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HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001 AASLD ABSTRACTS 491A 1275 ISOLATION AND CHARACTERISATION OF BILIARY EPITHELIAL CELLS FROM DIAGNOSTIC AND STAGING BIOPSIES IN EXTRAHE- PATIC BILIARY ATRESIA. Diana M Flynn Dr, Queen Elizabeth Hospital, Birmingham United Kingdom; Jean De Ville Mr, Birmingham Childrens Hosp, Birmingham United Kingdom; Sarbjit Nijaar Dr, Birmingham Univ, Birming- ham United Kingdom; Deirdre A Kelly Dr, Birmingham Childrens Hosp, Bir- mingham United Kingdom; Alastair J Strain Prof, Heather A Crosby Dr, Bir- mingham Univ, Birmingham United Kingdom Introduction: Extrahepatic bfliary atresia (EHBA) is the commonest paediatric liver disorder leading to transplantation, but is of unknown aetiology. Study of pathgenesis is limited by lack of an animal model and availability of human tissue. Our previous work has shown that end stage liver is a poor source of biliary epithelial cells in EHBA and that cell isolation from liver biopsies has not been possible in children under the age of 7 years. Aim: To isolate and culture biliary epithelial cells from paediatric EHBA liver biopsy specimens for cell[ culture and molecular studies of pathogenesis. Method: Liver biopsies we:re obtained by Menghini or wedge biopsy when clinicafly indicated, or at transplantation. Fragments (1-2ram) were plated onto plastic or fibronectin- coated wells, allowed to adhere, then fed with medium containing 10% fetal calf serum and HGF. Phenotype was determined by immunohistochemistry and RT-PCR. Results: Biopsies (n=33) were taken from 31 patients with EHBA (15; at diagnosis and/or Kasai procedure; 11 staging biopsies and 5 at time of transplantation) with median age 0.Syrs (range 4 weeks to 18 years); M:F 1:1. Normal (n=7) and diseased (n=30) controls had median age 3 years (range 5 weeks to 18 years) and M:F 3:1. Adhesion of biopsy fragments was improved by ]prior coating of wells with fibronectin. In EHBAbiliary epithelial cells were isolated from all ages in 44% of Menghini biopsies and 75% of wedge biopsies, corapared to 70% and 72% of all diseases and 100% of normal biopsies. Poorer growth was seen from female patients in EHBA (F=30%;M=80%) but not in controls (F=75%;M=73%). Cells stained positive for the biliary cell markers CK19 up to passage 3 but lost HEAl25 following one passage. RT-PCR showed that ceils expressed Jagged 1 mRNA, and those isolated from EHBA expressed NolLch 3 mRNA, comparable to immunostaining of frozen sections with Jaggedl and Notch3 in EHBA. Conclusion: Biliary epithelial cells can be suc- cessfully isolated and cultured from paediatric liver biopsies at all ages. This is a novel and useful tool for comparing cell populations from different stages in paediatric disease, where access to biliary epithelial cells has so far been lim- itedL. 1276 EXPRESSION OF HYPOXIA INDUCIBLE FACTOR ALPHA (HIF-lc~) IS INCREASED DURING ACUTE LIVER ALLOGRAFT REJECTION. Toby Richards, Deborah M Stroka, Deslie Nell, Melanie Leuener, Anne Williams, Daniel Candinas, Univ Hosp Birmingham, Birmingham Uk Background: Hypoxia Inducible Factor 1 (H/F-l) is a heterodimeric oxygen responsive basic-helix-loop-helix transcription factor consisting of an alpha and beta subunit. Both are members of the PAS family of transcription factors, which control a variety of critical embryonic and physiological events. HIF-1 transcriptional activity is determined by regulated expression of the HIF-1 alpha subunit, the beta subunit being constitutively expressed. HIF-1 mediates adaptive responses to hypoxia by regulating the expression of target genes controlling erythropoesis, angiogenesis and glucose homeostasis. HIF-1 ~e pro- tein expression is also increased in certain forms of cancer and in inflamma- tion. The role of HIF-1 in acute allograft rejection is not known. In this study we investigated the pattern of HIF-le~ expression during the process of acute liver allograft rejection. Methods: HIF-lc¢ expression was analysed by immu- nohistochemistry in Iiver transplant biopsy specimens. All biopsies were col- lected prospectively from patients undergoing transplant biopsies as defined by clinical requirements. Two blinded observers scored the immunohisto- chemistal staining and results were analysed by ANOVA. Morphological changes were assessed by light microscopy. Results: Weak HIF-lo~ protein expression contained to hepatocytes was observed in biopsy samples showing either non-specific posttranplant changes. There was a significantly (p=0.007) increased staining for H1F-I~ in samples showing acute rejection. This was again contained to hepatocytes and did not show a zonal expression pattern. Conclusions: The transcription factor HIF-1 is involved in the mechanism of acute allograft rejection suggesting either a response to local intragraft hypoxia or modulation of HIF-lcr protein expression by local proinflammatory cyto- kines. 