Domenico Alvaro

Abstract Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non-invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlighted.

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies with features of biliary tract differentiation. CCA is the second most common primary liver tumour and the incidence is increasing worldwide. CCA has high mortality owing to its aggressiveness, late diagnosis and refractory nature. In May 2015, the "European Network for the Study of Cholangiocarcinoma" (ENS-CCA: www.enscca.org or www.cholangiocarcinoma.eu) was created to promote and boost international research collaboration on the study of CCA at basic, translational and clinical level. In this Consensus Statement, we aim to provide valuable information on classifications, pathological features, risk factors, cells of origin, genetic and epigenetic modifications and current therapies available for this cancer. Moreover, future directions on basic and clinical investigations and plans for the ENS-CCA are highlighted.

A genome-wide association screen for primary biliary cirrhosis risk alleles was performed in an Italian cohort. The results from the Italian cohort replicated IL12A and IL12RB associations, and a combined meta-analysis using a Canadian dataset identified newly associated loci at SPIB (P = 7.9 x 10(-11), odds ratio (OR) = 1.46), IRF5-TNPO3 (P = 2.8 x 10(-10), OR = 1.63) and 17q12-21 (P = 1.7 x 10(-10), OR = 1.38).

Tom Karlsen and colleagues report an association study for primary sclerosing cholangitis (PSC), a severe liver disease, using the Immunochip array. They identify nine loci newly associated with PSC and examine pleiotropy with other autoimmune disorders. Primary sclerosing cholangitis (PSC) is a severe liver disease of unknown etiology leading to fibrotic destruction of the bile ducts and ultimately to the need for liver transplantation1,2,3. We compared 3,789 PSC cases of European ancestry to 25,079 population controls across 130,422 SNPs genotyped using the Immunochip4. We identified 12 genome-wide significant associations outside the human leukocyte antigen (HLA) complex, 9 of which were new, increasing the number of known PSC risk loci to 16. Despite comorbidity with inflammatory bowel disease (IBD) in 72% of the cases, 6 of the 12 loci showed significantly stronger association with PSC than with IBD, suggesting overlapping yet distinct genetic architectures for these two diseases. We incorporated association statistics from 7 diseases clinically occurring with PSC in the analysis and found suggestive evidence for 33 additional pleiotropic PSC risk loci. Together with network analyses, these findings add to the genetic risk map of PSC and expand on the relationship between PSC and other immune-mediated diseases.

Multipotent stem/progenitors are present in peribiliary glands of extrahepatic biliary trees from humans of all ages and in high numbers in hepato-pancreatic common duct, cystic duct, and hilum. They express endodermal transcription factors (e.g., Sox9, SOX17, FOXA2, PDX1, HES1, NGN3, PROX1) intranuclearly, stem/progenitor surface markers (EpCAM, NCAM, CD133, CXCR4), and sometimes weakly adult liver, bile duct, and pancreatic genes (albumin, cystic fibrosis transmembrane conductance regulator [CFTR], and insulin). They clonogenically expand on plastic and in serum-free medium, tailored for endodermal progenitors, remaining phenotypically stable as undifferentiated cells for months with a cell division initially every ≈36 hours and slowing to one every 2-3 days. Transfer into distinct culture conditions, each comprised of a specific mix of hormones and matrix components, yields either cords of hepatocytes (express albumin, CYP3A4, and transferrin), branching ducts of cholangiocytes (expressing anion exchanger-2-AE2 and CFTR), or regulatable C-peptide secreting neoislet-like clusters (expressing glucagon, insulin) and accompanied by changes in gene expression correlating with the adult fate. Transplantation into quiescent livers of immunocompromised mice results in functional human hepatocytes and cholangiocytes, whereas if into fat pads of streptozocin-induced diabetic mice, results in functional islets secreting glucose-regulatable human C-peptide. Conclusion: The phenotypes and availability from all age donors suggest that these stem/progenitors have considerable potential for regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes. (HEPATOLOGY2011;)

Livers are comprised of maturational lineages of cells beginning extrahepatically in the hepato-pancreatic common duct near the duodenum and intrahepatically in zone 1 by the portal triads. The extrahepatic stem cell niches are the peribiliary glands deep within the walls of the bile ducts; those intrahepatically are the canals of Hering in postnatal livers and that derive from ductal plates in fetal livers. Intrahepatically, there are at least eight maturational lineage stages from the stem cells in zone 1 (periportal), through the midacinar region (zone 2), to the most mature cells and apoptotic cells found pericentrally in zone 3. Those found in the biliary tree are still being defined. Parenchymal cells are closely associated with lineages of mesenchymal cells, and their maturation is coordinated. Each lineage stage consists of parenchymal and mesenchymal cell partners distinguishable by their morphology, ploidy, antigens, biochemical traits, gene expression, and ability to divide. They are governed by changes in chromatin (e.g., methylation), gradients of paracrine signals (soluble factors and insoluble extracellular matrix components), mechanical forces, and feedback loop signals derived from late lineage cells. Feedback loop signals, secreted by late lineage stage cells into bile, flow back to the periportal area and regulate the stem cells and other early lineage stage cells in mechanisms dictating the size of the liver mass. Recognition of maturational lineage biology and its regulation by these multiple mechanisms offers new understandings of liver biology, pathologies, and strategies for regenerative medicine and treatment of liver cancers.

In the last 15 years, the intrahepatic biliary tree has become the object of extensive studies, which highlighted the extraordinary biologic properties of cholangiocytes involved in bile formation, proliferation, injury repair, fibrosis, angiogenesis, and regulation of blood flow. Proliferation is a "typical" property of cholangiocytes and is key as a mechanism of repair responsible for maintaining the integrity of the biliary tree. Cholangiocyte proliferation occurs virtually in all pathologic conditions of liver injury where it is associated with inflammation, regeneration, and repair, thus conditioning the evolution of liver damage. Interestingly, proliferating cholangiocytes acquire the phenotype of neuroendocrine cells, and secrete different cytokines, growth factors, neuropeptides, and hormones, which represent potential mechanisms for cross talk with other liver cells. Many studies suggest the generation of a neuroendocrine compartment in the injured liver, mostly constituted by cells with cholangiocyte features, which functionally conditions the progression of liver disease. These insights on cholangiocyte pathophysiology will provide new potential strategies for the management of chronic liver diseases. The purpose of this review is to summarize the recent findings on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte biology.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist.

Background and Aims Lipopolysaccharides (LPS) is increased in nonalcoholic fatty liver disease (NAFLD), but its relationship with liver inflammation is not defined. Approach and Results We studied Escherichia coli LPS in patients with biopsy‐proven NAFLD, 25 simple steatosis (nonalcoholic fatty liver) and 25 nonalcoholic steatohepatitis (NASH), and in mice with diet‐induced NASH. NASH patients had higher serum LPS and hepatocytes LPS localization than controls, which was correlated with serum zonulin and phosphorylated nuclear factor‐κB expression. Toll‐like receptor 4 positive (TLR4 + ) macrophages were higher in NASH than simple steatosis or controls and correlated with serum LPS. NASH biopsies showed a higher CD61 + platelets, and most of them were TLR4 + . TLR4 + platelets correlated with serum LPS values. In mice with NASH, LPS serum levels and LPS hepatocyte localization were increased compared with control mice and associated with nuclear factor‐κB activation. Mice on aspirin developed lower fibrosis and extent compared with untreated ones. Treatment with TLR4 inhibitor resulted in lower liver inflammation in mice with NASH. Conclusions In NAFLD, Escherichia coli LPS may increase liver damage by inducing macrophage and platelet activation through the TLR4 pathway.

Stem/progenitors have been identified intrahepatically in the canals of Hering and extrahepatically in glands of the biliary tree. Glands of the biliary tree (peribiliary glands) are tubulo-alveolar glands with mucinous and serous acini, located deep within intrahepatic and extrahepatic bile ducts. We have shown that biliary tree stem/progenitors (BTSCs) are multipotent, giving rise in vitro and in vivo to hepatocytes, cholangiocytes or pancreatic islets. Cells with the phenotype of BTSCs are located at the bottom of the peribiliary glands near the fibromuscular layer. They are phenotypically heterogeneous, expressing transcription factors as well as surface and cytoplasmic markers for stem/progenitors of liver (e.g. SOX9/17), pancreas (e.g. PDX1) and endoderm (e.g. SOX17, EpCAM, NCAM, CXCR4, Lgr5, OCT4) but not for mature markers (e.g. albumin, secretin receptor or insulin). Subpopulations co-expressing liver and pancreatic markers (e.g. PDX1(+)/SOX17(+)) are EpCAM(+/-), and are assumed to be the most primitive of the BTSC subpopulations. Their descendants undergo a maturational lineage process from the interior to the surface of ducts and vary in the mature cells generated: pancreatic cells in hepatopancreatic ducts, liver cells in large intrahepatic bile ducts, and bile duct cells along most of the biliary tree. We hypothesize that there is ongoing organogenesis throughout life, with BTSCs giving rise to hepatic stem cells in the canals of Hering and to committed progenitors within the pancreas. The BTSCs are likely to be central to normal tissue turnover and injury repair and to be key elements in the pathophysiology of liver, pancreas and biliary tree diseases, including oncogenesis.

(Cell Reports 18, 2780–2794; March 14, 2017) In the originally published version of this article, the sample names in Figure 5D were incorrectly ordered during assembly of the heatmap. The sample order has now been corrected online, and the corrected figure appears below. The authors regret this error. Also, due to a misunderstanding prior to publication, the TCGA network authors were not properly indexed in PubMed. This has now been corrected online.Figure 5ARID1A Is Hypermethylated and Has Low Expression in the IDH COCA (original)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular ProfilesFarshidfar et al.Cell ReportsMarch 14, 2017In BriefFarshidfar et al. present The Cancer Genome Atlas (TCGA) marker analysis of cholangiocarcinoma. Through multi-platform analyses, they identify a distinct subtype enriched for IDH mutants. This subtype shows increased mitochondrial and decreased chromatin modifier gene expression, including potential epigenetic silencing of ARID1A. Other IDH-mutant liver cancers molecularly resemble cholangiocarcinoma. Full-Text PDF Open Access

The overall objective of these guidelines is to provide evidence‐based recommendations for the diagnosis and management of immunoglobulin G4 (IgG4)‐related digestive disease in adults and children. IgG4‐related digestive disease can be diagnosed only with a comprehensive work‐up that includes histology, organ morphology at imaging, serology, search for other organ involvement, and response to glucocorticoid treatment. Indications for treatment are symptomatic patients with obstructive jaundice, abdominal pain, posterior pancreatic pain, and involvement of extra‐pancreatic digestive organs, including IgG4‐related cholangitis. Treatment with glucocorticoids should be weight‐based and initiated at a dose of 0.6–0.8 mg/kg body weight/day orally (typical starting dose 30‐40 mg/day prednisone equivalent) for 1 month to induce remission and then be tapered within two additional months. Response to initial treatment should be assessed at week 2–4 with clinical, biochemical and morphological markers. Maintenance treatment with glucocorticoids should be considered in multi‐organ disease or history of relapse. If there is no change in disease activity and burden within 3 months, the diagnosis should be reconsidered. If the disease relapsed during the 3 months of treatment, immunosuppressive drugs should be added.

Cholangiocarcinoma (CCA) is a rare and heterogeneous biliary cancer, whose incidence and related mortality is increasing. This study investigates the clinical course of CCA and subtypes (intrahepatic [iCCA], perihilar [pCCA], and distal [dCCA]) in a pan-European cohort.The ENSCCA Registry is a multicenter observational study. Patients were included if they had a histologically proven diagnosis of CCA between 2010-2019. Demographic, histomorphological, biochemical, and clinical studies were performed.Overall, 2,234 patients were enrolled (male/female=1.29). iCCA (n = 1,243) was associated with overweight/obesity and chronic liver diseases involving cirrhosis and/or viral hepatitis; pCCA (n = 592) with primary sclerosing cholangitis; and dCCA (n = 399) with choledocholithiasis. At diagnosis, 42.2% of patients had local disease, 29.4% locally advanced disease (LAD), and 28.4% metastatic disease (MD). Serum CEA and CA19-9 showed low diagnostic sensitivity, but their concomitant elevation was associated with increased risk of presenting with LAD (odds ratio 2.16; 95% CI 1.43-3.27) or MD (odds ratio 5.88; 95% CI 3.69-9.25). Patients undergoing resection (50.3%) had the best outcomes, particularly with negative-resection margin (R0) (median overall survival [mOS] = 45.1 months); however, margin involvement (R1) (hazard ratio 1.92; 95% CI 1.53-2.41; mOS = 24.7 months) and lymph node invasion (hazard ratio 2.13; 95% CI 1.55-2.94; mOS = 23.3 months) compromised prognosis. Among patients with unresectable disease (49.6%), the mOS was 10.6 months for those receiving active palliative therapies, mostly chemotherapy (26.2%), and 4.0 months for those receiving best supportive care (20.6%). iCCAs were associated with worse outcomes than p/dCCAs. ECOG performance status, MD and CA19-9 were independent prognostic factors.CCA is frequently diagnosed at an advanced stage, a proportion of patients fail to receive cancer-specific therapies, and prognosis remains dismal. Identification of preventable risk factors and implementation of surveillance in high-risk populations are required to decrease cancer-related mortality.This is, to date, the largest international (pan-European: 26 hospitals and 11 countries) observational study, in which the course of cholangiocarcinoma has been investigated, comparing the 3 subtypes based on the latest International Classification of Diseases 11th Edition (ICD-11) (i.e., intrahepatic [2C12], perihilar [2C18], or distal [2C15] affected bile ducts), which come into effect in 2022. General and tumor-type specific features at diagnosis, risk factors, biomarker accuracy, as well as patient management and outcomes, are presented and compared, outlining the current clinical state of cholangiocarcinoma in Europe.

Intrahepatic cholangiocarcinoma (iCCA) develops inside the liver, between bile ductules and the second-order bile ducts. It is the second most frequent primary liver cancer after hepatocellular carcinoma, and its global incidence is increasing. It is associated with an alarming mortality rate owing to its silent presentation (often leading to late diagnosis), highly aggressive nature and resistance to treatment. Early diagnosis, molecular characterisation, accurate staging and personalised multidisciplinary treatments represent current challenges for researchers and physicians. Unfortunately, these challenges are beset by the high heterogeneity of iCCA at the clinical, genomic, epigenetic and molecular levels, very often precluding successful management. Nonetheless, in the last few years, progress has been made in molecular characterisation, surgical management, and targeted therapy. Recent advances together with the awareness that iCCA represents a distinct entity amongst the CCA family, led the ILCA and EASL governing boards to commission international experts to draft dedicated evidence-based guidelines for physicians involved in the diagnostic, prognostic, and therapeutic management of iCCA.

<h3>Background & aims</h3> An estimated 38% of adults worldwide have non-alcoholic fatty liver disease (NAFLD). From individual impacts to widespread public health and economic consequences, the implications of this disease are profound. This study aimed to develop an aligned, prioritised fatty liver disease research agenda for the global health community. <h3>Methods</h3> Nine co-chairs drafted initial research priorities, subsequently reviewed by 40 core authors and debated during a three-day in-person meeting. Following a Delphi methodology, over two rounds, a large panel (R1 n=344, R2 n=288) reviewed the priorities, via Qualtrics XM, indicating agreement using a four-point Likert-scale and providing written feedback. The core group revised the draft priorities between rounds. In R2, panellists also ranked the priorities within six domains: epidemiology, models of care, treatment and care, education and awareness, patient and community perspectives, and leadership and public health policy. <h3>Results</h3> The consensus-built fatty liver disease research agenda encompasses 28 priorities. The mean percentage of ‘agree' responses increased from 78.3 in R1 to 81.1 in R2. Five priorities received unanimous combined agreement (‘agree' + ‘somewhat agree'); the remaining 23 priorities had >90% combined agreement. While all but one of the priorities exhibited at least a super-majority of agreement (>66.7% ‘agree'), 13 priorities had <80% ‘agree', with greater reliance on ‘somewhat agree' to achieve >90% combined agreement. <h3>Conclusions</h3> Adopting this multidisciplinary consensus-built research priorities agenda can deliver a step-change in addressing fatty liver disease, mitigating against its individual and societal harms and proactively altering its natural history through prevention, identification, treatment, and care. This agenda should catalyse the global health community's efforts to advance and accelerate responses to this widespread and fast-growing public health threat. <h3>Impact and implications</h3> An estimated 38% of adults and 13% of children and adolescents worldwide have fatty liver disease, making it the most prevalent liver disease in history. Despite substantial scientific progress in the past three decades, the burden continues to grow, with an urgent need to advance understanding of how to prevent, manage, and treat the disease. Through a global consensus process, a multidisciplinary group agreed on 28 research priorities covering a broad range of themes, from disease burden, treatment, and health system responses to awareness and policy. The findings have relevance for clinical and non-clinical researchers as well as funders working on fatty liver disease and non-communicable diseases more broadly, setting out a prioritised, ranked research agenda for turning the tide on this fast-growing public health threat.

Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples.At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models.Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment.Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC.Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Hypertransaminasemia is a frequent side effect during chenodeoxycholic administration for gallstone dissolution. Evidence suggests that this effect is not mediated by lithocholic acid, the intestinal metabolite of chenodeoxycholic acid, but that toxicity is due to the chenodeoxycholic acid itself. In vitro cytotoxicity of bile salts is positively proportional to their detergent effect, which is, on the other hand, related to their hydrophobic-hydrophilic balance. We hypothesize that in vivo also liver injury can occur when the liver is perfused by an high proportion of strongly detergent bile salts. The more detergent bile salts are unconjugated or glycine conjugated, while the lesser are taurine conjugated and sulfated. Within each class the following order of decreasing detergent power can be indicated: lithocholic greater than deoxycholic greater than chenodeoxycholic greater than cholic greater than ursodeoxycholic acid. Besides chronic exogenous administration of chenodeoxycholic or deoxycholic acids, conditions in which the liver is perfused by an high mass of highly detergent bile salts are those characterized by an enhanced intestinal biodegradation of bile salts. These conditions, which are common features of some chronic inflammatory bowel diseases, are frequently associated with liver damage. On the other hand, a normally detergent bile salt pool can become hepatotoxic for liver cells which have already been injured. In this respect, as already reported for increased sulfation, the increased proportion of taurine conjugates and the reduced formation of deoxycholic acid in liver cirrhosis can be regarded as protective mechanisms. Liver toxicity induced by bile salts' detergent action can be prevented by favouring tauroconjugation or reducing the intestinal degradation of bile salts or by administering poorly detergent bile salts.

Current protocols for differentiation of stem cells make use of multiple treatments of soluble signals and/or matrix factors and result typically in partial differentiation to mature cells with under- or overexpression of adult tissue-specific genes. We developed a strategy for rapid and efficient differentiation of stem cells using substrata of biomatrix scaffolds, tissue-specific extracts enriched in extracellular matrix, and associated growth factors and cytokines, in combination with a serum-free, hormonally defined medium (HDM) tailored for the adult cell type of interest. Biomatrix scaffolds were prepared by a novel, four-step perfusion decellularization protocol using conditions designed to keep all collagen types insoluble. The scaffolds maintained native histology, patent vasculatures, and ≈1% of the tissue's proteins but >95% of its collagens, most of the tissue's collagen-associated matrix components, and physiological levels of matrix-bound growth factors and cytokines. Collagens increased from almost undetectable levels to >15% of the scaffold's proteins with the remainder including laminins, fibronectins, elastin, nidogen/entactin, proteoglycans, and matrix-bound cytokines and growth factors in patterns that correlate with histology. Human hepatic stem cells (hHpSCs), seeded onto liver biomatrix scaffolds and in an HDM tailored for adult liver cells, lost stem cell markers and differentiated to mature, functional parenchymal cells in ≈1 week, remaining viable and with stable mature cell phenotypes for more than 8 weeks. Conclusion: Biomatrix scaffolds can be used for biological and pharmaceutical studies of lineage-restricted stem cells, for maintenance of mature cells, and, in the future, for implantable, vascularized engineered tissues or organs. (HEPATOLOGY 2011.)

Vascular endothelial growth factor (VEGF) is secreted by several epithelia and modulates cellular functions by autocrine and paracrine mechanisms. The role of VEGF in cholangiocyte pathophysiology is unknown. We evaluated the role of VEGF in the regulation of cholangiocyte proliferation in rats that underwent bile duct ligation.The expression of VEGF-A and VEGF-C and their receptors in cholangiocytes from normal and BDL rats was evaluated. Normal or BDL rats were treated with recombinant-VEGF-A or recombinant-VEGF-C or anti-VEGF antibodies, and proliferation of cholangiocytes was evaluated in situ by morphometry and in vitro by proliferating cell nuclear antigen immunoblots and MTS assay. In vitro, normal rat cholangiocyte cultures were stimulated with r-VEGF-A or r-VEGF-C and proliferation and signal transduction were evaluated.We found that (1) cholangiocytes express messenger RNA and protein for VEGF-A, VEGF-C, VEGF receptor 2 (VEGFR-2), and VEGF receptor 3 (VEGFR-3) and secrete VEGF; (2) secretion of VEGF and expression of VEGFR-2 and VEGFR-3 increases in BDL cholangiocytes; (3) blocking VEGF in vivo by anti-VEGF-A or anti-VEGF-C antibodies decreases cholangiocyte proliferation; (4) the in vivo administration of r-VEGF-A or r-VEGF-C induces cholangiocyte proliferation in normal rats; and (5) in vitro, VEGF-A increases normal rat cholangiocyte culture proliferation by activation of inositol 1,4,5-triphosphate/Ca2+/protein kinase C alpha and phosphorylation of Src/ERK1/2.Cholangiocytes secrete VEGF and express VEGFR-2 and VEGFR-3, all of which are amplified in BDL cholangiocytes. VEGF induces cholangiocyte proliferation by activation of inositol 1,4,5-triphosphate/[Ca2+]i/protein kinase C alpha and phosphorylation of Src/ERK1/2. VEGF mediates the adaptive proliferative response of cholangiocytes to cholestasis.

Cholangiocyte proliferation is triggered during extrahepatic bile duct obstruction induced by bile duct ligation, which is a common in vivo model used for the study of cholangiocyte proliferation and liver fibrosis. The proliferative response of cholangiocytes during cholestasis is regulated by the complex interaction of several factors, including gastrointestinal hormones, neuroendocrine hormones and autocrine or paracrine signalling mechanisms. Activation of biliary proliferation (ductular reaction) is thought to have a key role in the initiation and progression of liver fibrosis. The first part of this review provides an overview of the primary functions of cholangiocytes in terms of secretin-stimulated bicarbonate secretion – a functional index of cholangiocyte growth. In the second section, we explore the important regulators, both inhibitory and stimulatory, that regulate the cholangiocyte proliferative response during cholestasis. We discuss the role of proliferating cholangiocytes in the induction of fibrosis either directly via epithelial mesenchymal transition or indirectly via the activation of other liver cell types. The possibility of targeting cholangiocyte proliferation as potential therapy for reducing and/or preventing liver fibrosis, and future avenues for research into how cholangiocytes participate in the process of liver fibrogenesis are described.

<h2>Abstract</h2> Cholangiocarcinoma is commonly considered a rare cancer. However, if we consider the hepato-biliary system a single entity, cancers of the gallbladder, intra-hepatic and extra-hepatic biliary tree altogether represent approximately 30% of the total with incidence rates close to that of hepatocellular carcinoma, which is the third most common cause of cancer-related death worldwide. In addition, cholangiocarcinoma is characterized by a very poor prognosis and virtually no response to chemotherapeutics; radical surgery, the only effective treatment, is not frequently applicable because late diagnosis. Biomarkers for screening programs and for follow-up of categories at risk are under investigation, however, currently none of the proposed markers has reached clinical application. For all these considerations, cancers of the biliary tree system should merit much more scientific attention also because a progressive increase in incidence and mortality for these cancers has been reported worldwide. This manuscript deals with the most recent advances in the epidemiology, biology and clinical presentation of cholangiocarcinoma.

