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Background/aims: The majority of cholangiocarcinoma patients present with advanced incurable disease. Therefore development of new therapeutic modalities including adenoviral gene therapy is of paramount importance. We set out to identify tumour specific promoters which have low activity in human liver cells and retain their specificity in an adenoviral vector., Methods: mRNA levels of cyclo-oxygenase-2, cytokeratin-19, mucin-1, midkine and telomerase reverse transcriptase (TERT) were determined in human liver, cholangiocarcinoma (resection specimens and cell lines), primary human hepatocytes, cholangiocytes and endothelial cells by Reverse Transcriptase-quantitative PCR. The activity of candidate promoters in adenoviral vectors was then determined in cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes., Results: mRNA levels of all tested tumour markers were significantly higher in cholangiocarcinoma than in normal liver. Based on low expression in hepatocytes, either in combination with low expression in primary cholangiocytes or endothelial cells, the cytokeratin-19, mucin-1 and TERT promoters were selected for further analyses. In an adenoviral vector, the activity of the TERT or cytokeratin-19 promoters were low in normal human hepatocytes and cholangiocytes, and high in cholangiocarcinoma cell lines., Conclusions: The TERT and Cytokeratin-19 promoters are highly expressed in cholangiocarcinoma and seem suitable to restrict adenoviral gene therapy to these intra-hepatic tumours.

Background/aims: To bridge acute liver failure (ALF) patients to orthotopic liver transplantation, several bioartificial liver (BAL) systems have been developed. The bio-component of most BAL systems consists mainly of porcine hepatocytes. Plasma or blood of ALF patients is perfused through the BAL thereby contacting porcine hepatocytes. Xenogeneic BAL systems may suffer from hyperacute rejection similar to whole-organ xenotransplants. Hyperacute rejection is mediated by antibodies directed against Galalpha(1-3)Gal, a carbohydrate structure present on most mammalian cells. Galalpha(1-3)Gal is produced by the enzyme alpha1,3-galactosyltansferase (alphaGal-T). Conflicting data have been published concerning Galalpha(1-3)Gal expression on hepatocytes in intact porcine liver. We investigated whether isolated porcine hepatocytes express Galalpha(1-3)Gal., Methods: Immunofluorescence, flow cytometry, RT-PCR and enzyme activity assays were performed on freshly isolated and cultured porcine hepatocytes and liver biopsies. Anti-Galalpha(1-3)Gal antibodies were measured in plasma from patients treated with BAL by ELISA., Results: Isolated porcine hepatocytes express (alphaGal-T) at low levels and Galalpha(1-3)Gal is present in low quantities on these cells, in contrast to hepatocytes in situ. Furthermore, IgG and IgM anti-Galalpha(1-3)Gal are depleted from the plasma of ALF patients during BAL treatment., Conclusions: Isolation and culture of porcine hepatocytes induce Galalpha(1-3)Gal expression, which may elicit immunological responses potentially compromising BAL functionality.