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Hepatitis C virus (HCV) infection and alcohol abuse are leading causes of chronic liver disease and frequently coexist in patients. The unfolded protein response (UPR), a cellular stress response ranging along a spectrum from cytoprotection to apoptosis commitment, has emerged as a major contributor to human diseases including liver injuries. However, the literature contains conflicting reports as to whether HCV and ethanol activate the UPR and which UPR genes are involved. Here we have used primary human hepatocytes (PHH) to reassess this issue and address combined impacts. In this physiologically relevant model, either stressor activated a chronic complete UPR. However, the levels of UPR gene induction were only modest in the case of HCV infection. Moreover, when combined to the strong stressor thapsigargin, ethanol exacerbated the activation of pro-apoptotic genes whereas HCV tended to limit the induction of key UPR genes. The UPR resulting from HCV plus ethanol was comparable to that induced by ethanol alone with the notable exception of three pro-survival genes the expressions of which were selectively enhanced by HCV. Interestingly, HCV genome replication was maintained at similar levels in PHH exposed to ethanol. In conclusion, while both HCV and alcohol activate the hepatocellular UPR, only HCV manipulates UPR signalling in the direction of a cytoprotective response, which appears as a viral strategy to spare its own replication.

BACKGROUND & AIMS Equality of access to organ transplantation is a mandatory public health requirement. Referral from a local to a university hospital and then registration on the national waiting list are the two key steps enabling access to liver transplantation (LT). Although the latter procedure is well defined using the Model for End-stage Liver Disease score that improves equality of access, the former is mostly reliant on the practices of referring physicians. The aim of this study was to clarify the factors determining this initial step. METHODS This observational study included consecutive inpatients with cirrhosis of whatever origin in a cohort constituted between 2003 and 2008, using medical records and structured questionnaires concerning patient characteristics and the opinions of hospital clinicians. Candidates for LT were defined in line with these opinions. RESULTS Four hundred and thirty-three patients, mostly affected by alcoholic cirrhosis, were included, 21.0% of whom were considered to be candidates for LT. Factors independently associated with their candidature were: physician empathy [odds ratio (OR) = 10.8; 95% CI: 4.0-29.5], adherence to treatment (OR = 16.6; 95% CI: 3.7-75.2), geographical area (OR = 6.8; 95% CI: 2.2-21.3) and the patient's physiological age (OR = 2.3; 95% CI: 1.1-4.7). CONCLUSIONS Several subjective markers restrict the referral of patients from local hospitals to liver transplant centres. Their advancement to this second step is thus markedly weakened by initial subjectivity. The development of objective guidelines for local hospital physicians to assist them with their initial decision-making on LT is now necessary.

Nonalcoholic steatohepatitis (NASH) is a liver disease that complicates insulin-resistant states. This trial tested the efficacy and safety of rosiglitazone, an insulin-sensitizing agent, in patients with NASH.Sixty-three patients with histologically proven NASH were randomly assigned to receive rosiglitazone (4 mg/day for the first month and 8 mg/day thereafter; n = 32) or placebo (n = 31) for 1 year. Liver biopsy was performed at the end of treatment. End points were improvement in the histologic score of steatosis, normalization of serum transaminase levels, and improvement in necroinflammation and fibrosis.More patients treated with rosiglitazone than receiving placebo had improved steatosis (47% vs 16%; P = .014) and normalized transaminase levels (38% vs 7%; P = .005), although only half of patients responded. There was no improvement in other histologic lesions, including fibrosis, and a composite score of activity, the nonalcoholic fatty liver disease activity score. Improvement of steatosis correlated with reduction of transaminase levels (r = 0.36; P.005), improvement in insulin sensitivity (r = 0.34; P = .008), and increase in adiponectin levels (r = -0.54; P.01) but not with weight variations. Independent predictors of response were rosiglitazone treatment, the absence of diabetes, and massive steatosis. Weight gain was the main adverse effect (mean gain of 1.5 kg in the rosiglitazone group vs -1 kg in the placebo group; P.01), and painful swollen legs was the main reason for dose reduction/discontinuation. Serum hemoglobin level was slightly but significantly reduced. There was no hepatic toxicity.In patients with NASH, rosiglitazone improves steatosis and transaminase levels despite weight gain, an effect related to an improvement in insulin sensitivity. However, there is no improvement in other parameters of liver injury.

Background/Aim: The aim of this study was to determine whether the expression of CD25, CD28 and CD38 (which reflects the degree of T-cell activation) by peripheral blood mononuclear cells constitutes a useful means of measuring the immune status of liver transplant recipients. Methods: Fifty-two patients enrolled in a prospective randomized study comparing cyclosporine and tacrolimus as the principal immunosuppressive drugs were monitored prospectively. The expression of CD25, CD28 and CD38 was analyzed on CD3-, CD4- and CD8-positive cells from whole blood using flow cytometry. The prognostic value of baseline and day 14 measurements regarding acute rejection was examined using Kaplan–Meier estimates for univariate analyses and the Cox model for multivariate analyses. Results: The mean frequencies of CD28 and CD38-expressing T cells were significantly higher in patients with acute rejection (p = 0.01 and p = 0.001, respectively), whereas the frequency CD25-expressing T cells did not differ significantly. Under univariate analysis, baseline CD25 levels, the type of calcineurin inhibitor, as well as the CD28 and CD38 frequencies obtained at day 14 were associated with the subsequent development of acute rejection. Under multivariate analysis, only CD28 and CD38 frequencies obtained at day 14 were independently associated with acute rejection. Conclusions: The evaluation of CD28 and CD38 expression in peripheral blood lymphocytes is a simple marker that could be used routinely in clinical practice to assess the level of immunosuppression.

