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POSTERS triglycerides, oxLDL and sVCAM-1 and improves vascular function as reflected by FMD, without affecting cholestasis progression. Therefore, statin therapy should be considered in PBC patients with additional risk factors for cardiovascular disease. 1118 POSTTRANSCRIPTIONAL REGULATION OF THE SODIUM TAUROCHOLATE COTRANSPORTING POLYPEPTIDE (NTCP) BY ENDOCYTOSIS AND SUBSEQUENT LYSOSOMAL DEGRADATION C. Stross, K. Weissenberger, A. Helmer, B. Gorg, ¨ D. Haussinger, ¨ R. Kubitz. Gastroenterology, Hepatology and Infectiology, D¨ usseldorf, Germany E-mail: [email protected] Background and Aims: The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake system for conjugated bile salts in the liver. It is constitutively expressed in hepatocytes but its expression can be influenced by bile salts and proinflammatory stimuli, for example. At the posttransciptional level, exocytosis of Ntcp, which is partly stored in intracellular vesicles, has been shown and proposed to adjust transport capacity to varying serum bile acid concentrations. On the other hand, regulated endocytosis and subsequent degradation of Ntcp has not been demonstrated so far. Methods: Ntcp was cloned with a N-terminal FLAG- and a C-terminal EGFP-tag. Stably transfected HepG2 cells were generated and used for the study of Ntcp endocytosis via flow cytometry, TIRF (total internal reflection microscopy), conventional immunofluorescence and Western Blot analyses. Results: We could show dynamin dependent internalization of Ntcp in response to activation of protein kinase C (PKC). This process is selective, not affecting the Na+ K+ -ATPase and does not involve the actin cytoskeleton. It is most likely mediated by the classical (c) PKCs because phorbol-12-myristate-13-acetate (PMA) as well as the cPKC selective activator thymeleatoxin (TTX) lead to a 30–40% decrease in membrane expression. Furthermore, the cPKC selective inhibitor Go6976 ¨ abolished PMA induced endocytosis of Ntcp. After prolonged PKC activation (2–4 h) endocytosed Ntcp colocalizes with the lysosomal marker Lamp-1 whereas there is no colocalization with Rab11, a marker of the recycling compartment, at any time point. In line with a lysosomal targeting an approximately 26% reduction in protein levels is measured 4 h after PMA stimulation which is sensitive to bafilomycin A1. Conclusions: Activation of classical PKCs induces vesicular retrieval of Ntcp from the plasma membrane and subsequent lysosomal degradation. This mechanism may be relevant to rapidly adjust cellular transport capacity and to protect hepatocytes from toxic intracellular bile salt concentrations. 1119 A FUNCTIONAL VARIANT IN FC RECEPTOR-LIKE 3 (FCRL3) GENE IS ASSOCIATED WITH SUSCEPTIBILITY OF PRIMARY BILIARY CIRRHOSIS IN JAPANESE, BUT NOT IN ITALIAN A. Tanaka1 , H. Ohira2 , K. Kikuchi3 , A. Shibuya4 , S. Nezu5 , P. Invernizzi6 , H. Takikawa1 . 1 Teikyo University, School of Medicine, Tokyo, 2 Fukushima Medical University, School of Medicine, Fukushima, 3 Teikyo University, School of Medicine, 4 Kitazato University, School of Medicine, Kanagawa, 5 Kyorin University, School of Medicine, Tokyo, Japan; 6 IRRCS Istituto Clinico Humanitas, Milan, Italy E-mail: [email protected] Background and Aims: Recent findings demonstrated that a functional variant in the promoter region of Fc receptor-like 3 (FCRL3) gene is associated with the susceptibility of several autoimmune diseases, suggesting the presence of common FCRL3related pathological pathways shared by different autoimmune diseases. It is of interest, however, that this association has been demonstrated only in Japanese; studies in Caucasian failed to confirm this connection. We demonstrated that a functional S432

