Biliary Proliferation Research Articles

Wnt in liver development: Role in biliary cell survival and proliferation

Wnt/13-catenni pathway plays a sigmficant role during development and carcinogenesis. We have earlier detected presence of Wnt-1 as early as in embryonic day 10 livers (El0). We have also reported effect of 13-catenin inhibition on liver growth and lineage specification during liver development. In the present study we investigate direct effect of Wnt on liver development utilizing embryonic liver cultures and Wnt conditioned media. (Wnt-3a). El0 livers were isolated and cultured in an organ culture system. Livers were cultured in conditioned medium from control L929 cells or Win-conditioned media (L cells transfected with Wnt-3a) alone or in presence of Hepatocyte growth factor (HGF). At least 3 livers were cultured for each growth condition. Embryonic liver cultures grown in DMEM supplemented with 10% fetal calf serum (FCS) were utilized as positive controls. After 72 hours all organ cultures were harvested, llxed and processed and finally analyzed for histology, proliferation, apoptosis and lineage. Negative conditioned media grown embryonic liver cultures demonstrated loss of architecture, enhanced apoptosia and deficit proliferation. There were almost no viable cells observed in these cultures. In the presence of Wnt-conditioned media, embryonic liver cultures showed several islands of survived cells and demonstrated a ductular morphology. These cells displayed proliferation as seen by PCNA and Ki-67 posinvity. Also this growth condition displayed an overall decrease in TUNEL positivity especially in the ductular structures. These cells were strongly positive for CK-19 and negative for cr ein and c-kit. Some CK-19 positive cells were also positive for albumin. Embryonic livers cultured in presence of Wnt and HGF show phenotype resembling the positive control (cultures grown in 10% FCS). These cultures exhibited normal cellular architecture with active proliferation and decreased apoptosis and displaying stem cells, hepatocytes (immature & mature) and primitive bile ducts. We conclude that Wnt plays an important role in bihary commitment of the bipotential stem cells by providing them a survival advantage. It also induces transdifferentiation of mature hepatocytes into biliary cells as shown by the presence of some hepato-biliary intermediates. HGF and Wnt seem to be sufficient to maintain a normal embryonic liver culture growth and differentiation supporting stem cells, hepatocytes & bile ducts. Our results suggest an existence of a precise balance of these growth factors for normal liver development

Double-stranded RNA activates a p38 MAPK-dependent cell survival program in biliary epithelia.

Double-stranded RNA (dsRNA) is produced during replicative viral infection or genotoxic stress. Thus knowledge of the cellular response to dsRNA is necessary to understand the effects of DNA damage or viral infection in biliary epithelia. We assessed the effect of dsRNA on biliary epithelial cell proliferation and apoptosis and the role of the stress-activated p38 MAPK signaling pathway in these responses. dsRNA did not induce apoptosis or proliferation in Mz-ChA-1 human malignant cholangiocytes, but decreased cytotoxicity induced by camptothecin or tumor necrosis factor-related apoptosis inducing ligand and decreased activity of caspases 3, 8, and 9. Furthermore, dsRNA increased p38 MAPK and JNK kinase active site phosphorylation but had no effect on either MAPK kinase (MEK)1/2 or protein kinase R phosphorylation. Inhibition of p38 MAPK with SB-203580 increased basal caspase activity. Thus dsRNA stimulates a p38 MAPK-dependent cell-survival pathway in biliary epithelial cells that may modulate the response of the biliary epithelia to dsRNA produced during genotoxic injury or virus infection.

Hepatocyte growth factor promotes compensatory hypertrophy and makes major hepatectomy possible in liver cirrhosis

the presence of HGF and EGF. Levels of Jaggedl increased during cell culture as detected by Western blot analyses. Following TGF-alpha treatment we detected an early increase and a later decrease in the Jaggedl levels. Minimal Notchl-protein was detected in fresh hepatocytes followed by a transient increase and a sustained decrease. This increase was suppressed by TGF-alpha. Our results also show that Jaggedl is well expressed in rat liver and down-regulated 48 h after partial hepatectomy. This coincides with the peak of biliary proliferation and TGF-alpha levels after partial hepatectomy. Only minor changes were seen in Notchl levels after partial hepatectomy. The above results indicate that cell-cell interaction via Notch/Jagged signaling pathway varies as a result of cell proliferation or liver regeneration. We hypothesize that this pathway may negatively regulate cell proliferation during liver regeneration especially of the biliary component.

