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Niches containing stem/progenitor cells are present in different anatomical locations along the human biliary tree and within liver acini. The most primitive stem/progenitors, biliary tree stem/progenitor cells (BTSCs), reside within peribiliary glands located throughout large extrahepatic and intrahepatic bile ducts. BTSCs are multipotent and can differentiate towards hepatic and pancreatic cell fates. These niches’ matrix chemistry and other characteristics are undefined. Canals of Hering (bile ductules) are found periportally and contain hepatic stem/progenitor cells (HpSCs), participating in the renewal of small intrahepatic bile ducts and being precursors to hepatocytes and cholangiocytes. The niches also contain precursors to hepatic stellate cells and endothelia, macrophages, and have a matrix chemistry rich in hyaluronans, minimally sulfated proteoglycans, fetal collagens, and laminin. The microenvironment furnishes key signals driving HpSC activation and differentiation. Newly discovered third niches are pericentral within hepatic acini, contain Axin2+ unipotent hepatocytic progenitors linked on their lateral borders to endothelia forming the central vein, and contribute to normal turnover of mature hepatocytes. Their relationship to the other stem/progenitors is undefined. Stem/progenitor niches have important implications in regenerative medicine for the liver and biliary tree and in pathogenic processes leading to diseases of these tissues.

Increased cholangiocyte growth is critical for the maintenance of biliary mass during liver injury by bile duct ligation (BDL). Circulating levels of testosterone decline following castration and during cholestasis. Cholangiocytes secrete sex hormones sustaining cholangiocyte growth by autocrine mechanisms. We tested the hypothesis that testosterone is an autocrine trophic factor stimulating biliary growth. The expression of androgen receptor (AR) was determined in liver sections, male cholangiocytes, and cholangiocyte cultures [normal rat intrahepatic cholangiocyte cultures (NRICC)]. Normal or BDL (immediately after surgery) rats were treated with testosterone or antitestosterone antibody or underwent surgical castration (followed by administration of testosterone) for 1 wk. We evaluated testosterone serum levels; intrahepatic bile duct mass (IBDM) in liver sections of female and male rats following the administration of testosterone; and secretin-stimulated cAMP levels and bile secretion. We evaluated the expression of 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3, the enzyme regulating testosterone synthesis) in cholangiocytes. We evaluated the effect of testosterone on the proliferation of NRICC in the absence/presence of flutamide (AR antagonist) and antitestosterone antibody and the expression of 17β-HSD3. Proliferation of NRICC was evaluated following stable knock down of 17β-HSD3. We found that cholangiocytes and NRICC expressed AR. Testosterone serum levels decreased in castrated rats (prevented by the administration of testosterone) and rats receiving antitestosterone antibody. Castration decreased IBDM and secretin-stimulated cAMP levels and ductal secretion of BDL rats. Testosterone increased 17β-HSD3 expression and proliferation in NRICC that was blocked by flutamide and antitestosterone antibody. Knock down of 17β-HSD3 blocks the proliferation of NRICC. Drug targeting of 17β-HSD3 may be important for managing cholangiopathies.

