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• W1972305087 abstract "It has recently been shown that reactive bile ductules display neuroendocrine features, including immunoreactivity for the neural cell adhesion molecule (NCAM). In this study we have compared the immunohistochemical expression of NCAM with that of HEA-125 (biliary specific) and LKM-1 (hepatocyte specific) and other markers relevant to morphogenesis (Bcl-2, EMA) and cell proliferation (Ki-67) in cryostat sections from different chronic liver diseases and from fetal livers at different gestational ages. In parallel, viable NCAM-positive ductular cells were purified from collagenase digests of cirrhotic livers by immunomagnetic separation and characterized by immunocytochemistry and transmission electron microscopy. We demonstrated that reactive ductules with atypical morphology coexpressed NCAM and Bcl-2 and were found mainly in congenital diseases associated with ductal plate malformation and in primary cholangiopathies. On the contrary, reactive ductules with typical morphology were negative for NCAM/Bcl-2 and positive for EMA. Reactive ductules coexpressing NCAM/Bcl-2 were negative for the proliferation marker Ki-67 and appeared to be directly connected with periportal hepatocytes. In fetal livers NCAM/Bcl-2 was transiently expressed during the early developmental stages of ductal plate (10–16 weeks) and started to disappear as the ductal plate began duplicating. NCAM-positive ductal plate cells were Ki-67 negative, becoming positive in duplicated segments. Thus the histogenesis of ductular reactive cells seems to recapitulate the early stages of biliary ontogenesis. In primary cholangiopathies and ductal plate malformations, these cells do not appear to maturate further, and thus abundant ductular structures coexist with vanishing mature ducts. These NCAM-positive ductular cells were immunopurified from patients with chronic cholestatic liver diseases and showed ultrastructural features consistent with a less differentiated phenotype than mature cholangiocytes. These isolated cells represent a useful model for in vitro studies. It has recently been shown that reactive bile ductules display neuroendocrine features, including immunoreactivity for the neural cell adhesion molecule (NCAM). In this study we have compared the immunohistochemical expression of NCAM with that of HEA-125 (biliary specific) and LKM-1 (hepatocyte specific) and other markers relevant to morphogenesis (Bcl-2, EMA) and cell proliferation (Ki-67) in cryostat sections from different chronic liver diseases and from fetal livers at different gestational ages. In parallel, viable NCAM-positive ductular cells were purified from collagenase digests of cirrhotic livers by immunomagnetic separation and characterized by immunocytochemistry and transmission electron microscopy. We demonstrated that reactive ductules with atypical morphology coexpressed NCAM and Bcl-2 and were found mainly in congenital diseases associated with ductal plate malformation and in primary cholangiopathies. On the contrary, reactive ductules with typical morphology were negative for NCAM/Bcl-2 and positive for EMA. Reactive ductules coexpressing NCAM/Bcl-2 were negative for the proliferation marker Ki-67 and appeared to be directly connected with periportal hepatocytes. In fetal livers NCAM/Bcl-2 was transiently expressed during the early developmental stages of ductal plate (10–16 weeks) and started to disappear as the ductal plate began duplicating. NCAM-positive ductal plate cells were Ki-67 negative, becoming positive in duplicated segments. Thus the histogenesis of ductular reactive cells seems to recapitulate the early stages of biliary ontogenesis. In primary cholangiopathies and ductal plate malformations, these cells do not appear to maturate further, and thus abundant ductular structures coexist with vanishing mature ducts. These NCAM-positive ductular cells were immunopurified from patients with chronic cholestatic liver diseases and showed ultrastructural features consistent with a less differentiated phenotype than mature cholangiocytes. These isolated cells represent a useful model for in vitro studies. A common histopathological response to many forms of chronic liver diseases is an increase in the number of bile duct-like structures,1Popper H Schaffner F Stein R Ductular cell reaction in the liver in hepatic injury.J Mt Sinai Hosp. 1957; 24: 551-556PubMed Google Scholar, 2Masuko K Rubin E Popper H Proliferation of bile ducts in cirrhosis.Arch Pathol. 