Intoxicação espontânea por Senecio brasiliensis (Asteraceae) em ovinos no Rio Grande do Sul

Descreve-se a ocorrência de um surto de intoxicação espontânea por Senecio brasiliensis em ovinos em um estabelecimento do município de Mata, Estado do Rio Grande do Sul, em meados de janeiro de 1997. De um total de 94 ovinos, 51 (54,25%) animais adoeceram e 50 (53,2%) morreram. Esse rebanho permaneceu durante aproximadamente 7 meses (de junho de 1996 a janeiro de 1997) em piquetes de pastagem nativa onde havia grande quantidade de S. brasiliensis. O quadro clínico manifestado pelos animais afetados consistia em fotossensibilização, emagrecimento progressivo, apatia, fraqueza, perturbações neurológicas como depressão, andar a esmo e desequilibrado, icterícia e hemoglobinúria. Houve melhora das lesões de pele naqueles ovinos que desenvolveram fotossensibilização hepatógena depois que foram retirados do sol. As principais lesões macroscópicas observadas em 9 dos 10 ovinos necropsiados incluíam fígado diminuído de tamanho, firme, difusamente marrom amarelado ou esverdeado, com quantidades variáveis de nódulos de 1-3 mm de diâmetro, bem circunscritos, salientes na cápsula, amarelados, distribuídos aleatoriamente por todo o parênquima. A vesícula biliar estava repleta e preenchida por bile verde escura e espessa. Havia também derrames cavitários (hidropericárdio e ascite). Crise hemolítica aguda fatal associada à intoxicação crônica hepatógena por cobre foi observada em cinco ovinos. Além das lesões hepáticas macroscópicas já mencionadas, foi observada icterícia generalizada da carcaça, rins tumefeitos, friáveis, difusamente escurecidos ou com fino pontilhado enegrecido; a urina era marrom escura (hemoglobinúria). As principais lesões microscópicas foram observadas no fígado e consistiam em hepatomegalocitose, proliferação de ductos biliares (hiperplasia ductal) e fibrose periportal moderada acompanhada de infiltrado inflamatório mononuclear. Macrófagos carregados de pigmento acastanhado formavam aglomerados nas tríades portais ou estavam dispersos entre os hepatócitos remanescentes. O material armazenado no citoplasma desses macrófagos correspondia a ceróide e cobre, positivo nas técnicas de PAS e rodanina, respectivamente. Nos rins de cinco animais, havia nefrose hemoglobinúrica caracterizada por degeneração e necrose do epitélio tubular, presença de hemoglobina e hemossiderina no citoplasma das células epiteliais dos túbulos contorcidos e cilindros de hemoglobina na luz tubular. Evidência morfológica de encefalopatia hepática incluía degeneração esponjosa (status spongiosus) da substância branca do encéfalo. Achados ultra-estruturais no fígado incluíam graus variáveis de degeneração hepatocelular caracterizada pelo acúmulo de numerosas gotas lipídicas no citoplasma das células hepáticas e presença de lisossomos carregados de material eletrodenso que, na maioria dos casos, correspondia à lipofuscina-ceróide. Adicionalmente, havia discreta dilatação do retículo endoplasmático rugoso e moderada hiperplasia do retículo endoplasmático liso em algumas regiões do citoplasma dos hepatócitos. No epitélio dos túbulos contorcidos proximais do rim foi observado edema intracelular e diversas alterações mitocondriais de caráter degenerativo que incluíam tumefação, desorganização e ruptura das cristas, matriz finamente granular, acúmulo de gotículas de gordura e ruptura das membranas em alguns casos. Lisossomos contendo material fortemente eletrodenso foram observados em muitas células tubulares renais. O exame laboratorial de fragmentos de fígado e rim dos ovinos afetados revelou níveis elevados de cobre que variaram respectivamente de 369 ppm a 1248 ppm e 152 ppm a 687 ppm com base na matéria seca. O diagnóstico de intoxicação por Senecio brasiliensis baseou-se em dados epidemiológicos, clínicos, de necropsia, histopatológicos e laboratoriais.

