Journal of Hepatology - Articles in Press

The hepatitis C virus core protein indirectly induces alpha-smooth muscle actin expression in hepatic stellate cells via interleukin-8 - Uncorrected Proof

2010-03-08

Background & Aims: Progressive deposition of liver fibrosis is a common feature of chronic hepatitis associated with hepatitis C virus (HCV) infection, and it may eventually lead to cirrhosis and liver failure. Although this fibrogenic process appears to be linked to HCV protein expression and replication via indirect mechanisms, i.e., to be mediated by virally-driven inflammation, a direct role of HCV in inducing fibrosis deposition has never been entirely excluded.Methods: We established an in vitro system in which the human hepatic stellate cell line LX-2 was cultured in the presence of conditioned medium from human hepatoma Huh-7 cells transduced with a lentiviral vector expressing HCV core proteins of different genotypes.Results: Treatment of LX-2 cells with conditioned medium from Huh-7 cells expressing HCV core protein led to the activation of α-smooth muscle actin expression. Among the chemokines secreted by cells transduced with HCV core, interleukin-8 was identified as the strongest inducer of α-smooth muscle actin expression in LX-2 and primary hepatic stellate cells. This effect was accompanied by a decrease in cell migration and increased focal contact organisation.Conclusions: The expression of the HCV core in hepatocytes may contribute to the establishment of a profibrogenic microenvironment.

Quantitative longitudinal evaluations of hepatitis delta virus RNA and hepatitis B virus DNA shows a dynamic, complex replicative profile in chronic hepatitis B and D - Uncorrected Proof

2010-03-08

Background & Aims: This study presents a real-time reverse-transcription PCR (rt-RT-PCR) assay for hepatitis delta virus (HDV) RNA quantification, designed to clarify the interplay between HDV and hepatitis B virus (HBV) in chronic infection.Methods: Serum HDV-RNA and HBV-DNA were analysed by rt-RT-PCR in a cross-sectional study of 37 untreated chronic HDV patients, 25 of whom were also longitudinally studied.Results: In the cross-sectional study, both viruses were active in 15 (40.5%) patients and inactive in 4 (10.8%); HDV alone was active in 12 (32.4%) and HBV in 6 (16.2%). The longitudinal study showed seven replication profiles, with considerable fluctuating activity of one or both viruses, including alternating predominance. In 20% of cases, longitudinal HBV/HDV viral loads differed from cross-sectional results, indicating a risk of misinterpreting HBV/HDV interactions when assessing a single determination. Fluctuating HBV replication only increased in the presence of fluctuating HDV activity. HBsAg levels, stable in HBV single infection, fluctuated in HDV chronic infection. The results of both the cross-sectional and longitudinal study call into question the major suppressor effect of HDV over HBV, revealing an important role of HBV.Conclusions: Longitudinal evaluation of viremia shows a complex interaction between HBV and HDV and is essential to understand the pathophysiology of chronic HDV infection.

Use of desirability functions to evaluate health status in patients with cirrhosis - Uncorrected Proof

Chris Gennings, Douglas Heuman, Otis Fulton, Arun J. Sanyal — 2010-03-08

Background & Aims: There is a need for methods that provide a quantitative assessment of a patient’s global health status, especially as regards quality of life, prognosis and impact of therapeutic interventions. We propose that desirability functions, widely used in industrial quality control, can be adapted to obtain a numerical “wellness” score, incorporating numerous easily measured clinical parameters, that reflects a patient’s overall clinical status and prognosis on a scale from 0 (worst) to 1 (best).Methods: In this pilot study, we used this approach to develop a Cirrhosis Relative Wellness Index for use with patients with liver disease. We relied on expert opinion to select 10 parameters of interest and to determine the desirability function for each parameter. A composite index was then developed and tested using data from 109 cirrhotic subjects enrolled in the North American Study for the Treatment of Refractory Ascites (NASTRA). The index was independently validated using a separate database obtained from a review of records of 1342 cirrhotic patients referred for liver transplantation candidacy in the Veterans Health System between 1997 and 2008.Results: In both datasets, the Cirrhosis Relative Wellness Index was significantly associated with transplant-free survival using either a proportional hazards model or a logrank test (where the index was dichotomised).Conclusion: Desirability function modelling represents a promising new approach to quantitatively estimating global health status.

Post-natal paucity of regulatory T cells and control of NK cell activation in experimental biliary atresia - Uncorrected Proof

2010-03-08

Background & Aims: Although recent studies have identified important roles for T and NK cells in the pathogenesis of biliary atresia (BA), the mechanisms by which susceptibility to bile duct injury is restricted to the neonatal period are unknown.Methods: We characterised hepatic regulatory T cells (Tregs) by flow cytometry in two groups of neonatal mice challenged with rhesus rotavirus (RRV) at day 7 (no ductal injury) or day 1 of life (resulting in BA), determined the functional interaction with effector cells in co-culture assays, and examined the effect of adoptive transfer of CD4+ cells on the BA phenotype.Results: While day 7 RRV infection increased hepatic Tregs (Foxp3+ CD4+ CD25+) by 10-fold within 3days, no increase in Tregs occurred at this time point following infection on day 1. In vitro, Tregs effectively suppressed NK cell activation by hepatic dendritic cells and decreased the production of pro-inflammatory cytokines, including TNFα and IL-15, following RRV infection. In vivo, adoptive transfer of CD4+ cells prior to RRV inoculation led to increased survival, improved weight gain, decreased population of hepatic NK cells, and persistence of donor Tregs in the liver.Conclusions: (1) The liver is devoid of Tregs early after perinatal RRV infection; (2) Tregs suppress DC-dependent activation of naive NK cells in vitro, and Treg-containing CD4+ cells inhibit hepatic NK cell expansion in vivo. Thus, the post-natal absence of Tregs may be a key factor that allows hepatic DCs to act unopposed in NK cell activation during the initiation of neonatal bile duct injury.

Mitochondrial dysfunction precedes insulin resistance and hepatic steatosis and contributes to the natural history of non-alcoholic fatty liver disease in an obese rodent model - Uncorrected Proof

2010-03-04

Background & Aims: In this study, we sought to determine the temporal relationship between hepatic mitochondrial dysfunction, hepatic steatosis and insulin resistance, and to examine their potential role in the natural progression of non-alcoholic fatty liver disease (NAFLD) utilising a sedentary, hyperphagic, obese, Otsuka Long–Evans Tokushima Fatty (OLETF) rat model.Methods: OLETF rats and their non-hyperphagic control Long–Evans Tokushima Otsuka (LETO) rats were sacrificed at 5, 8, 13, 20, and 40weeks of age (n=6–8 per group).Results: At 5weeks of age, serum insulin and glucose and hepatic triglyceride (TG) concentrations did not differ between animal groups; however, OLETF animals displayed significant (p<0.01) hepatic mitochondrial dysfunction as measured by reduced hepatic carnitine palmitoyl-CoA transferase-1 activity, fatty acid oxidation, and cytochrome c protein content compared with LETO rats. Hepatic TG levels were significantly elevated by 8weeks of age, and insulin resistance developed by 13weeks in the OLETF rats. NAFLD progressively worsened to include hepatocyte ballooning, perivenular fibrosis, 2.5-fold increase in serum ALT, hepatic mitochondrial ultrastructural abnormalities, and increased hepatic oxidative stress in the OLETF animals at later ages. Measures of hepatic mitochondrial content and function including β-hydroxyacyl-CoA dehydrogenase activity, citrate synthase activity, and immunofluorescence staining for mitochondrial carbamoyl phosphate synthetase-1 progressively worsened and were significantly reduced at 40weeks in OLETF rats compared to LETO animals.Conclusions: Our study documents that hepatic mitochondrial dysfunction precedes the development of NAFLD and insulin resistance in the OLETF rats. This evidence suggests that progressive mitochondrial dysfunction contributes to the natural history of obesity-associated NAFLD.

Bile acids modulate glucocorticoid metabolism and the hypothalamic–pituitary–adrenal axis in obstructive jaundice - Uncorrected Proof

2010-03-04

Background & Aims: Suppression of the hypothalamic–pituitary–adrenal axis occurs in cirrhosis and cholestasis and is associated with increased concentrations of bile acids. We investigated whether this was mediated through bile acids acting to impair steroid clearance by inhibiting glucocorticoid metabolism by 5β-reductase.Methods: The effect of bile acids on glucocorticoid metabolism was studied in vitro in hepatic subcellular fractions and hepatoma cells, allowing quantitation of the kinetics and transcript abundance of 5β-reductase. Metabolism was subsequently examined in vivo in rats following dietary manipulation or bile duct ligation. Finally, indices of glucocorticoid metabolism were assessed in humans with obstructive jaundice.Results: In rat hepatic cytosol, chenodeoxycholic acid competitively inhibited 5β-reductase (Ki 9.19±0.40μM) and reduced its transcript abundance (in H4IIE cells) and promoter activity (reporter system, HepG2 cells).In Wistar rats, dietary chenodeoxycholic acid (1% w/w chow) inhibited hepatic 5β-reductase activity, reduced urinary excretion of 3α,5β-tetrahydrocorticosterone and reduced adrenal weight. Conversely, a fat-free diet suppressed bile acid levels and increased hepatic 5β-reductase activity, supplementation of the fat-free diet with CDCA reduced 5β-reductase activity, and urinary 3α,5β-reduced corticosterone. Cholestasis in rats suppressed hepatic 5β-reductase activity and transcript abundance.In eight women with obstructive jaundice, relative urinary excretion of 3α,5β-tetrahydrocortisol was significantly lower than in healthy controls.Conclusion: These data suggest a novel role for bile acids in inhibiting hepatic glucocorticoid clearance, of sufficient magnitude to suppress hypothalamic–pituitary–adrenal axis activity. Elevated hepatic bile acids may account for adrenal insufficiency in liver disease.

