- Email: [email protected]
Osmolyte-strategy of Ito-cells during their transformation into myofibroblasts
128
Posters
1 P/CO7/019 P53 MUTATIONS IN DYSPLASTIC AND NEOPLASTIC BILE DUCT EPITHELIUM IN PRIMARY SCLEROSING CHOLANGITIS . . S Rydm G. Vautier+.s Departments of Gastroenterology+ and Pathology*, Queens Medical Centre, Nottingham and Institute of Liver Studies, King’s College Hospital, London. UK. We have previously shown that ~53 mutations are common in cholangiocarcinomas arising in patients with sclerosing cholangitis but the site of mutations within the ~53 gene in these patients and the point in the progression from normal bile duct to cancer where these mutations occur has not been described. 21 cases of cholangiocarcinoma, 6 cases of biliary dysplasia not amounting to invasive carcinoma and 12 non-dysplastic bile duct samples were obtained from patients at the time of surgery or liver transplantation. p53 mutations were sought using single stranded polymorphism analysis and direct sequencing of exons 5,6,7 and 8 of the p53 gene using DNA extracted from parafin embedded material. 14/21 (66%) cholangiocarcinomas had mutations within the p53 gene. These were all point mutations but no clustering of mutations was seen at any specific codon. 3/6 (50%) dysplastic epithelial samples contained a ~53 point mutation but none of 12 nondysplastic epithelial samples had p53 gene mutations. This study confirms that ~53 mutations are common in cholangiocarcinomas associated with primary sclerosing cholangitis and shows that these gene alterations occur at a relatively early stage in neoplastic change in the biliary epithelium. This suggests that ~53 mutations may be a useful target for screening patients with primary sclerosing cholangitis for the development of cholangiocarcinoma.
I P/CO7/018
I
METHOTREXATE FOR TREATMENT OF PRIMARY CHOLANGITIS. A Placebo-controlled Cross-over Trial
!STUDYGFlXIElMMUNCXTIQIGIYIXGF44INlXA-OR EXlBA-HEpATLc CHoL4NGloCARCNoMAs. ARullizM.Wintx&RF~C.~P.BoulacSaee.B.LeBail, S&e cIAn&& palhobgique,H&&l F+z&@, ad Laboratoire de Pathologie-GREF, Universitt? Bordeaux 2, France. Cholangiocarcinomas (CC) are malignant tumors of the biliary epithelium which express epithelial markers such as cytokeratins (CK)19 and 7, epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA). In the liver, the CK7+/ CK20- profile of peripheral CC would be useful for their distinction from colorectal metastasis (CK7-/CK20+)*. However, CC represent a heterogeneous group of tumors due to their different location in the biliary tree and to various histological features. Therefore, we sought to investigate the immunophenotype of a series of 44 CC from different locations (periphery: 19, intrahepatic large ducts:6, convergence: 10, extrahepatic ductsg), and with various architecture, differentiation and mucosecretion. Tumors were immunostained for CKs KLl (pan-keratin), 7, 19, and 20, EMA and CEAp. Results were semi-quantitated and statistically analyzed. An intense staining (>50% positive cells) was obtained in most of the tumors for CK7 (96%) and KLl (86%), but only in 64% of cases for CK19, EMA and CEA; no relation was found between the intensity of these stainings and the location, architecture, differentiation nor grade of the tumors. CK20 was positive in 7 1% cases but rarely intense (21%). Proximal CC (convergence, extra- or intra-hepatic ducts) were more frequently CK20+ (88%), than peripheral CC (52%) (p=O.O03) but an intense staining could be observed in both groups (21 and 20%. respectively). In conclusion, whatever their location, CC can express not only a classical CK7+/CK20-, but also a CK7+/CK20+phenotype. However, CK20 positivity is less frequent in peripheral CC. *Mod Path01 1996,9:901-909.
1 P/C67/020 SCLEROSING
Rasmussen’. U. Taee-Jensen’. M. Vvber$, P. Schlicbtine3, 0. Schaffalitzkv de MucadeW. 0. Bonnevie’. Dept. M’, and Institute of Patholod, Aalborg Hospital, Dept. A3, State University Hospital, Dept. S4, Odense University Hospital Dept. BS, Frederiksberg Hospital, Denmark H.H.