We investigated the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF-1), and IGF-1R (receptor) in human cholangiocarcinoma and cholangiocarcinoma cell lines (HuH-28, TFK-1, Mz-ChA-1), evaluating the role of estrogens and IGF-1 in the modulation of neoplastic cell growth. ER-α, ER-β, IGF-1, and IGF-1R were expressed (immunohistochemistry) in all biopsies (18 of 18) of intrahepatic cholangiocarcinoma. ER-α was expressed (Western blot) only by the HuH-28 cell line (intrahepatic cholangiocarcinoma), whereas ER-β, IGF-1, and IGF-1R were expressed in the three cell lines examined. In serum-deprived HuH-28 cells, serum readmission induced stimulation of cell proliferation that was inhibited by ER and IGF-1R antagonists. 17β-Estradiol and IGF-1 stimulated proliferation of HuH-28 cells to a similar extent to that of MCF7 (breast cancer) but greater than that of TFK-1 and Mz-ChA-1, inhibiting apoptosis and exerting additive effects. These effects of 17β-estradiol and IGF-1 were associated with enhanced protein expression of ER-α, phosphorylated (p)-ERK1/2 and pAKT but with decreased expression of ER-β. Finally, transfection of IGF-1R anti-sense oligonucleotides in HuH-28 cells markedly decreased cell proliferation. In conclusion, human intrahepatic cholangiocarcinomas express receptors for estrogens and IGF-1, which cooperate in the modulation of cell growth and apoptosis. Modulation of ER and IGF-1R could represent a strategy for the management of cholangiocarcinoma. We investigated the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF-1), and IGF-1R (receptor) in human cholangiocarcinoma and cholangiocarcinoma cell lines (HuH-28, TFK-1, Mz-ChA-1), evaluating the role of estrogens and IGF-1 in the modulation of neoplastic cell growth. ER-α, ER-β, IGF-1, and IGF-1R were expressed (immunohistochemistry) in all biopsies (18 of 18) of intrahepatic cholangiocarcinoma. ER-α was expressed (Western blot) only by the HuH-28 cell line (intrahepatic cholangiocarcinoma), whereas ER-β, IGF-1, and IGF-1R were expressed in the three cell lines examined. In serum-deprived HuH-28 cells, serum readmission induced stimulation of cell proliferation that was inhibited by ER and IGF-1R antagonists. 17β-Estradiol and IGF-1 stimulated proliferation of HuH-28 cells to a similar extent to that of MCF7 (breast cancer) but greater than that of TFK-1 and Mz-ChA-1, inhibiting apoptosis and exerting additive effects. These effects of 17β-estradiol and IGF-1 were associated with enhanced protein expression of ER-α, phosphorylated (p)-ERK1/2 and pAKT but with decreased expression of ER-β. Finally, transfection of IGF-1R anti-sense oligonucleotides in HuH-28 cells markedly decreased cell proliferation. In conclusion, human intrahepatic cholangiocarcinomas express receptors for estrogens and IGF-1, which cooperate in the modulation of cell growth and apoptosis. Modulation of ER and IGF-1R could represent a strategy for the management of cholangiocarcinoma. Cholangiocarcinoma is a malignant tumor arising from the epithelial cells (cholangiocytes) lining the biliary tree and characterized by a poor prognosis and scarce response to current therapies.1Gores GJ A spotlight on cholangiocarcinoma.Gastroenterology. 2003; 125: 1536-1538Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 2Gores GJ Cholangiocarcinoma: current concepts and insights.Hepatology. 2003; 37: 961-969Crossref PubMed Scopus (255) Google Scholar The incidence and mortality for cholangiocarcinoma are increasing worldwide.3Blendis L Halpern Z An increasing incidence of cholangio-carcinoma: why?.Gastroenterology. 2004; 127: 1008-1009Abstract Full Text Full Text PDF PubMed Google Scholar Estrogens are positive growth modulators for normal and neoplastic cells expressing estrogen receptors (ERs).4Migliaccio A Castoria G Di Domenico M de Falco A Bilancio A Lombardi M Bottero D Varricchio L Nanayakkara M Rotondi A Auricchio F Sex steroid hormones act as growth factors.J Steroid Biochem Mol Biol. 2002; 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83: 31-35Crossref PubMed Scopus (90) Google Scholar, 5Koike S Sakai M Muramatsu M Molecular cloning and characterization of rat estrogen receptor.Nucleic Acids Res. 1987; 15: 2499-2513Crossref PubMed Scopus (469) Google Scholar, 6Kuiper GG Enmark E Pelto-Huikko M Nilsson S Gustafsson JA Cloning of a novel receptor expressed in rat prostate and ovary.Proc Natl Acad Sci USA. 1996; 93: 5925-5930Crossref PubMed Scopus (4270) Google Scholar, 7Mosselman S Polman J Dijkema R ERβ: identification and characterization of a novel human estrogen receptor.FEBS Lett. 1996; 392: 49-53Abstract Full Text PDF PubMed Scopus (2073) Google Scholar The role played by estrogens and their receptors in the growth of ER-positive neoplasms represents the basis for the pharmacological treatment and/or prevention of different cancers (mainly breast cancer) with ER antagonists.8Platet N Cathiard AM Gleizes M Garcia M Estrogens and their receptors in breast cancer progression: a dual role in cancer proliferation and invasion.Crit Rev Oncol Hematol. 2004; 51: 55-67Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 9Thomas T Gallo MA Thomas TJ Estrogen receptors as targets for drug development for breast cancer, osteoporosis and cardiovascular diseases.Curr Cancer Drug Targets. 2004; 4: 483-499Crossref PubMed Scopus (41) Google Scholar We have recently shown that 1) human and rat cholangiocytes express both ER-α and/or ER-β subtypes, 2) estrogens positively modulate cholangiocyte proliferation, and 3) ERs are overexpressed during cholangiocyte proliferation associated with human cholangiopathies.10Alvaro D Invernizzi P Onori P Franchitto A De Santis A Crosignani A Sferra R Ginanni-Corradini S Mancino MG Maggioni M Attili AF Podda M Gaudio E Estrogen receptors in cholangiocytes and the progression of primary biliary cirrhosis.J Hepatol. 2004; 41: 905-912Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 11Alvaro D Alpini G Onori P Perego L Svegliati Baroni G Franchitto A Baiocchi L Glaser SS Le Sage G Folli F Gaudio E Estrogens stimulate proliferation of intrahepatic biliary epithelium in rats.Gastroenterology. 2000; 119: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 12Alvaro D Alpini G Onori P Franchitto A Glaser SS Le Sage G Gigliozzi A Attili AF Gaudio E Effect of ovariectomy on the proliferative capacity of intrahepatic biliary epithelium.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Alvaro D Onori P Drudi Metalli V Svegliati-Baroni G Folli F Franchitto A Alpini G Mancino MG Attili AF Gaudio E Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat.Hepatology. 2002; 36: 297-304Crossref PubMed Scopus (83) Google Scholar Furthermore, studies in rat cholangiocytes indicate that estrogens interact with and potentiate the effect of growth factors on cholangiocyte proliferation.14Gigliozzi A Alpini G Baroni GS Marucci L Metalli VD Glaser SS Francis H Mancino MG Ueno Y Barbaro B Benedetti A Attili AF Alvaro D Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis.Gastroenterology. 2004; 127: 1198-1209Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 1515. Alvaro D, Metalli VD, Alpini G, Onori P, Franchitto A, Barbaro B, Glaser SS, Francis H, Cantafora A, Blotta I, Attili AF, Gaudio E: The intrahepatic biliary epithelium is a target of the growth hormone/insulin-like growth factor 1 axis. J Hepatol 43:875–883Google Scholar Specifically, by interacting at both receptor and postreceptor levels, 17β-estradiol markedly potentiates the proliferating effect of insulin-like growth factor 1 (IGF-1) on isolated rat cholangiocytes.1515. Alvaro D, Metalli VD, Alpini G, Onori P, Franchitto A, Barbaro B, Glaser SS, Francis H, Cantafora A, Blotta I, Attili AF, Gaudio E: The intrahepatic biliary epithelium is a target of the growth hormone/insulin-like growth factor 1 axis. J Hepatol 43:875–883Google Scholar Similar interactions between IGF-1 and estrogens modulate neoplastic cell growth of tumors expressing ERs, which may include breast, ovary, and endometrial cancers.16Helle SI The insulin-like growth factor system in advanced breast cancer.Best Pract Res Clin Endocrinol Metab. 2004; 18: 67-79Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 17Wimalasena J Meehan D Dostal R Foster JS Cameron M Smith M Growth factors interact with estradiol and gonadotropins in the regulation of ovarian cancer cell growth and growth factor receptors.Oncol Res. 1993; 5: 325-337PubMed Google Scholar, 18Hata H Hamano M Watanabe J Kuramoto H Role of estrogen and estrogen-related growth factor in the mechanism of hormone dependency of endometrial carcinoma cells.Oncology. 1998; 55: 35-44Crossref PubMed Scopus (17) Google Scholar Little information exists on the role of estrogens and IGF-1 in the modulation of growth and progression of cholangiocarcinoma. In the present study, we investigated the expression of ER and IGF-1R in human cholangiocarcinoma and human cholangiocarcinoma cell lines and evaluated the role of estrogens and IGF-1 in the modulation of neoplastic cell growth. Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. Media and serum for cell culture were obtained from Life Technologies, Inc. (Gaithersburg, MD). The IGF-1R blocking antibody αIR3 was obtained from Oncogene-DBA (DBA Italia, srl, Segrate, Milan, Italy). We investigated 18 patients (nine females, age 60 to 75 years, and nine males, age 63 to 73 years) with intrahepatic cholangiocarcinoma presenting as a single mass lesion within the liver. In 10 of 18 patients, US-guided liver biopsies were investigated, whereas in 8 of 18 patients (four female, four male) specimens were obtained after surgical resection (four patients) or liver transplantation (four patients). As normal controls, we investigated 10 liver biopsies with a normal histology from patients (five females, age 58 to 69 years, and five males, age 60 to 72 years) submitted to laparotomy. Liver fragments (0.5 cm) were fixed in 10% buffered formalin for 2 to 4 hours and embedded in low-temperature fusion paraffin (55 to 57°C), and 3- to 4-μm sections were stained with hematoxylin and eosin and Masson's trichrome. For immunohistochemistry, sections were mounted on glass slides coated with 0.1% poly-l-lysine. After deparaffination, endogenous peroxidase activity was blocked by a 30-minute incubation in methanolic hydrogen peroxide (2.5%). The endogen biotin was then blocked by Biotin Blocking System (DAKO, code X0590; DAKO, Copenhagen, Denmark) according to the instructions supplied by the vendor. Sections were hydrated in graded alcohol and rinsed in phosphate-buffered saline (PBS, pH 7.4) before applying the primary antibody. Sections were incubated overnight at 4°C with antibodies for cytokeratin 19 (monoclonal antibody CK-19; DAKO), proliferating cellular nuclear antigen (PCNA) (PC10; DAKO), ER-α [a cocktail of three monoclonal antibodies: SC-314, D12, F10 (33% of each); Santa Cruz Biotechnology. Inc., Santa Cruz, CA], ER-β (monoclonal antibody; GenTex, San Antonio, TX), IGF-1 (Santa Cruz), or IGF-1R (Santa Cruz). Samples were then rinsed with PBS for 5 minutes, incubated for 10 minutes at room temperature with secondary biotinylated antibody (DAKO LSAB Plus System; HRP, Milan, Italy), incubated with DAKO ABC (DAKO LSAB Plus System; HRP, Milan, Italy), and finally developed with 3-3′ diaminobenzidine. For all immunoreactions, negative controls were also included. Light microscopy and immunohistochemistry observation were taken by BX-5 1 light microscopy (Olympus, Tokyo, Japan) with a videocam (Spot Insight; Diagnostic Instrument, Inc., Sterling Heights, MI) and processed with an Image Analysis System (IAS; Delta Sistemi, Rome, Italy). Light microscopy and immunohistochemical observations were independently performed by three pathologists in a blind manner. ER-α and ER-β immunohistochemical expression was evaluated as previously described.11Alvaro D Alpini G Onori P Perego L Svegliati Baroni G Franchitto A Baiocchi L Glaser SS Le Sage G Folli F Gaudio E Estrogens stimulate proliferation of intrahepatic biliary epithelium in rats.Gastroenterology. 2000; 119: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 12Alvaro D Alpini G Onori P Franchitto A Glaser SS Le Sage G Gigliozzi A Attili AF Gaudio E Effect of ovariectomy on the proliferative capacity of intrahepatic biliary epithelium.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Alvaro D Onori P Drudi Metalli V Svegliati-Baroni G Folli F Franchitto A Alpini G Mancino MG Attili AF Gaudio E Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat.Hepatology. 2002; 36: 297-304Crossref PubMed Scopus (83) Google Scholar Briefly, six slides were analyzed per each specimen of normal liver or cholangiocarcinoma. Neoplastic or normal cholangiocytes were counted in a random, blinded manner in six nonoverlapping fields (magnification ×20) of each slide and the data expressed as percentage of positive cells. The use of human material has been approved by the local institutional review board. Mz-ChA-1 cells (gallbladder origin)19Knuth A Gabbert H Dippold W Klein O Sachsse W Bitter-Suermann D Prellwitz W Meyer zum Buschenfelde KH Biliary adenocarcinoma. Characterization of three new human tumor cell lines.J Hepatol. 1985; 1: 579-596Abstract Full Text PDF PubMed Scopus (171) Google Scholar were a gift from Dr. J.G. Fitz (University of Texas, Southwest Medical Center, Dallas, TX). HuH-28 (intrahepatic bile duct)20Kusaka Y Tokiwa T Sato J Establishment and characterization of a cell line from a human cholangiocellular carcinoma.Res Exp Med (Berl). 1988; 188: 367-375Crossref PubMed Scopus (45) Google Scholar and TFK-1 (extrahepatic bile duct)21Saijyo S Kudo T Suzuki M Katayose Y Shinoda M Muto T Fukuhara K Suzuki T Matsuno S Establishment of a new extrahepatic bile duct carcinoma cell line, TFK-1.Tohoku J Exp Med. 1995; 177: 61-71Crossref PubMed Scopus (131) Google Scholar cholangiocarcinoma cell lines were acquired from Cancer Cell Repository, Tohoku University, Tohoku, Japan. Mz-ChA-1, TFK-1, and HuH-28 cells were maintained in CRML 1066 medium containing 10% fetal bovine serum. The human HCC cell line Alex (PRF/PLC/5) was a gift from Prof. R. Mazzanti (University of Florence, Florence, Italy) and was maintained in Eagle's minimum essential medium containing 10% fetal bovine serum. The human colon carcinoma SW 480 cell line (a gift from Dr. E. Porfiri, Polytechnic University of Ancona, Ancona, Italy) was cultured in Leibovitiz's L15 medium containing 10% fetal bovine serum. Cell lines, cultured in the appropriate medium containing 10% fetal bovine serum, were deprived of serum for 48 hours. Then, cells were maintained in serum-deprived conditions for an additional 48 hours (controls = C) or exposed to serum, 17β-estradiol, IGF-1, and/or receptor antagonists for an additional 48 hours. Specifically, cell medium was replaced with fresh serum-free or serum-containing medium to which the tested agent was added. 17β-Estradiol and ICI 182,780 were dissolved in dimethyl sulfoxide whereas IGF-1 and αIR3 were dissolved in saline as a stock solution that was then added (dilution, 1:100,000) to serum-free culture medium. In these experimental conditions, proliferation, apoptosis, and immunoblots were evaluated as described below. Cell proliferation was assessed by a commercially available colorimetric cell proliferation assay (CellTiter 96 aqueous nonradioactive cell proliferation assay, MTS kit; Promega, Madison, WI), by following the manufacturer's instructions. Proliferation index was calculated as the ratio (multiplied × 100) between cell numbers in unstimulated and stimulated cultures as described.22Park J Tadlock L Gores GJ Patel T Inhibition of interleukin 6-mediated mitogen-activated protein kinase activation attenuates growth of a cholangiocarcinoma cell line.Hepatology. 1999; 30: 1128-1133Crossref PubMed Scopus (175) Google Scholar In selected experiments, proliferation was also evaluated by PCNA protein expression (Western blot) or by [3H]thymidine incorporation as previously described.23Kanno N LeSage G Phinizy JL Glaser SS Francis H Alpini G Stimulation of α2-adrenergic receptor inhibits cholangiocarcinoma growth through modulation of Raf-1 and B-Raf activities.Hepatology. 2002; 35: 1329-1340Crossref PubMed Scopus (50) Google Scholar In these latter experiments, [3H]thymidine was added into the culture medium (1 μCi/ml) for the last 2 hours of each treatment. Apoptosis was evaluated by a caspase 3 colorimetric assay kit (Sigma Chemical Co.), based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitro-anilide (Ac-DEVD-pNA) by caspase 3, resulting in the release of the p-nitroaniline (pNA). For this assay, cells were lysed in the appropriate lysis buffer provided by the vendor (50 mmol/L HEPES, pH 7.4, 5 mmol/L CHAPS, and 5 mmol/L dithiothreitol). The concentration of the pNA released from the substrate was calculated from the absorbance values at 405 nm. Caspase 3 activity resulting from the measured concentration of pNA [controls (48 hours plus 48 hours serum-free) = 1.66 ± 0.11 μmol pNA/minute/ml] was expressed as percent changes with respect to controls. For Western blot analysis, cells were solubilized in lysis buffer containing 15 mmol/L Tris-HCl (pH 7.4), 5 mmol/L ethylenediaminetetraacetic acid, 100 mmol/L NaCl, 1% Igepal, 2 mmol/L phenylmethyl sulfonyl fluoride, 2 mmol/L benzamidine, and 1% aprotinin on ice for 30 minutes. After centrifugation at 10,000 × g for 20 seconds at 4°C, the supernatant was recovered, and protein concentration was determined with the protein assay-dye reagent (Bio-Rad Laboratories GmbH, Segrate, Milan, Italy). Cell extracts (10 μg) were diluted in 6× LSB (Laemmli sample buffer) containing 0.3 mol/L 2-mercaptoethanol and resolved by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Western blotting was performed as described12Alvaro D Alpini G Onori P Franchitto A Glaser SS Le Sage G Gigliozzi A Attili AF Gaudio E Effect of ovariectomy on the proliferative capacity of intrahepatic biliary epithelium.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Alvaro D Onori P Drudi Metalli V Svegliati-Baroni G Folli F Franchitto A Alpini G Mancino MG Attili AF Gaudio E Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat.Hepatology. 2002; 36: 297-304Crossref PubMed Scopus (83) Google Scholar, 14Gigliozzi A Alpini G Baroni GS Marucci L Metalli VD Glaser SS Francis H Mancino MG Ueno Y Barbaro B Benedetti A Attili AF Alvaro D Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis.Gastroenterology. 2004; 127: 1198-1209Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 1515. Alvaro D, Metalli VD, Alpini G, Onori P, Franchitto A, Barbaro B, Glaser SS, Francis H, Cantafora A, Blotta I, Attili AF, Gaudio E: The intrahepatic biliary epithelium is a target of the growth hormone/insulin-like growth factor 1 axis. J Hepatol 43:875–883Google Scholar by using the following primary antibodies from Santa Cruz: anti-PCNA, specific mouse monoclonal antibody (1:300 dilution); anti-IGF-1, goat polyclonal antibody (1:100 dilution); anti-IGF-1Rb, rabbit polyclonal antibody (1:400 dilution); anti-ER-α rabbit polyclonal antibody (1:200 dilution); anti-ER-β rabbit polyclonal antibody (1:200 dilution); anti-tERK, rabbit polyclonal antibody (1:1000 dilution); anti-pERK, mouse monoclonal antibody (1:800 dilution); anti-AKT, mouse monoclonal antibody (1:200 dilution); and anti-pAKT, rabbit polyclonal antibody (1:300 dilution). As secondary antibodies, anti-mouse IgG peroxidase-conjugated (1:2000; Sigma), anti-rabbit IgG peroxidase-conjugated (1:10,000; Sigma), or anti-goat IgG peroxidase-conjugated (1:10,000; Sigma) antibody was used. The intensity of the bands was determined by scanning video densitometry (Ultra Violet Products, Cambridge, UK) and expressed as arbitrary densitometric units normalized to β-actin expression (ie, tested protein/β-actin × 100). Total cellular RNA was extracted by the Micro-Fast Track II kit (Invitrogen, San Diego, CA) according to the instructions of the vendor. Total RNA (1 μg) was used for first strand cDNA synthesis by AMV reverse transcriptase (Roche Diagnostics, Mannheim, Germany). PCR primers for ER-α, 5′-AAGGAGACTCGCTACTGT-3′ (sense) and 5′-TCAAAGATCTCCACCATGCC-3′ (anti-sense), were based on the published sequence.24Inoue S Hoshino S Miyoshi H Akishita M Hosoi T Orimo H Ouchi Y Identification of a novel isoform of estrogen receptor, a potential inhibitor of estrogen action, in vascular smooth muscle cells.Biochem Biophys Res Com. 1996; 219: 766-772Crossref PubMed Scopus (53) Google Scholar GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as housekeeping gene.25Alpini G Glaser S Robertson W Phinizy JL Rodgers R Caligiuri A LeSage G Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes.Am J Physiol. 1997; 273: G518-G529PubMed Google Scholar Primers were synthesized by Invitrogen. PCR conditions used were as follows: 30 cycles of 1 minute at 94°C, 1 minute at 57°C, and 2 minutes at 72°C. An 18-mer anti-sense phosphorothioate oligonucleotide (S-ODN) targeted against codons 2 to 7 of the prepropeptide IGF-I receptor sequence,26Muller M Dietel M Turzynski A Wiechen K Antisense phosphorothioate oligodeoxynucleotide down-regulation of the insulin-like growth factor 1 receptor in ovarian cancer cells.Int J Cancer. 1998; 77: 567-571Crossref PubMed Scopus (38) Google Scholar, 27Ullrich A Gray A Tam AW Yang-Feng T Tsubokawa M Collins C Henzel W Le Bon T Kathuria S Chen E Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity.EMBO J. 1986; 5: 2503-2512Crossref PubMed Scopus (1642) Google Scholar sense, and mismatch control oligonucleotides were obtained from Invitrogen (Invitrogen S.R.L San Giuliano Milanese, Milan, Italy). The sequences were 5′-TCCTCCGGAGCCAGACTT-3′ (anti-sense), 5′-AAGTCTGGCTCCGGAGGA-3′ (sense), 5′-TGAGCCCTCCTCCGTAGA-3′ (mismatch primer 1) and 5′-CTCTGAGCCAGACGTCTC-3′ (mis-match primer 2). HuH-28 cells were kept in CMRL 1066 medium with penicillin-streptomycin-glutamine 1% + 10% fetal bovine serum (Invitrogen). Per protocol instructions, 1 day before transfection, HuH-28 cells were plated in growth medium (10% fetal bovine serum + 0.5% antibiotics) to obtain 50% confluency at the time of transfection. Phosphorothioate oligonucleotides were transfected into cells using oligofectamine reagent (Invitrogen). Per protocol instructions, the cells were washed two times with serum-free medium and then incubated with S-ODN-oligofectamine solution at 37°C in a CO2 incubator for 4 hours (serum-free medium). Then, a medium containing 30% serum and 1% antibiotics was added to the cells without removing the transfection mixture, and after 2 days (30% serum) the protein expression of IGF-1R and PCNA (proliferation marker) was analyzed. Data are presented as arithmetic mean ± SEs. Statistical analysis was conducted by using one-way analysis of the variance with pair-wise comparison by the Fisher's protected least-significant difference test. In all cases, P < 0.05 was considered significant. Cholangiocytes of intrahepatic bile ducts of normal human liver (n = 10 biopsies) were all negative at the immunohistochemical analysis for ER-α, ER-β, IGF-1, and IGF-1R (Figure 1A and Figure 2A). In contrast, all 18 human intrahepatic cholangiocarcinomas showed a marked positivity for both ER-α and ER-β, which involves 81.0 ± 3.5% and 82.5 ± 3.7% cells, respectively, with a staining located at both the cytoplasmic and nuclear level (Figure 1B). The 18 biopsies of human cholangiocarcinoma showed an intense positivity for both IGF-1 and IGF-1R, which involved 60.8 ± 2.8% and 64.4 ± 3.2% cells, respectively, with staining predominantly located at the cytoplasmic level (Figure 2B).Figure 2Immunohistochemistry for IGF-1 and IGF-1R in human normal liver and cholangiocarcinoma. A: Intrahepatic bile ducts of the normal liver were negative at immunohistochemical analysis for IGF-1 (left) and IGF-1R (right). B: Biopsies of human cholangiocarcinoma showing an intense positivity for both IGF-1 and IGF-1R at cytoplasmatic level (arrows). Light microscopy. Original magnifications, ×20.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Western blot analysis shows (Figure 3A) that ER-α was expressed by the HuH-28 (human intrahepatic) cell line but not by the TFK-1 (human extrahepatic) or Mz-ChA-1 (human gallbladder) cell lines (five independent experiments). In contrast, by qualitative RT-PCR the message for ER-α was also detected in TFK-1 and Mz-ChA-1 cells (Figure 3B). Therefore, Western blot data should be indicative for a very low level of ER-α protein expression in these two cell lines. In comparison with the MCF7 breast cancer cell line used as control, the expression (Western blot) of ER-α was significantly lower (P < 0.05, five independent experiments; Figure 3A) in HuH-28 cells. ER-β was similarly expressed in HuH-28 (intrahepatic) and in MCF7 breast cancer (positive control) cell lines but highly expressed in both TFK-1 (human extrahepatic) and Mz-ChA-1 (human gallbladder) cell line (P < 0.05 versus MCF7 or HuH-28, five independent experiments; Figure 3A). The protein mass of IGF-1 and IGF-1R (five independent experiments; Figure 3A) was similar in HuH-28 (intrahepatic) and TFK-1 (human extrahepatic) cholangiocarcinoma cell lines without significant differences with respect to cell lines derived from human hepatocellular carcinoma (Alex) or human colon carcinoma (SW480) used as positive controls. In contrast, the Mz-ChA-1 (gallbladder) cell line showed a protein mass of IGF-1 and IGF-1R significantly lower (P < 0.01, five independent experiments; Figure 3A) than all of the other cell lines investigated. Because the HuH-28 (intrahepatic) cell line, similar to human intrahepatic cholangiocarcinoma, expresses the protein for both ER-α and ER-β, we mainly focused on this cell line to evaluate the role and mechanism by which estrogens and IGF-1 modulate cell proliferation and apoptosis. For this purpose, HuH-28 cells were deprived of serum for 48 hours, a maneuver that elicited a 65.6 ± 8% decrease of proliferation index (10 independent experiments) and a 45 ± 5% (10 independent experiments) increase of apoptosis. Serum-deprived HuH-28 cells were left without serum (controls) or exposed to serum, 17β-estradiol, IGF-1, and/or receptor antagonists for an additional 48 hours. In controls, 48 + 48 hours serum-free starvation elicited a 75.4 ± 6% (10 independent experiments) decrease of proliferation index and a 54 ± 4% (10 independent experiments) increase of apoptosis with respect to HuH-28 cells cultured in serum-containing media. [3H]Thymidine incorporation decreased from 7560 ± 586 dpm/mg protein to 3050 ± 209 dpm/mg protein after 48 hours (P < 0.01) and to 2035 ± 197 dpm/mg protein after 48 + 48 hours (P < 0.05 versus 48 hours) serum-free starvation (10 independent experiments for each protocol). The significant [3H]thymidine incorporation demonstrates that a certain rate of proliferation still persists in HuH-28 cells starved without serum for 48 or 48 + 48 hours. We also performed flow cytometry analysis of propidium iodide-stained HuH-28 cells (not shown), indicating that after 48 hours of serum-free starvation HuH-28 cells do not completely arrest in G1 phase, but at least 24% of cells are still under proliferation (S phase). When serum-deprived HuH-28 cells were exposed for 48 hours to the ER antagonists tamoxifen (1 μmol/L, 10 independent experiments) and ICI 182,780 (1 μmol/L, 10 independent experiments) or to the IGF-1R blocking antibody αIR3 (1 μg/ml, 10 independent experiments), no significant changes in proliferation index were seen (Figure 4A). Readmission of serum for 48 hours induced a marked increase of proliferation (+67 ± 7%, 10 independent experiments) which was inhibited (P < 0.01) for ∼80% (10 independent experiments) by the two ER antagonists, tamoxifen and ICI 182,780, and to a higher extent (93% inhib

A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche.CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium.CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n=23) and correlated with a high level of serum cancer antigen 19.9 (n=17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n=12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect.CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease.Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.

Hepatic progenitor cells (HPCs) play a major role in liver repair and regeneration. We evaluated HPC involvement in pediatric nonalcoholic fatty liver disease (pNAFLD). Thirty biopsies of consecutive children and adolescents with untreated NAFLD (19 with nonalcoholic steatohepatitis [NASH] and 11 without NASH) were studied using immunohistochemistry and immunofluorescence. HPCs and HPC-expressing adipokines (e.g., adiponectin, resistin, and glucagon-like peptide 1 [GLP-1]) were counted and correlated with steatosis, inflammation, hepatocyte ballooning, fibrosis, and NAFLD activity score (NAS). The HPC compartment was expanded in pNAFLD, especially in children with NASH, and was independently associated with degree of fibrosis (r = 0.303; P = 0.033). NASH livers were also characterized by increased hepatocyte apoptosis, cell-cycle arrest, and an expanded pool of intermediate hepatocytes. Adiponectin expression in HPCs of pNAFLD patients was down-regulated with respect to the healthy liver, and this expression was inversely correlated with NAS score (r = -0.792; P < 0.001) and steatosis (r = -0.769; P < 0.001). Resistin expression in HPCs increased in pNAFLD and was related to degree of fibrosis (r = 0.432; P < 0.05). GLP-1 was overexpressed in HPCs of pNAFLD patients, and GLP-1 expression was related to degree of steatosis (r = 0.577; P < 0.05) and NAS (r = 0.594; P < 0.01).HPC activation is involved in the response of the liver to oxidative stress in pNAFLD and is correlated with fibrosis and the progression toward NASH. HPCs express adiponectin, resistin, and GLP-1, which become available to resident liver cells and are strongly associated with the severity of NAFLD. These results may have important pathophysiological implications in the modulation of hepatic insulin resistance and the progression of liver injury.

