Impact of hepatitis C virus and alcohol, alone and combined, on the unfolded protein response in primary human hepatocytes.

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.

Evaluation of laboratory tests for cirrhosis and for alcohol use, in the context of alcoholic cirrhosis.

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.

Subjective parameters markedly limit the referral of transplantation candidates to liver transplant centres.

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.

Quality indicators for colonoscopy procedures: a prospective multicentre method for endoscopy units.

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.

Evolutionary history of hepatitis C virus genotype 5a in France, a multicenter ANRS study.

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.

Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes.

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.

Analysis of gene transcription in sera during chronic hepatitis C infection.

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 induces the activation and collagen production of cultured human intrahepatic fibroblasts via the STAT-6 pathway.

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<0.05) and elevated collagen levels in supernatants (P=0.01 vs untreated cells). Therefore, IL-4 exerts profibrotic effects by activating hIHFs and inducing collagen production and secretion. This effect requires IL4-R binding and STAT-6 activation. IL-4 may thus be involved in accelerated course of fibrogenesis during recurrent hepatitis C.

CD25, CD28 and CD38 expression in peripheral blood lymphocytes as a tool to predict acute rejection after liver transplantation.

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.

Rosiglitazone for nonalcoholic steatohepatitis: one-year results of the randomized placebo-controlled Fatty Liver Improvement with Rosiglitazone Therapy (FLIRT) Trial.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Fulminant autoimmune hepatitis after successful interferon treatment in an HIV-HCV co-infected patient.

Hepatitis C (HCV) treatment using interferon-alpha (IFN-alpha) 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.

Elevated interleukin-4 expression in severe recurrent hepatitis C virus after liver transplantation.

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.

Interleukin-4 induces human hepatocyte apoptosis through a Fas-independent pathway.

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<0.001 vs. untreated control). IL-4 reduced the mitochondrial membrane potential, activated Bid and Bax, and augmented caspase 3, 8, and 9 activity. STAT6 blockade inhibited IL-4-induced apoptosis. Expression of Fas and Fas ligand was unaffected when HepG2 cells and hepatocytes were cultured with IL-4, and Fas/FasL pathway blockade failed to inhibit IL-4-induced apoptosis. These results show that IL-4 induces apoptosis of human hepatocytes through IL-4 receptor binding, STAT6 activation, decreased mitochondrial membrane potential, and increased caspase activation, independently of the Fas pathway. IL-4 might thus contribute to the progression of severe liver graft damage.

A novel, sensitive, and specific RT-PCR technique for quantitation of hepatitis C virus replication.

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 x 10(5) 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.

Hepatitis delta virus super-infection in a co-infected patient with the human immunodeficiency virus type 1 and a surface antigen-negative hepatitis B virus variant.

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 as a rare presentation of gastric cancer]. / Méningite carcinomateuse révélant un carcinomé gastrique à cellules indépendantes.

INTRODUCTION: Carcinomatous meningitis is a rare complication of gastric cancer. CASE: 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. DISCUSSION: 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.

Impaired expression of the peroxisome proliferator-activated receptor alpha during hepatitis C virus infection.

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.