- Email: [email protected]
273 FLUORESCENCE IN SITU HYBRIDIZATION (FISH) FOR THE DIAGNOSIS OF MALIGNANT PANCREATOBILIARY TRACT STRICTURES IN PATIENTS WITH JAUNDICE
S108
Poster Session − Thursday, April 23
with their originating tissues. Our data indicate that downregulation of the examined junctional proteins is characteristic of all biliary tract cancers, in contrast with claudins that were shown to be selectively preserved, or even upregulated, in specific types of biliary carcinoma. Hence, downregulation of ZO-1, occludin, and E-cadherin is supposed to be a common event in biliary carcinogenesis. Financial support: OTKA/49559 and 75468. 272 PHOSPHATIDYLINOSITIDE 3-KINASE (PI3K) MODULATES THE CANALICULAR TRANSPORTER INTERNALIZATION AND THE SECRETORY FAILURE INDUCED BY ESTRADIOL 17b-GLUCURONIDO (E17G): POSSIBLE ROLE FOR AKT A.C. Boaglio, A.E. Zucchetti, E.J. S´anchez Pozzi, A.D. Mottino, F.A. Crocenzi, M.G. Roma. Institute of Experimental Physiology (IFISE-CONICET), Fac. of Biochemical & Pharmacological Sciences − University of Rosario, Rosario, Argentina E-mail: [email protected] E17G is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion Bsep and Mrp2; this may justify its causal association with pregnancy-induced cholestasis. PI3K mediates cholestatic events, e.g. taurolithocholateinduced cholestasis (JBC 278: 17810, 2003). Since common pathomechanisms exist between both kinds of cholestasis, we assessed here whether PI3K is involved in E17G cholestatic effects. E17G (200 mM) activated PI3K from 20 min onwards, as assessed by the phosphorylation of the final PI3K effector Akt in primary cultured hepatocytes. When preadministered to isolated rat hepatocyte couplets (IRHCs), the PI3K inhibitor wortmannin (WM; 100 nM) prevented partially the reduction induced by E17G (12.5–800 mM) in the proportion of IRHCs accumulating apically the fluorescent Bsep and Mrp2 substrates cholyllysylfluorescein and glutathionylmethylfluorescein, respectively. The alternative PI3K inhibitor LY294002 (50 mM) and the “Akt inhibitor” (Calbiochem® #124005; 20 mM) showed similar protective effects. Immunostaining of Bsep and Mrp2 in IRHC followed by confocal microscopy and image analysis revealed that E17G induces endocytic internalization of the transporters. This redistribution was extensively prevented by WM. To evaluate this phenomenon in a more physiological model, we assessed the anticholestatic effect of WM in perfused rat livers. A bolus, intraportal injection of E17G (2 mmol) induced an acute decrease in bile flow within 10 min, which did not recover during the remaining perfusion period (Figure 1). WM (100 nM) did not prevent this initial decay, but greatly accelerated the recovery to normality of bile flow. A similar behaviour was observed for the biliary excretion of the Bsep and Mrp2 substrates [3 H]taurocholate and glutathione, respectively.
Figure 1.
We conclude that PI3K/Akt signalling pathway is involved in the the biliary secretory failure induced by E17G by producing a sustained internalization of canalicular transporters. PI3K/Akt may therefore be a potential therapeutic target in cholestatic phenomena associated to estrogens. 273 FLUORESCENCE IN SITU HYBRIDIZATION (FISH) FOR THE DIAGNOSIS OF MALIGNANT PANCREATOBILIARY TRACT STRICTURES IN PATIENTS WITH JAUNDICE M. Sartori1 , M. Orsello1 , F. Montino1 , M. Ballar`e1 , M. Balzarini1 , E. Garello1 , R. Boldorini2 , A. Paganotti2 , S. Andorno1 , M. Del Piano1 . 1 Gastroenterology Unit. AOU Maggiore della Carit` a., 2 Department of Medical Sciences, Universit`a del Piemonte Orientale ‘Amedeo Avogadro’, Novara, Italy E-mail: [email protected] Aims: The aim of this study was to assess the relative sensitivities and specificities of FISH and routine cytology for the detection of malignancy in biliary tract strictures in patients with extrahepatic jaundice. Methods: Brush cytological specimens from 32 consecutive jaundiced patients (20 males and 12 females, mean age 68±11 years) undergoing endoscopic retrograde cholangiopancreatography for pancreatobiliary strictures were examined by cytology and FISH. With FISH, a case was considered positive for malignancy if five or more cells exhibited polysomy. Results: Twenty-two of 32 patients had surgical pathologic and/or clinical evidence of malignancy (17 cholangiocarcinomas, 5 pancreatic carcinomas) and 10 had benign strictures. Conventional cytology had 59% sensitivity but 100% specificity (PPV 100%, NPV 52%). FISH showed a very high sensitivity (95%) while maintaining 90% specificity (PPV 95%, NPV 90%). Conventional cytology and FISH sensitivity, specificity, PPV, NPV for the detection of cholangiocarcinoma were respectively 58%, 100%, 100%, 58% and 94%, 90%, 94%, 90%. Conclusions: FISH increases the sensitivity for the diagnosis of malignant pancreatobiliary tract strictures in comparison with conventional cytology while maintaining good specificity. The excellent diagnostic efficacy of both conventional cytology and FISH in our patient series could possibly be due to the recruitment of jaundiced subjects with advanced malignant disease. 