J. Salmerón

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

UNLABELLEDnThis 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.nnnCONCLUSIONSnCC 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.

Low-dosage prophylactic vancomycin in central-venous catheters for neonates

Neonatal infectious pathology remains one of the main causes of morbidity and mortality in this age group. The introduction of plasticized catheters for the administration of medication, fluidotherapy and parenteral nutrition was a significant advance in treatment of patients at risk, but also led to the appearance of infectious complications. Negative coagulase staphylococcus is the principal pathogen in most neonatal intensive care units. Recent studies have examined the prophylactic use of vancomycin in preterm babies receiving parenteral nutrition. We have evaluated the efficacy of this procedure, applied via the central venous catheters employed for all neonates, within the intensive care unit over a period of one year. Prophylactic vancomycin administered via the catheters significantly reduced the incidence of Gram-positive infections, despite the presence within this group of a greater number of septic risk factors than in the control group.

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.

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-DQB1u2009*u20090301 allele have a combined response probability of 69%, while the remaining 31% do not respond, probably because the HCV immunodominant epitope (IE) against the DQB1u2009*u20090301 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-DQB1u2009*u20090301+, 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 (Pu2009=u20090.07), mutations/haplotypes (Pu2009=u20090.01) and polymorphic sites (Pu2009=u20090.02) than non-SVR. Three polymorphic sites were associated with the non-SVR patients: haplotype 7 (L5P); haplotype 11 (L7P); and haplotype 15, (L15S) (Pu2009=u20090.02). The in vitro study (nu2009=u20097) 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 (Pu2009=u20090.039). However, in the remaining 3/7 patients (Group 2) this pattern was not observed (Pu2009=u20090.7). Our findings suggest that HLA-DQB1u2009*u20090301+ 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.

P1200 EFFECTIVENESS OF TRIPLE THERAPY WITH BOCEPREVIR OR TELAPREVIR IN A MULTICENTRE CLINICAL PRACTICE COHORT OF HCV TREATMENT-EXPERIENCED PATIENTS WITH ADVANCED HEPATIC FIBROSIS. SVR-12W AFTER TREATMENT

P1200 EFFECTIVENESS OF TRIPLE THERAPY WITH BOCEPREVIR OR TELAPREVIR IN A MULTICENTRE CLINICAL PRACTICE COHORT OF HCV TREATMENT-EXPERIENCED PATIENTS WITH ADVANCED HEPATIC FIBROSIS. SVR-12W AFTER TREATMENT C. Fernandez, P. Munoz de Rueda, S. Alonso, M. Prieto, A. Martin-Alvarez, J. Pons, J. Pascasio, M. Serra, M. Romero, P. Conde, I. Carmona, M. Diago, M. Testillano, J. NavarroJarabo, J. Sanchez Ruano, J. Sousa, F. Nogueras, R. Granados, G. Sanchez Antolin, R. Andrade, H. Hallal, M. Marti Arribas, M. del Moral, J. Salmeron. Servicio de Aparato Digestivo, HU Fundacion de Alcorcon, Madrid, Unidad de Apoyo a la Investigacion, HU San Cecilio, Ciber de Enfermedades Hepaticas y Digestivas (CIBERehd), Granada, Unidad de Gestion Cĺinica de Enfermedades Digestivas, H La Fe, Valencia, Unidad de Hepatoloǵia, H Virgen de Arrixaca, Murcia, Unidad de Hepatoloǵia, H Virgen del Rocio, Sevilla, H Cĺinico de Valencia, Valencia, H Nuestra Senora de Valme, CIBERehd, Sevilla, H Virgen de la Concha, Zamora, Servicio de Aparato Digestivo, HU Virgen de la Macarena, Sevilla, H General de Valencia, Valencia, HU de CRUCES, Bilbao, Vizcaya, Unidad de Aparato Digestivo, Agencia Sanitaria Costa del Sol, Marbella, Malaga, Servicio de Aparato Digestivo, Complejo Hospitalario de Toledo, Toledo, Servicio de Digestivo, HU Virgen de las Nieves, Granada, HU Gran Canaria Dr. Negŕin, Las Palmas de Gran Canaria, Unidad de Hepatologia, HU Ŕio Hortega, Valladolid, HU Virgen de la Victoria, Malaga, H Morales Meseguer, Murcia, HU Salamanca, Salamanca, Unidad de Gestion Cĺinica de Aparato Digestivo, HU San Cecilio, Ciber de Enfermedades Hepaticas y Digestivas (Ciberehd), Granada, Spain E-mail: fsalmero@ugr.es