Background. Chronic hepatitis C virus (HCV) is usually associated with high levels of hepatic interleukin (IL)-2 and low levels of IL-4 transcripts. HCV frequently recurs after liver transplantation, and its course is accelerated in this setting. We compared in situ expression of IL-2 and IL-4 in transplanted and nontransplanted patients with HCV. Methods. A total of 74 liver biopsy specimens were studied; 52 came from transplanted patients, 38 of whom were HCV-positive (17 mild and 21 severe cases of recurrent HCV) and 22 came from nontransplanted patients, 17 of whom were HCV-positive (7 mild and 10 severe cases of HCV). The expression of IL-2 and IL-4 mRNA and IL-4 protein was studied using the reverse transcriptase polymerase chain reaction and immunohistochemical methods, respectively. Results. IL-2 transcript levels were significantly higher in severe than in mild HCV in both liver graft recipients and nontransplanted patients. However, IL-2 levels were higher in nontransplanted than in transplanted patients. IL-4 transcripts and protein were preferentially detected in graft recipients with severe recurrent HCV. Conclusion. IL-4 expression is elevated in severe recurrent HCV and may play a role in the progression of hepatic lesions after liver transplantation.

IL-4 is overexpressed in liver grafts during severe recurrent hepatitis C and rejection. Hepatocyte apoptosis is involved in both these phenomena. We therefore examined the proapoptotic effect of IL-4 on HepG2 cells and human hepatocytes in vitro, together with the underlying mechanisms. We first measured IL-4 receptor expression, STAT6 activation by IL-4, and STAT6 inhibition by an anti-IL-4 antibody or by STAT6 siRNA transfection. We then focused on the pathways involved in IL-4-mediated apoptosis and the role of STAT6 activation in apoptosis initiation. The IL-4 receptor was expressed on both cell types, and STAT6 was activated by IL-4. Both anti-IL-4 and STAT-6 siRNA inhibited this activation. IL-4 induced apoptosis of both HepG2 cells (P=0.008 vs. untreated control) and human hepatocytes (P

Transient elastometry is a noninvasive procedure used to measure fibrosis when patients are diagnosed with liver disease; it might be used to monitor changes over time. We investigated whether there are short-term variations in stiffness measurements that are not attributable to changes in fibrosis by studying patients with stable liver disease.We performed a retrospective analysis of 531 paired liver stiffness measurements made by Fibroscan when the study began (LSM1) and at follow-up (LSM2), more than 1 day and less than 1 year apart, from 432 stable (for body mass index, waist circumference, and alcohol consumption), untreated, immunocompetent patients with chronic liver disease (from January 2006 through March 2009). Variations between the first and follow-up measurements were expressed as absolute (LSM2-LSM1, kPa) or relative ([LSM2-LSM1]/LSM1*100) or as changes in fibrosis stage.There was20% variation in 49.7%,30% in 34.3%, and50% in 12.2% of paired measurements; this variation was constant across the spectrum of LSM1 values. The variations produced a 1-fibrosis stage difference in 31.5% of pairs and a ≥ 2-stage difference in 9.8% of pairs. Patients with LSM17 kPa had increased probability of having a different stage of fibrosis at LSM2, compared with patients with LSM17 kPa. Factors associated with variation included measurements made by 2 different operators or at least 1 non-senior operator, ratios of interquartile range:median values, significant fibrosis (≥ 7 kPa) at LSM1, baseline body mass index, or a 2-fold difference in level of alanine aminotransferase between measurements. When the analyses were restricted to measurements made by the same operator, the variation was slightly reduced; fibrosis stage differed between measurements for only 34.3% of cases.Operator-related and patient-related factors produce significant variations in liver stiffness measurements made by transient elastometry, limiting its use in monitoring patients. These variations are unrelated to disease progression. The lowest levels of variation occur in measurements made in patients with no or early-stage fibrosis or by a single experienced operator.

Alcohol consumption has a major impact on the natural history of chronic hepatitis C virus (HCV) infection, although the underlying mechanisms are still debated. We designed a clinical study to evaluate the impact of alcohol abuse on both viral load and expression of low-density lipoprotein receptor (LDLR) and CD81 expression. Thirty-eight consecutive HCV-infected patients were enrolled. Group 1 (n = 18),or =10 g alcohol/day, group 2 (n = 8),or =30 g alcohol/day, group 3 (n = 12),or =30 g alcohol/day. Receptors expression was measured by flow cytometry analysis in peripheral blood mononuclear cells (PBMC) and by specific real-time retrotranscription polymerase chain reaction (RT-PCR) in the liver. Serum viral load was evaluated by quantification of both HCV genomic RNA and total core antigen. The hepatic viral load was assessed by real-time RT-PCR. Serum HCV-RNA and total core antigen were significantly correlated, and were higher, albeit not significantly, in group 3 than in group 1. Alcohol consumption had no effect on expression of HCV putative receptors in PBMC, except for CD81, which was upregulated on monocytes in group 2. In the liver, viral load and levels of LDLR transcripts were significantly higher in group 3 than in group 1. Remarkably, a significant positive correlation was found between LDLR transcripts and HCV-RNA (r2 = 0.83, P10(-3)). Finally, in vitro experiments suggested that the effect of ethanol on LDLR expression was indirectly mediated by both tumour necrosis factor-alpha and interleukin-1beta. In conclusion, this study is the first to support a role for LDLR in the natural infection by HCV in man.

The detection of negative-strand hepatitis C virus (HCV) RNA is a hallmark of replication. A highly sensitive and specific method is required to quantify the very low level of replication inherent to in vitro infection systems. Based on reverse transcription with a tagged primer in the 5′ non-coding region of the HCV genome, followed by a nested PCR with a second round of real-time PCR, a novel method is described with improved sensitivity for negative-strand HCV RNA quantification. The lower detection level was 25 copies per reaction of negative-strand HCV RNA, even in the presence of 1 × 105 copies of positive-strand HCV RNA. This protocol was applied to the detection of negative HCV strand RNA in the liver of HCV-infected patients as well as in primary human hepatocytes infected in vitro. In both models, and particularly in each of three, independent in vitro infection experiments, this assay permitted the quantitation of HCV replication. J. Med. Virol. 79:155–160, 2007. © 2006 Wiley-Liss, Inc.