variant, C/T −169 (rs7528684) at the promoter region of FCRL3, was associated with susceptibility of PBC only in Japanese, but not in Italian populations. In this study we investigated whether another polymorphic site, G>A −110 (rs11264799), of FCRL3 gene is also associated with PBC. Patients and Methods: We enrolled 232 patients with PBC and 230 age- and sex-matched controls in Japanese, and 216 PBC/180 controls in Italian. The diagnosis of PBC was made according to serum biochemistry, presence of serum AMA and histological studies of the biopsied liver specimens. Genetic DNA was extracted from peripheral mononuclear blood cells. The FCRL3 genotyping at C/T −169 and G>A −110 was performed by melting analysis using LightCycler System (Roche Diagnostics). The association was statistically analyzed using Fisher’s exact test. Results: As shown previously, the frequency of allele C at C/T −169 was significantly higher in PBC (OR = 2.398 [1.626–3.534], p = 6.5×10−6 ), suggesting a strong association of the functional variant in FCRL3 gene with PBC in Japanese, while no significant association with this polymorphism was detected in Italian. As for G>A −110 site, the frequency of A/A, A/G, and G/G at −110 was 41/42/149 in PBC and 24/56/150 in controls in Japanese, thus the genotype A/A significantly higher in PBC (OR = 1.842 [1.073–3.164], p = 0.025), while the frequency of allele A was comparable, 124/464 in PBC and 104/460 in controls (p = 0.147). On the other hand, the frequency of A/A as well as allele A was similar between PBC and controls in Italian (p = 0.092 and 0.123, respectively). Conclusions: The G>A −110 variant in FCRL3 gene is also associated with the susceptibility of PBC only in Japanese, yet the association is more evident at C/T −169. 1120 BICARBONATE SECRETION OF MOUSE CHOLANGIOCYTES INVOLVES Na+ –HCO3 − COTRANSPORT IN ADDITION TO Na+ -INDEPENDENT Cl− /HCO3 − EXCHANGE I. Uriarte1 , J.M. Banales1 , E. Saez ´ 1 , F. Arenas1 , R.P.J. Oude Elferink2 , 1 1 1 J. Prieto , J.F. Medina . Gene Therapy and Hepatoly-Liver Unit, CIMA-University of Navarra, Pamplona, Spain; 2 Laboratory of Experimental Hepatology, AMC Liver Center-Academic Medical Center, Amsterdam, The Netherlands E-mail: [email protected] Background and Aim: Bicarbonate secretion from cholangiocytes participates in the appropriate modifications of bile along the biliary tract. The Na+ -independent Cl− /HCO3 − anion exchanger AE2/Ae2 mediates HCO3 − secretion in human and rat cholangiocytes, being also involved in intracellular pH regulation. In Ae2a,b -deficient mice, pHi is increased in lymphocytes and fibroblasts, whereas it is surprisingly normal in cholangiocytes. Here, we analyze the mechanisms for HCO3 − secretion in cultured Ae2a,b +/+ and Ae2a,b −/− mouse cholangiocytes. Methods: We isolated intrahepatic bile-duct units from both wildtype and knockout mice to established pure long-term cultures of respective cholangiocytes. Cells were loaded with BCECFAM for microfluorimetric determination of pHi variations upon perfusion maneuvers (mainly with propionate and isothionatebased buffers). Equivalent base fluxes (JOH − ) were calculated under basal conditions and following stimulation with HCO3 − secretion agonists (cAMP, ACh, ATP). Voltage-clamp experiments were performed in Ussing chambers to measure Cl− currents in polarized mouse cholangiocytes. Results: Cl− -withdrawal maneuvers showed that Ae2a,b +/+ , but not Ae2a,b −/− mouse cholangiocytes can display Cl− /HCO3 − exchange, indicating that such an exchange is entirely mediated by Ae2 in normal mouse cholangiocytes (as previously demonstrated for both human and rat cholangiocytes). But simultaneous withdrawal of Cl− and Na+ revealed that mouse cholangiocytes possess an additional transport activity for HCO3 − secretion, not observed