Although a number of medical treatments for primary sclerosing cholangitis (PSC) have been evaluated, no one has proven effective. This study was a multicentre, double-blind, randomisedcross-over design betweenmethotrexate (mtx)(lO mg/m* body area/wk in one dose) and placebo during a 2-year period. Inclusion criteria: I) Alkaline phosphatase > 6 times upper normal limit (>I650 U/L) on at least two occasions, more than 4 weeks apart within the last year and/or 2) alkaline phosphatase > 3 times upper normal limit and one or more of the following criteria: a) pruritus, b) pain in the upper right abdomen, c) jaundice (bilirubin > 2 times upper normal limit), d) suppurative cholangitis. e) weightloss (> 5%), and fJ fibrosis (liver biopsy). At baseline, after 12 and 24 months ERC, liver biopsy, liver tests (bilirubin, ALP, ASAT, albumin, prothrombin), hematological tests, SR, creatinine, autoantibodies/immunog~obuIins, chest X-ray and lung function test were done. Every second month liver tests, hematological tests, creatinine and symptom scores by visuel analogue scale (fatigue, pruritus, and right-upperquadrant pain) were done. Results: During the first 12 months of the study period, eight patients recieved placebo and five active treatment. Drop-outs: two patients died after 18 and 22 months because of liver failure and sepsis, two patients because of non-compliance (after 16 and 12 months). During mtx-therapy we did not observe any known adverse events related to the study-medication as measured by the safety parameters (heamatology, lungfunction). The mtxdose in this study was 15-20 mg/wk. A cross-over (Splitplot ANOVA) analysis did not reveal any statistical differences between mtx and placebo according to liver tests, symptomscore, lever histological stage, and ERC changes. No carry-over- or time-effect was observed. Conclusion: In this 2-year double-blind-cross-over study, methotrexate did not seem to be effective. This is in accordance with other studies. Whether methotrexate might be effective in long-term studies or in association with other immunosuppresives is unknown.
1
1
OSMOLYTE-STRATEGY OF ITO-CELLS DURING THEIR TRANSFORMATION INTO MYOFIBROBLASTS.
-
I..’
Medzinische Universitiuklimk, Klinik Mwrenstr 5, D-40225 Diisseldorf
HPussinge Wr Gastmenterologie,
Hepatologle
und Infektiologle,
Introduction: Organic osmolytes are compounds, which are specifically accumulated in or released from cells in response to cell shrinkage or swelling, respectively in order to maintain cell volume. Anisoosmotic cell volume changes were identified as a critical parameter in the regulation of cell functions. The present investigation was undertaken to enlighten a possible osmolyte-strategy in non-activated and activated Ito-cells, which play an important role in the pathogenesis of liver diseases. &&Q& Ito-cells were isolated from male Sprague Dawley rats by pronaaecollagenax perfusion and separated by single Nycodenz gradient. They were cultured in DMEM plus 10% FCS. For measuring osmolyte-uptake 2,7 and I4 days old cells were incubated in hyper-, normo- and hypoosmotic media for 16 h and then betaine, taurine, or myo-inositol was given. For shldies of osmolyte-ef?lux 2 and 14 days old cells were preloaded with osmolytes (betaine, taurine or myo-inositol) under hyperosmotic conditions, and then efflux was measured after hypoosmotic exposure. In parallel a-smooth muscle actin was Investigated by immunhistochemishy and quantified on Western blots for correlation of osmolyte-strategy and state of transformation. ti Hyperosmotic (hypoosmotic) exposure of Ito-cells after 2,7 and I4 days of cultivation led to an increase (decrease) in osmolyte transport into the cells, whereby taurine was prefemed, when compared to betaine and myo-inositol. The uptake of myo-inositol reached a maximum until day 7 of cultivation. In contrast the uptake of betaine and taurine increased until day 14 (2-fold and 6-fold, compared to day 2), in parallel to the transformation of Ito-cells into myofibroblasts, shown by an increasing expression of a-smooth muscle actin, as parameter of Ito-cell transformation. In Ito-cells no a-smooth muscle actin could be detected after 2 days, whereas 7 days old cells showed a distinct signal, which was markedly increased at day I4 of cultivation. when Ito-cells were preloaded with osmolytes, lowering of the extracellular osmolarity was followed by a rapid osmolyte emox from the cells, concerning betaine, taurine and myo-inositol. Conclusions: The results indicate, that betaine, taurme, and myo-inositol acts as osmolytes in non-activated Ito-cells as well as in actwated cells. In correlation to the level of a-smooth muscle actin expression, osmolyte-uptake was markedly enhanced. This could serve as a farther parameter for the status of Ito-cell transformation.
