Analysis of Liver Repair Mechanisms in Alagille Syndrome and Biliary Atresia Reveals a Role for Notch Signaling
2007; Elsevier BV; Volume: 171; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2007.070073
ISSN1525-2191
AutoresLuca Fabris, Massimiliano Cadamuro, Maria Guido, Carlo Spirlı̀, Romina Fiorotto, M. Colledan, Giuliano Torre, Daniele Alberti, Aurelio Sonzogni, Lajos Okolicsànyi, Mario Strazzabosco,
Tópico(s)Congenital Anomalies and Fetal Surgery
ResumoPatients with Alagille syndrome (AGS), a genetic disorder of Notch signaling, suffer from severe ductopenia and cholestasis, but progression to biliary cirrhosis is rare. Instead, in biliary atresia (BA) severe cholestasis is associated with a pronounced “ductular reaction” and rapid progression to biliary cirrhosis. Given the role of Notch in biliary development, we hypothesized that defective Notch signaling would influence the reparative mechanisms in cholestatic cholangiopathies. Thus we compared phenotype and relative abundance of the epithelial components of the hepatic reparative complex in AGS (n = 10) and BA (n = 30) using immunohistochemistry and computer-assisted morphometry. BA was characterized by an increase in reactive ductular and hepatic progenitor cells, whereas in AGS, a striking increase in intermediate hepatobiliary cells contrasted with the near absence of reactive ductular cells and hepatic progenitor cells. Hepatocellular mitoinhibition index (p21waf1/Ki67) was similar in AGS and BA. Fibrosis was more severe in BA, where portal septa thickness positively correlated with reactive ductular cells and hepatic progenitor cells. AGS hepatobiliary cells failed to express hepatic nuclear factor (HNF) 1β, a biliary-specific transcription factor. These data indicate that Notch signaling plays a role in liver repair mechanisms in postnatal life: its defect results in absent reactive ductular cells and accumulation of hepatobiliary cells lacking HNF1β, thus being unable to switch to a biliary phenotype. Patients with Alagille syndrome (AGS), a genetic disorder of Notch signaling, suffer from severe ductopenia and cholestasis, but progression to biliary cirrhosis is rare. Instead, in biliary atresia (BA) severe cholestasis is associated with a pronounced “ductular reaction” and rapid progression to biliary cirrhosis. Given the role of Notch in biliary development, we hypothesized that defective Notch signaling would influence the reparative mechanisms in cholestatic cholangiopathies. Thus we compared phenotype and relative abundance of the epithelial components of the hepatic reparative complex in AGS (n = 10) and BA (n = 30) using immunohistochemistry and computer-assisted morphometry. BA was characterized by an increase in reactive ductular and hepatic progenitor cells, whereas in AGS, a striking increase in intermediate hepatobiliary cells contrasted with the near absence of reactive ductular cells and hepatic progenitor cells. Hepatocellular mitoinhibition index (p21waf1/Ki67) was similar in AGS and BA. Fibrosis was more severe in BA, where portal septa thickness positively correlated with reactive ductular cells and hepatic progenitor cells. AGS hepatobiliary cells failed to express hepatic nuclear factor (HNF) 1β, a biliary-specific transcription factor. These data indicate that Notch signaling plays a role in liver repair mechanisms in postnatal life: its defect results in absent reactive ductular cells and accumulation of hepatobiliary cells lacking HNF1β, thus being unable to switch to a biliary phenotype. Primary cholangiopathies are characterized by chronic ongoing damage to the biliary epithelium. Proliferation of reactive ductules, inflammation, and portal