Points essentiels • La cirrhose est une complication grave dont l’incidence augmente chez les patients co-infectes VIH-VHC ou VHB en raison des progres dans les traitements anti-VIH (Haart). L’existence d’une co-infection par le VIH accelere en outre la progression de la fibrose de l’hepatite. • Le diagnostic de cirrhose est important afin de mettre en place des mesures preventives, par la realisation d’une biopsie hepatique ou de tests non invasifs de fibrose maintenant largement etudies (FibroTest, FibroScan, etc.). • La gravite de la cirrhose doit alors etre appreciee avec depistage des complications : evaluation de l’insuffisance hepatocellulaire (scores de Child-Pugh, MELD), de l’hypertension portale (endoscopie digestive haute) et du carcinome hepatocellulaire (echographie et dosage de l’α-fœtoproteine). Des precautions en termes de prescriptions medicamenteuses associees doivent etre respectees. • Les traitements specifiques des hepatites virales B ou C sont possibles a ce stade de cirrhose, bien que plus difficiles a mettre en œuvre notamment pour le VHC (resultats influences par le genotype, risque supplementaire de complications de type acidose lactique ou de decompensation hepatique). • La prise en charge des complications de l’hypertension portale doit s’envisager comme chez le non-infecte par le VIH. Le traitement du carcinome hepatocellulaire reste decevant et la transplantation hepatique, bien que realisable chez ces patients, reste a evaluer.

Most individuals exposed to hepatitis C virus (HCV) become chronically infected and are predisposed to liver disease. The mechanisms underlying viral persistence and disease progression are unknown. A role for the HCV NS5A protein in viral replication and interferon resistance has been demonstrated. To identify mechanisms affected by NS5A, we analyzed the gene expression of Huh7 cells expressing NS5A and control cells using oligonucleotide microarrays. A set of 103 genes (43 up-regulated, 60 down-regulated) whose expression was modified by at least twofold was selected. These included genes involved in cell adhesion and motility, calcium homeostasis, lipid transport and metabolism, and genes regulating immune responses. The finding of modulated expression of genes related to the TGF-beta superfamily and liver fibrosis was observed. Interestingly, both the tumor necrosis factor and lymphotoxin beta receptors were down-regulated by NS5A. Similar data were obtained following expression of four NS5A mutants obtained from patients who were not responsive or were sensitive to interferon therapy. Through computational analysis, we determined that 39 of the 43 genes up-regulated by NS5A contained one or more nuclear factor kappaB (NF-kappaB) binding sites within their promoter region. Using the Gibbs sampling method, we also detected enrichment of NF-kappaB consensus binding sites in the upstream regions of the 43 coexpressed genes. Activation of NF-kappaB by NS5A was subsequently demonstrated in luciferase reporter assays. Adenovirus-mediated expression of IkappaBalpha reverted NS5A mediated up-regulation of gene expression. In conclusion, this study suggests a role of NS5A and NF-kappaB in HCV pathogenesis and related liver disease. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

A link between chronic hepatitis C virus (HCV) infection and low-grade B-cell lymphomas has been suggested by epidemiological studies. Marginal zone lymphomas (MZLs) including splenic lymphomas with villous lymphocytes are among the most frequently reported subgroups in the setting of chronic HCV infection. In this study, we examined the effect of antiviral treatment in eight patients with HCV-associated MZL. We found that five out of eight patients have responded to interferon alpha and ribavirin. In some cases, hematologic responses were correlated to virologic responses. In addition, we report a case of large granular lymphocyte leukemia occurring in association with MZL and HCV, and responding to interferon and ribavirin. We suggest that there is an etiologic link between HCV and antigen-driven lymphoproliferative disorders.

Background. The aim of this study was to evaluate calcineurin activity and interleukin-2 (IL-2) expression by peripheral blood cells as a means of assessing the immune status of liver transplant recipients. Methods. Twenty-one patients were studied in a randomized study comparing cyclosporine and tacrolimus as the main immunosuppressive drug. Calcineurin activity was determined after separation of phosphorylated and dephosphorylated products of a calcineurin specific peptide substrate by high performance liquid chromatography (HPLC). Intracellular IL-2 expression was measured by flow cytometry. In 34 additional patients, intracellular IL-2 expression was prospectively measured. Results. Calcineurin activity fell after transplantation. Values were marginally higher in patients with acute rejection (P=0.059). The percentage of IL-2-producing T cells fell after transplantation. This percentage did not differ between patients with and without rejection. In contrast, the proportion of IL-2-producing CD8+ T cells was higher in patients with acute rejection than in patients free of acute rejection (P=0.003). Moreover, pretransplantation IL-2 expression by CD8+ T cells was higher in patients who subsequently developed acute rejection, suggesting that IL-2 production may be constitutively higher in those patients. The results obtained in the 34 additional patients confirmed these results. Conclusions. These data suggest that quantification of intracellular IL-2 in CD8+ T cells may be a useful index of immune status in liver transplant recipients. Preoperative IL-2 levels might serve to individually tailor the immunosuppressive regimen.

Background The molecular mechanisms involved in the immunosuppressive properties of bile salts are partly unknown. Methods The aim of the study was to compare the effects of bile salts to those of various compounds with a steroid structure, or straight-chain hydrocarbons of different lengths and polar groups in the human mixed lymphocyte reaction. Results We showed a significant correlation between the effects of bile salts and a low critical micellar concentration, a high surface activity index, and the absence of conjugation. In addition to mixed lymphocyte reaction (MLR) inhibition, chenodeoxycholate (CDC) inhibit ConA-induced IL2 production without any effect on IL2 R expression. Fusidate, a negatively charged steroid, with physical properties comparable to those of deoxycholate, had similar effects. Cetyltrimethylammonium bromide (CTAB), which exhibited a very low critical micellar concentration, inhibited mixed lymphocyte reaction in an extent comparable to cyclosporin A. In contrast, aliphatic compounds with critical micellar concentrations in the same range as bile salts but with a lower molecular area had no effect. Conclusion Amphiphilic negatively charged molecules inhibit T-cell proliferation to an extent that is dependent upon their hydrophobicity. These results may be explained, at least in part, by a modification in the cell membrane lipid bilayer structure.

