610 FUNCTIONAL STUDY OF THE INTESTINAL BARRIER IN PATIENTS WITH CIRRHOSIS AND PORTAL HYPERTENSION

POSTERS macrophages during conditions of excess iron by blocking the iron exporter ferroportin. In addition, IL-6 mediated upregulation of hepcidin during inflammation is the major mechanism of the socalled ‘anemia of chronic disease’. We here identify H2 O2 which is co-released by inflammatory cells as potent transcriptional activator of hepcidin independent of upstream regulators IL-6 and BMP6. Material and methods: H2 O2 release (1–8 mM) by immune cells was mimicked using purified enzymes glucose oxidase and catalase as recently described (GOX/CAT system). Huh7 hepatoma cells were treated with either IL-6 or BMP6 alone, or in combination with steady state H2 O2 over 24 h. Hepcidin regulation was assessed by quantitative real time PCR. Members of the intracellular signaling cascade such as STAT3 were assessed by Western blotting. Promotor studies were performed using hepcidin-promoter constructs with various deletions fused to luciferase as reporter gene with Renilla as control reporter gene. Results: Steady state non-toxic H2 O2 concentrations comparable to those by inflammatory cells rapidly and drastically upregulate hepcidin in a dose-dependent way in Huh7 cells by a factor of 10. In addition, H2 O2 further potentiates IL6 and BMP6 mediated upregulation of hepcidin 5-fold. Interestingly, induction was also not blunted by confounding hypoxia (2%). The H2 O2 mediated hepcidin response is an early response reaching the maximum mRNA-concentration already after 6 h. Promoter studies identified the STAT3 element as major promoter region of H2 O2 mediated hepcidin induction. Indeed, phosphorylation of STAT3 was confirmed under these conditions using Western blotting. Conclusions: Our studies establish H2 O2 as as an important regulatory link between systemic iron regulation and oxidative stress. Impaired H2 O2 -hepcidin signaling in chronic liver diseases could contribute to hepatic accumulation of iron in cirrhotic patients. 609 SMALL INTESTINE INFLAMMATION IN RATS WITH CCL4 CIRRHOSIS: ROLE OF ENTERIC BACTERIA L. Munoz ˜ 1,2 , M.J. Borrero1 , M. Ubeda1,2 , M. Lario1 , D. D´ıaz1 , 1,4 ´ J. Monserrat1 , L. Lledo´ 3 , M. Alvarez-Mon , A. Albillos1,2,5 . 1 Department of Medicine, University of Alcal´ a, Alcal´ a de Henares, 2 CIBERehd, Instituto de Salud Carlos III, Madrid, 3 Department of Microbiology and Parasitology, University of Alcal´ a, 4 University 5 Hospital Pr´ıncipe de Asturias, Alcal´ a de Henares, University Hospital Ram´ on y Cajal, IRYCIS, Madrid, Spain E-mail: [email protected] Increases in enteric bacterial load and intestinal permeability are among the factors that promote bacterial translocation to the mesenteric lymph nodes (MLN) in cirrhosis. The effect of these factors on intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) has not yet been addressed in cirrhosis. T-cell activating and trafficking to the gut have been incriminated in the increased intestinal permeability observed in chronic inflammatory bowel conditions. Aims: To study: i. the distribution and activation status of T-cells and monocytes/ macrophages in IELs and LPLs in cirrhotic rats, and ii. the contribution of luminal enteric bacteria to the observed abnormalities. Methods: Small intestinal IELs and LPLs were isolated from rats with CCl4 -cirrhosis (n = 18), controls (n = 8), and cirrhotic rats after bowel decontamination with antibiotics (n = 6). Cell populations were identified by four-colour flow cytometry. Gut bacterial translocation was defined as MLN culture positivity. Results: Compared with controls, cirrhotic rats showed increased (p < 0.01) numbers (cells/cm small intestine) of T-cells (174781±73443 vs 105826±27346) and monocytes/macrophages (CD11bbright+ CD3− CD45RA− NKR-P1Abright− ) (11438±6723 vs 4368

