Endothelin-1 is synthesized and inhibits cyclic adenosine monophosphate- dependent anion secretion by an autocrine/paracrine mechanism in gallbladder epithelial cells.

Ion and fluid transport across the biliary epithelium contributes to bile secretion. Since endothelin (ET)-1 affects ion transport activities and is released by human gallbladder- derived biliary epithelial cells in primary culture, we examined the expression of ET peptides and ET receptors and the influence of ET-1 on ion transport in this epithelium ex vivo. In freshly isolated gallbladder epithelial cells, preproET-1, -2, and -3 mRNAs were detected by reverse transcription PCR and ET-1 isopeptide was identified by chromatography. The cells also displayed ET receptor mRNAs and high-affinity binding sites for ET-1, mostly of the ETB type. Electrogenic anion secretion across intact gallbladder mucosa was stimulated by forskolin, secretin, and exogenous ATP, as assessed by short-circuit current (Isc) increases in Ussing-type chambers. ET-1 inhibited forskolin- and secretin-induced changes in Isc, without affecting baseline Isc or ATP-induced changes. Accordingly, ET-1 significantly reduced the accumulation of intracellular cAMP elicited by forskolin and secretin in the epithelial cells, and this effect was abolished by pertussis toxin. This is the first evidence that ET-1 is synthesized and inhibits, via a Gi protein-coupled receptor, cAMP-dependent anion secretion in human gallbladder epithelium, indicating a role in the control of bile secretion by an autocrine/paracrine mechanism.

Accumulation of azithromycin and roxithromycin in tracheal epithelial fetal cell lines expressing wild type or mutated cystic fibrosis transmembrane conductance regulator protein (CFTR).

Macrolides are accumulated in phagocytes, partially via an active transport system; the membrane carrier is not identified but many data indicate a link with the P-glycoprotein family which includes the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. We have used two epithelial cell lines which express either wild-type (N cells) or mutated (homozygous deltaF508) (F cells) CFTR to study the cellular accumulation of two macrolides (azithromycin and roxithromycin). Adherent cells were incubated with the radiolabeled drugs before extensive washings and counting. Azithromycin was better (about 2-fold) accumulated in F cells up to 60 min but then plateaued, whereas accumulation continued without saturation over 3 hours in N cells. Roxithromycin was also better (1.5-fold) accumulated in F cells at 15 and 30 min, but there were no differences at further incubation times. Macrolide efflux from loaded N and F cells, and the susceptibilities of the carrier systems (entry and efflux) to various pharmacologic agents were similar to those previously observed with phagocytes. These data suggest that the macrolide carriers (for entry and efflux) are not strictly specific for phagocytes and that the CFTR protein plays a role in macrolide uptake.

Identification of decorin proteoglycan in bovine tracheal serous cells in culture and localization of decorin mRNA in situ.

Bovine tracheal submucosal gland serous cells in culture synthesize and secrete proteoglycans and not mucin glycoconjugates. We are interested in the characterization and role of these proteoglycans in airway secretions. The major [35S]methionine-labeled proteoglycan present is identified as the small chondroitin/dermatan sulfate proteoglycan decorin (PG II. PG40). Consistent with its identity as decorin this proteoglycan showed average apparent molecular weights of 75,000 to 130,000 with a core protein of an average, M(r) of about 40,000 and with glycosaminoglycan chains sensitive to chondroitinase ABC lyase of an average M(r) of about 25,000. These data were obtained from gel chromatographic and SDS-PAGE analyses. Northern blot analysis and partial amino acid sequencing of the purified protein further confirmed its identity as decorin. In situ hybridization studies using a decorin riboprobe revealed no expression of decorin in the surface epithelium and only low levels of expression in submucosal gland epithelial cells of bovine tracheal tissue. However, high levels of expression were localized to cells which are peripheral to tracheal submucosal gland epithelial cells and which contact with the extracellular matrix.

Incompatible blood-group A determinants in tumoral mucins. Isolation of oligosaccharides having a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group at the non-reducing end.