fibrosis coexists with the progressive disappearance of the interlobular/septal bile ducts. Desmet1Desmet VJ Ludwig symposium on biliary disorders–part I. Pathogenesis of ductal plate abnormalities.Mayo Clin Proc. 1998; 73: 80-89Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar first proposed that this histological lesion, defined as “ductular reaction,” was the pacemaker of portal fibrosis and therefore the main mechanism for disease progression in cholangiopathies. Later it was recognized that ductular reactive cells possess distinctive features with respect to “quiescent cholangiocytes” and actively participate to portal inflammation, producing a vast array of cytokines and chemokines, growth factors, and inflammatory mediators that enable them to act as the main nodal point in the cross talk between the different cell components (fibroblasts, endothelial cells, and inflammatory cells) of the “hepatic reparative complex.”2Lazaridis KN Strazzabosco M Larusso NF The cholangiopathies: disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 3Strazzabosco M Fabris L Spirli C Pathophysiology of cholangiopathies.J Clin Gastroenterol. 2005; 39: S90-S102Crossref PubMed Scopus (135) Google Scholar A human disease that can be considered paradigmatic of this sequence of events is biliary atresia (BA). In this condition, the extrahepatic and major septal bile ducts fail to develop or are destroyed early after birth.4Perlmutter DH Shepherd RW Extrahepatic biliary atresia: a disease or a phenotype?.Hepatology. 2002; 35: 1297-1304Crossref PubMed Scopus (125) Google Scholar, 5Desmet VJ Congenital diseases of intrahepatic bile ducts: variations on the theme “ductal plate malformation.”.Hepatology. 1992; 16: 1069-1083Crossref PubMed Scopus (511) Google Scholar, 6Kahn E Biliary atresia revisited.Pediatr Dev Pathol. 2004; 7: 109-124Crossref PubMed Scopus (60) Google Scholar, 7Utterson EC Shepherd RW Sokol RJ Bucuvalas J Magee JC McDiarmid SV Anand R The Split Liver Research Group Biliary atresia: clinical profiles, risk factors, and outcomes of 755 patients listed for liver transplantation.J Pediatr. 2005; 147: 180-185Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 8Laurent J Gauthier F Bernard O Hadchouel M Odievre M Valayer J Alagille D Long-term outcome after surgery for biliary atresia. Study of 40 patients surviving for more than 10 years.Gastroenterology. 1990; 99: 1793-1797PubMed Scopus (130) Google Scholar Despite its heterogeneous etiology, BA represents a stereotypic pathological response, characterized by the generation of a vigorous ductular reaction with development of severe portal fibrosis. The condition rapidly progresses to biliary cirrhosis unless a Kasai operation is performed within the first few months after birth. Often, the Kasai procedure will only reduce the rate of progression, and the patient will receive a liver transplant a few years later.7Utterson EC Shepherd RW Sokol RJ Bucuvalas J Magee JC McDiarmid SV Anand R The Split Liver Research Group Biliary atresia: clinical profiles, risk factors, and outcomes of 755 patients listed for liver transplantation.J Pediatr. 2005; 147: 180-185Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 8Laurent J Gauthier F Bernard O Hadchouel M Odievre M Valayer J Alagille D Long-term outcome after surgery for biliary atresia. Study of 40 patients surviving for more than 10 years.Gastroenterology. 1990; 99: 1793-1797PubMed Scopus (130) Google Scholar Contrary to BA, patients with Alagille syndrome (AGS) suffer from deep jaundice and severe pruritus as a consequence of the cholestasis caused by congenital intrahepatic ductopenia.9Piccoli DA Spinner NB Alagille syndrome and the Jagged1 gene.Semin Liver Dis. 2001; 21: 525-534Crossref PubMed Scopus (99) Google Scholar, 10Emerick KM Rand EB Goldmuntz E Krantz ID Spinner NB Piccoli DA Features of Alagille syndrome in 92 patients: frequency and relation to prognosis.Hepatology. 