Arielle R. Rosenberg, Arnaud Carpentier, Philippe Podevin NOUVELLE permettra sans doute d’apporter quelques reponses grâce a l’analyse detaillee des afferences sensorielles recues par differents types de neurones. En effet, il est possible que d’autres types neuronaux, en particulier ceux presentant une dendrite apicale proeminente [13], ou ceux d’autres especes (organises en colonnes d’orientation), presentent une organisation spatiale differente de leurs afferences synaptiques, avec par exemple la presence de clusters capables de generer des potentiels d’action dendritiques [14]. Pour l’instant, seule l’activite d’une portion restreinte de l’arbre dendritique a pu etre enregistree (environ 10 %). Mais les progres constants en imagerie biphotonique permettent d’envisager que, dans un futur proche, il sera possible d’enregistrer l’activite de l’ensemble des afferences synaptiques que recoit un neurone et de mieux comprendre les mecanismes d’integration de l’information sensorielle [15]. ‡ Dendritic organization and functional characteristics of visually evoked inputs to cortical neurons in vivo

We have previously shown that cholestasis and bile acids inhibit 2', 5' oligoadenylate synthetase (OAS) activity in the liver and in primary hepatocyte cultures. Here, we assessed the influence of bile acids on interferon (IFN) pathway activation in three hepatoma cell lines. In HepG2 cells, bile acids (100-200 micromol/L) inhibited IFN-induced 2',5' OAS activity to an extent depending on their surface activity index. In Western blot analysis, IFN-induced expression of two major antiviral proteins, MxA and OAS p100, was reduced by 54% +/- 8% and 44% +/- 12%, respectively, when cells were preincubated for 4 hours with 100 micromol/L chenodeoxycholic acid (CDCA). In the same conditions, CDCA did not modify the IFN-induced signal transducers and activators of transcription (STAT)s tyrosine phosphorylation. In contrast, it reduced IFN-induced MxA promoter activity by 60%. The inhibitory effect of CDCA was not mediated by a 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA)-sensitive protein kinase C (PKC)-dependent pathway. Finally, using CHO cells stably expressing a functional human bile acid carrier (Na+-dependent taurocholate cotransporting polypeptide [NTCP]), we found that bile acid inhibition of the IFN pathway occurred in the range of more physiological concentrations (12-50 micromol/L). In summary, our results provide strong evidence that bile acids inhibit the induction of proteins involved in the antiviral activity of IFN. This might partly explain the lack of responsiveness to IFN therapy in some patients with advanced chronic viral liver diseases.

Background/Aims: The mechanisms involved in resistance to interferon alfa in patients with chronic hepatitis C are unclear. Both cirrhosis and cholestasis have been shown to be predictive of resistance. The aim of this study was to evaluate the influence of cholestasis and bile acids on 2′, 5′-oligoadenylate synthetase and natural killer activities, which are both involved in the antiviral activity of interferon. Methods: 2′, 5′-Oligoadenylate synthetase activity was evaluated in spleen, liver, and isolated hepatocytes from bile duct-ligated rats, and the effect of bile acids in vitro on interferon-induced 2′,5′-oligoadenylate synthetase and natural killer activities was examined in fresh mononuclear cells from healthy subjects. Results: Cholestasis had a time-dependent inhibitory effect on 2′, 5′-oligoadenylate synthetase activity in liver, spleen, and isolated hepatocytes from cholestatic rats (−70%, 86%, and 70% relative to baseline, respectively). In vitro, endogenous bile acids had a concentration-dependent inhibitory effect on interferon-induced 2′,5′-oligoadenylate synthetase and natural killer activities, which was related to their structure. This inhibitory effect correlated with the surface activity index. Conclusions: Cholestasis and bile acids diminish the biological activity of interferon and natural killer activity. The results suggest a decrease in the antiviral defenses in cholestatic conditions.

Endogenous bile acids such as chenodeoxycholic acid have been shown to display a suppressive effect in vitro on mononuclear cell activation. We investigated the signal transduction pathway involved in the effect of chenodeoxycholic acid on monocyte procoagulant activity, a model of monocyte activation. Chenodeoxycholic acid (25 to 250 μmol/L) had a concentration-dependent inhibitory effect on procoagulant activity expressed by endotoxin-stimulated mononuclear cells, with half-maximal and maximal inhibition occurring at concentrations of 100 and 250 μmol/L, respectively. The inhibitory effect of chenodeoxycholic acid was (a) closely mimicked by 4β-phorbol 12β-myristate 13α-acetate (PMA), a protein kinase C activator, but not by forskolin or dibutyryl cyclic AMP, two activators of the protein kinase A-dependent pathway; (b) prevented by staurosporine, a potent protein kinase C inhibitor; (c) partially abolished in protein kinase C-depleted cells; and (d) observed in conditions under which chenodeoxycholic acid, like PMA, significantly increased (41%) protein kinase C activity, as assessed by phosphorylation of exogenous (histone III-S) and endogenous (37-kD protein) substrates. In conclusion, our results (a) provide clear evidence of a marked inhibitory effect of chenodeoxycholic acid on monocyte activation, suggesting a potential role of primary endogenous bile acids in the immune defect associated with cholestasis; and (b) indicate that the inhibition of monocyte activation by chenodeoxycholic acid is mediated by way of protein kinase C activation (HEPATOLOGY 1994;19:1164–1170.)