1964; 78: 421-431PubMed Google Scholar, 3Desmet VJ Roskams T Van Eyken P Ductular reaction in the liver.Pathol Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar paralleled by an inflammatory cell infiltrate and periportal fibrosis. This ductular reaction is thought to act as a pacemaker in the development of the progressive fibrotic process that leads to liver cirrhosis.1Popper H Schaffner F Stein R Ductular cell reaction in the liver in hepatic injury.J Mt Sinai Hosp. 1957; 24: 551-556PubMed Google Scholar, 3Desmet VJ Roskams T Van Eyken P Ductular reaction in the liver.Pathol Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 4Roskams T Rosenbau J De Vos R David G Desmet VJ Heparan sulfate proteoglycan expression in chronic cholestatic human liver diseases.Hepatology. 1996; 24: 524-532Crossref PubMed Google Scholar After liver damage, bile ductular cells produce a number of mediators, such as interleukin-6,5Matsumoto K Fuji H Michalopoulos G Fung JJ Demetris AJ Human biliary epithelial cells secrete and respond to cytokines and hepatocyte growth factors in vitro: interleukin-6, hepatocyte growth factor and epidermal growth factor promote DNA synthesis in vitro.Hepatology. 1994; 20: 376-382Crossref PubMed Scopus (122) Google Scholar, 6Yasoshima M Kono N Sugawara H Katayanagi K Harda K Nakanuma Y Increased expression of interleukin-6 and tumor necrosis factor-alpha in pathologic biliary epithelial cells: in situ and culture study.Lab Invest. 1998; 78: 89-100PubMed Google Scholar transforming growth factor-β,7Milani S Herbst H Schuppan D Stein H Surrenti C Transforming growth factor β1 and β2 are differentially expressed in fibrotic liver disease.Am J Pathol. 1991; 139: 1221-1229PubMed Google Scholar endothelin-1,8De Groen PC, Vroman B, LaRusso NF: Physiologic regulation of cholangiocyte proliferation in vitro. Proceeding of the Digestive Disease Week 1996, May 19–22, San Francisco, CAGoogle Scholar monocyte chemotactic protein-1,9Marra F De Franco R Grappone C Pinzani M Milani S Pastacaldi S Laffi G Gentilini P Expression of monocyte chemotactic protein-1 during active hepatic fibrogenesis in humans.Ital J Gastroenterol. 1996; 20: 300-306Google Scholar platelet-derived growth factor,10Adams DH Biliary epithelial cells: innocent victims or active participants in immuno-mediated liver diseases?.J Lab Clin Med. 1996; 128: 528-530Abstract Full Text PDF PubMed Scopus (10) Google Scholar tumor necrosis factor-α,11Berg PA Klein R Rocken M Cytokines in primary biliary cirrhosis.Semin Liver Dis. 1997; 17: 115-123Crossref PubMed Scopus (51) Google Scholar nitric oxide,12Vos TA Gouw ASH Klok PA Havinga R Van Goor H Huitema S Roelofsen H Kuipers F Jansen PLM Moshage H Differential effects of nitric oxide synthase inhibitors on endotoxin-induced liver damage in rats.Gastroenterology. 1997; 113: 1323-1333Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar and parathyroid hormone-related peptide,13Roskams T Campos RV Drucker DJ Desmet VJ Reactive human bile ductules express parathyroid hormone-related peptide.Histopathology. 1993; 23: 11-19Crossref PubMed Scopus (48) Google Scholar which is probably involved in paracrine communications with mesenchymal and inflammatory cells.Currently, four types of ductular reaction are described: typical, atypical, cholangiolar, and oval cell.14Sirica AE Ductular hepatocytes.Histol Histopathol. 