1999; 29: 822-829Crossref PubMed Scopus (534) Google Scholar Progression to liver cirrhosis, however, is slower than in BA, and these patients rarely develop severe manifestations of portal hypertension and transplantation is eventually indicated because of failure to thrive, itching, and hypercholesterolemia. AGS is caused by mutations in the genes encoding Jagged1, a ligand of the Notch receptors,11Oda T Elkahloun AG Pike BL Okajima K Krantz ID Genin A Piccoli DA Meltzer PS Spinner NB Collins FS Chandrasekharappa SC Mutations in the human Jagged1 gene are responsible for Alagille syndrome.Nat Genet. 1997; 16: 235-242Crossref PubMed Scopus (968) Google Scholar, 12Li L Krantz ID Deng Y Genin A Banta AB Collins CC Qi M Trask BJ Kuo WL Cochran J Costa T Pierpont ME Rand EB Piccoli DA Hood L Spinner NB Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.Nat Genet. 1997; 16: 243-251Crossref PubMed Scopus (1045) Google Scholar or the Notch-2 receptor itself.13McDaniell R Warthen DM Sanchez-Lara PA Pai A Krantz ID Piccoli DA Spinner NB NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway.Am J Hum Genet. 2006; 79: 169-173Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar There are four Notch receptors, and they can interact with a number of different ligands (Jagged1, Jagged2, Delta-like1, Delta-like3, and Delta-like4).14Gridley T Notch signaling and inherited disease syndromes.Hum Mol Genet. 2003; 12: R9-R13Crossref PubMed Google Scholar These interactions regulate intracellular pathways involved in cell fate decisions15Crosnier C Attie-Bitach T Encha-Razavi F Audollent S Soudy F Hadchouel M Meunier-Rotival M Vekemans M JAGGED1 gene expression during human embryogenesis elucidates the wide phenotypic spectrum of Alagille syndrome.Hepatology. 2000; 32: 574-581Crossref PubMed Scopus (136) Google Scholar during embryonic development of many organs, including the liver. The clinical phenotype in AGS is, in fact, characterized by a wide range of extrahepatic manifestations9Piccoli DA Spinner NB Alagille syndrome and the Jagged1 gene.Semin Liver Dis. 2001; 21: 525-534Crossref PubMed Scopus (99) Google Scholar, 14Gridley T Notch signaling and inherited disease syndromes.Hum Mol Genet. 2003; 12: R9-R13Crossref PubMed Google Scholar, 16Krantz ID Piccoli DA Spinner NB Clinical and molecular genetics of Alagille syndrome.Curr Opin Pediatr. 1999; 11: 558-564Crossref PubMed Scopus (81) Google Scholar in association with severe ductopenia and cholestasis. Experimental studies in mutant mice and zebrafish17Flynn DM Nijjar S Hubscher SG de Goyet Jde V Kelly DA Strain AJ Crosby HA The role of Notch receptor expression in bile duct development and disease.J Pathol. 2004; 204: 55-64Crossref PubMed Scopus (68) Google Scholar, 18Kodama Y Hijikata M Kageyama R Shimotohno K Chiba T The role of notch signaling in the development of intrahepatic bile ducts.Gastroenterology. 2004; 127: 1775-1786Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 19Lorent K Yeo SY Oda T Chandrasekharappa S Chitnis A Matthews RP Pack M Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy.Development. 2004; 131: 5753-5766Crossref PubMed Scopus (179) Google Scholar and human genetic studies showed that Notch signaling is required for the biliary tree development during ductal plate remodeling.17Flynn DM Nijjar S Hubscher SG de Goyet Jde V Kelly DA Strain AJ Crosby HA The role of Notch receptor expression in bile duct development and disease.J Pathol. 