999 HUMAN MONOCYTE SUBSETS ACCUMULATE INTRAHEPATICALLY IN LIVER CIRRHOSIS AND DIFFERENTIALLY INTERACT WITH HEPATIC STELLATE CELLS

POSTERS phosphodiesterases were assessed in Sk-ChA-1 cells by Quantitative Real-Time PCR, western blot and immunohistochemistry. Results: Cyst fluid treatment of human cholangiocytes raises intracellular cAMP levels and cholangiocyte proliferation, which are significantly reduced by lanreotide treatment. We found that somatostatin receptor 5 is the most abundantly expressed somatostatin receptor in human cholangiocytes and induced by lanreotide treatment. There is a high basal expression of the cAMP metabolizing enzyme PDE4D in cholangiocytes which is significantly induced by lanreotide treatment. Blocking of SST5 with 8 nM BIM23056 or inhibition of PDE4D activity with 10 mM rolipram reverses the beneficial effect of lanreotide. Conclusions: Lanreotide significantly reduces PCLD patient cyst fluid-induced proliferation in a mechanism that depends on the somatostatin receptor 5 and the phosphodiesterase PDE4D. Therefore, reducing intracellular cAMP levels and cholangiocyte proliferation by the induction of PDE4D enzyme activity is an attractive novel target for the treatment of polycystic liver disease. 997 HESPERIDIN ATTENUATES LIPOPOLYSACCHARIDEINDUCED FULMINANT HEPATIC FAILURE IN D-GALACTOSAMINE-SENSITIZED MICE J.-Y. Wan1 , X. Gong2 , F.-L. Luo3 , H.-Z. Li4 , L. Zhang5 . 1 Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, 2 Department of Anatomy, 3 Department of Pharmacy, The first Affiliated Hospital of Chongqing Medical University, 4 Department of Pharmacology, 5 Department of Pathophysiology, Chongqing Medical University, Chongqing, China E-mail: [email protected] Background and Aims: Fulminant hepatic failure (FHF) remains an extremely poor prognosis and high mortality; better treatments are urgently needed. Hesperidin, a flavanone glycoside isolated from Poncirus trifoliate, has been described to exhibit anti-inflammatory activities in several inflammatory models. However, the effects of Hesperidin on FHF are poorly understood. The present study was undertaken to investigate whether hesperidin is efficacious against Lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced FHF in mice and its potential mechanisms. Methods: Hesperidin (30, 100 and 150 mg/kg/d) was pretreated orally once daily for 3 days before LPS/D-GalN injected in mice. The mortality, hepatic tissue histology, plasma levels of Tumor necrosis factor-alpha (TNF-alpha) and alanine aminotransferase (ALT) and aspartate aminotransferase (AST), hepatic tissue TNFalpha, myeloperoxidas (MPO) activity, caspase-3 activity and hepatocellular apoptosis were measured. Western blotting analysis of phosphor-inhibit kappa B (I-kappaB), phospho-p38 mitogenactivated protein kinase (phospho-p38 MAPK), phospho-c-jun N-terminal kinase (phospho-JNK) and phospho-extracellular signal regulated kinase (phospho-ERK) were determined. Besides, heme oxygenase-1 (HO-1) protein and activity in liver tissues was assayed. Results: Our data showed that administering hesperidin to mice reduced mortality and improved liver injury induced by LPS/D-GalN in a dose-dependent manner. In addition, TET dose-dependently inhibited LPS/D-GalN-induced NF-kappaB and MAPKs activation, TNF-alpha production, myeloperoxidas (MPO) activity, caspase-3 activation and hepatocellular apoptosis. Moreover, hesperidin dosedependently increased HO-1 protein expression and activity. Further, these protective effects of hesperidin against LPS/D-GalNinduced FHF were blocked by ZnPP IX, a HO-1 inhibitor. Conclusions: These results suggest that hesperidin has remarkable hepatoprotective effects on LPS/D-GalN-induced FHF and the possible mechanism is related to up-regulation of protein, which lead to inhibiting NF-kappaB and MAPKs, decreasing TNF-alpha production.

