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The expression of activation-induced cytidine deaminase, B-aggressive lymphoma, cyclin D1 and serine/threonine kinase 15 genes, among others, is increased in B cells from patients with chronic hepatitis C virus (HCV) infection. It is unknown whether the level of expression of these genes in B cells is increased in patients with hepatitis C who have achieved a sustained virological response (SVR) but who have persistent, detectable HCV RNA, so-called occult infection. Eighty-three patients who achieved and SVR, 27 with detectable HCV and 56 without detectable HCV RNA, 28 chronic hepatitis C patients and 32 healthy controls were studied. RNA was extracted from B cells, and gene expression levels were measured by RT-PCR. Patients with chronic HCV and those who achieved an SVR (with and without persistent low-level HCV RNA) showed a statistically significant higher expression compared to healthy controls, of activation-induced cytidine deaminase (P = 0.004, P < 0.001 and P = 0.002, respectively), B-aggressive lymphoma (P < 0.001, P = 0.001 and P = 0.006) and cyclin D1 (P = 0.026, P = 0.001; P = 0.038). For activation-induced cytidine deaminase patients with an SVR and 'occult infection' had a statistically significantly higher expression level than patients with and SVR without 'occult infection' (P = 0.014). The higher expression levels found for activation-induced cytidine deaminase, together with other genes indicates that these B lymphomagenesis-related genes are upregulated following HCV therapy and this is more marked when HCV can be detected in PBMCs.

Background & Aims Hepatitis C virus (HCV)-RNA detection in peripheral blood mononuclear cells (PBMCs) after recovery from HCV infection, is a type of occult HCV infection although is unclear how the viral persistence in PBMCs affects HCV-specific T-cell responses. The aim of this study was to investigate if cellular immune responses are modified by HCV persistence in PBMCs. Methods HCV-specific CD4+ and CD8+ T-cell responses against six HCV peptides, situated within the non-structural (NS) proteins NS3, NS4b and NS5b, were measured by flow cytometry-through intracellular detection of gamma interferon (IFN-γ) or interleukin 4 (IL-4) and CD69 expression- in 27 sustained virological responders (SVR): 13 with and 14 without occult HCV infection in PBMCs, detected by strand-specific real-time PCR. Ten healthy individuals and 14 chronically infected patients with viraemia, were included as controls. Results SVR without occult infection showed a higher percentage of activated CD4+ cells against peptides belonging to NS3 (p124, p153) and NS5b (p257, p294), activated CD8+ cells against NS3 (p124, p153, p158) and NS5b-p294, as well as an elevated percentage of CD4+ cells releasing IFN-γ + IL-4 against NS3-p153, and by CD8+ cells against NS3 (p124, p153). SVR without occult infection showed a higher percentage of activation and release of IFN-γ + IL-4 by both cell subpopulations than the two group of controls, in contrast to SVR with occult infection. Conclusion The lower HCV-specific T-cell response found in SVR with occult infection indicates that the immune response may be impaired when the virus persists in PBMCs.

The hepatitis A virus cellular receptor 1 (HAVCR1) gene is highly polymorphic, and several variants have been associated with susceptibility to allergic and autoimmune diseases. The HAVCR1 gene region was identified as a candidate for hepatitis C virus (HCV) natural clearance in a genotyping study of selected immune response genes in both European-American and African-American populations. The aim of the present study was to explore the influence of HAVCR1 in the outcome of HCV infection in the Spanish population. Three cohorts, consisting of 354 subjects with persistent HCV infection (285 with persistent HCV monoinfection and 69 with natural clearance), 182 coinfected HIV/HCV patients, and 320 controls, were included. Samples were genotyped in several polymorphic positions, insertion/deletion variants in exon 4 and tag single nucleotide polymorphisms (SNPs), in order to define previously described HAVCR1 haplotypes (haplotypes A to D). No statistically significant differences were observed with spontaneous resolution of infection or with viral clearance after treatment. Nevertheless, different rates of infection by viral genotypes (G's) were observed among the HAVCR1 haplotypes. Individuals bearing haplotype C had the highest viral G1 infection rate when compared to individuals bearing other haplotypes (75.82% versus 57.72%, respectively; corrected P value [ P c ], 3.2 × 10 −4 ; odds ratio [OR], 2.30; 95% confidence interval [CI], 1.51 to 3.47). Thus, HAVCR1 could be involved in susceptibility or resistance to infection by a particular HCV genotype.

