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BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is an orphan hepatobiliary disorder associated with inflammatory bowel disease (IBD). We aimed to estimate the risk of disease progression based on distinct clinical phenotypes in a large international cohort of patients with PSC. METHODS: We performed a retrospective outcome analysis of patients diagnosed with PSC from 1980 through 2010 at 37 centers in Europe, North America, and Australia. For each patient, we collected data on sex, clinician-reported age at and date of PSC and IBD diagnoses, phenotypes of IBD and PSC, and date and indication of IBD-related surgeries. The primary and secondary endpoints were liver transplantation or death (LTD) and hepatopancreatobiliary malignancy, respectively. Cox proportional hazards models were applied to determine the effects of individual covariates on rates of clinical events, with time-to-event analysis ascertained through Kaplan-Meier estimates. RESULTS: Of the 7121 patients in the cohort, 2616 met the primary endpoint (median time to event of 14.5 years) and 721 developed hepatopancreatobiliary malignancy. The most common malignancy was cholangiocarcinoma (n = 594); patients of advanced age at diagnosis had an increased incidence compared with younger patients (incidence rate: 1.2 per 100 patient-years for patients younger than 20 years old, 6.0 per 100 patient-years for patients 21-30 years old, 9.0 per 100 patient-years for patients 31-40 years old, 14.0 per 100 patient-years for patients 4150 years old, 15.2 per 100 patient-years for patients 51-60 years old, and 21.0 per 100 patient-years for patients older than 60 years). Of all patients with PSC studied, 65.5% were men, 89.8% had classical or large-duct disease, and 70.0% developed IBD at some point. Assessing the development of IBD as a time-dependent covariate, Crohn's disease and no IBD (both vs ulcerative colitis) were associated with a lower risk of LTD (unadjusted hazard ratio [HR], 0.62; P

Primary sclerosing cholangitis (PSC) is an enigmatic disease with scarce therapeutic options, potentially evolving in life-threatening complications, including cholangiocarcinoma (CCA). The clinical management of PSC remains challenging and will benefit from identification of outcome indicators to assess the quality of care and of the outcomes. This study aims at: A) identifying Outcome Indicators (OIs) for PSC, and B) validating OIs in a real-life context, by performing an interim analysis of the VBMH study. METHODS. A) A panel of experts generated a list of OIs using a modified version of the Delphi method. B) OIs with the highest RAND/UCLA score were tested in an ongoing multicentric and prospective study (VBMH). RESULTS. Five OIs were identified on the basis of the highest rating values (close to 9) and the lowest disagreement indexes (next to 0). They include: annual incidence of acute cholangitis episodes (OI#1); mortality rate for patients not yet listed for LTx (OI#2); improvement in patient reported quality of life measured by EQ-VAS (a visual assessment scale ranging from 0 to 100 where the patient points out his present-day health status) and EQ-5D (assessment of 5 domains that measure daily performance: mobility, self care, anxiety/depression, usual activities and pain/discomforts) (OI#3); number of patients died for CCA or colorectal cancer (OI#4); incidence and/or worsening of osteoporosis, expressed as T-score differential over a 2-year interval (OI#5). In the validation study, 63 consecutive patients with PSC enrolled in 3 liver centres in Northern Italy were evaluated for a 24-month follow-up period. For each OI, the following values were reported: OI#1) cumulative incidence of 5.2%, resulting in 0.029 cholangitis/patient; OI#2 and OI#4) no patients died without being listed for transplantation or because of cancer during the study time; OI#3) 38.9 and 19.4% of patients showed an improvement in EQ-VAS and EQ-5D, respectively; OI#5) 3% of patients developed or worsened osteoporosis. In CONCLUSION, five OIs for PSC were identified using the Delphi method and received a highly shared consensus. Albeit the study population is small (consistent with the rarity of PSC) and the follow-up time is short as compared to the long natural history of the disease, these OIs have proven to be easy to collect and to work appropriately. Therefore, they are suitable to be extended to specialized centres involved in PSC management to further validate their clinical usefulness in a larger scale. Patient reported outcomes showed improvements in a significant proportion of patients, indicating the importance to refer these patients to specialized centers

Cholangiocytes are the epithelial cells lining the bile ducts. Within the wide functional heterogeneity displayed by cholangiocytes through the different portions of the biliary tree, ductular cells of the finest peripheral ramifications, in strict contact with the canals of Hering, possess a reactive phenotype. This phenotype is essential for generating the hepatic reparative system, a multicellular complex including epithelial, mesenchymal and inflammatory cells, whereby they extensively communicate by mutually exchanging a variety of paracrine signals. Herein, we review the most recent insights on the molecular mechanisms underlying the ability of reactive cholangiocytes to orchestrate the development of this sort of ‘multiethnic society’, whose persistent activation is a critical determinant of liver disease progression

Introduction: Primary sclerosing cholangitis (PSC) is an enigmatic disease with scarce therapeutic options. The clinical management of PSC remains challenging and may benefit from Outcome Indicators (OI) to assess the quality of care. Aims: This study aims to: (A) identify OIs for PSC, and (B) validate OIs in a clinical context. Methods: (A) A panel of experts generated a list of OIs by Delphi method. (B) OIs with the highest RAND/UCLA score were tested in an ongoing multicentric, prospective study (Value-based Medicine in Hepatology, VBMH). Results: Five OIs were identified having the highest rating values and low disagreement indexes: annual rate of acute cholangitis episodes (OI#1); mortality rate for patients not yet listed for liver transplantation (OI#2); rate of quality of life improvement, measured by EQ-VAS and EQ-5D (OI#3); number of patients died for cholangiocarcinoma and colo-rectal carcinoma (OI#4); incidence and/or worsening of osteoporosis (OI#5). In the validation study, 63 consecutive patients with PSC enrolled in 3 tertiary centres in Northern Italy were evaluated for a median 24-months follow-up period. For each OI, the following values were reported: (OI#1) cumulative incidence of 5.2%, resulting in 0.029 cholangitis/patient; (OI#2, OI#4) no patients died without being listed for transplantation or because of cancer during study time; (OI#3) 38.9 and 19.4% of patients showed an improvement in EQ-VAS and EQ-5D parameters, respectively; (OI#5) 3% of patients developed or worsened osteoporosis. Conclusions: Five OIs for PSC were identified reporting high consensus. Albeit the study population is small (as in the case of rare diseases) and the follow-up time is short as compared to the long natural history of the disease, these OIs have proven to be easy to collect and to work appropriately. Therefore, they are suitable to be extended to specialized centres involved in PSC management to further validate their clinical usefulness.