127'7 IMPACT OF SELECTIVE INACTIVATION OF TRANSFORMING GROWTH FACTORq8 TYPE II RECEPTOR GENE (TGFBR2) DURING VIRAL HEPATITIS. Eric R Lemmer, Elizabeth A Conner, NCI, Bethesda, MD; Per Leveen, Lund University Hospital, Lund Switzerland; Valentina M Factor, Tyjen L Tsai, Chang-Goo Huh, NCI, Bethesda, MD; Stefan Karlsson, Lund University Hospital, Lund Switzerland; Snorri S Thorgeirsson, NCI, Bethesda, MD Transforming growth factor-t3 (TGF-j8) signaling is mediated via type I (TGF- /3RI) and type II (TGF-I3RIt) serine/threonine kinase-containing receptors, and plays a central role in the biology and pathobiology of the liver. Homozy- gous Tgflor2 mutation causes embryonic lethality around day El0.5 due to yolk sac clefects. "Floxed" conditional mutant mice were thus generated (by P.L.) to target exon 4 (kinase and transmembrane coding regions) of Tgfbr2. Successful generalized Cre/loxP recombination in vivo was demonstrated by quantitative PCP, using floxed Tgfbr2 mice crossed with mice expressing Cre under control of the inducible Mxl promoter. This analysis showed 95% efficiency of Tgfbr2 disruption in the liver following induction, and the crossings produced the same lethal inflammatory disorder as the conventional knockout of the TGF-]31 gene. Selective inactivation of Tgfbr2 in the liver was achieved using a Cre-expressing "gutless" adenoviral vector (Ad-Cre) given by tail vein injec- tion. Staining with X-gal following administration of a lacZ-expressing adeno- virus (Ad-lacZ) showed near 100% efficiency of infection of hepatocytes. Ad- Cre given at a standard dose (1.6x109 pfu) caused death by 72 hours from fulrninant liver failure due to massive hepatic necrosis. This severe liver injury appeared to be a strain effect rather than a gene effect. Lower doses of Ad vector (3.2x108 pfu) were well tolerated and resulted in high efficiencies of hepatic infection. Liver toxicity was still significant, however, and the mice developed diffuse hepatic parenchymal inflammation, which was maximal around one week. Thereafter, the parenchymal inflammation appeared to settle, but with the concurrent development of progressive portal inflammation. By two weeks there was intense portal inflammation, including prominent lymphoid folli- cles, and the liver histopathology was reminiscent of hepatitis C virus infec- tion. Currently, Tgflor2 flox mice are being treated with repeated monthly adjuvant doses of Ad vector (1.8x108 pfn) in an attempt to produce a chronic viral hepatitis in the presence of gene inactivation in the liver. Such a model may allow the study of the role of specific genes in the liver during chronic viral hepatitis and hepatocarcinogenesis. 1278 POSSIBLE ROLE IN FACTOR XA REGULATION OF HGF/SF AND HGF/ NK1 ASSOCIATED ACTIVITY. Peter Pediaditakis, Satdarshan S Monga, George K Michalopoulos, University of Pittsburgh, Pittsburgh, PA HGF/SF is a pluripotent factor that can act as a mitogen, morphogen and motogen with numerous different cell types. In the liver, HGF/SF is integrally involved in liver regeneration following partial hepatectomy (PHX), carbon tetrachloride poisoning, or Fas induced liver death. The full-length isoform of HGF/SF is a 728 aa single chain molecule. For HGF/SF to exhibit any activity, it must be proteolytically cleaved. The domain structure of the full-length HGF/SF from N-terminus to C-terminus consists of a hairpin loop followed by four kringle domains and a pseudo-protease domain. In addition to the full- length form, 3 additional isoforms have been discovered and all are a result of differential splicing. The first is termed deleted HGF/SF and is a nearly identi- cal to full-length HGF/SF except for