Cell-mediated immunity is impaired during cholestasis, and there is evidence for the involvement of endogenous bile acids. The aim of this study was to evaluate the effects of individual bile acids on immunity and to determine whether monocytes are a target. The effects of bile acids on the procoagulant activity of human monocytes, a lymphocyte-dependent model of monocyte activation, were assessed. Chenodeoxycholic acid, one of the main human primary bile acids, had a concentration-dependent inhibitory effect on procoagulant activity expressed by endotoxin-stimulated mononuclear cells, with half-maximal and maximal inhibitions at 100 and 250 microM, respectively. The inhibitory concentrations were similar for the procoagulant activity of unstimulated mononuclear cells and for endotoxin-stimulated isolated monocytes. In contrast, ursodeoxycholic acid, a bile acid which has beneficial effects in cholestatic diseases, had no significant inhibitory effects at concentrations up to 250 microM. These results indicate that endogenous bile acids tend to inhibit monocyte activation, suggesting a potential role for primary endogenous bile acids in the immune defect associated with cholestasis; ursodeoxycholic acid, which is devoid of effects on the immune system, may potentially reverse cholestasis-induced immunodeficiency.

The epidemic history of HCV genotype 5a is poorly documented in France, where its prevalence is very low, except in a small central area, where it accounts for 14.2% of chronic hepatitis C cases. A Bayesian coalescent phylogenetic investigation based on the E1 envelope gene and a non-structural genomic segment (NS3/4) was carried out to trace the origin of this epidemic using a large sample of genotype 5a isolates collected throughout France. The dates of documented transmissions by blood transfusion were used to calibrate five nodes in the phylogeny. The results of the E1 gene analysis showed that the best-fitting population dynamic model was the expansion growth model under a relaxed molecular clock. The rate of nucleotide substitutions and time to the most recent common ancestors (tMRCA) of genotype 5a isolates were estimated. The divergence of all the French HCV genotype 5a strains included in this study was dated to 1939 [95% HPD: 1921–1956], and the tMRCA of isolates from central France was dated to 1954 [1942–1967], which is in agreement with epidemiological data. NS3/4 analysis provided similar estimates with strongly overlapping HPD values. Phylodynamic analyses give a plausible reconstruction of the evolutionary history of HCV genotype 5a in France, suggesting the concomitant roles of transfusion, iatrogenic route and intra-familial transmission in viral diffusion.

Summary The clinical evolution of type I multiple endocrine neoplasia (MEN I) was studied in 45 patients among a consecutive series of 172 with Zollinger-Ellison syndrome (ZES). These 172 patients were seen in our hospital between 1959 and 1989. Diarrhea was half as frequent in ZES-MEN I as in sporadic ZES cases. At diagnosis, mean basal acid output and serum gastrin levels in MEN I patients (28.8 ± 6.6 mmoVh and 587 ± 487 pg/ml, respectively) were not different from those observed in the others with sporadic ZES. Laparotomy was performed in all 36 patients with no diffuse liver involvement to attempt the removal of gastrinomas. Twenty-nine patients had adenomas, located in the pancreas in 21, in the duodenal wall in 3, and in both in 5. Adenomas were multiple in 23 cases (78%). No tumor was found in seven patients. Twenty-nine of the 36 operated patients were tumor-free after surgery; 7 died in the postoperative period between 1959 and 1970. Median follow-up of the 38 other patients was 95 months (range 17–278 months). Among the 24 patients without residual tumor at discharge (group I), biological and or morphological evidence of a persistent or recurrent source of gastrin was obtained in 22. Among the 14 patients with residual tumor (group II), an increase in tumor size was seen in 5 after a median of 27 months (range 9–36 months), while no change occurred in 9 after 54 months (3-100 months). Actuarial survival curves were not different, either in group I versus group I1 patients (67 and 72% at 5-year follow-up, respectively) or in MEN I versus sporadic ZES patients. Apparently, complete resection of primary tumor did not reduce the incidence of subsequent liver metastases. In all, 21 of the 45 patients had malignant gastrinomas (47%), consisting of liver metastases in 14 (31%), metastatic lymph nodes in 11 (24%), and lung metastases in 2 (4%). Monitoring of fundic argyrophil cells disclosed hyperplasia in 13 of the 14 MEN I patients (92%), and 5 had invasive carcinoid tumors. Taken together, these results prompt us to recommend that in ZES-MEN I patients, surgery should be avoided and oxyntic mucosa regularly monitored.

Background & Aims Although hepatitis C virus (HCV) can be grown in the hepatocarcinoma-derived cell line Huh-7, a cell-culture model is needed that supports its complete, productive infection cycle in normal, quiescent, highly differentiated human hepatocytes. We sought to develop such a system. Methods Primary cultures of human adult hepatocytes were inoculated with HCV derived from Huh-7 cell culture (HCVcc) and monitored for expression of hepatocyte differentiation markers and replication of HCV. Culture supernatants were assayed for HCV RNA, core antigen, and infectivity titer. The buoyant densities of input and progeny virus were compared in iodixanol gradients. Results While retaining expression of differentiation markers, primary hepatocytes supported the complete infectious cycle of HCV, including production of significant titers of new infectious progeny virus, which was called primary-culture–derived virus (HCVpc). Compared with HCVcc, HCVpc had lower average buoyant density and higher specific infectivity; this was similar to the characteristics of virus particles associated with the very-low-density lipoproteins that are produced during in vivo infection. These properties were lost after re-culture of HCVpc in poorly differentiated Huh-7 cells, suggesting that authentic virions can be produced only by normal hepatocytes that secrete authentic very-low-density lipoproteins. Conclusions We have established a cell-culture–based system that allows production of infectious HCV in physiologically relevant human hepatocytes. This provides a useful tool for the study of HCV interactions with its natural host cell and for the development of antiviral therapies.