1995; 10: 433-456PubMed Google Scholar, 15Sell S Comparison of liver progenitor cells in human atypical ductular reactions with those seen in experimental models of liver injury.Hepatology. 1998; 27: 317-331Crossref PubMed Scopus (124) Google Scholar Atypical ductular reaction is characterized by an anastomosing network of ductules, with poorly defined lumina, lined by flattened cells with a scant cytoplasm; atypical ductules are most often localized at the peripheral zone of the portal space. This occurs in most forms of chronic cholestatic liver injury, such as in primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and chronic extrahepatic biliary obstruction. Available data indicate that whereas typical reactive ductules derive from proliferation of the preexisting well-differentiated biliary epithelial cells,16Slott PA Liu MJ Tavoloni N Origin, pattern and mechanism of bile duct proliferation following biliary obstruction in the rat.Gastroenterology. 1990; 99: 466-477Abstract PubMed Google Scholar atypical ductules arise from ductular metaplasia of hepatocytes14Sirica AE Ductular hepatocytes.Histol Histopathol. 1995; 10: 433-456PubMed Google Scholar, 17Van Eyken P Desmet VJ Development of intrahepatic bile ducts, ductular metaplasia of hepatocytes, and cytokeratin expression in various types of human hepatic neoplasms.in: Sirica AE The Role of Cell Types in Hepatocarcinogenesis. CRC Press, Boca Raton, FL1992: 227-263Google Scholar and/or from activation of a facultative bipotential stem cell,14Sirica AE Ductular hepatocytes.Histol Histopathol. 1995; 10: 433-456PubMed Google Scholar, 18Fausto N Liver stem cells.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter DA Shafritz DA The Liver: Biology and Pathobiology. Raven Press, New York1994: 1501-1518Google Scholar the actual identity of which in humans has not yet been determined. These cells, capable of differentiating into biliary epithelial cells and hepatocytes, are thought to be located in close proximity to the terminal bile ductules and to sustain liver regeneration under conditions such as fulminant hepatic failure that compromise the proliferative capability of hepatocytes and cholangiocytes.The study of the biological characteristics of reactive ductular cells may lead to a better understanding of both the dynamics of epithelial cell populations in the liver and some basic liver pathophysiological mechanisms. The phenotypic profile of ductular cells has been partly characterized by histological studies: they are anchored on a basement membrane and can express major histocompatibility complex (MHC) class I proteins19Rouger P Poupon R Gane P Mallissen B Darnis F Salmon C Expression of blood group antigens including HLA markers in human adult liver.Tissue Antigens. 1986; 27: 78-86Crossref PubMed Scopus (41) Google Scholar, 20Nakanuma Y Saski M Expression of blood group-related antigens in the intrahepatic biliary tree and hepatocytes in normal livers and various hepatobiliary diseases.Hepatology. 1989; 10: 174-178Crossref PubMed Scopus (101) Google Scholar and cell adhesion molecules, such as carcinoembryonic antigen21Gerber MA Thung SN Shen S Stromeyer FW Ishak KG Phenotypic characterization of hepatic proliferation: antigenic expression by proliferating epithelial cells in fetal liver, massive hepatic necrosis, and nodular transformation of the liver.Am J Pathol. 1983; 110: 70-74PubMed Google Scholar and intercellular adhesion molecule-1.22Adams DH Hubscher SG Shaw J Johnson GD Babbs C Rothlein R Neuberger JM Increased expression of intercellular adhesion molecule 1 on bile ducts in primary biliary cirrhosis and primary sclerosing cholangitis.Hepatology. 1991; 14: 426-431Crossref PubMed Scopus (140) Google Scholar, 23Dillon P Belchis D Tracy T Cilley R Hafer L Krummel T Increased expression of intercellular adhesion molecules in biliary atresia.Am J Pathol. 