- Email: [email protected]
Hepatocyte progenitor cells in massive hepatic necrosis
Poster Sessions
38
Results: Comparing 8d spheroids with Monolayer, significant (>2.5 fold) changes in expression were seen in >300 genes. Hemeoxygenase-1 protein at d8 was doubled cf. M or d1.5. MAPKinases activated by hypoxia, MEKK3 and MKK3b, were least abundant at d8. A -2.5 fold increase of Hypoxia-inducible factor protein (HII-la) was observed in d1.5 and M cf. d8. Genes with HII-la response elements showed an increase in d15 cf. d8, e.g. VEGF (3.22.fold) and Enolase (3.40.fold). Catalase specific activity (unitslminlmg protein) was increased at d8 (0.017 & 0.002) cf. monolayer (0.0075 & 0.001). Glutathione-s-transferase activity decreased at d15 (0.0016 & 0.0003) cf. d8 (0.0041 & 0.001 unitslminlmg protein). Conclusion: Stress protein expression with enhanced differentiated function may be an adaptive response to increased metabolic activity. Hypoxia related stress may be contributing to diminished levels of cell function observed at later time points of 3-D culture.
I 108
FACTORS ASSOCIATED FIBROSIS
FOLLOWING
WITH RAPID PROGRESSION LIVER TRANSPLANTATION
OF FOR
HEPATITIS C J. Contreras’,
l? Marcellin’, V. Tral-Smayra2, V. Paradis2, C. Francoz’, M. Vidaud4, K. Prakash3, C. Degott2, .I. Belghiti3, D. Valla’, F. Durand’. ‘Service D’Hepatologie And INSERM lJ481; 2Service D’Anatomie Pathologique; ‘Service D’Chirurgie Hepatobiliaire; 4Service De Biochimie, Hopital Beaujon, Clichy, France The progression of fibrosis in transplanted patients with HCV-recurrence is highly variable in different groups of patients. Aims. The aim of this study was to analyse the donor, recipient and virologic factors that might influence the progression of fibrosis in patients transplanted for HCV infection. Methods: A retrospective analysis of 72 patients (58 males) transplanted for HCV-related cirrhosis with a minimum follow-up of 6 months was done. There were 33 patients with HCC. The demographic data, biochemical factors, post-transplant treatment and the immunosuppressive regimen used were analysed. All the liver biopsies (n=137) were analysed and the fibrosis progression index was calculated (FPI=score fibrosis METAVIR/years). Univariate and multivariate analysis were performed to assess the factors responsible for rapid progression of fibrosis. Results: The average FPI was 0.8~tO.6 (O-3) U/yr. Two groups were identified, with the FPI less than 0.8 U/yr (n=37) or more than 0.8 U/yr (n=35). In univariate analysis, HCC (p=O.O5), sex of the recipient (p=O.O04), the donor’s age (p=O.OOS) and the tacrolimus immunosuppression (p=O.OOOl) were significantly associated with FPI. In multivariate analysis donor’s age, recipient’s sex and immunosuppression with tacrolimus (p=O.O04, p=O.Ol and p=O.OOl respectively) were significantly associated with FPI. Conclusions: The age of the donor, male sex of the recipient, use of tacrolimus for immunosuppression are associated with rapid progression of fibrosis. Further studies are required to assess the optimal immunosuppressive regimen in order to reduce the risk for progression of fibrosis in HCV transplanted.
