606 A genomic and proteomic study of obesity-related non-alcoholic fatty liver disease (NAFLD)

Category 9: Alcoholic Liver Disease, NAFLD and Drug-Induced Liver Disease the three groups for each of the four antigens was as follows; AcA: 1 = 0.25±0.17; 2 = 0.32±0.20; 3 = 0.35±0.24 (p=0.49, ANOVA); MDA: 1 = 0.71±0.17; 2 = 0.71±0.28; 3 = 0.81±0.37 (p=0.68); HER: 1 = 0.14 ±0.06; 2 = 0.15±0.09; 3 = 0.15±0.06 (p=0.90); CYP2E1: 1 = 0.66 ±0.28; 2 = 0.68±0.40; 3 = 0.94±0.42; score 1 v 3 p=0.03, score 1&2 v 3 p=0.03. Conclusions: Lymphocyte infiltration is a variable feature of liver biopsies in ALD. In contrast to the allo-antigens studied, titres of autoantibodies against CYP2E1 correlate with the degree of lymphocyte infiltration. These data suggest that autoimmune responses play a significant role in the pathogenesis of ALD.

604 PARENTERAL IRON ENHENCES LIVER INJURY IN METHIONINE CHOLINE DEFICIENT DIET INDUCED NON-ALCOHOLIC STEATOHEPATITIS IN RATS

V. Tahan 1 , A. Sonsuz 2 , N. Imeryuz 1 , S. Uraz 1 , S. Akpulat 3 , M. Yuksel 4 , D. Ozcelik 5 , E. Avsar 1 , C. Ataizi Celikel 3 , G. Haklar 4 , N. Tozun 1 . 1 Marmara University Medical Faculty, Department of Gastroenterology, Marmara, Turkey; 2 Istanbul University, Cerrahpasa Medical Faculty, Department of Hepatology, Istanbul, Turkey; 3 Marmara University Medical Faculty, Department of Pathology, Marmara, Turkey; 4 Marmara University Medical Faculty, Department of Biochemistry, Marmara, Turkey; 5 Istanbul University, Cerrahpasa Medical Faculty, Department of Biophysics, Istanbul, Turkey Objective: Inflammation and fibrosis superimposing on steatosis characterize non-alcoholic steatohepatitis (NASH). High serum and liver iron concentrations were found in some NASH patients, suggesting a role for iron as a co-factor aggravating injury and histopathological features. Aim: To investigate the effects of parenteral iron in methionine choline deficient diet (MCDD) induced NASH model. Methods: Wistar rats were divided into 4 groups; Group 1 (n=7): Controldiet (MCDD + methionine and-choline) + parenteral saline, Group 2 (n=7): Control-diet+parenteral iron, Group 3 (n=10): MCDD + parenteral saline, Group 4 (n=10): MCDD + parenteral iron. Iron-dextran 100-mg/kg was administered intra-muscularly in groups 2 and 4, while groups 1 and 3 were given im saline. Groups 1 and 2 were given Control-diet, while groups 3 and 4 were given MCDD, for 4 weeks. After-decapitation, blood and tissue samples were obtained for histopathology (steatosis, inflammation, and fibrosis), serum-ALT, alkaline phosphatase (ALP), total bilirubin (TB), liver-tissue ROS (Luminol and Lucigenin). Data were given as median (range), analyzed by Kruskal Wallis (KW), ANOVA (Dunns test) and Chi Square tests. Results: All findings are shown in Table 1 below. Conclusions: Iron injection alone did not affect the liver pathology of the rats fed with control diet (groups 1-2). While MCDD administration resulted in pathologic changes typical for NASH (groups 3-4). Parenteral iron superimposing on steatohepatitis induced by MCDD aggravated steatosis and fibrosis histopathologically and biochemically (e.g. higher ALT levels) whereas iron injection did not change inflammatory activity significantly. Our results are the first experimental finding supporting the role of iron as a second hit in NASH pathogenesis.