2004; 204: 55-64Crossref PubMed Scopus (68) Google Scholar, 18Kodama Y Hijikata M Kageyama R Shimotohno K Chiba T The role of notch signaling in the development of intrahepatic bile ducts.Gastroenterology. 2004; 127: 1775-1786Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 20Louis AA Van Eyken P Haber BA Hicks C Weinmaster G Taub R Rand EB Hepatic jagged1 expression studies.Hepatology. 1999; 30: 1269-1275Crossref PubMed Scopus (74) Google Scholar Notch signaling seems to control hepatoblasts and mature hepatocytes transdifferentiation into cholangiocytes21Tanimizu N Miyajima A Notch signaling controls hepatoblast differentiation by altering the expression of liver-enriched transcription factors.J Cell Sci. 2004; 117: 3165-3174Crossref PubMed Scopus (232) Google Scholar, 22Nishikawa Y Doi Y Watanabe H Tokairin T Omori Y Su M Yoshioka T Enomoto K Transdifferentiation of mature rat hepatocytes into bile duct-like cells in vitro.Am J Pathol. 2005; 166: 1077-1088Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar by altering the expression of liver-enriched transcription factors. Interestingly, changes in Jagged1 and Notch expression have been reported also in the course of chronic liver diseases.23Nijjar SS Crosby HA Wallace L Hubscher SG Strain AJ Notch receptor expression in adult human liver: a possible role in bile duct formation and hepatic neovascularization.Hepatology. 2001; 34: 1184-1192Crossref PubMed Scopus (94) Google Scholar Mechanisms regulating the regenerative and reparative response to biliary damage determine the long-term outcome of cholangiopathies. Both mature cholangiocytes and hepatocytes are able to proliferate in response to damage, but in most cholangiopathies, ductular reaction dominates the histological picture. Three epithelial phenotypes can be recognized,24Roskams TA Theise ND Balabaud C Bhagat G Bhathal PS Bioulac-Sage P Brunt EM Crawford JM Crosby HA Desmet V Finegold MJ Geller SA Gouw AS Hytiroglou P Knisely AS Kojiro M Lefkowitch JH Nakanuma Y Olynyk JK Park YN Portmann B Saxena R Scheuer PJ Strain AJ Thung SN Wanless IR West AB Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.Hepatology. 2004; 39: 1739-1745Crossref PubMed Scopus (597) Google Scholar ie, hepatic progenitor cells (HPCs), intermediate hepatobiliary cells (IHBCs), and reactive ductular cells (RDCs). They can be distinguished by their morphology and pattern of expression of cytokeratin-7 (CK7), a cytoskeletal protein that is absent in mature hepatocytes. HPCs behave as a bipotential transit-amplifying compartment able to differentiate into cells committed toward the hepatocellular (IHBC) or biliary lineage24Roskams TA Theise ND Balabaud C Bhagat G Bhathal PS Bioulac-Sage P Brunt EM Crawford JM Crosby HA Desmet V Finegold MJ Geller SA Gouw AS Hytiroglou P Knisely AS Kojiro M Lefkowitch JH Nakanuma Y Olynyk JK Park YN Portmann B Saxena R Scheuer PJ Strain AJ Thung SN Wanless IR West AB Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.Hepatology. 2004; 39: 1739-1745Crossref PubMed Scopus (597) Google Scholar, 25Sell S Heterogeneity and plasticity of hepatocyte lineage cells.Hepatology. 2001; 33: 738-750Crossref PubMed Scopus (386) Google Scholar (RDC). The cell components of this “hepatic reparative complex” eventually mature into differentiated bile ducts or hepatocytes or may regress by apoptosis if liver damage ceases. In human diseases, all three cellular phenotypes coexist (Figure 1), and their relative enrichment depends on the specific conditions, such as cholangiocyte versus hepatocyte damage and degree of hepatocellular mitoinhibition. These mechanisms, however, have been studied mostly in experimental models of obstructive cholestasis and acute liver injury3Strazzabosco M Fabris L Spirli C Pathophysiology of cholangiopathies.J Clin Gastroenterol. 