To further characterize the genetic basis of primary biliary cirrhosis (PBC), we genotyped 2426 PBC patients and 5731 unaffected controls from three independent cohorts using a single nucleotide polymorphism (SNP) array (Immunochip) enriched for autoimmune disease risk loci. Meta-analysis of the genotype data sets identified a novel disease-associated locus near the TNFSF11 gene at 13q14, provided evidence for association at six additional immune-related loci not previously implicated in PBC and confirmed associations at 19 of 22 established risk loci. Results of conditional analyses also provided evidence for multiple independent association signals at four risk loci, with haplotype analyses suggesting independent SNP effects at the 2q32 and 16p13 loci, but complex haplotype driven effects at the 3q25 and 6p21 loci. By imputing classical HLA alleles from this data set, four class II alleles independently contributing to the association signal from this region were identified. Imputation of genotypes at the non-HLA loci also provided additional associations, but none with stronger effects than the genotyped variants. An epistatic interaction between the IL12RB2 risk locus at 1p31and the IRF5 risk locus at 7q32 was also identified and suggests a complementary effect of these loci in predisposing to disease. These data expand the repertoire of genes with potential roles in PBC pathogenesis that need to be explored by follow-up biological studies.

Susceptibility to primary biliary cirrhosis (PBC) is strongly associated with human leukocyte antigen (HLA)-region polymorphisms. To determine if associations can be explained by classical HLA determinants, we studied Italian, 676 cases and 1440 controls, genotyped with dense single-nucleotide polymorphisms (SNPs) for which classical HLA alleles and amino acids were imputed. Although previous genome-wide association studies and our results show stronger SNP associations near DQB1, we demonstrate that the HLA signals can be attributed to classical DRB1 and DPB1 genes. Strong support for the predominant role of DRB1 is provided by our conditional analyses. We also demonstrate an independent association of DPB1. Specific HLA-DRB1 genes (*08, *11 and *14) account for most of the DRB1 association signal. Consistent with previous studies, DRB1*08 (P=1.59 × 10(-11)) was the strongest predisposing allele, whereas DRB1*11 (P=1.42 × 10(-10)) was protective. Additionally, DRB1*14 and the DPB1 association (DPB1*03:01; P=9.18 × 10(-7)) were predisposing risk alleles. No signal was observed in the HLA class 1 or class 3 regions. These findings better define the association of PBC with HLA and specifically support the role of classical HLA-DRB1 and DPB1 genes and alleles in susceptibility to PBC.Genes and Immunity advance online publication, 10 May 2012; doi:10.1038/gene.2012.17.

Background and Aims: Hepatocellular carcinoma (HCC) is a worldwide public health problem, accounting for increasing mortality and high costs. Aim of our study was to identify and test outcome indicators (OIs) in HCC, in light of their potential use in policy decision models. Methods: A panel of experts identified a list of OIs using a modified Delphi method; three of these OIs with the highest RAND/UCLA scores were tested in a prospective multicenter observational study (Value Based Medicine in Hepatology, VBMH). During 18 months, 711 HCC patients were enrolled and prospectively followed. Median follow-up time was 14 months. Results: The first OI was survival after 1–3–5 years stratified for BCLC stage or treatment (OI#1). One-year survival for BCLC stage 0/A, B, C, D was 93%, 86%, 50%, 26% respectively. One-year survival of 288 patients who had the first treatment during the study time was 88% for liver transplantation, 97% surgical resection, 100% ablation and 89% for TACE. The remaining two OIs meant to evaluate the appropriateness of treatments. Worsening of BCLC and/or CPT score after loco-regional therapy or surgical resection recorded at three months (OI#2), occurred in 16% of cases (76% after TACE). Very early HCC recurrence (within 6 months) after curative treatments (OI#3) happened in 15% of patients treated (20% after ablation). Conclusions: This set of outcome indicators proved their feasibility in a large cohort of patients and could serve as a benchmark for healthcare providers to move towards a value-based approach in the management of HCC.

Background and Aims: Liver disease (LD) is a major cause of morbidity and mortality worldwide. Development of outcome indicators (OIs) provides health care policy makers with objective criteria that can be used to generate a virtuous competition to improve value of care. Aim of our study was to generate and test a set of health care OIs for the major LDs. Methods: Using a modified Delphi method, 7 expert panels identified a set of OIs according to experience and scientific evidence (as of 2010). Each OI was rated in a two-step process using the RAND 9-point agreement scale. Median scores were computed for each OI. After the second rating, a disagreement index (DI) was calculated to identify and accept (if DI≤1) OIs with median rating ≥7. The final set of selected OIs was tested through an ongoing prospective multicenter observational study involving three tertiary centers in Lombardy, Italy. Results: A total of 51 OIs were identified by the focus groups. We recruited 3213 consecutive liver patients, of whom 91% had at least one follow-up visit after a median follow-up time of 15 months. Among these patients, 1752 were cirrhotic and 711 were affected by HCC. During observation time, 156 patients were transplanted and 210 patients died. All the identified OIs were successfully tested in the clinical setting showing excellent performance and correlation with natural history information. Conclusions: This study provides a set of validated outcomes indicators that can be used to monitor the performance of referral centers and improve the value and sustainability of care.