1994; 145: 263-267PubMed Google Scholar Furthermore, recent data indicate that reactive ductules display neuroendocrine features, including immunoreactivity for chromogranin A and for the neural cell adhesion molecule (NCAM).24Roskams T Van den Oord JJ De Vos R Desmet VJ Neuroendocrine features of reactive bile ductules in cholestatic liver disease.Am J Pathol. 1990; 137: 1019-1025PubMed Google Scholar NCAM is a surface glycoprotein, belonging to the immunoglobulin superfamily, that mediates cell-cell (homophilic) and cell-matrix (heterophilic. interactions during the development of the nervous system,25McClain DA Edelman GM A neural cell adhesion molecule from human brain.Proc Natl Acad Sci USA. 1982; 79: 6380-6384Crossref PubMed Scopus (46) Google Scholar, 26Hoffman S Edelman GM Kinetics of homophilic binding by embryonic and adult forms of the neural cell adhesion molecule.Proc Natl Acad Sci USA. 1983; 80: 5762-5766Crossref PubMed Scopus (515) Google Scholar, 27Werz W Schachner M Adhesion of neural cells to extracellular matrix constituents. Involvement of glycosaminoglycans and cell adhesion molecules.Dev Brain Res. 1988; 43: 225-234Crossref Scopus (43) Google Scholar, 28Lanier LL Testi R Bindl J Phillips JH Identity of Leu-19 (CD 56) leukocyte differentiation antigen and neural cell adhesion molecule.J Exp Med. 1989; 169: 2233-2238Crossref PubMed Scopus (421) Google Scholar lung, and gut epithelia.29Edelman GM Crossin KL Cell adhesion molecules: implications for a molecular histology.Annu Rev Biochem. 1991; 60: 155-190Crossref PubMed Scopus (651) Google ScholarGiven the role played by NCAM in morphogenetic processes, we have investigated its immunohistochemical expression and that of a number of markers relevant to morphogenesis and cell proliferation on the biliary epithelium in a wide variety of chronic hepatobiliary diseases and in fetal livers at different gestational ages. In particular, the B-cell leukemia lymphoma-2 protein (Bcl-2), a protooncogene product localized to the mitochondrial inner membrane,30Hockenbery DM Nunez G Milliman C Schreiber RD Korsmeyer SJ Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death.Nature. 1990; 348: 334-336Crossref PubMed Scopus (3523) Google Scholar is of potential interest: its ability to block apoptosis is required in the developmental processes of new epithelial structures.31Lu Q-L Poulsom R Wong L Hanby AM BCL-2 expression in adult and embryonic non-haematopoietic tissues.J Pathol. 1993; 169: 431-437Crossref PubMed Scopus (299) Google Scholar, 32LeBrun DP Warnke RA Cleary ML Expression of bcl-2 in fetal tissues suggests a role in morphogenesis.Am J Pathol. 1993; 142: 743-753PubMed Google Scholar Our results indicate that in congenital diseases related to ductal plate malformation and primary diseases of the biliary tree, atypical ductules retain less differentiated immunophenotypic features, such as coexpression of NCAM and Bcl-2, similar to those transiently expressed during the embryonic development of the biliary system. Furthermore, based on a positive selection of cells expressing NCAM, we have devised a method to purify a viable population of atypical ductular cells from cirrhotic human liver; ultrastructural studies of these isolated cells further support the less differentiated biliary phenotypic traits of NCAM-positive cells.Materials and MethodsImmunohistochemistrySource of TissueHuman tissue samples from hepatectomy specimens were snap frozen in liquid nitrogen and stored at −70°C. Normal liver tissue (n = 10) was obtained from graft reductions for pediatric liver transplant recipients. Diseased liver tissue was obtained at the time of the orthotopic liver transplantation; this included parenchymal liver cirrhosis (chronic autoimmune hepatitis, n = 5; cryptogenic cirrhosis, n = 5. alcoholic liver cirrhosis, n = 5), primary immune-mediated cholangiopathies (PBC, n = 12; PSC, n = 8), and congenital cholangiopathies (extrahepatic bile duct atresia, n = 5), congenital diseases of bile ducts related to ductal plate malformation (Caroli's disease, n = 2; polycystic liver disease, n = 2). Diagnosis was