrizonal and porto-periportal area involvement and the existence of liver biopsies without steatosis. CD68+ microgranulomas constitute an unequivocal marker of lobular inflammation in surgical biopsies and of lesion severity, which is gender-related.

Genetic variants of interferon-stimulated genes and IL-28B as host prognostic factors of response to combination treatment for chronic hepatitis C.

Chronic hepatitis C (CHC) is a worldwide health problem that is highly related to liver fibrosis, cirrhosis, and hepatocellular carcinoma. The achievement of response to the current standard of care-pegylated interferon plus ribavirin-has recently been described to be associated with single-nucleotide polymorphisms (SNPs) near the IL-28B gene. Additionally, baseline expression levels of genes involved in interferon (IFN)-stimulated genes (ISGs) have been found to be related to treatment outcome. In the present study, 285 patients were genotyped for 63 SNPs within genes of the IFN signaling pathway (IPGs) and ISGs. Two ISG polymorphisms-OASL rs12819210 (odds ratio (OR)=2.1, P=0.03) and IFIT1 rs304478 (OR=2.5, P=0.01)-were found to be independent predictive factors of sustained virological response (SVR) after adjusting for other clinical covariates. Furthermore, the predictive value of IL-28B SNP was notably improved by simultaneous genotyping of rs12819210 and rs304478, particularly in patients with the worst prognosis (viral genotype 1, area under the curve (AUC)=0.74). In conclusion, ISG SNPs could constitute a valuable tool for individualizing CHC therapy.

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.

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-2alpha 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 < or = 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.

Induction doses of interferon-alpha-2a in combination with ribavirin and/or amantadine for the treatment of chronic hepatitis C in non-responders to interferon monotherapy: a randomized trial.

The benefit of the triple therapy (interferon + amantadine + ribavirim) is still unknown. The efficacy of induction doses of interferon-alpha-2a monotherapy or in combination with ribavirin and/or amantadine was evaluated in interferon non-responders with chronic hepatitis C. A total of 378 patients were randomized. All the groups received the same doses and duration of interferon-alpha-2a: (i) interferon 9 MUI/day for 4 weeks and then 3 MUI/3 t.i.w. for 44 weeks (n = 53); (ii) interferon in combination with amantadine 100 mg twice daily for 48 weeks (n = 111); (iii) interferon in combination with ribavirin 1000-1200 mg (n = 106); (iv) interferon in combination with amantadine and ribavirin (n = 108). Baseline parameters were similar in the four groups. Sustained virological and biochemical responses were 13%, 6%, 18% and 22% respectively. No significant differences were found between double ribavirin arm vs triple therapy, but the difference was significant between interferon-amantadine (P = 0.008) and triple therapy (P = 0.0005). Hence, the induction doses of interferon in combination with ribavirin or ribavirin plus amantadine showed encouraging results in patients with chronic hepatitis C who were resistant to interferon. However, triple therapy is not superior to double.

Hepatitis post-transfusional en neonatos encamados en una Unidad de Cuidados Intensivos Neonatal / No disponible

Objetivo: estudiar la importancia y significado del aumento de transaminasas en neonatos que ingresaron en una Unidad de Cuidados Intensivos Neonatales y su relación con la transfusión sanguínea. Métodos: seguimiento prospectivo de 209 pacientes de los cuales 177 completaron el seguimiento: 129 transfundidos y 48 no transfundidos (57 nacidos a término y 120 prematuros). Se de- terminaron la ALT, AST y GGT mensualmente hasta los seis meses de edad y hasta los seis meses de la última transfusión. Al final del seguimiento y cuando había un aumento de transaminasas se investigó el virus de la hepatitis A, B, C, G, TT, citomegalovirus, Epstein-Barr, herpes 1-2 y toxoplasma. La serología viral también se estudió en todas las madres y en los niños positivos en sus donantes. Resultados: ciento veintinueve neonatos (73%) recibieron 461 U/concentrados-hematíes (3,6 ± 3 U/paciente). Cincuenta y cuatro pacientes (30,5%) presentaron un aumento de ALT, 46 (36%) transfundidos y 8 (17%) no transfundidos (p < 0,05). Las variables independientes asociadas con este aumento de las transaminasas fueron: la infección con el virus G, la nutrición parenteral durante más de 12 días y, con casi significación estadística, la transfusión. Veinte pacientes (11,3%) tenían un aumento de ALT superior a 2,5 veces el valor normal: 18 (14%) transfundidos, 2 (4%) no transfundidos (p = 0,106). Sólo el virus G y el virus TT se relacionaron con la transfusión; el curso clínico fue asintomático, aunque la mayoría quedaron crónicamente infectados. Conclusión: en el seguimiento de los neonatos graves, el aumento de las transaminasas es frecuente. Las transfusiones son seguras para la mayoría de los virus hepatotropos, pero es posible la transmisión de los virus G y TT (AU)

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Post-transfusional hepatitis in neonates hospitalized in a Neonatal Intensive Care Unit.