Origin and Structural Evolution of the Early Proliferating Oval Cells in Rat Liver

We have analyzed the histological changes in rat liver after 2-acetylaminofluorene (AAF) administration. The data demonstrate that AAF-induced oval cells were preferentially generated by proliferation of the terminal biliary ductules that we suggest constitute the primary hepatic stem cell niche. The oval cells formed ductular structures, representing an extension of the canals of Hering. This histological organization provides continuous bile drainage of the hepatocytes and uninterrupted blood flow in the sinusoids. The oval cell ductules are surrounded by a continuous basement membrane that is intermittently disrupted by processes of stellate cells that form direct cell-cell contact with the oval cells. Although both AAF treatment and bile duct ligation results in proliferation of biliary epithelial cells, the mechanism(s) responsible for the proliferation of the biliary epithelium seems to differ in the two models. In contrast to the biliary proliferation stimulated by bile ligation, AAF-induced oval cell proliferation as well as the capacity of these cells to differentiate into hepatocytes, bile epithelial cells and possibly other cell lineages can be blocked by administration of dexamethasone.

Biliary dysplasia, cell proliferation and nuclear DNA-fragmentation in primary sclerosing cholangitis with and without cholangiocarcinoma.

To study the extent of biliary dysplasia, and the degree of cell proliferation and apoptosis in bile duct cells (BDC) from patients with primary sclerosing cholangitis (PSC), with and without cholangiocarcinoma (CC). Specimens of liver tissue from 16 patients suffering from PSC and CC, and 16 patients with end-stage PSC without cancer, were investigated. Histological evaluation of presence of biliary dysplasia and bile duct proliferation was made. Immunohistochemistry, applying antibodies against Ki-67, p53 and bcl-2, was used. Nuclear DNA fragmentation was assessed by in situ DNA labelling (ApopTag). The numbers of positive cells expressed as a percentage of the total number of BDCs constituted the labelling index (LI). Bile duct dysplasia was significantly more frequent in nontumorous liver tissue from patients with PSC and CC than from patients having end-stage PSC without cancer (P < 0.05). Patients with biliary dysplasia had a higher frequency of marked bile duct proliferation (P < 0.01) than patients without dysplasia. In tumour tissue, the LI for Ki-67 positive nuclei was more than four times the LI of nuclear DNA fragmentation (P < 0.01). Ki-67, bcl-2, p53 or DNA fragmentation were not significantly different in nontumorous liver tissue from patients with and without CC. Immunohistochemical staining for p53 was positive in 75% of the tumours, whilst in nontumorous tissue no such overexpression was found. PSC patients with CC more often display biliary dysplasia than those with end-stage PSC, indicating that biliary dysplasia may be a precancerous stage in PSC. Additionally, p53 mutation seems to be a late event in tumour development, since no p53 expression was found in the premalignant areas with nontumorous BDC.

Protein expression of double-stranded RNA-activated protein kinase (PKR) in intrahepatic bile ducts in normal adult livers, fetal livers, primary biliary cirrhosis, hepatolithiasis and intrahepatic cholangiocarcinoma.

The protein expression of double-stranded RNA-activated protein kinase (PKR) in intrahepatic bile ducts has not been investigated. Immunohistochemistry and a semiquantitative scoring method in normal liver and biliary diseases were used for the investigation. In "normal" adult livers (n=10), intrahepatic bile ducts were negative for PKR. In normal fetal livers (n=25), primitive biliary epithelia were almost negative for PKR. In primary biliary cirrhosis (PBC) (n=30), damaged bile ducts were frequently positive for PKR, while uninvolved bile ducts were negative. In hepatolithiasis (n=27), proliferated bile ducts were positive for PKR, and the PKR score correlated with the degree of proliferation. In cholangiocarcinoma (CC) (n=44), PKR expression was frequently noted, and the PKR score correlated with good differentiation of CC, being highest in well-differentiated CC and lowest in poorly-differentiated CC. The PKR score decreased in the following order: CC (mean PKR score=3.96), hepatolithiasis (2.56), PBC (1.60), normal fetal liver (0.40), and normal adult livers (0.00). The PKR expression in hepatocytes was "baseline" in normal adult livers, while moderately increased in fetal livers, PBC, hepatolithiasis and CC. Although the significance of these data is unclear, they suggest (i) that PKR is absent in bile ducts in normal adult and fetal livers, (ii) that PKR in bile duct cells newly emerges or increases in PBC, hepatolithiasis, and CC, (iii) that PKR accumulates in damaged bile ducts in PBC, (iv) that PKR increases in parallel with biliary cell proliferation in hepatolithiasis, and (v) that PKR expression correlates with differentiation in CC. PKR expression in intrahepatic bile ducts seems to be associated with inflammation or cell proliferation of the bile duct cells.