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2-4 microm(2), embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl(-)/HCO(3)(-) AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl(-)/HCO(3)(-) exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.

Treatment guidelines recommend a stepwise approach to primary biliary cholangitis: all patients begin treatment with ursodeoxycholic acid (UDCA) monotherapy and those with an inadequate biochemical response after 12 months are subsequently considered for second-line therapies. However, as a result, patients at the highest risk can wait the longest for effective treatment. We determined whether UDCA response can be accurately predicted using pretreatment clinical parameters.We did logistic regression analysis of pretreatment variables in a discovery cohort of patients in the UK with primary biliary cholangitis to derive the best-fitting model of UDCA response, defined as alkaline phosphatase less than 1·67 times the upper limit of normal (ULN), measured after 12 months of treatment with UDCA. We validated the model in an external cohort of patients with primary biliary cholangitis and treated with UDCA in Italy. Additionally, we assessed correlations between model predictions and key histological features, such as biliary injury and fibrosis, on liver biopsy samples.2703 participants diagnosed with primary biliary cholangitis between Jan 1, 1998, and May 31, 2015, were included in the UK-PBC cohort for derivation of the model. The following pretreatment parameters were associated with lower probability of UDCA response: higher alkaline phosphatase concentration (p<0·0001), higher total bilirubin concentration (p=0·0003), lower aminotransferase concentration (p=0·0012), younger age (p<0·0001), longer interval from diagnosis to the start of UDCA treatment (treatment time lag, p<0·0001), and worsening of alkaline phosphatase concentration from diagnosis (p<0·0001). Based on these variables, we derived a predictive score of UDCA response. In the external validation cohort, 460 patients diagnosed with primary biliary cholangitis were treated with UDCA, with follow-up data until May 31, 2016. In this validation cohort, the area under the receiver operating characteristic curve for the score was 0·83 (95% CI 0·79-0·87). In 20 liver biopsy samples from patients with primary biliary cholangitis, the UDCA response score was associated with ductular reaction (r=-0·556, p=0·0130) and intermediate hepatocytes (probability of response was 0·90 if intermediate hepatocytes were absent vs 0·51 if present).We have derived and externally validated a model based on pretreatment variables that accurately predicts UDCA response. Association with histological features provides face validity. This model provides a basis to explore alternative approaches to treatment stratification in patients with primary biliary cholangitis.UK Medical Research Council and University of Milan-Bicocca.

Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)-to-distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG, OCT4, and SOX2), proliferation (Ki67), self-replication (SALL4), 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). Radial-axis lineages start in PBGs near the ducts' fibromuscular layers with stem cells and end at the ducts' lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum-free Kubota's Medium, proliferating for months as undifferentiated cells, whereas pancreas-derived cells underwent only approximately 8-10 divisions, then partially differentiated towards an islet fate. Biliary tree-derived cells proved precursors of pancreas' committed progenitors. Both could be driven by three-dimensional conditions, islet-derived matrix components and a serum-free, hormonally defined medium for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immunocompromised mice, chemically rendered diabetic, resulted in secretion of human C-peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree-derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life-long pancreatic organogenesis.

Primary sclerosing cholangitis (PSC) is characterised by fibro-stenosing strictures involving extrahepatic and/or large intrahepatic bile ducts. Mechanisms leading to bile duct injury are poorly understood. We aimed to study the biliary tree stem cell compartment located in peribiliary glands of extrahepatic and large intrahepatic bile ducts and its role in the pathogenesis of biliary fibrosis in PSC.Specimens containing extrahepatic or large intrahepatic bile ducts were obtained from normal liver (n=6), liver explants from patients with PSC (n=11), and primary biliary cirrhosis (n=6). Specimens were processed for histology, immunohistochemistry and immunofluorescence.In PSC samples, progressive hyperplasia and mucinous metaplasia of peribiliary glands were observed in large ducts with fibrosis, but not in inflamed ducts without fibrosis. Peribiliary gland hyperplasia was associated with progressive biliary fibrosis and the occurrence of dysplastic lesions. Hyperplasia of peribiliary glands was determined by the expansion of biliary tree stem cells, which sprouted towards the surface epithelium. In PSC, peribiliary glands and myofibroblasts displayed enhanced expression of Hedgehog pathway components. Peribiliary glands in ducts with onion skin-like fibrosis expressed epithelial-to-mesenchymal transition traits associated with components of Hedgehog pathway, markers of senescence and autophagy.The biliary tree stem cell compartment is activated in PSC, its activation contributes to biliary fibrosis, and is sustained by the Hedgehog pathway. Our findings suggest a key role for peribiliary glands in the progression of bile duct lesions in PSC and could explain the associated high risk of cholangiocarcinoma.

Cholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13(+) CSCs characterized mixed-intrahepatic, whereas LGR5(+) characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro, CSCs expressing epithelial markers formed a higher number of spheroids than CD13(+) or CD90(+) CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90(+) and CD13(+) cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell-based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers.

Abstract The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs), cancers occurring increasingly in children to young adults, is poorly understood. We present a transplantable tumour line, maintained in immune-compromised mice, and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays, spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model, with parallel studies on 27 primary hFL-HCC tumours, provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple, normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells—newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies.

The secretin/secretin receptor (SR) axis is up‐regulated by proliferating cholangiocytes during cholestasis. Secretin stimulates biliary proliferation by down‐regulation of let‐7a and subsequent up‐regulation of the growth‐promoting factor, nerve growth factor (NGF). It is not known whether the secretin/SR axis plays a role in subepithelial fibrosis observed during cholestasis. Our aim was to determine the role of the secretin/SR axis in activation of biliary fibrosis in animal models and human primary sclerosing cholangitis (PSC). Studies were performed in wild‐type (WT) mice with bile duct ligation (BDL), BDL SR −/− mice, or Mdr2 −/− mouse models of cholestatic liver injury. In selected studies, the SR antagonist (Sec 5‐27) was used to block the secretin/SR axis. Biliary proliferation and fibrosis were evaluated as well as secretion of secretin (by cholangiocytes and S cells), expression of markers of fibrosis, transforming growth factor‐β1 (TGF‐β1), transforming growth factor‐β1 receptor (TGF‐β1R), let‐7a, and downstream expression of NGF. Correlative studies were performed in human control and PSC liver tissue biopsies, serum, and bile. SR antagonist reduced biliary proliferation and hepatic fibrosis in BDL WT and Mdr2 −/− mice. There was decreased expression of let‐7a in BDL and Mdr2 −/− cholangiocytes that was associated with increased NGF expression. Inhibition of let‐7a accelerated liver fibrosis was attributed to cholestasis. There was increased expression of TGF‐β1 and TGF‐β1R. Significantly higher expression of secretin, SR, and TGF‐β1 was observed in PSC patient liver samples compared to healthy controls. In addition, there was higher expression of fibrosis genes and remarkably decreased expression of let‐7a and increased expression of NGF compared to the control. Conclusion : The secretin/SR axis plays a key role in regulating the biliary contribution to cholestasis‐induced hepatic fibrosis. (H epatology 2016;64:865‐879)

The diagnosis of primary sclerosing cholangitis (PSC) is difficult due to the lack of sensitive and specific biomarkers, as is the early diagnosis of cholangiocarcinoma (CC), a complication of PSC. The aim of this study was to identify specific serum miRNAs as diagnostic biomarkers for PSC and CC. The levels of 667 miRNAs were evaluated in 90 human serum samples (30 PSC, 30 CC and 30 control subjects) to identify disease-associated candidate miRNAs (discovery phase). The deregulated miRNAs were validated in an independent cohort of 140 samples [40 PSC, 40 CC, 20 primary biliary cirrhosis (PBC) and 40 controls]. Receiver operating characteristic (ROC) curves were established and only miRNAs with an area under the curve (AUC) > 0·70 were considered useful as biomarkers. In the discovery phase we identified the following: 21 miRNAs expressed differentially in PSC, 33 in CC and 26 in both in comparison to control subjects as well as 24 miRNAs expressed differentially between PSC and CC. After the validation phase, miR-200c was found to be expressed differentially in PSC versus controls, whereas miR-483-5p and miR-194 showed deregulated expression in CC compared with controls. We also demonstrate a difference in the expression of miR-222 and miR-483-5p in CC versus PSC. Combination of these specific miRNAs further improved the specificity and accuracy of diagnosis. This study provides a basis for the use of miRNAs as biomarkers for the diagnosis of PSC and CC.

<h2>ABSTRACT</h2><h3>Background</h3> In Italy, the spread of the COVID-19 pandemic has stressed the entire healthcare system and required a huge re-organization of many Divisions, including those of Gastroenterology. <h3>Aims</h3> to assess the impact of COVID-19 pandemic on Gastroenterology Divisions across Italy. <h3>Methods</h3> All members of the Italian Society of Gastroenterology (SIGE) were invited to answer a web-based survey. <h3>Results</h3> Data of 121 hospitals from all 20 Italian regions were analyzed. Overall, 10.7% Gastroenterology Divisions have been converted to Covid Units. Outpatients consultations, endoscopic and ultrasound procedures were limited to urgencies and oncology indications in 85.1%, 96.2% and 72.2% of Units, respectively, and 46.7% of them suspended the screening for colorectal cancer. Moreover, 72.2% of the staff received a training for use of personal protective equipment, although 45.5% did not have sufficient devices for adequate replacement. Overall, 132 healthcare workers in 41 Gastroenterology Divisions were found to be infected. <h3>Conclusion</h3> This is the first study to evaluate, at a country level, the impact of COVID-19 outbreak on Gastroenterology Divisions. Substantial changes of practice and reduction of procedures have been recorded in the entire country. The long-term impact of such modifications is difficult to estimate but potentially very risky for many digestive diseases.

The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis.Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice.Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression.THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA.Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) with liver metastases is perceived to have a poor prognosis, but the American Joint Committee on Cancer (AJCC) classifies them as early stage in the absence of lymph nodes or extrahepatic spread.Patients with iCCA from the European Network for the Study of Cholangiocarcinoma (ENS-CCA) and Surveillance, Epidemiology, and End Results (SEER) registries with survival/staging (AJCC v.7) data were eligible. Modified staging was used (mAJCC v.7): group A: stages I-III (excluding T2bN0); group B: stage IVa (excluding T2bN1M0); group C: liver metastases (T2bN0/1); and group D: stage IVb (extrahepatic metastases). Survival analysis (Kaplan-Meier and Cox regression) was performed in an ENS-CCA training cohort (TC) and findings internally (ENS-CCA iVC) and externally (SEER) validated. The aim was to assess whether liver metastases (group C) had a shorter survival compared to other early stages (group A) to propose a modified version of AJCC v.8 (mAJCC v.8). A total of 574 and 4,171 patients from the ENS-CCA and SEER registries were included. Following the new classification, 19.86% and 17.31% of patients from the ENS-CCA and SEER registries were reclassified into group C, respectively. In the ENS-CCA TC, multivariable Cox regression was adjusted for obesity (p = 0.026) and performance status (P < 0.001); patients in group C (HR, 2.53; 95% CI, 1.18-5.42; P = 0.017) had a higher risk of death (vs. group A). Findings were validated in the ENS-CCA iVC (HR, 2.93; 95% CI, 2.04-4.19; P < 0.001) and in the SEER registry (HR, 1.88; 95% CI, 1.68-2.09; P < 0.001).iCCA with liver metastases has a worse outcome than other early stages of iCCA. Given that AJCC v.8 does not take this into consideration, a modification of AJCC v.8 (mAJCC v.8), including "liver metastases: multiple liver lesions, with or without vascular invasion" as an "M1a stage," is suggested.

Obeticholic acid (OCA) is the second-line treatment approved for patients with primary biliary cholangitis (PBC) and an inadequate response or intolerance to ursodeoxycholic acid. We aimed to evaluate the effectiveness and safety of OCA under real-world conditions.Patients were recruited into the Italian PBC Registry, a multicentre, observational cohort study that monitors patients with PBC at national level. The primary endpoint was the biochemical response according to Poise criteria; the secondary endpoint was the biochemical response according to normal range criteria, defined as normal levels of bilirubin, alkaline phosphatase (ALP), and alanine aminotransferase (ALT) at 12 months. Safety and tolerability were also assessed.We analysed 191 patients until at least 12 months of follow-up. Median age was 57 years, 94% female, 61 (32%) had cirrhosis, 28 (15%) had histologically proven overlap with autoimmune hepatitis (PBC-AIH). At 12 months, significant median reductions of ALP (-32.3%), ALT (-31.4%), and bilirubin (-11.2%) were observed. Response rates were 42.9% according to Poise criteria, and 11% by normal range criteria. Patients with cirrhosis had lower response than patients without cirrhosis (29.5% vs. 49.2%, p = 0.01), owing to a higher rate of OCA discontinuation (30% vs. 12%, p = 0.004), although with similar ALP reduction (29.4% vs. 34%, p = 0.53). Overlap PBC-AIH had a similar response to pure PBC (46.4% vs. 42.3%, p = 0.68), with higher ALT reduction at 6 months (-38% vs. -29%, p = 0.04). Thirty-three patients (17%) prematurely discontinued OCA because of adverse events, of whom 11 experienced serious adverse events. Treatment-induced pruritus was the leading cause of OCA discontinuation (67%).Effectiveness and safety of OCA under real-world conditions mirror those in the Poise trial. Patients with cirrhosis had lower tolerability. Overlap PBC-AIH showed higher ALT reduction at 6 months compared with patients with pure PBC.Obeticholic acid (OCA) was shown to be effective in more than one-third of patients not responding to ursodeoxycholic acid in a real-world context in Italy. Patients with cirrhosis had more side effects with OCA, and this led to suspension of the drug in one-third of patients. OCA was also effective in patients who had overlap between autoimmune hepatitis and primary biliary cholangitis.

Dermatologic TherapyVolume 34, Issue 6 e15153 SHORT REPORTOpen Access Cutaneous adverse reactions after COVID-19 vaccines in a cohort of 2740 Italian subjects: An observational study Teresa Grieco, Teresa Grieco Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorPatrizia Maddalena, Patrizia Maddalena Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorAlvise Sernicola, Corresponding Author Alvise Sernicola [email protected] orcid.org/0000-0002-1985-0326 Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, Italy Correspondence Alvise Sernicola, Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, Italy. Email: [email protected]Search for more papers by this authorRovena Muharremi, Rovena Muharremi Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorStefania Basili, Stefania Basili Department of Translational and Precision Medicine, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorDomenico Alvaro, Domenico Alvaro Department of Translational and Precision Medicine, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorRoberto Cangemi, Roberto Cangemi Department of Translational and Precision Medicine, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorAlfredo Rossi, Alfredo Rossi Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorGiovanni Pellacani, Giovanni Pellacani Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this author Teresa Grieco, Teresa Grieco Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorPatrizia Maddalena, Patrizia Maddalena Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorAlvise Sernicola, Corresponding Author Alvise Sernicola [email protected] orcid.org/0000-0002-1985-0326 Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, Italy Correspondence Alvise Sernicola, Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, Italy. Email: [email protected]Search for more papers by this authorRovena Muharremi, Rovena Muharremi Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorStefania Basili, Stefania Basili Department of Translational and Precision Medicine, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorDomenico Alvaro, Domenico Alvaro Department of Translational and Precision Medicine, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorRoberto Cangemi, Roberto Cangemi Department of Translational and Precision Medicine, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorAlfredo Rossi, Alfredo Rossi Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this authorGiovanni Pellacani, Giovanni Pellacani Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, "Sapienza" University of Rome, Rome, ItalySearch for more papers by this author First published: 07 October 2021 https://doi.org/10.1111/dth.15153Citations: 19AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract An in-depth characterization of the incidence, morphology, and onset of COVID-19-vaccines cutaneous adverse reactions is currently lacking. The existing literature on COVID-19 vaccination-related cutaneous adverse reactions largely focused on messenger RNA vaccines and mainly included type 1 hypersensitivity reactions, such as urticaria and angioedema. Other cutaneous manifestations are still poorly characterized and have been classified as delayed hypersensitivity rash. Our prospective observational study on a sample of 2740 subjects who underwent the COVID-19 vaccination aimed at defining the prevalence of cutaneous adverse reactions and at identifying their timing of onset and their correlation with the administered dose. Vaccine-related cutaneous adverse reactions occurred in 50 subjects. Patients were asked to complete a questionnaire on the type of COVID-19 vaccine received, the time of onset of cutaneous reactions, and the dates of administration. Out of 2740 individuals who received the COVID-19 vaccination, 50 were diagnosed with cutaneous adverse reactions to vaccine, after the first dose in 28 patients, after the second in 20, and after both in two. We reported localized injection site erythema in 12 patients and generalized cutaneous reactions in 38 patients. Our study shows that cutaneous adverse reactions to COVID-19 vaccination are not common and most often occur after the first dose, recurring infrequently after the second dose. These reactions are usually easily manageable and, even in severe generalized cases, oral antihistamines and corticosteroids were sufficient for resolution. Therefore, except for immediate hypersensitivity reactions, cutaneous adverse reactions do not represent a contraindication to the completion of the vaccination cycle. 1 INTRODUCTION Global concern for the COVID-19 outbreak determined the urgency to develop effective vaccines. The first COVID-19 vaccine, Comirnaty™, was granted conditional marketing approval (CMA) on December 21, 2020.1 Authorization for Spikevax™ (previously COVID-19 Vaccine Moderna), for Vaxzevria™ (previously COVID-19 Vaccine AstraZeneca) and for COVID-19 Vaccine Janssen followed on January 6, 2021, January 29, 2021 and March 11, 2021, respectively.2, 3 Vaccination schedules involve a single dose for COVID-19 Vaccine Janssen and two doses administered via intramuscular route for the other vaccines; the interval between doses is 21 days for Comirnaty™, 28 days for Spikevax™, and 12 weeks for Vaxzevria™. During the first phase of vaccination, priority was given to healthcare providers and elderly people. Currently, pivotal phase 3 randomized placebo-controlled trials provided data on the safety of the abovementioned vaccines, including reports of local injection-site reactions, known as "COVID-19 vaccine arm" and consisting in erythema, edema, and pain to the site of injection.4 The existing literature on COVID-19 vaccination-related cutaneous adverse reactions (CARs) has largely focused on mRNA vaccines (Comirnaty™ and Spikevax™)5 and mainly included type 1 hypersensitivity reactions, such as urticaria and angioedema. Other cutaneous manifestations are still poorly characterized and have been classified as delayed hypersensitivity rash.6, 7 Except for the recent American Registry systematic report of 414 CARs,7 a deeper characterization of the incidence, morphology, and onset of COVID-19-vaccines CARs is lacking.5, 8 The objective of our study was, first, to define the prevalence of CARs in our sample, second, to identify the timing of reactions and their correlation with the administered doses (first, second, or both). A third objective was to characterize the cutaneous symptoms and, finally, to report our management of COVID-19 vaccine adverse reactions. 2 MATERIALS AND METHODS From January to July 2021, we conducted a prospective observational study on a total of 2740 subjects who underwent the COVID-19 vaccination and provided informed consent to use of their details. 2040 (74%) were health care workers and 700 (26%) were patients referring to the Department of Dermatology and Allergology of Policlinico Umberto I Hospital-Sapienza University of Rome. 2481 (91%) of subjects received Comirnaty™, 222 (8%), Vaxzevria™, and 37 (1%) Spikevax™. No subject was administered COVID-19 vaccine Janssen that was locally unavailable at the time of the study. Vaccine-related CARs occurred in 50 subjects; a thorough skin examination was performed in all patients; in seven cases histology and immunohistochemistry were also carried out to better define the diagnosis. In addition, patients were asked to complete a questionnaire on the type of COVID-19 vaccine received, the time of onset of cutaneous reactions, and the dates of administration. Comorbidities and concomitant therapies were also investigated. Serology and blood tests were carried out to investigate autoimmunity and inflammatory status. 3 RESULTS Out of 2740 individuals who received the COVID-19 vaccination during our observation period, 50 were diagnosed with CARs to vaccine, corresponding to an incidence of 31.28 cases/1000 people–year. Of these, 20 (40%) were males and 30 (60%) were females. Mean patients age was 47 (range: 22–76), and all patients were Caucasian. Thirty patients (60%) had received Comirnaty™, 16 (32%) Vaxzevria™, and 4 (8%) Spikevax™. In 28 patients (56%) CARs followed the first dose administration; in 20 patients (40%) and two patients (4%) CARs followed the second dose administration or both doses, respectively. Out of 28 patients manifesting CARs after the first dose, eight (29%) had received Comirnaty™, 16 (57%) Vaxzevria™, and four (14%) Spikevax™. Patients manifesting CARs either after the second dose or after both doses had received Comirnaty™ (Table 1). TABLE 1. Demographic and clinical characteristics of 50 patients developing CARs, out of 2740 observed subjects that received COVID-19 vaccination (prevalence 1.8%) Age 22;76 years (mean = 47) Sex Female: 30 (60%) Male: 20 (40%) Vaccine Comirnaty™ 30 (60%) Vaxzevria™ 16 (32%) Spikevax™ 4 (8%) Onset After 1st dose 28 (56%): Comirnaty™: 8 (16%) Vaxzevria™: 16 (32%) Spikevax™: 4 (8%) After 2nd dose 20 (40%): Comirnaty™: 20 (100%) After 1st and 2nd dose 2 (4%): Comirnaty™: 2 (100%) Latency <7 days 34 (68%) 7–14 days 13 (26%) >14 days 3 (6%) Comorbidities Hashimoto's thyroiditis 6 (12%) Anaphylaxis and previous ADR to vaccines 4 (8%) Bronchial asthma 2 (4%) Chronic kidney disease 1 (2%) Coronaropathy 1 (2%) Total 14 (28%) Abbreviations: ADR, adverse drug reaction; CAR, cutaneous adverse reaction. CARs occurred within 7 days from vaccine administration in 34 patients (68%), within 7–14 days in 13 patients (26%) and after more than 14 days in three patients (6%). They consisted of localized injection-site erythema in 12 patients (24%) and generalized cutaneous reactions in 38 patients (76%); urticarial rashes and/or angioedema (n = 14; 28%), generalized pruritus (n = 5; 10%), toxic erythema (n = 4; 8%), erythema multiforme (n = 3; 6%), pityriasis rosea-like eruption (n = 3; 6%), Stevens-Johnson syndrome (n = 1; 2%), morbilliform drug exanthema (n = 1; 2%), lymphomatoid drug eruption resembling PLEVA (n = 1; 2%), erythema nodosum (n = 1; 2%), late onset atopic dermatitis (n = 1; 2%), annular lichen planus (n = 1; 2%), pseudo-chilblain relapsing with necrotic features at the second dose (n = 1; 2%), filler injection-site reaction (n = 1; 2%), and genital fixed drug eruption (n = 1; 2%) (Table 2). TABLE 2. Characterization of cutaneous adverse reactions to COVID-19 vaccines. The timepoints after vaccination and related vaccine doses are reported for each reaction CARs Frequency Timepoint (days) Dose Urticarial rash/angioedema 14 (28%) <7, 7–14 1,2 Local injection site erythema 12 (24%) <7,7–14 1,2 Generalized pruritus 5 (10%) 7–14 2 Toxic erythema 4 (8%) <7 2 Erythema multiforme 3 (6%) 7–14 1 Pityriasis rosea-like eruption 3 (6%) >14 1 Other 9 (18%) <7, 7–14 1, 2 Note: Other includes Stevens-Johnson syndrome, morbilliform drug exanthema, lymphomatoid drug reaction resembling PLEVA, erythema nodosum, late onset atopic dermatitis, annular lichen planus, pseudo-chilblains, filler injection site reaction, and genital fixed drug eruption. Abbreviation: CAR, cutaneous adverse reaction. Anamnesis revealed that 14 patients (28%) had comorbidities: Hashimoto's thyroiditis (six patients), past episodes of anaphylaxis and previous adverse reactions to other vaccines (four patients), chronic kidney disease (one patient), coronaropathy (one patient), and bronchial asthma (two patients). No patients reported using anti-inflammatory drugs after vaccine administration. Accurate history obtained from each case allowed us to rule out other plausible causes for the observed reactions. This led us to exclude a case of thoracic herpes zoster in an elderly female patient following the second dose of Comirnaty™: herpes zoster is commonly observed in the elderly and the relationship to the vaccine could not be demonstrated. 4 DISCUSSION Our results showed that CARs related to COVID-19 vaccines Comirnaty™, Spikevax™, and Vaxzevria™ are not very common, occurring in about 1.8% of individuals. A limitation of our study was that the majority of the population sample included health care workers; the incidence of CARs—especially local injection-site reactions—may have been underestimated, due to many physicians not seeking dermatological consultation in the presence of mild cutaneous reactions. Patients manifesting CARs were mainly females (60%); however, this may be due to selection bias, since females made up 60% of our population sample. Moreover, age of our sample was not very high, as only 6.5% of subjects were aged above 65 years. CARs were chiefly caused by Comirnaty™ (60%), followed by Vaxzevria™ (32%) and Spikevax™ (8%). These percentages are influenced by the prevalence of vaccines administered in our study group; 91% of subjects, including all healthcare workers and elderly vaccinated in our hospital, received Comirnaty™, while 8% and 1% were administered Vaxzevria™ and Spikevax™, respectively. The most common generalized CARs were angioedema and urticarial rashes (28%) that in one patient presented with peculiar involvement of flexural areas and significant photosensitivity. Persistent generalized pruritus without any cutaneous manifestation (10%) and toxic erythema (8%), which is a typical drug-related rash,9 were also frequent. Less common manifestations consisted in pityriasis rosea-like eruption,10 erythema multiforme, Stevens-Johnson syndrome, morbilliform drug exanthema, lymphomatoid drug reaction resembling PLEVA,11 erythema nodosum, annular lichen planus, genital fixed drug eruption, pseudo-chilblain, and filler injection-site reaction. Interestingly, one 60-year-old patient presented with a generalized eczematous eruption suggestive of late onset atopic dermatitis.12 Of note, CARs mainly followed the first dose administration (60%), with recurrences after the second dose in only 4% cases; this should encourage patients developing cutaneous reactions after the first dose to complete the vaccination cycle. Immediate hypersensitivity reactions, occurring within 4 h after first dose administration, represent a contraindication to the second dose.13 None of our patients developed this type of reactions, so they all completed the vaccination cycle. In conclusion, our study shows that CARs related to COVID-19 vaccination are not very common and most often occur after the first dose administration. Furthermore, they infrequently recur after the second dose. CARs are usually easily manageable; in severe generalized CARs oral therapy with antihistamines and low dose corticosteroids was sufficient for the resolution of manifestations. None of our patients required hospitalization. Therefore, except for immediate hypersensitivity reactions,14 CARs do not represent a contraindication to the completion of the vaccination cycle.15 ACKNOWLEDGEMENT Open Access Funding provided by Universita degli Studi di Roma La Sapienza within the CRUI-CARE Agreement. [Correction added on May 23, 2022, after first online publication: CRUI funding statement has been added.] CONFLICT OF INTEREST The authors have no competing interests to disclose. AUTHOR CONTRIBUTIONS Teresa Grieco was responsible for conceptualization and writing of the original draft; Patrizia Maddalena, Alvise Sernicola, and Rovena Muharremi were responsible for investigation and writing of the original draft; Stefania Basili, Domenico Alvaro, Roberto Cangemi were responsible for investigation and conceptualization; Alfredo Rossi was responsible for investigation and supervision; Giovanni Pellacani was responsible for supervision and project administration. All authors contributed to review and editing of the manuscript. Open Research DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. REFERENCES 1Polack FP, Thomas SJ, Kitchin N, et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med. 2020; 383: 2603- 2615. 2Baden LR, El Sahly HM, Essink B, et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med. 2021; 384: 403- 416. 3Cavaleri M, Enzmann H, Straus S, Cooke E. The European medicines Agency's EU conditional marketing authorisations for COVID-19 vaccines. Lancet. 2021; 397: 355- 357. 4Johnston MS, Galan A, Watsky KL, Little AJ. Delayed localized hypersensitivity reactions to the Moderna COVID-19 vaccine. JAMA Dermatol. 2021; 157: 716- 720. 5Farinazzo E, Ponis G, Zelin E, et al. Cutaneous adverse reactions after m-RNA COVID-19 vaccine: early reports from Northeast Italy. J Eur Acad Dermatol Venereol. 2021; 35(9): e548– e551. 6Alpalhão M, Maia-Silva J, Filipe P. Severe acute respiratory syndrome coronavirus 2 vaccines and cutaneous adverse reactions: a review. Dermatitis. 2021; 32: 133- 139. 7McMahon DE, Amerson E, Rosenbach M, et al. Cutaneous reactions reported after Moderna and Pfizer COVID-19 vaccination: a registry-based study of 414 cases. J Am Acad Dermatol. 2021; 85: 46- 55. 8Larson V, Seidenberg R, Caplan A, et al. Clinical and histopathological spectrum of delayed adverse cutaneous reactions following COVID -19 vaccination. J Cutan Pathol. 2021. https://doi.org/10.1111/cup.14104 9Pulitzer MP, Nolan KA, Oshman RG, Phelps RG. CD30+ lymphomatoid drug reactions. Am J Dermatopathol. 2013; 35: 343- 350. 10Cyrenne BM, Al-Mohammedi F, DeKoven JG, Alhusayen R. Pityriasis rosea-like eruptions following vaccination with BNT162b2 mRNA COVID-19 vaccine. J Eur Acad Dermatol Venereol. 2021; 35(9): e546– e548. 11Bowers S, Warshaw EM. Pityriasis lichenoides and its subtypes. J Am Acad Dermatol. 2006; 55: 557- 572. 12Chello C, Carnicelli G, Sernicola A, et al. Atopic dermatitis in the elderly Caucasian population: diagnostic clinical criteria and review of the literature. Int J Dermatol. 2020; 59: 716- 721. 13Kounis NG, Koniari I, de Gregorio C, et al. Allergic reactions to current available COVID-19 vaccinations: pathophysiology, causality, and therapeutic considerations. Vaccine. 2021; 9: 221. 14Restivo V, Candore G, Barrale M, et al. Allergy to polyethilenglicole of anti-SARS CoV2 vaccine recipient: a case report of young adult recipient and the management of future exposure to SARS-CoV2. Vaccine. 2021; 9: 412. 15Stingeni L, Bianchi L, Zalaudek I, et al. Adverse cutaneous and mucous reactions from anti SARS-CoV-2 vaccines: recommendations from the Italian Society of Dermatology (SIDeMaST). Ital J Dermatol Venereol. 2021; 156: 115- 117. Citing Literature Volume34, Issue6November/December 2021e15153 ReferencesRelatedInformation