made by clinical, serological, and histological criteria. Fetal livers (n = 10) from human embryos and fetuses from the 10th to 16th weeks of gestation were obtained, after informed consent, from legal voluntary abortions.Classification of Intrahepatic Bile DuctsIn adult livers, intrahepatic biliary epithelium was categorized according to the method of Ludwig.33Ludwig J New concepts in biliary cirrhosis.Semin Liver Dis. 1987; 7: 293-301Crossref PubMed Scopus (104) Google Scholar Major ducts were accompanied by a hepatic artery branch and comprised both interlobular ducts (luminal diameter between 50 and 100 μm, lined by cuboidal cells) and septal ducts (luminal diameter greater than 100 μm, lined by a columnar epithelium). Bile ductules were the smallest conduits (less than 50 μm in diameter), were lined by cuboidal epithelial cells, and were not accompanied by the small branches of portal vein and hepatic artery. Typical or atypical reactive ductules were respectively classified according to the following histological criteria: lumen patency (discernible or no discernible lumen), morphology of the lining epithelium (cuboidal or flattened), and ductule profile (well-formed or irregular anastomosing plexuses).In fetal livers development of biliary epithelium was categorized as reported by Van Eyken34Van Eyken P Sciot R Callea F Van der Steen K Moerman P Desmet VJ The development of the intrahepatic bile ducts in man: a keratin-immunohistochemical study.Hepatology. 1988; 8: 1586-1595Crossref PubMed Scopus (267) Google Scholar and Shah.35Shah KD Gerber MA Development of intrahepatic bile ducts in humans.Arch Pathol Lab Med. 1990; 114: 597-600PubMed Google Scholar During the early stages of embryonic development (from 6 to 8 weeks), hepatoblasts adjacent to mesenchyme around the largest portal vein branches close to the liver hilum alter their phenotype toward bile duct-type cells, giving rise to the primordial ductal plate (9–10th weeks).Alkaline Phosphatase ImmunostainingSerial 5-μm cryostat sections were fixed in acetone at room temperature, and immunohistochemistry was performed by the alkaline phosphatase method, using monoclonal antibodies against human epithelial antigen-125 (HEA-125), NCAM, epithelial membrane antigen (EMA), and B-cell leukemia lymphoma-2 protein (Bcl-2) (see also Table 1). The working dilution of each antibody was determined in preliminary tests. After preliminary blocking with normal rabbit serum (1:10; Dako), sections were incubated with primary antibodies for 1 hour at room temperature. After incubation with the primary antibody, sections were washed three times before incubation with rabbit anti-mouse immunoglobulins (1:25; Dako) for 45 minutes. After further washing, sections were incubated with alkaline phosphatase mouse anti-alkaline phosphatase (APAAP) (1:50; Dako) for 45 minutes. Antibody binding was detected using fast red substrate (Sigma Chemical Co., Poole, UK), and sections were counterstained with Mayer's hematoxylin (Sigma). In controls, primary antibody was omitted. The sections were assessed by three independent observers (LF, RJ, SGH) according to the classification based on bile duct morphology given above.Table 1Primary Antibodies Used for Immunohistochemistry and Biological Significance of the Corresponding MarkersMarkersSourceIsotypeOptimal dilutionBiological significanceHEA-125Progen Biotechnik GmbhIgG1 (mouse)1:100 (APAAP) 1:50 (IF)34-kd epithelial surface glycoprotein (egp34) biliary lineage-specific42 homologous to nidogen72NCAMDakoIgG2a (mouse)1:20 (APAAP) 1:10 (IF)Family of cell surface sialoglycoproteins mediating homophilic and heterophilic interactions in neuroectodermally derived tissues25–28EMADakoIgG2a (mouse)1:20 (APAAP) 1:10 (IF)Group of 250–400-kd glycosylated membrane proteins, present in a variety of epithelia of both normal and neoplastic tissues73; a late biliary maturation marker57Bcl-2DakoIgG1 (mouse)1:40 (APAAP) 1:20 (IF)Protein localized to the mitochondrial inner membrane, able to block apoptosis30; it is up-regulated in processes requiring protection from apoptosis, such as metaplasia63,64Ki-67Immunotech (MIB-1 clone)IgG2a (mouse)1:40 (IF)Nuclear antigen associated with the cell cycle, expressed throughout all phases except G036LKM-1See references38Ballardini G Landi P Busachi GA Bianchi FB Pisi E HBsAg-induced hypertrophic smooth endoplasmic reticulum as a target for liver-kidney microsomal (LKM) antibodies.Clin Exp Immunol. 