I
109
HEPATOCYTE
PROGENITOR
CELLS IN MASSIVE
HEPATIC
NECROSIS
C.E.H. Craig’, A. Quaglia’, K. Savage’, M.W. Lowdel12, C. Seldon3, R. Sim’, .I. North2, H.G. Prentice2, H. Hodgson3, A.l? Dhillon’. ‘Department Of Histopathology, Royal Free And University College Medical School, London, UK; 2Department Of Haematology, Royal Free And University College Medical School, London, UK; ‘Departments Of Medicine, Royal Free And University College Medical School, London, UK Background: Until now the morphological appearance of putative progenitor cells in adult human livers has not been characterized. These cells are expected to be abundant in regenerating livers from cases of massive hepatic necrosis (MHN). We studied the morphological and immunohisto-
chemical characteristics of hepatic progenitor cells (HPC) in explants from six of these cases. Methods: For the purposes of this study we defined HPCs as blast-like cells that expressed markers associated with stem cells but not markers for lymphocytes or mast cells. We performed histological examination and immunohistochemical staining for CD34, CD1 17 (c-kit), CD133, CD45, mast cell tryptase, cytokeratins and HepParl antigens. Results: HPCs were found scattered within necrotic areas and within the periportal and portal infiltrate associated with regenerating ductules. The HPCs were round or oval shaped cells measuring 7-12 km in diameter. The nuclei were round and hyperchromatic with rare mitoses. They had a moderate amount of basophilic or amphophilic cytoplasm with an occasional recognizable Golgi apparatus giving an immunoblastoid or plasmacytoid appearance. The HPCs expressed CD117 and some co-expressed CD133. They were CD45 and mast cell tryptase negative. CD117 expression was also seen in ductules and metaplastic (duct&r) hepatocytes. Many ductules showed expression of CD133. There were no non-endothelial CD34 positive HPCs. Conclusion: We have identified previously unknown blast-like nonhepatocyte/non-biliaryrylnon-mast cell CD1 17+/CD133+ putative hepatic precursor cells in explant livers obtained from six patients with massive hepatic necrosis. ACKNOWLEDGEMENT: We wish to thank the Liver Transplant Team of the Royal Free Hospital, without whom this work would not have been possible.
I 110
CLINICAL
AND HISTOLOGICAL
TRANSPLANTATION HEMOCHROMATOSIS
FOLLOW-UP
AFTER
LIVER
(LT) FOR HEREDITARY (HH)
J.C. Duclos-Vallee’,
B. Roche’, l? Chanson2, F. Yilmaz3, F. Saliba’, D. Castaing’, H. Bismuth’, D. Samuel’, C. Guettier3. ‘Centre Hepato-Biliaire, Hospital l?Brousse EA 3541, Villejuif France; 2Service D’endoctinologie, Hospital Bicetre, Kremlin Bicetre, France; ‘Laboratory Of Pathology, Hospital ITBrowse EA 3541, Villejuif; France
Very few data concerning the post-transplant cam-se of patients transplanted for HH, are available, especially about the recurrence of iron overload in the liver graft. Aim: To evaluate the post-transplant cam-se of patients transplanted in our unit for HH with special regard to iron overload. Patients and methods: 9 patients, all males, mean age at time of LT: 54.5 ~t10.5 years (30.65), with a mean follow-up after LT of 10.8 & 3.4 (5-15) years were studied. Diagnosis of HH was assessed on the usual criteria including mutations of HFE gene. LT was performed for liver tumor (n=5) and end-stage liver disease (n=4). Post-transplant clinical events and biochemical data (serum iron, tranfenin saturation, serum ferritin) were collected. Native liver and post-transplant biopsies were reviewed for the following parameters: iron overload assessed on Perls’staining, fibrosis, activity, rejection and steatosis. Results: Iron overload was present only in 4 native livers because of iterative phlebotomies. Post-transplant liver biopsies showed slight to moderate iron overload in 4 patients respectively at 1,2,5,9 years post LT, associated in 1 case with elevation of serum ferritin (687 kg/L) at 2 years post-LT. Hemosiderin deposits were present in hepatocytes without obvious systematization. In the 5 other patients, no iron deposit was observed in liver graft even after 10 years follow-up. Three patients developed extra-hepatic cancers: lung, colonic adenocarcinoma and basocelullar carcinoma. Conclusions: Recurrence of liver iron deposits is inconstant within the 10 first post-LT years in patients transplanted for HH but may be observed very early after LT. A high incidence of de nova cancers is observed in these patients. These findings suggest a longer closer follow-up for iron overload survey and cancer screening in these patients.