Cholangiocarcinoma (CCA) is a malignant tumour arising from the biliary system. In Europe, this tumour frequently presents as a sporadic cancer in patients without defined risk factors and is usually diagnosed at advanced stages with a consequent poor prognosis. Therefore, the identification of biomarkers represents an utmost need for patients with CCA. Numerous studies proposed a wide spectrum of biomarkers at tissue and molecular levels. With the present paper, a multidisciplinary group of experts within the European Network for the Study of Cholangiocarcinoma discusses the clinical role of tissue biomarkers and provides a selection based on their current relevance and potential applications in the framework of CCA. Recent advances are proposed by dividing biomarkers based on their potential role in diagnosis, prognosis and therapy response. Limitations of current biomarkers are also identified, together with specific promising areas (ie, artificial intelligence, patient-derived organoids, targeted therapy) where research should be focused to develop future biomarkers.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA. Preclinical research is required to improve our understanding of cholangiocarcinoma (CCA). In this Consensus Statement, a task force of experts provides recommendations on the criteria for preclinical models of CCA to increase disease understanding and help to develop novel therapeutic approaches.

Intrahepatic cholangiocarcinoma is the second most frequent primary liver cancer after hepatocellular carcinoma. According to International Classification of Diseases-11 (ICD-11), intrahepatic cholangiocarcinoma is identified by a specific diagnostic code, different with respect to perihilar-CCA or distal-CCA. Intrahepatic cholangiocarcinoma originates from intrahepatic small or large bile ducts including the second-order bile ducts and has a silent presentation that combined with the highly aggressive nature and refractoriness to chemotherapy contributes to the alarming increasing incidence and mortality. Indeed, at the moment of the diagnosis, less than 40% of intrahepatic cholangiocarcinoma are suitable of curative surgical therapy, that is so far the only effective treatment. The main goals of clinicians and researchers are to make an early diagnosis, and to carry out molecular characterization to provide the patient with personalized treatment. Unfortunately, these goals are not easily achievable because of the heterogeneity of this tumor from anatomical, molecular, biological, and clinical perspectives. However, recent progress has been made in molecular characterization, surgical treatment, and management of intrahepatic cholangiocarcinoma and, this article deals with these advances.

We investigated the expression of estrogen receptor (ER) alpha and beta subtypes in cholangiocytes of normal and bile duct-ligated (BDL) rats and evaluated the role and mechanisms of estrogens in the modulation of cholangiocyte proliferation.ER-alpha and ER-beta were analyzed by immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blotting in normal and BDL rats. The effects of the ER antagonists tamoxifen and ICI 182,780 on cholangiocyte proliferation were evaluated.Cholangiocytes expressed both ER-alpha and ER-beta subtypes, whereas hepatocytes expressed only ER-alpha. In association with a marked cholangiocyte proliferation and with enhanced estradiol serum levels, the immunoreactivity for ER-alpha involved a 3-fold higher percentage of cholangiocytes in 3-week BDL than in normal rats; immunoreactivity for ER-beta showed a 30-fold increase. Western blot analysis showed that during BDL, the total amount of ER-beta in cholangiocytes was markedly increased (5-fold), whereas that of ER-alpha decreased slightly (-25%). Treatment with tamoxifen or ICI 182,780 of 3-week BDL rats inhibited cholangiocyte proliferation and induced overexpression of Fas antigen and apoptosis in cholangiocytes. In vitro, 17 beta estradiol stimulated proliferation of cholangiocyte, an effect blocked to the same extent by tamoxifen or ICI 182,780.This study suggests that estrogens and their receptors play a role in the modulation of cholangiocyte proliferation.

To investigate the role of the cholinergic system in regulation of cholangiocyte functions, we evaluated the effects of vagotomy on cholangiocyte proliferation and secretion in rats that underwent bile duct ligation (BDL rats).After bile duct ligation (BDL), the vagus nerve was resected; 7 days later, expression of M3 acetylcholine receptor was evaluated. Cholangiocyte proliferation was assessed by morphometry and measurement of DNA synthesis. Apoptosis was evaluated by light microscopy and annexin-V staining. Ductal secretion was evaluated by measurement of secretin-induced choleresis, secretin receptor (SR) gene expression, and cyclic adenosine 3',5'-monophosphate (cAMP) levels.Vagotomy decreased the expression of M3 acetylcholine receptors in cholangiocytes. DNA synthesis and ductal mass were markedly decreased, whereas cholangiocyte apoptosis was increased by vagotomy. Vagotomy decreased ductal secretion. Forskolin treatment prevented the decrease in cAMP levels induced by vagotomy, maintained cholangiocyte proliferation, and decreased cholangiocyte apoptosis caused by vagotomy in BDL rats. Cholangiocyte secretion was also maintained by forskolin.Vagotomy impairs cholangiocyte proliferation and enhances apoptosis, leading to decreased ductal mass in response to BDL. Secretin-induced choleresis of BDL rats was virtually eliminated by vagotomy in association with decreased cholangiocyte cAMP levels. Maintenance of cAMP levels by forskolin administration prevents the effects of vagotomy on cholangiocyte proliferation, apoptosis, and secretion.

HepatologyVolume 31, Issue 3 p. 555-561 Concise ReviewFree Access Functional heterogeneity of the intrahepatic biliary epithelium Noriatsu Kanno, Noriatsu Kanno Medical Physiology, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TXSearch for more papers by this authorGene LeSage, Gene LeSage Department of Internal Medicine, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TXSearch for more papers by this authorShannon Glaser, Shannon Glaser Division of Research and Education, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TXSearch for more papers by this authorDomenico Alvaro, Domenico Alvaro Division of Gastroenterology, University of Rome, “La Sapienza,” Rome, ItalySearch for more papers by this authorAssistant Professor Gianfranco Alpini Ph.D., Corresponding Author Assistant Professor Gianfranco Alpini Ph.D. falpini@mailbox.sw.org Department of Internal Medicine, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TX Medical Physiology, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TX Central Texas Veterans Health Care System, Temple, TXInternal Medicine and Medical Physiology, The Texas A & M University System Health Science Center, College of Medicine and Central Texas Veterans Health Care System, 1901 South 1st Street, Bldg. 147, Temple, TX 76504. fax: 254-771-5725===Search for more papers by this author Noriatsu Kanno, Noriatsu Kanno Medical Physiology, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TXSearch for more papers by this authorGene LeSage, Gene LeSage Department of Internal Medicine, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TXSearch for more papers by this authorShannon Glaser, Shannon Glaser Division of Research and Education, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TXSearch for more papers by this authorDomenico Alvaro, Domenico Alvaro Division of Gastroenterology, University of Rome, “La Sapienza,” Rome, ItalySearch for more papers by this authorAssistant Professor Gianfranco Alpini Ph.D., Corresponding Author Assistant Professor Gianfranco Alpini Ph.D. falpini@mailbox.sw.org Department of Internal Medicine, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TX Medical Physiology, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, TX Central Texas Veterans Health Care System, Temple, TXInternal Medicine and Medical Physiology, The Texas A & M University System Health Science Center, College of Medicine and Central Texas Veterans Health Care System, 1901 South 1st Street, Bldg. 147, Temple, TX 76504. fax: 254-771-5725===Search for more papers by this author First published: 30 December 2003 https://doi.org/10.1002/hep.510310302Citations: 111AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume31, Issue3March 2000Pages 555-561 ReferencesRelatedInformation

Background & Aims: The biliary tree is the target of cholangiopathies that are chronic cholestatic liver diseases characterized by loss of proliferative response and enhanced apoptosis of cholangiocytes, the epithelial cells lining the biliary tree. The endogenous factors that regulate cholangiocyte proliferation are poorly understood. Therefore, we studied the role of the neuroendocrine hormone serotonin as a modulator of cholangiocyte proliferation. Methods: The presence of the serotonin 1A and 1B receptors on cholangiocytes was evaluated. We then tested whether the activation of such receptors by the administration of the selective agonists modifies cholangiocyte proliferation and functional activity both in vivo and in vitro. In addition, the intracellular signal mediating the serotonin receptor action in cholangiocytes was characterized. We studied the expression and secretion of serotonin by cholangiocytes and the effects of the neutralization of the secreted hormone on the growth of the biliary tree. Results: Cholangiocytes express the serotonin 1A and 1B receptors. Their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis. Such changes are mediated by enhanced d-myo-inositol 1,4,5-triphosphate/Ca2+/protein kinase C signaling and the consequent inhibition of the adenosine 3′,5′-cyclic monophosphate/protein kinase A/Src/extracellular signal-regulated kinase 1/2 cascade. Cholangiocytes secrete serotonin, the blockage of which enhances cholangiocyte proliferation in the course of cholestasis. Conclusions: We observed the existence of an autocrine loop based on serotonin that limits the growth of the biliary tree in the course of chronic cholestasis. Our novel findings might open new approaches for the management of cholangiopathies. Background & Aims: The biliary tree is the target of cholangiopathies that are chronic cholestatic liver diseases characterized by loss of proliferative response and enhanced apoptosis of cholangiocytes, the epithelial cells lining the biliary tree. The endogenous factors that regulate cholangiocyte proliferation are poorly understood. Therefore, we studied the role of the neuroendocrine hormone serotonin as a modulator of cholangiocyte proliferation. Methods: The presence of the serotonin 1A and 1B receptors on cholangiocytes was evaluated. We then tested whether the activation of such receptors by the administration of the selective agonists modifies cholangiocyte proliferation and functional activity both in vivo and in vitro. In addition, the intracellular signal mediating the serotonin receptor action in cholangiocytes was characterized. We studied the expression and secretion of serotonin by cholangiocytes and the effects of the neutralization of the secreted hormone on the growth of the biliary tree. Results: Cholangiocytes express the serotonin 1A and 1B receptors. Their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis. Such changes are mediated by enhanced d-myo-inositol 1,4,5-triphosphate/Ca2+/protein kinase C signaling and the consequent inhibition of the adenosine 3′,5′-cyclic monophosphate/protein kinase A/Src/extracellular signal-regulated kinase 1/2 cascade. Cholangiocytes secrete serotonin, the blockage of which enhances cholangiocyte proliferation in the course of cholestasis. Conclusions: We observed the existence of an autocrine loop based on serotonin that limits the growth of the biliary tree in the course of chronic cholestasis. Our novel findings might open new approaches for the management of cholangiopathies. The intrahepatic biliary tree is the target of several human diseases defined as cholangiopathies,1Alpini G. McGill J.M. LaRusso N.F. The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (126) Google Scholar characterized by chronic cholestasis leading to liver failure.1Alpini G. McGill J.M. LaRusso N.F. The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (126) Google Scholar, 2Desmet V.J. Vanishing bile duct disorders.Prog Liver Dis. 1992; 10: 89-121PubMed Google Scholar Recent studies have shown that such disorders are responsible for more than 20% of the liver transplantations among adults and for the 50% among pediatric patients in the United States.3Annual report of the U.S. Organ Procurement and Transplantation Network and the Scientific Registry for Transplant Recipients. 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Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms.Hepatology. 2000; 32: 17-25Crossref PubMed Scopus (90) Google Scholar Cholangiocyte proliferation and the consequent growth of the intrahepatic biliary tree are coupled with enhanced secretin-induced choleresis and intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) synthesis.9Alpini G. Lenzi R. Sarkozi L. Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Crossref PubMed Scopus (288) Google Scholar, 11Kanno N. LeSage G. Glaser S. Alpini G. Regulation of cholangiocyte bicarbonate secretion.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G612-G625PubMed Google Scholar, 12Glaser S. Rodgers R. Phinizy J.L. Robertson W. Lasater J. Caligiuri A. Tretjak Z. LeSage G. Alpini G. Gastrin inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes.Am J Physiol Gastrointest Liver Physiol. 1997; 273: G1061-G1070Google ScholarThe neuroendocrine system plays a relevant role in the modulation of cholangiocyte biology.10Glaser S. Benedetti A. Marucci L. Alvaro D. Baiocchi L. Kanno N. Caligiuri A. Phinizy J.L. Chowdury U. Papa E. LeSage G. Alpini G. Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms.Hepatology. 2000; 32: 17-25Crossref PubMed Scopus (90) Google Scholar, 12Glaser S. Rodgers R. Phinizy J.L. Robertson W. Lasater J. Caligiuri A. Tretjak Z. LeSage G. Alpini G. Gastrin inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes.Am J Physiol Gastrointest Liver Physiol. 1997; 273: G1061-G1070Google Scholar, 13Alvaro D. Onori P. Metalli V.D. Svegliati-Baroni G. Folli F. Franchitto A. Alpini G. Mancino M.G. Attili A.F. Gaudio E. Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat.Hepatology. 2002; 36: 297-304Crossref PubMed Scopus (83) Google Scholar, 14Alvaro D. Alpini G. Onori P. Franchitto A. Glaser S. Le Sage G. Gigliozzi A. Vetuschi A. Morini S. Attili A.F. Gaudio E. Effect of ovariectomy on the proliferative capacity of intrahepatic rat cholangiocytes.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 15Kanno N. Glaser S. Chowdhury U. Phinizy J.L. Baiocchi L. Francis H. LeSage G. Alpini G. Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of Ca2+-dependent protein kinase C-alpha.J Hepatol. 2001; 34: 284-291Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 16LeSage G. Marucci L. Alvaro D. Glaser S. Benedetti A. Marzioni M. Patel T. Francis H. Phinizy J.L. Alpini G. Insulin inhibits secretin-induced ductal secretion by activation of PKC alpha and inhibition of PKA activity.Hepatology. 2002; 36: 641-651Crossref PubMed Scopus (45) Google Scholar Insulin inhibits secretin-induced choleresis in BDL rats,16LeSage G. Marucci L. Alvaro D. Glaser S. Benedetti A. Marzioni M. Patel T. Francis H. Phinizy J.L. Alpini G. Insulin inhibits secretin-induced ductal secretion by activation of PKC alpha and inhibition of PKA activity.Hepatology. 2002; 36: 641-651Crossref PubMed Scopus (45) Google Scholar whereas the proliferative response of cholangiocytes to the BDL is markedly diminished in rats deprived of endogenous estrogens14Alvaro D. Alpini G. Onori P. Franchitto A. Glaser S. Le Sage G. Gigliozzi A. Vetuschi A. Morini S. Attili A.F. Gaudio E. Effect of ovariectomy on the proliferative capacity of intrahepatic rat cholangiocytes.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar or chronically treated with antiestrogens.13Alvaro D. Onori P. Metalli V.D. Svegliati-Baroni G. Folli F. Franchitto A. Alpini G. Mancino M.G. Attili A.F. Gaudio E. Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat.Hepatology. 2002; 36: 297-304Crossref PubMed Scopus (83) Google Scholar Gastrin decreases the growth and functions of the biliary tree after BDL10Glaser S. Benedetti A. Marucci L. Alvaro D. Baiocchi L. Kanno N. Caligiuri A. Phinizy J.L. Chowdury U. Papa E. LeSage G. Alpini G. Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms.Hepatology. 2000; 32: 17-25Crossref PubMed Scopus (90) Google Scholar, 12Glaser S. Rodgers R. Phinizy J.L. Robertson W. Lasater J. Caligiuri A. Tretjak Z. LeSage G. Alpini G. Gastrin inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes.Am J Physiol Gastrointest Liver Physiol. 1997; 273: G1061-G1070Google Scholar and blocks the growth of cholangiocarcinoma cell lines.15Kanno N. Glaser S. Chowdhury U. Phinizy J.L. Baiocchi L. Francis H. LeSage G. Alpini G. Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of Ca2+-dependent protein kinase C-alpha.J Hepatol. 2001; 34: 284-291Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Interestingly, it has also been observed that cholangiocytes from human livers affected by chronic cholestatic diseases show neuroendocrine features not present in normal livers.17Roskams T. van den Oord J.J. De Vos R. Desmet V.J. Neuroendocrine features of reactive bile ductules in cholestatic liver disease.Am J Pathol. 1990; 137: 1019-1025PubMed Google ScholarSerotonin is a neuroendocrine hormone secreted by enterochromaffin cells throughout the entire gastrointestinal tract.18Suzuki A. Naruse S. Kitagawa M. Ishiguro H. Yoshikawa T. Ko S.B. Yamamoto A. Hamada H. Hayakawa T. 5-Hydroxytryptamine strongly inhibits fluid secretion in guinea pig pancreatic duct cells.J Clin Invest. 2001; 108: 749-756PubMed Google Scholar, 19Li Y. Hao Y. Zhu J. Owyang C. Serotonin released from intestinal enterochromaffin cells mediates luminal non-cholecystokinin-stimulated pancreatic secretion in rats.Gastroenterology. 2000; 118: 1197-1207Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar The local release of this hormone leads to marked changes in the functions of gastrointestinal epithelial cells. For example, the application of serotoninergic agents enhances fluid and ion secretion of the intestinal mucosa,20Borman R.A. Burleigh D.E. Heterogeneity of 5-HT receptors mediating secretion in the human intestine.Ann N Y Acad Sci. 1997; 812: 224-225Crossref PubMed Scopus (25) Google Scholar, 21Franks C.M. Hardcastle J. Hardcastle P.T. Neural involvement in 5-hydroxytryptamine-induced net electrogenic ion secretion in the rat intestine in-vivo.J Pharm Pharmacol. 1996; 48: 411-416Crossref PubMed Scopus (16) Google Scholar whereas the paracrine release of serotonin by enterochromaffin cells mediates the pancreatic secretion induced by intestinal lumenal factors.19Li Y. Hao Y. Zhu J. Owyang C. Serotonin released from intestinal enterochromaffin cells mediates luminal non-cholecystokinin-stimulated pancreatic secretion in rats.Gastroenterology. 2000; 118: 1197-1207Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 22Li Y. Wu X.Y. Zhu J.X. Owyang C. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G916-G923PubMed Google Scholar In addition to its role as a regulator of secretive processes, serotonin has been found to modulate cell proliferation of vascular smooth muscle cells,23Azmitia E.C. Modern views on an ancient chemical serotonin effects on cell proliferation, maturation, and apoptosis.Brain Res Bull. 2001; 56: 413-424Crossref PubMed Scopus (386) Google Scholar valvular subendocardial cells,24Rajamannan N.M. Caplice N. Anthikad F. Sebo T.J. Orszulak T.A. Edwards W.D. Tajik J. Schwartz R.S. Cell proliferation in carcinoid valve disease a mechanism for serotonin effects.J Heart Valve Dis. 2001; 10: 827-831PubMed Google Scholar lymphocytes,25Aune T.M. Golden H.W. McGrath K.M. Inhibitors of serotonin synthesis and antagonists of serotonin 1A receptors inhibit T lymphocyte function in vitro and cell-mediated immunity in vivo.J Immunol. 1994; 153: 489-498PubMed Google Scholar kidney epithelial cells,23Azmitia E.C. Modern views on an ancient chemical serotonin effects on cell proliferation, maturation, and apoptosis.Brain Res Bull. 2001; 56: 413-424Crossref PubMed Scopus (386) Google Scholar or, in the gastrointestinal apparatus, hepatocytes.26Balasubramanian S. Paulose C.S. Induction of DNA synthesis in primary cultures of rat hepatocytes by serotonin possible involvement of serotonin S2 receptor.Hepatology. 1998; 27: 62-66Crossref PubMed Scopus (88) Google ScholarIt has been recently shown that serotonin is involved in the pathogenesis of certain clinical features of cholangiopathies, pruritus, and fatigue in particular.27Jones E.A. Bergasa N.V. The pathogenesis and treatment of pruritus and fatigue in patients with PBC.Eur J Gastroenterol Hepatol. 1999; 11: 623-631Crossref PubMed Scopus (45) Google Scholar, 28Swain M.G. Maric M. Improvement in cholestasis-associated fatigue with a serotonin receptor agonist using a novel rat model of fatigue assessment.Hepatology. 1997; 25: 291-294Crossref PubMed Scopus (86) Google Scholar In animal models of chronic cholestasis, this seems to be due to an enhanced release of serotonin in the central nervous system and its interactions with subtype 1 serotonin receptors.28Swain M.G. Maric M. Improvement in cholestasis-associated fatigue with a serotonin receptor agonist using a novel rat model of fatigue assessment.Hepatology. 1997; 25: 291-294Crossref PubMed Scopus (86) Google ScholarUnfortunately, nothing is known about the possible role of serotonin in the pathophysiology of chronic cholestasis. Therefore, in this investigation we posed the following questions: (1) Do cholangiocytes express functional serotonin 1A and 1B receptor subtypes? (2) What is the effect of the activation of these receptors on cholangiocyte proliferation and functional activity in the course of chronic cholestasis? (3) Which intracellular pathways are involved in the transduction of the serotoninergic intracellular signal? and (4) Is serotonin involved in the autocrine modulation of the cholangiocyte response to chronic cholestasis?Materials and methodsMaterialsReagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. The monoclonal mouse antibody against proliferating cellular nuclear antigen (PCNA) was obtained from DAKO (Kyoto, Japan). Intracellular cAMP and d-myo-inositol 1,4,5-triphosphate (IP3) levels were determined with radioimmunoassay (RIA) kits purchased from Amersham (Arlington Heights, IL). Serotonin receptor selective agonists and antagonists were purchased from Tocris (Ballwin, MO). Antibodies for immunoblotting and immunohistochemistry were purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA) unless otherwise indicated. The substrate for γ-glutamyltranspeptidase (γ-GT), N-(γ-l-glutamyl)-4-methoxy-2-naphthylamide, was purchased from Polysciences (Warrington, PA).Expression of the serotonin 1A and 1B receptor subtypes in cholangiocytesThe expression of serotonin receptors was evaluated by immunohistochemistry in formalin-fixed and paraffin-embedded liver sections. Sections were immersed in 0.01 mol/L citrate buffer (pH 6.0) and irradiated in a microwave oven for 15 minutes. Liver sections were then incubated with or without the polyclonal primary antibody (1:20 dilution in 1× phosphate-buffered saline) for 2 hours at room temperature, and the reactive sites were detected by using a DAKO LSAB kit (according to the manufacturer’s instructions) followed by incubation for 5 minutes with 1× phosphate-buffered saline containing 0.06% diaminobenzidine and 0.01% H2O2. Slides were counterstained with Harris hematoxylin.Immunofluorescence was performed in acetone-fixed fresh-frozen liver sections and incubated with either the serotonin 1A or 1B antibody (both diluted 1:20) at 4°C overnight. Thereafter, after several washings, the samples were incubated with Alexa488-labeled secondary antibody (diluted 1:100; Molecular Probes, Eugene, OR) for 2 hours at room temperature. Then, the samples were mounted with antifade (Molecular Probes). These samples were evaluated by using confocal laser microscopy (Bio-Rad, Hercules, CA) with adequate filters.The expression of the receptors was also evaluated by immunoblots both in whole cells and after subcellular fractionation to separate cholangiocyte basolateral and apical membranes. Basolateral and apical fractions were obtained as previously described.29Tietz P.S. Holman R.T. Miller L.J. LaRusso N.F. Isolation and characterization of rat cholangiocyte vesicles enriched in apical or basolateral plasma membrane domains.Biochemistry. 1995; 34: 15436-15443Crossref PubMed Scopus (63) Google Scholar, 30Glaser S. Alvaro D. Roskams T. Phinizy J.L. Stoica G. Francis H. Ueno Y. Barbaro B. Marzioni M. Mauldin J. Rashid S. Mancino M.G. LeSage G. Alpini G. Dopaminergic inhibition of secretin-stimulated choleresis by increased PKC-gamma expression and decrease of PKA activity.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G683-G694PubMed Google ScholarExperimental designIn vivo studiesMale Fischer 344 rats (150–175 g), purchased from Charles River Laboratories (Wilmington, MA), were kept in a temperature-controlled environment (20°C–22°C) with a 12-hour light/dark cycle and with free access to drinking water and standard rat chow.To study the effect of serotonin receptor agonist administration on cholangiocyte proliferation and functional activity, our studies were performed in (1) normal rats and rats with BDL7Alpini G. Glaser S. Ueno Y. Phinizy J.L. Rodgers R. Francis H. Baiocchi L. Holcomb L. Caligiuri A. LeSage G. Bile acid feeding induces cholangiocyte proliferation and secretion evidence for bile acid-regulated ductal secretion.Gastroenterology. 1999; 116: 179-186Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 9Alpini G. Lenzi R. Sarkozi L. Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Crossref PubMed Scopus (288) Google Scholar (for cholangiocyte purification or liver sections) or bile duct incannulation (BDI; for bile collection) for 1 week9Alpini G. Lenzi R. Sarkozi L. Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Crossref PubMed Scopus (288) Google Scholar and (2) normal, BDL, or BDI rats that, immediately after BDL or BDI, were treated by intraperitoneal (IP) implanted Alzet (Cupertino, CA) osmotic minipumps with saline, 8-hydroxy-2-(di-n-propylamino)-tertralin (DPAT) (a serotonin 1A receptor agonist19Li Y. Hao Y. Zhu J. Owyang C. Serotonin released from intestinal enterochromaffin cells mediates luminal non-cholecystokinin-stimulated pancreatic secretion in rats.Gastroenterology. 2000; 118: 1197-1207Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 23Azmitia E.C. Modern views on an ancient chemical serotonin effects on cell proliferation, maturation, and apoptosis.Brain Res Bull. 2001; 56: 413-424Crossref PubMed Scopus (386) Google Scholar; 1 nmol · kg−1 · h−1), anpirtoline (a serotonin 1B receptor agonist31de Almeida R.M. Miczek K.A. Aggression escalated by social instigation or by discontinuation of reinforcement (“frustration”) in mice: inhibition by anpirtoline: a 5HT1B receptor agonist.Neuropsychopharmacology. 2002; 27: 171-181Crossref PubMed Scopus (121) Google Scholar; 1 nmol · kg−1 · h−1), or 8-hydroxy-DPAT and anpirtoline together for 1 week. 8-Hydroxy-DPAT and anpirtoline are known to have a very high affinity for their receptors: endogenous ligands do not represent an obstacle for their binding to the receptors.32Currie P.J. Braver M. Mirza A. Sricharoon K. Sex differences in the reversal of fluoxetine-induced anorexia following raphe injections of 8-OH-DPAT.Psychopharmacology. 2004; 172: 359-364Crossref PubMed Scopus (25) Google Scholar To study the effect of the blockage of endogenous serotonin on cholangiocyte proliferation, normal or BDL rats were treated by IP injections with a serotonin-neutralizing antibody (12 μg/d; Biomeda, Foster City, CA) or nonimmune serum for 1 week.The animals were fasted overnight before each experiment.7Alpini G. Glaser S. Ueno Y. Phinizy J.L. Rodgers R. Francis H. Baiocchi L. Holcomb L. Caligiuri A. LeSage G. Bile acid feeding induces cholangiocyte proliferation and secretion evidence for bile acid-regulated ductal secretion.Gastroenterology. 1999; 116: 179-186Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Before each procedure, animals were anesthetized with sodium pentobarbital (50 mg/kg, IP). Study protocols were performed in compliance with institutional guidelines.In vitro studiesTo show that the effects of the serotonin receptor agonists on cholangiocyte proliferation are due to a specific interaction with serotonin receptors on cholangiocytes, pure cholangiocytes from 1-week BDL rats were incubated at 37°C with (1) 0.2% bovine serum albumin (BSA; control) for 5 hours; (2) 0.2% BSA for 1 hour followed by 8-hydroxy-DPAT (a serotonin 1A receptor agonist; 10 μmol/L)22Li Y. Wu X.Y. Zhu J.X. Owyang C. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G916-G923PubMed Google Scholar, 23Azmitia E.C. Modern views on an ancient chemical serotonin effects on cell proliferation, maturation, and apoptosis.Brain Res Bull. 2001; 56: 413-424Crossref PubMed Scopus (386) Google Scholar, 25Aune T.M. Golden H.W. McGrath K.M. Inhibitors of serotonin synthesis and antagonists of serotonin 1A receptors inhibit T lymphocyte function in vitro and cell-mediated immunity in vivo.J Immunol. 1994; 153: 489-498PubMed Google Scholar, 33Middlemiss D.N. Fozard J.R. 8-Hydroxy-2-(di-n-propylamino)-tetralin discriminates between subtypes of the 5-HT1 recognition site.Eur J Pharmacol. 1983; 90: 151-153Crossref PubMed Scopus (826) Google Scholar for 4 hours with 0.2% BSA; (3) (S)-WAY 100135 dihydrochloride (a serotonin 1A receptor antagonist; 10 μmol/L)22Li Y. Wu X.Y. Zhu J.X. Owyang C. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G916-G923PubMed Google Scholar, 23Azmitia E.C. Modern views on an ancient chemical serotonin effects on cell proliferation, maturation, and apoptosis.Brain Res Bull. 2001; 56: 413-424Crossref PubMed Scopus (386) Google Scholar for 1 hour followed by 8-hydroxy-DPAT for 4 hours with 0.2% BSA; (4) 0.2% BSA for 1 hour followed by anpirtoline (a serotonin 1B receptor agonist; 10 μmol/L)22Li Y. Wu X.Y. Zhu J.X. Owyang C. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G916-G923PubMed Google Scholar, 31de Almeida R.M. Miczek K.A. Aggression escalated by social instigation or by discontinuation of reinforcement (“frustration”) in mice: inhibition by anpirtoline: a 5HT1B receptor agonist.Neuropsychopharmacology. 2002; 27: 171-181Crossref PubMed Scopus (121) Google Scholar for 4 hours with 0.2% BSA; (5) SB 216641 hydrochloride (a serotonin 1B receptor antagonist; 10 μmol/L)22Li Y. Wu X.Y. Zhu J.X. Owyang C. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G916-G923PubMed Google Scholar, 34Przegalinski E. Siwanowicz J. Nowak E. Papla I. Filip M. Role of 5-HT(1B) receptors in the sensitization to amphetamine in mice.Eur J Pharmacol. 2001; 422: 91-99Crossref PubMed Scopus (41) Google Scholar for 1 hour followed by anpirtoline for 4 hours with 0.2% BSA; or (6) 0.2% BSA for 1 hour followed by 8-hydroxy-DPAT and anpirtoline together for 4 hours with 0.2% BSA.To show that the effects of the serotonin 1A and 1B receptor agonists on cholangiocyte proliferation are mediated by the cAMP/protein kinase A (PKA)/Src pathway cells from 1-week BDL rats were also incubated with (1) dybutyryl-cAMP (cAMP agonist; 500 μmol/L)35Roma M.G. Milkiewicz P. Elias E. Coleman R. Control by signaling modulators of the sorting of canalicular transporters in rat hepatocyte couplets role of the cytoskeleton.Hepatology. 2000; 32: 1342-1356Crossref PubMed Scopus (57) Google Scholar for 30 minutes followed by 8-hydroxy-DPAT or anpirtoline for 4 hours; (2) PP2 (a Src inhibitor; 1 μmol/L)13Alvaro D. Onori P. Metalli V.D. Svegliati-Baroni G. Folli F. Franchitto A. Alpini G. Mancino M.G. Attili A.F. Gaudio E. Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat.Hepatology. 2002; 36: 297-304Crossref PubMed Scopus (83) Google Scholar for 30 minutes followed by dybutyryl-cAMP for 30 minutes followed afterward by 8-hydroxy-DPAT or anpirtoline for 4 hours; (3) dybutyryl-cAMP for 30 minutes followed by 0.2% BSA for 4 hours (control); (4) PP2 for 30 minutes followed by 0.2% BSA for 4 hours (control); or (5) PP2 for 30 minutes followed by dybu