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POSTERS in control experiments with rat cholangiocytes. Propionate-based maneuvers indicated that this supplemental Na+ -driven HCO3 − secreting activity is Cl− -independent, consistent with a Na+ -HCO3 − cotransport (NBC). NBC activity is greater in Ae2a,b −/− than Ae2a,b +/+ mouse cholangiocytes and membrane-depolarization experiments showed that it is electrogenic. Consistent with the potential role of Slc4a4/Nbc1 as the involved transporter, Ae2a,b −/− mouse cholangiocytes showed upregulated expression of this electrogenic NBC carrier. While Ae2-mediated Cl− /HCO3 − exchange in Ae2a,b +/+ mouse cholangiocytes is stimulated by cAMP and ACh, the NBC activity is downregulated by cAMP and ATP in Ae2a,b −/− mouse cholangiocytes. Ae2a,b −/− mouse cholangiocytes polarized in Ussing chambers showed decreased (though not abolished) cAMPdependent Cl− current and increased ATP-dependent/Ca2+ -activated Cl− secretion, which run in parallel with decreased CFTR mRNA expression and increased intracellular Ca2+ levels. Conclusion: Bicarbonate secretion in mouse cholangiocytes involves two differentially regulated activities: Ae2-mediated Cl− /HCO3 − exchange and Na+ -HCO3 − cotransport. 1121 INCREASED TGF-B3 PRODUCTION IN PRIMARY BILIARY CIRRHOSIS A. Voumvouraki1,2 , M. Tsardi3 , M. Koulentaki1 , O. Sfakianaki2 , M. Fragaki2 , G. Notas2,4 , E. Kouroumalis1,2 . 1 Gastroenterology, University Hospital of Heraklion, 2 Liver Research Laboratory, 3 Pathology, 4 Experimental Endocrinology, University of Crete Faculty of Medicine, Heraklion, Greece E-mail: [email protected] Background and Aims: TGF-beta is considered the main activator of hepatic stellate cells for extracellular matrix production during liver fibrogenesis. However the role of its 3 isoforms in different liver diseases has not been studied so far. Methods: Blood was collected from 62 patients with Primary Biliary Cirrhosis (PBC, 35 stages I-II and 27 stages III-IV) before and after 6 months of UDCA treatment and from 34 patients with viral cirrhosis, 44 patients with chronic hepatitis C (CHC) and 60 healthy controls. Furthermore blood was collected from the hepatic vein from 15 PBC patients before and after 6 months of UDCA treatment and 17 patients with viral cirrhosis. TGFb1, TGFb2 and TGFb3 were measured in serum with commercially available ELISA’s. Tissue samples from PBC patients and disease controls were also stained for TGFb1, TGFb2 and TGFb3. Results: TGFb1 was decreased in cirrhotic patients (viral 16.9±1.1 ng/ml, PBC I-II 19.6±1.4, III-IV 14.7±1.6, p < 0.001) compared to controls (29.8±1.6 ng/ml, chronic CHC 23.2±1.8). TGFb2 was increased in patients with viral cirrhosis but not in PBC and CHC. However TGFb3 was increased only in PBC (I-II 106.2±10.1 pg/ml, III-IV 178.6±17.3 pg/ml, p < 0.001) and decreased in viral cirrhosis (p < 0.05) compared to controls (53.9±4.7). UDCA treatment did not have any effect on all isoform’s levels. The levels of TGFb isoforms measured from the hepatic vein samples were similar to the peripheral blood samples. Immunocytochemistry showed that the production of the first two isoforms was mainly from the cells of the hepatic sinusoids and lymphocytes while TGFb3 was over-expressed in PBC patient hepatocytes. Conclusions: The three isoforms of TGFb are regulated differently in different types of cirrhosis. Furthermore, increased TGFb3 seems to be a specific characteristic of PBC.