Fibrous obliterative lesions of veins contribute to progressive fibrosis in chronic liver allograft rejection

Fibrosis in liver allografts undergoing chronic rejection (CR) is variable and poorly understood. The temporal and spatial relationships of venous, arterial, and biliary lesions were studied to clarify their potential contributions to graft fibrosis. The severity, prevalence, and morphology of intimal lesions of vessels were analyzed and compared with the fibrosis stage. Three groups were found; group 1 (n = 5) with no hepatic vein (HV) lesions, group 2 (n = 5) with HV lesions only, and group 3 with lesions of both HV and portal veins (PV). The earliest lesion to develop, in 71% of grafts, was concentric intimal thickening of small HV. This was significantly more severe and frequent in grafts from group 3. With increasing frequency and severity of small HV sclerosis, fibrosis developed in medium/large veins. The morphology of larger vessel lesions suggested organized thrombus. Centrilobular fibrosis was significantly more severe in group 3 and developed unpredictably and sometimes rapidly. Conversely, portal fibrosis scores were significantly higher in grafts with ductular proliferation and did not correlate with venous lesions. This suggests that in CR, veno-occlusive-like lesions develop commonly in terminal hepatic venules, probably caused by immune-mediated damage. In only a proportion, with increased frequency and severity of the lesions, stasis and thrombosis in portal and larger veins occur and could result in loss of hepatic and portal venous outflow, which leads to ischemia and fibrosis. The stage of fibrosis did not correlate with foam-cell arteriopathy. A second pathway of portal fibrosis occurs in patients with longstanding biliary proliferation. (Hepatology2000;32:1240-1247.)

Expression of collagens type I and IV, osteonectin and transforming growth factor beta-1 (TGFβ1) in biliary atresia and paucity of intrahepatic bile ducts during infancy

Background/Aims: Biliary atresia and paucity of intrahepatic bile ducts are the main causes of neonatal cholestasis leading to hepatic fibrosis. Fibrotic evolution is slow in paucity of bile ducts as compared to the rapid progression to biliary cirrhosis in biliary atresia when cholestasis persists despite hepatoportoenterostomy. Our aim was to compare the expression of collagens type I and IV,α-smooth muscle actin, osteonectin and transforming growth factor β1 in biliary atresia and paucity of bile ducts. Methods: Liver biopsies were obtained in 12 children with biliary atresia and in five with paucity of bile ducts. Collagens type I and IV, α-smooth muscle actin were detected with immunostaining. Collagens type I and IV, osteonectin and transforming growth factor β1 mRNAs were detected by in situ hybridization. Results: Expression of mRNA and proteins was roughly parallel. In ductular proliferation areas of biliary atresia: (1) the expression of collagens type I and IV and osteonectin was increased, and was localized to periductular myofibroblasts; (2) transforming growth factor β1 was expressed around biliary ductules, probably in inflammatory cells, and also in biliary cells. Osteonectin expression was also increased in the lobules. In paucity of bile ducts, there was no overexpression of collagens type I and IV and transforming growth factor β1, except in the only child with marked fibrosis. However, osteonectin expression was enhanced at the periphery of the lobules, even when fibrosis was mild or absent. Conclusions: These findings suggest that in biliary atresia ductular proliferation areas are the site of a marked production of extracellular matrix proteins in periductular myofibroblasts, probably secondary to transforming growth factor β1 production by inflammatory cells and by biliary cells. The weak expression of transforming growth factor β1 could explain the slow progression of fibrosis in paucity of bile ducts.

