- Email: [email protected]
3 ACTIVATED INTESTINAL MACROPHAGES IN LIVER CIRRHOSIS PRODUCE NITRIC OXIDE AND DISRUPT INTESTINAL BARRIER FUNCTION
*p < 0.001, significant difference vs control (RVR and subgroups not tested); HCV RNA <25 IU/mL undetectable at 1 Week 4 (rapid virologic response [RVR]) and 2 24 weeks after planned end-of-treatment (sustained virologic response [SVR]); Pbo, placebo; PR, peginterferon a-2a/ribavirin; TMCx/PRy, TMC435 administered for x weeks and P/R administered for a total of y weeks,
3/16 (19) 2/23 (9) 10/27 (37) 10/17 (59) 19/22 (86) 23/26 (89) 7/17 (41) 18/24 (75) 24/27 (89) 9/17 (53) 15/23 (65) 20/26 (77) 9/16 (56) 11/23 (48) 23/26 (89) 6/16 (38) 16/23 (70) 24/27 (89)
8/18 (44) 12/22 (55) 20/26 (77)
0/16(0) 0/23(0) 1/27(4) 15/66 (23) 7/17(41) 15/22(68) 21/26(81) 52/65 (80)* 7/17(41) 16/24(67) 23/27(85) 49/68 (72)* 6/17(35) 15/23(65) 20/26(77) 44/66 (67)* 8/16(50) 9/23(39) 21/26(81) 43/65 (66)* 5/16(31) 15/23(65) 24/27(89) 46/66 (70)*
4/18(22) 14/22(64) 17/26(65) 40/66 (61)*
1/66(2) 43/65(66) 46/68(68) 41/66(62) 35/66(53) 38/65(59)
TMC12/PR48 TMC24/PR48 TMC48/PR48 TMC12/PR48 TMC24/PR48 TMC48/PR48 Pbo48/PR48 100 mg 100 mg 100 mg 150 mg 150 mg 150 mg (N = 66) (N = 66) (N = 65) (N = 66) (N = 66) (N = 68) (N = 65)
44/66(67)
RVR11 for total population Prior Null Responder Prior Partial Responder Prior Relapser SVR242 for total population Prior Null Responder Prior Partial Responder Prior Relapser
Table 1
Response, n/N (%)
ORAL PRESENTATIONS
Conclusion: Following treatment with TMC435 plus P/R, patients who failed previous P/R treatment exhibited significantly higher SVR24 rates compared with placebo, including difficult-to-treat prior null-responders with cirrhosis. TMC435 was well tolerated in this population. 3 ACTIVATED INTESTINAL MACROPHAGES IN LIVER CIRRHOSIS PRODUCE NITRIC OXIDE AND DISRUPT INTESTINAL BARRIER FUNCTION J. du Plessis1 , L. Roos1 , T. Slavik2,3 , P.I. Stivaktas4,5 , M. Nieuwoudt4 , S.G. van Wyk1 , W.A. Vieira6 , E. Pretorius7 , M. Beukes8 , S.W. van der Merwe1,9 . 1 Immunology, Hepatology and GI Research Laboratory, University of Pretoria, 2 Ampath Pathology Laboratories, 3 Anatomical Pathology, 4 Immunology, MRC Unit of Inflammation and Immunity, University of Pretoria, 5 Tshwane Academic Division of the National Health Laboratory Service, 6 Anatomy, 7 Physiology, 8 Biochemistry, University of Pretoria, Pretoria, South Africa; 9 Department of Hepatology, University Hospitals KU Leuven, Leuven, Belgium E-mail: [email protected] Introduction: Bacterial infections commonly occur in cirrhosis. Culture positive infections in addition to the presence of bacterial products and bacterial DNA alter the natural history of cirrhosis that can lead to loss of liver function and decompensation. Most S2
infections result from bacterial translocation (BT) from the intestine. The mechanisms and molecular pathways associated with BT are unknown. The aims of this study were to determine macrophage phenotype and molecular pathways associated with bacterial translocation as well as the structural integrity of the epithelial barrier at the level of the duodenum in liver cirrhosis. Patients and Methods: Fourty-four patients with NASH/ASH liver cirrhosis (decompensated n = 29, compensated n = 15 and controls n = 19) undergoing upper endoscopy were included in the study. Duodenal biopsies and serum samples were collected. Intestinal macrophages were characterized by flow cytometry and immunohistochemistry. NO production was measured in the supernatant of cultured biopsies. Gene expression analysis was performed on RNA isolated from duodenal samples assessing the expression of 84 inflammatory genes. Inflammatory cytokine determination was done in serum and culture supernatant by means of customized cytometric bead arrays. The duodenal wall was assessed by electron microscopy and the expression of tight junction proteins were assessed by qRT-PCR and western blot analysis. Results: Increased plasma LPS levels were observed in decompensated liver cirrhosis. Patients with decompensated cirrhosis also demonstrated increased numbers of activated