±1858) among LPLs. This T-cell expansion affected both Th(CD3+ CD4+ ) (1.5-fold) and Tc- (CD3+ TCRab+ CD8ab+ ) (3.5-fold) LPLs. T-cells from LPLs showed signs of activation, including increased numbers of recently activated Th-cells (CD3+ CD4+ CD134+ ) and NKTcells (CD3+ NKR-P1A+ ). Besides, LPLs from cirrhotic rats showed a higher (p < 0.05) frequency of IFNg-secreting Th- (34±5% vs 24±1%) and Tc-cells (58±8% vs 37±2%). In addition, LPLs from cirrhotic rats showed a marked (p < 0.01) increase in regulatory populations, such as gdT-cells (CD3+TCRgd+) (6.0-fold) and Treg (CD3+ CD4+ CD25+ FoxP3+ ) (2.3-fold). The IELs of cirrhotic rats showed similar behaviour. Intestinal mucosal immune activation was more intense in cirrhotic rats showing bacterial translocation (72%). Gut decontamination significantly reduced the faecal load of aerobic bacteria and signs of T-cell activation in the intestinal mucosa of cirrhotic rats. Conclusions: Cirrhosis is associated with a state of immune activation of the intestinal mucosa, which is probably induced by persistent enteric bacterial pressure. The adequate response of immunoregulatory mechanisms maintains mucosal inflammation at the subclinical level. 610 FUNCTIONAL STUDY OF THE INTESTINAL BARRIER IN PATIENTS WITH CIRRHOSIS AND PORTAL HYPERTENSION E. Nadal1 , A. Buda2 , D. Pizzuti2 , L. Nai2 , P. Burra1 , M. Senzolo1 . 1 Multivisceral Transplant Unit, Department of Surgical and Gastroenterological Sciences, 2 Gastroenterology, Department of Surgical and Gastroenterological Sciences, University Hospital of Padua, Padua, Italy E-mail: [email protected] Background: Patients with liver cirrhosis and portal hypertension are characterized by several abnormalities of intestinal function. Resulting alterations in the intestinal barrier can increase bacterial translocation. Aim: To assess the functional alterations of the intestinal barrier in patients with cirrhosis, to correlate these alterations with the presence of ascites, and to explore the possible role of b-blockers in ameliorating these abnormalities. Methods: 20 patients with liver cirrhosis (10 with ascites) not treated with b-blockers were enrolled. 10 subjects without gastric abnormalities at EGDS were included as controls. All patients and controls underwent lactulose/mannitol (L/M) permeability test and Ussing chamber method to assess intestinal mucosal barrier function. Expression of tight junction (TJ) proteins Zona occludens-1 (ZO-1) and Occludin were evaluated by immunohistochemistry in duodenal biopsy specimens; confocal endomicroendoscopy “in vivo” was performed to study microcirculatory and morphology/ histology changes. These evaluations were repeated at least 2 months after starting b-blockers in 2 patients. Results: Mean±SD L/M urine ratio was significantly higher in cirrhotic patients than in controls (0.026vs0.014, p = 0.001); L/M test was more frequently abnormal in patients with than without ascites (5/10 vs 2/10). Cirrhotics with altered L/M test showed a significantly lower transepithelial resistance at Ussing Chamber compared to those without L/M test alteration (11.7±2.3 vs 18.4±6.3, p = 0.026) and controls (11.7±2.3 vs 31.13±5.6, p < 0.001). Immunofluorescence assay demonstrated a significant reduction of Occludin expression in cirrhotics, more pronounced in those with L/M test alteration. Confocal laser endomicroscopy in cirrhotics revealed loss of normal villi architecture, microvascular dilatation and congestion with extravasation of fluorescein into the extracellular matrix, indicating endothelial spillage. Two patients with altered L/M test and ascites repeated the evaluation after starting b-blockers, and showed significant amelioration of intestinal permeability, transepithelial resistance and reduction of hypervascularization at confocal endomicroscopy.

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POSTERS Conclusions: Patient with liver cirrhosis are characterized by the loss of intestinal barrier integrity, especially in the presence of ascites. This is associated with an abnormal expression and distribution of TJ protein Occludin and modification of intestinal microcirculation. Therapy with non selective beta-blockers could ameliorate these abnormalities correcting functional alterations and possibly reducing bacterial translocation. 611 PROGRESSION OF HEMODYNAMIC CHANGES IN LIVER CIRRHOSIS. A MATHEMATICAL APPROACH L. Noiret1,2 , S. Baigent3 , R. Jalan2 . 1 CoMPLEX, University College London, 2 Liver Failure Group, University College London – Royal Free Hospital, 3 Mathematics, University College London, London, UK E-mail: [email protected] Background and Aims: Systemic and hemodynamic changes are a consistent feature associated with severity of liver cirrhosis classified using the Child–Pugh score and this leads to a major redistribution of blood flow. Clinical measurements provide a clear trend and order of magnitude of change for individual tissue, but fail to provide a coordinated picture as the disease progresses. A mathematical model simulating the evolution of distribution of blood flow subsequent to change in resistance was built. Methods: A lumped model of systemic circulation was implemented. Only key components were represented: hepatic artery, splanchnic bed, porto-systemic (PS) collaterals, portal vein, liver, and one component representing “other organs” (brain, muscle, kidney, etc). Relationships between flow and pressure were described using Ohm’s law. Different levels of cardiac output (CO) and several scenarios for PS collaterals implemented. Choice of parameter values and selection of scenarios for blood flow distribution were based on literature review. Results: To observe a significant change in the distribution of blood flow, a decrease in splanchnic resistance was required. Decreased splanchnic resistance led to an increase in flow through the splanchnic bed, and therefore to a decreased perfusion of other organs (% CO) (see Table 1). Absolute perfusion could be maintained through an increase cardiac output, but at the cost of worsening HVPG. Hepatic perfusion depended on the degree of PS collaterals allowed: 40% to 75% of splanchnic blood might by pass the liver. Table 1: Simulation results CO MAP HVPG PS shunt PV (ml/min) (mmHg) (mmHg) ml/min ml/min (%CO) (%CO) Normal Value 5500 (literature) Early Child A 5500

