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.

Laboratory tests can play an important role in assessment of alcoholic patients, including for evaluation of liver damage and as markers of alcohol intake. Evidence on test performance should lead to better selection of appropriate tests and improved interpretation of results. We compared laboratory test results from 1578 patients between cases (with alcoholic cirrhosis; 753 men, 243 women) and controls (with equivalent lifetime alcohol intake but no liver disease; 439 men, 143 women). Comparisons were also made between 631 cases who had reportedly been abstinent from alcohol for over 60 days and 364 who had not. ROC curve analysis was used to estimate and compare tests' ability to distinguish patients with and without cirrhosis, and abstinent and drinking cases. The best tests for presence of cirrhosis were INR and bilirubin, with areas under the ROC curve (AUCs) of 0.91 ± 0.01 and 0.88 ± 0.01, respectively. Confining analysis to patients with no current or previous ascites gave AUCs of 0.88 ± 0.01 for INR and 0.85 ± 0.01 for bilirubin. GGT and AST showed discrimination between abstinence and recent drinking in patients with cirrhosis, including those without ascites, when appropriate (and for GGT, sex-specific) limits were used. For AST, a cut-off limit of 85 units/L gave 90% specificity and 37% sensitivity. For GGT, cut-off limits of 288 units/L in men and 138 units/L in women gave 90% specificity for both and 40% sensitivity in men, 63% sensitivity in women. INR and bilirubin show the best separation between patients with alcoholic cirrhosis (with or without ascites) and control patients with similar lifetime alcohol exposure. Although AST and GGT are substantially increased by liver disease, they can give useful information on recent alcohol intake in patients with alcoholic cirrhosis when appropriate cut-off limits are used.

Marillier, Maude PharmD; Batisse, Anne PharmD; Edel, Yves MD; Nguyen, An-Hung MD; Chevallier, Cécile PharmD; Pfau, Grégory PharmD; Podevin, Philippe MD; Djezzar, Samira MD Author Information

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.

advantage of our HCV culture system in primary human adult hepatocytes (PHH), which, contrary to the widely used hepatocarcinoma-derived Huh-7 sublines, retain the liver metabolism of ethanol and secrete authentic VLDL and LVP.METHODS: PHH were infected with the HCV strain JFH1 or a chimeric virus derived thereof and treated with increasing doses of ethanol (0-100 mM) for 2 weeks.Cultures were monitored for HCV genome replication (negative strand RT-qPCR), viral load (clinically used test), production of infectious virus (titration by focus-formation assay), and VLDL secretion (apolipoprotein B ELISA).The density of viral particles was assessed by isopycnic iodixanol ultracentrifugation.RESULTS: Ethanol exposure of HCV-infected PHH caused a time-and dose-dependent increase in the viral load, comparable to that reported in clinical studies.Most strikingly, it caused an even greater increase in the infectious titer but did not significantly affect the viral genome replication, thus pointing to an effect on virus morphogenesis.This effect was not seen in Huh-7.5.1 cells treated in parallel, suggesting that it involves the liver metabolism of ethanol.The higher specific infectivity of HCV particles produced by PHH in the presence of ethanol correlated with lower mean buoyant density, consistent with triglyceride enrichment.Finally, in either HCV-infected or naïve PHH, addition of ethanol caused a time-dependent increase in VLDL secretion.CONCLUSION: This study in the relevant PHH model reveals that ethanol via its metabolites increases the production of HCV particles of lowest buoyant density and highest infectivity, i.e., LVP, most likely due to the impact of ethanol on triglyceride metabolism that results in increased VLDL secretion.Drugs targeting this host metabolic pathway may be useful in difficult-to-treat alcoholic patients, often poorly compliant with therapy and therefore at risk for resistance if treated by direct antivirals only.VP & CH: equal contribution.

Background and Aims: Healthcare professionals are required to conduct quality control of endoscopy procedures, and yet there is no standardised method for assessing quality. The topic of the present study was to validate the applicability of the procedure in daily practice, giving physicians the ability to define areas for continuous quality improvement.Methods: In ten endoscopy units in France, 200 patients per centre undergoing colonoscopy were enrolled in the study. An evaluation was carried out based on a prospectively developed checklist of 10 quality-control indicators including five dependent upon and five independent of the colonoscopy procedure.Results: Of the 2000 procedures, 30% were done at general hospitals, 20% at university hospitals, and 50% in private practices. The colonoscopies were carried out for a valid indication for 95.9% (range 92.5-100). Colon preparation was insufficient in 3.7% (range 1-10.5). Colonoscopies were successful in 95.3% (range 81-99). Adenoma detection rate was 0.31 (range 0.17-0.45) in successful colonoscopies.Conclusion: This tool for evaluating the quality of colonoscopy procedures in healthcare units is based on standard endoscopy and patient criteria. It is an easy and feasible procedure giving the ability to detect suboptimal practice and differences between endoscopy-units. It will enable individual units to assess the quality of their colonoscopy techniques.