Re-emerging interest in hepatitis delta: New insights into the dynamic interplay between HBV and HDV - Uncorrected Proof

Heiner Wedemeyer — 2010-03-03

Delta hepatitis is still a major global health problem affecting 15–20 million individuals world-wide . The implementation of vaccine programs against hepatitis B was initially associated with a decline in hepatitis D virus (HDV) infections in Southern Europe during the 1980s and 1990s . However, several studies published over the last few years have shown that the prevalence of hepatitis delta remains rather high in Europe – in particular in immigrant populations. Between 8% and 12% of HBsAg-positive individuals tested anti-HDV positive in France , Germany , Italy , the United Kingdom , and Turkey . A common feature of all these studies was that the majority HDV-infected individuals were born in highly endemic areas such as Eastern Turkey, Central Asia, or Africa. HDV is also prevalent in South America, in particular in the Amazonian region of Western Brazil , and the Western pacific population .

Personalized molecular targeted therapy in advanced, recurrent hepatocellular carcinoma after liver transplantation: A proof of principle - Uncorrected Proof

2010-03-03

Background & Aims: The advent of molecular medicine that targets specific pathways is changing the therapeutic approach to hepatocellular carcinoma. For several aberrantly activated pathways in hepatocarcinoma, surrogate markers of activation can be assessed by immunohistochemistry, although associations with in vivo response to targeted therapies are still lacking.Methods: A patient who presented with hepatic and extra-hepatic hepatocarcinoma recurrence 11years after liver transplantation was assessed for β-catenin, pERK and pS6 in primary and secondary tumor specimens, in order to define a possible activation of the Wnt, Ras/MAPK and Akt/mTOR pathways and design a personalized targeted therapy in absence of alternative treatment options. Moreover, mutation analysis of the β-catenin gene (CTNNB1) and DNA microsatellite analyses were performed.Results: The identification of the same mutation in the β-catenin gene, as well as the same microsatellite pattern in tumor tissues taken 11years apart, proved that the observed hepatocarcinoma was a true recurrence. Nuclear β-catenin and pS6 in tumor cells was positive, whereas pERK was positive only in the peritumoral endothelium. This pattern of immunohistochemistry, after failure of sorafenib alone, lead to the choice to add the mTOR inhibitor, everolimus, to sorafenib. Three months later a 50% tumor reduction was observed, and after 6months a further reduction of tumor vital components was confirmed, while a grade II gastrointestinal bleeding episode occurred.Conclusions: A personalized approach aimed to treat recurrent hepatocarcinoma is possible through analysis of tumoral molecular pathways. Partial success of the selected combination of sorafenib and everolimus supports the pivotal role of mTOR signalling and highlights the importance of reliable biomarkers to route the best molecular-based therapeutic options in HCC.

Mutations in hepatitis C virus genotype 1b and the sensitivity of interferon-ribavirin therapy after liver transplantation - Uncorrected Proof

2010-03-03

Background & Aims: The results of post-transplant antiviral therapy for recurrent hepatitis C virus (HCV) are poor, and significant pre-transplant predictors for sustained viral response (SVR) have not yet been identified.Methods: Pegylated interferon/ribavirin therapy was performed for more than 48weeks in 50 patients who underwent liver transplantation (LT) for HCV genotype 1-related liver disease. Of these, 22 patients achieved SVR. The predictive potential of the viral mutations, including amino acids (aa) 70 and 91 in the Core region, interferon sensitivity-determining region (ISDR, aa 2209–2248) and interferon/ribavirin resistance-determining region (IRRDR, aa 2334–2379) in NS5A, was evaluated.Results: In 16 patients, the sequences in the pre- and post-transplant samples were the same, except for aa 70 in the Core of 1 patient. The SVR achievement ratio was significantly lower in the Non-double wild (DW) at aa 70 and 91, the ISDR<2 and IRRDR<6 groups than in the DW (30% vs. 65%, p=0.015), the ISDR⩾2 (35% vs. 69%, p=0.035) and IRRDR⩾6 (25% vs. 78%, p<0.001) groups, respectively. Predictive scoring with these three items provides a newly established and significant predictor for SVR after LT (p=0.015).Conclusion: DW, ISDR⩾2 and IRRDR⩾6 were found to be significant predictors for SVR after LT. In addition, it is possible that the establishment of a new scoring system consisting of these three factors may be a useful marker to predict interferon sensitivity for recurrent HCV after LT.

The let-7 family of microRNAs inhibits Bcl-xL expression and potentiates sorafenib-induced apoptosis in human hepatocellular carcinoma - Uncorrected Proof

2010-03-03

Background & Aims: Bcl-xL, an anti-apoptotic member of the Bcl-2 family, is over-expressed in human hepatocellular carcinoma, conferring a survival advantage to tumour cells. The mechanisms underlying its dysregulation have not been clarified. In the present study, we explored the involvement of microRNAs that act as endogenous sequence-specific suppressors of gene expression.Methods: The expression profiles of microRNAs in Huh7 hepatoma cells and primary human hepatocytes were compared by microarray analysis. The effect of let-7 on Bcl-xL expression was examined by Western blot and a reporter assay. The involvement of let-7 microRNAs in human tissues were analysed by western blot and reverse transcription-PCR.Results: Microarray analysis followed by in silico target prediction identified let-7 microRNAs as being downregulated in Huh7 hepatoma cells in comparison with primary human hepatocytes as well as possessing a putative target site in the bcl-xl mRNA. Over-expression of let-7c or let-7g led to a clear decrease of Bcl-xL expression in Huh7 and HepG2 cell lines. Reporter assays revealed direct post-transcriptional regulation involving let-7c or let-7g and the 3′-untranslated region of bcl-xl mRNA. Human hepatocellular carcinoma tissues with low expression of let-7c displayed higher expression of Bcl-xL protein than those with high expression of let-7c, suggesting that low let-7 microRNA expression contributes to Bcl-xL over-expression. Finally, expression of let-7c enhanced apoptosis of hepatoma cells upon exposure to sorafenib, which downregulates expression of another anti-apoptotic Bcl-2 protein, Mcl-1.Conclusions: let-7 microRNAs negatively regulate Bcl-xL expression in human hepatocellular carcinomas and induce apoptosis in cooperation with an anti-cancer drug targeting Mcl-1.

Impact of peginterferon and ribavirin therapy on hepatocellular carcinoma: Incidence and survival in hepatitis C patients with advanced fibrosis - Uncorrected Proof

2010-03-03

Background & Aims: Hepatocellular carcinoma (HCC) currently represents the major cause of liver-related death in patients with hepatitis C virus (HCV)-related cirrhosis. We assessed the influence of combination therapy on the risk of HCC, liver-related complications (ascites, variceal bleeding), and liver-related death (or liver transplantation).Methods: Three hundred seven chronic hepatitis C patients with bridging fibrosis (n=127) or cirrhosis (n=180) were evaluated by Cox regression analysis. Sustained virological response (SVR) was defined as undetectable serum HCV RNA at 24weeks after treatment.Results: An SVR developed in 33% of patients. The SVR rates were not different between patients with bridging fibrosis (37%) and those with cirrhosis (30%), p=0.186. During a median follow-up of 3.5years (range 1–18years) after the last treatment, the incidence rates per 100 person-years of HCC, liver-related complications, and liver-related death, were 1.24, 0.62, and 0.61 among SVR patients, respectively, and 5.85, 4.16, and 3.76 among non-SVR patients, respectively (log-rank test, p<0.001). According to multivariate analysis, non-SVR was an independent predictor of HCC (HR 3.06; 95% CI=1.12–8.39), liver-related complications (HR 4.73; 95% CI: 1.09–20.57), and liver-related death (HR 3.71; 95% CI=1.05–13.05).Conclusions: SVR is achieved in one-third of patients with HCV-related cirrhosis treated with peginterferon and ribavirin. An SVR has a strong independent positive influence on the incidence of HCC and on the prognosis of these patients.

Unexpected effects of fasting on murine lipid homeostasis – Transcriptomic and lipid profiling - Uncorrected Proof

2010-03-03

Background & Aims: Starvation induces massive perturbations in metabolic pathways involved in energy metabolism, but its effect on the metabolism of lipids, particularly cholesterol, is little understood.Methods: A comparative genomic analysis of the gut and the liver in response to fasting was performed, with intestinal perfusion and lipid profiling of the plasma, bile, liver, intestinal tissue, perfusate and faeces in FVB mice.Results: The expression profiles suggested increased cholesterol trafficking in the liver and decreased trafficking in the small intestine. Plasma cholesterol concentrations significantly increased, and triglycerides decreased in fasting. Surprisingly, in prolonged fasting, the biliary bile salt and lipid output rates increased, with increased hepatic and intestinal lipid turnover, and enhanced trans-intestinal cholesterol excretion. In contrast, faecal sterol loss declined sharply. To investigate whether the increased biliary phospholipid secretion could nourish the intestinal epithelium, we studied the histology of the small intestines upon fasting in multidrug resistant protein 2 deficient mice with scarce biliary phospholipids. Their adaptive biliary response to fasting was lost, while the shortage of biliary phospholipids strongly induced apoptosis and proliferation in the small intestine and increased the number of mucin-producing cells.Conclusion: Even with no dietary fat, lipid levels remain remarkably constant in the murine liver and intestines during prolonged fasting. The biliary system, always assumed to be coupled to the postprandial response, shows a paradoxical increase in activity. We hypothesise that biliary lipids are mobilised to supply the enterocytes with luminal fuel and to stabilise transport systems in the intestine for ensuring a rapid recovery when the food supply resumes.