Background Inevitable hepatitis C virus (HCV) recurrence after liver transplantation is a major barrier to the survival of a transplanted liver. It may be promoted by immunosuppression and the emergence of CD4+CD25+ regulatory T cells (Treg). Treg cells can mediate the induction and maintenance of immunological self-tolerance as well as transplant tolerance. We investigated the effects of cyclosporine (CsA), a widely used immunosuppressive agent, on human CD4+CD25+ Treg cells. Methods Human CD4+CD25+ cells isolated from healthy donors were cultured in the presence of 40 or 400 ng/mL CsA. The suppressive activity of Treg was assessed in mixed leukocyte reactions (MLR) using CD25+ and autologous activated peripheral blood mononuclear cells (PBMC). Phenotype analysis (flow cytometric, Q-PCR) and cytokine production (ELISA) of Treg cells were then performed on cultures. Results CsA (40 or 400 ng/mL) inhibited the proliferative capacity of PBMC and CD4+CD25+ Treg in a dose-dependent manner. Interestingly, addition of 40 ng/mL CsA in MLR impaired the suppressive activity of CD4+CD25+ cells, whereas a higher dose of CsA had no effect on Treg function. It appears that a therapeutic dose of CsA (40 ng/mL) did not change the phenotype of CD4+CD25+ T cells, but altered Treg activity by switching the regulatory to an inflammatory cytokine profile. Conclusion CsA significantly impaired the function of CD4+CD25+ Treg cells by inducing interleukin-2 (IL-2) and interferon-γ (IFN-γ) secretion. The present studies suggested that CsA may block the induction of immune tolerance and decrease the risk of hepatitis C recurrence.

International audience; Immune response failure during HCV infection has been associated with the activity of regulatory T cells. Hepatitis C-related cirrhosis is the main reason for liver transplantation. However, 80% of transplanted patients present an accelerated recurrence of the disease. This study assessed the involvement of regulatory T-cell subsets (CD4+CD25+ cells: 'Treg' and CD49b+CD18+ cells: 'T regulatory-1' cells), in the recurrence of HCV after liver transplantation, using transcriptomic analysis, ELISA assays on serum samples and immunohistochemistry on liver biopsies from liver recipients 1 and 5 years after transplantation. Three groups of patients were included: stable HCV-negative recipients and those with mild and severe hepatitis C recurrence. At 5 years, Treg markers were overexpressed in all HCV+ recipients. By contrast, Tr1 markers were only overexpressed in patients with severe recurrence. At 1 year, a trend toward the overexpression of Tr1 was noted in patients evolving toward severe recurrence. IL-10 production, a characteristic of the Tr1 subset, was enhanced in severe recurrence at both 1 and 5 years. These results suggest that Tr1 are enhanced during severe HCV recurrence after liver transplantation and could be predictive of HCV recurrence. High levels of IL-10 at 1 year could be predictive of severe recurrence, and high IL-10 producers might warrant more intensive management.

International audience; Alternative, non-invasive techniques are necessary to monitor the progression of liver disease during chronic hepatitis C. Firstly, because serum is the most accessible material for studies using qPCR in microplates, gene transcription was compared in 219 selected genes involved in the pathogenesis of hepatitis C virus (HCV) infection between sera, PBMCs and liver samples collected simultaneously from five patients infected chronically. Secondly, using sera, gene profiles were compared between HCV-infected patients (n = 10) and healthy controls (n = 10). In addition, the influence of alcohol intake was examined in patients infected with HCV genotype-1. Firstly, amplifiable mRNAs were obtained in all samples. After amplification, significant correlations were observed between: liver versus serum; liver versus PBMCs; and serum versus PBMCs (r(2) = 0.37, r(2) = 0.54, r(2) = 0.49, respectively). A comparison of gene transcription by gene involved in T- and B-cell markers, adhesion molecules, apoptosis, liver matrix turnover and inflammation, revealed comparable, significant correlations between serum and liver, (r(2) = 0.30, r(2) = 0.60, r(2) = 0.51, r(2) = 0.51, r(2) = 0.26, and r(2) = 0.61 respectively). Secondly, a quantitative analysis of gene expression in sera between genotype-1b-infected patients and healthy controls revealed that 41 genes involved closely in T-cell activation and apoptosis were over-expressed significantly in patients infected with HCV. In these patients, alcohol consumption was associated with an increased expression of six genes involved in the inflammatory response, together with a decrease of genes associated with dendritic cell function. It is concluded that in patients infected with HCV, serum can be used to evaluate expression of liver genes. Further prospective studies are clearly needed to validate the initial results and to define the relevant genes.

Interleukin-4 (IL-4) is overexpressed in liver grafts in a context of severe recurrent hepatitis C, during which the development of fibrosis is dramatically accelerated. In this study, we examined the effects of IL-4 on the activation and collagen production of cultured human intrahepatic (myo)fibroblasts (hIHFs), and investigated the underlying mechanisms. The myofibroblastic nature of cells was evaluated morphologically using activation markers (smooth muscle alpha-actin, vimentin and prolyl 4-hydroxylase). Quiescent hIHFs were obtained by cell incubation in serum-free medium or cell culture on Matrigel. We first analyzed IL-4 receptor expression, STAT-6 activation by IL-4, and STAT-6 inhibition by an anti-IL-4 antibody or by STAT-6 small-interfering RNA (siRNA) transfection. We then focused on collagen production, using quantitative real-time PCR to analyze the effect of IL-4 on the mRNA expression of collagens I, III and IV, and on collagen levels in supernatants of hIHFs, using the Sircol collagen assay. hIHFs cultured in plastic wells appeared to be morphologically activated. The expression of activation markers was reduced by serum deprivation or culture on Matrigel, and restored by IL-4 incubation. The IL-4 receptor was expressed by hIHFs, and STAT-6 was activated following incubation with IL-4. Both anti-IL-4 antibody and STAT-6 siRNA transfection inhibited this activation. The treatment of hIHFs with IL-4 increased the mRNA expression of collagens I, III and IV (P

Summary Hepatitis C (HCV) treatment using interferon-α (IFN-α) and ribavirin is recommended in HIV/HCV co-infected patients to prevent liver cirrhosis and liver-related death. However, in addition to its antiviral activity, IFN is a pleiotropic cytokine able to synergistically amplify T-cell autoreactivity. Here, we report for the first time the induction of a subfulminant autoimmune hepatitis (AIH) after four months of a successful treatment of HCV-1b infection using peg-IFN and ribavirin, in a 48-year-old woman co-infected with HIV. Diagnosis was assessed according to the international AIH scoring system, including liver biopsy and confirmed by positive response to steroid challenge.

