G. Svegliati Baroni

Practice guidelines for the diagnosis and management of nonalcoholic fatty liver disease. A decalogue from the Italian Association for the Study of the Liver (AISF) Expert Committee.

We report the evidence-based Italian Association for the Study of Liver guidelines for the appropriate diagnosis and management of patients with nonalcoholic fatty liver disease in clinical practice and its related research agenda. The prevalence of nonalcoholic fatty liver disease varies according to age, gender and ethnicity. In the general population, the prevalence of nonalcoholic fatty liver disease is about 25% and the incidence is of two new cases/100 people/year. 2-3% of individuals in the general population will suffer from nonalcoholic steatohepatitis. Uncomplicated steatosis will usually follow a benign course. Individuals with nonalcoholic steatohepatitis, however, have a reduced life expectancy, mainly owing to vascular diseases and liver-related causes. Moreover, steatosis has deleterious effects on the natural history of HCV infection. Nonalcoholic fatty liver disease is usually diagnosed in asymptomatic patients prompted by the occasional discovery of increased liver enzymes and/or of ultrasonographic steatosis. Medical history, complete physical examination, etiologic screening of liver injury, liver biochemistry tests, serum lipids and insulin sensitivity tests should be performed in every patient. Occult alcohol abuse should be ruled out. Ultrasonography is the first-line imaging technique. Liver biopsy, the gold standard in diagnosis and prognosis of nonalcoholic fatty liver disease, is an invasive procedure and its results will not influence treatment in most cases but will provide prognostic information. Assessment of fibrosis by composite scores, specific laboratory parameters and transient elastography might reduce the number of nonalcoholic fatty liver disease patients requiring liver biopsy. Dieting and physical training reinforced by behavioural therapy are associated with improved nonalcoholic fatty liver disease. Diabetes and the metabolic syndrome should be ruled out at timed intervals in nonalcoholic fatty liver disease. Nonalcoholic steatohepatitis patients should undergo periodic evaluation of cardiovascular risk and of advancement of their liver disease; those with nonalcoholic steatohepatitis-cirrhosis should be evaluated for early diagnosis of hepatocellular carcinoma.

Cell proliferation following extrahepatic biliary obstruction: Evaluation by immunohistochemical methods

The aim of the present investigation was to conduct an immunohistochemical study using bromodeoxyuridine (BrdU) incorporation as a marker of S-phase cells and cytokeratins as markers of biliary epithelial cells, in bile-duct-ligated rats at intervals of 1, 3, 7, 14 and 21 days after total biliary obstruction. Data obtained using only BrdU incorporation by S-phase nuclei were compared with those obtained by the simultaneous demonstration of S-phase nuclei and cytoplasmic cytokeratins. The labelling index of parenchymal liver cells and of biliary epithelial cells was evaluated as an index of the cellular growth pattern after total biliary obstruction. Our data show that following total biliary obstruction: (a) cell proliferation follows a similar pattern for biliary epithelial cells hepatocytes with a peak on the 3rd day; (b) the labelling index is significantly higher in biliary epithelial cells than in hepatocytes; and (c) sequential immunohistochemical staining using cytokeratin as a marker allows better identification of biliary epithelial cells, especially when the ductular lumen is not clearly outlined, or in isolated biliary cells which appear as components of the wall of the ducts of Hering.

Proliferation of Hepatic Stellate Cells and Lipid Peroxidation: Changes Due to Polyphenols.

Hepatic fibrosis represents the final steps of chronic liver injury and consists of excessive deposition of extracellular matrix components. Hepatic stellate cells (HSC) are responsible for this event when, through a process of activation, they proliferate and acquire a myofibroblatic phenotype. In this article, we review the role of oxidative stress in liver fibrosis. Moreover we also demonstrate that lipid peroxidation products, directly or released by damaged hepatocytes, stimulate HSC proliferation and collagen synthesis. This effect can be inhibited by resveratrol, a novel polyphenolic antioxidant.

P353 ACTIVATION OF THE DEVELOPMENTAL PATHWAY Ngn-3/miR-7a REGULATES CHOLANGIOCYTES PROLIFERATION IN RESPONSE TO INJURY

Background and Aims: The activation of the biliary stemcell signalling pathway Hes-1/PDX-1 in mature cholangiocytes determines cell proliferation. Neurogenin-3 (Ngn-3) is required for pancreas development and for ductal cell neogenesis. PDX-1dependent activation of Ngn-3 initiates the differentiation program, by inducting microRNA (miR)-7 expression. We aimed to verify whether Ngn-3 regulates cholangiocyte proliferation. Methods: Expression levels of Ngn-3 and miR-7 isoforms were tested in cholangiocytes from normal and cholestatic livers. Ngn-3 was knocked down in vitro by siRNA. In vivo, wild type (WT) and Ngn-3-heterozygous (+/−) mice were subjected to Bile Duct Ligation (BDL) for 2 weeks. Results: In the liver, Ngn-3 is expressed in cholangiocytes of mice subjected to BDL and of patients affected by PSC, but not in normal conditions. Expression of miR-7a-1 and miR-7a-2 isoforms, but not miR-7b, was increased in BDL cholangiocytes as compared to normal ones. In vitro, Ngn-3 siRNA neutralized the increases in cell proliferation and in the expression of IGF-1 (a pro-proliferative effector) and miR-7a, but not of PDX-1 or VEGF, observed after exposure to FBS or exendin-4. Anti-sense miR-7 neutralized the FBS or exendin-4 induced increases in cell proliferation but not in PDX-1 and Ngn-3 synthesis. In vivo, increases in bile duct mass and collagen deposition induced by BDL were significantly reduced in Ngn-3 mice. Conclusions: Ngn-3-dependent activation of miR-7a is a determinant of cholangiocyte proliferation. These findings indicate that the re-acquisition of a molecular profile typical of organ development is essential for the biological response to injury by mature cholangiocytes.