177

605 DECREASED PORTAL BLOOD FLOW MEASURED BY DOPPLER IS ASSOCIATED WITH SEVERITY OF LIVER FIBROSIS IN ALCOHOLICS

G. Vanbiervliet 1 , F. Le Breton 2 , E. Marine-Barjoan 1 , C. Tran 3 , E. Gelsi 1 , R. Anty 1 , R. Salame 1 , M.C. Saint-Paul 2 , P.M. Huet 1 , J.N. Bruneton 3 , A. Tran 1 . 1 Service D’hepatologie, Hopital L’Archet 2, Nice, France; 2 Service D’anatomo-Pathologie, Hopital Pasteur, Nice, France; 3 Service De Radiologie, Hopital L’Archet 2, Nice, France Objectives: The aim of this study was to determine the diagnosis accuracy of ultrasound-Doppler for liver fibrosis in alcoholics. Methods: 102 alcoholics (68 men, 34 woman, mean age 49.5 ± 8.5) were included. All patients consumed over 80 g of ethanol per day for more than five years and were negative for HBsAg and anti-HCV. A liver biopsy and an abdominal ultrasound-Doppler were performed in all patients. The severity of liver fibrosis was assessed semiquantatively using a score derived from METAVIR. Two groups were determined: mild (F0-F1) and significant fibrosis (F2-F4). Hepatic and splenic length, portal vein diameter, ascite, nodular liver surface, respiratory variation of the splenic vein, patent umbilical vein, portal flow, hepatic arterial pulsatility and resistance, pulsatility index of superior mesenteric artery were measured in all patients (JNB). Results: 55 patients had low fibrosis and 46 significant fibrosis. Lower hepatic arterial pulsatility, higher hepatic and splenic length, presence of ascites, patent umbilical vein and nodular liver surface were associated with significant fibrosis (p<0.05). There was a significant inverse correlation between portal flow and severity of fibrosis (p<0.01). In multivariate analysis, portal flow (OR 2.8 IC95% 1.1-9.9) and nodular liver surface (OR 17.8 IC95% 2.1-154) were associated with significant fibrosis (p<0.001). For a threshold value of portal flow of 400ml/mn, the diagnosis accuracy of significant fibrosis was 71%. Conclusions: Portal flow measured by Doppler allows to correctly classify significant fibrosis in 7 out of 10 alcoholics.

606 A GENOMIC AND PROTEOMIC STUDY OF OBESITY-RELATED NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD)

K. Schlauch 2 , L. O’Reilly 2 , T. Born 2 , K. Ziegler 2 , J. Ong 1 , H. Elariny 1 , F. Gorreta 2 , L. Del Giacco 2 , A. Younoszai 1 , G. Grant 2 , V. Chandhoke 2 , Z. Goodman 3 , Z. Younossi 1 . 1 Center For Liver Diseases, Inova Fairfax Hospital, Falls Church VA, USA; 2 Center For The Study of Genomics In Liver Disease, George Mason University, Manassas VA, USA; 3 Armed Forces Institute of Pathology, Washington DC, USA Gene and protein expression patterns may explain the differential progression of the NAFLD subtypes. Aim: To assess genomic-proteomic differences of NAFLD using microarray and Surface Enhanced Laser Desorption/Ionization – Time of Flight Mass Spectrometry (SELDI-TOF MS) techniques. Methods: RNA was extracted from snap-frozen liver specimens of biopsyproven NAFLD and cDNA probes were manufactured, hybridized with customized microarray cDNA chips and scanned using GSI Lumonics ScanArray-5000. Each patient’s serum was incubated on a hydrophobic

Abstract 604 – Table 1 ALT 1) Control diet+saline (n=7) 2) Comtrol diet+iron (n=7) 3) MCDD+saline (n=10) 4) MCDD+iron (n=10) pKW *Chi square.