2005; 39: S90-S102Crossref PubMed Scopus (135) Google Scholar rather than in human cholangiopathies. Given the role of Notch signaling in biliary differentiation, we hypothesized that defects in this pathway may generate an imbalance in the cellular elements involved in the regenerative/reparative responses to liver damage. To this aim, using a number of phenotypic markers, we have compared the relative abundance and phenotype of the different epithelial components of the hepatic reparative complex in a congenital deficiency of Notch signaling (AGS) versus a stereotyped response to perinatal biliary damage (BA). We found important differences between these two cholestatic cholangiopathies. In contrast with BA, AGS was characterized by the absence of ductular reactive cells, by the accumulation of IHBCs that do not express the biliary-specific transcription factor HNF1β and by less advanced fibrosis. These changes are consistent with a profound effect of Notch signaling on liver repair mechanisms during postnatal life. Consistent with the prominent role of the ductular reaction in portal fibrosis, these changes affect the type and extent of liver fibrosis, and the clinical course of the two diseases. Frozen samples of AGS (n = 10) and BA-transplanted (BA-Tx) (n = 17) liver tissue were obtained from explants of patients undergoing liver transplantation at the Ospedali Riuniti di Bergamo, Bergamo, Italy. Additional BA samples were obtained at the time of Kasai operation (BA-Kasai, n = 13), at the Ospedali Riuniti di Bergamo. Normal liver (NL) tissue (n = 2) was obtained from potential liver donors, both males aged 49 and 18 years, respectively, whose liver grafts could not be transplanted because of iatrogenic lesions. All diagnoses were based on clinical and laboratory data and on histopathological examination of histological samples.4Perlmutter DH Shepherd RW Extrahepatic biliary atresia: a disease or a phenotype?.Hepatology. 2002; 35: 1297-1304Crossref PubMed Scopus (125) Google Scholar, 6Kahn E Biliary atresia revisited.Pediatr Dev Pathol. 2004; 7: 109-124Crossref PubMed Scopus (60) Google Scholar, 16Krantz ID Piccoli DA Spinner NB Clinical and molecular genetics of Alagille syndrome.Curr Opin Pediatr. 1999; 11: 558-564Crossref PubMed Scopus (81) Google Scholar The demographic, clinical, and biochemical characteristics of patients from the three disease groups are reported in Table 1. Liver tissue was snap-frozen in liquid nitrogen-cooled isopentane and stored at −80°C. Informed consent and local ethical committee approval were obtained before tissue collection.Table 1Demographic and Clinical Characteristics of Disease GroupsDiseaseNo. of casesAge (years)*Median and range.Sex (M/F)PruritusAGS101.66†P < 0.05. (0.94 to 16.78)4/69/10 (90%)BA-Tx170.81 (0.50 to 27.70)9/88/17 (47%)BA-Kasai130.21†P < 0.05. (0.08 to 0.94)4/9NoTot bil (mg/dl)*Median and range.ALP (<140 U/L)*Median and range.γGT (<35 U/L)*Median and range.Indication to OLTx22.35‡P < 0.01. (8.3 to 85.1)691 (120 to 1092)434.5§P < 0.05. (37 to 1530)Growth retardation: 9/10 (90%)Ascites + impaired protein synthesis: 1/10 (10%)12.6 (0.6 to 44.7)432 (98 to 750)151§P < 0.05.¶P < 0.01. (17 to 1164)Growth retardation: 12/17 (70%)Ascites: 6/17 (35%)Impaired protein synthesis: 2/17 (12%)Recurrent cholangitis: 2/17 (12%)Severe portal hypertension: 2/17 (12%)9‡P < 0.01. (6.9 to 14)464 (181 to 990)421¶P < 0.01. (200 to 1127)ALP, alkaline phosphate; γGT, γ-glutamil transpeptide; OLTX, orthotopic liver transplantation; Tot bil, total bilirubin.