a 5 aa deletion in the first kringle domain. The two other splice variants identified are HGF/NK1 and HGF/NK2. Each of these isoforms contains the hairpin loop followed by either the first kringle or the first two kringles respectively. HGF/NK1 has been described as both an agonist and antagonist depending on the cell type while HGF/NK2 has consis- tently been described as an antagonist. In this work, we have found that both HGF/SF as well as HGF/NK1 are capable of being processed by coagulation factor Xa (FXa). In the case of HGF/NK1, FXa makes one discrete cleavage at the C terminal peptide bond of R105. With regard to HGF/SF, FXa cleaves not only at R105 but also at the previously described activation site of pro-HGF/SF (R494). In our primary hepatocyte culture system, HGF/SF and HGF/NK1 both possess comparable mitogenic activity, but HGF/SF is more potent. Fol- lowing processing by FXa, HGF/NKI activity is abolished while the HGF/SF activity is largely unaffected. Addition of heparin to the reaction accelerates the proteolysis it but does not restore activity to HGF/NK1. We conclude that in the body, this may be a mechanism by which HGF/NK1 associated activity can be destroyed. The bond at R105 may be more susceptible to cleavage in a wound environment where many serine proteases are active. This would allow HGF/SF to continue to function (subsequent to activation) while abolishing the more unregulated activity of HGF/NK1 (which is constitutively active).
Transcript of document
HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001 AASLD ABSTRACTS 491A
1275
ISOLATION AND CHARACTERISATION OF BILIARY EPITHELIAL CELLS FROM DIAGNOSTIC AND STAGING BIOPSIES IN EXTRAHE- PATIC BILIARY ATRESIA. Diana M Flynn Dr, Queen Elizabeth Hospital, Birmingham United Kingdom; Jean De Ville Mr, Birmingham Childrens Hosp, Birmingham United Kingdom; Sarbjit Nijaar Dr, Birmingham Univ, Birming- ham United Kingdom; Deirdre A Kelly Dr, Birmingham Childrens Hosp, Bir- mingham United Kingdom; Alastair J Strain Prof, Heather A Crosby Dr, Bir- mingham Univ, Birmingham United Kingdom
Introduction: Extrahepatic bfliary atresia (EHBA) is the commonest paediatric liver disorder leading to transplantation, but is of unknown aetiology. Study of pathgenesis is limited by lack of an animal model and availability of human tissue. Our previous work has shown that end stage liver is a poor source of biliary epithelial cells in EHBA and that cell isolation from liver biopsies has not been possible in children under the age of 7 years. Aim: To isolate and culture biliary epithelial cells from paediatric EHBA liver biopsy specimens for cell[ culture and molecular studies of pathogenesis. Method: Liver biopsies we:re obtained by Menghini or wedge biopsy when clinicafly indicated, or at transplantation. Fragments (1-2ram) were plated onto plastic or fibronectin- coated wells, allowed to adhere, then fed with medium containing 10% fetal calf serum and HGF. Phenotype was determined by immunohistochemistry and RT-PCR. Results: Biopsies (n=33) were taken from 31 patients with EHBA (15; at diagnosis and/or Kasai procedure; 11 staging biopsies and 5 at time of transplantation) with median age 0.Syrs (range 4 weeks to 18 years); M:F 1:1. Normal (n=7) and diseased (n=30) controls had median age 3 years (range 5 weeks to 18 years) and M:F 3:1. Adhesion of biopsy fragments was improved by ]prior coating of wells with fibronectin. In EHBA biliary epithelial cells were isolated from all ages in 44% of Menghini biopsies and 75% of wedge biopsies, corapared to 70% and 72% of all diseases and 100% of normal biopsies. Poorer growth was seen from female patients in EHBA (F=30%;M=80%) but not in controls (F=75%;M=73%). Cells stained positive for the biliary cell markers CK19 up to passage 3 but lost HEAl25 following one passage. RT-PCR showed that ceils expressed Jagged 1 mRNA, and those isolated from EHBA expressed NolLch 3 mRNA, comparable to immunostaining of frozen sections with Jaggedl and Notch3 in EHBA. Conclusion: Biliary epithelial cells can be suc- cessfully isolated and cultured from paediatric liver biopsies at all ages. This is a novel and useful tool for comparing cell populations from different stages in paediatric disease, where access to biliary epithelial cells has so far been lim- itedL.