Human immunodeficiency virus infection has a major impact on the natural history of chronic hepatitis B and favours the emergence of viral mutants. We describe an acute hepatitis D virus superinfection in an HIV-1-infected patient under HAART treatment who was previously a chronic carrier of a surface negative HBV variant resistant to lamivudine.

Carcinomatous meningitis is a rare complication of gastric cancer.A 46 year-old man with an unremarkable medical history was hospitalized for treatment of suspected meningitis. Despite treatment, his clinical state rapidly worsened and he died without regaining consciousness, shortly after transfer to the ICU. Autopsy showed that he had anchorage-independent cell gastric carcinoma, with simultaneous lymphatic, pulmonary, cutaneous and meningeal metastases.The rising incidence of secondary meningeal lesions in the last two decades is probably associated with the treatment-related improvement in life expectancy. Prognosis is very grave, and median survival time is only several weeks. Carcinomatous meningitis is difficult to diagnosis, especially as the first sign of a primary tumor. This rare presentation of gastric cancer indicates diffuse metastatic spread and extremely poor prognosis.

International audience; BACKGROUND AND AIMS: Liver inflammation, fibrosis, and dyslipidemia are common features in patients with chronic hepatitis C virus (HCV) infection. Because peroxisome proliferator-activated receptor alpha (PPARalpha) is highly expressed in the liver and is involved in the regulation of lipid metabolism and inflammation, we sought to determine whether HCV infection may locally impair PPARalpha expression and activity. METHODS: PPARalpha expression was investigated in liver biopsy specimens of 86 untreated patients with HCV infection and controls, by using real-time polymerase chain reaction (PCR), Western blot analysis, and immunohistochemistry. PPARalpha activity was assessed by quantification of the key gene target carnitine palmitoyl acyl-CoA transferase 1 (CPT1A) messenger RNA (mRNA).The influence of HCV core protein on PPARalpha mRNA expression was analyzed in vitro by real-time PCR in HCV core-expressing HepG2 cells activated with the PPARalpha ligand fenofibric acid. RESULTS: Hepatic concentrations of PPARalpha and CPT1A expressed by hepatocytes were impaired profoundly in the livers of untreated patients with HCV infection compared with controls. A mean decrease of 85% in PPARalpha mRNA expression paralleled with a lack of CPT1A mRNA induction also were observed in HCV core-expressing HepG2 cells compared with controls. CONCLUSIONS: HCV infection is related to altered expression and function of the anti-inflammatory nuclear receptor PPARalpha. These results identify hepatic PPARalpha as one mechanism underlying the pathogenesis of HCV infection, and as a new therapeutic target in traditional treatment of HCV-induced liver injury.

The impact of hepatitis C virus NS5A protein mutations on interferon alfa (IFN-alpha) signaling pathway, cell proliferation, and viability is an important issue that is still under debate. We have therefore combined transient and stable expression in a human hepatocytic cell line (Huh7) of 3 full-length NS5A sequences, isolated from patients with or without response to IFN-alpha therapy. Expression of all 3 NS5A-reduced IFN-alpha global antiviral activity on both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) replication. We did not show, however, an effect of these 3 NS5A proteins on double-stranded RNA-dependent kinase (PKR) expression and activity as well as colocalization and coimmunoprecipitation between NS5A and PKR. We also failed to show an effect of the 3 NS5A mutants tested on cell proliferation and viability. Overall, our results support an important role of NS5A in controlling IFN-alpha antiviral activity; they show, however, that PKR-independent mechanisms are implicated, at least in liver-derived cells.

Hepatitis C virus (HCV) is a major etiological agent of chronic liver disease and hepatocellular carcinoma (HCC). We demonstrate herewith that HCV core proteins encoded by sequences isolated from HCC tumor tissues, but not those derived from their non-tumor counterparts in the same liver, co-localise in vitro and in vivo and co-immunoprecipitate with PKR in hepatocytic Huh7 cells. We show that this association in fact augments the autophosphorylation of PKR and the phosphorylation of the translation initiation factor eIF2alpha, which are two markers of PKR activity. The present study therefore identifies a novel model of virus-cell interactions whereby a viral protein, the HCV core, activates PKR activity.

Whereas bile acids in excess depress the cell-mediated immune response, their effects on the humoral response have been little investigated. The aim of this study was to investigate the effects of bile acids on immunoglobulin production. Human peripheral blood mononuclear cells were stimulated for 5 days by Staphylococcus aureus Cowan I (SAC-I). Immunoglobulins, were measured in the supernatants and cell lysates using ELISA. We found that bile acids inhibited IgM production in a dose-dependent manner. The inhibitory effects of 50 muM chenodeoxycholic acid (CDCA) and its glyco- and tauro-conjugates (62, 53 and 51%, respectively) were stronger than those of ursodeoxycholic acid (UDCA) and its conjugates (45, 40 and 34%, respectively). The inhibition of IgG production by CDCA and UDCA was weak (23 and 12%, respectively, at 50 LM). IgA production was not modified. The inhibition of intracellular IgM concentration paralleled that observed in the secreted compartment. By contrast, CDCA enhanced intracellular concentration of IgG. In the absence of significant necrosis or apoptosis, CDCA-mediated inhibition of SAC-I-induced IgM production was significantly correlated to the ability of the bile acid to inhibit cell proliferation (r=0.98; p 0.05). In conclusion, we showed that hydrophobic bile acids strongly depress the primary humoral response. This effect resulted from both an inhibition of cell proliferation, and to a lesser extent from a deficient exocytosis of immunoglobulins. (C) 2001 Elsevier Science Inc. All rights reserved.