1981; 43: 599-604PubMed Google Scholar, 39Bianchi FB Biagini G Ballardini G Cenacchi G Faccani A Pisi E Laschi R Liotta L Garbisa S Basement membrane production by hepatocytes in chronic liver disease.Hepatology. 1984; 4: 1167-1172Crossref PubMed Scopus (78) Google ScholarIgG (human) FITC-conjugatedNeat (IF)P450IID6, a cytochrome P-450 monooxygenase localized to the smooth endoplasmic reticulum; hepatocyte-specific40Homberg JC Abuaf N Bernard O Islam S Alvarez F Khalil SH Poupon R Darnis F Lévy VG Grippon P Opolon P Bernuau J Benhamou JP Alagille D Chronic active hepatitis associated with antiliver/kidney microsome antibody type 1: a second type of “autoimmune” hepatitis.Hepatology. 1987; 7: 1333-1339Crossref PubMed Scopus (526) Google Scholar, 41Manns MP Griffin KJ Sullivan KF Johnson EF LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase.J Clin Invest. 1991; 88: 1370-1378Crossref PubMed Scopus (421) Google Scholar Open table in a new tab Double ImmunostainingDouble immunostaining was performed on selected sections with different combinations of the following antibodies (see Table 1). HEA-125, NCAM, EMA, Bcl-2, and Ki-67 (MIB-1 clone, a nuclear antigen associated with the cell cycle),36Hall PA Levison DA Review: assessment of cell proliferation in histological material.J Clin Pathol. 1990; 43: 184-192Crossref PubMed Scopus (411) Google Scholar and Texas red and fluorescein isothiocyanate (FITC) (anti-IgG1 or anti-IgG2a, 1:50. Europath, Bude, UK) conjugates as secondary fluorescent antibodies. When primary antibodies belonged to different subclasses (such as HEA-125 and NCAM, HEA-125 and EMA, HEA-125 and Ki-67, NCAM and Bcl-2. conventional simultaneous labeling could be used: the two primary antibodies were mixed, incubated together for 30 minutes, and then visualized with a mixture of Texas red and FITC conjugates. When primary antibodies shared the same subclass (such as HEA-125 and Bcl-2, NCAM and EMA, NCAM and Ki-67), staining was performed using the reverse sequential indirect method, in which the sequence of addition of primary antibodies in duplicate immunostaining experiments is reversed.37Johnson GD Walker L Reversed sequential double immunostaining—an indirect (antiglobulin) method for identifying cellular subsets using monoclonal antibodies of the same isotype.J Immunol Methods. 1986; 95: 149-152Crossref PubMed Scopus (5) Google Scholar FITC-conjugated human IgG from a liver kidney microsome-1 (LKM-1)-positive patient (0.1 mg/ml in phosphate-buffered saline (PBS)) was used as a hepatocellular marker, because it homogeneously and specifically decorates the hepatocyte smooth endoplasmic reticulum by reacting with a cytochrome P-450 monooxygenase isoform.38Ballardini G Landi P Busachi GA Bianchi FB Pisi E HBsAg-induced hypertrophic smooth endoplasmic reticulum as a target for liver-kidney microsomal (LKM) antibodies.Clin Exp Immunol. 1981; 43: 599-604PubMed Google Scholar, 39Bianchi FB Biagini G Ballardini G Cenacchi G Faccani A Pisi E Laschi R Liotta L Garbisa S Basement membrane production by hepatocytes in chronic liver disease.Hepatology. 1984; 4: 1167-1172Crossref PubMed Scopus (78) Google Scholar, 40Homberg JC Abuaf N Bernard O Islam S Alvarez F Khalil SH Poupon R Darnis F Lévy VG Grippon P Opolon P Bernuau J Benhamou JP Alagille D Chronic active hepatitis associated with antiliver/kidney microsome antibody type 1: a second type of “autoimmune” hepatitis.Hepatology. 