998 CENTROMERE PROTEIN A IS OVEREXPRESSED IN HEPATOCELLULAR CARCINOMA AND INVOLVED IN CELL CYCLE REGULATION Y. Li, Z. Zhu, S. Zhang, D. Yu, H. Yu, L. Liu, X. Cao, L. Wang, H. Gao, M. Zhu. Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai, China E-mail: [email protected] Background and Aims: Centromere protein A (CENP-A) plays important roles in cell cycle regulation and genetic stability. The association of CENP-A with hepatocarcinogenesis remains unknown. The aim of this study is to further confirm the status of CENP-A in liver cancer cells and to investigate the role of CENP-A in cell cycle regulation in hepatocarcinogenesis. Methods: Plasmids expressing the full length CENP-A or short interfering RNA targeting CENP-A were constructed and transfected into HepG2 cells. The biological activities, the expression of genes associated with cell cycle and apoptosis in these transfectants were analyzed by a series of assays. Additionally, the expression of CENP-A and clinicopathological features of hepatocellular carcinomas were analyzed. Results: CENP-A was overexpressed at mRNA and protein levels in hepatoma HepG2 cells and human hepatocellular carcinoma tissues. Small RNA interference targeting CENP-A partially inhibited HepG2 growth in vitro and in vivo by blocking cell cycle at G0-G1 phase through mediation of P21WAF1 and SKP-2, and by promoting apoptosis of HepG2 cells as indicated by the changes of MDM2 and Bcl-2/Bax. CENP-A overexpression was positively correlated with a high Ki-67 index, P53 protein, histological grade in human hepatocellular carcinomas. Conclusions: To the best of our knowledge, this is the first study showing overexpression of CENP-A in hepatoma cell lines and human HCC tissues. CENP-A might serve as a cell proliferation marker in cell cycle regulation and a potential therapeutic target for hepatocellular carcinoma. 999 HUMAN MONOCYTE SUBSETS ACCUMULATE INTRAHEPATICALLY IN LIVER CIRRHOSIS AND DIFFERENTIALLY INTERACT WITH HEPATIC STELLATE CELLS H.W. Zimmermann1 , S. Seidler1 , J. Nattermann2 , N. Gassler3 , C. Hellerbrand4 , R. Weiskirchen5 , C. Trautwein1 , F. Tacke1 . 1 Medizinische Klinik III, Universit¨ atsklinikum Aachen, Aachen, 2 Universit¨ at Bonn, Bonn, 3 Institut f¨ ur Pathologie, Universit¨ atsklinikum Aachen, Aachen, 4 Universit¨ at Regensburg, Regensburg, 5 Institut f¨ ur Klinische Chemie und Pathobiochemie, Universit¨ atsklinikum Aachen, Aachen, Germany E-mail: [email protected] Background and Aims: Monocyte-derived macrophages critically perpetuate inflammatory responses after liver injury as a prerequisite for organ fibrosis. Experimental murine models identified an essential role for the CCR2-dependent infiltration of classical Gr1/Ly6C+ monocytes in hepatic fibrosis. The relevance of monocyte subpopulations for human liver fibrosis is unknown. We therefore addressed the role of circulating and intrahepatic CD14++ CD16− and CD14+ CD16+ monocytes in patients with chronic liver diseases. Materials and Methods: Circulating blood monocyte subsets were studied by FACS in 226 chronic liver disease patients and 184 healthy controls. Liver specimens were analyzed by FACS and immunofluorescence for the presence and activation of monocyte/macrophage subsets. Moreover, isolated monocyte subsets were functionally characterized in vitro as well as cocultured with primary human hepatic stellate cells (HSC) to elucidate possible interactions between both cell types.

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S385

POSTERS Results: Circulating monocytes are significantly expanded in patients with chronic liver diseases with a marked increase of the non-classical CD14+ CD16+ subset that shows an activated phenotype in patients and correlates with proinflammatory cytokines, chemokines and clinical progression. Correspondingly, CD14+ CD16+ monocytes/macrophages massively accumulate in the fibrotic/cirrhotic liver and account for about 50% of all liver macrophages in cirrhotic in comparison to 10% in normal or early fibrotic tissue. Monocyte-related chemokine pathways (i.e. CCL2, CCL3, CCL4, CCL5, CX3 CL1) are differentially activated in patients in the circulation and liver, alongside up-regulated chemokine receptors on monocytes. Functionally, non-classical CD14+ CD16+ monocytes release abundant proinflammatory cytokines upon isolation, whereas the classical CD14++ CD16− subset primarily provides the chemokine CCL2 (MCP-1) and anti-inflammatory IL10. Co-culture experiments of primary HSC and monocyte subsets demonstrated that only classical CD14++ CD16− monocytes are able to exert direct fibrogenic stimuli on HSC by inducing collagen-1 and acta2 mRNA transcripts. In turn, HSC differentially affect the phenotype of monocyte subsets upon co-culture by regulating the expression of activation markers and chemokine receptors. Conclusions: Our data thereby reveal differential functional contributions of the non-classical and classical human monocyte subset for the perpetuation of intrahepatic inflammation and profibrogenic HSC activation, respectively. The modulation of monocyte-subset recruitment into the liver and their subsequent differentiation may offer novel opportunities for therapeutic interventions in human liver fibrosis. 1000 ROLE OF CALCIUM-CALMODULIN PATHWAY IN ESTRADIOL17b-GLUCURONIDE INDUCED IMPAIRMENT OF CANALICULAR SECRETION IN ISOLATED RAT HEPATOCYTE COUPLETS A. Zucchetti, F.D. Toledo, I.R. Barosso, E.J. Ochoa, F.A. Crocenzi, E.J. Sanchez ´ Pozzi. Instituto de Fisiolog´ıa Experimental, Rosario, Argentina E-mail: zucchetti@ifise-conicet.gov.ar The endogenous estradiol metabolite, estradiol 17b-D-glucuronide (E17G), induces an acute cholestasis in rat liver due in part to retrieval of canalicular transporters such as the bile salt export pump (Bsep, Abcc11) and the multidrug resistance associated protein 2 (Mrp2, Abcc2), in a process that involves estrogen receptor (J. Hepatol 50: s103, 2009). Calmodulin interacts with estrogen receptor and potentiates estrogen activation of the receptor (Cell Signal 19: 439–443, 2007). The aim of this study was to evaluate the involvement of calcium-calmodulin pathway in E17G-induced changes in canalicular excretion. Methods: Canalicular transport activities: Isolated rat hepatocytes couplets were cultured for 5 h, exposed to verapamyl (V, calcium channel blocker, 10 mM), trifluoroperazin (T, Calcium-calmodulin inhibitor, 10 mM) or W7 (W, Calcium-calmodulin kinase II [CaMKII] inhibitor, 100 mM) for 15 min and then incubated with E17G (100 mM) for 20 min. Finally, all preparations were incubated with cholyl-lysylfluorescein (CLF, fluorescent bile salt substrate of Bsep) or CMFDA (intracellularly converted in gluthationmethylfluorescein [GMF], fluorescent substrate of Mrp2). Couplets accumulating CLF or GMF in their vacuole were counted in a fluorescent microscope and informed as a percentage (%AC). CaMKII activation: Isolated hepatocyte were cultivated in collagen sandwich for 5 days, exposed to E17G (200 mM) for 15 and 30 min and then lysed. Western blot of the samples were performed using antibodies against total and phosphorylated CaMKII. The ratio of the densitometry of phosphorylated/total CaMKII bands was used as estimation of kinase activation. Results were expressed as mean±SD and compared by ANOVA followed by Student-Newman-Keuls test. Results (n = 3): See the tables. S386