Two glycopeptide fractions were obtained from pseudomyxomatous mucins secreted by an ovarian cystadenocarcinoma from a female having blood-group B, and by an appendix tumor from a male having blood-group O. The carbohydrate and amino acid content of these fractions suggests the presence of numerous carbohydrate side-chains linked through O-glycosyl bonds to a peptide core rich in threonine and proline. The two glycopeptide fractions exhibit compatible B- and H-blood-group activities. They are reactive towards Dolichos biflorus lectin and human anti-A agglutinins, and so exhibit an incompatible A activity. Alkali-borohydride degradation of Pronase-digested glycopeptides gave dialyzable oligosaccharides that were purified and shown to possess 2-acetamido-2-deoxygalactitol at the terminal reducing-end. 2-Acetamido-2-deoxyglucose, galactose, fucose, and neuraminic acid were absent, or present, in variable proportions. Four oligosaccharides containing 2-acetamido-2-deoxy-D-galactose residues were reactive towards Dolichos biflorus lectin and human anti-A agglutinins, indicating the presence, at the nonreducing end, of a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group, responsible for blood-group A activity.

Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells.

Scaffold proteins form multiprotein complexes that are central to the regulation of intracellular signaling. The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is highly expressed at the plasma membrane of normal biliary epithelial cells and binds epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with oncogenic properties. This study investigated EBP50-EGFR interplay in biliary cancer. We report that in a collection of 106 cholangiocarcinomas, EBP50 was delocalized to the cytoplasm of tumor cells in 66% of the cases. Ectopic expression of EBP50 was correlated with the presence of satellite nodules and with the expression of EGFR, which was at the plasma membrane, implying a loss of interaction with EBP50 in these cases. In vitro, loss of interaction between EBP50 and EGFR was mimicked by EBP50 depletion using a small interfering RNA approach in human biliary carcinoma cells co-expressing the two proteins at their plasma membrane, and in which interaction between EBP50 and EGFR was validated. EBP50 depletion caused an increase in EGFR expression at their surface, and a sustained activation of the receptor and of its downstream effectors (extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3) in both basal and EGF-stimulated conditions. Cells lacking EBP50 showed epithelial-to-mesenchymal transition-associated features, including reduction in E-cadherin and cytokeratin-19 expression, induction of S100A4 and of the E-cadherin transcriptional repressor, Slug, and loss of cell polarity. Accordingly, depletion of EBP50 induced the disruption of adherens junctional complexes, the development of lamellipodia structures and the subsequent acquisition of motility properties. All these phenotypic changes were prevented upon inhibition of EGFR tyrosine kinase by gefitinib. These findings indicate that loss of EBP50 at the plasma membrane in tumor cells may contribute to biliary carcinogenesis through EGFR activation.

[Alpha-N-Acetylgalactosaminyl transferase activity in mucous ovarian tumors obtained from blood group O2 patients (author's transl)]. / Mise en èvidence d'une activité alpha-N-acetylgalactosaminyl-transfèrase dans les tumeurs secrétantes de sujets de groupe érythrocytaire O2.

We have demonstrated, by the transfer of (1-14C) GalN Ac from exogenous UDP[ (1-14C) GalN Ac into a glycopeptide H-acceptor, the presence of an alpha-N-acetylgalactosaminyltransferase in the wall and fluid of ovarian tumors. The transfer occurs in the absence of exogenous acceptor but to a greater degree in the tumor wall and fluid obtained from blood group O patients, thereby indicating the presence of preferential endogenous acceptor(s) in tumors of these subjects.

Glycoconjugates secreted by bovine tracheal serous cells in culture.