Genetic associations in Italian primary sclerosing cholangitis: Heterogeneity across Europe defines a critical role for HLA-C - Uncorrected Proof

2010-03-03

Background & Aims: The HLA complex on chromosome 6p21 is firmly established as a risk locus for primary sclerosing cholangitis (PSC). We aimed to exploit genetic differences between Northern Europe and Italy in an attempt to define a causative locus in this genetic region.Methods: Seventy-eight North-Italian PSC patients and 79 controls were included. We performed sequencing-based genotyping of HLA-C, HLA-B and HLA-DRB1. The major histocompatibility chain-related A (MICA) transmembrane microsatellite was analysed using PCR fragment length determination. The tumour necrosis factor-alpha (TNF-α)-308G→A polymorphism was genotyped with TaqMan®. Allele frequencies were compared with Chi-square tests. Uncorrected p-values <0.05 were considered statistically significant when replicating findings in previous studies. The p-values of novel associations were corrected for multiple comparisons (Bonferroni).Results: The frequency of the strong inhibitory HLA-C2 killer-immunoglobulin receptor (KIR) ligand variant was significantly reduced in PSC vs. controls (0.39 vs. 0.58, p=0.0006). Consequently, HLA-C1 homozygosity was associated with an increased risk of PSC (OR 3.1; 95% CI 1.4–6.7, p=0.004). Importantly, there were no significant associations with the HLA-Bw4 KIR ligand variant, at the neighbouring MICA locus or with TNF-α-308G→A. At HLA-DRB1, we confirmed positive and negative associations with DRB1*15 and DRB1*07, respectively, while there were no associations with the DRB1*03, *04 or *1301 alleles typically detected in PSC in Northern Europe.Conclusions: The strong inhibitory KIR ligand HLA-C2 protects against PSC development in all populations hitherto studied. Further studies on the role of natural killer cells and T-lymphocytes expressing KIRs in PSC pathogenesis are warranted.

In vivo silencing of Reptin blocks the progression of human hepatocellular carcinoma in xenografts and is associated with replicative senescence - Uncorrected Proof

2010-03-03

Background & Aims: We showed that Reptin is overexpressed in hepatocellular carcinoma (HCC). In vitro depletion of Reptin with siRNAs led to HCC cell growth arrest and apoptosis. We asked whether in vivo targeting of Reptin in established tumours had a therapeutic effect.Methods: We used lentiviral vectors to construct HuH7 and Hep3B cell lines with doxycycline (Dox)-dependent expression of Reptin (R2) or control shRNA (GL2). Cells were injected subcutaneously into immunodeficient mice, and Dox was given when tumours reached a volume of 250mm3.Results: In vitro, the growth of GL2−Dox, GL2+Dox, and R2−Dox cells was undistinguishable whereas that of R2+Dox cells stopped 4days after Dox treatment. The growth decrease was associated with increased apoptosis, and evidence of replicative senescence, as shown by staining for acid β-galactosidase and the presence of senescence-associated heterochromatin foci. In xenografted mice, R2+Dox tumour growth stagnated or even regressed with prolonged treatment in contrast with the GL2−Dox, GL2+Dox, and R2−Dox tumours that progressed steadily. The blockage of tumour progression was associated with the induction of senescence and reduced cell proliferation.Conclusions: In vivo Reptin depletion leads to tumour growth arrest. Reptin may prove a valuable target in HCC.

The scavenger receptors SRA-1 and SREC-I cooperate with TLR2 in the recognition of the hepatitis C virus non-structural protein 3 by dendritic cells - Uncorrected Proof

2010-03-03

Backgrounds & Aims: The hepatitis C virus NS3 protein is taken up by myeloid cells in a TLR2-independent manner and activates myeloid cells via TLR2. This study aimed to identify the endocytic receptor(s) involved in the uptake of NS3 by myeloid cells and its relation with TLR2.Methods: Inhibitors and transfected cells were used to identify the nature of the NS3-binding receptors expressed by myeloid cells. The cooperation between scavenger receptors (SRs) and TLR2 in the NS3-mediated activation of myeloid cells was evaluated using inhibitors, cells from TLR2−/− mice and confocal microscopy. The involvement of SRs in NS3 cross-presentation was evaluated in vitro using an NS3-specific human T-cell clone.Results: We observed that SRs are the main binding structures for NS3 on myeloid cells and identified the SRs SRA-1 and SREC-I as endocytic receptors for NS3. Moreover, both SRs and TLR2 cooperate in NS3-induced myeloid cell activation.Conclusion: This study highlights a central role for SRs in NS3 uptake and cross-presentation and demonstrates a tightly orchestrated cooperation between signalling and endocytic innate receptors in NS3 recognition.

Let-7g targets collagen type I α2 and inhibits cell migration in hepatocellular carcinoma - Uncorrected Proof

2010-03-02

Background & Aims: Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis mainly due to metastasis. MicroRNAs are endogenous small noncoding RNAs that regulate cellular gene expression and are functionally linked to tumourigenesis. Using microarray analysis, we recently identified 20 miRNAs associated with HCC metastasis. Here, we carried out further analyses on one of these microRNAs, let-7g, to determine whether it is functionally linked to HCC metastasis.Methods: Quantitative real-time polymerase chain reaction was used to determine the level of mature let-7g transcript in HCC clinical specimens and its correlation with patient survival. Ectopic expression of let-7g was carried out in HCC cell lines to assess its influence on cell growth, migration and invasion.Results: We confirmed that the level of let-7g was significantly lower in metastatic HCCs compared to metastasis-free HCCs. Moreover, low let-7g expression in a tumour was predictive of poor survival in HCC patients. Functional studies indicated that ectopic expression of let-7g significantly inhibits HCC cell migration and cell growth. In-silico analysis revealed members of soluble collagens as potential targets of let-7g. Consistently, the levels of type I collagen α2 (COL1A2) and let-7g were inversely correlated in HCC clinical specimens. COL1A2 was experimentally validated as a direct target of let-7g. Moreover, addition of COL1A2 counteracted the inhibitory effect of let-7g on cell migration.Conclusions: These results suggest that let-7g may suppress HCC metastasis partially through targeting COL1A2.

Impaired liver regeneration of steatotic rats after portal vein ligation: A particular emphasis on 99mTc-DISIDA scintigraphy and adiponectin signalling - Corrected Proof

2010-02-19

Background & Aims: Portal vein ligation (PVL) is increasingly employed prior to major hepatectomy in order to enhance the volume of the future liver remnant (FLR) and to avoid post-hepatectomy liver failure. The efficacy of PVL on subjects with non-alcohol fatty liver disease (NAFLD) is largely unknown.Methods: Sprague–Dawley rats fed with normal diet (control) and methionine-choline deficient diet (MCD) were used. The animals underwent PVL and were sacrificed at indicated time points.Results: Livers from MCD rats exhibited a decreased BrdU and Ki-67 labelling index, and an increased apoptotic index after PVL compared to normal rats; as a net effect, MCD rats exhibited a decrease in their restituted liver mass and redistributed volume ratio, compared to normal rats. Normal rats displayed similar serum levels of ICG15-R before and after PVL; whereas MCD rats displayed reduced ICG15-R after PVL. Using 99mTc-DISIDA scintigraphy examination, livers from MCD rats exhibited decreased HEF and prolonged TE1/2 of FLR after PVL, indicating deteriorating hepatocyte function despite of the shift in volume. The basal level of plasma TNFα, IL-1α, IL-1β, and IL-10 of MCD rats were significantly increased before PVL compared to normal rats; however their plasma level did not increase in response to PVL as in normal rats. Hepatic adiponectin mRNA surged in MCD rats after PVL, whereas its receptors, AdipoR1 and AdipoR2, were paradoxically down-regulated. PPARα, a down-stream molecule of AdipoR2 axis, was also decreased in MCD rats.Conclusions: Reduced regenerated liver mass and deteriorated hepatocyte function of the FLR from steatotic rats after PVL may be associated with deranged Kupffer cell-mediated cytokine expression and disrupted adiponectin signalling.

Vinyl chloride and the liver: Misrepresentation of epidemiological evidence - Uncorrected Proof

Giuseppe Mastrangelo, Diego Martines, Ugo Fedeli — 2010-02-18

In reviewing the epidemiological literature on vinyl chloride monomer (VCM) and hepatocellular carcinoma (HCC), Sherman incurred errors and misrepresentation of the overall evidence .