Background and Aims: Liver Cirrhosis (LC) is responsible for high morbidity, mortality and increasing costs. Aim of our study was to identify outcome indicators (OIs) able to measure quality and value of care in compensated (CC) and decompensated (DC) cirrhosis. Methods: A panel of experts identified a list of OIs using a modified Delphi method; seven of these OIs with the highest RAND/UCLA scores were then tested in a prospective multicenter observational study (Value Based Medicine in Hepatology, VBMH). During 18 months, 1751 patients with LC were enrolled (1004 CC, 747 DC). Results: Annual rate of decompensation (OI#1), annual incidence of first variceal bleeding (OI#2) and annual incidence of HCC (OI#3) in CC were 12%, 2% for low- and 3% for high-risk varices, and 4.3% (81% diagnosed in BCLC-A stage) respectively. One-year survival for CPT score A, B, C was 96%, 81% and 59% (OI#4) respectively, and 94%, 56% when stratified for MELD cut-off of 15 (OI#5). Among DC patients, 4% of them had an episode of variceal bleeding (6 weeks survival 90%, 32% recurrence) (OI#6), 3% had spontaneous bacterial peritonitis (6 weeks survival 86%, 13% recurrence) (OI#7). Conclusions: The seven outcome indicators identified in our study performed well when tested in a large cohort of patients and represent a reference tool to implement a value-based approach to liver diseases.

Genetically determined loss of fibrocystin function causes congenital hepatic fibrosis (CHF), Caroli disease (CD), and autosomal recessive polycystic kidney disease (ARPKD). Cystic dysplasia of the intrahepatic bile ducts and progressive portal fibrosis characterize liver pathology in CHF/CD. At a cellular level, several functional morphological and signaling changes have been reported including increased levels of 3'-5'-cyclic adenosine monophosphate (cAMP). In this study we addressed the relationships between increased cAMP and β-catenin. In cholangiocytes isolated and cultured from Pkhd1(del4/del4) mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser(675) -phosphorylation of β-catenin, its nuclear localization, and its transcriptional activity (western blot and TOP flash assay, respectively) along with a down-regulation of E-cadherin expression (immunocytochemistry and western blot); these changes were inhibited by the PKA blocker, PKI (1 μM). The Rho-GTPase, Rac-1, was also significantly activated by cAMP in Pkhd1(del4/del4) cholangiocytes. Rac-1 inhibition blocked cAMP-dependent nuclear translocation and transcriptional activity of pSer(675) -β-catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1(del4/del4) and was inhibited by: (1) PKI, (2) silencing β-catenin (siRNA), and (3) the Rac-1 inhibitor NSC 23766. Conclusion: These data show that in fibrocystin-defective cholangiocytes, cAMP/PKA signaling stimulates pSer(675) -phosphorylation of β-catenin and Rac-1 activity. In the presence of activated Rac-1, pSer(675) -β-catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1(del4/del4) cholangiocytes. β-Catenin-dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target. (Hepatology 2013)

BACKGROUND AND AIM Congenital Hepatic Fibrosis (CHF) is a genetic liver disease caused by mutations in PKHD1, a gene encoding for Fibrocystin, a ciliary protein expressed by cholangiocytes. CHF is characterized by biliary dysgenesia associated with progressive portal fibrosis and portal hypertension. In CHF mechanisms of portal fibrosis are unknown. We have recently reported that Pkhd1-/- mice present: 1)activation of b-catenin signaling in cystic cholangiocytes; 2)increased pericystic infiltrate of CD45+/Collagen type1 (Col1)+ cells, reminiscent of fibrocytes. Fibrocytes are monocyte-derived cells, involved in several fibrosing conditions, but their role in liver scarring is controversial. b-catenin is emerging as a regulator of inflammation, therefore we hypothesized that a b-catenin-dependent chemokine production by cholangiocytes drives recruitment of fibrocytes in Pkhd1-/- mice. METHODS In Pkhd1-/- mice we investigated: a)a panel of 32 cyto/chemokines in both apical and basolateral medium of cultured polarized cholangiocytes (Luminex); b)the effects of two different b-catenin inhibitors (ICG-001, 25uM, or quercetin, 50uM) on the expression of CXCL1 and CXCL10 (RT-PCR); c)the immunohistochemical expression of CD45/Col1 (fibrocyte markers) and aSMA (myofibroblast marker) in the portal inflammatory cells, and their correlation with portal fibrosis (Sirius-red) in liver samples at 1-12 months; d)the effects of cholangiocyte conditioned media (CM) and CXCL1+CXCL10 on WEHI265.1-monocyte chemoattraction (Boyden chamber) and transdifferentiation into fibrocytes (RT-PCR for COL1(A1)). WT mice served as controls. RESULTS Pkhd1-/- cholangiocytes secreted significantly higher basolateral levels of CXCL1 and CXCL10 as compared to WT. Expression of CXCL1 and CXCL10 was significantly inhibited in cells treated with ICG-001 and quercetin. Pkhd1-/- mice showed progressive fibrosis, but portal accumulation of aSMA+ cells was evident only after 9 months. In contrast, we observed an early peribiliary recruitment of CD45+/Col1+ cells, whose number strongly correlated with the Sirius-red area (r=0.89,p