Glycoconjugates secreted by bovine tracheal gland serous cells in culture were characterized after incorporation of radioactive precursor [1-14C]glucosamine and stimulation with isoproterenol. Under dissociative conditions, glycoconjugates eluted in both the void and included volumes on Sepharose Cl-4B. Fractionated by anion-exchange chromatography, the high-molecular-weight (Sepharose Cl-4B; V0) glycoconjugates gave two acidic fractions eluting at 0.5 and 2.0 M NaCl; low-molecular-weight glycoconjugates of the included volumes gave a neutral fraction and two acidic fractions eluting at 0.5 and 2.0 M NaCl. Based on chemical analysis and specific enzymatic digestions, the material eluting in the void volume was shown to contain hyaluronic acid and chondroitin sulfate proteoglycan. In addition, the presence of small amounts of galactose, fucose, sialic acid, glucosamine, and galactosamine suggest the presence of O-glycosidically linked glycoproteins in the void volume. The identification of galactosaminitol in beta-eliminated oligosaccharides from this material confirms this notion. The material eluting in the included volume was shown to contain N-linked glycoproteins with glycans of complex type in the neutral fraction and chondroitin sulfate proteoglycans in the two acidic fractions. Significant N-sulfation of amino sugars was detected in the 0.5 M acidic fraction, indicating the presence of heparan sulfate. Hyaluronic acid and chondroitin sulfate proteoglycan have recently been identified in tracheal secretions; our results suggest that these components originate at least in part from tracheal gland serous cells.

Regulation of secretion in cultured tracheal serous cells by protein kinases A and C.

We recently reported that cultured gland serous cells release chondroitin sulfate proteoglycans (CSPGs) in response to beta-adrenergic agonists. In this study, we analyzed this regulatory pathway and other cellular mechanisms responsible for CSPG secretion. We show the following. 1) Isoproterenol increased CSPG secretion in a concentration-dependent manner, with maximal stimulation (50%) obtained at 10(-5) M; at this concentration, the beta-agonist also stimulated protein kinase A (PKA) by 50%, whereas it increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) content by 300%. 2) Phenylephrine (10(-5) M), 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (1.6 x 10(-7) M), and A23187 (10(-6) M) also stimulated CSPG secretion; this stimulation was concomitant with protein kinase C (PKC) translocation from cytosol to membrane, was blocked by sphingosine (2 x 10(-5) M), and was additive with that elicited by isoproterenol. 3) All PKC activators potentiated the isoproterenol-induced increased in cAMP accumulation without modifying the activation of PKA elicited by the beta-agonist. Our results indicate that although the signaling pathways triggered by alpha- and beta-adrenergic agonists converge at the level of adenylate cyclase in tracheal serous cells, PKA and PKC independently regulate CSPG secretion.

Bile acid transport and regulating functions in the human biliary epithelium.

Whether bile acids regulate biliary epithelial cell (BEC) secretory functions in human is poorly known. The purpose of the study was to determine if human gallbladder-derived BEC exhibit bile acid transport activity that affect their secretory functions and to evaluate the influence of bile acid hydrophobicity in this response by comparing the effects of tauroursodeoxycholate (TUDC) and of taurochenodeoxycholate (TCDC). Expression of the apical sodium-dependent bile acid transporter (ASBT) and of the organic anion transporting polypeptide (OATP-A) was detected and associated with sodium-dependent and sodium-independent [(3)H]taurocholate uptake in BEC. Sodium-dependent uptake (K(m), 66 +/- 2.5 micromol/L; Vmax, 39.4 +/- 4.6 pmol/mg protein/min) was significantly higher than sodium-independent uptake. TCDC stimulated Cl(-) efflux and mucin secretion in cultured cells, and both effects were sodium-dependent. Both TCDC and TUDC were efficiently transported in BEC, as assessed by competitive uptake experiments. However, as compared with TCDC, TUDC induced significantly lower mucin secretion whereas there was no significant difference between TCDC- and TUDC-induced chloride efflux. Protein kinase C down-regulation caused a 70% reduction in TUDC-induced mucin secretion, but did not affect TCDC-induced secretion, which was mediated predominantly by Ca(2+)/calmodulin-dependent protein kinase II activation. These results provide evidence that bile acids may be transported mainly via ASBT in human gallbladder BEC and stimulate hydroelectrolytic and mucin secretion in these cells. Individual bile acids activate different signaling pathways leading to a different balance between mucin and chloride secretion. The differential effect of TUDC may cause a reduction in bile inspissation and provide a benefit in biliary disorders.

Regulation of mucin secretion in human gallbladder epithelial cells: predominant role of calcium and protein kinase C.