Summary. It is unknown whether hepatitis C virus (HCV)-specific cellular immune responses can develop in seronegative sexual partners of chronically HCV-infected patients and whether they have occult infection. Thirty-one heterosexual partners of patients with chronic HCV were studied, fifteen of them with HCV transmission risks. Ten healthy individuals and 17 anti-HCV seropositive patients, without viremia, were used as controls. Virus-specific CD4+ and CD8+ T-cell responses were measured by flow cytometry against six HCV peptides, situated within the nonstructural (NS) proteins NS3, NS4 and NS5, through intracellular detection of gamma interferon (IFN-γ) or interleukin 4 (IL-4) production and CD69 expression. Sexual partners had a higher production of IFN-γ and IL-4 by CD4+ cells against NS3-p124 (P = 0.003), NS5b-p257 (P = 0.005) and NS5b-p294 (P = 0.012), and CD8+ cells against NS3-p124 (P = 0.002), NS4b-p177 (P = 0.001) and NS3-p294 (P = 0.004) as compared with healthy controls. We observed elevated IFN-γ production by CD4+ T cells against NS5b-p257 (P = 0.042) and NS5b-p294 (P = 0.009) in the sexual partners with HCV transmission risks (sexual, professional and familial altogether) than in those without risks. RNA was extracted from peripheral blood mononuclear cells (PBMC), and detection of HCV-RNA positive and replicative (negative) strands was performed by strand-specific real-time PCR. In four sexual partners, the presence of positive and negative HCV- RNA strands in PBMC was confirmed. Hence, we found an HCV-specific cellular immune response as well as occult HCV infection in seronegative and aviremic sexual partners of chronically HCV-infected patients.

Genetic host factors may modify the course of the hepatitis C virus (HCV) infection. Very recently, a genome-wide scan that reported association of the IL28B locus with response to treatment in HCV infection was published. The aim of the current study was to investigate the relationship of this locus with outcome of HCV infection in a cohort constituted by a total of 731 Spanish individuals. From these, 284 were subjects with persistent infection, 69 were individuals who naturally cleared the virus, and 378 were noninfected subjects. Genotyping of the rs12979860 (C>T) in the IL28B locus was performed using a TaqMan 5′ allelic discrimination assay. The CC genotype was overrepresented among patients infected with viral genotypes non-1 (66.7% versus 39.1% in patients infected with viral genotype-1, P = 8.5 × 10−5, odds ratio [OR] = 0.32, 95% confidence interval [CI] 0.17-0.60); patients with spontaneous resolution of infection (72.5% versus 45.6% of the individuals with persistent infection, P = 6.2 × 10−5, OR = 0.32; 95%CI, 0.18-0.57); and lastly, patients with sustained response (60.2% versus 32.1% found in patients with nonsustained response, P = 3.1 × 10−5, OR = 0.31; 95%CI, 0.17-0.56). Conclusion: We have found different rates of viral genotype infection depending on the IL28B variant as well as an association of this locus with natural and treatment-mediated response. HEPATOLOGY 2010