Liver Cirrhosis (LC) is responsible for high morbidity, mortality and raising costs. Current guidelines set the standard of care for management of cirrhosis in clinical practice, but explicit outcome indicators (OIs) are lacking. If available, OIs could guide clinical care and decision making, so that efforts and resources can be properly allocated. Aim of our study was to generate and test a set of health care OIs for compensated (CC) and decompensated (DC) LC. This study is part of a larger effort (the V.B.M.H. study) to generate OIs for several liver diseases. An expert panel of hepatologists identified a set of OIs for LC according to experience and scientific evidence (as of 2010), and used a modified Delphi method to rate them through a RAND 9-point agreement scale. A final list of 7 indicators with median rating ≥7 and with disagreement index ≤1 was selected. Three OIs were designed for CC and 4 for DC. In the second phase of the study, the selected OIs were tested in clinical practice through a prospective multicenter observational study, involving three tertiary centers in Lombardy, Italy. A web-based EMR was used to collect data. 1732 LC patients were enrolled in 18 months: 984 CC (57%) and 748 DC (43%). 91% of these LC patients had at least two consultations in a 13 months median follow-up. The annual rate of decompensation in CC (OI#1) was 12%, decompensation being more frequent in HCV-related CC. The annual incidence of 1st variceal bleeding (VB) for low-risk varices was 2% and was null for high risk varices, indicating effective primary profilaxis (OI#2). Annual incidence of HCC was 4%, while the percentage of CC patients diagnosed at early stage HCC (BCLC 0/A) was 74%, reflecting the accuracy of oncologic surveillance (OI#3). In DC patients, survival at 1 year after the first episode of decompensation (ascites in 78% of cases) was 99% for CPT A, 87% for CPT B and 73% for CPT C (OI#4). 3% of DC patients had an episode of VB, with survival of 88% after 6 weeks from the VB episode (OI#5) and with recurrence rate of 27% (OI#6). 19 out of 748 DC patients (3%) had spontaneous bacterial peritonitis, with 79% survival after 6 weeks from the episode (OI#7). In conclusion, the selected OIs performed well in monitoring the rate of decompensation in CC and the accuracy of surveillance for HCC; in DC, OIs were able to capture survival and the efficacy of management of major complications. This study represents the first attempt to identify and test a set of value-based OIs for LC, and provides a reference tool for healthcare policy makers to improve quality of care in patients affected by LC

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

Liver diseases (LDs) have a high impact on morbidity, mortality and health-related quality of life (HRQoL). Different LDs may have different effects on patients’ HRQoL. The aim of our study was to assess the reliability and benefit of using a generic HRQoL questionnaire to evaluate the health status of patients with the major liver conditions: hepatitis B (HBV), hepatitis C (HCV), cirrhosis, hepatocellular carcinoma (HCC), autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), NAFLD/NASH and patients in the liver transplant list. A naturalistic, prospective, multicenter study has been conducted to generate and validate a set of health care outcomes indicators for the major liver conditions. LDs patients (age>18 years) were enrolled in 3 major Italian medical centers and are still being followed up (median f-up: 13 months). Within this study, socio-demographic, clinical and HRQoL were collected using the EQ-5D-3L, a generic instrument that enables HRQoL to compared within and between clinical conditions and with the general population. It generates a health profile made up of 5 domains (mobility, self care, anxiety/depression, usual activities and pain/discomfort), each one with three levels of severity. It also consists of a visual analogue scale (EQ-5D VAS) which measures overall HRQoL in a range from 0 (worst imaginable health state) to 100 (best imaginable health state). The baseline HRQoL data was analyzed dividing the patients in sub-groups according to the most recently diagnosed and most severe condition. The results reported below focus on the mean VAS. We enrolled 3,217 patients, 64.8% male, aged 19-91 (median=61) years; 95.0% of them filled in the EQ-5D at baseline. Patients in the HCC group were 22.6%, those with compensated cirrhosis were 21.2%, HCV 20.9%, decompensated cirrhosis 10.3%, HBV 9.5%. The HBV group reported the best HRQoL with a mean EQ-5D VAS of 77.8. NAFLD/NASH, HCV and PSC patients had a similar HRQoL with a mean EQ-5D VAS between 76.5 and 75.1. While, compensated cirrhosis and PBC had a slightly worsen values (74.5 and 74.0, respectively). HCC and decompensated cirrhosis showed a mean EQ-5D VAS of about 69.0. At least, AIH and listed for liver transplant patients reported the worst HRQoL levels than the other sub-groups (67.7 and 67.0, respectively). In conclusion, EQ-5D is well accepted by the patients and accurately reflects the changes in HRQoL related to the clinical severity of LDs. Understanding the different impact of LDs on the patients’ HRQoL could help physicians and decision makers to better estimate the burden of these conditions and to improve the quality of care

BACKGROUND: Monoethylglycinexylidide (MEGX) formation following lignocaine injection has recently been proposed as a simple dynamic liver function test based on a single measurement of its serum concentration. AIM: To determine the optimal sampling time for MEGX determination. METHODS: A modelling analysis of lignocaine and MEGX kinetics was performed in seven normals and in four patients with compensated liver cirrhosis; a similar study was performed in 74 cirrhotic patients, divided into two groups according to disease severity (Pugh score). RESULTS: Only the MEGX fractional formation rate (kf) and formation delay (tau) were significantly altered in cirrhotic patients compared to normals: kf = 0.15 +/- 0.03 vs. 0.32 +/- 0.10 min-1 (mean +/- s.d.); tau = 7.7 +/- 2.0 vs. 3.9 +/- 2.9 min-1. A good correlation was found between kf and late (r = 0.82) but not early (r = 0.63) serum MEGX formation, suggesting that late measurements for the clinical MEGX test are preferred. In the second part of our investigation, by discriminant analysis of MEGX test data for 74 cirrhotic patients, the late MEGX concentrations gave the best discrimination between the two classes. In particular, the 60 min MEGX concentration showed the best diagnostic accuracy (81%), sensitivity (75%) and specificity (84%). The association of this with other MEGX parameters, either singly or derived from the whole curve measurements, did not improve the performance of the method. CONCLUSION: The MEGX test, based on a single determination 60 min after lignocaine injection, may be regarded as a simple and sensitive quantitative liver function test

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 × 10−11, odds ratio (OR) = 1.46), IRF5-TNPO3 (P = 2.8 × 10−10, OR = 1.63) and 17q12-21 (P = 1.7 × 10−10, OR = 1.38).

BACKGROUND AND AIMS: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. APPROACH AND RESULTS: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2(−/−)) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2(−/−) mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2(−/−) mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2(−/−) mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2(−/−) mice, respectively. 5HT levels increase in Mdr2(−/−) mice and in PSC human patients compared to their controls and decrease in serum of Mdr2(−/−) mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2(−/−) mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. CONCLUSIONS: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.