OBJECTIVE: To assess the significance of increased serum transaminase levels in neonates admitted to a Neonatal Intensive Care Unit and its relationship with blood transfusion. METHODS: Follow-up prospective study of 209 patients; 177 completed follow-up, of whom 129 were transfused and 48 were not; 57 were born after full gestation and 120 were born prematurely. The activity of serum levels of ALT, AST, and GGT was measured monthly up to six months of age, and until six months after the last transfusion. At the end of follow-up, and whenever an increase in serum transaminase levels was detected, the viral agents of hepatitis A, B, C, G, TT, cytomegalovirus, Epstein-Barr, and herpes 1 and 2, and toxoplasma were studied. Viral serology was also carried out in mothers and in donors when children tested positive. RESULTS: One hundred twenty nine neonates (73%) received 461 U red blood cell transfusions (3.6 +/- 3 U/patient). ALT levels increased in 54 (30.5%) patients, of whom 46 (36%) were transfused and eight (17%) were not (p < 0.05). The independent variables were 'infection by G virus' and 'parenteral nutrition for more than 12 days'; the variable 'transfusion' was close to the limit for statistical significance. Twenty patients (11.3%) had increased serum ALT levels 2.5 times above the normal value: 18 (14%) were transfused and two (4%) were not (p = 0.106). Only the G and TT viruses were related with transfusion; patients remained asymptomatic, although most neonates were chronically infected. CONCLUSION: Follow-up showed that increased serum ALT levels are common among severely ill neonates. Blood transfusions are safe concerning most hepatotropic viruses, but transmission of viruses G and TT is possible.

Estudio de la replicación del virus de la hepatitis C y del virus de la hepatitis G en suero, hígado y células mononucleares de sangre periférica / Genomic and antigenomic chains of hepatitis C virus and hepatitis G virus in serum, liver and peripheral blood mononuclear cells

Objetivo: determinar los lugares de replicación del VHC y del VHG en pacientes con hepatitis crónica C y estudiar la interacción de ambos virus. Pacientes: se estudió el ARN-VHG en 272 pacientes con hepatitis crónica C. De éstos, 35 fueron positivos (grupo I). Se seleccionaron 23 pacientes con hepatitis crónica C y no coinfectados con el VHG (grupo II). Resultados: se estudiaron las cadenas genómica y antigenómica del VHC en los dos grupos y del VHG en el grupo I en muestras de suero, células mononucleares de sangre periférica y tejido hepático. En el grupo I se observó la genómica del VHC y VHG en un 86 y 100 por ciento respectivamente (ns) en las muestras de suero (n=35), y la antigenómica en un 17 y 23 por ciento (ns). En las muestras de células mononucleares (n=15), el 100 por ciento presentaba la cadena genómica del VHC y el 60 por ciento la del VHG (p<0,05); las antigenómicas se detectaron en un 13 y 33 por ciento respectivamente (ns). En hígado (n=25) las cadenas genómicas se observaron en un 100 y 12 por ciento respectivamente (p<0,001); la antigenómica del VHC se detectó en un 76 por ciento mientras que la del VHG no estaba presente (p<0,001). En el grupo II la cadena genómica del VHC se encontraba en un porcentaje muy elevado en todas las muestras, mientras que la antigenómica apareció en un 13 por ciento en suero y células mononucleares y en un 89 por ciento en hígado. Conclusiones: el VHC y el VHG tienen lugares distintos de replicación: mientras que el VHC se replica principalmente en el hígado, el VHG no es hepatotropo. Las células mononucleares podrían representar un lugar de replicación para el VHG y menos importante para el VHC. Por último, el VHG no modifica la replicación viral del VHC. (AU)

Genomic and antigenomic chains of hepatitis C virus and hepatitis G virus in serum, liver and peripheral blood mononuclear cells.