Biochemical parameters ALP TB

55 47-94 59 48-118 108.5 80-135 175.5 115-203 0.0001

195 155-343 303 196-728 897.5 537-1960 1059.5 430-1870 0.0001

0.11 0.6-0.2 0.11 0.3-0.39 0.34 0.25-0.67 0.35 0.22-0.5 0.0002

ROM Luminol

Lucigenin

Steatosis

28.6 16.6-35.1 52.7 23.2-78.4 16.65 11.3-147.2 99.9 41.6-152.3 0.0012

20.7 11.8-37.7 32.8 23.9-50.5 42.1 27-86.5 43.35 28-101.9 0.0075

0 0-0 0 0-0 0.6 0.37-0.84 0.9 0.48-0.93 0.001

Histology Inflammation Fibrosis 0 0-0 0 0-0 21.05 0.5-52 19 3.4-81.4 0.0001

NASH grade

0

0

0

0

0

2 1-2

3 0.048

2.5 1-4 0.03*

178

Poster Sessions

chip, washing off the unbound proteins and examined by SELDI-TOF. Comparisons were made between Non-alcoholic Steatohepatitis (NASH), Steatosis alone, Steatosis with non-specific inflammation and Controls using Mann-Whitney, Kruskal-Wallis and t-tests with Bonferroni corrections. Results: 84 non-diabetic patients (23 NASH, 10 Steatosis alone, 44 Steatosis with non-specific inflammation and 7 obese controls) with extensive clinical, pathologic and biochemical data were included. Analyses showed that insulin-like growth factor binding protein 1, glutathione S-transferase M4, B melanoma antigen, BTG family member 3 and ornithine aminotransferase were significantly differentially expressed between NAFLD subtypes (p-value<0.01). In comparing protein expression profiles between NASH, Steatosis alone, and Controls, two protein peaks (approximate molecular mass 8960 and 43430) were significantly differentially expressed (p-value<0.0003). Further identification of these proteins using ESI-LC/MS/MS is underway. Conclusions: Analysis of gene and protein expressions of the NAFLD spectrum reveals potential gene and protein expression profiles associated with the progressive form of NAFLD or NASH. These data may provide insight into the pathogenesis of NASH, development of new diagnostic tools and the identification of potential therapeutic targets.

Category 10: Miscellaneous

607 LONG-TERM IMMUNOGENICITY AND EFFICACY ASSESSMENT OF ANTI-HEPATITIS B VIRUS (HBV) VACCINATION IN ITALIAN CHILDREN

A. Mariano 1 , A. Mele 1 , T. Stroffolini 1 , L. Romano 2 , M.E. Tosti 1 , F. Marzolini 1 , R. Coppola 3 , M. Cuccia 4 , R. Mangione 5 , F. Negrone 6 , A. Parlato 7 , M. Quarto 8 , P. Ragni 9 , E. Zamparo 6 , C. Zotti 10 , A. Zanetti 2 . 1 Istituto Superiore Sanita, Rome, Italy; 2 Istituto Virologia, Universita Di Milano, Milano, Italy; 3 Istituto Igiene, Universita Di Cagliari, Cagliari, Italy; 4 Servizio Epidemiologia, ASL 3, Catania, Italy; 5 Servizio Igiene Pubblica, ASL 1, Licata, Italy; 6 Servizio Igiene Pubblica, ASL 2 Potenza, ASL 6, Pordenone, Italy; 7 Azienda Sanitaria Napoli 2, Quarto, Italy; 8 Istituto Igiene, Policlinico Di Bari, Bari, Italy; 9 Assessorato Sanita, Regione EmiliaRomagna, Bologna, Italy; 10 Isituto Igiene, Universita Di Torino, Torino, Italy Background: Evidence suggests that, thanks to persistence of immunological memory, anti-HBV vaccination can provide a long-lasting protection despite anti-HBs decreasing or loss. Starting from 1991, in Italy all infants receive a 3-dose schedule anti-HBV vaccination. Objectives of this study were to assess anti-HBV vaccine long-term efficacy and immunogenicity in Italian children and to investigate the emergence of vaccine-induced escape mutants. Methods: During 2003 sera of children born in 1992, vaccinated in infancy were collected. Anti-HBs and anti-HBc were determined; anti-HBcpositive samples were further tested for HBsAg and HBV-DNA. Children with anti-HBs <10mIU/ml were offered a booster-dose of vaccine. A subgroup of them will be examined for B-cell memory by spot-ELISA. Children who developed anti-HBs levels >100mIU/ml at check performed 15 days after the booster injection were considered as having an anamnestic response while those showing anti-HBs levels <100mIU/ml were offered to complete a new full-course of vaccination. Results: Eleven years after vaccination, 1 of 1260 children examined was anti-HBc positive (HBsAg and HBV-DNA negative), and 329 (26.1%) had anti-HBs <10mIU/ml. At present time, 226 of 329 children (68.7%) had a booster dose of vaccine and 185 (81.9%) of them showed an anamnestic response. The enrollment for boostering and B-cell memory analysis are in progress.