We investigated, in isolated bile duct units (IBDU) and cholangiocytes isolated from normal rat liver, the occurrence of acetylcholine (ACh) receptors, and the role and mechanisms of ACh in the regulation of the Cl-/HCO3- exchanger activity. The Cl-/HCO3- exchanger activity was evaluated measuring changes in intracellular pH induced by acute Cl- removal/readmission. M3 subtype ACh receptors were detected in IBDU and isolated cholangiocytes by immunofluorescence, immunoelectron microscopy, and reverse transcriptase PCR. M1 subtype ACh receptor mRNA was not detected by reverse transcriptase PCR and M2 subtype was negative by immunofluorescence. ACh (10 microM) showed no effect on the basal activity of the Cl-/HCO3- exchanger. When IBDU were exposed to ACh plus secretin, ACh significantly (P < 0.03) increased the maximal rate of alkalinization after Cl- removal and the maximal rate of recovery after Cl- readmission compared with secretin alone (50 nM), indicating that ACh potentiates the stimulatory effect of secretin on the Cl-/HCO3- exchanger activity. This effect of ACh was blocked by the M3 ACh receptor antagonist, 4-diphenyl-acetoxy-N-(2-chloroethyl)-piperidine (40 nM), by the intracellular Ca2+ chelator, 1,2-bis (2-Aminophenoxy)- ethane-N,N,N', N'-tetraacetic acid acetoxymethylester (50 microM), but not by the protein kinase C antagonist, staurosporine (0.1 microM). Intracellular cAMP levels, in isolated rat cholangiocytes, were unaffected by ACh alone, but were markedly higher after exposure to secretin plus ACh compared with secretin alone (P < 0.01). The ACh-induced potentiation of the secretin effect on both intracellular cAMP levels and the Cl-/HCO3- exchanger activity was individually abolished by two calcineurin inhibitors, FK-506 and cyclosporin A (100 nM).M3 ACh receptors are markedly and diffusively represented in rat cholangiocytes. ACh did not influence the basal activity of the Cl-/HCO3- exchanger, but enhanced the stimulation by secretin of this anion exchanger by a Ca2+-dependent, protein kinase C-insensitive pathway that potentiates the secretin stimulation of adenylyl cyclase. Calcineurin most likely mediates the cross-talk between the calcium and adenylyl cyclase pathways. Since secretin targets cholangiocytes during parasympathetic predominance, coordinated regulation of Cl-/HCO3- exchanger by secretin (cAMP) and ACh (Ca2+) could play a major role in the regulation of ductal bicarbonate excretion in bile just when the bicarbonate requirement in the intestine is maximal.

The effects of secretin on ion transport mechanisms involved in regulation of intracellular pH (pHi) and HCO3- excretion were characterized in bile duct epithelial (BDE) cells isolated from normal rat liver. pHi was measured with 2,7-bis(carboxy-ethyl)-5(6)-carboxy-fluorescein-acetomethylester (BCECF-AM) using a microfluorimetric method. Basal pHi of BDE was 7.04 +/- 0.06 in Hepes and 7.16 +/- 0.10 in KRB and was unaffected by secretin (50-200 nM). Recovery rates from an acid load in Hepes or in KRB media (with and without amiloride) were also not altered by secretin, indicating that Na+/H+ exchange and Na+/HCO3- cotransport were not affected by this hormone. After acute Cl- removal, pHi rose 0.24 +/- 0.08 pHU at a maximal rate of 0.125 +/- 0.06 pHU/min (H+ flux rates = 6.02 +/- 3.27 mM/min) and recovered after Cl- readmission (0.188 +/- 0.08 pHU/min; H+ flux rates = 11.82 +/- 5.34 mM/min). Pretreatment with 1 mM DIDS inhibited the effects of Cl- removal, while valinomycin, which induces cell depolarization, enhanced these effects, probably by stimulating electrogenic HCO3- influx. Secretin significantly increased both the maximal rate of alkalinization after Cl- removal (P < 0.012) and of pHi recovery after Cl- readmission (P < 0.025), indicating stimulation of Cl-/HCO3- exchange activity. These findings were reproduced with N6,2'-O-Dibutyryladenosine-3',5'-cyclic monophosphate (DBcAMP). The Cl- channel blocker 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB, 10 microM) significantly decreased the effects of secretin and DBcAMP on the pHi changes promoted by acute Cl- removal/readmission. These findings establish that secretin stimulates the activity of the Cl-/HCO3- exchanger in BDE cells, probably by activating Cl- channels via the intracellular messenger cAMP. This in turn depolarizes the cell, stimulating electrogenic Na+/HCO3- symport. The cell depolarization induced by Cl- channel activation should enhance HCO3- entrance through electrogenic Na+/HCO3- symport, which in turn stimulates the Cl-/HCO3- exchange. These mechanisms could account for secretin stimulated bicarbonate secretion in bile.

Genetic factors are critical in determining susceptibility to primary biliary cirrhosis (PBC), but there has not been a clear association with human leukocyte antigen (HLA) genes. We performed a multicenter case-control study and analyzed HLA class II DRB1 associations using a large cohort of 664 well-defined cases of PBC and 1992 controls of Italian ancestry. Importantly, healthy controls were rigorously matched not only by age and sex, but also for the geographical origin of the proband four grandparents (Northern, Central, and Southern Italy). After correction for multiple testing, DRB1*08 [odds ratio (OR), 3.3; 95% confidence interval (CI), 2.4-4.5] and DRB1*02 (OR 0.9; 95% CI 0.8-1.2) were significantly associated with PBC, whereas alleles DRB1*11 (OR 0.4; 95% CI 0.3-0.4) and DRB1*13 (OR 0.7; 95% CI 0.6-0.9) were protective. When subjects were stratified according to their grandparental geographical origin, only the associations with DRB1*08 and DRB1*11 were common to all three areas. Associated DRB1 alleles were found only in a minority of patients, whereas an additive genetic model is supported by the gene dosage effect for DRB1*11 allele and the interaction of DRB1*11,*13, and *08. Lastly, no significant associations were detected between specific DRB1 alleles and relevant clinical features represented by the presence of cirrhosis or serum autoantibodies. In conclusion, we confirm the role for HLA to determine PBC susceptibility and suggest that the effect of HLA is limited to patient subgroups. We suggest that a large whole-genome approach is required to identify further genetic elements contributing to the loss of tolerance in this disease. (HEPATOLOGY 2008;48:1906-1912.)

The scientific framework concerning estrogen effects on different tissues has expanded enormously during the last decades, when estrogen receptor (ER) subtypes were identified. Estrogens are not only essential for the female reproductive system, but they also control fundamental functions in other tissues including the cardiovascular system, bone, brain and liver. Recently, estrogens have been shown to target the biliary tree, where they modulate the proliferative and secretory activities of cholangiocytes, the epithelial cells lining bile ducts. By acting on both estrogen receptors (ER-alpha) and (ER-beta) subtypes, and by activating either genomic or non-genomic pathways, estrogens play a key role in the complex loop of growth factors and cytokines, which modulates the proliferative response of cholangiocytes to damage. Specifically, estrogens activate intracellular signalling cascades [ERK(1/2) (extracellular regulated kinases (1/2), PI3- kinase/AKT (phosphatidylinositol-3' kinase/AKT)] typical of growth factors such as insulin like growth factor (IGF1), nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), thus potentiating their action. In addition, estrogens stimulate the secretion of different growth factors in proliferating cholangiocytes. This review specifically deals with the recent advances related to the role and mechanisms by which estrogens modulate cholangiocyte functions in normal and pathological conditions.

To evaluate if increased cholangiocyte cAMP levels alone are sufficient to enhance cholangiocyte proliferation and secretion.Normal rats were treated in vivo with forskolin for two weeks. Cholangiocyte apoptosis, proliferation and secretion were evaluated. Purified cholangiocytes from normal rats were treated in vitro with forskolin in the absence or presence of Rp-cAMPs (a PKA inhibitor), PP2 (an Src inhibitor) or PD98059 (a MEK inhibitor). Subsequently, we evaluated cholangiocyte proliferation by determination of proliferating cellular nuclear antigen (PCNA) protein expression by immunoblots. We evaluated if the effects of forskolin on cholangiocyte functions are associated with changes in the cAMP/PKA/Src/MEK/ERK1/2 pathway.Chronic administration of forskolin to normal rats increased the number of ducts, cAMP levels, and secretin-induced choleresis compared to controls. Forskolin-induced increases in cholangiocyte proliferation and secretion were devoid of cholangiocyte necrosis, inflammation and apoptosis. In vitro, in pure isolated cholangiocytes, forskolin increased cholangiocyte proliferation, which was ablated by Rp-cAMPs, PP2 and PD98059. The effects of forskolin on cholangiocyte proliferation were associated with increased activity of PKA, Src Tyrosine 139 (Tyr 139) and ERK1/2.Modulation of the PKA/Src/MEK/ERK1/2 pathway may be important in the regulation of cholangiocyte growth and secretion observed in cholestatic liver diseases.

Background/Aims Estrogen receptors (ER) in cholangiocytes of primary biliary cirrhosis (PBC) patients and their relationship with cell proliferation and death were evaluated. Methods Liver biopsies from PBC patients with different histological stages were investigated by immunohistochemistry for ER-α and -β, cytokeratin-19, proliferating cellular nuclear antigen (PCNA), Fas and terminal deoxynucleotide transferase end labelling (TUNEL). Normal livers and livers from primary sclerosing cholangitis and alcoholic cirrhosis were investigated as controls. Results ER-α and -β were observed in cholangiocytes of PBC patients but not in normal liver. In PBC, positivity for ER-β was high (50–65%) in all histological stages while, positivity for ER-α increased from 1% in stage I to 12% in stage III (positivity correlated and co-localized in the same cell with PCNA). In stage IV of PBC, cholangiocytes were negative for ER-α in association with a lower PCNA positivity and with maximal degree of ductopenia. ER-α positivity in cholangiocytes of PBC patients was markedly lower than primary sclerosing cholangitis and alcoholic cirrhosis. Conclusions ER are expressed in PBC and other pathologies associated with cholangiocyte proliferation but not in normal subjects. The low expression of ER-α in PBC and their disappearance in the advanced histological stages suggests that an estrogenic deficiency could favour the evolution of this disease toward ductopenia. Estrogen receptors (ER) in cholangiocytes of primary biliary cirrhosis (PBC) patients and their relationship with cell proliferation and death were evaluated. Liver biopsies from PBC patients with different histological stages were investigated by immunohistochemistry for ER-α and -β, cytokeratin-19, proliferating cellular nuclear antigen (PCNA), Fas and terminal deoxynucleotide transferase end labelling (TUNEL). Normal livers and livers from primary sclerosing cholangitis and alcoholic cirrhosis were investigated as controls. ER-α and -β were observed in cholangiocytes of PBC patients but not in normal liver. In PBC, positivity for ER-β was high (50–65%) in all histological stages while, positivity for ER-α increased from 1% in stage I to 12% in stage III (positivity correlated and co-localized in the same cell with PCNA). In stage IV of PBC, cholangiocytes were negative for ER-α in association with a lower PCNA positivity and with maximal degree of ductopenia. ER-α positivity in cholangiocytes of PBC patients was markedly lower than primary sclerosing cholangitis and alcoholic cirrhosis. ER are expressed in PBC and other pathologies associated with cholangiocyte proliferation but not in normal subjects. The low expression of ER-α in PBC and their disappearance in the advanced histological stages suggests that an estrogenic deficiency could favour the evolution of this disease toward ductopenia.

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.

Abstract Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. Symptoms are usually evident after blockage of the bile duct by the tumor, and at this late stage, they are relatively resistant to chemotherapy and radiation therapy. Therefore, it is imperative that alternative treatment options are explored. We present novel data indicating that the metabolism of serotonin is dysregulated in cholangiocarcinoma cell lines, compared with normal cholangiocytes, and tissue and bile from cholangiocarcinoma patients. Specifically, there was an increased expression of tryptophan hydroxylase 1 and a suppression of monoamine oxidase A expression (enzymes responsible for the synthesis and degradation of serotonin, respectively) in cholangiocarcinoma. This resulted in an increased secretion of serotonin from cholangiocarcinoma and increased serotonin in the bile from cholangiocarcinoma patients. Increased local serotonin release may have implications on cholangiocarcinoma cell growth. Serotonin administration increased cholangiocarcinoma cell growth in vitro, whereas inhibition of serotonin synthesis decreases tumor cell growth both in vitro and in vivo. The data presented here represent the first evidence that serotonin metabolism is dysregulated in cholangiocarcinoma and that modulation of serotonin synthesis may represent an alternative target for the development of therapeutic strategies. [Cancer Res 2008;68(22):9184–93]

Estrogens may induce the proliferation of neoplastic cells by activating neo-angiogenesis.To evaluate the effect of estrogens on the expression of vascular endothelial growth factor (VEGF) and related receptors (VEGF-R) in human cholangiocarcinoma and the role played by VEGF in mediating the proliferative effects of estrogens.Seven biopsies of intra-hepatic cholangiocarcinoma and the HuH-28 cell lines were investigated. Cell proliferation was measured by both PCNA Western blot and MTS proliferation assay.By immunohistochemistry, biopsies of human cholangiocarcinoma stained positively for VEGF-A and VEGF-C and related receptors. HuH-28 cells expressed VEGF-A, -C, and VEGFR-1, -2, -3 and, their protein level was enhanced by 17beta-estradiol in association with the stimulation of cell proliferation. 17beta-Estradiol-stimulated proliferation of HuH-28 cells was blocked by 70% by VEGF-TRAP, a receptor-based VEGF inhibitor. 17beta-Estradiol induced the secretion of VEGF in the supernatant of HuH-28 cells. The stimulatory effect of 17beta-estradiol on the protein expression of VEGF-A, VEGF-C and VEGFR-1, -2, -3 was blocked by antagonists of ER (Ici182,780) or insulin-like growth factor 1-receptor (alphaIR3).With the limitations of experiments performed in a cell line, our study indicates that VEGF plays a major role in mediating the proliferative effects of estrogens on human cholangiocarcinoma.

Hepatocellular carcinoma and cholangiocarcinoma are primary liver cancers, both represent a growing challenge for clinicians due to their increasing morbidity and mortality. In the last few years a number of in vivo models of hepatocellular carcinoma and cholangiocarcinoma have been developed. The study of these models is providing a significant contribution in unveiling the pathophysiology of primary liver malignancies. They are also fundamental tools to evaluate newly designed molecules to be tested as new potential therapeutic agents in a pre-clinical set. Technical aspects of each model are critical steps, and they should always be considered in order to appropriately interpret the findings of a study or its planning. The purpose of this review is to describe the technical and experimental features of the most significant rodent models, highlighting similarities or differences between the corresponding human diseases. The first part is dedicated to the discussion of models of hepatocellular carcinoma, developed using toxic agents, or through dietary or genetic manipulations. In the second we will address models of cholangiocarcinoma developed in rats or mice by toxin administration, genetic manipulation and/or bile duct incannulation or surgery. Xenograft or syngenic models are also proposed.

Regenerative medicine is transitioning into clinical programs using stem/progenitor cell therapies for repair of damaged organs. We summarize those for liver and pancreas, organs that share endodermal stem cell populations, biliary tree stem cells (hBTSCs), located in peribiliary glands. They are precursors to hepatic stem/progenitors in canals of Hering and to committed progenitors in pancreatic duct glands. They give rise to maturational lineages along a radial axis within bile duct walls and a proximal-to-distal axis starting at the duodenum and ending with mature cells in the liver or pancreas. Clinical trials have been ongoing for years assessing effects of determined stem cells (fetal-liver-derived hepatic stem/progenitors) transplanted into the hepatic artery of patients with various liver diseases. Immunosuppression was not required. Control subjects, those given standard of care for a given condition, all died within a year or deteriorated in their liver functions. Subjects transplanted with 100-150 million hepatic stem/progenitor cells had improved liver functions and survival extending for several years. Full evaluations of safety and efficacy of transplants are still in progress. Determined stem cell therapies for diabetes using hBTSCs remain to be explored but are likely to occur following ongoing preclinical studies. In addition, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being used for patients with chronic liver conditions or with diabetes. MSCs have demonstrated significant effects through paracrine signaling of trophic and immunomodulatory factors, and there is limited evidence for inefficient lineage restriction into mature parenchymal or islet cells. HSCs' effects are primarily via modulation of immune mechanisms.

Proliferating cholangiocytes secrete and respond to neuroendocrine hormones, including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice.Cholestasis was induced in secretin knockout (Sct(-/-)) and wild-type (control) mice by bile duct ligation (BDL). At days 3 and 7 after BDL, control and Sct(-/-) mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected. Immunohistochemical, immunoblot, luciferase reporter, and real-time polymerase chain reaction assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells and in serum and bile.Secretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile after BDL in control mice. BDL Sct(-/-) mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor (VEGF) A and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA, and those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue.After liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in up-regulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases.

Background Very little data exist on the epidemiology of cholangiocarcinoma in Italy. Aim We focus on the descriptive epidemiology of cholangiocarcinoma in Italy. Methods Data on incidence were obtained from the Italian Association of Tumour Registries while mortality data were obtained from the Italian National Institute of Statistics. Results A progressive increase of incidence with age was seen for extra-hepatic, intra-hepatic and not otherwise specified cholangiocarcinoma. Crude incidence rates were higher for extra-hepatic cholangiocarcinoma than those for intra-hepatic cholangiocarcinoma and in men compared to women. An increasing incidence trend was observed, from 1988 to 2005, for both extra-hepatic- and intra-hepatic cholangiocarcinoma with a 3–6% yearly increase and with a rate of increase higher for men than for women and for intra-hepatic- than for extra-hepatic cholangiocarcinoma. For intra-hepatic cholangiocarcinoma, the mortality rates progressively increased from 0.15 per million in 1980 to 5.9 per million in 2003, when mortality for this cancer surpassed extra-hepatic cholangiocarcinoma. Mortality rates for extra-hepatic cholangiocarcinoma showed an increasing trend from 1980 to 1994 but, in contrast to intra-hepatic cholangiocarcinoma, a stable or slightly decreasing trend from 1995 to 2003 was observed. Conclusions In Italy, cholangiocarcinoma showed a progressive increase in incidence and mortality in the last two decades mainly in intra-hepatic cholangiocarcinoma.

Cholangiocarcinomas (CC) as well as gallbladder cancers are relatively rare and intractable diseases. Clinical, pathological, and epidemiological studies on these tumors have been under investigation. The current status and/or topics on biliary tract cancers have been reported in the East West Association of Liver Tumor (EWALT), held in Milano, Italy in 2015.All the authors, herein, specifcally reported the current status and leading-edge findings on biliary tract cancers as the following sequence: epidemiology of CC, surgical therapy for intrahepatic CC, surgical therapy for perihilar CC, surgical therapy for gallblad der cancer, chemotherapy for biliary tract cancers, and new histological features in CC.The present review article will update the knowledge on biliary tract cancers, en hancing the quality of daily clinical practice. However, many features about these cancers remain unknown; further studies are required to establish disease-specific optimal treatment strategies.