Canalicular transport activities

CLF %AC GMF %AC

Control

E17G

E17G+V

E17G+T

E17G+W

100±0 100±0

66±4a 65±4a

84±4a,b 81±11a,b

90±11b 95±6b

97±13b 94±1b

a

significantly different from Control (p < 0.01); b significantly different from E17G (p < 0.05). V, T and W did not affect %AC of the substrates. CaMKII activation Control

phosphorylated/total CaMKII (arbitrary units) a significantly

100±0

E17G 15 min

30 min

126±25

172±34a

different from Control (p < 0.05).

Conclusions: E17G activates calcium-calmodulin pathway as demonstrated by CaMKII activation, and this pathway participates in estrogen-induced alteration in canalicular secretion.

05e. VIRAL HEPATITIS: e. HEPATITIS B – CLINICAL (THERAPY NEW COMPOUNDS, RESISTANCE)

1001 PRAGMATIC ASSESSMENT OF LIVER FIBROSIS DURING METHOTREXATE THERAPY: COMPARISON OF PATIENTS WITH PSORIASIS, RHEUMATOID ARTHRITIS OR CROHN’S DISEASE B. Alby-Lepresle1 , J.-P. Cervoni1,2 , E. Monnet1 , F. Aubin3 , D. Wendling4 , E. Toussirot4 , I. Mermet3 , M. Nachury5 , E. Bertolini4 , F. Carbonnel5 , V. Bague2 , P. Cales ` 6 , V. Di Martino1 . 1 Service 3 d’H´epatologie, CHU Jean Minjoz, 2 CIC-BT, CHU Besancon, ¸ Service de Dermatologie, CHU Saint-Jacques, 4 Service de Rhumatologie, 5 6 Service de Gastroent´erologie, CHU Jean Minjoz, Besancon, ¸ Service d’H´epato-Gastroent´erologie, CHU Angers, Angers, France E-mail: [email protected] Background: Reported hepatotoxicity limits the use of methotrexate and underlines the need for of a non-invasive sequential and reliable evaluation of liver fibrosis. Aims: 1. To estimate by non-invasive methods the prevalence of significant liver fibrosis among patients treated with methotrexate for psoriasis, rheumatoid arthritis (RA) or Crohn’s disease (CD); 2. To assess the factors associated with significant fibrosis (≥F2). Methods: Prospective observation of consecutive patients followed in our institution between February 2008 and August 2009 for Psoriasis, PR, or CD. 189 variables were collected (questionnaire, clinical examination, Fibroscan® , blood sample, abdominal ultrasound), focused on risk factors for chronic liver disease or metabolic syndrome. Fibrosis was evaluated by 9 non-invasive tests (Fibroscan® , Forns, APRI, FPI Hepascore, FIB4, Fibrotest® , Angulo’s score, FibroMeters). Diagnosis reference of significant fibrosis was developed without liver biopsy by defining a “specific method” (concordance of tests) and “sensitive method” (at least one test indicating ≥F2). Multivariate analyses were performed to assess the factors associated with fibrosis ≥F2 or numeric values of Angulo’s score, FibroMeters-S, and Fibroscan® . The performance of 24 variables including the result of each test for the diagnosis of significant fibrosis was assessed using ROC curves. Results: 89 patients (41 psoriasis, 23 PR, 25 MCR) were enrolled including 57 receiving methotrexate. The prevalence of fibrosis ≥F2 according to the specific or the sensitive definitions was 7% and

Journal of Hepatology 2010 vol. 52 | S319–S457