Background Autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis represent the three major autoimmune liver diseases (AILDs). Their management is highly specialized, requires a multidisciplinary approach and often relies on expensive, orphan drugs. Unfortunately, their treatment is often unsatisfactory, and the care pathway heterogeneous across different centers. Disease-specific clinical outcome indicators (COIs) able to evaluate the whole cycle of care are needed to assist both clinicians and administrators in improving quality and value of care. Aim of our study was to generate a set of COIs for the three AILDs. We then prospectively validated these indicators based on a series of consecutive patients recruited at three tertiary clinical centers in Lombardy, Italy. Methods In phase I using a Delphi method and a RAND 9-point appropriateness scale a set of COIs was generated. In phase II the indicators were applied in a real-life dataset. Results Two-hundred fourteen patients were enrolled and followed-up for a median time of 54 months and the above COIs were recorded using a web-based electronic medical record program. The COIs were easy to collect in the clinical practice environment and their values compared well with the available natural history studies. Conclusions We have generated a comprehensive set of COIs which sequentially capture different clinical outcome of the three AILDs explored. These indicators represent a critical tool to implement a value-based approach to patients with these conditions, to monitor, compare and improve quality through benchmarking of clinical performance and to assess the significance of novel drugs and technologies. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Prognosis of cholangiocarcinoma, a devastating liver epithelial malignancy characterized by early invasiveness, remains very dismal, though its incidence has been steadily increasing. Evidence is mounting that in cholangiocarcinoma, tumor epithelial cells establish an intricate web of mutual interactions with multiple stromal components, largely determining the pervasive behavior of the tumor. The main cellular components of the tumor microenvironment (i.e. myofibroblasts, macrophages, lymphatic endothelial cells), which has been recently termed as ‘tumor reactive stroma’, are recruited and activated by neoplastic cells, and in turn, deleteriously mold tumor behavior by releasing a huge variety of paracrine signals, including cyto/chemokines, growth factors, morphogens and proteinases. An abnormally remodeled and stiff extracellular matrix favors and supports these cell interactions. Although the mechanisms responsible for the generation of tumor reactive stroma are largely uncertain, hypoxia presumably plays a central role. In this review, we will dissect the intimate relationship among the different cell elements cooperating within this complex ‘ecosystem’, with the ultimate goal to pave the way for a deeper understanding of the mechanisms underlying cholangiocarcinoma aggressiveness, and possibly, to foster the development of innovative, combinatorial therapies aimed at halting tumor progression. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Congenital Hepatic Fibrosis (CHF) is a disease of the biliary epithelium characterized by bile duct changes resembling ductal plate malformations and by progressive peribiliary fibrosis, in the absence of overt necroinflammation. Progressive liver fibrosis leads to portal hypertension and liver failure, however the mechanisms leading to fibrosis in CHF remain elusive. CHF is caused by mutations in PKHD1, a gene encoding for fibrocystin, a ciliary protein expressed in cholangiocytes. Using a fibrocystin-defective (Pkhd1(del4/del4) ) mouse, which is orthologous of CHF, we show that Pkhd1(del4/del4) cholangiocytes are characterized by a β-catenin-dependent secretion of a range of chemokines, including CXCL1, CXCL10 and CXCL12, which stimulate bone marrow-derived macrophage recruitment. We also show that Pkhd1(del4/del4) cholangiocytes, in turn, respond to proinflammatory cytokines released by macrophages by up-regulating αvβ6 integrin, an activator of latent local TGFβ1. While the macrophage infiltrate is initially dominated by the M1 phenotype, the profibrogenic M2 phenotype increases with disease progression, along with the number of portal myofibroblasts. Consistent with these findings, clodronate-induced macrophage depletion results in a significant reduction of portal fibrosis and portal hypertension as well as of liver cysts. our results show that fibrosis can be initiated by an epithelial cell dysfunction, leading to low-grade inflammation, macrophage recruitment and collagen deposition. These findings establish a new paradigm for biliary fibrosis and represent a model to understand the relationship between cell dysfunction, parainflammation, liver fibrosis and macrophage polarization over time. This article is protected by copyright. All rights reserved.

The acquisition of invasive functions by tumor cells is a first and crucial step toward the development of metastasis, which nowadays represents the main cause of cancer-related death. Cholangiocarcinoma (CCA), a primary liver cancer originating from the biliary epithelium, typically develops intrahepatic or lymph node metastases at early stages, thus preventing the majority of patients from undergoing curative treatments, consistent with their very poor prognosis. As in most carcinomas, CCA cells gradually adopt a motile, mesenchymal-like phenotype, enabling them to cross the basement membrane, detach from the primary tumor, and invade the surrounding stroma. Unfortunately, little is known about the molecular mechanisms that synergistically orchestrate this proinvasive phenotypic switch. Autocrine and paracrine signals (cyto/chemokines, growth factors, and morphogens) permeating the tumor microenvironment undoubtedly play a prominent role in this context. Moreover, a number of recently identified signaling systems are currently drawing attention as putative mechanistic determinants of CCA cell invasion. They encompass transcription factors, protein kinases and phosphatases, ubiquitin ligases, adaptor proteins, and miRNAs, whose aberrant expression may result from either stochastic mutations or the abnormal activation of upstream pro-oncogenic pathways. Herein we sought to summarize the most relevant molecules in this field and to discuss their mechanism of action and potential prognostic relevance in CCA. Hopefully, a deeper knowledge of the molecular determinants of CCA invasiveness will help to identify clinically useful biomarkers and novel druggable targets, with the ultimate goal to develop innovative approaches to the management of this devastating malignancy.