1987; 7: 1333-1339Crossref PubMed Scopus (526) Google Scholar, 41Manns MP Griffin KJ Sullivan KF Johnson EF LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase.J Clin Invest. 1991; 88: 1370-1378Crossref PubMed Scopus (421) Google Scholar LKM-1 was used in combination with HEA-12542Momburg F Moldenhauer G Hammerling GH Moller P Immunohistochemical study of the surface expression of a M4 34.000 human epithelium-specific glycoprotein in normal and malignant tissues.Cancer Res. 1987; 47: 2883-2891PubMed Google Scholar and NCAM,24Roskams T Van den Oord JJ De Vos R Desmet VJ Neuroendocrine features of reactive bile ductules in cholestatic liver disease.Am J Pathol. 1990; 137: 1019-1025PubMed Google Scholar taken as biliary cell markers. Sections were mounted in Diazabicyclo(2.2.2)octane (DABCO) to retard the fading of the fluorochromes. In controls reactivity of each conjugate with each primary antibody was tested to exclude the possibility of subclass cross-reactivity.Sections were analyzed by with a laser scanning confocal microscopy MCR-500 system, equipped with a krypton-argon laser (Bio-Rad), for coincident sites of reactivity. Fluorescence images were collected and analyzed in the dual-channel mode, with the laser lines at 488 and 568 nm. Under these conditions, single labeling appears red (Texas red) and green (FITC), whereas coincident labeling appears yellow. To measure the relative fluorescence intensity of antigen expression on reactive ductules, the fluorescence within a defined area occupied by biliary ductular cells was defined in bands of increasing intensity (from 0 to 255), where the highest values were shown in red (219–255) and the lowest appeared in blue (37–72); intensity below 36 was black and indicated unstained areas.Isolation of NCAM-Positive Ductular Cells from Human LiverSource of TissueNormal liver (n = 12) from the pediatric segmental transplantation procedures and cirrhotic liver (n = 12) from patients undergoing liver transplantation for PBC (n = 9) and PSC (n = 3) were used for cell isolation studies. Donor organs, perfused with University of Wisconsin surgical preservation fluid, and slices of cirrhotic liver, in tissue culture medium, were stored for up to 24 hours at 4°C before cell preparation.Purification of CellsApproximately 30 g of liver tissue was dissected, finely diced, and incubated at 37°C with 50 ml collagenase type 1A (1 mg/ml. Sigma Chemical Company). After 1–2 hours of incubation the digest was filtered through fine mesh (Sigma screen cup; Sigma Chemical Company), and the filtered tissue pieces were further diced to enhance the release of cells. Digested tissue pieces and filtrate were recombined and incubated for an additional 30 minutes to 1 hour at 37°C. After a total of 2–3 hours of digestion the tissue pieces were again filtered through fine mesh and the filtrate was washed three times in PBS. After the final wash the volume of filtrate was adjusted to 24 ml. Three milliliters of filtrate was overlaid on Percoll gradients, each composed of 3.5 ml 1.04 g/ml Percoll underlaid by 3.5 ml 1.09 g/ml Percoll and then centrifuged at 800 × g for 30 minutes at room temperature. After centrifugation, five separate fractions were evident. Each fraction was harvested separately, washed three times in PBS, and centrifuged at 800 × g for 30 minutes. Cell smears from the final cell pellet derived from each fraction were stained with the biliary specific HEA-125 and cytokeratin 19 (CK-19. antibodies, to establish where atypical cholangiocytes could be harvested. In addition, from each fraction, cells were immunopurified using HEA-125 as already described43Joplin R Strain AJ Neuberger JM Immuno-isolation and culture of biliary epithelial cells from normal human liver.In Vitro Cell Dev Biol. 1989; 25: 1189-1192Crossref PubMed Scopus (82) Google Scholar, 44Joplin R Strain AJ Neuberger JM Biliary epithelial cells from the liver of patients with primary biliary cirrhosis: isolation, characterization, and short-term culture.J Pathol. 