A study of graft versus host disease using bile duct implants under the kidney capsule.

BACKGROUND/AIMS: Graft versus host disease (GVHD) following histoincompatible bone marrow transplantation may be modelled experimentally using irradiated metallothionein promoter-H-2Kb transgenic mice (MET-Kb mice), reconstituted with syngeneic non-transgenic spleen and lymph node (LN) cells. In this model, inflammation peaks at 3 weeks post-reconstitution, but resolves by 3 months, and is focussed on portal tracts and bile ducts (BD). The aim of this study was to determine if transgene-expressing hepatocytes play a role in the immune response, why portal tracts are selectively targeted, and which cell types are involved. METHODS: Intrahepatic BD (IHBD) with attached hepatocytes, or extrahepatic BD (EHBD) devoid of hepatocytes, were isolated from MET-Kb mice and implanted under the kidney capsule of transgenic (syngeneic) and congenic (allogeneic) mice. Three weeks post-implantation, BD were scored histologically for rejection or survival, and stained for various cell-surface molecules. RESULTS: Generally, IHBD survived better than EHBD, and T cells were the predominant infiltrating cell type in both implants. Both types of implants undergoing rejection expressed intercellular adhesion molecule-1 (ICAM-1) and leukocyte function antigen-1 (LFA-1) at high density; BD and the underlying kidney parenchyma also expressed class I and II major histocompatibility complex (MHC). CONCLUSIONS: The rejection of both groups of implants by congenic recipients suggests that BD from MET-Kb mice express the transgene, but the reason for the selective targeting of portal tracts rather than transgene-expressing hepatocytes remains unclear. One possible explanation is that dendritic cells/antigen-presenting cells (DC/APC) in portal tracts, which express high levels of MHC and co-stimulatory molecules, are the primary targets, and that BD are infiltrated and destroyed as 'bystanders'.

A method for culturing and transplanting biliary epithelial cell from syrian golden hamster.

The present paper describes the establishment of a method for simultaneous culturing of biliary epithelial cells (BECs) from the gall bladder (GB), extrahepatic bile duct (EBD) and intrahepatic bile duct (IBD) of the hamster. GB, EBD and IBD were cut from the biliary tree after collagenase perfusion of the liver. These biliary segments were minced into fragments. The fragments were embedded in collagen gel and cultured in Dulbecco's modified Eagle medium/HamF12 medium containing 10% fetal bovine serum. The various cells subsequently spread from the fragments and formed cellular sheets. After the fragments and flattened cells were removed with the aid of a Pasteur pipette under phase-contrast microscopy, the sheets remaining were found to be composed of cuboidal cells. These cuboidal cells were shown to express gamma glutamyl transpeptidase and cytokeratin 7, which are known to be specific markers of BECs. Ultrastructurally, a large number of microvilli were observed on the luminal surface and junctional complex and interdigitation was identifiable on the lateral surfaces. BEC cultures were subcultured by digestion with collagenase and dispase and then dissociated by subsequent digestion in trypsin and ethylenediaminetetraacetic acid and then maintained on collagen gel for up to 8 weeks. After several passages, the BECs in culture eventually increased in size and showed vacuoles in the cytoplasm. They demonstrated irreversible growth arrest at 9 weeks. The BECs tended to form cystic structures when the BECs with collagen gel were transplanted into the interscapular fat pads of syngeneic hamsters. We established a method for culturing and transplanting biliary cells from syrian golden hamsters. This method may help to clarify the mechanism of hepatobiliary diseases.

Intrahepatic biliary strictures after liver transplantation.

PURPOSE: To evaluate the prevalence, cholangiographic features, causes, and management of intrahepatic biliary strictures in hepatic transplants. MATERIALS AND METHODS: Over a 12-year period, cholangiography was performed in 1,590 liver allografts. Confirmed cases of stricture were evaluated and correlated with clinical variables. RESULTS: Intrahepatic biliary strictures occurred in 130 of 1,590 grafts (8.2%). Strictures were multiple in 99 grafts (76.2%) and single in 31 (23.8%). Locations were the common hepatic duct bifurcation in 46 grafts (35.4%), the peripheral ducts in 44 (33.8%), and both in 40 (30.8%). Strictures caused mild to moderate bile duct dilatation in 72 grafts (55.4%), marked dilatation in 11 (8.5%), and obstruction in four (3.1%). Hepatic artery occlusion, pretransplantation primary sclerosing cholangitis, choledochojejunostomy, use of Euro-Collins organ preservation solution, cholangitis at liver biopsy, and young age were statistically significantly associated with strictures (P < .001). CONCLUSION: Strictures have multiple causes and may be an important indicator of underlying abnormalities. They often require interventional radiologic or surgical treatment.

Isolation, culture, and transplantation of intrahepatic biliary epithelial cells and oval cells.

Recent advances made in the isolation, culture, and transplantation of defined populations of intrahepatic biliary epithelial cells and oval cells have permitted direct analysis of the functions, growth properties, and differentiation potential of these respective cell types in their untransformed or transformed states. This review provides a current and comprehensive examination of the various approaches that have been taken to isolate and culture intrahepatic biliary epithelial cells from normal and cholestatic liver and oval cells from preneoplastic liver. Emphasis is placed on comparing the phenotypic features and growth properties of these various biliary cell types in vitro as well as on describing their transplantation into ectopic tissue sites. In addition, the oval cell is evaluated in terms of its potential role as a 'facultative stem cell' during hepatocarcinogenesis.

Characterization of a primary bile ductular cell culture from the livers of rats during extrahepatic cholestasis.

The establishment of novel bile ductular cell cultures was accomplished with the use of explants of a hyperplastic bile ductular tissue preparation obtained from rat livers at 10 to 15 weeks after bile duct ligation or a bile ductular cell fraction isolated from this tissue preparation by a procedure involving Percoll density gradient centrifugation. Observations made on these primary explant and monolayer bile ductular cell cultures were limited to the first 3 days of culture where the morphologic features of the bile ductular epithelium remained fairly well preserved, while fibroblast contamination was found to be very low. These cultured cells also retained over this period a high specific activity for the bile ductular cell marker enzyme gamma-glutamyl transpeptidase, as well as possessed measurable but decreasing specific activities for leucine aminopeptidase and alkaline phosphatase. Karyotypic analysis of the cultured monolayer cells further showed them to be diploid. In addition, preliminary transplantation studies demonstrated the presence of well-differentiated bile ductular-like structures following inoculation of the freshly isolated bile ductular cell fraction into the interscapular fat pads of recipient rats.