* Median and range.† P < 0.05.‡ P < 0.01.§ P < 0.05.¶ P < 0.01. Open table in a new tab ALP, alkaline phosphate; γGT, γ-glutamil transpeptide; OLTX, orthotopic liver transplantation; Tot bil, total bilirubin. Acetone-fixed, 4-μm-thick, serially cut frozen tissue sections were immunostained with antibodies against CK7, CK19, human epithelial antigen-125 (HEA-125), epithelial membrane antigen, and neural cell adhesion molecule; details are given in Table 2. Immunostaining was performed using a two-step procedure with EnVision (DAKO, Milan, Italy).26Cassiman D Libbrecht L Sinelli N Desmet V Denef C Roskams T The vagal nerve stimulates activation of the hepatic progenitor cell compartment via muscarinic acetylcholine receptor type 3.Am J Pathol. 2002; 161: 521-530Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Briefly, after 45 minutes incubation with primary antibodies, sections were sequentially incubated with the proper secondary horseradish peroxidase-labeled antibody (DAKO EnVision) for 30 minutes. DAKO EnVision polymer was used to improve immunoreactivity of the mouse primary antibodies. Immunohistochemical reactions were developed using 0.04 mg/ml 3,3-diaminobenzedine tetrahydrochloride and 0.01% H2O2 and counterstained with Gill's Hematoxylin (no. 2; Sigma-Aldrich, St. Louis, MO). In control sections, the primary antibody was omitted.Table 2Primary Antibodies Used for Immunohistochemistry and Biological Significance of the Corresponding MarkersAntibodyCloneHostDilutionIncubationSupplierBiological significanceCytokeratin 7 (CK7)OVTL-12/30Ms IgG1HRP 1:5; IF 1:5045′Acris, Hiddenhausen, GermanyCytoskeletal protein specific of biliary lineage; late expression during biliary ontogenesisCytokeratin 19 (CK19)RCK 108Ms IgG11:2045′DAKOCytoskeletal protein specific of biliary lineage; early expression during biliary ontogenesisHEA-125HEA-125Ms IgG11:10045′Progen Biotechnik, Heidelberg, Germany34-kd epithelial surface glycoprotein (egp34) biliary lineage-specific homologous to nidogenEpithelial membrane antigenE29Ms IgG2a1:2045′DAKOGroup of 250- to 400-kd glycosylated membrane proteins, present in a variety of epithelia of both normal and neoplastic tissues; expressed by mature biliary cellsNeural cell adhesion moleculeUJ13AMs IgG2a1:2045′DAKOFamily of cell surface sialo-glycoproteins mediating homophilic and heterophilic interactions in neuroectodermally derived tissues; expressed by immature biliary cellsLKM-1Hu IgG, FITC-conjugated1:101 hourRef. 28Fabris L Strazzabosco M Crosby HA Ballardini G Hubscher SG Kelly DA Neuberger JM Strain AJ Joplin R Characterization and isolation of ductular cells coexpressing neural cell adhesion molecule and Bcl-2 from primary cholangiopathies and ductal plate malformations.Am J Pathol. 2000; 156: 1599-1612Abstract Full Text Full Text PDF PubMed Scopus (111) Google ScholarCYP2D6, belongs to hepatocyte-specific cytochrome P450II superfamily; localized in the smooth endoplasmic reticulumKi67Ki-S5Ms IgG11:5045′DAKORequired for maintaining cell proliferation. Localized in the G1 phase in the perinuclear region, in later phases, it is also detected in the nuclear matrixp21waf1Ms IgG11:5045′Oncogene Science, Cambridge, MAInhibitor of cellular proliferation in response to DNA damage. It binds and inhibits cyclin-dependent kinase activity, thus preventing their phosphorylation and blocking cell cycle progressionBSEP (ABCB11)Goat polyclonal1:100OvernightSanta Cruz Biotechnology, Inc., Santa Cruz, CATransmembrane protein, selectively expressed by hepatocytes, involved in the ATP-dependent secretion of bile salts into canaliculiHNF1βGoat polyclonal1:300OvernightSanta Cruz BiotechnologyNuclear biliary-specific