Lactoferin (Lf) is an iron-binding glycoprotein belonging to the transferrin family and it is present at high levels in breast milk and colostrum. This protein has many known functions and it is a potential antibacterial, antiviral, immunostimulatory, antioxidant, and cancer preventive agent.It has been seen that a 105 kDa Lf receptor (LfR) specifically mediates the effects of Lf in several different cell types (1). In human cholangiopathies, cholangiocytes are able to proliferate and replace the cell loss restoring the integrity of damaged biliary epithelium. However, when cholangiocyte proliferation is severely impaired, the activation of facultative hepatic progenitor cells (HPCs) takes place (2). The aims of our study have been i) to investigate the expression of Lf and LfR in cholangiocytes and in HPCs both in rats and in humans; and ii) to evaluate the in vitro effects of bLf on cholangiocyte proliferation and on HPC activation. Liver specimens have been obtained from normal (N=5) and bile duct ligated (BDL) (N=5) rats; from normal patients (N=5) and from patients with primary biliary cirrhosis (PBC, N=5); Specimens were processed for histology, immunohistochemistry and immunofluorescence. Moreover for the in vitro study small and large cholangioytes from mouse, human non malignant cholangiocytes (H69), and HPCs treated or not with lactoferrin were used. Our results showed that: i) cholangiocytes and hepatic progenitor cells express lactoferrin and its receptor, ii) cholangiocytes and HPC proliferation is enhanced by lactoferrin; iii) the treatment with lactoferrin determine the commitment of HPCs towards cholangiocyte fate; this commitment is characterized by HPC morphological and phenotypical changes. Our current findings suggest that modulation of lactoferrin may be an important therapeutic tool for managing the proliferation of cholangiocyte and the activation of progenitor cell compartment in biliary disorders., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Glands of biliary tree (Peribiliary Glands: PBGs) contain a niche of heterogeneous endodermal-like stem/progenitors cells (Biliary Tree Stem/progenitor Cells: BTSCs) that can be easily induced to differentiate, in vitro and in vivo, towards pancreatic islets (1,2). Whether these cells may play a role in insulin production in diabetes is unknown. The aim of this study was to evaluate, in experimental diabetes, proliferation of PBGs and differentiation of BTSCs towards insulin-producing cells. Diabetes was generated in mice by intraperitoneal injection of a single dose of 200 mg/kg (High-Dose STZ group; N=12) or 120 mg/kg of STZ (Low-Dose STZ group; N=12). Control mice (N=17) were injected with citrate buffer (i.e. the STZ carrier). Glucose levels in blood and urine and HbA1c blood levels were measured. Liver, pancreas and extrahepatic biliary trees (EHBT) were en bloc microdissected and processed for histology, immunohistochemistry, immunofluorescence, and RT-PCR. Our results showed that PBGs proliferated in an experimental model of type-1 diabe- tes. Their proliferation was greatest at the hepato-pancreatic ampulla and correlated with a decreased pancreatic islets’ mass. The PBG proliferation was characterized by the expansion of the compartment of Sox9+ stem/progenitor cells that gave rise to insulin-producing cells. Insulin-producing cells were mostly located in the portion of EHBT closest to the duodenum, and their appearance was associated with the upregulation of MafA and Gli1 gene expression. PBG proliferation and the expansion of Sox-9+, insulin-producing cells were more evident after 90 than after 30 days STZ treatment. In conclusion, PBGs and associated BTSCs respond to diabetes with proliferation and differentiation towards insulin-producing cells. This indicates that PBG niche may rescue the pancreatic islet impairment in diabetes with important implications for the patho-physiology and complications of this disease., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Recently, among anticancer agents, of growing interest are the kinases inhibitors. Many of them are under clinical trials while some have been already approved by FDA as angiogenesis blockers (Vascular endothelial growth factor receptor 2 (VEGFR- 2) inhibitors), such as Sunitinib and Vandetanib. HDACI (histone deacetylase inhibitors) are another newest drug group recently approved for the treatment of Cutaneous T-cell lymphoma (CTCL) (Wagner et al., 2010) and several others HDACIs are in pre-clinical