OBJECTIVE: To determine HCV and HGV replication sites in patients with chronic hepatitis C and to study interaction between these two viruses. PATIENTS: HGV RNA was studied in 272 patients with chronic hepatitis C. Of these, 35 were positive (group I). Twenty-three patients with chronic hepatitis C not co-infected with HGV were selected (group II). RESULTS: Genomic and antigenomic chains of HCV were studied in both groups and those of HGV in group I in serum samples, peripheral blood mononuclear cells and liver tissue. In group I genomic chains of HCV and HGV were observed in 86 and 100%, respectively (ns), in serum samples (n = 35), and antigenomic chains in 17 and 23%, respectively (ns). In mononuclear cell samples (n = 15) 100% presented the genomic chain of HCV and 60% presented that of HGV (p < 0.05). Antigenomic chains were detected in 13 and 33%, respectively (ns). In liver tissue (n = 25) genomic chains were observed in 100 and 12%, respectively (p < 0.001); the antigenomic chain of HCV was detected in 76% while that of HGV was not present (p < 0.001). In group II genomic chains of HCV were found to be present in a very high percentage in all samples, while antigenomic chains appeared in 13% of serum and mononuclear cell samples and 89% of liver samples. CONCLUSIONS: HCV and HGV have different sites of replication: whereas HCV replicates mainly in the liver, HGV is not hepatotropic. Mononuclear cells could represent a replication site for HGV but they are less important for HCV. Lastly, HGV does not modify the viral replication of HCV.

[Coinfection with hepatitis G virus in chronic hepatitis C. Response to treatment with interferon alpha]. / Estudio de la coinfección por el virus de la hepatitis G en la hepatitis crónica C. Respuesta al tratamiento con interferón alfa.

BACKGROUND: It is thought that the cytopathic effect of HGV is not important. Nevertheless, the cytopathic effect on liver is less known in the cases of co-infection with HCV. The aim was to study the prevalence of co-infection in patients with chronic hepatitis C (CHC) and to analyse the clinical-epidemiological and histological data and the interferon (IFN) response. PATIENTS AND METHODS: We included 180 patients with CHC and the HGV-RNA was determined. RESULTS: The prevalence of co-infection was 12.2% (n = 22). No statistical differences were observed between the non co-infected and co-infected groups with regard to the age, sex, mechanism of transmission and alcohol abuse. Also, there were no differences in the hepatic biochemical, no organ-specific antibodies, histological lesions and Knodell index. The HCV biochemical response (BR) and virological response (VR) after 6 months post-IFN were the same in both groups (HGV negative: BR = 29%, VR = 12%; HGV positive: BR = 22%, VR = 18%). HGV was determined after 6 months posttreatment in the co-infected group (first cycle of IFN, n = 22; second cycle of IFN, n = 9): 12 (55%) were HGV-RNA negative and 5 (23%) HCV-RNA negative, (p = 0.021). When we compared the BR vs VR in this group, there were 12 HGV-RNA negative but only two had BR (NS). On the contrary, the BR was related to HCV-RNA negative (p = 0.023). CONCLUSION: The prevalence of HGV co-infection is important in our area (12.8%). The HGV does not increase the pathogenicity of HCV and does not change the IFN response, although the HGV is more IFN sensible than HCV. The determination of HGV is not necessary in patients with HCV.

[Retrospective study of the incidence of hepatitis G virus infection in transfused patients]. / Estudio retrospectivo de la incidencia de infección por el virus de la hepatitis G en los pacientes transfundidos.

BACKGROUND: Recently the parenteral transmission of hepatitis G virus (HGV) has been shown. The aim of the study was to investigate the incidence of post-transfusion HGV. PATIENTS AND METHODS: HGV (RNA-HGV and anti-HGVE2) were retrospectively studied in 140 transfused patients. RESULTS: 12 (8.6%) were infected after transfusion: 9 of 12 (75%) the RNA-HGV remained detectable after 6 months and 3 (25%) seroconverted to anti-HGVE2. No patient had post-transfusional hepatitis criteria. In 5 (42%) the transaminases levels were slightly increased. The clinical evolution was favourable. No significant differences were found between patients with or without HGV infection. CONCLUSIONS: HGV is an agent associated with transfusion but it carries a low pathogenic capability.