BACKGROUND & AIMS: The cellular mechanisms that regulate biliary mucin secretion in humans are unknown. To address this question, human gallbladder epithelial cells were used in primary culture. METHODS: [1-(14)C]-glucosamine-labeled glycoproteins secreted in vitro were analyzed and quantified after exposing cells to activators and inhibitors of the main transduction pathways and to potential biologically active secretagogues. RESULTS: Secreted glycoproteins showed characteristics of biliary mucins. Activators of adenosine 3',5'-cyclic monophosphate-dependent pathway as well as secretin and vasoactive intestinal polypeptide did not significantly modify mucin secretion. By contrast, ionomycin and phorbol-12-myristate 13-acetate increased mucin secretion by 292% +/- 48% and 134% +/- 19% over basal level, respectively. The effects of these two agents were additive and were mediated by a calcium-dependent pathway implicating Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-kinase II) and by the activation of protein kinase C (PKC), respectively, as ascertained by using inhibitors. Mucin secretion was stimulated by extracellular adenosine 5'-triphosphate via P2U receptors, cytosolic calcium increase, and PKC and by taurochenodeoxycholate via cytosolic calcium increase and Ca2+/CaM-kinase II. CONCLUSIONS: Mucin secretion in human gallbladder is regulated predominantly by calcium-dependent pathways implicating Ca2+/CaM-kinase II and PKC. Extracellular adenosine 5'-triphosphate and taurochenodeoxycholate may play a role in the regulation of biliary mucin secretion by activating these different signaling pathways.

Deregulated expression and function of CFTR and Cl- secretion after activation of the Ras and Src/PyMT pathways in Caco-2 cells.

We evaluated the role of the activated Ras and Src/PyMT (Polyoma Middle T) signaling pathways on the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in human colonic Caco-2 cell lines. Control vector-transfected Caco-2 cell monolayer preparations (Caco-2-H) responded to forskolin with an increase in short circuit current (Isc) mediated by CFTR. Furthermore, Caco-2-H cells responded to ATP, a reported stimulator of intracellular Ca2+ (Cai2+), and a potential source of adenosine-mediated elevation of cAMP. In contrast, Caco-2 cells transfected with PyMT (Caco-2-MT), expressing high levels of PKC, showed no sustained Isc response to forskolin or ATP. Pretreatment of Caco-2-MT cells with 2.5 microM phorbol 12-myristate 13-acetate (PMA) for 24 hr. effectively down-regulated PKC activity and restored expression of CFTR mRNA but failed to re-establish functional CFTR. These data suggest that, stable up-regulation of PKC alpha, consequent to activation of the Ras or Src/PyMT pathways, leads to an absence of CFTR expression and Cl- secretion mediated by either cAMP or Cai2+. Moreover, Cl- secretion in the colonic Caco-2 epithelial cell line is mediated primarily by CFTR and an alternate Cai(2+)-activated Cl- channel is not functional in these cells.

Expression of delta F508 cystic fibrosis transmembrane conductance regulator protein and related chloride transport properties in the gallbladder epithelium from cystic fibrosis patients.

Cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, functions as an adenosine 3', 5'-cyclic monophosphate (cAMP)-regulated chloride channel in the apical membrane of biliary epithelial cells, including gallbladder epithelial cells. It has been shown that triangle upF508, the most common CF mutation, impedes CFTR trafficking to the apical surface of epithelial cells. To elucidate the mechanisms of CF biliary disease, we examined structural features, CFTR expression, and chloride transport properties in gallbladder epithelial cells from nine triangle upF508 homozygous liver transplant recipients. Three CF patients had microgallbladders, characterized by severe histological abnormalities. Microgallbladder epithelial cells displayed aberrant immunolocalization of CFTR and of other normally apical proteins in the lateral domain of their plasma membrane and in their cytoplasm. This pattern was mimicked by chronic cholecystitis in non-CF patients. In the 6 remaining CF patients, CFTR was predominantly apical in the gallbladder epithelium, consistent with the detection of a fully glycosylated form by Western blot. In CF as compared with non-CF gallbladder epithelial cells in primary culture, chloride efflux was lower in response to cAMP and tended to be higher in response to exogenous adenosine 5'-triphosphate (ATP). The CF cells exhibited a residual cAMP-dependent chloride secretion that was inversely correlated with ATP-induced chloride secretion, and almost completely blunted in the cells derived from microgallbladders. Our results suggest that epithelial structural alterations aggravate triangle upF508 CFTR mislocalization in the gallbladder epithelium. The associated decrease in residual cAMP-dependent chloride secretion may contribute to biliary damage despite the up-regulation of alternative chloride transport pathways.