In their letter,1 Nakanuma and Sato provide evidence that peribiliary glands (PBGs) contain cells implicated in the origin of intraductal papillary neoplasms of the bile duct. This fits well with the hypothesis that mucin-producing cholangiocarcinomas might arise from biliary tree stem/progenitor cells (BTSCs) located in the PBGs of large intra- and extrahepatic bile ducts.2 BTSCs are associated with mucin-producing cells within the liver and biliary tree.3 Figure 1 shows an example of a mucin-producing intrahepatic cholangiocarcinoma morphologically being the malignant counterpart of PBGs. Thus, intraductal papillary neoplasms could represent the preneoplastic lesions preceding the emergence of mucin-producing cholangiocarcinomas, supporting similarities between pancreatic and bile duct neoplasias. In response to injuries, pancreatic duct glands undergo a mucinous metaplasia that might lead to pancreatic cancer4; this could occur also in the biliary tree during pathologic processes with risk factors for cholangiocarcinoma, such as primary sclerosing cholangitis.5 An unresolved question has been which cells are the origin of cholangiocarcinomas. Stem cells are candidates, because their self-renewal and longevity facilitate the sequential accumulation of oncogenic mutations.6 Two stem-cell niches have been described within the liver: canals of Hering, containing hepatic stem cells, and intrahepatic PBGs, containing BTSCs.3, 7 The former has been involved in the pathogenesis of cholangiocarcinomas with mixed features,8 whereas the latter could be implicated in the carcinogenesis of mucin-producing cholangiocarcinomas. Mucin-producing intrahepatic cholangiocarcinoma should be considered as having a similar origin to hilar and extrahepatic cholangiocarcinomas, opening new perspectives in the classification of cholangiocarcinomas. Immunohistochemistry for epithelial cell adhesion molecule (EpCAM) counterstained with Periodic Acid Schiff (PAS). Original magnification: 20×. Comparison of normal peribiliary glands and mucin-producing intrahepatic cholangiocarcinoma. Mucin-producing intrahepatic cholangiocarcinoma is morphologically the malignant counterpart of peribiliary glands. Vincenzo Cardinale M.D.*, Yunfang Wang M.D., Ph.D. , Guido Carpino Ph.D. , Lola M. Reid Ph.D.¶, Eugenio Gaudio M.D.** §, Domenico Alvaro M.D.* **, * Department of Medico-Surgical Sciences and Biotechnologies, Polo Pontino, Sapienza University of Rome, Rome, Italy, The Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing, China, Department of Health Sciences, University of Rome “Foro Italico”, Rome, Italy, § Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza University of Rome, Rome, Italy, ¶ Department of Cell and Molecular Physiology, Program in Molecular Biology and Biotechnology, University of North Carolina School of Medicine, Chapel Hill, NC, ** Eleonora Lorillard Spencer-Cenci Foundation, Rome, Italy.

Cholangiocarcinoma (CCA) is a devastating liver tumour arising from malignant transformation of bile duct epithelial cells. Cancer stem cells (CSC) are a subset of tumour cells endowed with stem-like properties, which play a role in tumour initiation, recurrence and metastasis. In appropriate conditions, CSC form 3D spheres (SPH), which retain stem-like tumour-initiating features. Here, we found different expression of iron proteins indicating increased iron content, oxidative stress and higher expression of CSC markers in CCA-SPH compared to tumour cells growing as monolayers. Exposure to the iron chelator desferrioxamine decreased SPH forming efficiency and the expression of CSC markers and stem-like genes, whereas iron had an opposite effect. Microarray profiles in CCA samples (n = 104) showed decreased H ferritin, hepcidin and ferroportin expression in tumours respect to surrounding liver, whereas transferrin receptor was up-regulated. Moreover, we found a trend toward poorer outcome in CCA patients with elevated expression of ferritin and hepcidin, two major proteins of iron metabolism. These findings, which represent the first evidence of a role for iron in the stem cell compartment as a novel metabolic factor involved in CCA growth, may have implications for a better therapeutic approach.

This review focuses on the recent advances related to the heterogeneity of different-sized bile ducts with regard to the morphological and phenotypical characteristics, and the differential secretory, apoptotic and proliferative responses of small and large cholangiocytes to gastrointestinal hormones/peptides, neuropeptides and toxins. We describe several in vivo and in vitro models used for evaluating biliary heterogeneity. Subsequently, we discuss the heterogeneous proliferative and apoptotic responses of small and large cholangiocytes to liver injury and the mechanisms regulating the differentiation of small into large (more differentiated) cholangiocytes. Following a discussion on the heterogeneity of stem/progenitor cells in the biliary epithelium, we outline the heterogeneity of bile ducts in human cholangiopathies. After a summary section, we discuss the future perspectives that will further advance the field of the functional heterogeneity of the biliary epithelium.

Multipotent stem/progenitor cells are found in peribiliary glands throughout human biliary trees and are able to generate mature cells of hepato-biliary and pancreatic endocrine lineages. The presence of endodermal stem/progenitors in human gallbladder was explored.Gallbladders were obtained from organ donors and laparoscopic surgery for symptomatic cholelithiasis. Tissues or isolated cells were characterized by immunohistochemistry and flow cytometry. EpCAM+ (Epithelial Cell Adhesion Molecule) cells were immunoselected by magnetic microbeads, plated onto plastic in self-replication conditions and subsequently transferred to distinct serum-free, hormonally defined media tailored for differentiation to specific adult fates. In vivo studies were conducted in an experimental model of liver cirrhosis.The gallbladder does not have peribiliary glands, but it has stem/progenitors organized instead in mucosal crypts. Most of these can be isolated by immune-selection for EpCAM. Approximately 10% of EpCAM+ cells in situ and of immunoselected EpCAM+ cells co-expressed multiple pluripotency genes and various stem cell markers; other EpCAM+ cells qualified as progenitors. Single EpCAM+ cells demonstrated clonogenic expansion ex vivo with maintenance of stemness in self-replication conditions. Freshly isolated or cultured EpCAM+ cells could be differentiated to multiple, distinct adult fates: cords of albumin-secreting hepatocytes, branching ducts of secretin receptor+ cholangiocytes, or glucose-responsive, insulin/glucagon-secreting neoislets. EpCAM+ cells transplanted in vivo in immune-compromised hosts gave rise to human albumin-producing hepatocytes and to human Cytokeratin7+ cholangiocytes occurring in higher numbers when transplanted in cirrhotic mice.Human gallbladders contain easily isolatable cells with phenotypic and biological properties of multipotent, endodermal stem cells.

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.

Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are human primary cholangiopathies characterized by the damage of mature cholangiocytes and by the appearance of ductular reaction (DR) as the results of hepatic progenitor cell activation. This study evaluated the differences in progenitor cell niche activation between these two cholangiopathies. Liver tissue was obtained from healthy liver donors (n = 5) and from patients with PSC (n = 20) or PBC (n = 20). DR, progenitor cell phenotype, and signaling pathways were investigated by IHC analysis and immunofluorescence. Our results indicated that DR was more extended, appeared earlier, and had a higher proliferation index in PBC compared with PSC. In PBC, DR was strongly correlated with clinical prognostic scores. A higher percentage of sex determining region Y-box (SOX)9+ and cytokeratin 19+ cells but fewer features of hepatocyte fate characterized progenitor cell activation in PBC versus PSC. Lower levels of laminin and neurogenic locus notch homolog protein 1 but higher expression of wingless-related integration site (WNT) family pathway components characterize progenitor cell niche in PSC compared with PBC. In conclusion, progenitor cell activation differs between PSC and PBC and is characterized by a divergent fate commitment and different signaling pathway predominance. In PBC, DR represents a relevant histologic prognostic marker.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory cholangiopathy frequently complicated by cholangiocarcinoma (CCA). Massive proliferation of biliary tree stem/progenitor cells (BTSCs), expansion of peribiliary glands (PBGs), and dysplasia were observed in PSC. The aims of the present study were to evaluate the involvement of PBGs and BTSCs in CCA which emerged in PSC patients. Specimens from normal liver (n = 5), PSC (n = 20), and PSC‐associated CCA (n = 20) were included. Samples were processed for histology, immunohistochemistry, and immunofluorescence. In vitro experiments were performed on human BTSCs, human mucinous primary CCA cell cultures, and human cholangiocyte cell lines (H69). Our results indicated that all CCAs emerging in PSC patients were mucin‐producing tumors characterized by PBG involvement and a high expression of stem/progenitor cell markers. Ducts with neoplastic lesions showed higher inflammation, wall thickness, and PBG activation compared to nonneoplastic PSC‐affected ducts. CCA showed higher microvascular density and higher expression of nuclear factor kappa B, interleukin‐6, interleukin‐8, transforming growth factor β, and vascular endothelial growth factor‐1 compared to nonneoplastic ducts. CCA cells were characterized by a higher expression of epithelial‐to‐mesenchymal transition (EMT) traits and by the absence of primary cilia compared to bile ducts and PBG cells in controls and patients with PSC. Our in vitro study demonstrated that lipopolysaccharide and oxysterols (PSC‐related stressors) induced the expression of EMT traits, the nuclear factor kappa B pathway, autophagy, and the loss of primary cilia in human BTSCs. Conclusion : CCA arising in patients with PSC is characterized by extensive PBG involvement and by activation of the BTSC niche in these patients, the presence of duct lesions at different stages suggests a progressive tumorigenesis.

Cholangiocarcinoma (CCA) is the second most common primary liver cancer, characterized by a poor prognosis and resistance to chemotherapeutics. The progressive increase in CCA incidence and mortality registered worldwide in the last two decades and the need to clarify various aspects of clinical management have prompted the Italian Association for the Study of the Liver (AISF) to commission the drafting of dedicated guidelines in collaboration with a group of Italian scientific societies. These guidelines have been formulated in accordance with the Italian National Institute of Health indications and developed by following the GRADE method and related advancements.

Cholangiopathies, such as primary sclerosing cholangitis, biliary atresia, and cholangiocarcinoma, have limited experimental models. Not only cholangiocytes but also other hepatic cells including hepatic stellate cells and macrophages are involved in the pathophysiology of cholangiopathies, and these hepatic cells orchestrate the coordinated response against diseased conditions. Classic two‐dimensional monolayer cell cultures do not resemble intercellular cell‐to‐cell interaction and communication; however, three‐dimensional cell culture systems, such as organoids and spheroids, can mimic cellular interaction and architecture between hepatic cells. Previous studies have demonstrated the generation of hepatic or biliary organoids/spheroids using various cell sources including pluripotent stem cells, hepatic progenitor cells, primary cells from liver biopsies, and immortalized cell lines. Gene manipulation, such as transfection and transduction can be performed in organoids, and established organoids have functional characteristics which can be suitable for drug screening. This review summarizes current methodologies for organoid/spheroid formation and a potential for three‐dimensional hepatic cell cultures as in vitro models of cholangiopathies.

<h2>Abstract</h2><h3>Background</h3> The SARS-CoV-2 pandemic has had a huge impact on healthcare systems, resulting in many routine diagnostic procedures either being halted or postponed. <h3>Aims</h3> To evaluate whether the diagnoses of colorectal, gastric and pancreatic cancers have been impacted by the SARS-CoV-2 pandemic in Italy. <h3>Methods</h3> A survey designed to collect the number of histologically-proven diagnoses of the three cancers in gastroenterology services across Italy from January 1 to October 31 in 2017–2020. Non-parametric ANOVA for repeated measurements was applied to compare distributions by years and macro-areas. <h3>Results</h3> Compared to 2019, in 2020 gastric cancer diagnoses decreased by 15.9%, CRC by 11.9% and pancreatic by 9.9%. CRC distributions showed significant differences between all years, stomach cancer between 2018 and 2020 and 2019–2020, and pancreatic cancer only between 2017 and 2019. The 2019–2020 comparison showed fewer CRC diagnoses in the North (-13.7%), Center (-16.5%) and South (-4.1%), fewer stomach cancers in the North (-19.0%) and South (-9.4%), and fewer pancreatic cancers in the North (-14.1%) and Center (-4.7%), with an increase in the South (+12.3%). Distributions of CRC and gastric cancer were significantly different between all years in the North. <h3>Conclusions</h3> This survey highlights the concerning effects of the COVID-19 pandemic on the diagnostic yield of gastroenterology services for stomach, colorectal and pancreatic cancers in Italy.

Cholangiocarcinoma (CCA) is a very aggressive cancer showing the presence of high cancer stem cells (CSCs). Doublecortin-like kinase1 (DCLK1) has been demonstrated as a CSC marker in different gastroenterological solid tumors. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA).Specimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5 [leucine-rich repeat-containing G protein-coupled receptor], CD [clusters of differentiation] 90, EpCAM [epithelial cell adhesion molecule], CD133, and CD13), and primary cell cultures were prepared. DCLK1 expression was analyzed in CCA cell cultures by real-time quantitative PCR, western blot, and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2-IN-1) on cell proliferation (MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, cell population doubling time), apoptosis, and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and real-time quantitative PCR. DCLK1 serum concentration was analyzed by enzyme-linked immunosorbent assay. We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCALGR5+ and in iCCACD133+ cells compared with unsorted cells. LRRK2-IN-1 showed an anti-proliferative effect in a dose-dependent manner. LRRK2-IN-1 markedly impaired cell proliferation, induced apoptosis, and decreased colony formation capacity and colony size in both iCCA and pCCA compared with the untreated cells. In situ analysis confirmed that DCLK1 is present only in tumors, and not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of patients with iCCA (high), pCCA (high), HCC (low), and cirrhosis (low), but it was almost undetectable in healthy controls.DCLK1 characterizes a specific CSC subpopulation of iCCACD133+ and pCCALGR5+ , and its inhibition exerts anti-neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis.

Liver fibrosis holds a relevant prognostic meaning in primary biliary cholangitis (PBC). Noninvasive fibrosis evaluation using vibration-controlled transient elastography (VCTE) is routinely performed. However, there is limited evidence on its accuracy at diagnosis in PBC. We aimed to estimate the diagnostic accuracy of VCTE in assessing advanced fibrosis (AF) at disease presentation in PBC.We collected data from 167 consecutive treatment-naïve PBC patients who underwent liver biopsy (LB) at diagnosis at six Italian centers. VCTE examinations were completed within 12 weeks of LB. Biopsies were scored by two blinded expert pathologists, according to the Ludwig system. Diagnostic accuracy was estimated using the area under the receiver operating characteristic curves (AUROCs) for AF (Ludwig stage ≥III). Effects of biochemical and clinical parameters on liver stiffness measurement (LSM) were appraised. The derivation cohort consisted of 126 patients with valid LSM and LB; VCTE identified patients with AF with an AUROC of 0.89. LSM cutoffs ≤6.5 and >11.0 kPa enabled to exclude and confirm, respectively, AF (negative predictive value [NPV] = 0.94; positive predictive value [PPV] = 0.89; error rate = 5.6%). These values were externally validated in an independent cohort of 91 PBC patients (NPV = 0.93; PPV = 0.89; error rate = 8.6%). Multivariable analysis found that the only parameter affecting LSM was fibrosis stage. No association was found with BMI and liver biochemistry.In a multicenter study of treatment-naïve PBC patients, we identified two cutoffs (LSM ≤6.5 and >11.0 kPa) able to discriminate at diagnosis the absence or presence, respectively, of AF in PBC patients, with external validation. In patients with LSM between these two cutoffs, VCTE is not reliable and liver biopsy should be evaluated for accurate disease staging. BMI and liver biochemistry did not affect LSMs.

Common precursors for the liver, biliary tree, and pancreas exist at an early stage of development in the definitive endoderm forming the foregut. We have identified and characterised endodermal stem/progenitor cells with regenerative potential persisting in the adult human duodenum.Human duodena were obtained from organ donors, and duodenal submucosal gland cells were isolated after removal of the mucosa layer. Cells were cultured on plastic or as organoids and were transplanted into severe combined immunodeficient (SCID) mouse livers.In situ studies of submucosal glands in the human duodenum revealed cells expressing stem/progenitor cell markers that had unique phenotypic traits distinguishable from intestinal crypt cells. Genetic signature studies indicated that the cells are closer to biliary tree stem cells and to definitive endodermal cells than to adult hepatocytes, supporting the interpretation that they are endodermal stem/progenitor cells. In vitro, human duodenal submucosal gland cells demonstrated clonal growth, capability to form organoids, and ability to acquire functional hepatocyte traits. In vivo, transplanted cells engrafted into the livers of immunocompromised mice and differentiated to mature liver cells. In an experimental model of fatty liver, human duodenal submucosal gland cells were able to rescue hosts from liver damage by supporting repopulation and regeneration of the liver.A cell population with clonal growth and organoid formation capability, which has liver differentiation potency in vitro and in vivo in murine experimental models, is present within adult duodenal submucosal glands. These cells can be isolated, do not require reprogramming, and thus could potentially represent a novel cell source for regenerative medicine of the liver.Cell therapies for liver disease could represent an option to support liver function, but the identification of sustainable and viable cell sources is critical. Here, we describe a cell population with organoid formation capability and liver-specific regenerative potential in submucosal glands of the human duodenum. Duodenal submucosal gland cells are isolated from adult organs, do not require reprogramming, and could rescue hepatocellular damage in preclinical models of chronic, but not acute, liver injury. Duodenal submucosal gland cells could represent a potential candidate cell source for regenerative medicine of the liver, but the determination of cell dose and toxicity is needed before clinical testing in humans.

Fragments of small interlobular bile ducts averaging 20 microns in diameter can be isolated from rat liver. These isolated bile duct units form luminal spaces that are impermeant to dextran-40 and expand in size when cultured in 10 microM forskolin for 24-48 hr. Secretion is Cl- and HCO3- dependent and is stimulated by forskolin > dibutyryl cAMP > secretion but not by dideoxyforskolin, as assessed by video imaging techniques. Secretin stimulates Cl-/HCO3- exchange activity, and intraluminal pH increases after forskolin administration. These studies establish that small polarized physiologically intact interlobular bile ducts can be isolated from rat liver. These isolated bile duct units should be useful preparations for assessing the transport properties of small bile duct segments, which are the primary site of injury in cholestatic liver disorders, known as "vanishing bile duct syndromes."