and clinical stages. Interestingly, many scientific reports indicate the synergistic effects of the HDACI and VEGFR-2 inhibitors combination. Lately, has been proposed and showed that cycle oxygenases (COXs) inhibitors could reduce the adverse side effects upon HDACI administration. Furthermore, the anticancer effectiveness of COX-2 inhibitors in the treatment of gastric cancer animal models has been demonstrated (Thiel et al., 2011). Taking consideration of these observations we aim to study a new antiproliferative therapy with the combined use of three potentially synergic drug classes: HDACI, inhibitors of VEGFR-2 and COX-2. The antitproliferative evaluation of the more promising molecules will be evaluated both in vitro, in colangiocarcinoma and hepatocarcinoma cell lines trough MTT proliferation assay and Caspase3 Elisa kit, and in vivo models. Along with individual compound assays, synergistic effects will be also determined. The main findings derived from individual compound assay studies will be integrated and used to set-up the best combination of compounds for a multi-target approach that we expect to be highly effective against cancer., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Peribiliary glands (PBGs) in bile duct walls (Carpino et al., 2012), and pancreatic duct glands (PDGs) associated with pancreatic ducts contain a continuous, ramifying network of cells in overlapping maturational lineages (Wang et al., 2013). Our aims have been to investigate the presence of stem/progenitor cells within glands associated with bile and pancreatic ducts. Our results showed that proximal (PBGs)-todistal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG, OCT4, and SOX2) and proliferation (PCNA), and early hepato-pancreatic commitment (SOX9, SOX17, PDX1, and LGR5), transitioning to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3, MUC6, and insulin). Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, proliferating for months as undifferentiated cells; on the other hand, pancreas-derived cells underwent only approximately 8-10 divisions, then partially differentiated towards an islet fate. Both could be driven for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neo-islets, including glucose regulatability. Implantation of these neo-islets into epididymal fat pads of immunocompromised diabetic mice was able to alleviate hyperglycemia by the secretion of glucose-regulated human C-peptide., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Cholangiocytes are the cells lining the biliary tree from canals of Hering to larger bile ducts. By morphology, they are divided in small and large cholangiocytes, which result heterogeneous at functional and proliferative levels. Proliferating cholangiocytes acquire a neuroendocrine phenotype, modulated by several factors including neurotrophins. Brain Derivated Neurotrophic Factor (BDNF) is a neurotrophin expressed in the nervous system and also in different types of epithelial and progenitor cells. The aim of the present study is to detect the expression of BDNF and of its two receptors (TrKB and p75NT, or p75NTR) in normal and bile duct ligated (BDL) rat livers. In normal and BDL livers, BDNF and its receptors are expressed by small and large cholangiocytes and by hepatic progenitors cells. In cholangiocytes, the expression of BDNF and of its receptors changes after different BDL timing. After one or two weeks of BDL, both BDNF and TrKB and p75NT receptors are highly expressed, while after BDL for three weeks BDNF expression is drastically reduced and p75NT receptor prevails on TrKB. The expression of BDNF and of its receptors correlates with the proliferation rate of biliary tree during BDL. Indeed, after one or two weeks of BDL, proliferation prevails on apoptosis, whereas after BDL for three weeks, apoptosis prevails on proliferation. Our morphological results strongly suggest that BDNF plays a role in the remodeling of biliary tree during cholestasis and that it may be involved in the pathophysiology of cholestasic liver diseases., Italian Journal of Anatomy and Embryology, Vol 119, No 2 (2014)