Conclusions: This study evidences HB-vaccine long-term immunogenicity as, 11 years after vaccination, 74% of children still have protective antibodies and 81.9% of those with anti-HBs <10mIU/ml show an anamnestic response when boostered. Moreover the vaccine is highly efficacious as only one of 1260 resulted anti-HBc positive.

608 IDENTIFICATION OF THE ENZYMATIC FUNCTION OF THE HISTIDINE TRIAD (HIT) PROTEIN HINT2

J. Martin 1 , K. Schmidt 1 , C. Brenner 2 , J.F. Dufour 1 . 1 Institute For Clinical Pharmacology, University of Bern, Bern, Switzerland; 2 Genetics and Biochemistry and Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon NH, USA We identified the first human mitochondrial HIstidine Triad protein (HIT) protein, Hint2, which is predominantly expressed in the liver. Previously described HIT proteins, Hint1 and the yeast homolog Hnt1, are AMP-NH2 hydrolases, which regulate Cdk7. Hint1 is also purported to be a PKC inhibitor, but the results are inconclusive. Fhit, another HIT protein, which is disrupted in many types of cancer, increases the expression of the proapoptotic regulator Bak. Our aim was to determine whether Hint2 shares biochemical properties with other HIT homologs. Methods: 1) Expression of Hint2 in bacteria and purification by AMPagarose affinity chromatography. 2) Overlay, immunoprecipitation and phosphorylation assay with PKCs-enriched rat brain preparations. 3) Hydrolytic activity for a range of adenosine substrates. 4) Site directed mutagenesis of the third histidine of the HIT domain (Hint2H149A). 5) Stable transfection of HepG2 cells with Hint2, Hint2H149A and vector. 6) Western blots for proteins of the Bcl family. Results: The best substrate for Hint2 was adenosine-5 -phosphoraminidate. The AMP-lysine hydrolase activity of Hint2 was 10-fold higher than that of Hint1 (kcat = 0.0223±0.0031 s-1 vs 0.00193±0.00013 s-1) and lost with Hint2H149A. Neither overlays, nor co-immunoprecipitations detected an interaction with PKCs. Hint2 did not inhibit phosphorylation of histone. In HepG2 cells, overexpression of Hint2 and Hint2H149A did not change the expression of Bcl-XL, phosphoBcl-2, Bad, Bak and Bax. Conclusions: Hint2 is an AMP-lysine hydrolase and this function depends on the histidine triad domain. Hint2 is not a PKC inhibitor. These data suggest that Hint2 may remove adenylyl adducts from mitochondrial proteins.

609 LIVER INJURY DURING OBSTRUCTIVE SLEEP APNEA F. Tanne 1 , L. Serfaty 1 , F. Gagnadoux 2 , B. Fleury 2 , D. Wendum 3 , O. Chazouilleres 1 , B. Lebeau 2 , R. Poupon 1 . 1 Service D’Hepatologie, Hopital Saint-Antoine, Paris, France; 2 Service De Pneumologie, Hopital Saint-Antoine, pARIS, France; 3 Service D’Anatomopathologie, Hopital Saint-Antoine, pARIS, France Because of overweight, patients with obstructive sleep apnea (OSA) are at risk of fatty liver. Several data suggest that OSA per se could be a risk factor of liver injury: 1) ischaemic hepatitis during severe OSA has been reported, 2) OSA is an independent risk factor for insulin resistance*. Therefore, we investigated liver injuries and potential mechanisms in a cohort of patients with OSA. Methods: Nocturnal polysomnographic recording was performed in 205 consecutive patients with clinical suspicion of OSA. Serum ALT, AST, GGT and aGST levels were measured in all patients. Liver biopsy was proposed to patients with elevated liver enzymes. Intrahepatic hypoxemia was assessed by using HIFAb and VEGFAb on liver biopsy specimen. Results: 29% of patients had severe OSA (apnea-hypopnea index>50/h), 51% moderate OSA (1030 (OR 2.8) and severe OSA (OR 2.4). In 18/39 patients with elevated liver enzymes, liver biopsy showed steatohepatitis in 14, associated with fibrosis in 9, and normal histology in 4 cases. Serum insulin level, insulin resistance (HOMA), severity of steatosis