1276
EXPRESSION OF HYPOXIA INDUCIBLE FACTOR ALPHA (HIF-lc~) IS INCREASED DURING ACUTE LIVER ALLOGRAFT REJECTION. Toby Richards, Deborah M Stroka, Deslie Nell, Melanie Leuener, Anne Williams, Daniel Candinas, Univ Hosp Birmingham, Birmingham Uk
Background: Hypoxia Inducible Factor 1 (H/F-l) is a heterodimeric oxygen responsive basic-helix-loop-helix transcription factor consisting of an alpha and beta subunit. Both are members of the PAS family of transcription factors, which control a variety of critical embryonic and physiological events. HIF-1 transcriptional activity is determined by regulated expression of the HIF-1 alpha subunit, the beta subunit being constitutively expressed. HIF-1 mediates adaptive responses to hypoxia by regulating the expression of target genes controlling erythropoesis, angiogenesis and glucose homeostasis. HIF-1 ~e pro- tein expression is also increased in certain forms of cancer and in inflamma- tion. The role of HIF-1 in acute allograft rejection is not known. In this study we investigated the pattern of HIF-le~ expression during the process of acute liver allograft rejection. Methods: HIF-lc¢ expression was analysed by immu- nohistochemistry in Iiver transplant biopsy specimens. All biopsies were col- lected prospectively from patients undergoing transplant biopsies as defined by clinical requirements. Two blinded observers scored the immunohisto- chemistal staining and results were analysed by ANOVA. Morphological changes were assessed by light microscopy. Results: Weak HIF-lo~ protein expression contained to hepatocytes was observed in biopsy samples showing either non-specific posttranplant changes. There was a significantly (p=0.007) increased staining for H1F-I~ in samples showing acute rejection. This was again contained to hepatocytes and did not show a zonal expression pattern. Conclusions: The transcription factor HIF-1 is involved in the mechanism of acute allograft rejection suggesting either a response to local intragraft hypoxia or modulation of HIF-lcr protein expression by local proinflammatory cyto- kines.