Cholangiocytes are the cells lining the biliary tree from canals of Hering to larger bile ducts. At morphological level, we can distinguish small and large cholangiocytes, which result heterogeneous also at functional and proliferative levels (Alpini et al., 1998). Proliferating cholangiocytes are modulated by several factors including neurotrophins (Alvaro et al., 2008). BDNF is a neurotrophin expressed in the nervous system but produced also by different types of epithelial cells and by progenitor cells of neuronal and mesenchymal origin (Prakash et al., 2010). Our aim was to investigate the expression of BDNF in the biliary epithelium. The expression of BDNF and of its two receptors (TrkB and p75NT) was detected through immunohistochemistry and immunofluorescence in the biliary epithelium of normal and bile duct ligation (BDL) rat liver and of human cholastatic liver diseases. BDNF and its two receptors are expressed by small, large cholangiocytes and by Hepatic Progenitor Cells (HPC), both in normal and BDL rat livers. During BDL, the expression of BDNF and of its receptors correlates with the proliferation rate of small and large cholangiocytes. Indeed, during first two weeks of BDL, BDNF, TrkB and p75NT are highly expressed and proliferation prevails on apoptosis. After three weeks of BDL, BDNF and TrkB are slightly expressed while p75NT expression remain high and apoptosis prevails on proliferation. In vitro studies confirmed that BDNF and its receptors are expressed by small and large rat cholangiocytes. Also in human samples BDNF and its receptors are expressed in biliary epithelium, with a different intensity during chronic cholestasis. Our results suggest that BDNF plays a role in the remodeling of biliary tree during experimental cholestasis and cholestasic liver diseases., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Background: Secretin stimulates bicarbonate secretion in large ducts by interaction with receptors by a cAMP-dependent pathway [1]. In BDL rats, the growth of large (but not small) cholangiocytes is activated by the cAMP/ERK1/2/Elk-1 pathway; and is associated with increased secretin receptor (SR) expression [2]. The aim of our study was to define the role of secretin in experimental models of cholestatic mice. Methods: We used (SEC+/+) (wild-type, WT) and SEC-/- male mice that underwent sham surgery or BDL for 7 days treated with saline or secretin. Then, we used: (i) liver sections to evaluate secretin protein expression, cholangiocyte growth, apoptosis, the presence of VEGF-A/C and NGF; (ii) purified and immortalized small and large BDL cholangiocytes to measure PCNA protein expression and the same previous growth factors. Results: In vivo, we demonstrated that: (i) secretin is expressed in normal liver by large bile ducts; and (ii) secretin expression increases in large bile ducts following BDL. Isolated large cholangiocytes from BDL WT mice express higher levels of the mRNA for secretin and secrete greater amounts of this hormone compared to normal large cholangiocytes. Knockout of the secretin gene reduced BDL-induced increase in large IBDM compared to WT BDL mice, an event that was associated with enhanced apoptosis of large cholangiocytes and decreased expression of VEGF-A/C and NGF. Concomitant with reduced large IBDM there was increased small IBDM in SEC-/- KO BDL mice compared to WT BDL mice. After prolonged treatment, secretin increased the biliary expression of PCNA, VEGF-A/C and NGF. Conclusions: The study introduces the novel concept that cholangiocytes express and secrete secretin and that manipulation of this gene may be an important target for the management of biliary disorders., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Reduction of biliary serotonin N-acetyltransferase (AANAT) expression and melatonin administration/secretion in cholangiocytes increases biliary proliferation and the expression of SR, CFTR and Cl(-)/HCO3 (-) AE2. The balance between biliary proliferation/damage is regulated by several autocrine neuroendocrine factors including vascular endothelial growth factor-A/C (VEGF-A/C). VEGFs are secreted by several epithelia, where they modulate cell growth by autocrine and paracrine mechanisms. No data exists regarding the effect of AANAT modulation on the expressions of VEGFs by cholangiocytes.In this study, we evaluated the effect of local modulation of biliary AANAT expression on the cholangiocytes synthesis of VEGF-A/C.The decrease in AANAT expression and subsequent lower melatonin secretion by cholangiocytes was associated with increased expression of VEGF-A/C. Overexpression of AANAT in cholangiocyte lines decreased the expression of VEGF-A/C.Modulation of melatonin synthesis may affect the expression of VEGF-A/C by cholangiocytes and may modulate the hepatic microvascularization through the regulation of VEGF-A/C expression regulating biliary functions.