Cholangiocarcinoma (CCA) is a highly aggressive epithelial malignancy still carrying a dismal prognosis, owing to early lymph node metastatic dissemination and striking resistance to conventional chemotherapy. Although mechanisms underpinning CCA progression are still a conundrum, it is now increasingly recognized that the desmoplastic microenvironment developing in conjunction with biliary carcinogenesis, recently renamed tumor reactive stroma (TRS), behaves as a paramount tumor-promoting driver. Indeed, once being recruited, activated and dangerously co-opted by neoplastic cells, the cellular components of the TRS (myofibroblasts, macrophages, endothelial cells and mesenchymal stem cells) continuously rekindle malignancy by secreting a huge variety of soluble factors (cyto/chemokines, growth factors, morphogens and proteinases). Furthermore, these factors are long-term stored within an abnormally remodeled extracellular matrix (ECM), which in turn can deleteriously mold cancer cell behavior. In this review, we will highlight evidence for the active role played by reactive stromal cells (as well as by the TRS-associated ECM) in CCA progression, including an overview of the most relevant TRS-derived signals possibly fueling CCA cell aggressiveness. Hopefully, a deeper knowledge of the paracrine communications reciprocally exchanged between cancer and stromal cells will steer the development of innovative, combinatorial therapies, which can finally hinder the progression of CCA, as well as of other cancer types with abundant TRS, such as pancreatic and breast carcinomas.

The biliary tree is an arborizing system of intra- and extrahepatic conduits connecting the liver to the intestine. The biliary tree has a complex tridimensional structure, encompassing bile ducts of different sizes, morphologies, and functions. The most studied function of biliary epithelial cells (cholangiocytes) is to regulate the hydration and alkalinity of the primary bile secreted by hepatocytes. An increasing number of evidence highlight the ability of cholangiocyte to undergo changes in phenotype, proliferation, and secretory activity in response to liver damage. Cholangiocytes are involved in biliary innate immunity; altered biliary innate immunity plays a role in a number of biliary diseases, including genetic cholangiopathies, such as cystic fibrosis-related liver disease. In addition, cholangiocytes may behave as antigen-presenting cells and secrete immunoglobulins as well as several antimicrobial peptides. Thus, cholangiocytes, by participating actively to the immune and inflammatory responses, represent a first defense line against liver injury from different causes. In fact, cholangiocytes possess a number of sensing receptors for pathogen-associated molecular patterns (PAMPs), such as Toll-like receptors (TLRs), which modulate their proinflammatory behavior. Derangements of the signals controlling these mechanisms are at the basis of the pathogenesis of different cholangiopathies, often extending beyond the classically recognized immune-mediated (primary biliary cirrhosis, primary sclerosing cholangitis), as in cystic fibrosis liver disease.

In severe liver injury, ductular reactions (DRs) containing bipotential hepatic progenitor cells (HPCs) branch from the portal tract. Neural cell adhesion molecule (NCAM) marks bile ducts and DRs, but not mature hepatocytes. NCAM mediates interactions between cells and surrounding matrix; however, its role in liver development and regeneration is undefined. Polysialic acid (polySia), a unique posttranslational modifier of NCAM, is produced by the enzymes, ST8SiaII and ST8SiaIV, and weakens NCAM interactions. The role of polySia with NCAM synthesizing enzymes ST8SiaII and ST8SiaIV were examined in HPCs in vivo using the choline-deficient ethionine-supplemented and 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet models of liver injury and regeneration, in vitro using models of proliferation, differentiation, and migration, and by use of mouse models with gene defects in the polysialyltransferases (St8sia 2+/-4+/-, and St8sia2-/-4-/-). We show that, during liver development, polySia is required for the correct formation of bile ducts because gene defects in both the polysialyltransferases (St8sia2+/-4+/- and St8sia2-/-4-/- mice) caused abnormal bile duct development. In normal liver, there is minimal polySia production and few ductular NCAM+ cells. Subsequent to injury, NCAM+ cells expand and polySia is produced by DRs/HPCs through ST8SiaIV. PolySia weakens cell-cell and cell-matrix interactions, facilitating HGF-induced migration. Differentiation of HPCs to hepatocytes in vitro results in both transcriptional down-regulation of polySia and cleavage of polySia-NCAM. Cleavage of polySia by endosialidase (endoN) during liver regeneration reduces migration of DRs into parenchyma.PolySia modification of NCAM+ ductules weakens cell-cell and cell-matrix interactions, allowing DRs/HPCs to migrate for normal development and regeneration. Modulation of polySia levels may provide a therapeutic option in liver regeneration.

Nuclear expression of the calcium-binding protein S100A4 is a biomarker of increased invasiveness in cholangiocarcinoma, a primary liver cancer with scarce treatment opportunities and dismal prognosis. In this study, we provide evidence that targeting S100A4 nuclear import by low-dose paclitaxel, a microtubule-stabilizing agent, inhibits cholangiocarcinoma invasiveness and metastatic spread. Administration of low-dose paclitaxel to established (EGI-1) and primary (CCA-TV3) cholangiocarcinoma cell lines expressing nuclear S100A4 triggered a marked reduction in nuclear expression of S100A4 without modifying its cytoplasmic levels, an effect associated with a significant decrease in cell migration and invasiveness. While low-dose paclitaxel did not affect cellular proliferation, apoptosis, or cytoskeletal integrity, it significantly reduced SUMOylation of S100A4, a critical posttranslational modification that directs its trafficking to the nucleus. This effect of low-dose paclitaxel was reproduced by ginkolic acid, a specific SUMOylation inhibitor. Downregulation of nuclear S100A4 by low-dose paclitaxel was associated with a strong reduction in RhoA and Cdc42 GTPase activity, MT1-MMP expression, and MMP-9 secretion. In an SCID mouse xenograft model, low-dose metronomic paclitaxel treatment decreased lung dissemination of EGI-1 cells without significantly affecting their local tumor growth. In the tumor mass, nuclear S100A4 expression by cholangiocarcinoma cells was significantly reduced, whereas rates of proliferation and apoptosis were unchanged. Overall, our findings highlight nuclear S100A4 as a candidate therapeutic target in cholangiocarcinoma and establish a mechanistic rationale for the use of low-dose paclitaxel in blocking metastatic progression of cholangiocarcinoma. Cancer Res; 76(16); 4775–84. ©2016 AACR.