P214 SPECIFIC MUTATIONS (L5P; L7P; L15S) IN THE IMMUNODOMINANT EPITOPE HCV-NS3 (AA 1253–1272) AGAINST HLA-DQB1*0301, PROVOKE NON-RESPONSE TO ANTIVIRAL TREATMENT WITH PEGIFN/RBV IN PATIENTS WITH CHC-1

Background and Aims: Recent studies indicate that metformin has growth inhibitory and antiangiogenic effects, reducing the risk of some tumors. In this work, we have investigated the molecular mechanisms underlying HCC development promoted by HCV infection. Methods: Huh7.5 and HepG2 cells were grown in supplemented DMEM culture medium. Huh7.5 cells were infected with JFH1 particles (one particle/cell). Human hepatocytes were prepared from the liver biopsies obtained from 3 patients and hepatocyte isolation was based on the two-step collagenase procedure and treated with metformin (2 mM) for 48 hours. Gene expression was analyzed by qPCR.Proteins were analyzed by western-blot using primary antibodies against AKT, MTOR, PTEN and PTP1B. Results: HepG2 and Huh7.5 cell-growth was affected by metformin treatment at the indicated concentrations. A significant reduction (20%-Huh7.5 and 30%-HepG2) in cell confluence was seen after 48 and 72 hours of metformin treatment, respectively. This effect was observed to be dose-dependent. In Huh7.5 cultures, MAP3K (1.5±0.3 fold), AKT (2.3±0.5), PI3K (1.7±0.3), PTEN1 (1.8±0.2), TPT (3.3±0.6) and PTPN2 (2.3±0.5) gene expression were upregulated. Metformin treatment downregulated PTP1B and PTEN protein expression in JFH1-infected cells compared to Huh7.5 noninfected cells (control). MTOR protein expression was increased. In primary hepatocytes treated with metformin, PTP1B and PTEN proteins were also found down-regulated, together with AKT and mTOR.Metformin activated apoptosis through caspase-3. Conclusions: PTP1B and PTEN, involved in tumor progression were found down-regulated in HCV infection and primary hepatocytes treated with metformin, suggesting a role for these proteins in HCC development. Akt/mTOR pathway is activated in HCV infected cells and was found down-regulated after metformin treatment. These findings could explain the tumor suppressor effect exerted by metformin.

1162 ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN INTERFERON STIMULATED GENES WITH CHRONIC HEPATITIS C TREATMENT OUTCOME

insulin resistance and hepatic steatosis. Steatosis may increase susceptibility to apoptosis, inflammation and fibrosis by triggering hepatocytes to up-regulate CD95/Fas. We investigated this hypothesis and potential role of adipocytokines in modulating the progression of liver disease in patients with HCV-4. Methods: In 147 HCV patients and 89 controls we measured serum adiponectin, HMW adiponectin, leptin, TNF-a, IL-6 and CK-18. Liver biopsies were evaluated for steatosis/inflammation/ fibrosis, adiponectin mRNA/protein, AdipoR1/-R2 mRNA and phosphoenolpyruvate carboxykinase (PEPCK) gene expression; and adiponectin and CD95 immunoreactivity. The potential associations with hepatic steatosis and fibrosis were analyzed. Results: CD95 immunoreactivity and adiponectin immunoreactivity were readily detected in all biopsies examined and scored as grade 3 in 24 (16.3%) of patients, and as bright in 20 (13.6%) of patients, respectively. Adiponectin immunostaining within the liver correlated positively with the intensity of hepatic CD95/Fas immunostaining within the liver (r = 424; p =0.001). A significant association between high serum adiponectin and HMW adiponectin levels with CD95/Fas immunoreactivity (r = −0.16, p = 0.04, r = 0.21, p = 0.001; respectively), but not with adiponectin immunoreactivity in the liver was also identified. Adiponectin and HMW adiponectin were negatively correlated with the expression of AdipoR1, but positively correlated with the expression of AdipoR2. Hepatocyte CD95/Fas up-regulation correlated with steatosis, inflammation and fibrosis (p = 0.001). Significant correlation of serum adiponectin, HMW adiponectin and AdipoR1 and 2 mRNA expression, as well as liver adiponectin immunostaining within the liver were identified with steatosis. mRNA transcription for PEPCK was also significantly correlated with the amount of steatosis. A positive association between adiponectin and HMW adiponectin and hepatic inflammation was identified. This correlation remained significant even after following adjusting for age, gender and BMI (r = 0.17; p = 0.03; r = 0.45; p = 0.0001) respectively. Factors independently associated with the stage of fibrosis were HOMA-IR, inflammation score and age. Conclusions: Our findings in HCV-4 infection shows that adiponectin correlates with the different stages of liver injury. Steatosis up-regulates hepatocyte CD95/Fas and thus increases apoptosis, which facilitates inflammation and fibrosis. These findings may offer potential clues for future therapeutic intervention.