- Email: [email protected]
O135 INHIBITION OF INTESTINAL BILE ACID ABSORPTION BY ASBT INHIBITOR A4250 PROTECTS AGAINST BILE ACID-MEDIATED CHOLESTATIC LIVER INJURY IN MICE
ORAL PRESENTATIONS Table 3 (abstract O134). Biliary epithelial cell (BEC) activation, % expression (n = 5)
ICAM-1 HLA-DR CD40
CD4+CD28−ve
CD4+CD28+ve
CD8+CD28−ve
CD8+CD28+ve
33.43±16 52.1±15.6 33.22±18.3
9.34±4.2 33.54±6.06 30.1±16.6
28.4±13.4 46.59±17.01 46.9±14.3
16.17±5.7 49.9±14.2 34.4±16.5
Table 4 (abstract O134). CD28−ve frequency (after 21 days in culture) a
% CD28*−ve
a
Significance
Untreated
1,25(OH)2 D3
TNFa
TNFa+1,25(OH)2 D3
22.94±6.34
1.96±0.8
37.6±5.2
3.66±1.29
*p < 0.05 (Untreated vs 1,25(OH)2 D3 **p < 0.01 (1,25(OH)2 D3 vs TNFa) *p < 0.05 (TNFa vs TNFa+1,25(OH)2 D3 )
All cells were treated initially with aCD3/aCD28 beads and IL-2 (50 U/ml) was supplemented in culture.
Conclusions: A high proportion of CD28−ve T cells are present in PSC liver. Vitamin D abrogates the pathogenic effects of CD28−ve cells representing a therapeutic opportunity. O135 INHIBITION OF INTESTINAL BILE ACID ABSORPTION BY ASBT INHIBITOR A4250 PROTECTS AGAINST BILE ACID-MEDIATED CHOLESTATIC LIVER INJURY IN MICE 1 A. Baghdasaryan1 , P. Jha1 , M. Muller ¨ , N. Auer1 , A. Deutschmann2 , 2 3 3 C. Zohrer ¨ , I. Pahlman ˚ , H. Graffner , P. Fickert2 , M. Trauner1 . 1 Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, 2 Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; 3 Albireo, Gothenburg, Sweden E-mail: [email protected]
Background and Aims: Approximately 95% of biliary bile acids (BAs) are reabsorbed in the gut and circulate back to the liver for further secretion into bile. Therefore, pharmacological inhibition of the ileal apical sodium-dependent BA transporter (ASBT/SLC10A2) may protect against BA-mediated liver injury in cholestasis. Methods: 8-week-old Mdr2−/− (Abcb4−/− ) mice (model of cholestatic liver injury and sclerosing cholangitis) received either a diet supplemented with A4250 (0.01% w/w) – a highly potent and selective ASBT inhibitor – or a chow diet and liver injury was assessed after 4 weeks. Bile flow and composition were analyzed after 1 week. As a complementary model, wild type mice received A4250-supplemented diet over 1 week before and after common bile duct ligation (CBDL). Results: Compared with chow-fed Mdr2−/− mice A4250 significantly reduced serum ALT (111±22 vs. 484±214 U/L), ALP (122±9 vs. 210±47 U/L) and serum BAs (25±6 vs. 86±30 mmol/L), inhibited proinflammatory (Tnf-a, Vcam-1, Mcp-1) and profibrogenic (Col1a1, Col1a2) gene expression and reduced bile duct proliferation (mRNA and immunohistochemistry for K19). Furthermore, A4250 significantly reduced bile flow (0.9±0.2 vs. 1.7±0.2 ml/gLW/min) and biliary BA output (3.1±1.6 vs. 35.6±13.0 mmol/L/gLW/min), which correlated with Bsep repression, while Ntcp and Cyp7a1 were induced. In CBDL mice, A4250 reduced serum ALP (711±212 vs. 1278±355 U/L, p < 0.05) and BA levels (168.8±57.7 vs. 249.8±141.3 mmol/L n.s.). Conclusions: Pharmacological ASBT inhibition attenuates cholestatic liver injury by reducing biliary BA output.
O136 A NOVEL, HYBRID RECOMBINANT AAV-piggyBac TRANSPOSON VECTOR PERMITS ROBUST LONG-TERM PHENOTYPE CORRECTION OF THE PROGRESSIVE FAMILIAL INTRAHEPATIC CHOLESTASIS TYPE 3 MOUSE MODEL IN VIVO S.M. Siew1,2 , S.C. Cunningham1 , I.E. Alexander1 . 1 Gene Therapy Research Unit, Children’s Medical Research Institute, 2 Department of Gastroenterology, The Children’s Hospital at Westmead, Sydney, NSW, Australia E-mail: [email protected] Background and Aims: Efficient liver-targeted gene transfer using recombinant adeno-associated viral (rAAV) vectors has led to promising therapeutic efficacy in pre-clinical models and clinical trials. However, delivery of conventional, predominantly nonintegrating rAAV vectors to juvenile mice results in loss of persistent transgene expression in the liver due to rapid liver growth and hepatocyte proliferation. The piggyBac transposon system permits stable genomic modification of mammalian cells, but targeted delivery is relatively inefficient in vivo. Aim: To develop a hybrid vector, utilising high transduction efficiency of rAAV together with piggyBac transposase-mediated somatic integration to stably express human ABCB4 under a hepatocyte-specific promoter/enhancer in vivo, and to correct chronic liver disease in a murine model of Progressive Familial Intrahepatic Cholestasis type 3, lacking canalicular phosphatidylcholine translocation.
Figure: Comparison of biliary phosphatidylcholine concentrations.