Posters
1 P/CO7/019 P53 MUTATIONS IN DYSPLASTIC AND NEOPLASTIC BILE DUCT EPITHELIUM IN PRIMARY SCLEROSING CHOLANGITIS . . S Rydm G. Vautier+.s Departments of Gastroenterology+ and Pathology*, Queens Medical Centre, Nottingham and Institute of Liver Studies, King’s College Hospital, London. UK. We have previously shown that ~53 mutations are common in cholangiocarcinomas arising in patients with sclerosing cholangitis but the site of mutations within the ~53 gene in these patients and the point in the progression from normal bile duct to cancer where these mutations occur has not been described. 21 cases of cholangiocarcinoma, 6 cases of biliary dysplasia not amounting to invasive carcinoma and 12 non-dysplastic bile duct samples were obtained from patients at the time of surgery or liver transplantation. p53 mutations were sought using single stranded polymorphism analysis and direct sequencing of exons 5,6,7 and 8 of the p53 gene using DNA extracted from parafin embedded material. 14/21 (66%) cholangiocarcinomas had mutations within the p53 gene. These were all point mutations but no clustering of mutations was seen at any specific codon. 3/6 (50%) dysplastic epithelial samples contained a ~53 point mutation but none of 12 nondysplastic epithelial samples had p53 gene mutations. This study confirms that ~53 mutations are common in cholangiocarcinomas associated with primary sclerosing cholangitis and shows that these gene alterations occur at a relatively early stage in neoplastic change in the biliary epithelium. This suggests that ~53 mutations may be a useful target for screening patients with primary sclerosing cholangitis for the development of cholangiocarcinoma.
I P/CO7/018
I
METHOTREXATE FOR TREATMENT OF PRIMARY CHOLANGITIS. A Placebo-controlled Cross-over Trial
!STUDYGFlXIElMMUNCXTIQIGIYIXGF44INlXA-OR EXlBA-HEpATLc CHoL4NGloCARCNoMAs. ARullizM.Wintx&RF~C.~P.BoulacSaee.B.LeBail, S&e cIAn&& palhobgique,H&&l F+z&@, ad Laboratoire de Pathologie-GREF, Universitt? Bordeaux 2, France. Cholangiocarcinomas (CC) are malignant tumors of the biliary epithelium which express epithelial markers such as cytokeratins (CK)19 and 7, epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA). In the liver, the CK7+/ CK20- profile of peripheral CC would be useful for their distinction from colorectal metastasis (CK7-/CK20+)*. However, CC represent a heterogeneous group of tumors due to their different location in the biliary tree and to various histological features. Therefore, we sought to investigate the immunophenotype of a series of 44 CC from different locations (periphery: 19, intrahepatic large ducts:6, convergence: 10, extrahepatic ductsg), and with various architecture, differentiation and mucosecretion. Tumors were immunostained for CKs KLl (pan-keratin), 7, 19, and 20, EMA and CEAp. Results were semi-quantitated and statistically analyzed. An intense staining (>50% positive cells) was obtained in most of the tumors for CK7 (96%) and KLl (86%), but only in 64% of cases for CK19, EMA and CEA; no relation was found between the intensity of these stainings and the location, architecture, differentiation nor grade of the tumors. CK20 was positive in 7 1% cases but rarely intense (21%). Proximal CC (convergence, extra- or intra-hepatic ducts) were more frequently CK20+ (88%), than peripheral CC (52%) (p=O.O03) but an intense staining could be observed in both groups (21 and 20%. respectively). In conclusion, whatever their location, CC can express not only a classical CK7+/CK20-, but also a CK7+/CK20+phenotype. However, CK20 positivity is less frequent in peripheral CC. *Mod Path01 1996,9:901-909.
1 P/C67/020 SCLEROSING
Rasmussen’. U. Taee-Jensen’. M. Vvber$, P. Schlicbtine3, 0. Schaffalitzkv de MucadeW. 0. Bonnevie’. Dept. M’, and Institute of Patholod, Aalborg Hospital, Dept. A3, State University Hospital, Dept. S4, Odense University Hospital Dept. BS, Frederiksberg Hospital, Denmark H.H.