Differential Susceptibility to Hepatic Inflammation and Proliferation in AXB Recombinant Inbred Mice Chronically Infected with Helicobacter hepaticus

Helicobacter hepaticus is a naturally occurring pathogen of mice and has been used to develop models of chronic hepatitis, liver cancer, and, more recently, inflammatory bowel disease, in selected mouse strains. A/JCr mice are particularly susceptible to H. hepaticus-induced hepatitis and subsequent development of liver neoplasms, whereas C57BL/6 mice are resistant. In this study, we inoculated nine AXB recombinant inbred (RI) mouse strains, derived from A/J and C57BL/6 mice, with H. hepaticus to determine the genetic basis of resistance to Helicobacter-induced liver disease. Mice were surveyed 14 months after inoculation by culture and PCR for H. hepaticus colonization of the liver and cecum, and microscopic morphometric evaluations of the liver were performed to quantify and correlate the severity of inflammation, apoptosis, and proliferation. Analysis of variance of hepatic inflammation demonstrated significant variation among the RI strains (P < 0.0001), and the strain distribution pattern suggested a multigenic basis of disease resistance. Quantitative trait analysis using linear regression suggested possible linkage to loci on mouse chromosome 19. Hepatocellular and biliary epithelial apoptosis and proliferation indices, including proliferation of oval cells, were markedly increased and correlated with severity of inflammation. Prevalence of hepatic neoplasia was also increased in susceptible RI strains. These findings demonstrate a genetic basis for susceptibility to Helicobacter-induced disease and provide insight into its pathogenesis.

C-erbB-2 protein is expressed in hepatolithiasis and cholangiocarcinoma.

The c-erbB-2 proto-oncogene encodes a transmembrane protein which is highly homologous to epidermal growth factor receptor. Overexpression of this c-erbB-2 protein has been reported in many human carcinomas, including breast carcinoma. However, there have been few studies of the expression of c-erbB-2 in cholangiocarcinoma and hepatolithiasis, a condition occasionally associated with cholangiocarcinoma. In this study, we evaluated immunoreactivity for the c-erbB-2 protein in human cholangiocarcinomas (n = 47), hepatolithiasis (n = 20), fetal livers (n = 36) and normal adult livers (n = 6). In normal adult livers and fetal livers, expression of c-erbB-2 protein could not be detected in hepatocytes or intrahepatic biliary cells. In hepatolithiasis, there was overexpression of c-erbB-2 in 15/20 (75%). The expression was found with a membranous pattern on the proliferated intrahepatic bile ducts and proliferated intrahepatic peribiliary glands around the bile ducts containing stones. Hepatocytes were negative for c-erbB-2 protein. Moreover, the biliary cell expression of the c-erbB-2 protein correlated significantly with Ki67 labelling index. On the other hand, aberrant expression of c-erbB-2 was found in 33/47 (70%) cholangiocarcinomas. The c-erbB-2 expression in cholangiocarcinomas did not correlate with Ki67 labelling index or p53 expression. These results indicate that aberrant expression of c-erbB-2 protein is found in cholangiocarcinoma and also in noncancerous biliary proliferative lesions such as hepatolithiasis. These findings also suggest that c-erbB-2 oncogene participates not only in cholangiocarcinogenesis but also in biliary cell proliferation in non-neoplastic conditions.

Dissimilarity in aflatoxin dose-response relationships between DNA adduct formation and development of preneoplastic foci in rat liver