transcription factor involved in the Jagged1/Notch signal pathway and responsible for the biliary commitment in liver developmentHNF4αK9218Ms IgG2a1:100OvernightR&D Systems, Minneapolis, MNNuclear transcription factor involved in liver and kidney development, selectively expressed by hepatocytesHNF6Rabbit polyclonal1:200OvernightSanta Cruz BiotechnologyNuclear transcription factor expressed by both hepatocytes and cholangiocytes and involved in liver development and morphogenesisIF, immunofluorescence; Ms, mouse; Hu, human. Open table in a new tab IF, immunofluorescence; Ms, mouse; Hu, human. To study the balance between proliferation and mitoinhibition of hepatocytes, the ratio between Ki67 and p21waf1 was determined as described27Clouston AD Powell EE Walsh MJ Richardson MM Demetris AJ Jonsson JR Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis.Hepatology. 2005; 41: 809-818Crossref PubMed Scopus (311) Google Scholar(Table 2). To differentiate hepatocytes from IHBCs, a double-peroxidase immunostaining was performed in acetone-fixed cryosections using CK7. Briefly, after incubation with the primary antibody Ki67 or p21waf1, sections were then incubated with DAKO EnVision for 30 minutes and developed with 3,3-diaminobenzedine tetrahydrochloride, producing a brown staining. Specimens were then sequentially rinsed in distilled water for 15 minutes and 1 mol/L phosphate-buffered saline for 15 minutes, and incubated with anti-CK7 antibody for 45 minutes first and then with DAKO EnVision for 30 minutes; TrueBlue (KPL, Gaithersburg, MD) substrate was used to obtain blue staining of CK7-positive cells. Staining for the hepatocyte nuclear factors HNF4α, HNF6, and HNF1β was performed (see Table 2 for details) to study the biliary and hepatocellular differentiation of the reactive cellular elements in AGS compared with BA. To identify IHBCs and RDCs, double immunostaining for CK7 and HNF1β was performed in selected samples according to the procedure previously described for Ki67 or p21waf1, where CK7 was developed with TrueBlue (KPL) and HNF1β with 3,3-diaminobenzedine tetrahydrochloride. To study coexpression of hepatocyte and cholangiocyte markers in the different reactive cellular elements, dual immunofluorescence staining was performed in selected acetone-fixed cryosections matching anti-CK7 antibody with either anti-LKM-1 fluorescein isothiocyanate (FITC)-conjugated antibody28Fabris L Strazzabosco M Crosby HA Ballardini G Hubscher SG Kelly DA Neuberger JM Strain AJ Joplin R Characterization and isolation of ductular cells coexpressing neural cell adhesion molecule and Bcl-2 from primary cholangiopathies and ductal plate malformations.Am J Pathol. 2000; 156: 1599-1612Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar (courtesy of G. Ballardini, Department of Internal Medicine, S. Orsala-Malpighi, Bologna, Italy) or with anti-bile salt export pump (BSEP) antibody29Sidler Pfändler MA Hochli M Inderbitzin D Meier PJ Stieger B Small hepatocytes in culture develop polarized transporter expression and differentiation.J Cell Sci. 2004; 117: 4077-4087Crossref PubMed Scopus (32) Google Scholar(Table 2). Following 1-hour incubation at room temperature with LKM-1 FITC-conjugated antibody or overnight incubation with BSEP, tissue sections were rinsed and then incubated with the secondary antibody FITC-conjugated rabbit anti-goat (dilution, 1:20; incubation, 30 minutes; DAKO). In both cases, tissue sections were then incubated for 45 minutes with the CK7 antibody, detected by the secondary antibody Texas Red-conjugated horse anti-mouse (dilution, 1:20; incubation, 30 minutes; Vector Laboratories, Burlingame, CA). Slides were mounted in glycerol