The place of liver transplantation in the treatment of severe alcoholic hepatitis - Uncorrected Proof

Parul Dureja, Michael R. Lucey — 2010-02-18

Although most patients with alcoholic liver disease experience positive outcomes following liver transplantation, data on the outcome after liver transplantation in-patients with severe alcoholic hepatitis are limited. Furthermore, predicting which patients with severe alcoholic hepatitis will maintain sobriety after transplantation is especially difficult. We review the arguments in favour and against extending liver transplantation to selected patients with severe alcoholic hepatitis. In conclusion, we propose that liver transplantation should be a rescue option for occasional patients with severe alcoholic hepatitis who meet the following criteria: those with severe alcoholic hepatitis that has failed medical management, who fulfil all other standard criteria for transplantation, including a thorough psychosocial assessment, yet who are unlikely to survive a mandatory 6-month abstinence period.

Primary biliary cirrhosis: A 2010 update - Uncorrected Proof

Raoul Poupon — 2010-02-18

Primary biliary cirrhosis is a chronic inflammatory autoimmune disease that mainly targets the cholangiocytes of the interlobular bile ducts in the liver. The condition primarily affects middle-aged women. Without treatment, PBC generally progresses to cirrhosis and eventually liver failure over a period of 10–20years. PBC is a rare disease with prevalence of less than 1/2000. PBC is thought to result from a combination of multiple genetic factors and superimposed environmental triggers. The contribution of the genetic predisposition is evidenced by the familial clustering. Several risk factors, including exposure to infectious agents and chemical xenobiotics, have been suggested. Ursodeoxycholic acid (UDCA) is currently the only FDA-approved medical treatment for PBC. When administered at doses of 13–15mg/kg/day, a majority of patients with PBC have a normal life expectancy without additional therapeutic measures. One out of three patients does not adequately respond to UDCA therapy and may need additional medical therapy and/or liver transplantation. This review summarises current knowledge on the epidemiology, ethiopathogenesis, clinical, and therapeutic aspects of PBC.

Foxa1 and Foxa2 regulate bile duct development in mice - Uncorrected Proof

Mario Strazzabosco — 2010-02-18

Commentary on: Foxa1 and Foxa2 regulate bile duct development in mice. Li Z, White P, Tuteja G, Rubins N, Sackett S, Kaestner KH. J Clin Invest 2009;119:1537–45Abstract: The forkhead box proteins A1 and A2 (Foxa1 and Foxa2) are transcription factors with critical roles in establishing the developmental competence of the foregut endoderm and in initiating liver specification. Using conditional gene ablation during a later phase of liver development, we show here that deletion of both Foxa1 and Foxa2 (Foxa1/2) in the embryonic liver caused hyperplasia of the biliary tree. Abnormal bile duct formation in Foxa1/2-deficient liver was due, at least in part, to activation of IL-6 expression, a proliferative signal for cholangiocytes. The glucocorticoid receptor is a negative regulator of IL-6 transcription; in the absence of Foxa1/2, the glucocorticoid receptor failed to bind to the IL-6 promoter, causing enhanced IL-6 expression. Thus, after liver specification, Foxa1/2 are required for normal bile duct development through prevention of excess cholangiocyte proliferation. Our data suggest that Foxa1/2 function as terminators of bile duct expansion in the adult liver through inhibition of IL-6 expression.

Surrogate endpoints and non-inferiority trials in chronic viral hepatitis - Uncorrected Proof

Ivan Gentile — 2010-02-18

We read with great interest the recent article by Dr. Garattini and Dr. Bertelè . We would just like to comment on the authors’ statement that “Quality of life, morbidity and mortality should always be the primary hard end-points for evaluating new drugs”. We believe that this rule should be applied with caution in the field of chronic viral hepatitis because of the slow progression of this disease and the availability of surrogate virological end-points. For example, we know that the quality of life and survival of chronic hepatitis B patients can be improved if HBV replication is suppressed in a sustained manner . Also in HCV-related chronic liver disease, a sustained virological response, namely HCV clearance 6months after treatment withdrawal, is associated with increased survival and a low rate of progression towards decompensation or hepatocellular carcinoma . A trial comparing the effects of different drugs in the setting of HBV- or HCV-related chronic hepatitis with survival as end-point would be considered poorly ethical. Indeed, it would take several decades to demonstrate the superiority of one drug over another, and patients would be deprived of the best treatment strategy for a prolonged period. Obviously, toxicity and adverse events must be carefully monitored in trials with a surrogate endpoint. Therefore, we agree with Dr. Garattini and Dr. Bertelè that “in some cases an endpoint that closely correlates with a hard end-point can be used as a surrogate” and suggest that this is the case of treatment of chronic viral hepatitis.

No inflammation? No cancer! Clear HBV early and live happily - Uncorrected Proof

Erica Villa, Giovanna Fattovich — 2010-02-18

COMMENTARY ON: Clearance of Hepatitis B Surface Antigen and Risk of Hepatocellular Carcinoma in a cohort Chronically Infected with Hepatitis B Virus.Simonetti J et al. Hepatology [ahead of print].Abstract: Some individuals who are chronically infected with hepatitis B virus (HBV) eventually lose hepatitis B surface antigen (HBsAg). Hepatocellular carcinoma (HCC) has been demonstrated to occur in a few patients after loss of HBsAg. Neither factors associated with loss of HBsAg nor the incidence of HCC thereafter have been clearly elucidated. We performed a prospective population-based cohort study in 1271 Alaska native persons with chronic HBV infection followed for an average of 19.6years to determine factors associated with loss of HBsAg and risk of developing HCC thereafter. HBsAg loss occurred in 158 persons for a rate of HBsAg clearance of 0.7%/year. Older age, but not sex, was associated with clearance of HBsAg, and loss of HBsAg was not associated with any particular HBV genotypes (A–D, and F) found in this population. Participants were followed for an average of 108.9months after HBsAg loss. Six patients, two with cirrhosis and four without, developed HCC a mean of 7.3years after HBsAg clearance (range, 2.0–15.5years). The incidence of HCC after clearance of HBsAg was 36.8 per 100,000 per year (95% CI 13.5–80.0) which was significantly lower than the rate in those who remained HBsAg-positive (195.7 cases per 100,000 person-years of follow-up [95% CI 141.1–264.5; P<0.001). After loss of HBsAg, HBV DNA was detected in the sera of 28 (18%) of those who cleared a median of 3.6years after clearance. Conclusion: HCC can occur in persons with chronic hepatitis B who have lost HBsAg, even in the absence of cirrhosis. These persons should still be followed with periodic liver ultrasound to detect HCC early.Simonetti J. et al. Hepatology. 2009 Nov 30. [Epub ahead of print].Reprinted with permission of John Wiley & Sons, Inc.

Selective rather than routine approach to endosopic retrograde cholangio-pancreatography in diagnosis of biliary atresia - Uncorrected Proof

Nedim Hadzic — 2010-02-18

Petersen and colleagues describe their experience in the “routine” use of endoscopic retrograde cholangio-pancreatography (ERCP) in children awaiting explorative laparotomy (EL) for suspected biliary atresia (BA) . They report that almost 25% of the infants have avoided surgery after documenting a patent biliary tree. The quoted specificity of ERCP in diagnosing BA was 73% .

Corrigendum to “Antibody-targeted myofibroblast apoptosis reduces fibrosis during sustained liver injury” [J Hepatol 49 (2008) 88–98)] - Uncorrected Proof

2010-02-18

A panel from B was incorrectly transposed into D. B is correct as published, D should be as given below.

Are viral or host factors predictive of response to interferon–ribavirin in transplant patients with hepatitis C? - Uncorrected Proof

Bruno Roche, Didier Samuel — 2010-02-18

In Europe and the United States, hepatitis C virus (HCV)-related end-stage liver disease is the main indicator of the need for liver transplantation (LT). Recurrence of hepatitis C in the graft is universal after LT in HCV RNA-positive candidates, leading to lobular or chronic active hepatitis in most patients and to cholestatic hepatitis in a minority of them. Cirrhosis occurs in 20–30% of transplant recipients within 5–10years of transplantation and at least 10% of patients will require re-transplantation for hepatitis C graft failure. Following transplantation, a significant increase in viral load is observed as the HCV follows a more aggressive course in immunocompromised patients when compared to competent patients. As a result, patient and graft survival are lower in HCV-positive recipients than in HCV-negative recipients . Antiviral therapy based on pegylated interferon (Peg-IFN) in combination with ribavirin (RBV) is primarily given when there is histological evidence of recurrent HCV disease. However, as in the non-transplant setting, its efficacy is lower in HCV genotype 1 infected patients. The reported sustained virological response (SVR) rate with Peg-IFN and RBV in genotype 1 patients ranges from 12% to 40% (mean 28.7%) and is lower than in non-transplant patients . Factors potentially responsible for the reduced virological response to Peg-IFN and RBV therapy in the setting of LT are: prior non-response to (Peg) IFN and RBV before transplantation, high prevalence of genotype 1, immunosuppression, intolerance, and side effects of IFN or RBV. Tolerability is a major issue, with 70–80% of patients requiring dose reduction and 10–40% requiring drug discontinuation . Most studies show improvements in biochemical and necroinflammatory activity in virological responders. Less consistent improvements in fibrosis scores have been reported, possibly due to the stage of fibrosis at the initiation of therapy, with more advanced stages being less reversible . A survival benefit was also reported in virological responders . Predictive factors associated with SVR are: initial viral kinetics and more specifically viral response assessed at 4 and 12weeks of therapy, non-1 genotype, adherence to therapy, baseline viremia, and baseline fibrosis stage .