Earlier work in this laboratory and that carried out by others demonstrated that after a single dose of aflatoxin B 1 (AFB) the resulting liver AFB-DNA adduct levels were directly proportional to dose. Earlier work also showed that after ten daily doses the AFB dose-response relationship with γ-glutamyl transpeptidase (GGT) positive preneoplastic foci measured at 3 months was sublinear, with a threshold at a dose of about 150 μg/kg body weight/day. The objective of this study is to determine the factors influencing the shift in AFB dose-response between AFB-DNA adducts and GGT foci. Male Fisher 344 weanling rats were orally administered one or ten doses of AFB ranging from 50 to 350 μg/kg body weight/day. The animals were killed 2 or 24 h after the first AFB dose, or after the tenth AFB dose. The first and tenth doses were tritiated in these animals and 3H-AFB-guanine adducts isolated from liver DNA were measured by HPLC. Another group was killed 3 months after receiving ten doses in order to measure GGT foci development. AFB-guanine adduct levels were directly proportional to dose after the first dose, but after the tenth dose were much lower in the 200–350 μg/kg groups than after a single dose. The GGT foci response confirmed earlier work concerning a sublinear response. Among the individual animals in the 200–350 μg/kg groups there was a positive relationship, after controlling for dose, between GGT foci development and weight gained both during dosing ( P = 0.018) and also to a lesser extent during the early promotional period ( P = 0.066). Enzyme activity levels of GGT in liver homogenates were higher in the highest dose groups and reflected biliary proliferation rather than histological GGT stained foci. Urinary levels of AFB metabolites changed proportions in the high dosage multiply dosed animals reflecting alteration in AFB metabolism or excretion. The differences between the linear adduct and the sublinear foci dose-response curves may be the result of non-adduct effects of higher multiple AFB doses on foci formation including acute cytotoxicity, altered AFB metabolism and disposition, enhanced weight gains, or shortened foci latency but not through enhanced guanine adduct levels. Other studies that showed a linear relationship between AFB dose and liver tumor development used continuous feeding of maximal doses an order of magnitude less than the lowest dose in this study and thus avoided acutely toxic effects. We hypothesize that liver tumor development may mirror foci response in a 10-dose AFB regimen with doses above 100 μ/kg due to acute toxicity effects.

Detection of transforming growth factor-α protein and messenger RNA in hepatobiliary diseases by immunohistochemical and in situ hybridization techniques

Transforming growth factor-α (TGF-α) is a cytokines related to cell proliferation and transformation. Immunoreactive TGF-α protein is expressed in regenerating hepatocytes and interlobular bile ducts as well as in hepatocellular carcinoma. Although TGF-α is thought to play an important role in the intrahepatic biliary tree, its role in cellular physiology is poorly understood. This study investigates the expression of TGF-α and its messenger RNA (mRNA) in various hepatobiliary diseases. The authors showed by immunohistochemistry that TGF-α and its receptor, epidermal growth factor receptor (EGFR), were expressed in interlobular bile ducts, proliferating bile ductules, and most hepatocytes in various hepatobiliary liver tissues. They also showed by Western blot analysis that TGF-α protein was present in hepatic bile samples obtained from patients with obstructive jaundice. In situ hybridization showed that TGF-α mRNA was localized in hepatocytes of some pathological liver tissues, but it was absent in biliary epithelial cells of the same tissues. These findings suggest that TGF-α protein is produced by hepatocytes, and hepatocyte stimulation occurred as autocrine growth regulation. The release of TGF-α into hepatic bile caused biliary proliferation and transformation through EGFR, present on the existing cell surface membrane of biliary epithelial cells.

Biliary proliferation and adaptation in furan-induced rat liver injury and carcinogenesis.

Distinctive intrahepatic biliary adaptation responses occur in the liver of rats subjected to select hepatotoxic and/or carcinogenic treatments with the nongenotoxic cholangiocarcinogenic agent furan. Specifically, metaplastic small intestinal-like glands closely resembling in their cellular composition the crypts of Lieberkühn of normal rat small intestine were selectively derived from putative hyperplastic bile ductule-like progenitor structures in the right and caudate liver lobes of young adult Fischer-344 male rats given furan by gavage at a daily dose of 30-45 mg/kg body weight, 5 times weekly, over a 2-6-wk treatment period. Longer term chronic administration of furan at 30 mg/kg/day for 9-19 wk resulted in the preferential development of primary hepatic adenocarcinomas, which arose at 70-100% incidences from right/caudate liver lobes and which were characterized by small intestine mucosal cell differentiation. Interestingly, the neoplastic glands of these "intestinal-type" hapatic tumors demonstrated strongly positive immunochemical reactions for both hepatocyte growth factor/scatter factor and its c-met encoded receptor and were immunohistochemically positive for transforming growth factor beta 1 (TGF-beta 1) and for mannose-6-phosphate/insulin-like growth factor II receptor, implicated in the activation of latent TGF-beta 1. In contrast, a different pattern of aberrant bile ductular cell differentiation was noted to occur in the atrophied right liver lobe of moribund Fischer-344 male rats that were chronically exposed over a 10-14-day period to a severely hepatotoxic dose of furan (60 mg/kg/day). Under this latter experimental condition, rare yet distinct cholangiolar-like structures composed of biliary epithelial cells and typically a single ductular hepatocytic cell in various stages of maturation specifically formed in association with an extensive hyperplastic bile ductular reaction. Very similar cholangiolar-like structures also appeared in areas of preexisting hyperplastic bile ductule tissue at 3-5 days following the administration of a single hepatonecrogenic dose of CCl4 to rats that 4-6 wk earlier had been subjected to a bile duct ligation. In addition, a novel rat model was developed in which furan combined in a unique synergistic manner with bile duct ligation to induce the replacement of almost all of liver with well-differentiated hyperplastic bile ductules without evidence of differentiation along either metaplastic small intestine mucosal cell or ductular hepatocyte lineages. Bile ductular epithelial cell isolated from bile duct-ligated/furan-treated rats were further observed to be organized in the form of bile duct-like structures in vitro under specific conditions of primary cell culture and in vivo following their cell transplantation into the inguinal fat pads of sygeneic recipient rats. Overall, these findings serve to exemplify the remarkable plasticity that may be exhibited by certain proliferating biliary cell populations in liver in response to specific types of severe hepatic injury and/or during cholangiocarcinogenesis.