274 DEFICIENCY OF NIEMANN-PICK C1 PROTEIN EXPRESSION PROTECTS AGAINST DIET-INDUCED GALLSTONE FORMATION IN MICE M.G. Morales, L. Amigo, J. Castro, F. Nervi, A. Rigotti, S. Zanlungo. Departamento de Gastroenterologia, Facultad de Medicina, Pontificia Universidad Catolica de Chile, Santiago, Chile E-mail: [email protected] Background and Aims: Receptor-mediated endocytosis is a relevant mechanism for uptake of lipoprotein cholesterol in the liver. Since Niemann-Pick C1 (NPC1) protein is a key component for the intracellular distribution of cholesterol obtained from lipoproteins endocytosis, it may play a critical role in controlling cholesterol biliary secretion and gallstone formation induced by a lithogenic diet. Methods: We studied biliary cholesterol secretion, gallbladder lipid composition, and gallstone formation in NPC1-deficient, NPC1 (−/−), mice fed a lithogenic diet (containing 1.5% cholesterol and 0.5% cholic acid) compared with control animals under the same diet. Results: The lipid secretion response to the lithogenic diet was impaired in NPC1 (−/−) mice, leading to a decrease in cholesterol output and to an increase in hepatic cholesterol concentration compared to lithogenic diet fed wild-type mice. In contrast, the hepatic gene expression response to the lithogenic diet was not impaired in the NPC1 (−/−) mice, leading to an increase in the mRNA levels of the cholesterol canalicular transporters Abcg5 and Abcg8. A decreased cholesterol saturation index was found in gallbladder bile of NPC1 (−/−) mice. Consequently, NPC1 (−/−) mice fed a lithogenic diet had a drastically lower frequency of gallbladder cholesterol crystals and reduced prevalence of gallstones.
Poster Session − Thursday, April 23
with their originating tissues. Our data indicate that downregulation of the examined junctional proteins is characteristic of all biliary tract cancers, in contrast with claudins that were shown to be selectively preserved, or even upregulated, in specific types of biliary carcinoma. Hence, downregulation of ZO-1, occludin, and E-cadherin is supposed to be a common event in biliary carcinogenesis. Financial support: OTKA/49559 and 75468. 272 PHOSPHATIDYLINOSITIDE 3-KINASE (PI3K) MODULATES THE CANALICULAR TRANSPORTER INTERNALIZATION AND THE SECRETORY FAILURE INDUCED BY ESTRADIOL 17b-GLUCURONIDO (E17G): POSSIBLE ROLE FOR AKT A.C. Boaglio, A.E. Zucchetti, E.J. S´anchez Pozzi, A.D. Mottino, F.A. Crocenzi, M.G. Roma. Institute of Experimental Physiology (IFISE-CONICET), Fac. of Biochemical & Pharmacological Sciences − University of Rosario, Rosario, Argentina E-mail: [email protected] E17G is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion Bsep and Mrp2; this may justify its causal association with pregnancy-induced cholestasis. PI3K mediates cholestatic events, e.g. taurolithocholateinduced cholestasis (JBC 278: 17810, 2003). Since common pathomechanisms exist between both kinds of cholestasis, we assessed here whether PI3K is involved in E17G cholestatic effects. E17G (200 mM) activated PI3K from 20 min onwards, as assessed by the phosphorylation of the final PI3K effector Akt in primary cultured hepatocytes. When preadministered to isolated rat hepatocyte couplets (IRHCs), the PI3K inhibitor wortmannin (WM; 100 nM) prevented partially the reduction induced by E17G (12.5–800 mM) in the proportion of IRHCs accumulating apically the fluorescent Bsep and Mrp2 substrates cholyllysylfluorescein and glutathionylmethylfluorescein, respectively. The alternative PI3K inhibitor LY294002 (50 mM) and the “Akt inhibitor” (Calbiochem® #124005; 20 mM) showed similar protective effects. Immunostaining of Bsep and Mrp2 in IRHC followed by confocal microscopy and image analysis revealed that E17G induces endocytic internalization of the transporters. This redistribution was extensively prevented by WM. To evaluate this phenomenon in a more physiological model, we assessed the anticholestatic effect of WM in perfused rat livers. A bolus, intraportal injection of E17G (2 mmol) induced an acute decrease in bile flow within 10 min, which did not recover during the remaining perfusion period (Figure 1). WM (100 nM) did not prevent this initial decay, but greatly accelerated the recovery to normality of bile flow. A similar behaviour was observed for the biliary excretion of the Bsep and Mrp2 substrates [3 H]taurocholate and glutathione, respectively.