Focus - Uncorrected Proof

Daniel Shouval — 2010-02-18

Extra hepatic biliary atresia is a relatively common cholestatic liver disease affecting one in 10–20,000 newborns which develop an obstructive cholangiopathy. The etio-pathogenesis of this entity is not fully understood. A number of mechanisms have been proposed to explain the rapid development of the characteristic destructive cholangiopathy which requires immediate surgical intervention, preferably within 4–8weeks after birth. These include among others, an infectious etiology, i.e., by group C Rota virus, cytomegalic virus infection, immune mediated injury as a result of immune dysregulation, a graft versus host induced cholangiocyte injury by maternal chimeric lymphocytes and a genetic etiology which leads to an undefined susceptibility to environmental infectious agents or toxins.

Hepatitis B surface antigen levels during the natural history of chronic hepatitis B: A perspective on Asia - Corrected Proof

2010-02-17

Background & Aims: Data from clinical trials suggest a potential role for on-treatment monitoring of serum HBsAg titres during interferon-alpha (pegIFN) therapy in predicting virological responses. However, baseline HBsAg titres during the natural history of chronic hepatitis B (CHB) have not been well-characterized. We aimed to define the serum HBsAg titres during the different phases of CHB in a cohort of Asian patients infected with either genotype B or C HBV.Methods: Two-hundred and twenty patients were classified into immune-tolerant (IT), immune-clearance (IC), non/low-replicative (LR) or hepatitis B e antigen negative hepatitis (ENH) phases. Serum HBsAg was quantified using the ARCHITECT platform (Abbott Laboratories, Chicago, USA). Correlation of HBsAg titre with HBV DNA and serum ALT within each phase of infection was performed.Results: Median HBsAg titres were different between each phase of CHB (p=0.001): IT (4.53 log10IU/ml), IC (4.03 log10IU/ml), LR (2.86 log10IU/ml), and ENH (3.35 log10IU/ml). HBsAg titres were highest in the IT phase, and lowest in the LR phase. In general, median HBsAg titres were similar between genotypes B and C HBV. Serum HBsAg titres only correlated with HBV viral load in the IC phase. No correlation between the serum HBsAg level and ALT was observed.Conclusions: This study demonstrated significant differences in median baseline serum HBsAg titres across the different phases of CHB. These results provide further insight into the HBV viral life cycle in the setting of the various phases of CHB. Baseline HBsAg quantification may help refine future treatment algorithms for both immune-modulator therapy and oral nucleos(t)ide analogue therapy.

New therapeutic targets in HCC: Reptin ATPase and HCC senescence - Uncorrected Proof

Carmen Berasain — 2010-02-17

Cellular senescence is a process characterized by permanent cell cycle arrest. It is the consequence of the finite proliferative capacity of normal cells (replicative senescence) and the response to stress and damage from exogenous and endogenous sources. Several years ago it was shown that senescence is an innate tumour suppressive mechanism associated with the activation of oncogenes, which limits the progression of pre-malignant lesions . As a consequence, escape from senescence is a prerequisite for the progression to malignancy. There are three main mechanisms that trigger cellular senescence and that must be quelled in cancer cells: activation of the p53 pathway, upregulation of the CDKN2A locus and telomere shortening . Genetic and epigenetic aberrations in any or all of these pathways are common marks of all types of human cancer. However, the senescence resistance associated with transformed cells is reversible , and senescence-inducing drugs could represent an ideal opportunity to increase the arsenal of anti-cancer weapons . In the present issue of The Journal of Hepatology the work of Ménard et al. describes a new example of tumour progression blockage linked to the induction of replicative senescence in a xenograft model of liver cancer .

Treating hepatitis C in patients with cirrhosis: The effort is worth it - Uncorrected Proof

Xavier Forns, Jordi Bruix — 2010-02-17

The efficacy of antiviral therapy for chronic hepatitis C (CHC) has improved significantly in the last decade. Currently, with the use of pegylated interferon and ribavirin, around 50% of treated patients will achieve a sustained virological response (SVR) . However, there are certain groups of patients in whom treatment is still a challenge. One of these groups is constituted of individuals with advanced fibrosis or cirrhosis. Several reasons explain the difficulty in treating these patients. First, the efficacy of antiviral therapy in this group is significantly lower than in patients with mild disease. The presence of cirrhosis has not always been an independent factor for non-response in most randomized clinical trials, and the explanation for this is most likely due to the small proportion of individuals with bridging fibrosis or cirrhosis included in these studies. Recent data obtained from a large number of individuals included in three randomized international studies show that SVR rates decreased progressively from 60% in genotype 1/4 patients without advanced fibrosis to 51% in those with bridging fibrosis and 33% in those with cirrhosis (p<0.01); the same figures for genotype 2/3 patients were 76%, 61% and 57%, respectively (p<0.01). Data from the HALT-C study further support that SVR rates are significantly lower in patients with advanced fibrosis and particularly with cirrhosis . Apart from a decreased efficacy, patients with advanced liver disease are older in comparison to individuals with mild chronic hepatitis and thus, tolerance to therapy is poor. Furthermore, some adverse events (such as neutropenia, anemia or thrombocytopenia) occur more frequently due to the presence of portal hypertension. Importantly, co-morbidities such as diabetes, hypertension or depression are more prevalent in these individuals. Some of these co-morbidities (such as the presence of diabetes) seem to directly influence the rates of viral clearance; other associated diseases decrease treatment adherence or represent relative contraindications that make antiviral therapy particularly difficult. All together, treating physicians are less motivated to indicate antiviral treatment in patients with CHC and advanced liver disease.

Palmitoleate attenuates palmitate-induced Bim and PUMA up-regulation and hepatocyte lipoapoptosis - Corrected Proof

2010-02-15

Background & Aims: Saturated free fatty acids induce hepatocyte lipoapoptosis. This lipotoxicity involves an endoplasmic reticulum stress response, activation of JNK, and altered expression and function of Bcl-2 proteins. The mono-unsaturated free fatty acid, palmitoleate is an adipose-derived lipokine, which suppresses free fatty acid-mediated lipotoxicity by unclear mechanisms. Herein we examined the mechanisms responsible for cytoprotection.Methods: We employed isolated human and mouse primary hepatocytes, and the Huh-7 and Hep 3B cell lines for these studies. Cells were incubated in presence and absence of palmitate (16:0), stearate (18:0), and or palmitoleate (16:1, n-7).Results: Palmitoleate significantly reduced lipoapoptosis by palmitate or stearate in both primary cells and cell lines. Palmitoleate accentuated palmitate-induced steatosis in Huh-7 cells excluding inhibition of steatosis as a mechanism for reduced apoptosis. Palmitoleate inhibited palmitate induction of the endoplasmic reticulum stress response as demonstrated by reductions in CHOP expression, eIF2-α phosphorylation, XBP-1 splicing, and JNK activation. Palmitate increased expression of the BH3-only proteins PUMA and Bim, which was attenuated by palmitoleate. Consistent with its inhibition of PUMA and Bim induction, palmitoleate prevented activation of the downstream death mediator Bax.Conclusions: These data suggest palmitoleate inhibits lipoapoptosis by blocking endoplasmic reticulum stress-associated increases of the BH3-only proteins Bim and PUMA.

Reperfusion stress induced during intermittent selective clamping accelerates rat liver regeneration through JNK pathway - Corrected Proof

2010-02-15

Background & Aims: Liver resection includes temporal vascular inflow occlusion resulting in ischemia/reperfusion injury in the remnant liver. Here, we developed a rat model of selective lobe occlusion to isolate reperfusion stress from ischemia and to analyze its effect on liver regeneration.Methods: Left lateral and median lobes of liver were either mobilized or subjected twice for 10min to ischemia followed by 5min reperfusion prior to resection while the regenerative lobes were only subjected to reperfusion.Results: Although intermittent reperfusion stress induced higher levels of serum transaminases, analysis of cell cycle regulators revealed accelerated regenerative response compared to standard partial hepatectomy. The G0/G1 transition occurred before tissue resection, as evidenced by c-fos, junB, and IL-6 induction. Following hepatectomy, Cyclin D1 up-regulation, G1/S transition, and cell division occurred earlier than normal. Unexpectedly, liver mobilization, a component of the clamping procedure, also resulted in earlier G1/S transition. The shortened G1-phase was driven by the c-Jun N-terminal Kinase pathway and was associated with an oxidative stress response as evidenced by the expression of inducible nitric oxide synthase.Conclusion: Intermittent selective clamping of lobes to be resected induced reperfusion stress on remnant liver that was beneficial for liver regeneration, suggesting this procedure could be applied in clinical practice.