Biliary Epithelial Cell Proliferation Following α-Naphthylisothiocyanate (ANIT) Treatment: Relationship to Bile Duct Obstruction

Biliary Epithelial Cell Proliferation Following α-Naphthylisothiocyanate (ANIT) Treatment: Relationship to Bile Duct Obstruction. Kossor, D. C., Goldstein, R. S., Ngo, W., DeNicola, D, B., Leonard, T. B., Dulik, D. M., and Meunier, P. C. (1995). Fundam. Appl. Toxicol. 26, 51-62.These studies were designed to evaluate the importance of bile duct obstruction in the pathogenesis of α-naphthylisothiocyanate (ANIT)-induced biliary epithelial cell (BEC) hyperplasia in rats. Hepatobiliary function and morphology were evaluated in adult male Sprague-Dawley rats 16, 24, 48, 72, 120, and 168 hr after a single oral dose of ANIT (0, 25, 75, or 150 mg/kg). After 75 or 150 mg/kg ANIT, multifocal bile duct obstruction was observed at 48 and 72 hr and preceded BEC hyperplasia which occurred at 120 and 168 hr. BEC proliferation, reflected by 5-bromo-2′-deoxyuridine (BrdU) incorporation, occurred at doses and at time points coinciding with BEC necrosis and/or bile duct obstruction. In contrast, 25 mg/kg ANIT produced minimal BEC damage and no evidence of bile duct obstruction or BEC hyperplasia. In a separate experiment, BEC proliferation was evaluated following bile duct ligation or ANIT treatment (150 mg/kg). The onset and peak of BEC proliferation occurred 24 and 48 hr, respectively, following bile duct obstruction resulting from either ligation or ANIT treatment. Furthermore, BEC proliferation occurred at all levels of the biliary tree in both bile duct-ligated and ANIT-treated rats. These data indicate that (a) dose-response curves for ANIT-induced bile duct obstruction and BEC hyperplasia are similar; (b) ANIT-induced BEC proliferation and bile duct obstruction precedes BEC hyperplasia; (c) BEC proliferation occurred at doses/timepoints associated with BEC damage and bile duct obstruction; and (d) once ANIT-induced bile duct obstruction occurs, the spatial and temporal aspects of BEC proliferation are comparable to those following biliary obstruction induced by bile duct ligation. Collectively, these data suggest that ANIT-induced BEC hyperplasia is secondary to intrahepatic bile duct obstruction.

Biliary Epithelial Cell Proliferation Following α-Naphthylisothiocyanate (ANIT) Treatment: Relationship to Bile Duct Obstruction

Biliary Epithelial Cell Proliferation Following α-Naphthylisothiocyanate (ANIT) Treatment: Relationship to Bile Duct Obstruction

Occult Celiac Disease Associated with Lymphocytic Sclerosing Cholangitis

A 60-year-old male with dermatitis herpetiformis and a previously treated lymphoma involving an inguinal lymph node developed abnormal liver chemistry tests. Because of intermittent diarrhea, additional studies revealed lymphocytic colitis and occult celiac disease that responded to a gluten-free diet. A liver biopsy done to explore persistently abnormal liver chemistry tests showed a portal tract-centred inflammatory process characterized by biliary ductal proliferation, epithelial lymphocytosis and concentric lamellar fibrosis. Quantitation of immunoglobulins was normal and antimitochondrial antibodies were negative. Retrograde cholangiograms showed radiological features typical of primary sclerosing cholangius. The epithelial lymphocycosis reported in gastric, small and large intestinal mucosa of some patients with celiac disease may also be present in the biliary ductal columnar epithelium. This report provides additional evidence that celiac disease may be a far more extensive pathological process.