Cholangiocytes are ephitilial cells which line the biliary tree and that in normal condition are mitotically quiescient. Under experimental conditions, such as bile duct ligation (BDL) or in some human cholangiopathies (eg, primary biliary cirrhosis-PBC) cholangiocytes undergo a marked proliferation. A number of factors including Vascular endothelial growth factor (VEGF) and Follicle-stimulating hormone (FSH) regulate biliary tree growth. Melatonin is secreted by the pineal gland and other tissues where it acts as a local regulator of growth, as in the gastrointestinal tract. Melatonin exerts its effects through two membrane receptors (MT1 and MT2) and is synthesized from tryptophan by four enzymes: tryptophan-hydroxylase (TPH), aromatic-amino acid decarboxylase (AADC), serotonin N-acetyltransferase (AANAT) and acetilserotonin- O-methyltransferase (ASMT). Melatonin acts by modulating the expression of Clock genes in the pineal gland. In the liver melatonin reduces oxidative damage. Little information exists about the effects of melatonin on biliary epithelium, therefore the aim of this study was to evaluate the role of melatonin in the regulation of proliferation of biliary epithelium. To this end, we used normal and BDL rats treated or not with: i) melatonin, ii) Arilalkilamina-N-acetyltransferase (AANAT) mistatch and iii) AANAT Vivo Morpholino for 7days and iiii) nude mice injected with the cholangiocarcinoma cell line (Mz-Cha-1) treated or not with melatonin. On liver samples of the experimental models we evaluated: i) the expression of melatonin, the melatonin receptors (MT1 and MT2) and Clock genes, cholangiocytes proliferation and apoptosis, ii) basal and secretin Cyclic adenosine monophosphate (cAMP) levels and Protein Chinase A (PKA) phosphorylation. In addition, we evaluated the expression of melatonin, its receptors and AANAT also in fragments of normal human liver, cholangiocarcioma and PBC. In vitro, large mouse cholangiocytes (LMC), normal human cholangiocytes (H69) and cholangiocarcinoma cell line (Mz-Cha-1) were treated with melatonin 10-11 M for 48h, plus or minus Luzindole (MT1 antagonist) or cis-4-Phenyl-2-propionamidotetralin(4P-PDOT) (MT1 and MT2 antagonist); Finally, we evaluated the effects of AANAT overexpression in LMC and in Mz-Cha-1 on proliferation, apoptosis and cAMP pathway. The results obtained showed that: cholangiocytes expess AANAT, melatonin and its receptors MT1 and MT2 and Clock genes. Chronic administration of melatonin in vivo decreased cholangiocytes proliferation after BDL, and the down-regulation of AANAT in Normal and BDL AANAT Morpholino treated rats increased proliferation. In vitro, melatonin decreased LMC proliferation, decreased which is blocked by Luzindole but not by (4P-PDOT). Overexpression of AANAT in LMC decreased cholangiocytes proliferation, and the intracellular cAMP levels. The expression of AANAT and melatonin decreased in cholangiocarcinoma, instead increased is the expression of MT1 and MT2. In nude mice injected with CCA cell lines the tumor decreased after treatment with melatonin. Overexpression of AANAT in Mz-Cha-1 decreased proliferation and increased apoptosis. In PBC the expression of AANAT, melatonin and its receptors increased if compared to normal. Based on the results obtained, we can say that melatonin plays an important role in the regulation of cholangiocytes proliferation during cholestatic liver diseases and cholangiocarcinoma.

The intrahepatic biliary epithelium is formed by different-sized cells called large and small cholangiocytes (Alpini et al., 1996). Large cholangiocytes exert their functions by activation of cAMP, while Ca2+-dependent signalling regulates the function of small cholangiocytes (Francis et al., 2007). Histamine interacts with four receptors, H1–H4HRs (Onori et al 2010). H1HR acts by IP3/Ca2+-dependent mechanism, whereas H2HR stimulates cAMP signalling pathway. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1–H4HRs was evaluated in liver sections and in isolated and cultured cholangiocytes (normal rat intrahepatic cholangiocyte culture, NRIC). Normal rats were treated with H1-H4HRs agonists for 1 week. Small and large cholangiocytes express H1-H4HR. . Histamine and the H1HR agonist increased small Bile Duct Mass (BDM), whereas histamine and the H2HR agonist increased large BDM. H1HR agonists stimulated IP(3) levels and PKCα phosphorylation, whereas H2HR agonist increased cAMP levels and PKA phosphorylation. The activation of differential signaling mechanisms targeting small and large cholangiocytes may be important for biliary regeneration during pathologies affecting different-sized bile ducts., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine/paracrine mechanisms (Alpini et al., 1994). Melatonin inhibits biliary growth and secretin-stimulated choleresis