The outcome of chronic hepatitis C virus infection varies, depending on viral and host factors. Those mechanisms involved in the control of the innate and adaptive response could have an influence on the outcome of infection. The PTPN22 gene encodes an intracellular lymphoid-specific phosphatase (Lyp) with a lymphocyte activating downregulatory effect. A single-nucleotide polymorphism (SNP) C1858T located on this gene has been associated with autoimmune diseases and bacterial infections. The aim of this study was to assess whether the PTPN22 C1858T polymorphism is related to the outcome of hepatitis C viral infection. A total of 69 patients with spontaneous viral clearance (SVC), 281 patients with chronic hepatitis C (CHC), and 1036 individuals not infected with hepatitis C (NIC) were included in this study. Patients with CHC were stratified according to Scheuer score of hepatic fibrosis from F0-F2 (n = 200) and F3-F4 (n = 81), and according to their response to therapy in patients with sustained responses (SR; n = 103) and non-sustained response (NSR; n = 104). Genotyping of the C1858T polymorphism was performed using TaqMan probes. No statistically significant differences in the distribution of PTPN22 C1858T polymorphism were observed upon comparison of patient group with the NIC group. Also, when the different patient groups were compared to one another, no statistically significant differences were detected: the SVC with the CHC group (10.2% versus 12.5%; p = 0.6), the F0-F2 with the F3-F4 group (11.5% versus 14.8%; p = 0.5), and the NSR with the SR group (11.5% versus 14.6%; p = 0.4). Our results do not support a major role of this polymorphism of the PTPN22 gene in the outcome of chronic hepatitis C virus infection in the Spanish population.

Summary Particular alleles of human leucocyte antigen (HLA) and immunoglobulin gamma (GM) and immunoglobulin kappa (KM) allotypes (polymorphic determinants of IgG heavy chains and κ-type light chains, respectively) are associated with the outcome of several infections. To examine their role in the outcome of hepatitis C virus (HCV) infection, we genotyped 50 individuals with resolved and 117 with persistent HCV infection. None of the GM, KM or HLA-C genotypes by themselves were associated with the resolution or persistence of HCV infection. However, particular combinations of HLA and GM genotypes were associated significantly with the outcome of HCV infection. Subjects with the HLA C1C1 genotype, in the absence of GM ff, were more than seven times [odds ratio (OR) 7·15] as likely to have persistent infection as the subjects who lacked both these genotypes. The presence of GM ff, in the absence of HLA C1C2, was associated with the resolution of infection (OR 0·27). The absence of GM fz, in the presence of HLA C2C2, was also associated with the resolution of infection (OR 0·27). Compared to the subjects who lacked both these genotypes, subjects with GM fz, in the absence of HLA C1C2, were almost four times as likely to have persistent infection (OR 3·91); similarly, subjects with HLA C1C2, in the absence of GM fz, were almost three times as likely to have persistent infection (OR 2·80). These results show, for the first time, interactive effects of GM and HLA genotypes in the outcome of HCV infection.

To determine whether the different tumour necrosis factor alpha (TNF-alpha) promoter gene polymorphisms are involved in the development of steatosis in chronic hepatitis C.One hundred and thirty patients (89 men and 41 women; mean age 42.5 +/- 12.3 years) with chronic hepatitis C were included. Insulin resistance was measured according to the Homeostasis model assessment (HOMA IR). Serum leptin levels were also obtained and the body mass index and fat mass were calculated. Liver biopsy was carried out in all the patients, and steatosis was measured as one of four stages (0 to 3): stage 0, no steatosis; stage 1,25% of hepatocytes with steatosis; stage 2, 25-50%; and stage 3,50%. DNA samples were obtained in order to describe the polymorphisms at the TNF-alpha promoter gene position.Fifty-nine of the 130 (45.38%) patients had different degrees of steatosis, while 71/130 (54.62%) were not steatosic. Six of the 59 (10.2%) patients with steatosis presented mutations at the -238 position of the TNF-alpha promoter region, while 5/71 (7.0%) patients without steatosis also showed mutations at this position (P=NS). Seventeen of the 59 (28.8%) steatosic patients showed a mutation at the -308 position, while 16/71 (22.5%) without steatosis also had this mutation (P=NS). Insulin resistance, beta cells reserve, insulin and leptin levels showed no differences between patients with or without mutations at the promoter region of the TNF-alpha gene.TNF-alpha mutations do not seem to play any role in the development of steatosis in chronic hepatitis C.