Hepatitis B surface antigen (HBsAg) levels in the natural history of hepatitis B virus (HBV)-infection: A European perspective - Corrected Proof

2010-02-15

Background & Aims: The quantifiable level of HBsAg has been suggested as a predictor of treatment response in chronic hepatitis B. However, there is limited information on HBsAg levels considering the dynamic natural course of HBV-infection. This study aimed to determine HBsAg levels in the different phases of HBV-infection in European HBsAg-positive patients.Methods: 226 HBV-monoinfected patients, not undergoing antiviral therapy, were analyzed in a cross-sectional study. Patients were categorized according to the phase of HBV-infection: HBeAg(+) immune tolerance phase (IT, n=30), immune clearance phase (IC, n=48), HBeAg(−) low-replicative phase (LR, n=68), HBeAg(−) hepatitis (ENH, n=68), and acute hepatitis B (n=12). HBsAg was quantified and correlated with HBV-DNA, HBV-genotypes and clinical parameters. In addition, 30 LR-patients were followed longitudinally.Results: HBsAg levels were higher in IT-patients and IC-patients compared to LR-patients and ENH-patients (4.96/4.37/3.09/3.87-log10IU/ml, p<0.001). HBsAg showed a strong correlation with HBV-DNA during acute hepatitis B (R=0.79, p<0.01). Correlation of HBsAg and HBV-DNA was weak or missing when analyzing different phases of persistent HBV-infection separately. However, associations between HBsAg and HBV-DNA were observed in patients infected with HBV-genotype D but not with HBV-genotype A. LR-patients with HBV-reactivation during follow-up (increase of HBV-DNA >2000IU/ml) showed >3-fold higher baseline HBsAg levels with a NPV of 95% for an HBsAg cut-off of 3500IU/ml.Conclusions: HBsAg levels show significant differences during the natural course of HBV-infection and between HBV-genotypes. These findings may have important implications for understanding the natural history of HBV-infection and for using quantitative HBsAg as a diagnostic tool, i.e. as a marker for predicting HBV-reactivation.

IGF activation in a molecular subclass of hepatocellular carcinoma and pre-clinical efficacy of IGF-1R blockage - Corrected Proof

2010-02-15

Background & Aims: IGF signaling has a relevant role in a variety of human malignancies. We analyzed the underlying molecular mechanisms of IGF signaling activation in early hepatocellular carcinoma (HCC; BCLC class 0 or A) and assessed novel targeted therapies blocking this pathway.Methods: An integrative molecular dissection of the axis was conducted in a cohort of 104 HCCs analyzing gene and miRNA expression, structural aberrations, and protein activation. The therapeutic potential of a selective IGF-1R inhibitor, the monoclonal antibody A12, was assessed in vitro and in a xenograft model of HCC.Results: Activation of the IGF axis was observed in 21% of early HCCs. Several molecular aberrations were identified, such as overexpression of IGF2 –resulting from reactivation of fetal promoters P3 and P4-, IGFBP3 downregulation and allelic losses of IGF2R (25% of cases). A gene signature defining IGF-1R activation was developed. Overall, activation of IGF signaling in HCC was significantly associated with mTOR signaling (p=0.035) and was clearly enriched in the Proliferation subclass of the molecular classification of HCC (p=0.001). We also found an inverse correlation between IGF activation and miR-100/miR-216 levels (FDR<0.05). In vitro studies showed that A12-induced abrogation of IGF-1R activation and downstream signaling significantly decreased cell viability and proliferation. In vivo, A12 delayed tumor growth and prolonged survival, reducing proliferation rates and inducing apoptosis.Conclusions: Integrative genomic analysis showed enrichment of activation of IGF signaling in the Proliferation subclass of HCC. Effective blockage of IGF signaling with A12 provides the rationale for testing this therapy in clinical trials.

Relevance of the mTOR signaling pathway in the pathophysiology of splenomegaly in rats with chronic portal hypertension - Corrected Proof

2010-02-03

Background & Aims: Splenomegaly is a frequent hallmark of portal hypertension that, in some cases, can be very prominent and cause symptoms like abdominal pain, splenic infarction, and cytopenia. This study characterizes the pathogenetic mechanisms leading to spleen enlargement in portal hypertensive rats and focuses on mTOR pathway as a potential modulator of splenomegaly in portal hypertension.Methods: Characterization of splenomegaly was performed by histological, hematological, immunohistochemical and Western blot analyses in rats with portal hypertension induced by portal vein ligation, and compared with sham-operated animals. The contribution of the mTOR signaling pathway to splenomegaly was determined in rats with fully developed portal hypertension and control rats by treatment with rapamycin or vehicle.Results: Our results illustrate that splenomegaly in portal hypertensive rats arises as a consequence of the interplay of several factors, including not only spleen congestion, as traditionally thought, but also enlargement and hyperactivation of the splenic lymphoid tissue, as well as increased angiogenesis and fibrogenesis. Since mTOR signaling plays a central role in immunological processes, angiogenesis and fibrogenesis, we next determined the involvement of mTOR on splenomegaly. Interestingly, mTOR signaling was overactivated in the spleen of portal hypertensive rats, and mTOR blockade by rapamycin profoundly ameliorated splenomegaly, causing a 44% decrease in spleen size. This effect was most likely accounted for the inhibitory action of rapamycin on lymphocyte proliferation, neovascularization and fibrosis.Conclusions: These findings shed light on the pathogenesis of splenomegaly in portal hypertension, and identify mTOR signaling as a potential target for therapeutic intervention in this disease.

Serum sodium, renal function and survival of patients with end-stage liver disease - Corrected Proof

2010-02-03

Background & Aims: Serum creatinine, a component of the model for end-stage liver disease (MELD), is an important prognostic indicator in patients with end-stage liver disease (ESLD). In addition, serum sodium has recently been recognized as an important predictor of mortality in patients with ESLD. We investigate the role of serum creatinine and sodium and glomerular filtration rate (GFR) as determinants of survival in patients with ESLD.Methods: A prospective database was utilized to identify all adults listed for primary liver transplantation (LTx) at the Mayo Clinic, Rochester, between 1990 and 1999. GFR was measured by iothalamate clearance.Results: Among 837 patients listed for LTx, 660 had complete data including measured GFR. There was a significant association between GFR and survival after adjustment for MELD, with a linear rise in the risk of death as GFR decreased between 60 and 20ml/min/1.73m2. Multivariable models showed that GFR is superior to creatinine in predicting mortality – a model consisting of total bilirubin (hazard ratio (HR)=2.17, p<0.01), INR (HR=3.26, p<0.01) and GFR (HR=0.42, p<0.01) was superior to MELD (chi-square 65.6 vs. 59.4, c-statistic 0.792 vs. 0.780). Serum sodium did not contribute to survival prediction when accurately measured GFR data were available.Conclusions: Serum concentrations of creatinine and sodium in patients with end-stage liver disease reflect a reduction in renal function, the underlying event that decreases survival.

Regulation of the human bile acid UDP-glucuronosyltransferase 1A3 by the farnesoid X receptor and bile acids - Corrected Proof

2010-02-03

Background & Aims: Cholestasis is a serious complication of many liver diseases leading to increased serum bile acids (BA) and their conjugates. Chenodeoxycholic (CDCA) acid is a substrate of the human hepatic UDP-glucuronosyltransferase (UGT) 1A3. UGT1A3 may, therefore, be a BA-inducible gene relevant to BA regulation.Methods: BA and human bile were used to induce UGT1A3 in HepG2 cells. Genomic DNA was analyzed by PCR amplification and sequencing. Transcriptional regulation was studied by DNA mutagenesis, RT-PCR, luciferase reporter gene constructs and electrophoretic mobility shift assays (EMSA).Results: CDCA differentially induced UGT1A3 but not UGT1A4 expression. Bile from ursodeoxycholic acid (UDCA)-treated and untreated patients differentially induced UGT1A3. A farnesoid X receptor (FXR) half-site DNA motif was identified in the UGT1A3 5′ upstream region. The FXR inducer GW4064 activated UGT1A3 transcription, and electrophoretic mobility shift assays identified UGT1A3 as a FXR target gene.Conclusions: Transcriptional regulation of the human bile acid and xenobiotic UGT1A3 by its substrate CDCA and FXR is shown. CDCA glucuronidation can be controlled by feed back inhibition proceeding via the glucuronidation of CDCA. UDCA does not induce UGT1A3 transcription. Since UGT1A3 is significantly induced by xenobiotics this physiologically links xenobiotic and bile acid metabolism to cholestasis.

Characterization of ribavirin uptake systems in human hepatocytes - Corrected Proof

Yukina Fukuchi, Tomomi Furihata, Misato Hashizume, Minami Iikura, Kan Chiba — 2010-02-03

Background & Aims: The purpose of this study was to identify the major ribavirin uptake transporter(s) in human hepatocytes and to determine if these previously unidentified transporters are involved in hepatic ribavirin uptake. Furthermore, we aimed to address what causes the difference in uptake levels among human hepatocytes.Methods: Profiles of ribavirin uptake and nucleoside transporter mRNA expression in Caucasian hepatocytes (HH268, HH283 and HH291) were characterized by transport assay and reverse transcription-polymerase chain reaction (RT-PCR). The 5′-side of the SLC29A1 gene structure was characterized by determination of transcription start sites and by RT-PCR.Results: Equilibrative nucleoside transporter 1 (ENT1)-mediated uptake was exclusively involved in ribavirin uptake in HH268 and HH283 and was responsible for the largest ribavirin uptake fraction in HH291. The level of ENT1-mediated uptake in HH291 was higher than that in HH268 and HH283. Characterization of the SLC29A1 gene structure revealed the existence of several ENT1 mRNA isoforms in the human liver, and the levels of four ENT1 mRNA isoforms in HH291 were higher than those in HH268 or HH283. No ENT2-mediated uptake was observed in any hepatocyte lines. Na+-dependent uptake was detected only in HH291; however, mRNA levels of concentrative nucleoside transporters (CNTs) were at trace levels in all hepatocyte lines.Conclusions: ENT1, but not ENT2 or CNTs, is a major ribavirin uptake transporter in human hepatocytes. The different ENT1-mediated ribavirin uptake levels in different hepatocyte lines are associated with different expression levels of specific isoforms of ENT1 mRNAs. Furthermore, an unidentified Na+-dependent ribavirin transport system might exist in human hepatocytes.