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.

BACKGROUND:Experimental data strongly suggest that in animal and probably in man, noradrenergic and serotoninergic become uncoupled during repeated consumption of drugs of abuse, strongly suggesting that different drugs share common mechanisms for drug-dependence. Using cocaine-dependence as model of strong addiction we speculate that careful analysis of psychic adjustments in patients who experience prolonged abstinence could be a useful tool for patient's care. AIM:The aim of this retrospective study was first to establish similarities in patients' histories concerning modes of entrance, circumstances favouring the stopping, and modality of withdrawal. Secondly, we analysed the different ways used by subjects to substitute their cocaine-dependence. PATIENTS AND METHODS:Cocaine-dependent subjects who had succeeded in supporting abstinence for at least 12 months without consumption were evaluated retrospectively by a face-to face interview. RESULTS:We obtained a list of circumstances associated with entries and exit from cocaine-dependence. Second, when seeking for similarities in addictive behaviour, before and after, between cocaine users, we proposed to classify patients according to the strength in their addictive dominant trait between strong, moderate, mild, or absence of addictive behaviour. For didactic aims, purposes are illustrated by clinical vignettes. CONCLUSIONS:This retrospective study allows us to clear arbitrarily four types of psychical modifications associated with prolonged abstinence in cocaine-dependent patients. Prospective clinical studies are clearly needed to standardize and to validate these clinical criteria.

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.

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

Introduction: La qualité de la préparation colique avant la coloscopie est à prendre en considération puisque une préparation insuffisante est une cause fréquente des echecs de coloscopie totale et a un impact sur le taux de détection des polypes. Le but de notre travail était d'évaluer l'impact de la qualité de la préparation colique sur le taux de détection de polypes et le taux de coloscopiesincomplètes lors d'une étude multicentrique prospective réalisée dans 11 centres d'endoscopie privés, semi-privés, militaires et publiques.

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

Introduction: L'EPP est une obligation légale et une nécessité dans le domaine de l'endoscopie digestive. Cette EPP doit évaluer l'ensemble des procédures rentrant en jeu avant, pendant et après l'acte. Une méthode simple et reproductible d'EPP a été développée afin d'évaluer la qualité de la coloscopie [1]. Le but de ce travail prospectif a été d'évaluer la faisabilité de cette méthode dans des centres d'endoscopie de CHU, de CHG, de PS-PH et dans des cliniques privées.

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 C&A (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-gamma (IFN-gamma) secretion. The present studies suggested that CsA may block the induction of immune tolerance and decrease the risk of hepatitis C recurrence.

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 genetype-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. J. Med. Virol. 81:473-480,2009. (C) 2009 Wiley-Liss Inc.

Background and Aims: Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by progressive iron overload through increased intestinal absorption.Phlebotomy, the preferred treatment, can prevent or reverse some complications of iron overload, such as hepatic damage; however, compliance is variable and some patients are poor candidates because of underlying medical disorders and/or poor venous access.Thus, if an oral iron chelator such as deferasirox (Exjade ® ) proves to be tolerable and effective, HH patients will have an alternative treatment option.Methods: This is an inter-patient dose-escalation study of deferasirox (5, 10, 15 and 20 mg/kg) administered daily for 24 weeks to C282Y HFE homozygous HH patients with a pre-treatment serum ferritin (SF) value of 300-2000 ng/mL, transferrin saturation 45% and no known history of cirrhosis.A 6-month extension trial is ongoing.The primary endpoint is the incidence and severity of adverse events (AEs).Secondary endpoints include change in SF, time to SF normalization (<100 ng/mL), longitudinal course of SF, and pharmacokinetics of deferasirox.Results: Enrollment has ended and 48 patients (32 men, 16 women; mean age 50.4 years) with a mean of 3.1 years since HH diagnosis have been treated with deferasirox 5 (n = 11), 10 (n = 15) or 15 mg/kg/day (n = 22) for at least 24 weeks.Bayesian analysis and medical review were performed between dose escalations.Meaningful reductions in SF were observed across the first three dose groups and escalation to 20 mg/kg/day was not undertaken.Mean SF reductions during the 6-month core were 176, 478 and 442 ng/mL in the 5, 10 and 15 mg/kg/day groups, respectively.AEs in the core were dose dependent and consistent with the known safety profile of deferasirox.Noteworthy laboratory abnormalities of ALT >3× baseline, and serum creatinine 1.33× baseline and upper limit of normal on two consecutive occasions, occurred in eight and three patients, respectively.All have resolved with dose cessation or modification.Conclusions: These preliminary results from the core trial suggest that deferasirox doses of 5, 10 and 15 mg/kg/day are effective at reducing iron burden in HH patients with an acceptable safety profile.Larger studies are planned to confirm this effect.