Oncogene-mediated propagation of tracheal epithelial cells from two cystic fibrosis fetuses with different mutations. Characterization of CFT-1 and CFT-2 cells in culture.

Primary tracheal epithelial cells obtained from two fetuses with cystic fibrosis (CF) were successfully transfected with a plasmid vector recombined with the large T oncogene of SV40. The resulting tracheal cells were propagated in culture for up to 25 passages and retained the mutations of the CF genes carried by the two fetuses, one heterozygous for the S549N and N1303K substitutions (CFT-1 cells), and the other homozygous for the most common deletion delta F508 (CFT-2 cells). The transfected cells: (a) expressed the SV40 large T oncogene, as determined by immunofluorescence and Northern blot analysis; (b) retained typical epithelial morphology, as assessed by the presence of microvilli, desmosomes, gap junctions, and cytokeratin expression; (c) were fully responsive to the cAMP-stimulating agents isoproterenol, forskolin and vasoactive intestinal peptide for cAMP production and PKA activation; (d) do not produce any tumour in the athymic nude mice; (e) were diploid and tetraploid with a normal chromosomal complement at early passages, and (f) exhibited the abnormal regulation of chloride conductance characteristic of CF. These results indicate that CFT-1 and CFT-2 cells constitute a suitable model for: (a) comparison of the maturation and function of the CFTR protein mutated in the two nucleotide-binding domains; (2) analysis of the biochemical defect in CF epithelial airway cells, (c) development of new therapeutic agents, and correction of the CF defect by gene replacement therapy in vitro.

Expression of cystic fibrosis transmembrane conductance regulator in human gallbladder epithelial cells.

BACKGROUND: Hepatobiliary complications in cystic fibrosis result predominantly from lesions of the biliary epithelium. These abnormalities affect the intrahepatic as well as extrahepatic bile ducts and the gallbladder. The protein cystic fibrosis transmembrane conductance regulator (CFTR), the gene product defective in cystic fibrosis, functions as a cAMP-activated chloride channel in the plasma membrane. As such, it may represent an important driving force for fluid transport across the epithelium. EXPERIMENTAL DESIGN: The purpose of this study was to investigate the expression of CFTR in human gallbladder epithelial cells and to examine the chloride ion transport properties of these cells. Immunolocalization was performed on tissue sections. The reverse transcription-PCR was used to analyze the expression of CFTR mRNA in freshly isolated and cultured gallbladder epithelial cells. The CFTR protein was detected by Western blotting and immunoprecipitation. The chloride ion transport properties of the cells were determined by 36Cl efflux studies. RESULTS: The CFTR protein was immunodetected in human gallbladder in situ and localized predominantly to the apical membrane of epithelial cells. High levels of CFTR mRNA and protein were maintained in gallbladder epithelial cells in primary cultured. Glycosylated forms of CFTR were present as confirmed by treatment with N-glycanase. Chloride efflux was stimulated by Ca(++)-dependent pathways but more intensely by cAMP-dependent pathways. Stimulation of chloride efflux by agonist of the cAMP-pathway was inhibited by diphenylamine carboxylic acid, a chloride channel blocker. Two physiologically active peptides--acting via cAMP, vasoactive intestinal peptide, and secretin--also stimulated chloride efflux in vitro. CONCLUSIONS: Our results are consistent with a high expression of endogenous functional CFTR protein in human gallbladder epithelial cells. Physiologically active peptides, vasoactive intestinal peptide and secretin, stimulate chloride conductance in these cells. These findings indicate that CFTR play an important role in the pathophysiology of the biliary epithelium, including the gallbladder epithelium.

CFTR gene transfer corrects defective glycoconjugate secretion in human CF epithelial tracheal cells.