Antiviral effect of entecavir in chronic hepatitis B: Influence of prior exposure to nucleos(t)ide analogues - Corrected Proof

2010-02-03

Background & Aims: Entecavir is a potent inhibitor of viral replication in nucleos(t)ide analogue (NA)-naïve chronic hepatitis B patients, but data on the efficacy in NA-experienced subjects are limited.Methods: In a multi-center cohort study we investigated 161 chronic hepatitis B patients (34% NA-experienced) treated with entecavir monotherapy.Results: During a median follow-up of 11 (3–23)months, 82 (79%) of 104 NA-naïve patients achieved virologic response (VR), defined as HBV DNA <80IU/ml, and none of the patients (0%) developed genotypic entecavir-resistance. VR was demonstrated in 31 (54%) of 57 NA-experienced patients during a median follow-up of 12 (3–31)months. Patients with lamivudine-resistant mutations at the start of entecavir monotherapy had a reduced probability of achieving VR compared to lamivudine-naïve patients (HR 0.14; 95% CI 0.04–0.58; p=0.007). Antiviral efficacy was not decreased by prior treatment with lamivudine when lamivudine-resistance had never developed (HR 0.81; 95% CI 0.43–1.52; p=0.52). Prior adefovir therapy without development of adefovir-resistance (HR 0.84; 95% CI 0.43–1.64; p=0.61) and presence of adefovir-resistance (HR 0.86; 95% CI 0.27–2.71; p=0.80) did not influence antiviral response to entecavir. Switching to a tenofovir-containing treatment regimen resulted in viral load decline in patients with entecavir-resistance associated mutations.Conclusions: Entecavir proved to be efficacious in NA-naïve patients. The antiviral efficacy of entecavir was not influenced by prior treatment with adefovir or presence of adefovir-resistance. Entecavir should not be used in patients with previous lamivudine-resistance, yet it may still be an option in lamivudine-experienced patients in case lamivudine-resistance never developed.

High sustained virologic response rates in children with chronic hepatitis C receiving peginterferon alfa-2b plus ribavirin - Corrected Proof

2010-02-03

Background & Aims: Pegylated interferon (PEG-IFN) alfa-2b plus ribavirin (RBV) is the standard of care for adults with chronic hepatitis C but was not approved for the treatment of children at the time of this study. The aim of this study was to evaluate the efficacy and safety of PEG-IFN alfa-2b plus RBV in children.Methods: Children and adolescents ages 3–17years were treated with PEG-IFN alfa-2b (60μg/m2/week) plus RBV (15mg/kg/day). The duration of therapy was 24weeks for genotype (G) 2 and G3 patients with low viral load (<600,000IU/ml) and 48weeks for G1, G4, and G3 with high viral load (⩾600,000IU/ml). The primary end point was sustained virologic response (SVR), defined as undetectable hepatitis C virus (HCV) RNA 24weeks after completion of therapy.Results: SVR was attained by 70 (65%) children. Genotype was the main predictor of response: G1, 53%; G2/3, 93%; G4, 80%. SVRs were similar in younger and older children. Baseline viral load was the main predictor of response in the G1 cohort. No new safety signals were identified, and adverse events (AEs) were generally mild or moderate in severity. Dose was modified because of AEs in 25% of children; 1 child discontinued because of an AE (thrombocytopenia). No serious AEs related to study drugs were reported.Conclusion: Therapy with PEG-IFN alfa-2b plus RBV in children and adolescents with chronic hepatitis C offers favorable efficacy, reduced injection frequency, and an acceptable safety profile.

Evolution of full-length HBV sequences in chronic hepatitis B patients with sequential lamivudine and adefovir dipivoxil resistance - Corrected Proof

2010-02-02

Background and Aims: The aim of this study was to determine the evolution of full-length hepatitis B virus (HBV) sequences in chronic hepatitis B (CHB) patients with sequential lamivudine (LAM) and adefovir (ADV) resistance.Methods: The full-length genomes of HBV were sequenced from 11 CHB patients before LAM treatment and at the emergence of LAM- and ADV-resistant HBV.Results: Besides the known LAM-resistant polymerase gene mutations, 10 of 11 patients who had LAM-resistant HBV variants had additional amino acid changes in the reverse transcriptase (RT) domain, and ADV therapy reversed these additional changes to pre-LAM therapy status. Furthermore, new amino acid changes in the RT domain, distinct from the known ADV-resistant HBV variants, were selected at the emergence of ADV resistance in six of 11 patients. Seven patients had amino acid changes within the known T-cell or B-cell epitopes of HBV surface and core antigens at the emergence of LAM and/or ADV resistance. The frequency of pre-S deletions between nucleotide 3037–56 was higher at the emergence of ADV resistance compared with that at the emergence of LAM resistance (7/11 vs. 1/11; p=0.024). Combined LAM–ADV resistance was detected in one of 11 patients. This patient had resistant mutations to both drugs on the same viral genome by molecular cloning (5/24 polymerase gene clones).Conclusions: In addition to the known LAM- and ADV-resistant mutations accompanying the emergence of LAM and ADV resistance, the changes of nucleotide or amino acid sequences occurred commonly in the HBV surface antigen or RT domain and were scattered along the full-length HBV genomes.

Non-invasive assessment of hepatic steatosis: Prospective comparison of the accuracy of imaging examinations - Corrected Proof

2010-02-02

Background & Aims: Despite increasing use of various imaging examinations for non-invasive assessment of hepatic steatosis (HS), their relative accuracy is unknown. The objective of this study is to prospectively compare the accuracy of computed tomography (CT), dual gradient echo magnetic resonance imaging (DGE-MRI), proton magnetic resonance spectroscopy (1H-MRS), and ultrasonography (US) for the diagnosis and quantitative estimation of HS.Methods: A total of 161 consecutive potential living liver donors underwent US (performed by two independent radiologists, US1 and US2), CT, DGE-MRI, 1H-MRS, and liver biopsy on the same day. Using the histologic degree of HS as the reference standard, we compared the diagnostic performance of US1, US2, CT, DGE-MRI, and 1H-MRS for diagnosing HS⩾5% and HS⩾30% and compared the accuracy of CT, DGE-MRI, and 1H-MRS in the quantitative estimation of HS.Results: DGE-MRI and 1H-MRS significantly outperformed CT and US for the diagnosis of HS⩾5%. DGE-MRI showed a tendency of higher accuracy than the other examinations for diagnosing HS⩾30%. The cross-validated sensitivity and specificity of DGE-MRI at the optimal cut-off were 76.7% and 87.1%, respectively, for diagnosing HS⩾5% and 90.9% and 94%, respectively, for diagnosing HS⩾30%. The cross-validated Bland–Altman 95% limits of agreement between the estimated degree of HS on imaging examinations and the histologic degree of HS, were the narrowest with DGE-MRI, yielding −12.7% to 12.7%.Conclusions: Among CT, DGE-MRI, 1H-MRS, and US, DGE-MRI is the most accurate method for the diagnosis and quantitative estimation of HS. Therefore, DGE-MRI may be the preferred imaging examination for the non-invasive assessment of HS.

A new role for an old marker, HBsAg - Corrected Proof

Maurizia Rossana Brunetto — 2010-02-01

Detection in the serum of the “Australia antigen”, namely hepatitis B surface antigen (HBsAg), was the Nobel prize discovery that identified hepatitis B virus (HBV) about 40years ago; to this day HBsAg remains the hallmark of overt HBV infection . HBsAg circulates in a wide array of particulate forms: competent virions (42nm, Dane particles), 20nm diameter filaments of variable length and 20–22nm spherical defective particles, corresponding to empty viral envelopes . Serum HBsAg results from the different combinations of three proteins (small, medium and large), either glycosylated or not, that are specified by a single open reading frame providing 3 carboxy-terminal colinear HBsAg proteins of different length. The small (S) protein (226 amino acids) is expressed at the highest levels, predominates in both virions and subviral particles and is secreted without cleavage of amino acid residues during translocation because of its self-assembling capacity with host-derived lipids in the cell ER . The middle (M) protein (containing 55 extra residues of the pre-S2 domain) is regulated by the same promoter and is similarly secreted, whereas the transcription of the large protein (L) is regulated by a specific but weaker promoter (pre-S1) . Hepatitis B virus large surface protein (L-HBs) containing both the pre-S2 region and the 108–119 additional residues of the pre-S1 domain, is an essential component of both virions and filaments, and represents 10–20% of their envelope proteins. In contrast, the L-HBs represents only 2% of the 22nm spherical particles . The complexity of HBsAg production and secretion is known since the early studies that showed a larger excess of both filaments and spherical subviral particles was present in highly viremic HBeAg positive carriers as compared to low viremic anti-HBe positive carriers, in whom the decline of filaments paralleled that of virions whereas spherical particles remained in moderate excess . Thus, subviral HBsAg particles exceed virions by a variable factor of 102–105 and can accumulate up to concentrations of several hundred micrograms per milliliter of serum .