duodenal macrophages expressing CD33+ /CD14+ /Trem-1+ , synthesizing iNOS and functionally secreting NO. Upregulation at the mRNA level of IL-8, CCL-2, CCL-13, NOS2 was found and increased IL-8, and IL-6 levels were detected in the serum and culture supernatant of patients with cirrhosis. In addition the epithelial barrier was not structurally altered by electron microscopy but increased expression of the “pore” forming tight junction Claudin-2 levels were detected by western blot. Conclusions: This study shows the presence of activated CD14+ Trem-1+ iNOS+ intestinal macrophages, and increased levels of NO, IL-6 and claudin-2 levels in the duodenum of patients with decompensated liver cirrhosis, suggesting that these factors enhance intestinal permeability to bacterial products. 4 DIFFERENT ROLES OF JNK1 AND JNK2 IN THE DEVELOPMENT OF NASH AND HCC F.J. Cubero1 , G. Zhao1 , Y.A. Nevzorova1 , M. Hatting1 , O. Drvarov1 , M. Al Masaoudi1 , N. Gassler2 , C. Liedtke1 , C. Trautwein1 . 1 Department of Medicine III, Unirversity Hospital RWTH Aachen, 2 Institute of Pathology, University Hospital RWTH Aachen, Aachen, Germany E-mail: [email protected] Background: Hepatocellular carcinoma (HCC) is the third largest cause of mortality wordwide. Nuclear factor (NF)-úB and the JNKs are pivotal regulators of tumor promotion. Indeed, ablation of the IKKg/Nemo subunit in hepatocytes (NemoDhepa ) of NF-úB triggers non-alcoholic steatohepatitis (NASH) and spontaneous HCC. Furthermore, phosphorylation of JNK in NemoDhepa mice suggests that the JNK signaling pathway is essential in the progression of the disease. Aims: To elucidate the role of the JNK genes in the development of hepatitis, steatosis, fibrosis and tumor initiation and development in a spontaneous model of NASH and HCC. Methods: We generated NemoDhepa /JNK1−/− and NemoDhepa /JNK2−/− and their respective controls and characterized their phenotype during progression of liver disease. Results: Genetic inactivation of JNK1 exacerbated the spontaneous hepatocyte death of NemoDhepa mice as observed by increased TUNEL positive cells, Caspase-3/8 activity and cleaved Caspase-3 expression. NemoDhepa /JNK1−/− livers revealed mitotic chaos associated with overexpression of cell cycle parameters such as
Journal of Hepatology 2012 vol. 56 | S1–S20
3/16 (19) 2/23 (9) 10/27 (37) 10/17 (59) 19/22 (86) 23/26 (89) 7/17 (41) 18/24 (75) 24/27 (89) 9/17 (53) 15/23 (65) 20/26 (77) 9/16 (56) 11/23 (48) 23/26 (89) 6/16 (38) 16/23 (70) 24/27 (89)
8/18 (44) 12/22 (55) 20/26 (77)
0/16(0) 0/23(0) 1/27(4) 15/66 (23) 7/17(41) 15/22(68) 21/26(81) 52/65 (80)* 7/17(41) 16/24(67) 23/27(85) 49/68 (72)* 6/17(35) 15/23(65) 20/26(77) 44/66 (67)* 8/16(50) 9/23(39) 21/26(81) 43/65 (66)* 5/16(31) 15/23(65) 24/27(89) 46/66 (70)*
4/18(22) 14/22(64) 17/26(65) 40/66 (61)*
1/66(2) 43/65(66) 46/68(68) 41/66(62) 35/66(53) 38/65(59)
TMC12/PR48 TMC24/PR48 TMC48/PR48 TMC12/PR48 TMC24/PR48 TMC48/PR48 Pbo48/PR48 100 mg 100 mg 100 mg 150 mg 150 mg 150 mg (N = 66) (N = 66) (N = 65) (N = 66) (N = 66) (N = 68) (N = 65)
44/66(67)
RVR11 for total population Prior Null Responder Prior Partial Responder Prior Relapser SVR242 for total population Prior Null Responder Prior Partial Responder Prior Relapser
Table 1
Response, n/N (%)
ORAL PRESENTATIONS
Conclusion: Following treatment with TMC435 plus P/R, patients who failed previous P/R treatment exhibited significantly higher SVR24 rates compared with placebo, including difficult-to-treat prior null-responders with cirrhosis. TMC435 was well tolerated in this population. 3 ACTIVATED INTESTINAL MACROPHAGES IN LIVER CIRRHOSIS PRODUCE NITRIC OXIDE AND DISRUPT INTESTINAL BARRIER FUNCTION J. du Plessis1 , L. Roos1 , T. Slavik2,3 , P.I. Stivaktas4,5 , M. Nieuwoudt4 , S.G. van Wyk1 , W.A. Vieira6 , E. Pretorius7 , M. Beukes8 , S.W. van der Merwe1,9 . 1 Immunology, Hepatology and GI Research Laboratory, University of Pretoria, 2 Ampath Pathology Laboratories, 3 Anatomical Pathology, 4 Immunology, MRC Unit of Inflammation and Immunity, University of Pretoria, 5 Tshwane Academic Division of the National Health Laboratory Service, 6 Anatomy, 7 Physiology, 8 Biochemistry, University of Pretoria, Pretoria, South Africa; 9 Department of Hepatology, University Hospitals KU Leuven, Leuven, Belgium E-mail: [email protected] Introduction: Bacterial infections commonly occur in cirrhosis. Culture positive infections in addition to the presence of bacterial products and bacterial DNA alter the natural history of cirrhosis that can lead to loss of liver function and decompensation. Most S2
