D. Alvaro

New insights into liver stem cells

Hepatic progenitor cells are bi-potential stem cells residing in human and animal livers that are able to differentiate towards the hepatocytic and the cholangiocytic lineages. In adult livers, hepatic progenitor cells are quiescent stem cells with a low proliferating rate, representing a reserve compartment that is activated only when the mature epithelial cells of the liver are continuously damaged or inhibited in their replication, or in cases of severe cell loss. Hepatic progenitor cell activation has been described in various acute and chronic liver diseases. Their niche is composed by numerous cells such as Hepatic Stellate Cells, endothelial cells, hepatocytes, cholangiocytes, Kupffer cells, pit cells and inflammatory cells. All these cells, numerous hormones and growth factors could interact and cross-talk with progenitor cells influencing their proliferative and differentiative processes. Hepatic progenitor cells and their niche could represent, in the near future, a target for therapeutic approaches to liver disease based on cell-specific drug delivery systems. Isolation and transplantation of hepatic progenitor cells could represent a new approach for therapy of end-stage chronic liver diseases, as they offer many advantages to transplantation of mature hepatocytes. The possibility of applying stem cell therapy to liver diseases will represent a major goal in this field.

Relationships between bile salts hydrophilicity and phospholipid composition in bile of various animal species

Bile salts and phospholipids from bile of chicken, dog, sheep, rat, ox, pig, guinea-pig and man were analyzed by high-performance liquid chromatography. Bile salts showed marked differences in their hydrophilic properties, owing to hydroxyl structure and type of conjugation. Phospholipids were generally similar, containing 90-95% of phosphatidylcholine which was made of molecular species containing palmitic acid in the sn-1 position. The comparative analysis of bile salts and phosphatidylcholines profile demonstrated that bile salts hydrophilicity influences the quantity of phosphatidylcholine in bile but not the quality.

Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

To elucidate mechanisms of glucagon-induced bicarbonate-rich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pHi) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pHi, significantly stimulates the Cl-/HCO3- exchange activity. The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3- exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3- exchange was individually blocked by two specific and selective inhibitors of protein kinase A, Rp-cAMPs (10 microM) and H-89 (30 microM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl- channel blocker, NPPB (10 microM), showed no effect on either the basal or the glucagon-stimulated Cl-/HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 microM), completely blocked the glucagon stimulation of the Cl-/HCO3- exchange; however, this effect was achieved through a significant inhibition of the glucagon-stimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3- exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pHi or at acid pHi values. In contrast, glucagon, at basal pHi, stimulated the Na(+)-HCO3- symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3- exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis.

Influence of tauroursodeoxycholic and taurodeoxycholic acids on hepatic metabolism and biliary secretion of phosphatidylcholine in the isolated rat liver

Studies were carried out using an isolated rat liver system to define: the contribution of exogenous phosphatidylcholine (PC) to biliary phospholipid secretion; and its hepatic metabolism during perfusion of the livers with conjugated bile salts with different hydrophilic/hydrophobic properties. A tracer dose of sn-1-palmitoyl-sn-2-[14C]linoleoylPC was injected as a bolus into the recirculating liver perfusate, under constant infusion of 0.75 mumol/min of tauroursodeoxycholate or taurodeoxycholate. The effects on bile flow, biliary lipid secretion, 14C disappearance from the perfusate and its appearance in bile, as well as hepatic and biliary biotransformation were determined. With both the bile salts, about 40% of the [14C]PC was taken up by the liver from the perfusate over 100 min. During the same period less than 2% of the given radioactivity was secreted into bile. More than 95% of the 14C recovered in bile was located within the identical injected PC molecular species. The biliary secretion of labeled as well as unlabeled PC, however, was significantly higher in livers perfused with taurodeoxycholate than tauroursodeoxycholate, while the reverse was observed with respect to bile flow and total bile salt secretion. The exogenous PC underwent extensive hepatic metabolization which appeared to be influenced by the type of bile salt perfusing the liver. After 2 h perfusion, the liver radioactivity was found, in decreasing order, in PC, triacylglycerol, phosphatidylethanolamine and diacylglycerol. In addition, the specific activity of triacylglycerol was significantly higher in tauroursodeoxycholate than in taurodeoxycholate-perfused livers (P less than 0.025), while the reverse was true for the specific activity of hepatic PC (P less than 0.01). Because taurodeoxycholate and tauroursodeoxycholate showed opposite effects on both biliary lipid secretion and hepatic PC biotransformations, we conclude that the hepatic metabolism of glycerolipids is influenced by the physiochemical properties of bile salts.

Hepatic 3α-dehydrogenation and 7α-hydroxylation of deoxycholic acid in the guinea-pig

The metabolic fate of exogenous deoxycholate administered either intraperitoneally or intragastrically to male Hartley guinea-pigs was investigated. Two animals received a constant infusion of [24-14C]deoxycholate through an intraperitoneal catheter for 2 hr. Bile was quantitatively collected in 30 min samples during infusion and for 2 additional hours. Each bile sample was analyzed for composition and radioactivity. Five animals received for 15 days, through an intragastric catheter, 35 mg/kg/day of deoxycholate. The biliary bile acid composition was compared with that of a sham-operated control group. The studies with both animal models indicated that guinea-pigs, as the only species so far known, extensively oxidize deoxycholate to form 3-oxo,12 alpha-hydroxy-cholanic acid, which is secreted in bile mostly conjugated with glycine. In addition a small fraction (approx. 7%) of the administered deoxycholate is 7 alpha hydroxylated to form cholic acid. The metabolites being more hydrophilic than administered deoxycholate, it is suggested that guinea-pig liver counteracts the adverse increase in bile acids detergency, which follows deoxycholate administration, by converting most of the latter into less detergent compounds.