CD81, the scavenger receptor-BI (SR-BI) and the low-density lipoprotein receptor (LDLR) are involved in peripheral blood mononuclear cells (PBMCs) hepatitis C virus (HCV) entry. To investigate if these molecules are altered by HCV, 20 controls and 66 patients: 37 untreated and 29 sustained virological responders, were studied. CD81 and LDLR expression, measured the percentage of cells expressing the HCV-receptors and their mean fluorescence intensity (MFI), was analyzed on lymphocytes and monocytes, as well as SR-BI on monocytes by flow cytometry. RNA was extracted from PBMCs and detection of the HCV-RNA positive and negative strands was performed by strand-specific RT-PCR. A statistically significant increase of CD81 expression was observed on lymphocytes, a higher percentage of LDLR on lymphocytes and monocytes, as well as SR-BI on monocytes was found in the patients as compared to the controls (P

Natural killer (NK) cells are key components of the innate antiviral immune response. NK cell function is regulated by the interaction of major histocompatibility complex class I molecules with NK inhibitory receptors. The aim of this study was to investigate the role of the HLA-C/KIR pair in hepatitis C virus clearance in our population. A total of 196 hepatitis C virus-infected patients (65 resolved and 131 with persistent infection) were included in the study. Genotyping of HLA-C was carried out using polymerase chain reaction followed by a reverse sequence-specific oligonucleotide probe detection system. NK receptor-specific polymerase chain reaction typing of KIR2DL1, KIR2DL2, and KIR2DL3 was performed on the same patient group. Frequencies of the KIR2DL2 gene and the KIR2DL2/KIR2DL2 genotype were lower among patients with persistent infection (32.3% vs 45.4% among resolved, P = 0.01, OR = 0.57, 95% CI = 0.36–0.91; and 16.2% vs 32.3% among resolved, P = 0.02, OR = 0.41, 95% CI = 0.19–0.87). Nevetheless, the frequency of the KIR2DL3 gene was higher among patients with persistent infection (66.9% vs 54.6% among resolved P = 0.02, OR = 1.68, 95% CI = 1.07–2.65). Trends toward lower frequencies of the HLA-C2C2 genotype and NK-HLA interactions with strong and moderate affinity among the patients with persistent infection were also observed.

Background and aims: The solute carrier family 11 member 1 (SLC11A1) gene (formerly Nramp1) encodes for the protein solute carrier family 11, member 1. It affects susceptibility and clinical outcome of autoimmune and infectious diseases. We investigated the possible role of the functional polymorphism located in the promoter region of SLC11A1 and tumour necrosis factor (TNF) genes in the progression of fibrosis in chronic hepatitis C. Methods: A total of 242 Caucasian Spanish patients with biopsy proven chronic hepatitis C and 194 healthy control subjects were genotyped for SLC11A1 and TNF promoter polymorphisms. Results: No significant differences in the distribution of frequencies among patient and control groups were observed. The SCL11A1 homozygous 2/2 genotype was rarely detected among patients showing advanced fibrosis (2/82; 2.4%) but was highly represented in those with mild fibrosis (29/160; 18.1%; odds ratio (OR) 8.85 (95% confidence interval (CI) 1.9–55.2, p c = 0.002). In patients carrying allele 3 of SLC11A1, the presence of −238 TNF A/G was associated with advanced fibrosis (14/26 (53.8%) v 68/216 (31.4%); OR 2.53 (95% CI 1.03–6.23); p = 0.02). Conclusions: SLC11A1 gene promoter polymorphism could influence fibrosis progression in chronic hepatitis C in that the homozygous genotype 2/2 exerts a protective effect against cirrhosis development. Also, the combination of TNF −238 A/G and the presence of allele 3 is conducive to progression to pre-cirrhotic or cirrhotic stages of the disease.