Background/Aims: The pathophysiology of chronic hepatitis C and the mechanisms of resistance to interferon α are poorly understood. The aim of this work was to assess the influence of HCV infection and the viral genotype on lymphocyte production of 2′,5′ oligo-adenylate synthetase activity and monocyte production of TNFα and IL1β. Methods: Mononuclear cells from 50 consecutive patients were studied after 6 months of interferon treatment. Patients with persistent viremia (PCR-positive, elevated ALT, n =39) were compared with the PCR-negative patients with normal ALT activity ( n =11) of similar age and sex ratio. Results: Cells from the viremic patients showed lower basal and stimulated 2′,5′ oligo-adenylate synthetase activity, and a lower in vitro response capacity to human recombinant interferon. In contrast, no difference was observed in basal and stimulated TNFα or IL1β production between the two groups. In the PCR-positive patients the viral genotype had no significant influence on the response of mononuclear cells to interferon or endotoxin. Conclusions: These results show that the presence of HCV in blood is associated with an elective defect in interferon system activation, independently of the viral genotype.

Resume Introduction Les cancers gastriques sont rarement compliques de meningite carcinomateuse. Observation Un homme de 46 ans a ete hospitalise pour la prise en charge d’une meningite. Rapidement, son etat s’est aggrave et il etait transfere en reanimation ou il est decede sans avoir repris conscience. L’examen autopsique a montre un carcinome gastrique a cellules independantes, avec metastases synchrones ganglionnaires, pulmonaires, cutanees et neuro-meningees. Commentaires L’augmentation de l’incidence des lesions secondaires meningees depuis les deux dernieres decennies serait en relation avec l’allongement de la survie sous traitement. Le pronostic est tres sombre et la mediane de survie ne depasse pas quelques semaines sous traitement. La meningite carcinomateuse est de diagnostic difficile, surtout lorsqu’elle est revelatrice de la tumeur primitive. Ce mode de presentation est exceptionnel en cas de cancer gastrique. Il temoigne d’une diffusion metastatique et signe un pronostic extremement pejoratif.

Many drugs are suspected of inducing liver injury, but the mechanisms of this effect have been studied with only a few drugs. Moreover, for the most studied hepatotoxins, the mechanisms are still incompletely understood. In addition to its theoretical interest, determination of the mechanism of hepatotoxicity may have therapeutic consequences: recommendations or contraindications of drug utilization concerning the target population, specific monitoring for serum transaminase activities, and possibility of rechallenge. We conclude that a better understanding of the mechanisms of toxicity is necessary. In vitro and in vivo methods may be initiated to identify persons who have a biochemical or genetic basis for hypersensitivity to drugs.

The difficult and controversial diagnostic and therapeutic management of patients having gastrinoma or insulinoma with multiple endocrine neoplasia type I (MEN-I) has been discussed by reference to the literature and a personal series of 45 gastrinoma/MEN-I patients followed consecutively at Bichat Hospital. In both gastrinoma/ and insulinoma/MEN-I patients, anatomic distribution and morphology of tumoral process(es) are usually multiple, diffuse, of small size, and associated with endocrine cell hyperplasia and even nesidioblastosis. These features enhance the difficulty of tumor localization and eradication. Despite the dramatic development of modern medical imagery and surgical experience, the real possibility, on a long-term basis, of curing the patients from their disease remains limited, especially in the gastrinoma/MEN-I patients. In the latter group, according to our experience, persistence or recurrence of the disease after surgery is usual, and metachronous hepatic metastasis development is frequently observed when the follow-up is long enough. Patients with liver metastases, however, seem to undergo a more indolent course than sporadic gastrinoma cases. In insulinoma/MEN-I patients, removal of the functionally dominant islet cell area(s) is essential. Various preoperative and intraoperative localization techniques allow efficacious selective pancreatic surgery in many cases. The latter refinements and the promises of long-acting somatostatin analogs, if confirmed, might restrict to exceptional circumstances the indication of near-total or total pancreatectomy.

Cell-mediated immunity and macrophage activity, especially that of Kupffer cells, are impaired during cholestasis. Some evidence exists that bile acids play a role in these immune defects. The purpose of this study was to evaluate the effects of individual bile acids on immunity and to determine whether monocytes could be a target. We assessed the effects of chenodeoxycholic acid, an endogenous bile acid, ursodeoxycholic acid, which has been shown to partially correct the immunological abnormalities observed in primary biliary cirrhosis, and their tauroconjugates on the production of interleukin-1, interleukin-6 and tumor necrosis factor-alpha. Chenodeoxycholic acid had a dose-dependent inhibitory effect on interleukin-1 (inhibitory concentration 50% = 60 mumol/L), interleukin-6 (inhibitory concentration 50% = 80 mumol/L) and tumor necrosis factor-alpha (inhibitory concentration 50% = 80 mumol/L) production; inhibition was almost complete at 250 mumol/L. In contrast, ursodeoxycholic acid had lesser or minimal inhibitory effects (inhibitory concentration 50% = 100 mumol/L for interleukin-1 and above 200 mumol/L for interleukin-6 and tumor necrosis factor-alpha). The inhibitory effects of taurochenodeoxy-cholic acid and tauroursodeoxycholic acid were similar to those of chenodeoxycholic acid and ursodeoxycholic acid, respectively. Ursodeoxycholic acid did not reverse the chenodeoxycholic acid-induced inhibition of interleukin-6 or tumor necrosis factor-alpha production. In conclusion, chenodeoxycholic acid exerts strong inhibitory effects on monocyte activity in vitro, whereas the effects of ursodeoxycholic acid are minor.