Although a number of medical treatments for primary sclerosing cholangitis (PSC) have been evaluated, no one has proven effective. This study was a multicentre, double-blind, randomisedcross-over design betweenmethotrexate (mtx)(lO mg/m* body area/wk in one dose) and placebo during a 2-year period. Inclusion criteria: I) Alkaline phosphatase > 6 times upper normal limit (>I650 U/L) on at least two occasions, more than 4 weeks apart within the last year and/or 2) alkaline phosphatase > 3 times upper normal limit and one or more of the following criteria: a) pruritus, b) pain in the upper right abdomen, c) jaundice (bilirubin > 2 times upper normal limit), d) suppurative cholangitis. e) weightloss (> 5%), and fJ fibrosis (liver biopsy). At baseline, after 12 and 24 months ERC, liver biopsy, liver tests (bilirubin, ALP, ASAT, albumin, prothrombin), hematological tests, SR, creatinine, autoantibodies/immunog~obuIins, chest X-ray and lung function test were done. Every second month liver tests, hematological tests, creatinine and symptom scores by visuel analogue scale (fatigue, pruritus, and right-upperquadrant pain) were done. Results: During the first 12 months of the study period, eight patients recieved placebo and five active treatment. Drop-outs: two patients died after 18 and 22 months because of liver failure and sepsis, two patients because of non-compliance (after 16 and 12 months). During mtx-therapy we did not observe any known adverse events related to the study-medication as measured by the safety parameters (heamatology, lungfunction). The mtxdose in this study was 15-20 mg/wk. A cross-over (Splitplot ANOVA) analysis did not reveal any statistical differences between mtx and placebo according to liver tests, symptomscore, lever histological stage, and ERC changes. No carry-over- or time-effect was observed. Conclusion: In this 2-year double-blind-cross-over study, methotrexate did not seem to be effective. This is in accordance with other studies. Whether methotrexate might be effective in long-term studies or in association with other immunosuppresives is unknown.
1
1
OSMOLYTE-STRATEGY OF ITO-CELLS DURING THEIR TRANSFORMATION INTO MYOFIBROBLASTS.
-
I..’
Medzinische Universitiuklimk, Klinik Mwrenstr 5, D-40225 Diisseldorf
HPussinge Wr Gastmenterologie,
Hepatologle
und Infektiologle,
Introduction: Organic osmolytes are compounds, which are specifically accumulated in or released from cells in response to cell shrinkage or swelling, respectively in order to maintain cell volume. Anisoosmotic cell volume changes were identified as a critical parameter in the regulation of cell functions. The present investigation was undertaken to enlighten a possible osmolyte-strategy in non-activated and activated Ito-cells, which play an important role in the pathogenesis of liver diseases. &&Q& Ito-cells were isolated from male Sprague Dawley rats by pronaaecollagenax perfusion and separated by single Nycodenz gradient. They were cultured in DMEM plus 10% FCS. For measuring osmolyte-uptake 2,7 and I4 days old cells were incubated in hyper-, normo- and hypoosmotic media for 16 h and then betaine, taurine, or myo-inositol was given. For shldies of osmolyte-ef?lux 2 and 14 days old cells were preloaded with osmolytes (betaine, taurine or myo-inositol) under hyperosmotic conditions, and then efflux was measured after hypoosmotic exposure. In parallel a-smooth muscle actin was Investigated by immunhistochemishy and quantified on Western blots for correlation of osmolyte-strategy and state of transformation. ti Hyperosmotic (hypoosmotic) exposure of Ito-cells after 2,7 and I4 days of cultivation led to an increase (decrease) in osmolyte transport into the cells, whereby taurine was prefemed, when compared to betaine and myo-inositol. The uptake of myo-inositol reached a maximum until day 7 of cultivation. In contrast the uptake of betaine and taurine increased until day 14 (2-fold and 6-fold, compared to day 2), in parallel to the transformation of Ito-cells into myofibroblasts, shown by an increasing expression of a-smooth muscle actin, as parameter of Ito-cell transformation. In Ito-cells no a-smooth muscle actin could be detected after 2 days, whereas 7 days old cells showed a distinct signal, which was markedly increased at day I4 of cultivation. when Ito-cells were preloaded with osmolytes, lowering of the extracellular osmolarity was followed by a rapid osmolyte emox from the cells, concerning betaine, taurine and myo-inositol. Conclusions: The results indicate, that betaine, taurme, and myo-inositol acts as osmolytes in non-activated Ito-cells as well as in actwated cells. In correlation to the level of a-smooth muscle actin expression, osmolyte-uptake was markedly enhanced. This could serve as a farther parameter for the status of Ito-cell transformation.