1990; 162: 255-260Crossref PubMed Scopus (39) Google Scholar and further characterized by immunocytochemistry for CK-19, NCAM, and EMA. The supernatant and pellicle (fraction 1) that floated as a layer on top of the gradient were composed mainly of cell debris and hepatocytes. Some HEA-125/CK-19-positive cells were present, but cells in this fraction had very low viability (<20%). Cells equilibrating in 1.04 g/ml Percoll (fraction 2) and as a layer on top of 1.09 g/ml Percoll (fraction 3) were composed of single cells and cell aggregates, which were positive for HEA-125 and CK-19 and were >95% viable. In particular, fraction 2 was composed of single cells or small aggregates of 2–10 cells each. All were positive for CK-19 and, in cirrhotic liver, many (>50%) were positive for NCAM and negative for EMA. In contrast, HEA-125-positive cells equilibrating in fraction 3 were predominantly small to large aggregates of around 10–100 cells each. These cells were positive for CK-19 and EMA (>95% positive) but were largely negative for NCAM (<5% positive). Very few cells were present in 1.09 g/ml Percoll (fraction 4), whereas the pellet (fraction 5. appeared to be composed predominantly of erythrocytes. These preliminary studies indicate that atypical ductular cells fractionate predominantly in 1.04 g/ml Percoll, whereas mature cholangiocytes pass through this fraction and can be harvested from the layer of cells floating on 1.09 g/ml Percoll. Therefore, fractions 2 and 3 were pooled together for further sequential immunomagnetic separation of biliary cells expressing NCAM and HEA-125. Pooled fractions 2 and 3 were incubated with anti-NCAM (37°C for 30 minutes) followed by bead-conjugated rat anti-mouse IgG2 (Dynal, Oslo, Norway) (4°C for 30 minutes). After magnetic harvest of NCAM-positive cells, a subset of biliary epithelial cells was p" @default.
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• W1972305087 title "Characterization and Isolation of Ductular Cells Coexpressing Neural Cell Adhesion Molecule and Bcl-2 from Primary Cholangiopathies and Ductal Plate Malformations" @default.
• W1972305087 cites W1805419512 @default.
• W1972305087 cites W1963985392 @default.
• W1972305087 cites W1967031305 @default.
• W1972305087 cites W1976630370 @default.
• W1972305087 cites W1980254549 @default.
• W1972305087 cites W1981442614 @default.
• W1972305087 cites W1986076862 @default.
• W1972305087 cites W1993767935 @default.
• W1972305087 cites W2004082968 @default.
• W1972305087 cites W2007771924 @default.
• W1972305087 cites W2013727967 @default.
• W1972305087 cites W2014102565 @default.
• W1972305087 cites W2016008430 @default.
• W1972305087 cites W2017492932 @default.
• W1972305087 cites W2022273331 @default.
• W1972305087 cites W2023469977 @default.
• W1972305087 cites W2028427669 @default.
• W1972305087 cites W2029714766 @default.
• W1972305087 cites W2030552215 @default.
• W1972305087 cites W2046378363 @default.
• W1972305087 cites W2048716965 @default.
• W1972305087 cites W2055565800 @default.
• W1972305087 cites W2057257609 @default.
• W1972305087 cites W2057613283 @default.
• W1972305087 cites W2060474446 @default.
• W1972305087 cites W2064259553 @default.
• W1972305087 cites W2081843085 @default.
• W1972305087 cites W2084767118 @default.
• W1972305087 cites W2084857174 @default.
• W1972305087 cites W2085979160 @default.
• W1972305087 cites W2090557152 @default.
• W1972305087 cites W2091942765 @default.
• W1972305087 cites W2106125634 @default.
• W1972305087 cites W2120646999 @default.
• W1972305087 cites W2126405738 @default.
• W1972305087 cites W2130818367 @default.
• W1972305087 cites W2132286068 @default.
• W1972305087 cites W2136218955 @default.
• W1972305087 cites W2139537685 @default.
• W1972305087 cites W2147754142 @default.
• W1972305087 cites W2156480888 @default.
• W1972305087 cites W2164606734 @default.
• W1972305087 cites W2168362482 @default.
• W1972305087 cites W2172160601 @default.
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