38
Results: Comparing 8d spheroids with Monolayer, significant (>2.5 fold) changes in expression were seen in >300 genes. Hemeoxygenase-1 protein at d8 was doubled cf. M or d1.5. MAPKinases activated by hypoxia, MEKK3 and MKK3b, were least abundant at d8. A -2.5 fold increase of Hypoxia-inducible factor protein (HII-la) was observed in d1.5 and M cf. d8. Genes with HII-la response elements showed an increase in d15 cf. d8, e.g. VEGF (3.22.fold) and Enolase (3.40.fold). Catalase specific activity (unitslminlmg protein) was increased at d8 (0.017 & 0.002) cf. monolayer (0.0075 & 0.001). Glutathione-s-transferase activity decreased at d15 (0.0016 & 0.0003) cf. d8 (0.0041 & 0.001 unitslminlmg protein). Conclusion: Stress protein expression with enhanced differentiated function may be an adaptive response to increased metabolic activity. Hypoxia related stress may be contributing to diminished levels of cell function observed at later time points of 3-D culture.
I 108
FACTORS ASSOCIATED FIBROSIS
FOLLOWING
WITH RAPID PROGRESSION LIVER TRANSPLANTATION
OF FOR
HEPATITIS C J. Contreras’,
l? Marcellin’, V. Tral-Smayra2, V. Paradis2, C. Francoz’, M. Vidaud4, K. Prakash3, C. Degott2, .I. Belghiti3, D. Valla’, F. Durand’. ‘Service D’Hepatologie And INSERM lJ481; 2Service D’Anatomie Pathologique; ‘Service D’Chirurgie Hepatobiliaire; 4Service De Biochimie, Hopital Beaujon, Clichy, France The progression of fibrosis in transplanted patients with HCV-recurrence is highly variable in different groups of patients. Aims. The aim of this study was to analyse the donor, recipient and virologic factors that might influence the progression of fibrosis in patients transplanted for HCV infection. Methods: A retrospective analysis of 72 patients (58 males) transplanted for HCV-related cirrhosis with a minimum follow-up of 6 months was done. There were 33 patients with HCC. The demographic data, biochemical factors, post-transplant treatment and the immunosuppressive regimen used were analysed. All the liver biopsies (n=137) were analysed and the fibrosis progression index was calculated (FPI=score fibrosis METAVIR/years). Univariate and multivariate analysis were performed to assess the factors responsible for rapid progression of fibrosis. Results: The average FPI was 0.8~tO.6 (O-3) U/yr. Two groups were identified, with the FPI less than 0.8 U/yr (n=37) or more than 0.8 U/yr (n=35). In univariate analysis, HCC (p=O.O5), sex of the recipient (p=O.O04), the donor’s age (p=O.OOS) and the tacrolimus immunosuppression (p=O.OOOl) were significantly associated with FPI. In multivariate analysis donor’s age, recipient’s sex and immunosuppression with tacrolimus (p=O.O04, p=O.Ol and p=O.OOl respectively) were significantly associated with FPI. Conclusions: The age of the donor, male sex of the recipient, use of tacrolimus for immunosuppression are associated with rapid progression of fibrosis. Further studies are required to assess the optimal immunosuppressive regimen in order to reduce the risk for progression of fibrosis in HCV transplanted.