Cholangiocarcinomas: New Insights from the Discovery of Stem Cell Niches in Peribiliary Glands of the Biliary Tree

Peribiliary glands (PBGs) are located in the large intrahepatic and extrahepatic bile ducts. Although they were described many years ago, their functions have been elucidated only in the last couple of years when our group demonstrated that PBGs are niches of multipotent stem/progenitor cells of endodermal origin. These cells express genes of multipotency and can be rapidly differentiated in vitro into hepatocytes, cholangiocytes, and endocrine pancreatic cells. PBGs share common features, in terms of stem/progenitor cell niches, with pancreatic duct glands and colon crypts, glandular structures representing in the adult life the endodermal remnants of fetal life. PBG stem/progenitor cells participate in the renewal of surface biliary epithelium and are active players in chronic pathologies of the biliary tree as well as in cholangiocarcinomas (CCA). Specifically, a large amount of recent evidence indicates that the pure mucin-CCA originates from PBGs; this could explain the similarities with pancreatic ductal adenocarcinoma and colorectal cancer, which also originate from transformed gland cells. In this paper, we summarized our recent findings concerning structure and functions of PBGs with the implications for liver pathophysiology and, specifically, for cancers of the biliary tree.

P44 TUMORIGENIC POTENTIAL OF CANCER STEM CELLS (CSCS) ISOLATED FROM HUMAN CHOLANGIOCARCINOMA (CCA) SUBTYPES

A. Torrice ∗ ,2, G. Carpino 1, A. Fraveto 2, A. Renzi 3, M.C. Bragazzi 2, F. Giuliante 4, A.M. Derose 2, V. Cardinale 2, R. Gentile 2, P. Onori 3, C. Napoletano 5, A. Franchitto 3, A. Cantafora 2, G. Grazi 2, E. Gaudio 2, D. Alvaro 2 1Health Science, University of Rome “Foro Italico”, Rome, Italy; 2Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy; 3SAIMLAL, Sapienza, University of Rome, Rome, Italy; 4Surgery, Hepatobiliary Unit, Catholic University of The Sacred Heart School of Medicine, Rome, Italy; 5Experimental Medicine, Sapienza University of Rome, Rome, Italy

880 ESTROGEN RECEPTOR β SELECTIVE AGONISTS EXERT ANTI-CANCER ACTIVITY ON EXPERIMENTAL INTRA-HEPATIC CHOLANGIOCARCINOMA: AN IN VITRO AND IN VIVO STUDY

is lacking in the literature. The aim of this study is to evaluate the evolution of AHC in haemodialysis patients and the response to IFNamonotherapy in those patients with chronic evolution. Patients and Methods: Nineteen consecutive haemodialysis patients with AHC (12 male, median age 57 years) were observed from 1994 to 2009 and enrolled in this study. Diagnosis of AHC was based on well-documented seroconversion for anti-HCV in all patients. No patient had symptomatic disease. Median time between initiating haemodialysis and acute HCV infection was 28.5 months (range 2−96). Patients with HCV RNA clearance within 12 weeks from the AHC onset were considered self-limiting patients whereas patients with HCV RNA still detectable at this time point were considered to have a chronic evolution. Results: 16/19 haemodialysis patients with AHC were followed for a median period of 25 months (range 12–109), while 3/19 patients were lost to follow-up. The evolution of AHC was assessed after 12−24 weeks from onset of disease; at the end of this observation period, HCV RNA was still detectable in 14/16 (87.5%) patients with a chronic evolution, while 2/16 (12.5%) patients were HCV RNA negative and were considered to have a spontaneous resolution. Of the 14 chronic evolution patients, 13 underwent IFN monotherapy: 12 were treated with a recombinant IFNa2b dosage of 3−5 MU three times weekly for 6 months and one patient received PEG-IFNa2a 135mcg/week for 6 months. Two patients prematurely discontinued therapy after 2 and 5 months (1 for side effects and one for acute cytomegalovirus infection). Overall, a sustained virologic response (SVR) was observed in 10/13 patients (77%); in particular, 10/11 patients (91%) who completed a 6-month course of therapy obtained a SVR compared to neither of the two drop-out patients. The only non-responder patient was retreated with IFNa monotherapy for 12 months achieving SVR. Conclusions: In haemodialysis patients, acute hepatitis C presents a higher rate of chronicity when compared to that observed in the general population. Treatment with IFNa in the early stage of infection is generally well-tolerated and is associated with a high rate of sustained virologic response, even in patients on dialysis.

922 MULTIPOTENT STEM CELLS RESIDE IN HUMAN EXTRAHEPATIC BILE DUCTS AND CAN GIVE RISE TO HEPATOCYTES, CHOLANGIOCYTES AND PANCREATIC ISLET CELLS

1 Department Human Anatomy, “Sapienza” University of Rome, Italy 2 Department Health Science, University “Foro Italico” Rome, Italy 3 Department Clinical Medicine, “Sapienza” University of Rome, Italy 4 Polo Pontino, “Sapienza”, University of Rome, Italy 5 Department “P. Stefanini”, “Sapienza” University of Rome, Italy 6 UNC School of Medicine, Chapel Hill, NC, USA 7 Diabetes Research Institute, Miami Medical Center, USA