infections result from bacterial translocation (BT) from the intestine. The mechanisms and molecular pathways associated with BT are unknown. The aims of this study were to determine macrophage phenotype and molecular pathways associated with bacterial translocation as well as the structural integrity of the epithelial barrier at the level of the duodenum in liver cirrhosis. Patients and Methods: Fourty-four patients with NASH/ASH liver cirrhosis (decompensated n = 29, compensated n = 15 and controls n = 19) undergoing upper endoscopy were included in the study. Duodenal biopsies and serum samples were collected. Intestinal macrophages were characterized by flow cytometry and immunohistochemistry. NO production was measured in the supernatant of cultured biopsies. Gene expression analysis was performed on RNA isolated from duodenal samples assessing the expression of 84 inflammatory genes. Inflammatory cytokine determination was done in serum and culture supernatant by means of customized cytometric bead arrays. The duodenal wall was assessed by electron microscopy and the expression of tight junction proteins were assessed by qRT-PCR and western blot analysis. Results: Increased plasma LPS levels were observed in decompensated liver cirrhosis. Patients with decompensated cirrhosis also demonstrated increased numbers of activated duodenal macrophages expressing CD33+ /CD14+ /Trem-1+ , synthesizing iNOS and functionally secreting NO. Upregulation at the mRNA level of IL-8, CCL-2, CCL-13, NOS2 was found and increased IL-8, and IL-6 levels were detected in the serum and culture supernatant of patients with cirrhosis. In addition the epithelial barrier was not structurally altered by electron microscopy but increased expression of the “pore” forming tight junction Claudin-2 levels were detected by western blot. Conclusions: This study shows the presence of activated CD14+ Trem-1+ iNOS+ intestinal macrophages, and increased levels of NO, IL-6 and claudin-2 levels in the duodenum of patients with decompensated liver cirrhosis, suggesting that these factors enhance intestinal permeability to bacterial products. 4 DIFFERENT ROLES OF JNK1 AND JNK2 IN THE DEVELOPMENT OF NASH AND HCC F.J. Cubero1 , G. Zhao1 , Y.A. Nevzorova1 , M. Hatting1 , O. Drvarov1 , M. Al Masaoudi1 , N. Gassler2 , C. Liedtke1 , C. Trautwein1 . 1 Department of Medicine III, Unirversity Hospital RWTH Aachen, 2 Institute of Pathology, University Hospital RWTH Aachen, Aachen, Germany E-mail: [email protected] Background: Hepatocellular carcinoma (HCC) is the third largest cause of mortality wordwide. Nuclear factor (NF)-úB and the JNKs are pivotal regulators of tumor promotion. Indeed, ablation of the IKKg/Nemo subunit in hepatocytes (NemoDhepa ) of NF-úB triggers non-alcoholic steatohepatitis (NASH) and spontaneous HCC. Furthermore, phosphorylation of JNK in NemoDhepa mice suggests that the JNK signaling pathway is essential in the progression of the disease. Aims: To elucidate the role of the JNK genes in the development of hepatitis, steatosis, fibrosis and tumor initiation and development in a spontaneous model of NASH and HCC. Methods: We generated NemoDhepa /JNK1−/− and NemoDhepa /JNK2−/− and their respective controls and characterized their phenotype during progression of liver disease. Results: Genetic inactivation of JNK1 exacerbated the spontaneous hepatocyte death of NemoDhepa mice as observed by increased TUNEL positive cells, Caspase-3/8 activity and cleaved Caspase-3 expression. NemoDhepa /JNK1−/− livers revealed mitotic chaos associated with overexpression of cell cycle parameters such as
Journal of Hepatology 2012 vol. 56 | S1–S20