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651 Intrahepatic CD4+ lymphocytes trapping during tolerance induction using mushroom derived formulations: A possible role for the liver in tolerance induction
06. Autoimmune and chronic eholestatic liver diseases and/or alkaline phosphatases (n 20). The main causes of liver damage (except overweight) were ruled out. Among them, 26 patients underwent a liver biopsy. Twenty-nine women referred to our hospital for infertility were used as control. All women had karyotype analysis and fluorescence in situ hybridization was performed in case of karyotype abnormality. Results: Overweight (BMI > 25 kg/m2) was present in 77% of the patients. Abnormal karyotype was observed in 27 patients (88%) and one control (3%, p < 0.0001). The karyotype study revealed X structural abnormalities in 5 patients: translocation in one, X-ring in one and iso-chromosomy of the X long arm in three. Other abnormalities present in 24 patients included complete monosomy in one (46X0) and mosaicism in 23 (45,X0/46,XX or 45X0/46,XX, 47,XXX). We considered the diagnosis of mosaicism when the proportion of X0 cells was equal or superior to 7%. Liver histology was always abnormal and showed isolated or associated changes already described in TS: macrovesicular steatosis (n 20), architectural distortion (n 17), including 12 cases of nodular regenerative hyperplasia, biliary lesions (n 13) including lymphocytic cholangitis (n 9), ductular proliferation (n 8) and ductopenia (n 1). In conclusion, in women with short stature (generally overweighted) and unexplained elevated liver enzymes, chromosome X abnormalities are extremely frequent. The liver lesions observed are similar to those of TS. Anatomical changes of the liver might be a consequence of the deletion of one or more genes located on chromosome X.
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DEMONSTRATION OF THE TRANSITION OF INTRAHEPATIC BILIARY EPITHELIAL CELLS TO FIBROBLASTS DURING CHRONIC INFLAMMATORY LIVER DISEASES
K.A. Rygiell~ H. Robertson 1, A.D. Burt 2, D.E.J. Jones 3, J.A. Kirby 1.
1SARS, 2SCLS, 3SCMS, Medical School, University of Newcastle upon Tyne, UK Background and Aims: Intrahepatic bile ducts are lost during chronic inflammatory liver diseases. Recent studies have shown that the loss of tubules observed during renal fibrosis can be caused by epithelial to mesenchymal transition (EMT) in which tubular epithelial cells lose differentiated function and develop an invasive fibroblast phenotype. This study was designed to determine whether intrahepatic biliary epithelial cells can undergo a similar phenotypic transition. Methods: Liver sections from patients with primary biliary cirrhosis, primary sclerosing cholangitis and alcoholic liver disease were used for immunohistochemical identification of biliary epithelium (cytokeratin 19; CK19), cytotoxic T cells (CD8), EMT (S100A4, and a-smooth muscle actin; aSMA) and intracellular signal transduction mediated by phosphorylated (p)Smad2/3. In further studies, human intrahepatic biliary epithelial cells (HIBEC) were cultured and some cells were stimulated with TGF[J for 72 hours. After morphological examination the cells were fixed and processed for immunofluorescence detection of CK19, S100A4, aSMA and pSmad2/3. Results: Biliary epithelium in normal liver and resting HIBEC expressed CK19, but epithelial cells in neither preparation showed expression of the early and late EMT markers S100A4 and aSMA. However, sections of diseased liver showed intraepithelial expression of S 100A4 associated with nuclear localisation of the TGF[J-induced signalling molecule pSmad2/3. These cells were adjacent to infiltrating CD8+ T cells and were also spatially associated with invasive myofibroblasts which expressed aSMA. Treatment of cultured HIBEC with TGF[J stimulated a morphological transition to bipolar, fibroblastic cells. Scanning laser confocal microscopy demonstrated that TGF[J also caused phosphorylation and nuclear localisation of Smad2/3 together with induction of both S100A4 and aSMA. Conclusions: This study demonstrates that the epithelial cells lining small and medium-sized bile ducts can undergo EMT during chronic inflammatory liver diseases. Furthermore, this process can be driven by TGF[J and the resulting mesenchymal cells can differentiate to a mature,
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myofibroblast phenotype. In kidney it is known that EMT can be reversed by treatment with bone morphogenetic protein-7; this raises interesting therapeutic possibilities for chronic fibrosis of the liver.