T-15 Interim analysis of the Italian Master-Entas cohort study: entecavir can improve liver function in patients with chronic hepatitis B and liver cirrhosis

ance and side effects. Median age was 55 years (24–83) and median time to the first visit was 3.5 months. Forty-four per cent had liver cirrhosis. Seventeen patients were NUC experienced (8 LAM, 5 ADV+LAM, 1 LdT+LAM, 2 ADV, 1 TNF), 1/17 NUC experienced patients started a combination treatment with TDF-ETV. Cumulative rates of undetectable hepatitis B virus DNA were 62.8%, 83.1%, 89% and 89% and normalization of ALT was 59.2%, 78.7%, 87.1% and 81.2% at 6, 12, 18 and 24 months of follow-up. Thirty-nine patients were HBeAg positive and 17 achieved negative HBeAg with anti-HBe seroconversion in 15 cases (38.4%). HBsAg loss was observed in 5 patients. Two patients switched to TDF, in one case because of virologic failure and in the second one because of partial virologic response. Drug resistance mutations were evaluated in 10 patients at the first visit showing in a LAM-ADV experienced, patient mutation A181T and in the second LAM experienced patient, mutation A181V. During follow-up, drug resistance mutations were assessed in 15 patients, identifying A181V-T184S in a subject (LAM-ADV experienced) and L180M-M250V-M204V in the patient presenting virological failure (LAM experienced). Treatment compliance was in 96.3% of the patients, in 9/11 not compliant subjects undetectable HBV-DNA was achieved. The study of Entecavir treatment in field practice confirms its efficacy in suppressing HBV replication with rare drug resistance mutations and its safety in patients with chronic hepatitis and liver cirrhosis.

36 PDX-1/HES-1 INTERACTIONS DETERMINE CHOLANGIOCYTE PROLIFERATIVE RESPONSE TO INJURY: THE SIGNIFICANCE FOR SCLEROSING CHOLANGITIS

in PLINK v1.07. For selected SNPs, previously published summary statistics (Melum et al., Nat. Gen. 2011) were used to perform a meta-analysis. Results: Significant association (P < 8.5×10−4) corrected for multiple testing (Bonferroni method) was observed for three SNPs at 10p15 and one SNP at 4q27 (Table 1). In addition, nominal significance (P < 0.05) was seen for 10/28 SNPs at 10p15 and 9/27 SNPs at 4q27. Genome-wide significance (P < 5×10−8) was observed for rs4147359 (10p15) in the combined analysis.

Differential expression of ras isoforms in hepatic stellate cells

DIFFERENTIAL EXPRESSION OF RAS ISOFORMS IN HEPATIC STELLATE CELLS E Ridolfi, A. Benedetti, A. Di Sario, S. Saccomanno, L. Marucci, E. Bendia, A.M. Jezequel, G. Sve~liati Baroni Dept. of Gastroenterology and Inst. of Exp. Pathology, University of Ancona, Ancona, Italy. Ras proteins represent the products of three different Ras genes (H-, K-, and N-ms) and are plasma membrane-associated GTPases that function as relay switches transducing biological information from extracellular signals to the nucleus. One or the other isoform may predominate in a different cell type where they play a key role in transforming activity and regulating cell proliferation. Hepatic stellate cell (HSC) proliferation close to area of necrosis represent a key event in liver fibrosis and PDGF represents the main mitogen. Aims of the present study ware thus:l)to evaluate the expression of Ras isoforms in activated HSC;2)to determine the effect of blocking Ras signaling on HSC proliferation;3)to determine the intracellular pathways which transduce extracellular signal to the nucleus via Ras proteins. By immunoprecipitation, the H-Ras isoform only was detected in total HSC lysate. This was confirmed by immunohistochemisffy by which the H-Ras isofonn was detected preferentially in the cytoplasmic area. PDGF-stimulated HSC showed a 4-fold increase in cell proliferation as determined by BrdU incorporation in S-phase nuclei. This was blocked by chaetomellic acid (a H-Ras processing inhibitor) which reduced cell proliferation down to control value starting at 2 pM. In addition, chaetomellic acid reduced PDGF-induced ERKll2 phosphorylation while had no effect on 70 kD

P0340 : Clinical patterns of hepatocellular carcinoma (HCC) in non alcoholic fatty liver disease (NAFLD): A multicenter case-control study

6 kinase (a downstream component of the PI3Kinase pathway) activation as determined by Western blot. This study thus indicates that PDGF-induced HSC proliferation is driven by H-Res. Since a non toxic inhibition of Ras processing has been obtained in vivo, H-Ras represents a plausible target for the experimental therapy of hepatic fibrosis. [ P/C03/38 ]