We demonstrate that in immortalized normal human tracheal epithelial cells (NT-1 and 56FHTE8o-) 14C-labeled glycoconjugate secretion may be regulated independently by agonists of the protein kinase A (PKA) and protein kinase C (PKC) signaling pathways. In contrast, in immortalized cystic fibrosis (CF) human tracheal epithelial cells (CFT-1 and CFT-2), regulation is defective for agonists specific for the PKA but not for the PKC pathway. To characterize the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in regulated glycoconjugate secretion, we examined the effect of adenovirus-mediated gene transfer of CFTR to CF and control cells. Forty-eight hours after infection, at a multiplicity of infection of 50 plaque-forming units per cell, high levels of CFTR mRNA were detected by reverse transcription-polymerase chain reaction, and de novo synthesis of CFTR protein was demonstrated by immunoblotting. Gene transfer to CF cells restored defective adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretion not only of chloride but also of glycoconjugates. Taken together, these results argue for a role for CFTR in cAMP-mediated glycoconjugate secretion.

Regulation of electrogenic anion secretion in normal and cystic fibrosis gallbladder mucosa.

Fluid and ion transport across biliary epithelium contributes to bile flow. Alterations of this function may explain hepatobiliary complications in cystic fibrosis (CF). We investigated electrogenic anion transport across intact non-CF and CF human gallbladder mucosa in Ussing-type chambers. In non-CF tissues, baseline transmural potential difference (PD), short-circuit current (Isc), and resistance (R) were -2.2 +/- 0.3 mV (lumen negative), 40.7 +/- 7.8 microA/cm2, and 66.5 +/- 9.6 Omega. cm2, respectively (n = 14). The addition of forskolin (10(-5) mol/L) to the apical and basolateral baths and that of adenosine 5'-triphosphate (ATP) (10(-4) mol/L) to the apical bath induced significant increases in Isc by 8.0 +/- 1.4 and 10.3 +/- 1.8 microA/cm2, respectively. Depletion of bathing solutions in Cl- and HCO3- significantly reduced baseline Isc and the forskolin- and ATP-induced increases in Isc. Anion secretion was stimulated by extracellular ATP via P2Y2 purinoceptors, as indicated by the effects of different nucleotides on Isc and on 36Cl efflux in cultured gallbladder epithelial cells. This effect was mediated by cytosolic calcium increase and Ca2+/calmodulin-dependent protein kinase II, as ascertained by using inhibitors. In CF preparations, basal PD and Isc were lower than in non-CF, and the response to forskolin was abolished, whereas the response to ATP was enhanced (P <.05 for all). We conclude that electrogenic anion secretion occurs in human gallbladder mucosa under basal state and is stimulated by an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent pathway mediated by cystic fibrosis transmembrane conductance regulator (CFTR), and by exogenous ATP via a CFTR-independent pathway that is up-regulated in CF and involves P2Y2 purinoceptors and a calcium-dependent pathway.

Permissiveness of human biliary epithelial cells to infection by hepatitis C virus.

The cellular tropism of hepatitis C virus (HCV) is an important but much debated issue. Permissivity to HCV of biliary cells has never been demonstrated. In this context, we used gallbladder epithelial cells (GBEC) as a model of the more proximal biliary epithelium. These cells were isolated from HCV-positive and -negative individuals and cultured for up to 40 days. Biliary cells from HCV-negative subjects were infected in vitro with various inocula. The retention of GBEC functional characteristics was assessed by the expression of cystic fibrosis transmembrane conductance regulator (CFTR). All 12 GBEC tested from HCV-negative patients were successfully infected by HCV. This was assessed by: 1) the detection of HCV-RNA positive and negative strands; 2) the detection of the viral capsid by immunofluorescence; and 3) the combination of single-strand conformation polymorphism (SSCP) and HVR1 sequence analysis demonstrating the distinct majoritary HCV genomes in serum and in GBEC. The level of HCV RNA in cell extracts and supernatants was low, but HCV infection was highly reproducible. Our results expand those showing the cellular tropism of HCV, and demonstrate the sensitivity of biliary cells to HCV infection. This might have an important impact in terms of pathogenesis and pathological features of HCV infection. In addition, given the easy access to these cells and the high reproducibility of in vitro infection, they should constitute an important tool for studies aimed at analyzing the issue of HCV penetration and neutralizing antibodies.