Whether liver epithelial cells contribute to the development of hepatic scarring by undergoing epithelial-to-mesenchymal transition (EMT) is a controversial issue. Herein, we revisit the concept of EMT in cholangiopathies, a group of severe hepatic disorders primarily targeting the bile duct epithelial cell (cholangiocyte), leading to progressive portal fibrosis, the main determinant of liver disease progression. Unfortunately, therapies able to halt this process are currently lacking. In cholangiopathies, fibrogenesis is part of ductular reaction, a reparative complex involving epithelial, mesenchymal, and inflammatory cells. Ductular reactive cells (DRC) are cholangiocytes derived from the activation of the hepatic progenitor cell compartment. These cells are arranged into irregular strings and express a “reactive” phenotype, which enables them to extensively crosstalk with the other components of ductular reaction. We will first discuss EMT in liver morphogenesis and then highlight how some of these developmental programs are partly reactivated in DRC. Evidence for “bona fide” EMT changes in cholangiocytes is lacking, but expression of some mesenchymal markers represents a fundamental repair mechanism in response to chronic biliary damage with potential harmful fibrogenetic effects. Understanding microenvironmental cues and signaling perturbations promoting these changes in DRC may help to identify potential targets for new antifibrotic therapies in cholangiopathies.

SummarySeveral cholangiopathies result from a perturbation of developmental processes. Most of these cholangiopathies are characterised by the persistence of biliary structures with foetal configuration. Developmental processes are also relevant in acquired liver diseases, as liver repair mechanisms exploit a range of autocrine and paracrine signals transiently expressed in embryonic life. We briefly review the ontogenesis of the intra- and extrahepatic biliary tree, highlighting the morphogens, growth factors, and transcription factors that regulate biliary development, and the relationships between developing bile ducts and other branching biliary structures. Then, we discuss the ontogenetic mechanisms involved in liver repair, and how these mechanisms are recapitulated in ductular reaction, a common reparative response to many forms of biliary and hepatocellular damage. Finally, we discuss the pathogenic aspects of the most important primary cholangiopathies related to altered biliary development, i.e. polycystic and fibropolycystic liver diseases, Alagille syndrome.

In most cholangiopathies, liver diseases of different etiologies in which the biliary epithelium is the primary target in the pathogenic sequence, the central mechanism involves inflammation. Inflammation, characterized by pleomorphic peribiliary infiltrate containing fibroblasts, macrophages, lymphocytes, as well as endothelial cells and pericytes, is associated to the emergence of "reactive cholangiocytes." These biliary cells do not possess bile secretory functions, are in contiguity with terminal cholangioles, and are of a less-differentiated phenotype. They have acquired several mesenchymal properties, including motility and ability to secrete a vast number of proinflammatory chemo/cytokines and growth factors along with de novo expression of a rich receptor machinery. These functional properties enable reactive cholangiocytes to establish intimate contacts and to mutually exchange a variety of paracrine signals with the different mesenchymal cell types populating the portal infiltrate. The extensive crosstalk between the epithelial and mesenchymal compartments is the driver of liver repair mechanisms in cholangiopathies, ultimately evolving toward portal fibrosis. Herein, the authors first review the properties of the different cell types involved in their interaction, and then analyze the underlying molecular mechanisms as they relate to liver repair in cholangiopathies.

Cholangiocarcinoma is an aggressive, strongly chemoresistant liver malignancy. Leukemia inhibitory factor (LIF), an IL-6 family cytokine, promotes progression of various carcinomas. To investigate the role of LIF in cholangiocarcinoma, we evaluated the expression of LIF and its receptor (LIFR) in human samples. LIF secretion and LIFR expression were assessed in established and primary human cholangiocarcinoma cell lines. In cholangiocarcinoma cells, we tested LIF effects on proliferation, invasion, stem cell-like phenotype, chemotherapy-induced apoptosis (gemcitabine+cisplatin), expression levels of pro-apoptotic (Bax) and anti-apoptotic (Mcl-1) proteins, with/without PI3K inhibition, and of pSTAT3, pERK1/2, pAKT. LIF effect on chemotherapy-induced apoptosis was evaluated after LIFR silencing and Mcl-1 inactivation. Results show that LIF and LIFR expression were higher in neoplastic than in control cholangiocytes; LIF was also expressed by tumor stromal cells. LIF had no effects on cholangiocarcinoma cell proliferation, invasion, and stemness signatures, whilst it counteracted drug-induced apoptosis. Upon LIF stimulation, decreased apoptosis was associated with Mcl-1 and pAKT up-regulation and abolished by PI3K inhibition. LIFR silencing and Mcl-1 blockade restored drug-induced apoptosis. In conclusion, autocrine and paracrine LIF signaling promote chemoresistance in cholangiocarcinoma by up-regulating Mcl-1 via a novel STAT3- and MAPK-independent, PI3K/AKT-dependent pathway. Targeting LIF signaling may increase CCA responsiveness to chemotherapy.

In addition to its well-established role in embryo development, epithelial-to-mesenchymal transition (EMT) has been proposed as a general mechanism favoring tumor metastatization in several epithelial malignancies. Herein, we review the topic of EMT in cholangiocarcinoma (CCA), a primary liver cancer arising from the epithelial cells lining the bile ducts (cholangiocytes) and characterized by an abundant stromal reaction. CCA carries a dismal prognosis, owing to a pronounced invasiveness and scarce therapeutic opportunities. In CCA, several reports indicate that cancer cells acquire a number of EMT biomarkers and functions. These phenotypic changes are likely induced by both autocrine and paracrine signals released in the tumor microenvironment (cytokines, growth factors, morphogens) and intracellular stimuli (microRNAs, oncogenes, tumor suppressor genes) variably associated with specific disease mechanisms, including chronic inflammation and hypoxia. Nevertheless, evidence supporting a complete EMT of neoplastic cholangiocytes into stromal cells is lacking, and the gain of EMT-like changes by CCA cells rather reflects a shift towards an enhanced pro-invasive phenotype, likely induced by the tumor stroma. This concept may help to identify new biomarkers of early metastatic behavior along with potential therapeutic targets.