95

4

94

7

Child A

6050

92

9

Child B

6600

88

11

Late B Early C 7150

84

13

0 (0%) 0 (0%) 710 (12%) 1490 (23%) 2685 (38%)

1045 (19%) 1104 (20%) 1020 (17%) 1002 (15%) 547 (8%)

Liver ml/min (%CO)

Kidney ml/min (%CO)

Brain ml/min (%CO)

Muscle ml/min (%CO)

1402 (25%) 1449 (26%) 1366 (23%) 1332 (20%) 863 (12%)

1045 (19%) 1033 (19%) 1014 (17%) 964 (15%) 919 (13%)

935 (17%) 924 (17%) 907 (15%) 862 (13%) 822 (11%)

660 (12%) 653 (12%) 640 (11%) 609 (9%) 580 (8%)

Conclusion: Systemic vasodilation is not necessary to observe a hyperdynamic state. Progression of liver disease is associated with a failure to fully compensate the splanchnic dilation by an increase in cardiac output. Monitoring the evolution of patient hemodynamic state may be used as a key marker for predicting its outcome.

612 MIR-16 REVERSES ACTIVATED PHENOTYPE OF HEPATIC STELLATE CELL BY TRANSCRIPTOMIC REGULATION Q. Pan1 , J. Wang2 , Y.S. He3 , J.G. Fan1 . 1 Department of Gastroenterology, XinHua Hospital, School of Medicine, Shanghai Jiaotong University, 2 Department of Pharmacology, Second Military Medical University, 3 Department of Cardiology/Institution of Cardiovascular Research, RuiJin Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China E-mail: [email protected] Background and Aims: miR-16 takes a critical place during the activation of hepatic stellate cells (HSCs). Although multiple targets have been recognized, the global effect of miR-16 is still remained to be clarified. Methods: Quiescent, activated HSCs were isolated from normal and CCl4 -induced fibrotic rats, respectively. miR-16 restoration was then carried out in activated HSCs by lentivirus containing pre-rno-miR16. Being compared with controls, the impact of miR-16 on activated HSCs was further detected by high-throughout hybridization and transcriptome analysis. According to the transcriptomic characteristics, cyclin D1 expression, cell cycle and CCK8 assay were employed to evaluate the regulatory role of miR-16 on HSCs’ proliferation. Expression of smad-2 and downstream genes (collagen type I, III) was also measured to reflect its action on extracellular matrix (ECM) production. Moreover, the apoptosisrelated property of miR-16 was assessed by Bcl-2 level, caspase 3/7 activity and flow cytometry. Results: Pre-rno-miR-16 administration significantly up-regulated the miR-16 level in activated HSCs. As a result, (1) Adipogenic genes, which characterized quiescent status of HSCs, hold the growingup tendency in expression. (2) Descended fibrosis-inducing cytokines, collagens and ascended matrix metallopeptidases facilitated the normalization of ECM production/zymohydrolysis. (3) Cell-cycle-inhibitor up-regulation and cell-cycle-mediator down-regulation retarded the activation-based over-growth. (4) Increased transcription of mitochondrial apoptosis-mediating genes attenuated the apoptosis resistance. Being consistent with transcriptomic findings, both mRNA and protein loss of cyclin D1 were documented in miR-16-treated HSCs, which in turn lead to G0 /G1 block and proliferation inhibition. In addition, miR-16 treatment gave rise to smad-2 depression post-transcriptionally, and greatly reduced the supernatant concentration of collagen I, III. Another effect of miR-16 lied in the lowered level of Bcl-2. Elevation of caspase 3/7 ratio, and then apoptosis rate, occurred resultantly within activated HSCs. Conclusion: miR-16 may reverse the phenotype of activated HSCs by inducing quiescent-like pattern of transcriptome. 613 NEBIVOLOL DETERIORATES PORTAL HEMODYNAMICS IN CIRRHOTIC RATS BY INCREASING SPLANCHNIC BLOOD FLOW T. Reiberger1 , B.A. Payer1 , B. Angermayr1,2 , V. Fuhrmann1 , 1 P. Schwabl1 , B. Jager ¨ , T. Hummel1 , T. Horvatits1 , M. Mitterhauser3 , M. Peck-Radosavljevic1 , Vienna Experimental Portal Hypertension Study Group. 1 Internal Medicine III, Div. of Gastroenterology & Hepatology, Medical University of Vienna, Vienna, 2 Internal Medicine II, Div. of Gastroenterology & Hepatology, Zentralklinikum St. P¨ olten, St. P¨ olten, 3 Nuclear Medicine, Medical University of Vienna, Vienna, Austria E-mail: [email protected] Introduction: Portal hypertension (P), as a function of Ohm’s law (P = R·I) is influenced by intrahepatic resistance (R) and splanchnic blood flow (I). Non-selective beta-blockers (NSBB) decrease splanchnic blood flow and NO-donors (e.g. ISMN) decrease intrahepatic resistance, thus both drugs are used for treatment of portal hypertension. We evaluated the effects of Nebivolol, a third

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Journal of Hepatology 2011 vol. 54 | S209–S361