Cholangiocytes are the epithelial cells which line the intrahepatic biliary tree, a network of interconnecting ducts of increasing diameter from the duct of Hering to the extrahepatic bile ducts (1.Roberts SK Ludwig J Larusso NF The pathobiology of biliary epithelia.Gastroenterology. 1997; 112: 269-279Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 2.Strazzabosco M New insights into cholangiocyte physiology.J Hepatol. 1997; 27: 945-952Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 3.Kanno N LeSage G Glaser S Alvaro D Alpini G Functional heterogeneity of the intrahepatic biliary epithelium.Hepatology. 2000; 31: 555-561Crossref PubMed Scopus (111) Google Scholar, 4.Boyer JL Vanishing Bile Duct Syndrome - from bench to bed side.in: Alvaro D Benedetti A Strazzabosco M Vanishing Bile Duct Syndrome. Kluwer Academic Publishers, London1997: 240-246Google Scholar, 5.Alvaro D Biliary epithelium: a new chapter in cell biology.It J Gastroenterol Hepatol. 1999; 31: 78-83PubMed Google Scholar, 6.Alpini G Lenzi R Sarkozi L Tavoloni N Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Crossref PubMed Scopus (289) Google Scholar). Cholangiocytes determine the final bile composition through a series of secretory and absorptive processes regulated by a number of hormones and neuropeptides (2.Strazzabosco M New insights into cholangiocyte physiology.J Hepatol. 1997; 27: 945-952Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 3.Kanno N LeSage G Glaser S Alvaro D Alpini G Functional heterogeneity of the intrahepatic biliary epithelium.Hepatology. 2000; 31: 555-561Crossref PubMed Scopus (111) Google Scholar, 4.Boyer JL Vanishing Bile Duct Syndrome - from bench to bed side.in: Alvaro D Benedetti A Strazzabosco M Vanishing Bile Duct Syndrome. Kluwer Academic Publishers, London1997: 240-246Google Scholar, 5.Alvaro D Biliary epithelium: a new chapter in cell biology.It J Gastroenterol Hepatol. 1999; 31: 78-83PubMed Google Scholar). The intrahepatic bile ducts are the target of damage in a group of chronic cholestatic liver diseases recently classified as Vanishing Bile Duct Syndromes (4.Boyer JL Vanishing Bile Duct Syndrome - from bench to bed side.in: Alvaro D Benedetti A Strazzabosco M Vanishing Bile Duct Syndrome. Kluwer Academic Publishers, London1997: 240-246Google Scholar, 7.Desmet V Roskams T Van Eyken P Histopathology of vanishing bile duct diseases.Adv Clin Pathol. 1998; 2: 87-99PubMed Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar). These diseases are characterized by the progressive disappearance of intrahepatic bile ducts, which leads to a severe ductopenic condition in the terminal stages (7.Desmet V Roskams T Van Eyken P Histopathology of vanishing bile duct diseases.Adv Clin Pathol. 1998; 2: 87-99PubMed Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar). The residual bile ducts tend to proliferate as a compensatory mechanism (8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar). Thus, the course of these diseases is characterized by a balance between damage (loss) of bile ducts and compensatory proliferation of the residual ducts. The terminal decompensated stages are characterized by the inefficacy of proliferation to balance for the loss of intrahepatic bile ducts. Therefore, a therapeutic strategy designed to support efficacious cholangiocyte proliferation could delay progression to ductopenia. This represents a challenge for the future.The aim of this review is to focus on current knowledge of the regulation and dysregulation of cholangiocyte proliferation.Cholangiocyte ProliferationA peculiar property of cholangiocytes is the capacity to proliferate, as evidenced in specific experimental conditions as well as in different human pathological conditions (1.Roberts SK Ludwig J Larusso NF The pathobiology of biliary epithelia.Gastroenterology. 1997; 112: 269-279Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 2.Strazzabosco M New insights into cholangiocyte physiology.J Hepatol. 1997; 27: 945-952Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 3.Kanno N LeSage G Glaser S Alvaro D Alpini G Functional heterogeneity of the intrahepatic biliary epithelium.Hepatology. 2000; 31: 555-561Crossref PubMed Scopus (111) Google Scholar, 4.Boyer JL Vanishing Bile Duct Syndrome - from bench to bed side.in: Alvaro D Benedetti A Strazzabosco M Vanishing Bile Duct Syndrome. Kluwer Academic Publishers, London1997: 240-246Google Scholar, 5.Alvaro D Biliary epithelium: a new chapter in cell biology.It J Gastroenterol Hepatol. 1999; 31: 78-83PubMed Google Scholar, 6.Alpini G Lenzi R Sarkozi L Tavoloni N Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Crossref PubMed Scopus (289) Google Scholar, 7.Desmet V Roskams T Van Eyken P Histopathology of vanishing bile duct diseases.Adv Clin Pathol. 1998; 2: 87-99PubMed Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar, 10.Uchida T Peters RL The nature and origin of proliferated bile ductules in alcoholic liver.Am J Clin Pathol. 1983; 79: 326-333Crossref PubMed Scopus (74) Google Scholar, 11.Knowles DM Wolff M Focal nodular hyperplasia of the liver: a clinicopathologic study and review of the literature.Hum Pathol. 1976; 7: 533-545Abstract Full Text PDF PubMed Scopus (142) Google Scholar, 12.Miller DJ Keeton DG Webber BL Pathol FF Saunders SJ Jaundice in severe bacterial infection.Gastroenterology. 1976; 71: 94-97Abstract Full Text PDF PubMed Scopus (98) Google Scholar). Neoplastic proliferation leads to cholangiocarcinoma, which is one of the worst malignancies (13.Celli A Que FG Dysregulation of apoptosis in the cholangiopathies and cholangiocarcinoma.Semin Liver Dis. 1998; 2: 177-185Crossref Scopus (59) Google Scholar).Cholangiocyte proliferation is usually classified as type I (“typical”), type II(“atypical”) and type III (oval cell) proliferation. Type I or “typical” cholangiocyte proliferation is a hyperplastic reaction resulting in an increased number of intrahepatic bile ducts, which, however, remain confined to portal spaces (7.Desmet V Roskams T Van Eyken P Histopathology of vanishing bile duct diseases.Adv Clin Pathol. 1998; 2: 87-99PubMed Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar). The proliferating cholangiocytes form a well-differentiated three-dimensional network of tubular structures with a well-defined lumen (7.Desmet V Roskams T Van Eyken P Histopathology of vanishing bile duct diseases.Adv Clin Pathol. 1998; 2: 87-99PubMed Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar). In the rat, “typical” cholangiocyte proliferation is observed after bile duct ligation (BDL) (6.Alpini G Lenzi R Sarkozi L Tavoloni N Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Crossref PubMed Scopus (289) Google Scholar), 70% hepatectomy (14.LeSage G Glaser S Gubba S Robertson WE Phinizy JL Lasater J et al.Regrowth of the rat biliary tree after 70% partial hepatectomy is coupled to increased secretin-induced ductal bile secretion.Gastroenterology. 1996; 111: 1633-1644Abstract Full Text PDF PubMed Scopus (123) Google Scholar), chronic α-naphthylisothiocyanate feeding (15.Goldfarb S Singer EJ Popper H Experimental cholangitis due to a-naphthylisothiocyanate (ANIT).Am J Path. 1962; 40: 585-598Google Scholar), chronic L-proline treatment (16.Vacanti JP Folkman J Bile duct enlargement by infusion of L-proline: potential significance in biliary atresia.J Pediatr Surg. 1979; 14: 814-818Abstract Full Text PDF PubMed Scopus (18) Google Scholar), prolonged oral administration of lithocholate (LCA), chenodeoxycholate and taurocholate (TCA) (17.Palmer RH Ruban Z Production of bile duct hyperplasia and gallstones by lithocholic acid.J Clin Invest. 1966; 45: 1255-1267Crossref PubMed Scopus (90) Google Scholar, 18.Alpini G Glaser S Ueno Y Phinizy JL Rodgers R Francis H et al.Bile acid feeding induces cholangiocyte proliferation and secretion: evidence for bile acid-regulated ductal secretion.Gastroenterology. 1999; 116: 179-186Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In humans, a “typical” proliferation may be observed in acute severe obstructive cholestasis (19.James L Lygidakis NJ van Eyken P Tanka AK Bosch KS Ramaekers FC et al.Application of keratin immunocytochemistry and sirius red staining in evaluation of intrahepatic changes with acute extrahepatic cholestasis due to hepatic duct carcinoma.Hepato-Gastroenterology. 1989; 36: 151-155PubMed Google Scholar) as well as in the early phases of chronic cholestatic liver diseases (7.Desmet V Roskams T Van Eyken P Histopathology of vanishing bile duct diseases.Adv Clin Pathol. 1998; 2: 87-99PubMed Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar). It is quite accepted that the “typical” ductal proliferation results from the elongation of pre-existing bile ducts located within portal areas. This conclusion arises from a number of observations: i) in the BDL rat proliferating ducts retain immunohistochemical, ultrastructural and functional characteristics of their normal counterparts and appear, morphologically, as elongation of pre-existing ducts (6.Alpini G Lenzi R Sarkozi L Tavoloni N Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Crossref PubMed Scopus (289) Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar); ii) thymidine labeling studies were consistent with a proliferation of pre-existing ducts (20.Slott PA Liu MH Tavoloni N Origin, pattern, and mechanism of bile duct proliferation following biliary obstruction in the rat.Gastroenterology. 1990; 99: 466-477Abstract Full Text PDF PubMed Google Scholar, 21.Grisham JW A morphological study of deoxyribonucleic acid synthesis and cell proliferation, in regenerating rat liver: autoradiography with thymidine-H3.Cancer Res. 1962; 22: 842-849PubMed Google Scholar); iii) in humans with extrahepatic biliary obstruction of recent onset, proliferating parenchymal and bile duct cells express their respective normal cytokine profile (19.James L Lygidakis NJ van Eyken P Tanka AK Bosch KS Ramaekers FC et al.Application of keratin immunocytochemistry and sirius red staining in evaluation of intrahepatic changes with acute extrahepatic cholestasis due to hepatic duct carcinoma.Hepato-Gastroenterology. 1989; 36: 151-155PubMed Google Scholar). However, in young rats submitted to BDL, proliferating ductular cells show characteristics of progenitor cells given the expression of full length α-fetoprotein and stem cell factor, c-kit, thus indicating that, in “typical” proliferation as well, the participation of a stem cell compartment cannot be completely ruled out (22.Omori M Evarts RP Omori N Hu Z Marsden ER Thorgeirsson SS Expression of α-feto-protein and stem cell factor/c-kit system in BDL young rats.Hepatology. 1997; 25: 1115-1122Crossref PubMed Scopus (75) Google Scholar).Type II or “atypical” ductular proliferation occurs in rodents in association with oval cell proliferation after chronic administration of chemicals such as D-galactosamine (23.Kuhlmann WD Wurster K Correlation of histology and alpha 1-fetoprotein resurgence in rat liver regeneration after experimental injury by galactosamine.Virchows Arch A Pathol Anat Histol. 1980; 387: 47-57Crossref PubMed Scopus (35) Google Scholar) and carbon tetrachloride (CCl4) (24.LeSage GD Benedetti A Glaser S Marucci L Tretjak Z Caligiuri A et al.Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes from normal rat liver.Hepatology. 1999; 29: 307-319Crossref PubMed Scopus (99) Google Scholar). In humans, it occurs after massive hepatic necrosis, in alcoholic liver diseases, long-standing extrahepatic biliary obstruction, focal nodular hyperplasia and chronic cholestatic liver diseases [(i.e. primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC)] (7.Desmet V Roskams T Van Eyken P Histopathology of vanishing bile duct diseases.Adv Clin Pathol. 1998; 2: 87-99PubMed Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar, 25.Desmet VJ Current problems in diagnosis of biliary disease and-cholestasis.Semin Liv Dis. 1986; 6: 233-245Crossref PubMed Scopus (38) Google Scholar, 26.Fausto N Webber EM Liver regeneration.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter D Shafritz DA The Liver: Biology and Pathobiology. 3rd ed. Raven Press, Ltd, New York1994: 1059-1084Google Scholar, 27.Nagore N Howe S Boxer L Scheuer PJ Liver cell rosettes: structural differences in cholestasis and hepatitis.Liver. 1989; 9: 43-51Crossref PubMed Scopus (32) Google Scholar). The process is characterized by an irregular proliferation of intrahepatic bile ducts which are not confined to portal areas but spread into periportal and parenchymal regions as wedge-shaped extensions creating an irregular portal-parenchymal interface(“biliary piecemeal necrosis”). These proliferating ductular structures are arranged in a three-dimensional network of tortuous and irregular conduits that do not possess a well-defined lumen and are associated with edema and neutrophil infiltration (7.Desmet V Roskams T Van Eyken P Histopathology of vanishing bile duct diseases.Adv Clin Pathol. 1998; 2: 87-99PubMed Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar, 25.Desmet VJ Current problems in diagnosis of biliary disease and-cholestasis.Semin Liv Dis. 1986; 6: 233-245Crossref PubMed Scopus (38) Google Scholar, 26.Fausto N Webber EM Liver regeneration.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter D Shafritz DA The Liver: Biology and Pathobiology. 3rd ed. Raven Press, Ltd, New York1994: 1059-1084Google Scholar, 27.Nagore N Howe S Boxer L Scheuer PJ Liver cell rosettes: structural differences in cholestasis and hepatitis.Liver. 1989; 9: 43-51Crossref PubMed Scopus (32) Google Scholar). This implies that the neoformed bile ducts are often functionally inefficient, andthe lack of anatomic contact with the canalicular spaces and peribiliary plexus around them further supports this concept. At variance with “typical” proliferation, in “atypical” proliferation, transitional or intermediate cells with phenotypical characteristics of both hepatocytes and cholangiocytes have been documented (28.Wegmann R Corcos V Caroli J Histoenzymologie des ductules biliaires chez l'embryon humain normal et au cours des cirrhoses humaines.Arch Mal Appar Dig. 1967; 54: 215-228Google Scholar). This favors the view that this type II hyperplasia originates from ductular metaplasia or transformation of hepatic liver cell cords (i.e., retrodifferentiation of hepatocytes into cholangiocytes) and not from replication of pre-existing cholangiocytes (9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar, 25.Desmet VJ Current problems in diagnosis of biliary disease and-cholestasis.Semin Liv Dis. 1986; 6: 233-245Crossref PubMed Scopus (38) Google Scholar, 26.Fausto N Webber EM Liver regeneration.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter D Shafritz DA The Liver: Biology and Pathobiology. 3rd ed. Raven Press, Ltd, New York1994: 1059-1084Google Scholar, 27.Nagore N Howe S Boxer L Scheuer PJ Liver cell rosettes: structural differences in cholestasis and hepatitis.Liver. 1989; 9: 43-51Crossref PubMed Scopus (32) Google Scholar). Three-dimensional reconstruction studies support this concept (29.Yamada S Howe s Scheuer PJ Three-dimensional reconstruction of biliary pathways in primary biliary cirrhosis: a computer-assisted study.J Pathol. 1987; 152: 317-323Crossref PubMed Scopus (36) Google Scholar). As demonstrated by Hillan et al. (30.Hillan KJ Burt AD George WD Macsween RN Grifiths MR Bradley JA Intrasplenic hepatocytes transplantation in rats with exprerimental liver injury: morphological and morphometric studies.J Pathol. 1989; 159: 67-73Crossref PubMed Scopus (30) Google Scholar), when hepatocytes are transplanted into the spleen of BDL rats, but not normal rats, they can give rise to bile duct structures, suggesting that a pattern of hormones/growth factors during BDL may trigger the ductal metaplasia of hepatocytes. The alternative hypothesis that the “atypical” proliferation may arise from a stem cell compartment has also been considered (31.Scarpelli DG Multipotent developmental capacity of cells in the adult animal.Lab Invest. 1985; 52: 331-333PubMed Google Scholar). Independently of their origin, proliferating cholangiocytes, especially in the “atypical” proliferation, acquire phenotypical features of a neuroendocrine epithelium: 1) expression of neuroendocrine markers (chromogranin A, glycolipid A2-B4, S-100 protein, neural cell adhesion molecule) and acquisition of neuroendocrine granules (8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 32.Roskams T Van den Oord JJ De Vos R Desmet VJ Neuroendocrine features or reactive bile ductules in cholestatic liver disease.Am J Pathol. 1990; 137: 1019-1025PubMed Google Scholar); 2) expression of parathyroid hormonerelated peptide (PTHrP), which is encoded by a growth factor regulated “early response” gene and which is involved in the growth and differentiation of the cell (33.Roskams T Campos RV Drucker DJ Desmet V Reactive human bile ductules express parathyroid hormone-related peptide.Histopathology. 1993; 23: 11-19Crossref PubMed Scopus (48) Google Scholar); 3) increased expression of and response to endothelin (34.Caligiuri A Glaser S Rodgers R Phinizy JL Robertson W Papa E et al.Endothelin 1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes.Am J Physiol. 1998; 275: G835-G846PubMed Google Scholar); 4) enhanced response to hormones/neuropeptides such as secretin, somatostatin and acetylcholine (1.Roberts SK Ludwig J Larusso NF The pathobiology of biliary epithelia.Gastroenterology. 1997; 112: 269-279Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 2.Strazzabosco M New insights into cholangiocyte physiology.J Hepatol. 1997; 27: 945-952Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 3.Kanno N LeSage G Glaser S Alvaro D Alpini G Functional heterogeneity of the intrahepatic biliary epithelium.Hepatology. 2000; 31: 555-561Crossref PubMed Scopus (111) Google Scholar, 4.Boyer JL Vanishing Bile Duct Syndrome - from bench to bed side.in: Alvaro D Benedetti A Strazzabosco M Vanishing Bile Duct Syndrome. Kluwer Academic Publishers, London1997: 240-246Google Scholar, 5.Alvaro D Biliary epithelium: a new chapter in cell biology.It J Gastroenterol Hepatol. 1999; 31: 78-83PubMed Google Scholar, 6.Alpini G Lenzi R Sarkozi L Tavoloni N Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Crossref PubMed Scopus (289) Google Scholar).Type III ductular hyperplasia, also called “oval cell” proliferation, occurs in the early stages of carcinogenesis in rat liver (35.Petropoulos CJ Yaswen P Panzica M Fausto N Cell lineages in liver carcinogenesis: possible clues from studies of the distribution of alpha-fetoprotein RNA sequences in cell populations isolated from normal, regenerating, and preneoplastic rat livers.Cancer Res. 1985; 43: 5762-5768Google Scholar, 36.Sirica AE Cihla HP Isolation and partial characterizations of oval and hyperplastic bile ductular cell-enriched populations from the livers of carcinogen and noncarcinogen-treated rats.Cancer Res. 1984; 44: 3454-3466PubMed Google Scholar, 37.Farber E Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetyl-aminofluorene, and 3′-methyl-4-dimethyl-aminoazobenzene.CancerRes. 1956; 16: 142-148Google Scholar, 38.Farber E The sequential analysis of cancer induction with chemicals.Acta Pathol Japonica. 1981; 31: 1-11PubMed Google Scholar) and is caused by a number of chemicals including ethionine and 2-acetyl-aminofluorene. This type of proliferation induces the formation of disorganized tubular structures with a poorly defined duct lumen which randomly spread into hepatic lobules, creating a distorted hepatic architecture (7.Desmet V Roskams T Van Eyken P Histopathology of vanishing bile duct diseases.Adv Clin Pathol. 1998; 2: 87-99PubMed Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar). Oval cells represent a subpopulation of nonparenchymal cells which are heterogeneous with regard to proteins and cell surface markers and which are unable to form recognizable ductular structures. Oval cells express phenotypes of parenchymal or neoplastic cells (i.e., α-fetoprotein, albumin, and G-6-PO4) (7.Desmet V Roskams T Van Eyken P Histopathology of vanishing bile duct diseases.Adv Clin Pathol. 1998; 2: 87-99PubMed Google Scholar, 8.Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Path Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 9.Desmet V Roskams T Van Eyken P Pathology of the biliary tree in cholestasis: ductular reaction.in: Manns MP Boyer JL Jansen PLM Reichen J Cholestatic Liver Diseases. Kluwer Academic Publishers, London1998: 143-154Google Scholar, 35.Petropoulos CJ Yaswen P Panzica M Fausto N Cell lineages in liver carcinogenesis: possible clues from studies of the distribution of alpha-fetoprotein RNA sequences in cell populations isolated from normal, regenerating, and preneoplastic rat livers.Cancer Res. 1985; 43: 5762-5768Google Scholar, 36.Sirica AE Cihla HP Isolation and partial characterizations of oval and hyperplastic bile ductular cell-enriched populations from the livers of carcinogen and noncarcinogen-treated rats.Cancer Res. 1984; 44: 3454-3466PubMed Google Scholar, 37.Farber E Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetyl-aminofluorene, and 3′-methyl-4-dimethyl-aminoazobenzene.CancerRes. 1956; 16: 142-148Google Scholar, 38.Farber E The sequential analysis of cancer induction with chemicals.Acta Pathol Japonica. 1981; 31: 1-11PubMed Google Scholar) and may differentiate into hepatocytes, bile duct epithelial cells, enterocytes and exocrine pancreatic cells (35.Petropoulos CJ Yaswen P Panzica M Fausto N Cell lineages in liver carcinogenesis: possible clues from studies of the distribution of alpha-fetoprotein RNA sequences in cell populations isolated from normal, regenerating, and preneoplastic rat livers.Cancer Res. 1985; 43: 5762-5768Google Scholar, 36.Sirica AE Cihla HP Isolation and partial characterizations of oval and hyperplastic bile ductular cell-enriched populations from the livers of carcinogen and noncarcinogen-treated rats.Cancer Res. 1984; 44: 3454-3466PubMed Google Scholar, 37.Farber E Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetyl-aminofluorene, and 3′-methyl-4-dimethyl-aminoazobenzene.CancerRes. 1956; 16: 142-148Google Scholar, 38.Farber E The sequential analysis of cancer induction with chemicals.Acta Pathol Japonica. 1981; 31: 1-11PubMed Google Scholar).Regulation of Cholangiocyte ProliferationSeveral growth factors, hormones and neuropetides as well as bile salts (BS) are involved in the regulation of cholangiocyte proliferation (Table 1). In the BDL rat, early studies suggested that the increased pressure in the biliary tree triggers duct proliferation (6.Alpini G Lenzi R Sarkozi L Tavoloni N Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Crossref PubMed Scopus (289) Google Scholar). However, by using the model of selective ligation of lobar ducts, Polimeno et al. (39.Polimeno L Azzarone A Zeng QH Panella C Subbotin V Carr B et al.Cell proliferation and oncogene expression after bile duct ligation in the rat: evidence of a specific growth effect on bile duct cells.Hepatology. 1995; 21: 1070-1088PubMed Google Scholar) showed that proliferation also occurs in the non-ligated lobes, suggesting that humoral factors play a more important role.TABLE 1Regulation of cholangiocyte proliferationStimulationInhibitionCytokines/growth factorsIL1α, IL6, TGF-α TNF-α, EGF, HGF, IGF-1TGF-β1, TGF-β2Hormones/neuropeptidesEstrogens, ACh, PTHrPSomatostatin, gastrinBile saltsLCA, TLCA, TCAUDCA, TUDCAIL=interleukin; TGF=transforming growth factor; TNF=tumor necrosis factor; EGF=epidermal growth factor; HGF=hepatocyte growth factor; IGF=insulin-like growth factor; ACh=acetylcholine; PTHrP=parathyroid hormone-related peptide; LCA=lithocholate; TLCA=taurolithocholate; TCA=taurocholate; UDCA=ursodeoxycholate; TUDCA=tauroursodeoxycholate. Open table in a new tab Growth factors and cytokinesStudies on the role and effect of growth factors in modulating cholangiocyte proliferation have been performed in vitro by using cell cultures, or in vivo in the BDL rat, in the model of partial hepatectomy or by studying cholangiocyte proliferation after injury.Epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF1), the interleukin-6 (IL-6)/gp-80 ligand/receptor system, IL 1α, and tumor necrosis factor α (TNFα) stimulate in vitro proliferation of cholangiocyte or cholangiocyte cell lines (40.Joplin R Hishida T Tsubouchi H Daikuhara Y Ayres R Neuberger JM et al.Human intrahepatic biliary epithelial cells proliferate in vitro in response to human hepatocyte growth factor.J Clin Invest. 1992; 90: 1284-1289Crossref PubMed Scopus (129) Google Scholar, 41.Matsumoto K Fuji H Michalopoulos G Fung JJ Demetris AJ Human biliary epithelial cells secrete and respond to cytokines and hepatocyte growth factor in vitro: interleukin-6, hepatocyte growth factor factor promote DNA synthesis in vitro.Hepatology. 1994; 20: 376-382Crossref PubMed Scopus (122) Google Scholar).In the BDL model, selective and “typical” cholangiocyte proliferation is associated with the increased intrahepatic expression of EGF, IL-6, basic fibroblast growth factor, and transforming growth factor β (TGFβ) (42.Liu Z Sakamoto T Ezure T Yokomuro S Mu

The aim of this study was to explore the intracellular signaling pathways involved in the stimulatory effects of estrogens on cholangiocyte proliferation. We investigated the tyrosine kinase-receptor pathway by evaluating the protein expression of total and phosphorylated mitogen-activated protein kinase (MAPK) isoform p44/p42 (e.g., extracellular signal-regulated kinase [ERK]1/2), the steroid-receptor coactivator Src and Shc (Src-homology/collagen protein). The study was performed in 3-week-old bile duct-ligated (BDL) rats, BDL rats treated with the antiestrogens, tamoxifen or Ici 182,780, and normal control rats. Proliferation was also evaluated in normal purified cholangiocytes treated with 17 beta estradiol in the presence or absence of tamoxifen, Ici 182,780, ERK, or Src inhibitors. After bile duct ligation, cholangiocyte proliferation was associated with a marked immunohistochemical nuclear positivity for phosphorylated (p)-ERK1/2, which was inhibited by in vivo treatment with tamoxifen or Ici 182,780. Protein expression of total and p-ERK1/2, and Shc in cholangiocytes isolated from BDL rats was markedly increased compared with controls and was inhibited by in vivo treatment with antiestrogens. In vitro, 17 beta estradiol-induced proliferation of isolated normal cholangiocyte was associated with increased (P <.01) protein expression of p-ERK1/2, Src, and Shc. Specific inhibitors of ER (Ici 182,780), ERK (U0125), and Src (PP2) inhibited in vitro 17 beta estradiol-induced cholangiocyte proliferation. In conclusion, this study showed that estrogens induced cholangiocyte proliferation by activating the Src/Shc/ERK pathway. This might suggest that pharmacologic modulation of ER, ERK, and/or Src could be proposed for the treatment of human pathology characterized by dysregulation of cholangiocyte proliferation.

Cholangiocyte proliferation and loss through apoptosis occur in cholestatic liver diseases. Our aim was to determine the mechanisms of apoptosis in an animal model of ductal hyperplasia. Rats were fed alpha-naphthylisothiocyanate (ANIT) for 2 wk and subsequently fed normal chow for 1, 2, and 4 wk. Proliferation was assessed in sections by morphometry and in small and large cholangiocytes by proliferating cellular nuclear antigen immunoblots and measurement of cAMP levels. Apoptosis and reactive oxygen species (ROS) levels were also assessed. ANIT feeding increased small and large cholangiocyte proliferation and apoptosis. Cessation of ANIT feeding was associated with decreased proliferation and a further increase in apoptosis in small and large cholangiocytes. Cholangiocytes from ANIT-fed rats or exposed to ANIT in vitro showed increased apoptosis and ROS generation. ANIT-induced duct injury results in enhanced proliferation and apoptosis in small and large cholangiocytes. The mechanism of ANIT-induced apoptosis may be due to ROS generation induced directly by ANIT. Our model has implications for understanding the pathophysiology of cholangiopathies (characterized by the coexistence of cholangiocyte apoptosis and proliferation).

We studied the role of gastrin in regulating cholangiocyte proliferation induced by bile duct ligation (BDL). In purified cholangiocytes, we evaluated (1) for the presence of cholecystokinin-B (CCK-B)/gastrin receptors, (2) the effect of gastrin on d -myo-Inositol 1,4,5-triphosphate (IP3) levels, and (3) the effect of gastrin on DNA synthesis and adenosine 3′, 5′-monophosphate (cAMP) levels in the absence or presence of CCK-A (L-364,718) and CCK-B/gastrin (L-365,260) receptor inhibitors, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetxymethyl ester) (BAPTA/AM; an intracellular Ca2+ chelator), and 2 protein kinase C (PKC) inhibitors, 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporin. To evaluate if gastrin effects on cholangiocyte proliferation are mediated by the isoform PKCα, we evaluated (1) for the presence of PKCα in cholangiocytes and (2) the effect of gastrin on the PKCα protein expression in a triton-soluble (containing cytoplasm + membrane) and a triton-insoluble (containing cytoskeleton) fraction. To evaluate the effects of gastrin in vivo, immediately following BDL, gastrin or bovine serum albumin (BSA) was infused by minipumps for 7 days to rats and we measured cholangiocyte growth and cAMP levels. We found CCK-B/gastrin receptors on cholangiocytes. Gastrin increased IP3 levels. Gastrin inhibited DNA synthesis and cAMP synthesis in cholangiocytes. Gastrin effects on cholangiocyte functions were blocked by L-365,260, BAPTA/AM, H7, and staurosporin but not by L-364,718. Gastrin induced translocation of PKCα from cholangiocyte cytoskeleton to membrane. In vivo, gastrin decreased cholangiocyte growth and cAMP synthesis compared with controls. We concluded that gastrin inhibits cholangiocyte growth in BDL rats by interacting with CCK-B/gastrin receptors through a signal transduction pathway involving IP3, Ca2+, and PKCα.

We evaluated cytotoxic effects of different unconjugated and glycine- and taurine-conjugated bile salts (BS) against bile duct epithelial cells in isolated bile ductule fragments and isolated perfused rat liver. Ultrastructural morphometric studies were performed in polarized rat bile ductule fragments exposed in vitro to increasing concentrations (10-100 micromol/L) of lithocholate (LCA), deoxycholate (DCA), chenodeoxycholate (CDCA), cholate (CA), ursodeoxycholate (UDCA), their taurine-conjugates, and glycoconjugates of cholic (GCA) or chenodeoxycholic acid (GCDCA) for 20, 30, or 75 minutes. To evaluate the cytotoxicity of unconjugated hydrophobic bile salts against biliary epithelium (BDE) in the whole liver, livers were isolated from rats with impaired taurine-conjugation capacity (beta-alanine treatment) and perfused for 70 minutes with 2 micromol/min LCA (n = 6), CDCA (n = 6), CA (n = 6), or 0.5 micromol/min tauro-LCA (n = 4). In isolated bile ductule fragments, hydrophobic unconjugated bile salts (LCA, CDCA, DCA) induced a marked damage of intracellular organelles, mainly mitochondria. The damage started at a concentration of 10 micromol/L and became prominent at concentrations higher than 50 micromol/L. No damage of the apical and basolateral membrane was seen and tight junctions appeared intact. UDCA, taurine and glycoconjugated bile salts failed to induce any evident ultrastructural alteration. In taurine-depleted isolated livers, perfused with LCA, CDCA, or CA, bile duct epithelial cells showed no evidence of intracellular damage, despite the increased biliary excretion of unconjugated BS. Marked alterations of the apical cell membrane were seen only in livers perfused with LCA and in isolated segments of the biliary epithelium. In contrast with biliary epithelium, hepatocytes showed prominent subcellular damage with CA and CDCA, and profound alterations of the canalicular membrane with LCA and tauro-LCA. We have shown that, in vitro, BDE cells are not damaged by taurine- or glycine-conjugated BS, but they are very sensitive to cytotoxicity of hydrophobic unconjugated BS. Such sensitivity is not present in the whole liver, probably because of the specificity of BS transport processes, the microvascular architecture of the bile ductal system, and the presence in bile of a physiological surfactant, such as phospholipids.

The peribiliary plexus plays a fundamental role in supporting the secretory and absorptive functions of biliary epithelium. Little information is available on the rearrangement of the peribiliary plexus during conditions associated with ductular proliferation. This study investigated the chronological modulation of bile duct and peribiliary plexus proliferation after common bile duct ligation in the rat.Light microscopy and scanning electron microscopy vascular corrosion cast technique was used to study the architecture of the peribiliary plexus in rats with 1, 2, and 4 weeks of common bile duct ligation or in sham-operated controls.After 1 week of common bile duct ligation, no evident change of hepatic microvasculature was observed despite significant proliferation of bile ducts. After 2 and 4 weeks, significant microvasculature proliferation was observed extending from the peribiliary plexus of bile tracts. Vascular proliferation coincides with the extension of portal tract connective tissue. No evidence of vascular proliferation or other morphological modifications was present at the level of sinusoids around the portal tracts.After common bile duct ligation, the peribillary plexus undergoes marked proliferation, thus supporting the increased nutritional and functional demands from the proliferated bile ductal system. However, the proliferation of the peribillary plexus only occurs after that of the bile ductal system.

We evaluated the expression of neurotrophins in rat cholangiocytes and the role and mechanisms by which nerve growth factor (NGF) modulates cholangiocyte proliferation.The expression of neurotrophins and their receptors was investigated by immunohistochemistry in liver sections and reverse-transcription polymerase chain reaction and immunoblots in isolated cholangiocytes. In vitro, the effect of NGF on cholangiocyte proliferation and signal transduction was investigated by immunoblotting for proliferating cell nuclear antigen, phosphorylated AKT (p-AKT), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated c-jun-N-terminal kinase, and phosphorylated p38. In vivo, rats that had undergone bile duct ligation (BDL) were treated with an anti-NGF antibody to immunoneutralize NGF and bile duct mass, proliferation, apoptosis, and inflammation were investigated by immunohistochemistry.NGF and its TrkA receptor were expressed by normal rat cholangiocytes and up-regulated following BDL. Cholangiocytes secrete NGF, and secretion is increased in proliferating BDL cholangiocytes. In vitro, NGF stimulated cholangiocyte proliferation, which was associated with enhanced p-AKT and p-ERK1/2 expression. NGF proliferation in vitro was partially blocked by the MEK inhibitor (UO126) and completely ablated by the phosphatidylinositol 3-kinase inhibitor (wortmannin). In vitro, NGF and estrogens have an additive effect on cholangiocyte proliferation by acting on phosphorylated TrkA and p-ERK1/2. In vivo, immunoneutralization of NGF decreased bile duct mass in BDL rats, which was associated with depressed proliferation and enhanced apoptosis and with increased portal inflammation.Cholangiocytes secrete NGF and express NGF receptors. NGF induces cholangiocyte proliferation by activating the ERK and, predominantly, the phosphatidylinositol 3-kinase pathway and exerts an additive effect in combination with estrogens on proliferation.

We studied the effect of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), in the regulation of cholangiocarcinoma growth. We determined the in vitro effect of GABA on the proliferation of the cholangiocarcinoma cell lines (Mz-ChA-1, HuH-28, and TFK-1) and evaluated the intracellular pathways involved. The effect of GABA on migration of Mz-ChA-1 cells was also evaluated. In vivo, Mz-ChA-1 cells were s.c. injected in athymic mice, and the effects of GABA on tumor size, tumor cell proliferation, apoptosis, collagen quantity, and the expression of vascular endothelial growth factor-A (VEGF-A) and VEGF-C (cancer growth regulators) were measured after 82 days. GABA decreased in vitro cholangiocarcinoma growth in a time-dependent and dose-dependent manner, by both cyclic AMP/protein kinase A- and D-myo-inositol-1,4,5-thriphosphate/Ca(2+)-dependent pathways, leading to down-regulation of extracellular signal-regulated kinase 1/2 phosphorylation. Blocking of GABA(A), GABA(B), and GABA(C) receptors prevented GABA inhibition of cholangiocarcinoma proliferation. GABA inhibited Mz-ChA-1 cell migration and, in vivo, significantly decreased tumor volume, tumor cell proliferation, and VEGF-A/C expression whereas increasing apoptosis compared with controls. An increase in collagen was evident in GABA-treated tumors. GABA decreases biliary cancer proliferation and reduces the metastatic potential of cholangiocarcinoma. GABA may represent a therapeutic agent for patients affected by malignancies of the biliary tract.

1. Bile salts and phospholipids from bile of chicken, dog, sheep, rat, ox, pig, guinea-pig and man were analyzed by high-performance liquid chromatography. 2. Bile salts showed marked differences in their hydrophilic properties, owing to their hydroxyl structure and type of conjugation. 3. Phospholipids were generally similar, containing 90–95% of phosphatidylcholine which was made of molecular species containing palmitic acid in the sn-1 position. 4. 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.