Methods: Neonatal Abcb4−/− mice were injected intraperitoneally with 5×1011 vector genomes of the hybrid vector, co-administered with a rAAV expressing piggyBac transposase (pBase+ ), and were Journal of Hepatology 2014 vol. 60 | S45–S66
S57
ICAM-1 HLA-DR CD40
CD4+CD28−ve
CD4+CD28+ve
CD8+CD28−ve
CD8+CD28+ve
33.43±16 52.1±15.6 33.22±18.3
9.34±4.2 33.54±6.06 30.1±16.6
28.4±13.4 46.59±17.01 46.9±14.3
16.17±5.7 49.9±14.2 34.4±16.5
Table 4 (abstract O134). CD28−ve frequency (after 21 days in culture) a
% CD28*−ve
a
Significance
Untreated
1,25(OH)2 D3
TNFa
TNFa+1,25(OH)2 D3
22.94±6.34
1.96±0.8
37.6±5.2
3.66±1.29
*p < 0.05 (Untreated vs 1,25(OH)2 D3 **p < 0.01 (1,25(OH)2 D3 vs TNFa) *p < 0.05 (TNFa vs TNFa+1,25(OH)2 D3 )
All cells were treated initially with aCD3/aCD28 beads and IL-2 (50 U/ml) was supplemented in culture.
Conclusions: A high proportion of CD28−ve T cells are present in PSC liver. Vitamin D abrogates the pathogenic effects of CD28−ve cells representing a therapeutic opportunity. O135 INHIBITION OF INTESTINAL BILE ACID ABSORPTION BY ASBT INHIBITOR A4250 PROTECTS AGAINST BILE ACID-MEDIATED CHOLESTATIC LIVER INJURY IN MICE 1 A. Baghdasaryan1 , P. Jha1 , M. Muller ¨ , N. Auer1 , A. Deutschmann2 , 2 3 3 C. Zohrer ¨ , I. Pahlman ˚ , H. Graffner , P. Fickert2 , M. Trauner1 . 1 Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, 2 Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; 3 Albireo, Gothenburg, Sweden E-mail: [email protected]
Background and Aims: Approximately 95% of biliary bile acids (BAs) are reabsorbed in the gut and circulate back to the liver for further secretion into bile. Therefore, pharmacological inhibition of the ileal apical sodium-dependent BA transporter (ASBT/SLC10A2) may protect against BA-mediated liver injury in cholestasis. Methods: 8-week-old Mdr2−/− (Abcb4−/− ) mice (model of cholestatic liver injury and sclerosing cholangitis) received either a diet supplemented with A4250 (0.01% w/w) – a highly potent and selective ASBT inhibitor – or a chow diet and liver injury was assessed after 4 weeks. Bile flow and composition were analyzed after 1 week. As a complementary model, wild type mice received A4250-supplemented diet over 1 week before and after common bile duct ligation (CBDL). Results: Compared with chow-fed Mdr2−/− mice A4250 significantly reduced serum ALT (111±22 vs. 484±214 U/L), ALP (122±9 vs. 210±47 U/L) and serum BAs (25±6 vs. 86±30 mmol/L), inhibited proinflammatory (Tnf-a, Vcam-1, Mcp-1) and profibrogenic (Col1a1, Col1a2) gene expression and reduced bile duct proliferation (mRNA and immunohistochemistry for K19). Furthermore, A4250 significantly reduced bile flow (0.9±0.2 vs. 1.7±0.2 ml/gLW/min) and biliary BA output (3.1±1.6 vs. 35.6±13.0 mmol/L/gLW/min), which correlated with Bsep repression, while Ntcp and Cyp7a1 were induced. In CBDL mice, A4250 reduced serum ALP (711±212 vs. 1278±355 U/L, p < 0.05) and BA levels (168.8±57.7 vs. 249.8±141.3 mmol/L n.s.). Conclusions: Pharmacological ASBT inhibition attenuates cholestatic liver injury by reducing biliary BA output.
O136 A NOVEL, HYBRID RECOMBINANT AAV-piggyBac TRANSPOSON VECTOR PERMITS ROBUST LONG-TERM PHENOTYPE CORRECTION OF THE PROGRESSIVE FAMILIAL INTRAHEPATIC CHOLESTASIS TYPE 3 MOUSE MODEL IN VIVO S.M. Siew1,2 , S.C. Cunningham1 , I.E. Alexander1 . 1 Gene Therapy Research Unit, Children’s Medical Research Institute, 2 Department of Gastroenterology, The Children’s Hospital at Westmead, Sydney, NSW, Australia E-mail: [email protected] Background and Aims: Efficient liver-targeted gene transfer using recombinant adeno-associated viral (rAAV) vectors has led to promising therapeutic efficacy in pre-clinical models and clinical trials. However, delivery of conventional, predominantly nonintegrating rAAV vectors to juvenile mice results in loss of persistent transgene expression in the liver due to rapid liver growth and hepatocyte proliferation. The piggyBac transposon system permits stable genomic modification of mammalian cells, but targeted delivery is relatively inefficient in vivo. Aim: To develop a hybrid vector, utilising high transduction efficiency of rAAV together with piggyBac transposase-mediated somatic integration to stably express human ABCB4 under a hepatocyte-specific promoter/enhancer in vivo, and to correct chronic liver disease in a murine model of Progressive Familial Intrahepatic Cholestasis type 3, lacking canalicular phosphatidylcholine translocation.
Figure: Comparison of biliary phosphatidylcholine concentrations.
Methods: Neonatal Abcb4−/− mice were injected intraperitoneally with 5×1011 vector genomes of the hybrid vector, co-administered with a rAAV expressing piggyBac transposase (pBase+ ), and were Journal of Hepatology 2014 vol. 60 | S45–S66
S57