1122 ANXIETY AND DEPRESSION IN PATIENTS WITH AUTOIMMUNE HEPATITIS C. Weiler-Normann1 , I. Wahl2 , C. Wiegard1 , C. Glaubke1 , A.W. Lohse1 , M. Rose2 , C. Schramm1 . 1 I. Department of Medicine, 2 Clinic for Psychosomatic Medicine and Psychotherapy, University Medical Center Hamburg-Eppendorf, Hamburg, Germany E-mail: [email protected] Background and Aims: Patients with autoimmune hepatitis (AIH) seem to be more distressed than one would expect from their somatic condition. However, there is only little empirical evidence for a potential comorbidity of mental disorders in patients suffering from AIH. The aim of our study was to assess the occurrence of generalized anxiety disorder and depression in AIH-patients. Methods: Between 4/2008 and 7/2009, consecutive patients with a diagnosis of AIH seen at our specialized outpatient clinic were asked to answer the Patient Health Questionnaire, including modules for depression (PHQ-9) and generalized anxiety disorder (GAD-7). An adhoc-questionnaire was used to assess disease-specific concerns. Clinical data was drawn from the medical records. Results: 114 patients with definite AIH participated in the study, comprising 83% of all AIH patients treated during the recruitment period (age 50±18, 71% female). AIH-patients had significantly higher depression scores (PHQ-9: 6.2±5.7, categorical analysis following DSM IV; scale 0–27) than the German general population (n = 5096, 3.6±4.1). Also, they had a significantly higher level of anxiety (GAD-7: 4.3±4.6 (scale 0–21)) than the German general population (n = 5096; 2.9±3.4). In 34.5% of the participants, fear of having liver cirrhosis was predominant and 24.5% were afraid of being stigmatized as an alcoholic. According to our screening tools, 11.2% showed moderate or severe anxiety symptoms (GAD-7 >10). 9.7% fulfilled the criteria for a major depressive disorder (PHQ-9). Liver cirrhosis, biochemical disease activity (including ALT, gammaglobulins, IgG) or leucocyte count as a marker of inflammation were unrelated to the severity of psychological symptoms. Conclusions: Our results show for the first time, that AIH is associated with a significant prevalence of mental disorders including anxiety and depression as compared to the general population. Comorbid mental disorders call for our attention in order to improve the care of patients with AIH. 1123 PROTEIN KINASE C MEDIATED ENDOCYTOSIS OF SODIUM TAUROCHOLATE COTRANSPORTING POLYPEPTIDE (NTCP) AS A PROTECTIVE MECHANISM K. Weissenberger, C. Stross, A. Helmer, V. Keitel, D. Haussinger, ¨ R. Kubitz. Gastroenterology, Hepatology and Infectiology, D¨ usseldorf, Germany E-mail: [email protected] Background and Aims: The sodium taurocholate cotransporting polypeptide (Ntcp) is the main basolateral transporter for conjugated bile salts from portal blood. Ntcp critically regulates serum as well as intracellular bile salt concentrations in hepatocytes. During cholestasis Ntcp is downregulated, thereby reducing bile salt uptake which may protect hepatocytes from toxic bile salt concentrations. Methods: Ntcp was fused to an intracellular green fluorescent protein additionaly to the FLAG peptide on its extracellular domain and stably expressed in the human hepatoma cell line HepG2. Localization of Ntcp and cell responses to bile salts were evaluated by flow cytometric methods, immunofluorescence as well as live cell imaging. Results: Bile salt uptake of FLAG-Ntcp-EGFP HepG2 cells was equivalent to wild type HepG2 with a Km for taurocholate of 24 mM. Stimulation of the cells with the protein kinase C activator phorbol-

Journal of Hepatology 2010 vol. 52 | S319–S457

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