in cholestatic rats by interaction with melatonin recepror 1 (MT1) (Renzi et al., 2011). We will try to localize the key enzyme involved in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT), in cholangiocytes and, possibly, we will try to determine the effect of modulation of AANAT on the autocrine proliferative/secretory responses of cholangiocytes. In liver sections we found that: (i) AANAT is expressed by cholangiocytes and hepatocytes; (ii) the cholangiocytes expression of AANAT decreased in morpholino (AANAT down regulator)-treated rats; and (iii) the decrease in AANAT expression and subsequent lower melatonin secretion by cholangiocytes is associated with increased biliary proliferation and increased expression of Secretin Receptor (SR) and VEGFA/ C. In vitro, we observed that overexpression of AANAT in large cholangiocyte (LC) decreased proliferation and ablated secretin-stimulated biliary secretion. These results indicate that: in vivo down-regulation of biliary AANAT stimulates cholangiocyte proliferation by an autocrine loop, and in vitro overexpression of AANAT in LC decreases proliferation. Local targeting of AANAT in cholangiocytes may be important for the management of cholangiopathies., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Background and aims. Cholangiocytes, the cells that line biliary ducts, have the capacity to respond to a variety of hormones and neuropeptides, regulating their surrounding cell functions and proliferative activity1. There is no evidence that the sensory neuropeptide substance P (SP) regulates cholangiocyte hyperplasia interacting with its endogenous neurokinin 1 receptor (NK1-R)2. Methods. To shed light on this interaction, Wild-type (WT, +/+) and NK-1 receptor (NK-1R) knock-out (NK-1R -/-) mice underwent sham or BDL for 1 week. Then, we evaluated: (i) NK-1R expression by immunohistochemistry, (ii) necrosis, apoptosis, steatosis and the number of cholangiocytes positive for citokeratin-19 (CK-19) in liver sections; (iii) PCNA expression and PKA phosphorylation by western blots. In vitro, we determined the effects of SP on growth by MTS proliferation assay, cAMP intracellular levels and PKA phosphorylation by western blots. Results. Cholangiocytes express NK-1R with upregulation following BDL. In normal NK-1R (-/-) mice, there was higher apoptosis and scattered steatosis compared to controls. In NK-1R -/- BDL mice, there was a decrease in the number of CK-19-positive cholangiocytes and enhanced biliary apoptosis compared to controls. In cholangiocytes from BDL NK-1R -/- mice there was decreased PCNA expression and PKA phosphorylation. In vitro, SP increased cAMP levels, proliferation and PKA phosphorylation of cholangiocytes. Conclusion. In the present study we have demonstrated that modulation of SP, by NK-1R, such as other neuroendocrine factors, may offer a new target in the inhibition and in the regulation of biliary functions during cholangiopathies., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Background and aims. In the experimental model of bile duct ligated (BDL) rats, the growth of large cholangiocytes is regulated by activation of cAMP-dependent signalling pathway1-2. Melatonin, which is secreted from pineal gland as well as extrapineal tissues, regulates cell proliferation by interacting with its receptors (MT1 and MT2) via the modulation of cAMP levels and the expression of the CLOCK genes3. We studied the mechanism by which melatonin regulates the growth of the biliary epithelium. Methods. Normal and BDL rats were treated in vivo with melatonin for 7 days before evaluating: (i) intrahepatic bile duct mass (IBDM) in liver sections by immunohistochemistry for citokeratin-19 (CK-19), a specific marker of cholangiocytes; and (ii) expression of MT1, MT2 and CLOCK genes by immunohistochemistry and real time PCR. In vitro, large cholangiocytes were stimulated with melatonin in the absence/presence of luzindole (a MT1/MT2 antagonist) and 4-P-PDOT (a specific MT2 antagonist) before assessing cellular growth by MTS proliferation assay, cAMP levels by a RIA kit and ERK1/2 phosphorylation by western blots. Results. Cholangiocytes express MT1, MT2 and CLOCK genes that were all upregulated following BDL. Administration of melatonin to BDL rats decreased IBDM and the expression of all CLOCK genes. In cells, melatonin decreased the proliferation, cAMP levels and the activation of ERK2, decreases that were blocked by luzindole. Conclusion. Supporting other studies we found that melatonin-inhibition of biliary hyperplasia is associated with downregulation of CLOCK gene expression. These findings have important clinical implications since melatonin may be an important therapeutic tool for managing cholangiocyte hyperproliferation in biliary disorders., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011