127'7
IMPACT OF SELECTIVE INACTIVATION OF TRANSFORMING GROWTH FACTORq8 TYPE II RECEPTOR GENE (TGFBR2) DURING VIRAL HEPATITIS. Eric R Lemmer, Elizabeth A Conner, NCI, Bethesda, MD; Per Leveen, Lund University Hospital, Lund Switzerland; Valentina M Factor, Tyjen L Tsai, Chang-Goo Huh, NCI, Bethesda, MD; Stefan Karlsson, Lund University Hospital, Lund Switzerland; Snorri S Thorgeirsson, NCI, Bethesda, MD
Transforming growth factor-t3 (TGF-j8) signaling is mediated via type I (TGF- /3RI) and type II (TGF-I3RIt) serine/threonine kinase-containing receptors, and plays a central role in the biology and pathobiology of the liver. Homozy- gous Tgflor2 mutation causes embryonic lethality around day El0.5 due to yolk sac clefects. "Floxed" conditional mutant mice were thus generated (by P.L.) to target exon 4 (kinase and transmembrane coding regions) of Tgfbr2. Successful generalized Cre/loxP recombination in vivo was demonstrated by quantitative PCP, using floxed Tgfbr2 mice crossed with mice expressing Cre under control of the inducible Mxl promoter. This analysis showed 95% efficiency of Tgfbr2 disruption in the liver following induction, and the crossings produced the same lethal inflammatory disorder as the conventional knockout of the TGF-]31 gene. Selective inactivation of Tgfbr2 in the liver was achieved using a Cre-expressing "gutless" adenoviral vector (Ad-Cre) given by tail vein injec- tion. Staining with X-gal following administration of a lacZ-expressing adeno- virus (Ad-lacZ) showed near 100% efficiency of infection of hepatocytes. Ad- Cre given at a standard dose (1.6x109 pfu) caused death by 72 hours from fulrninant liver failure due to massive hepatic necrosis. This severe liver injury appeared to be a strain effect rather than a gene effect. Lower doses of Ad vector (3.2x108 pfu) were well tolerated and resulted in high efficiencies of hepatic infection. Liver toxicity was still significant, however, and the mice developed diffuse hepatic parenchymal inflammation, which was maximal around one week. Thereafter, the parenchymal inflammation appeared to settle, but with the concurrent development of progressive portal inflammation. By two weeks there was intense portal inflammation, including prominent lymphoid folli- cles, and the liver histopathology was reminiscent of hepatitis C virus infec- tion. Currently, Tgflor2 flox mice are being treated with repeated monthly adjuvant doses of Ad vector (1.8x108 pfn) in an attempt to produce a chronic viral hepatitis in the presence of gene inactivation in the liver. Such a model may allow the study of the role of specific genes in the liver during chronic viral hepatitis and hepatocarcinogenesis.
1278
POSSIBLE ROLE IN FACTOR XA REGULATION OF HGF/SF AND HGF/ NK1 ASSOCIATED ACTIVITY. Peter Pediaditakis, Satdarshan S Monga, George K Michalopoulos, University of Pittsburgh, Pittsburgh, PA
HGF/SF is a pluripotent factor that can act as a mitogen, morphogen and motogen with numerous different cell types. In the liver, HGF/SF is integrally involved in liver regeneration following partial hepatectomy (PHX), carbon tetrachloride poisoning, or Fas induced liver death. The full-length isoform of HGF/SF is a 728 aa single chain molecule. For HGF/SF to exhibit any activity, it must be proteolytically cleaved. The domain structure of the full-length HGF/SF from N-terminus to C-terminus consists of a hairpin loop followed by four kringle domains and a pseudo-protease domain. In addition to the full- length form, 3 additional isoforms have been discovered and all are a result of differential splicing. The first is termed deleted HGF/SF and is a nearly identi- cal to full-length HGF/SF except for a 5 aa deletion in the first kringle domain. The two other splice variants identified are HGF/NK1 and HGF/NK2. Each of these isoforms contains the hairpin loop followed by either the first kringle or the first two kringles respectively. HGF/NK1 has been described as both an agonist and antagonist depending on the cell type while HGF/NK2 has consis- tently been described as an antagonist. In this work, we have found that both HGF/SF as well as HGF/NK1 are capable of being processed by coagulation factor Xa (FXa). In the case of HGF/NK1, FXa makes one discrete cleavage at the C terminal peptide bond of R105. With regard to HGF/SF, FXa cleaves not only at R105 but also at the previously described activation site of pro-HGF/SF (R494). In our primary hepatocyte culture system, HGF/SF and HGF/NK1 both possess comparable mitogenic activity, but HGF/SF is more potent. Fol- lowing processing by FXa, HGF/NKI activity is abolished while the HGF/SF activity is largely unaffected. Addition of heparin to the reaction accelerates the proteolysis it but does not restore activity to HGF/NK1. We conclude that in the body, this may be a mechanism by which HGF/NK1 associated activity can be destroyed. The bond at R105 may be more susceptible to cleavage in a wound environment where many serine proteases are active. This would allow HGF/SF to continue to function (subsequent to activation) while abolishing the more unregulated activity of HGF/NK1 (which is constitutively active).