~-5"~ INTRAHEPATIC CD4+ LYMPHOCYTES TRAPPING DURING TOLERANCE INDUCTION USING M U S H R O O M DERIVED FORMULATIONS: A POSSIBLE ROLE FOR THE LIVER IN TOLERANCE INDUCTION M. Shuvy 1, T. Hershcovici2 , L. Zolotarov2, Y. Ilan 1. 1Internal medicine A,
2Liver Unit, Hadassah-Hebrew University Medical Cente~ Jerusalem, Israel Background: The liver is a site for lymphocyte clearance, and plays a role in determining the CD4+/CD8+ balance during peripheral tolerance induction. Liver-associated NKT lymphocytes play a regulatory role in various immune mediated disorders. Shitake is a mushroom with immune modulatory functions. Aims: To determine the effect of Shitake on liver-mediated immune regulation in a murine model of immune-mediated colitis. Methods: Four groups of mice were studied. Colitis was induced by intracolonic instillation of TNBS in groups A and B. Group A mice were fed daily with shitake extract (50 gg/mouse) for 9 days, and group B mice received daily bovine serum albumin. Groups C and D mice were similarly fed with daily shitake extract or bovine serum albumin respectively, without colitis induction. Mice were followed for macroscopic and microscopic colitis scores. The immune modulatory effect of Shitake was determined by FACS analysis of intrahepatic and intrasplenic lymphocytes for NKT, CD4 and CD8 markers, and by IFN? ELISPOT. Results: Administration of Shitake extract resulted in marked alleviation of colitis manifested by significant improvement of the macroscopic and microscopic scores, and by a remarkable decrease in IFN?-producing colonies in group A compared with group B (1.7 vs 3.7, respectively). This beneficial effect was associated with a significant increase of intrahepatic CD4+ lymphocyte trapping as manifested by a 57% increase in the intrahepatic/peripheral CD4+/CD8+ ratio (group A vs. B). A similar effect was observed when shitake was administered to naive animals [intrahepatic/ peripheral CD4+/CD8+ ratio increased by 76% in treated (group C) versus untreated animals (group D)]. The beneficial effect was associated with a 17% increase in the intrahepatic NKT cell number. Conclusions: Shitake-derived formulations affect liver-mediated immune regulation by altering NKT lymphocyte distribution and increasing intrahepatic CD4+ T lymphocyte trapping, thereby, leading to alleviation of immune mediated colitis.
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JUVENILE AUTOIMMUNE SCLEROSING CHOLANGITIS IS ASSOCIATED WITH POSSESSION OF HLA DRB1*13
J. Underhill 1, C. Liaskos2, D.P. Bogdanos2, P. Cheeseman2, G. MieliVergani 2, D. Vergani 2, Y. Ma 2. 1Institute of Liver Studies, King k College
Hospital, 2Institute of Liver Studies, King k College London School of Medicine at Kingk College Hospital, London, UK Background: Autoimmune sclerosing cholangitis (ASC) shares the histological features of interface hepatitis with autoimmune hepatitis type 1 (AIH-1) and type 2 (AIH-2), but has additional lesions of intra- and extrahepatic bile ducts. While HLA DRBI*03 and DRBI*07 have been defined as disease susceptibility genes in AIH-1 and AIH-2, respectively (J Hepatol 2002; 36(S1): 156), no HLA association for ASC has been reported. Aim: To investigate whether there is an association between HLA alleles and ASC. Patients and Methods: 144 patients were investigated (74 with AIH-1, 34 with AIH-2 and 36 with ASC, 92 female, median age 11.1 yrs, range 1 to 15.8). HLA class I and II genotyping was performed by PCPUSSP using Biotest kits (Dreiech, Germany). Over-representation of HLA A*01
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DEMONSTRATION OF THE TRANSITION OF INTRAHEPATIC BILIARY EPITHELIAL CELLS TO FIBROBLASTS DURING CHRONIC INFLAMMATORY LIVER DISEASES
K.A. Rygiell~ H. Robertson 1, A.D. Burt 2, D.E.J. Jones 3, J.A. Kirby 1.
1SARS, 2SCLS, 3SCMS, Medical School, University of Newcastle upon Tyne, UK Background and Aims: Intrahepatic bile ducts are lost during chronic inflammatory liver diseases. Recent studies have shown that the loss of tubules observed during renal fibrosis can be caused by epithelial to mesenchymal transition (EMT) in which tubular epithelial cells lose differentiated function and develop an invasive fibroblast phenotype. This study was designed to determine whether intrahepatic biliary epithelial cells can undergo a similar phenotypic transition. Methods: Liver sections from patients with primary biliary cirrhosis, primary sclerosing cholangitis and alcoholic liver disease were used for immunohistochemical identification of biliary epithelium (cytokeratin 19; CK19), cytotoxic T cells (CD8), EMT (S100A4, and a-smooth muscle actin; aSMA) and intracellular signal transduction mediated by phosphorylated (p)Smad2/3. In further studies, human intrahepatic biliary epithelial cells (HIBEC) were cultured and some cells were stimulated with TGF[J for 72 hours. After morphological examination the cells were fixed and processed for immunofluorescence detection of CK19, S100A4, aSMA and pSmad2/3. Results: Biliary epithelium in normal liver and resting HIBEC expressed CK19, but epithelial cells in neither preparation showed expression of the early and late EMT markers S100A4 and aSMA. However, sections of diseased liver showed intraepithelial expression of S 100A4 associated with nuclear localisation of the TGF[J-induced signalling molecule pSmad2/3. These cells were adjacent to infiltrating CD8+ T cells and were also spatially associated with invasive myofibroblasts which expressed aSMA. Treatment of cultured HIBEC with TGF[J stimulated a morphological transition to bipolar, fibroblastic cells. Scanning laser confocal microscopy demonstrated that TGF[J also caused phosphorylation and nuclear localisation of Smad2/3 together with induction of both S100A4 and aSMA. Conclusions: This study demonstrates that the epithelial cells lining small and medium-sized bile ducts can undergo EMT during chronic inflammatory liver diseases. Furthermore, this process can be driven by TGF[J and the resulting mesenchymal cells can differentiate to a mature,
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myofibroblast phenotype. In kidney it is known that EMT can be reversed by treatment with bone morphogenetic protein-7; this raises interesting therapeutic possibilities for chronic fibrosis of the liver.