The aim of this study was to assess the influence of host genetic factors on response to combination therapy for chronic hepatitis C infection.Patients with biopsy-proved chronic hepatitis C infection were treated with interferon alone (n = 143) or combined therapy of interferon + ribavarin (n = 105; 46 treatment naïve, 59 relapsers). Human leukocyte antigen (HLA) class I was determined by microlymphocytotoxicity and class II by polymerase chain reaction-single specific oligonucleotide. The two biallelic tumor necrosis factor-alpha promoter polymorphisms were studied by a polymerase chain reaction-amplification refractory mutation system. Other variables measured were viral genotype, hepatitis C virus RNA load, liver function tests, and ferritin concentration.Univariate analysis indicated that patients bearing HLA B44+, DRB1*03, infected by genotype non-1, with higher concentrations of transaminases and shorter duration of infection showed a higher sustained response (SR) rate than those on combination therapy. HLA class II and TNF-alpha promoter polymorphisms were not related to SR. In multivariate analysis, non-1 genotype (OR 2.42, 95% CI 1.12-5.55, p = 0.026) and HLA B44+ (OR 4.84, 95% CI 1.3-17.8, p = 0.017) were the independent variables associated with SR. However, HLA B44+ was not associated with SR in patients treated with interferon alone.HLA class I B44 is related to a higher rate of SR in combination therapy but not in interferon monotherapy, whereas HLA class II, tumor necrosis factor-alpha -238A or -308A seem not to influence response to the antiviral therapy. These findings may be of value in therapy selection for hepatitis C-infected patients.

In patients with compensated liver cirrhosis the clinical repercussions of detecting subclinical hepatic encephalopathy (SHE) are unclear. We present a long-term follow-up study in cirrhotic patients to examine the relationship between SHE and subsequent episodes of overt hepatic encephalopathy.A total of 63 cirrhotic patients were studied by Number Connection Test and auditory evoked potentials. We determined glutamine, ammonia, zinc, glutamate, urea, and ratio of branched chain amino acids to aromatic amino acids, and Child-Pugh classification.Of 63 patients, 34 (53%) exhibited SHE. Nineteen out of 63 (30%) developed overt hepatic encephalopathy during follow-up. Hepatic encephalopathy in follow-up was related to alcoholic etiology, ammonia, glutamine, zinc, ratio of branched chain amino acids to aromatic amino acids, liver function, presence of esophageal varices, and detection of SHE (84% of patients who exhibited hepatic encephalopathy in follow-up showed SHE). In Cox-regression, glutamine levels, SHE, esophageal varices, and Child-Pugh class were the independent variables related to hepatic encephalopathy in follow-up.SHE (defined on the basis of number connection test or auditory evoked potentials alteration) could predict a subsequent episode of overt hepatic encephalopathy. Lower glutamine levels, presence of esophageal varices, and liver dysfunction were also related to the development of overt hepatic encephalopathy.

AIM: To assess whether CCL2 or interactions between this chemokine and its receptor (CCR2) are associated with outcomes of chronic hepatitis C and with responses to antiviral therapy. METHODS: Two hundred and eighty-four patients with chronic hepatitis C and 193 non-infected matched controls were included in this study. Patients were categorized according to their Scheuer score of hepatic fibrosis as F0-F2 (n = 202) or F3-F4 (n = 82) and according to their response to anti-Hepatitis C virus (HCV) therapy as sustained response (SR, n = 101) or non-sustained response (NSR, n = 98). Genotyping of the -2518 (A/G) CCL2 was performed using PCR-RFLP, genotyping of the 190 (A/G) CCR2 using a PCR-ARMS system, and genotyping of the rs3138042 (G/A) CCR2 using Taqman probes. RESULTS: Univariate analyses identified 4 parameters (infection duration time, viral genotype, gender and AST levels) that tended to influence fibrosis and 7 parameters (CCL2G, CCL2ACCR2A, viremia levels, fibrosis stage, viral genotype, infection duration time and AST levels) that significantly influenced or tended to influence response to treatment. Multivariate analysis identified gender and AST levels as parameters that independently influenced fibrosis stage and viral genotype and infection duration time were the two parameters that independently influenced response to treatment. CONCLUSION: Our results indicate that the mutations studied in the gene pair CCL2/CCR2 do not play a major role in the outcome and response to treatment for HCV infection in the Spanish population.