I
109
HEPATOCYTE
PROGENITOR
CELLS IN MASSIVE
HEPATIC
NECROSIS
C.E.H. Craig’, A. Quaglia’, K. Savage’, M.W. Lowdel12, C. Seldon3, R. Sim’, .I. North2, H.G. Prentice2, H. Hodgson3, A.l? Dhillon’. ‘Department Of Histopathology, Royal Free And University College Medical School, London, UK; 2Department Of Haematology, Royal Free And University College Medical School, London, UK; ‘Departments Of Medicine, Royal Free And University College Medical School, London, UK Background: Until now the morphological appearance of putative progenitor cells in adult human livers has not been characterized. These cells are expected to be abundant in regenerating livers from cases of massive hepatic necrosis (MHN). We studied the morphological and immunohisto-
chemical characteristics of hepatic progenitor cells (HPC) in explants from six of these cases. Methods: For the purposes of this study we defined HPCs as blast-like cells that expressed markers associated with stem cells but not markers for lymphocytes or mast cells. We performed histological examination and immunohistochemical staining for CD34, CD1 17 (c-kit), CD133, CD45, mast cell tryptase, cytokeratins and HepParl antigens. Results: HPCs were found scattered within necrotic areas and within the periportal and portal infiltrate associated with regenerating ductules. The HPCs were round or oval shaped cells measuring 7-12 km in diameter. The nuclei were round and hyperchromatic with rare mitoses. They had a moderate amount of basophilic or amphophilic cytoplasm with an occasional recognizable Golgi apparatus giving an immunoblastoid or plasmacytoid appearance. The HPCs expressed CD117 and some co-expressed CD133. They were CD45 and mast cell tryptase negative. CD117 expression was also seen in ductules and metaplastic (duct&r) hepatocytes. Many ductules showed expression of CD133. There were no non-endothelial CD34 positive HPCs. Conclusion: We have identified previously unknown blast-like nonhepatocyte/non-biliaryrylnon-mast cell CD1 17+/CD133+ putative hepatic precursor cells in explant livers obtained from six patients with massive hepatic necrosis. ACKNOWLEDGEMENT: We wish to thank the Liver Transplant Team of the Royal Free Hospital, without whom this work would not have been possible.
I 110
CLINICAL
AND HISTOLOGICAL
TRANSPLANTATION HEMOCHROMATOSIS
FOLLOW-UP
AFTER
LIVER
(LT) FOR HEREDITARY (HH)
J.C. Duclos-Vallee’,
B. Roche’, l? Chanson2, F. Yilmaz3, F. Saliba’, D. Castaing’, H. Bismuth’, D. Samuel’, C. Guettier3. ‘Centre Hepato-Biliaire, Hospital l?Brousse EA 3541, Villejuif France; 2Service D’endoctinologie, Hospital Bicetre, Kremlin Bicetre, France; ‘Laboratory Of Pathology, Hospital ITBrowse EA 3541, Villejuif; France
Very few data concerning the post-transplant cam-se of patients transplanted for HH, are available, especially about the recurrence of iron overload in the liver graft. Aim: To evaluate the post-transplant cam-se of patients transplanted in our unit for HH with special regard to iron overload. Patients and methods: 9 patients, all males, mean age at time of LT: 54.5 ~t10.5 years (30.65), with a mean follow-up after LT of 10.8 & 3.4 (5-15) years were studied. Diagnosis of HH was assessed on the usual criteria including mutations of HFE gene. LT was performed for liver tumor (n=5) and end-stage liver disease (n=4). Post-transplant clinical events and biochemical data (serum iron, tranfenin saturation, serum ferritin) were collected. Native liver and post-transplant biopsies were reviewed for the following parameters: iron overload assessed on Perls’staining, fibrosis, activity, rejection and steatosis. Results: Iron overload was present only in 4 native livers because of iterative phlebotomies. Post-transplant liver biopsies showed slight to moderate iron overload in 4 patients respectively at 1,2,5,9 years post LT, associated in 1 case with elevation of serum ferritin (687 kg/L) at 2 years post-LT. Hemosiderin deposits were present in hepatocytes without obvious systematization. In the 5 other patients, no iron deposit was observed in liver graft even after 10 years follow-up. Three patients developed extra-hepatic cancers: lung, colonic adenocarcinoma and basocelullar carcinoma. Conclusions: Recurrence of liver iron deposits is inconstant within the 10 first post-LT years in patients transplanted for HH but may be observed very early after LT. A high incidence of de nova cancers is observed in these patients. These findings suggest a longer closer follow-up for iron overload survey and cancer screening in these patients.