~-5"~ INTRAHEPATIC CD4+ LYMPHOCYTES TRAPPING DURING TOLERANCE INDUCTION USING M U S H R O O M DERIVED FORMULATIONS: A POSSIBLE ROLE FOR THE LIVER IN TOLERANCE INDUCTION M. Shuvy 1, T. Hershcovici2 , L. Zolotarov2, Y. Ilan 1. 1Internal medicine A,
2Liver Unit, Hadassah-Hebrew University Medical Cente~ Jerusalem, Israel Background: The liver is a site for lymphocyte clearance, and plays a role in determining the CD4+/CD8+ balance during peripheral tolerance induction. Liver-associated NKT lymphocytes play a regulatory role in various immune mediated disorders. Shitake is a mushroom with immune modulatory functions. Aims: To determine the effect of Shitake on liver-mediated immune regulation in a murine model of immune-mediated colitis. Methods: Four groups of mice were studied. Colitis was induced by intracolonic instillation of TNBS in groups A and B. Group A mice were fed daily with shitake extract (50 gg/mouse) for 9 days, and group B mice received daily bovine serum albumin. Groups C and D mice were similarly fed with daily shitake extract or bovine serum albumin respectively, without colitis induction. Mice were followed for macroscopic and microscopic colitis scores. The immune modulatory effect of Shitake was determined by FACS analysis of intrahepatic and intrasplenic lymphocytes for NKT, CD4 and CD8 markers, and by IFN? ELISPOT. Results: Administration of Shitake extract resulted in marked alleviation of colitis manifested by significant improvement of the macroscopic and microscopic scores, and by a remarkable decrease in IFN?-producing colonies in group A compared with group B (1.7 vs 3.7, respectively). This beneficial effect was associated with a significant increase of intrahepatic CD4+ lymphocyte trapping as manifested by a 57% increase in the intrahepatic/peripheral CD4+/CD8+ ratio (group A vs. B). A similar effect was observed when shitake was administered to naive animals [intrahepatic/ peripheral CD4+/CD8+ ratio increased by 76% in treated (group C) versus untreated animals (group D)]. The beneficial effect was associated with a 17% increase in the intrahepatic NKT cell number. Conclusions: Shitake-derived formulations affect liver-mediated immune regulation by altering NKT lymphocyte distribution and increasing intrahepatic CD4+ T lymphocyte trapping, thereby, leading to alleviation of immune mediated colitis.
•
JUVENILE AUTOIMMUNE SCLEROSING CHOLANGITIS IS ASSOCIATED WITH POSSESSION OF HLA DRB1*13
J. Underhill 1, C. Liaskos2, D.P. Bogdanos2, P. Cheeseman2, G. MieliVergani 2, D. Vergani 2, Y. Ma 2. 1Institute of Liver Studies, King k College
Hospital, 2Institute of Liver Studies, King k College London School of Medicine at Kingk College Hospital, London, UK Background: Autoimmune sclerosing cholangitis (ASC) shares the histological features of interface hepatitis with autoimmune hepatitis type 1 (AIH-1) and type 2 (AIH-2), but has additional lesions of intra- and extrahepatic bile ducts. While HLA DRBI*03 and DRBI*07 have been defined as disease susceptibility genes in AIH-1 and AIH-2, respectively (J Hepatol 2002; 36(S1): 156), no HLA association for ASC has been reported. Aim: To investigate whether there is an association between HLA alleles and ASC. Patients and Methods: 144 patients were investigated (74 with AIH-1, 34 with AIH-2 and 36 with ASC, 92 female, median age 11.1 yrs, range 1 to 15.8). HLA class I and II genotyping was performed by PCPUSSP using Biotest kits (Dreiech, Germany). Over-representation of HLA A*01