supplemented with 5% 1,4-diazabicyclo[2.2.2]octane (Sigma-Aldrich) to avoid fluorescence bleaching. Peroxidase immunostainings were analyzed with a Nikon Eclipse E800 microscope (Nikon, Milan, Italy). Images were collected using a digital camera (Coolpix 995; Nikon), stored by the Fotostation 4.5 software (FotoWare, Oslo, Norway) and analyzed by the Photoshop 5.0 software (Adobe, San Jose, CA) and by the UTHSCSA Image Tool 3.0 (University of Texas, San Antonio, TX). Computer-assisted morphometric analysis was used to quantify RDCs and IHBCs. The different epithelial elements were categorized according to the definition proposed by Roskams et al.24Roskams TA Theise ND Balabaud C Bhagat G Bhathal PS Bioulac-Sage P Brunt EM Crawford JM Crosby HA Desmet V Finegold MJ Geller SA Gouw AS Hytiroglou P Knisely AS Kojiro M Lefkowitch JH Nakanuma Y Olynyk JK Park YN Portmann B Saxena R Scheuer PJ Strain AJ Thung SN Wanless IR West AB Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.Hepatology. 2004; 39: 1739-1745Crossref PubMed Scopus (597) Google Scholar RDCs were defined as CK7- and CK19-positive cells with biliary phenotype arranged in irregularly shaped structures.3Strazzabosco M Fabris L Spirli C Pathophysiology of cholangiopathies.J Clin Gastroenterol. 2005; 39: S90-S102Crossref PubMed Scopus (135) Google Scholar, 25Sell S Heterogeneity and plasticity of hepatocyte lineage cells.Hepatology. 2001; 33: 738-750Crossref PubMed Scopus (386) Google Scholar IHBCs were defined as cell with morphology and size intermediate between hepatocyte and cholangiocyte, with a peculiar pattern of CK7 immunoreactivity, faint on the cytoplasm and reinforced at the plasma membrane.24Roskams TA Theise ND Balabaud C Bhagat G Bhathal PS Bioulac-Sage P Brunt EM Crawford JM Crosby HA Desmet V Finegold MJ Geller SA Gouw AS Hytiroglou P Knisely AS Kojiro M Lefkowitch JH Nakanuma Y Olynyk JK Park YN Portmann B Saxena R Scheuer PJ Strain AJ Thung SN Wanless IR West AB Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.Hepatology. 2004; 39: 1739-1745Crossref PubMed Scopus (597) Google Scholar In digital images of 10 nonoverlapping random fields taken at ×200, the cytokeratin-positive area (CK19 for RDCs and CK7 for IHBCs) was calculated as the percentage of pixels above the threshold value with respect to the total pixels per field; IHBC area was then calculated by subtracting the CK19 to the CK7 area. HPCs were counted as small, oval, or spindle-shaped cells with scant cytoplasm and oval nucleus, alone or in small clamps, localized in the parenchyma or at the portal interface and recognizable by CK19 immunoreactivity.24Roskams TA Theise ND Balabaud C Bhagat G Bhathal PS Bioulac-Sage P Brunt EM Crawford JM Crosby HA Desmet V Finegold MJ Geller SA Gouw AS Hytiroglou P Knisely AS Kojiro M Lefkowitch JH Nakanuma Y Olynyk JK Park YN Portmann B Saxena R Scheuer PJ Strain AJ Thung SN Wanless IR West AB Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.Hepatology. 2004; 39: 1739-1745Crossref PubMed Scopus (597) Google Scholar, 30Libbrecht L Roskams T Hepatic progenitor cells in human liver diseases.Semin Cell Dev Biol. 2002; 13: 389-396Crossref PubMed Scopus (177) Google Scholar The number of HPCs was counted by two independent observers (L.F. and A.S.) in five nonoverlapping random fields observed at ×200. The proliferative index of hepatocyte and IHBCs was calculated counting the number of nuclei positive for Ki67 in five nonoverlapping random fields taken at ×400. The hepatocyte replicative arrest ratio was expressed according to Clouston27Clouston AD Powell EE Walsh MJ Richardson MM Demetris AJ Jonsson JR Fibrosis corre
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