Figure 1.
We conclude that PI3K/Akt signalling pathway is involved in the the biliary secretory failure induced by E17G by producing a sustained internalization of canalicular transporters. PI3K/Akt may therefore be a potential therapeutic target in cholestatic phenomena associated to estrogens. 273 FLUORESCENCE IN SITU HYBRIDIZATION (FISH) FOR THE DIAGNOSIS OF MALIGNANT PANCREATOBILIARY TRACT STRICTURES IN PATIENTS WITH JAUNDICE M. Sartori1 , M. Orsello1 , F. Montino1 , M. Ballar`e1 , M. Balzarini1 , E. Garello1 , R. Boldorini2 , A. Paganotti2 , S. Andorno1 , M. Del Piano1 . 1 Gastroenterology Unit. AOU Maggiore della Carit` a., 2 Department of Medical Sciences, Universit`a del Piemonte Orientale ‘Amedeo Avogadro’, Novara, Italy E-mail: [email protected] Aims: The aim of this study was to assess the relative sensitivities and specificities of FISH and routine cytology for the detection of malignancy in biliary tract strictures in patients with extrahepatic jaundice. Methods: Brush cytological specimens from 32 consecutive jaundiced patients (20 males and 12 females, mean age 68±11 years) undergoing endoscopic retrograde cholangiopancreatography for pancreatobiliary strictures were examined by cytology and FISH. With FISH, a case was considered positive for malignancy if five or more cells exhibited polysomy. Results: Twenty-two of 32 patients had surgical pathologic and/or clinical evidence of malignancy (17 cholangiocarcinomas, 5 pancreatic carcinomas) and 10 had benign strictures. Conventional cytology had 59% sensitivity but 100% specificity (PPV 100%, NPV 52%). FISH showed a very high sensitivity (95%) while maintaining 90% specificity (PPV 95%, NPV 90%). Conventional cytology and FISH sensitivity, specificity, PPV, NPV for the detection of cholangiocarcinoma were respectively 58%, 100%, 100%, 58% and 94%, 90%, 94%, 90%. Conclusions: FISH increases the sensitivity for the diagnosis of malignant pancreatobiliary tract strictures in comparison with conventional cytology while maintaining good specificity. The excellent diagnostic efficacy of both conventional cytology and FISH in our patient series could possibly be due to the recruitment of jaundiced subjects with advanced malignant disease. 274 DEFICIENCY OF NIEMANN-PICK C1 PROTEIN EXPRESSION PROTECTS AGAINST DIET-INDUCED GALLSTONE FORMATION IN MICE M.G. Morales, L. Amigo, J. Castro, F. Nervi, A. Rigotti, S. Zanlungo. Departamento de Gastroenterologia, Facultad de Medicina, Pontificia Universidad Catolica de Chile, Santiago, Chile E-mail: [email protected] Background and Aims: Receptor-mediated endocytosis is a relevant mechanism for uptake of lipoprotein cholesterol in the liver. Since Niemann-Pick C1 (NPC1) protein is a key component for the intracellular distribution of cholesterol obtained from lipoproteins endocytosis, it may play a critical role in controlling cholesterol biliary secretion and gallstone formation induced by a lithogenic diet. Methods: We studied biliary cholesterol secretion, gallbladder lipid composition, and gallstone formation in NPC1-deficient, NPC1 (−/−), mice fed a lithogenic diet (containing 1.5% cholesterol and 0.5% cholic acid) compared with control animals under the same diet. Results: The lipid secretion response to the lithogenic diet was impaired in NPC1 (−/−) mice, leading to a decrease in cholesterol output and to an increase in hepatic cholesterol concentration compared to lithogenic diet fed wild-type mice. In contrast, the hepatic gene expression response to the lithogenic diet was not impaired in the NPC1 (−/−) mice, leading to an increase in the mRNA levels of the cholesterol canalicular transporters Abcg5 and Abcg8. A decreased cholesterol saturation index was found in gallbladder bile of NPC1 (−/−) mice. Consequently, NPC1 (−/−) mice fed a lithogenic diet had a drastically lower frequency of gallbladder cholesterol crystals and reduced prevalence of gallstones.