Histamine regulates many functions by binding to four histamine G-coupled receptor proteins (H1R, H2R, H3R and H4R). As H3R exerts their effects by coupling to Galpha(i/o) proteins reducing adenosine 3', 5'-monophosphate (cAMP) levels (a key player in the modulation of cholangiocyte hyperplasia/damage), we evaluated the role of H3R in the regulation of biliary growth. We posed the following questions: (1) Do cholangiocytes express H3R? (2) Does in vivo administration of (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH) (H3R agonist), thioperamide maleate (H3R antagonist) or histamine, in the absence/presence of thioperamide maleate, to bile duct ligated (BDL) rats regulate cholangiocyte proliferation? and (3) Does RAMH inhibit cholangiocyte proliferation by downregulation of cAMP-dependent phosphorylation of protein kinase A (PKA)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ets-like gene-1 (Elk-1)? The expression of H3R was evaluated in liver sections by immunohistochemistry and immunofluorescence, and by real-time PCR in cholangiocyte RNA from normal and BDL rats. BDL rats (immediately after BDL) were treated daily with RAMH, thioperamide maleate or histamine in the absence/presence of thioperamide maleate for 1 week. Following in vivo treatment of BDL rats with RAMH for 1 week, and in vitro stimulation of BDL cholangiocytes with RAMH, we evaluated cholangiocyte proliferation, cAMP levels and PKA, ERK1/2 and Elk-1 phosphorylation. Cholangiocytes from normal and BDL rats express H3R. The expression of H3R mRNA increased in BDL compared to normal cholangiocytes. Histamine decreased cholangiocyte growth of BDL rats to a lower extent than that observed in BDL RAMH-treated rats; histamine-induced inhibition of cholangiocyte growth was partly blocked by thioperamide maleate. In BDL rats treated with thioperamide maleate, cholangiocyte hyperplasia was slightly higher than that of BDL rats. In vitro, RAMH inhibited the proliferation of BDL cholangiocytes. RAMH inhibition of cholangiocyte growth was associated with decreased cAMP levels and PKA/ERK1/2/Elk-1 phosphorylation. Downregulation of cAMP-dependent PKA/ERK1/2/Elk-1 phosphorylation (by activation of H3R) is important in the inhibition of cholangiocyte growth in liver diseases.

We evaluated the morphological and functional features of hepatic cyst epithelium in adult autosomal dominant polycystic kidney disease (ADPKD). In six ADPKD patients, we investigated the morphology of cyst epithelium apical surface by scanning electron microscopy and the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF1), IGF1 receptors (IGF1-R), growth hormone receptor, the proliferation marker proliferating cell nuclear antigen, and pAKT by immunohistochemistry and immunofluorescence. Proliferation of liver cyst-derived epithelial cells was evaluated by both MTS proliferation assay and [(3)H]thymidine incorporation into DNA. The hepatic cyst epithelium displayed heterogeneous features, being normal in small cysts (<1 cm), characterized by rare or shortened cilia in 1- to 3-cm cysts, and exhibiting the absence of both primary cilia and microvilli in large cysts (>3 cm). Cyst epithelium showed marked immunohistochemical expression of ER, growth hormone receptor, IGF1, IGF1-R, proliferating cell nuclear antigen, and pAKT. IGF1 was 10-fold more enriched in the hepatic cyst fluid than in serum. Serum-deprived liver cyst-derived epithelial cells proliferated when exposed to 17beta-estradiol and IGF1 and when exposed to human cyst fluid. ER or IGF1-R antagonists inhibited the proliferative effect of serum readmission, cyst fluid, 17beta-estradiol, and IGF1. Our findings could explain the role of estrogens in accelerating the progression of ADPKD and may suggest a potential benefit of therapeutic strategies based on estrogen antagonism.

To discuss recent studies proposing new markers in serum or bile for the diagnosis and prognosis of cholangiocarcinoma (CCA), which could help in the differential diagnosis between malignant and benign biliary disorders or for the surveillance of disorders at risk, including primitive sclerosing cholangitis.In the last few years, efforts have been made to identify biomarkers with adequate diagnostic accuracy for CCA in serum or biological fluid. Studies have been focused on cytokines, growth factors or enzymes produced and secreted by CCA cells as well as on the proteomic analysis of serum and bile.The serum levels of interleukin 6, trypsinogen, mucin-5AC, soluble fragment of cytokeratin 19 and the platelet-lymphocyte ratio have been recently shown to help in the diagnosis of CCA with, in some cases, a prognostic value. As far as bile is concerned, the ratio of pancreatic elastase/amylase, mucin-4, minichromosome maintenance replication protein and insulin-like growth factor 1 have been explored, with the insulin-like growth factor 1 biliary concentration capable of completely discriminating CCA from benign biliary disorders and pancreatic cancer. We have also discussed advances in the proteomic of serum and bile, which seem promising in identifying new markers for CCA.

Caffeic acid phenethyl ester (CAPE) inhibits the growth of tumor cells and is a known inhibitor of nuclear factor kappa beta (NF-kappaB), which is constitutively active in cholangiocarcinoma (CCH) cells. We evaluated the effects of CAPE on CCH growth both in vitro and in vivo. Inhibition of NF-kappaB DNA-binding activity was confirmed in nuclear extracts treated with CAPE at 50, 40 and 20 microM. CAPE decreases the expression of NF-kappaB1 (p50) and RelA (p65). CAPE decreased the growth of a number of CCH cells but not normal cholangiocytes. Cell cycle decrease was seen by a decrease in PCNA protein expression and the number of BrdU-positive cells treated with CAPE at 20 microM compared to vehicle. Inhibition of growth and increased cell cycle arrest of Mz-ChA-1 cells by CAPE were coupled with increased apoptosis. Bax expression was increased, whereas Bcl-2 was decreased in cells treated with CAPE compared to vehicle. In vivo studies were performed in BALB/c nude (nu/nu) mice implanted subcutaneously with Mz-ChA-1 cells and treated with daily IP injections of DMSO or CAPE (10 mg/kg body weight in DMSO) for 77 days. Tumor growth was decreased and tumor latency was increased 2-fold in CAPE compared to vehicle-treated nude mice. In tumor samples, decreased CCH growth by CAPE was coupled with increased apoptosis. CAPE both in vivo and in vitro decreases the growth of CCH cells by increasing apoptosis. These results demonstrate that CAPE might be an important therapeutic tool in the treatment of CCH.

Cholangiocarcinoma is a strongly aggressive malignancy with a very poor prognosis. Effective therapeutic strategies are lacking because molecular mechanisms regulating cholangiocarcinoma cell growth are unknown. Furthermore, experimental in vivo animal models useful to study the pathophysiologic mechanisms of malignant cholangiocytes are lacking. Leptin, the hormone regulating caloric homeostasis, which is increased in obese patients, stimulates the growth of several cancers, such as hepatocellular carcinoma. The aim of this study was to define if leptin stimulates cholangiocarcinoma growth. We determined the expression of leptin receptors in normal and malignant human cholangiocytes. Effects on intrahepatic cholangiocarcinoma (HuH-28) cell proliferation, migration, and apoptosis of the in vitro exposure to leptin, together with the intracellular pathways, were then studied. Moreover, cholangiocarcinoma was experimentally induced in obese fa/fa Zucker rats, a genetically established animal species with faulty leptin receptors, and in their littermates by chronic feeding with thioacetamide, a potent carcinogen. After 24 weeks, the effect of leptin on cholangiocarcinoma development and growth was assessed. Normal and malignant human cholangiocytes express leptin receptors. Leptin increased the proliferation and the metastatic potential of cholangiocarcinoma cells in vitro through a signal transducers and activators of transcription 3-dependent activation of extracellular signal-regulated kinase 1/2. Leptin increased the growth and migration, and was antiapoptotic for cholangiocarcinoma cells. Moreover, the loss of leptin function reduced the development and the growth of cholangiocarcinoma. The experimental carcinogenesis model induced by thioacetamide administration is a valid and reproducible method to study cholangiocarcinoma pathobiology. Modulation of the leptin-mediated signal could be considered a valid tool for the prevention and treatment of cholangiocarcinoma.

In this paper we propose a novel, rapid and simple high-performance liquid chromatographic (HPLC) method for the identification and quantitation of individual phosphatidylcholine (PC) molecular species from natural mixtures. To overcome difficulties deriving from the lack of adequate standards and from the variability of the responses to UV spectrophotometric detectors currently used in HPLC analysis, we first fractionated and quantitated the major molecular species of a commercial egg PC by means of a preparative column. The identification of PC molecular species was confirmed by gas-liquid chromatographic analysis of fatty acids. We employed the fractions recovered from preparative HPLC to determine the detector calibration factors of the individual molecular species separated using an analytical, high-speed, reversed-phase HPLC column. The proposed method seems to be adequate for the analysis of PC from many biological sources. Its application to the analysis of human hepatic and gallbladder biliary PC is shown.

Large cholangiocytes secrete bicarbonate in response to secretin and proliferate after bile duct ligation by activation of cyclic adenosine 3', 5'-monophosphate signaling. The Ca(2+)-dependent adenylyl cyclase 8 (AC8, expressed by large cholangiocytes) regulates secretin-induced choleresis. Ca(2+)-dependent protein kinase C (PKC) regulates small cholangiocyte function. Because gamma-aminobutyric acid (GABA) affects cell functions by activation of both Ca(2+) signaling and inhibition of AC, we sought to develop an in vivo model characterized by large cholangiocyte damage and proliferation of small ducts. Bile duct ligation rats were treated with GABA for one week, and we evaluated: GABA(A), GABA(B), and GABA(C) receptor expression; intrahepatic bile duct mass (IBDM) and the percentage of apoptotic cholangiocytes; secretin-stimulated choleresis; and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation and activation of Ca(2+-)dependent PKC isoforms and AC8 expression. We found that both small and large cholangiocytes expressed GABA receptors. GABA: (i) induced apoptosis of large cholangiocytes and reduced large IBDM; (ii) decreased secretin-stimulated choleresis; and (iii) reduced ERK1/2 phosphorylation and AC8 expression in large cholangiocytes. Small cholangiocytes: (i) proliferated leading to increased IBDM; (ii) displayed activation of PKCbetaII; and (iii) de novo expressed secretin receptor, cystic fibrosis transmembrane regulator, Cl(-)/HCO(3)(-) anion exchanger 2 and AC8, and responded to secretin. Therefore, in pathologies of large ducts, small ducts replenish the biliary epithelium by amplification of Ca(2+)-dependent signaling and acquisition of large cholangiocyte phenotypes.

Very few studies assessed cholangiocarcinoma clinical characteristics.To evaluate the clinical characteristics of intra-hepatic (IH) and extra-hepatic (EH)-CCA.We performed a national survey based on a questionnaire.218 cholangiocarcinomas were observed (47% EH-CCA, 53% IH-CCA) with an age at the diagnosis higher for EH-CCA. Coexistence of cirrhosis or viral cirrhosis was more frequent in IH-CCA than EH-CCA. An incidental asymptomatic presentation occurred in 28% of IH-CCA vs 4% EH-CCA whilst, 74% EH-CCA vs 28% IH-CCA presented with jaundice. 91% of IH-CCA presented as a single intra-hepatic mass, whilst 50% of EH-CCA was peri-hilar. In the diagnostic work-up, 70% of all cholangiocarcinoma cases received at least 3 different imaging procedures. Tissue-proven diagnosis was obtained in 80% cholangiocarcinoma. Open surgery with curative intent was performed in 45% of IH-CCA and 29% EH-CCA. 18% IH-CCA vs 4% EH-CCA did not received treatment.In Italy IH-CCA is managed as frequently as EH-CCA. In comparison to EH-CCA, IH-CCA occurs at younger age and is more frequently associated with cirrhosis and with an incidental asymptomatic presentation. In contrast, most EH-CCAs are jaundiced at the diagnosis. Cholangiocarcinoma diagnostic management is cost- and time-consuming with curative surgical treatment applicable more frequently in IH-CCA.

Sex hormones regulate cholangiocyte hyperplasia in bile duct-ligated (BDL) rats. We studied whether follicle-stimulating hormone (FSH) regulates cholangiocyte proliferation. FSH receptor (FSHR) and FSH expression was evaluated in liver sections, purified cholangiocytes, and cholangiocyte cultures (NRICC). In vivo, normal female and male rats were treated with FSH or immediately after BDL with antide (a gonadotropin-releasing hormone antagonist blocking FSH secretion) or a neutralizing FSH antibody for 1 wk. We evaluated 1) cholangiocyte proliferation in sections and cholangiocytes and 2) changes in secretin-stimulated cAMP (functional index of cholangiocyte growth) levels, and ERK1/2 and Elk-1 phosphorylation. NRICC were stimulated with FSH before evaluation of proliferation, cAMP/IP 3 levels, and ERK1/2 and Elk-1 phosphorylation. To determine whether FSH regulates cholangiocyte proliferation by an autocrine mechanism, we evaluated the effects of 1) cholangiocyte supernatant (containing FSH) on NRICC proliferation and 2) FSH silencing in NRICC before measuring proliferation and ERK1/2 and Elk-1 phosphorylation. Cholangiocytes and NRICC express FSHR and FSH and secrete FSH. In vivo administration of FSH to normal rats increased, whereas administration of antide and anti-FSH antibody to BDL rats decreased 1) ductal mass and 2) secretin-stimulated cAMP levels, proliferation, and ERK1/2 and Elk-1 phosphorylation in cholangiocytes compared with controls. In NRICC, FSH increased cholangiocyte proliferation, cAMP levels, and ERK1/2 and Elk-1 phosphorylation. The supernatant of cholangiocytes increased NRICC proliferation, inhibited by preincubation with anti-FSH antibody. Silencing of FSH gene decreases cholangiocyte proliferation and ERK1/2 and Elk-1 phosphorylation. Modulation of cholangiocyte FSH expression may be important for the management of cholangiopathies.

In bile duct-ligated (BDL) rats, large cholangiocytes proliferate by activation of cAMP-dependent signaling. Melatonin, which is secreted from pineal gland as well as extrapineal tissues, regulates cell mitosis by interacting with melatonin receptors (MT1 and MT2) modulating cAMP and clock genes. In the liver, melatonin suppresses oxidative damage and ameliorates fibrosis. No information exists regarding the role of melatonin in the regulation of biliary hyperplasia. We evaluated the mechanisms of action by which melatonin regulates the growth of cholangiocytes. In normal and BDL rats, we determined the hepatic distribution of MT1, MT2, and the clock genes, CLOCK, BMAL1, CRY1, and PER1. Normal and BDL (immediately after BDL) rats were treated in vivo with melatonin before evaluating 1) serum levels of melatonin, bilirubin, and transaminases; 2) intrahepatic bile duct mass (IBDM) in liver sections; and 3) the expression of MT1 and MT2, clock genes, and PKA phosphorylation. In vitro, large cholangiocytes were stimulated with melatonin in the absence/presence of luzindole (MT1/MT2 antagonist) and 4-phenyl-2-propionamidotetralin (MT2 antagonist) before evaluating cell proliferation, cAMP levels, and PKA phosphorylation. Cholangiocytes express MT1 and MT2, CLOCK, BMAL1, CRY1, and PER1 that were all upregulated following BDL. Administration of melatonin to BDL rats decreased IBDM, serum bilirubin and transaminases levels, the expression of all clock genes, cAMP levels, and PKA phosphorylation in cholangiocytes. In vitro, melatonin decreased the proliferation, cAMP levels, and PKA phosphorylation, decreases that were blocked by luzindole. Melatonin may be important in the management of biliary hyperplasia in human cholangiopathies.

Background & Aims Biliary tree, liver, and pancreas share a common embryological origin. We previously demonstrated the presence of stem/progenitor cells of endodermal origin in the adult human extrahepatic biliary tree. This study evaluated the human foetal biliary trees as sources of stem/progenitor cells of multiple endodermal-derived mature fates. Methods Human foetal intrahepatic and extrahepatic biliary tree tissues and isolated cells were tested for cytoplasmic and surface markers of stem cells and committed progenitors, as well as endodermal transcription factors requisite for a liver versus pancreatic fate. In vitro and in vivo experiments were conducted to evaluate the potential mature fates of differentiation. Results Foetal biliary tree cells proliferated clonogenically for more than 1 month on plastic in a serum-free Kubota medium. After culture expansion, cells exhibited multipotency and could be restricted to certain lineages under defined microenvironments, including hepatocytes, cholangiocytes, and pancreatic islet cells. Transplantation of foetal biliary tree cells into the livers of immunodeficient mice resulted in effective engraftment and differentiation into mature hepatocytes and cholangiocytes. Conclusions Foetal biliary trees contain multipotent stem/progenitor cells comparable with those in adults. These cells can be easily expanded and induced in vitro to differentiate into liver and pancreatic mature fates, and engrafted and differentiated into mature cells when transplanted in vivo. These findings further characterise the development of these stem/progenitor cell populations from foetuses to adults, which are thought to contribute to liver and pancreas organogenesis throughout life. Biliary tree, liver, and pancreas share a common embryological origin. We previously demonstrated the presence of stem/progenitor cells of endodermal origin in the adult human extrahepatic biliary tree. This study evaluated the human foetal biliary trees as sources of stem/progenitor cells of multiple endodermal-derived mature fates. Human foetal intrahepatic and extrahepatic biliary tree tissues and isolated cells were tested for cytoplasmic and surface markers of stem cells and committed progenitors, as well as endodermal transcription factors requisite for a liver versus pancreatic fate. In vitro and in vivo experiments were conducted to evaluate the potential mature fates of differentiation. Foetal biliary tree cells proliferated clonogenically for more than 1 month on plastic in a serum-free Kubota medium. After culture expansion, cells exhibited multipotency and could be restricted to certain lineages under defined microenvironments, including hepatocytes, cholangiocytes, and pancreatic islet cells. Transplantation of foetal biliary tree cells into the livers of immunodeficient mice resulted in effective engraftment and differentiation into mature hepatocytes and cholangiocytes. Foetal biliary trees contain multipotent stem/progenitor cells comparable with those in adults. These cells can be easily expanded and induced in vitro to differentiate into liver and pancreatic mature fates, and engrafted and differentiated into mature cells when transplanted in vivo. These findings further characterise the development of these stem/progenitor cell populations from foetuses to adults, which are thought to contribute to liver and pancreas organogenesis throughout life.

Efforts to identify cell sources and approaches for cell therapy of liver diseases are ongoing, taking into consideration the limits recognized for adult liver tissue and for other forms of stem cells. In the present study, we described the first procedure of via hepatic artery transplantation of human fetal biliary tree stem cells in patients with advanced cirrhosis. The cells were immune-sorted from human fetal biliary tree by protocols in accordance with current good manufacturing practice (cGMP) and extensively characterized. Two patients with advanced liver cirrhosis (Child-Pugh C) have been submitted to the procedure and observed through a 12 months follow-up. The resulting procedure was found absolutely safe. Immuno-suppressants were not required, and the patients did not display any adverse effects correlated with cell transplantation or suggestive of immunological complications. From a clinical point of view, both patients showed biochemical and clinical improvement during the 6 month follow-up and the second patient maintained a stable improvement for 12 months. This report represents proof of the concept that the human fetal biliary tree stem cells are a suitable and large source for cell therapy of liver cirrhosis. The isolation procedure can be carried out under cGMP conditions and, finally, the infusion procedure is easy and safe for the patients. This represents the basis for forthcoming controlled clinical trials.

Genome-wide association studies (GWAS) have successfully identified several loci associated with primary biliary cirrhosis (PBC) risk. Pathway analysis complements conventional GWAS analysis. We applied the recently developed linear combination test for pathways to datasets drawn from independent PBC GWAS in Italian and Canadian subjects. Of the Kyoto Encyclopedia of Genes and Genomes and BioCarta pathways tested, 25 pathways in the Italian dataset (449 cases, 940 controls) and 26 pathways in the Canadian dataset (530 cases, 398 controls) were associated with PBC susceptibility (P<0.05). After correcting for multiple comparisons, only the eight most significant pathways in the Italian dataset had FDR <0.25 with tumor necrosis factor/stress-related signaling emerging as the top pathway (P=7.38 × 10⁻⁴, FDR=0.18). Two pathways, phosphatidylinositol signaling and hedgehog signaling, were replicated in both datasets (P<0.05), and subjected to two additional complementary pathway tests. Both pathway signals remained significant in the Italian dataset on modified gene set enrichment analysis (P<0.05). In both GWAS, variants nominally associated with PBC were significantly overrepresented in the phosphatidylinositol pathway (Fisher exact P<0.05). These results point to established and novel pathway-level associations with inherited predisposition to PBC that, on further independent replication and functional validation, may provide fresh insights into PBC etiology.

Secretin receptor (SR), only expressed by cholangiocytes, plays a key role in the regulation of biliary damage and liver fibrosis. The aim of this study was to determine the effects of genetic depletion of SR in Mdr2-/- mice on intrahepatic biliary mass, liver fibrosis, senescence, and angiogenesis. 12 wk SR-/-, Mdr2-/-, and SR-/-/Mdr2-/- mice with corresponding wild-type mice were used for the in vivo studies. Immunohistochemistry or immunofluorescence was performed in liver sections for (i) biliary expression of SR; (ii) hematoxylin and eosin; (iii) intrahepatic biliary mass by CK-19; (iv) fibrosis by Col1a1 and α-SMA; (v) senescence by SA-β-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-β1 levels were measured in serum and cholangiocyte supernatant by ELISA. In total liver, isolated cholangiocytes or HSCs, we evaluated the expression of fibrosis markers (FN-1 and Col1a1); senescence markers (p16 and CCL2); microRNA 125b and angiogenesis markers (VEGF-A, VEGFR-2, CD31, and vWF) by immunoblots and/or qPCR. In vitro, we measured the paracrine effect of cholangiocyte supernatant on the expression of senescent and fibrosis markers in human hepatic stellate cells (HHSteCs). The increased level of ductular reaction, fibrosis, and angiogenesis in Mdr2-/- mice was reduced in SR-/-/Mdr2-/- mice. Enhanced senescence levels in cholangiocytes from Mdr2-/- mice were reversed to normal in SR-/-/Mdr2-/- mice. However, senescence was decreased in HSCs from Mdr2-/- mice but returned to normal values in SR-/-/Mdr2-/- mice. In vitro treatment of HHSteCs with supernatant from cholangiocyte lacking SR (containing lower biliary levels of Sct-dependent TGF-β1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-β1 secretion was mediated by microRNA 125b. Our data suggest that differential modulation of angiogenesis-dependent senescence of cholangiocytes and HSCs may be important for the treatment of liver fibrosis in cholangiopathies.

Primary sclerosing cholangitis (PSC) is a genetically complex, inflammatory bile duct disease of largely unknown aetiology often leading to liver transplantation or death. Little is known about the genetic contribution to the severity and progression of PSC. The aim of this study is to identify genetic variants associated with PSC disease progression and development of complications.We collected standardised PSC subphenotypes in a large cohort of 3402 patients with PSC. After quality control, we combined 130 422 single nucleotide polymorphisms of all patients-obtained using the Illumina immunochip-with their disease subphenotypes. Using logistic regression and Cox proportional hazards models, we identified genetic variants associated with binary and time-to-event PSC subphenotypes.We identified genetic variant rs853974 to be associated with liver transplant-free survival (p=6.07×10-9). Kaplan-Meier survival analysis showed a 50.9% (95% CI 41.5% to 59.5%) transplant-free survival for homozygous AA allele carriers of rs853974 compared with 72.8% (95% CI 69.6% to 75.7%) for GG carriers at 10 years after PSC diagnosis. For the candidate gene in the region, RSPO3, we demonstrated expression in key liver-resident effector cells, such as human and murine cholangiocytes and human hepatic stellate cells.We present a large international PSC cohort, and report genetic loci associated with PSC disease progression. For liver transplant-free survival, we identified a genome-wide significant signal and demonstrated expression of the candidate gene RSPO3 in key liver-resident effector cells. This warrants further assessments of the role of this potential key PSC modifier gene.

Background and Aims Mechanisms underlying the repair of extrahepatic biliary tree (EHBT) after injury have been scarcely explored. The aims of this study were to evaluate, by using a lineage tracing approach, the contribution of peribiliary gland (PBG) niche in the regeneration of EHBT after damage and to evaluate, in vivo and in vitro , the signaling pathways involved. Approach and Results Bile duct injury was induced by the administration of 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) diet for 14 days to Krt19 Cre TdTomato LSL mice. Human biliary tree stem/progenitor cells (BTSC) within PBGs were isolated from EHBT obtained from liver donors. Hepatic duct samples (n = 10) were obtained from patients affected by primary sclerosing cholangitis (PSC). Samples were analyzed by histology, immunohistochemistry, western blotting, and polymerase chain reaction. DDC administration causes hyperplasia of PBGs and periductal fibrosis in EHBT. A PBG cell population (Cytokeratin19 ‐ /SOX9 + ) is involved in the renewal of surface epithelium in injured EHBT. The Wnt signaling pathway triggers human BTSC proliferation in vitro and influences PBG hyperplasia in vivo in the DDC‐mediated mouse biliary injury model. The Notch signaling pathway activation induces BTSC differentiation in vitro toward mature cholangiocytes and is associated with PBG activation in the DDC model. In human PSC, inflammatory and stromal cells trigger PBG activation through the up‐regulation of the Wnt and Notch signaling pathways. Conclusions We demonstrated the involvement of PBG cells in regenerating the injured biliary epithelium and identified the signaling pathways driving BTSC activation. These results could have relevant implications on the pathophysiology and treatment of cholangiopathies.

Two distinct stem/progenitor cell populations of biliary origin have been identified in the adult liver and biliary tree. Hepatic Stem/progenitor Cells (HpSCs) are bipotent progenitor cells located within the canals of Hering and can be differentiated into mature hepatocytes and cholangiocytes; Biliary Tree Stem/progenitor Cells (BTSCs) are multipotent stem cells located within the peribiliary glands of large intrahepatic and extrahepatic bile ducts and able to differentiate into hepatic and pancreatic lineages. HpSCs and BTSCs are endowed in a specialized niche constituted by supporting cells and extracellular matrix compounds. The actual contribution of these stem cell niches to liver and biliary tree homeostatic regeneration is marginal; this is due to the high replicative capabilities and plasticity of mature parenchymal cells (i.e., hepatocytes and cholangiocytes). However, the study of human liver and biliary diseases disclosed how these stem cell niches are involved in the regenerative response after extensive and/or chronic injuries, with the activation of specific signaling pathways. The present review summarizes the contribution of stem/progenitor cell niches in human liver diseases, underlining mechanisms of activation and clinical implications, including fibrogenesis and disease progression.

Currently, the only curative treatment for cholangiocarcinoma (CCA) is surgical resection, though this treatment is possible in less than 40% of patients. However, recent improvements in preoperative management have led to a higher number of patients who are candidates for this procedure. For unresectable patients, progress is ongoing in terms of locoregional and chemoradiation treatments and target therapies, especially in the definition of patient selection criteria. This is the second part of the Italian CCA guidelines, dealing with CCA treatment, that have been formulated in accordance with Italian National Institute of Health indications and developed according to the GRADE method and related advancements.