Florid duct lesion in primary biliary cirrhosis shows highly proliferative activities

Primary biliary cirrhosis is characterized by non-suppurative inflammation and destruction of the interlobular bile ducts (IBDs) (florid duct lesions). The present study attempted to analyze the cell kinetics of florid duct lesions using the histometry, immunostaining of proliferating cell nuclear antigen and by counting argyrophilic nucleolar organizer regions. Florid duct lesions disclosed nuclear stratification, pseudopapillary infoldings and tortuosity. These findings suggest increased proliferative activity of epithelial cells in these affected ducts. Proportion of proliferating cell nuclear antigen positive interlobular bile ducts (88.8 +/- 7.9%) and counts of argyrophilic nucleolar organizer regions in biliary epithelial cells (3.89 +/- 0.73) were increased in florid duct lesions relative to non-inflamed interlobular bile ducts (45.0 +/- 25.4% and 2.65 +/- 0.67, respectively) in primary biliary cirrhosis and also relative to interlobular bile ducts (21.8 +/- 8.6% and 2.25 +/- 0.09, respectively) in normal livers, which supports the above-mentioned suggestion. The increased outer diameter of these affected bile ducts which was demonstrated histometrically, may be due to biliary epithelial proliferation with variable luminal dilatation. The present study showed that an increased proliferative activity of biliary epithelial cells is one of the characteristics of florid duct lesions and results in an increase in the size of the affected bile ducts. It remains unclear, however, why proliferation and extensive destruction of biliary epithelial cells coexist in primary biliary cirrhosis.

Recognition of major histocompatibility complex antigens on cultured human biliary epithelial cells by alloreactive lymphocytes

We have developed an in vitro system to study the interactions between biliary epithelium and lymphocytes using cultured human biliary epithelial cells. No class II antigens were detected by immunoperoxidase staining of the normal biliary epithelial cells, but alloactivated lymphocyte culture supernatants were able to induce class II expression. The activity of the supernatants was blocked with an anti-gamma-interferon monoclonal antibody. In addition, recombinant human gamma-interferon alone induced the expression of class II antigens and increased the intensity of class I staining of cultured biliary epithelial cells. Biliary epithelial cell-induced proliferation of alloreactive T lymphocytes demonstrated that the major histocompatibility complex molecules carry functional lymphocyte-activating determinants. The recognition of major histocompatibility complex determinants was confirmed by monoclonal antibody-blocking studies and by stimulation of an alloreactive T-cell clone. However, the biliary epithelial cells were much less potent stimulators than arterial endothelial cells tested in the same assay system.

Biochemical Changes and Morphological Alterations of the Liver in Guinea‐Pigs after Administration of Simvastatin (HMG CoA Reductase‐Inhibitor)

Simvastatin is a potent competitive inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) which is the rate-limiting enzyme of cholesterol synthesis. In guinea-pigs, administration of a high oral dose of simvastatin (125 mg/kg/day at the beginning of the study) during 18 days had a major hepatotoxic effect whereas a lower oral dose (30 mg/kg/day) did not seem to cause any liver damage. A significant reduction in microsomal Cyt P 450 content was only observed on a high dose of simvastatin whereas HMG CoA reductase activity was reduced in the group with the low simvastatin dose. The hepatic microsomal aminopyrine N-demethylase activity remained unchanged in all groups. The liver lesion was hepatocellular necrosis accompanied in some animals by a biliary duct proliferation. It was associated with a 10-fold elevation in serum aspartate and alanine aminotransferase activities, as well as a great reduction in daily food intake and body weight (28%). The hepatotoxicity of simvastatin could result from the low basal content of HMG-CoA reductase in guinea-pig liver, the prolonged inhibition of mevalonate synthesis and probably, from the absence of HMG-CoA reductase enzyme de novo synthesis.