Focus - Corrected Proof

Daniel Shouva — 2010-02-01

For more than a decade, a large number of patients with chronic hepatitis B (CHB) have been treated with lamivudine or adefovir worldwide. While these anti-viral agents have contributed significantly towards the reduction of CHB, resistance to lamivudine and to a lesser degree to adefovir has emerged as a major barrier in the control of viral replication. The recently published 2009 EASL Clinical Practice Guideline: “Management of Chronic Hepatitis B” recommends treating naïve CHB patients that are not candidates for interferon therapy with “the most potent drugs with the optimal resistance profile, i.e. tenofovir or entecavir as first-line monotherapies”. These nucleos(t)ide analogues (NA) are also used in treating patients who developed resistance to lamivudine or adefovir usually in an “add on” strategy. Changing treatment from one monotherapy with an anti-viral agent to another may lead to the emergence of new resistance patterns as shown in patients with lamivudine resistant mutations who were switched to entecavir. In this issue of the Journal, Reijnders and co-workers report the results of a European multi-center study in which two groups of patients were treated with 0.5mg entecavir/day with a median follow-up of 11months (3–23). While 79% of 104 NA naïve patients achieved virologic response, control of HBV replication was obtained in only 54% of 57 NA experienced patients treated either with lamivudine or adefovir and switched to entecavir monotherapy. As expected and based on previous experience, patients with lamivudine resistant mutations at the start of entecavir therapy were prone to develop resistance to entecavir and had a reduced probability of achieving viral control as compared to lamivudine naïve patients. Yet, efficacy of entecavir in lamivudine experienced patients who did not develop these mutations remained unchanged. Baseline HBeAg negativity, high ALT, low serum HBV-DNA and absence of lamivudine resistant mutations were predictors for viral response to entecavir. In contrast, prior treatment with adefovir had no negative impact on anti-viral response after switching to entecavir monotherapy, irrespective of presence or absence of resistance to adefovir. These results confirm previous reports and add new information on the reduced potency and risk of administering entecavir to lamivudine experienced CHB patients with YMDD mutants. Yet it is important to note that the potency of entecavir monotherapy remained intact in adefovir experienced patients as well as in patients treated previously with lamivudine who had no history of lamivudine resistant mutations. This observation is reassuring for many of the CHB patients in the community who originally were treated with lamivudine without developing YMDD mutations and who were switched by their physicians to entecavir.

Renal function and MELD: Being direct is better - Uncorrected Proof

Stevan A. Gonzalez, James F. Trotter — 2010-02-01

Renal insufficiency is common in patients with end-stage liver disease (ESLD) and is strongly linked to survival in those awaiting liver transplantation. In liver patients, renal dysfunction is largely due to a circulatory disturbance characterized by a low systemic vascular resistance and decreased effective arterial volume leading to impaired renal excretion of solute-free water, dilutional hyponatremia and ultimately hepatorenal syndrome (HRS) . Once a patient develops type I HRS, the median survival without treatment is as low as 2weeks, while the median survival in type II HRS is approximately six months; both a significant decrease in survival compared with patients without HRS . The diagnosis and severity of HRS is dependent on two biochemical markers, serum sodium and creatinine, both of which are independently associated with an increased risk of mortality in cirrhosis . Their importance is underscored by inclusion in the most widely utilized predictive survival function, the model for end-stage liver disease (MELD) score or Na-MELD, where the addition of serum sodium significantly improves the performance of MELD. Whether sodium will be included into the MELD score (for prioritizing liver transplantation candidates) remains in question for two reasons. Serum sodium levels are easily manipulated with water ingestion and the administration of vaptans. In addition, hyponatremia may have a negative impact on post-transplant complications and survival, although recent data has brought this into question .

Serological Surveillance for hepatocellular carcinoma: Time to quit - Corrected Proof

M. Sherman — 2010-01-14

COMMENTARY ON: Des-gamma-carboxy Prothrombin and Alpha fetoprotein as Biomarkers for the Early Detection of Hepatocellular Carcinoma. Lok AS, Sterling RK, Everhart JE, Wright EC, Hoefs JC, Di Bisceglie AM, Morgan TR, Kim HY, Lee WM, Bonkovsky HL, Dienstag JL; HALT-C Trial Group. Reprinted from Gastroenterology. 2009 Oct 20 [Epub ahead of print], with permission from Elsevier. Abstract: Background & Aims. The outcome of patients with hepatocellular carcinoma (HCC) remains poor because of late diagnosis. The aim of this study was to compare the accuracy of alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) in the early diagnosis of HCC. Methods: Among 1031 patients randomized in the Hepatitis C Antiviral Long-term Treatment Against Cirrhosis (HALT-C) Trial, a nested case-control study of 39 HCC cases (24 early stage) and 77 matched controls was conducted to compare the performance of AFP and DCP. Testing was performed on sera from 12months prior (month-12) to the time of HCC diagnosis (month 0). Results: The sensitivity and specificity of DCP at month 0 was 74% and 86%, respectively, at a cut-off of 40mAU/ml and 43% and 100%, respectively, at a cutoff of 150mAU/ml. The sensitivity and specificity of AFP at month 0 was 61% and 81% at a cutoff of 20ng/ml and 22% and 100% at a cutoff of 200ng/ml. At month-12, the sensitivity and specificity at the low cutoff was 43% and 94%, respectively, for DCP and 47% and 75%, respectively, for AFP. Combining both markers increased the sensitivity to 91% at month 0 and 73% at month 12, but the specificity decreased to 74% and 71%, respectively. Diagnosis of early HCC was triggered by surveillance ultrasound in 14, doubling of AFP in 5, and combination of tests in 5 patients. Conclusions: Biomarkers are needed to complement ultrasound in the detection of early HCC, but neither DCP nor AFP is optimal.

Mechanisms of HBV-related hepatocarcinogenesis - Corrected Proof

Christine Neuveut, Yu Wei, Marie Annick Buendia — 2010-01-07

The hepatitis B virus (HBV) is a small enveloped DNA virus, which primarily infects hepatocytes and causes acute and persistent liver disease. Epidemiological studies have provided overwhelming evidence for a causal role of chronic HBV infection in the development of hepatocellular carcinoma, but the molecular mechanisms underlying virally-induced tumourigenesis remain largely debated. In the absence of a dominant oncogene encoded by the HBV genome, indirect roles have been proposed, including insertional activation of cellular cancer-related genes by HBV DNA integration, induction of genetic instability by viral integration or by the regulatory protein HBx, and long-term effects of viral proteins in enhancing immune-mediated liver disease. Recent genetic studies indicate that HBV-related tumours display a distinctive profile with a high rate of chromosomal alterations and low frequency of β-catenin mutations. This review will discuss the evidence implicating chronic HBV infection as a causal risk factor of primary liver cancer. It will also discuss the molecular mechanisms that are critical for the tumourigenic process due to long lasting infection with HBV.

The molecular basis of the failed immune response in chronic HBV: Therapeutic implications - Corrected Proof

Mala K. Maini, Anna Schurich — 2010-01-07

There is a pressing need to develop new immunotherapeutic interventions in chronic hepatitis B virus (HBV) infection in order to limit the high costs and risks of toxicity or the viral resistance associated with the maintenance of antiviral treatment. Here we review recent advances in our understanding of the molecular defects underlying the profound T cell depletion characteristic of these patients. We propose that T cells are driven to apoptosis by the combination of a persistent, high antigen load and excessive inhibitory signals encountered in the hepatic microenvironment. The feasibility of boosting sustained antiviral control by targeted reversal of key tolerising mechanisms is discussed.

The evaluation of renal function and disease in patients with cirrhosis - Corrected Proof

Claire Francoz, Denis Glotz, Richard Moreau, François Durand — 2010-01-07

The MELD score has shown that, besides markers of liver function, serum creatinine has a strong prognostic value in cirrhosis. However, even though creatinine has a good prognostic value, it is an inaccurate marker of renal function in cirrhosis. Creatinine and creatinine-based equations tend to overestimate glomerular filtration rate (GFR), and creatinine clearance from timed urine collection also overestimates GFR. Hence, clearance of exogenous markers such as iohexol remains the only reliable method for assessing precisely GFR in cirrhosis. Whereas these investigations are limited by their costs and complexity, and they can hardly be repeated at short intervals, serum cystatin C could be an alternative, although it needs further validation. Accurate markers and/or specific equations are therefore still needed to assess GFR in cirrhotic patients. Pre-renal failure and hepatorenal syndrome (HRS) are the main causes of acute renal failure in cirrhosis. Both result from decreased renal blood flow and both can result in acute tubular necrosis. HRS is not always fully reversible with liver transplantation possibly due to underlying chronic kidney damage. A number of cirrhotic patients with acute renal failure may also have chronic kidney damage (“acute-on-chronic renal failure”); furthermore, cirrhotic patients frequently have co-morbidities such as diabetes that may result in chronic impairment in renal function. Since conventional urinary markers are biased in cirrhosis, a biopsy is the only way to document and quantify renal lesions; moreover, transvenous route should be preferred to percutaneous route. In candidates for transplantation, attention should therefore be focused on vascular lesions which may represent a risk factor for nephrotoxicities induced by calcineurin-inhibitors.

Treatment of chronic hepatitis C in children: Is it necessary and, if so, in whom? - Corrected Proof

Anna Alisi, Donatella Comparcola, Valerio Nobili — 2010-01-07

Hepatitis C virus (HCV) infection continues to be an important global health problem. It is estimated that approximately 170 million people worldwide are infected with HCV , and although children are only a small portion of those infected and the incidence of new infections has decreased in recent years, HCV still contributes to chronic liver disease in childhood .