The diseases of the intrahepatic biliary tree are a large group of potentially evolutive congenital and acquired liver disorders affecting both the adult and pediatric populations. They represent a relevant cause of liver-related morbidity and mortality and an important indication for liver transplantation, particularly in children. While the practical approach to patients affected by biliary tree diseases has not significantly changed yet, the conceptual approach to the pathophysiology of cholangiopathies has witnessed important advances that will be discussed. The primary cell target of the pathogenetic sequence of these disorders is the biliary epithelium. Cholangiocytes have multifaceted functions, not limited to bile production. Their capability to secrete a range of different pro-inflammatory mediators, cytokines, and chemokines indicates a major role of cholangiocytes in the inflammatory reaction. Furthermore, paracrine secretion of growth factors and peptides mediates an extensive cross-talk with other liver cell types, including hepatocytes, stellate, and endothelial and inflammatory cells. Cholangiopathies share a number of pathogenetic mechanisms, including inflammation, cholestasis, fibrosis, apoptosis, altered development, and neoplastic transformation. These basic disease mechanisms will be discussed in detail, along with the distinct features of a number of cholangiopathies. Furthermore, an increase in the biliary cell compartment is a common response to many forms of liver injury, from cholangiopathies to viral and fulminant hepatitis. Elucidation of these pathophysiologic mechanisms will likely provide clues for future therapeutic strategies. Furthermore, understanding the role of cholangiocytes in liver regeneration/repair and the mechanisms of cholangiocyte activation and their relationship with liver progenitor cell will be of further interest.

Background/Aims In routine examination of liver biopsies isolated ductular hyperplasia (IDH) may be the only histopathological change. Here we describe the clinical and immunophenotypic features of a number of cases retrospectively identified reviewing consecutive liver biopsies from five Italian centers over 4 years. Methods We reviewed 1235 cases biopsied for chronic liver disease (1078 for viral hepatitis). Records of cases fulfilling the inclusion criteria for IDH were reviewed to identify possible aetiologies. Biopsies showing IDH and control biopsies were studied by immunohistochemistry for cytokeratin-7, epithelial-membrane-antigen (EMA), neural-cell-adhesion-molecule (NCAM), Ki-67. Results Out of 70 biopsies fulfilling IDH criteria, 16 (22.8%) were of unknown aetiology. Patients with idiopathic IDH (age 38.2±11 years) were asymptomatic with mild, long-lasting ALT and/or γGT increases. A significant increase of well-differentiated (EMA-positive; NCAM-negative) bile ductules localized at the portal interface and inside the lobule was found in idiopathic IDH. Conclusions Idiopathic IDH was present in 10% of adults biopsied for persistent mild liver function test abnormalities unrelated to viral hepatitis. In contrast with the ductular reaction seen in many forms of liver disease, it is characterized by well-differentiated hyperplastic ductules in absence of significant inflammation, and may represent a non-specific pattern of reaction to mild liver damages.

Background & Aims: The biliary epithelium is involved both in bile production and in the inflammatory/reparative response to liver damage. Recent data indicate that inflammatory aggression to intrahepatic bile ducts results in chronic progressive cholestasis. Methods: To understand the effects of nitric oxide on cholangiocyte secretion and biliary tract pathophysiology we have investigated: (1) the effects of proinflammatory cytokines on NO production and expression of the inducible nitric oxide synthase (NOS2), (2) the effects of NO on cAMP-dependent secretory mechanisms, and (3) the immunohistochemical expression of NOS2 in a number of human chronic liver diseases. Results: Our results show that: (1) tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, synergically stimulate NO production in cultured cholangiocytes through an increase in NOS2 gene and protein expression; (2) micromolar concentrations of NO inhibit forskolin-stimulated cAMP production by adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP)-dependent fluid secretion, and cAMP-dependent Cl − and HCO 3 − transport mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and anion exchanger isoform 2, respectively; (3) cholestatic effects of NO and of proinflammatory cytokines are prevented by NOS-2 inhibitors and by agents (manganese(III)-tetrakis(4-benzoic acid)porphyrin [MnTBAP], urate, trolox) able to block the formation of reactive nitrogen oxide species (RNOS); (4) NOS2 expression is increased significantly in the biliary epithelium of patients with primary sclerosing cholangitis (PSC). Conclusions: Our findings show that proinflammatory cytokines stimulate the biliary epithelium to generate NO, via NOS2 induction, and that NO causes ductular cholestasis by a RNOS-mediated inhibition of AC and of cAMP-dependent HCO 3 − and Cl − secretory mechanisms. This pathogenetic sequence may contribute to ductal cholestasis in inflammatory cholangiopathies. GASTROENTEROLOGY 2003;124:737-753

In human chronic liver disease, there is association between ductular reaction (DR) and fibrosis; yet, the mechanism triggering its onset and its role in scar formation remains unknown. Since we previously showed that osteopontin (OPN) is highly induced during drug-induced liver fibrosis, we hypothesised that OPN could drive oval cells (OC) expansion and DR and signal to hepatic stellate cells (HSC) to promote scarring.In vivo studies demonstrated increased OPN expression in biliary epithelial cells (BEC) and in OC in thioacetamide (TAA)-treated mice. OPN ablation protected mice from TAA and bile duct ligation-induced liver injury, DR and scarring. This was associated with greater hepatocyte proliferation, lower OC expansion and DR along with less fibrosis, suggesting that OPN could activate the OC compartment to differentiate into BEC, which could then signal to HSC to enhance scarring. Since TAA-treated wild-type mice and cirrhotic patients showed TGF-β(+) BEC, which were lacking in TAA-treated Opn(-/-) mice and in healthy human explants, this suggested that OPN could regulate TGF-β, a profibrogenic factor. In vitro experiments confirmed that recombinant OPN (rOPN) decreases hepatocyte proliferation and increases OC and BEC proliferation. To evaluate how BEC regulate collagen-I production in HSC, co-cultures were established. Co-cultured BEC upregulated OPN and TGF-β expression and enhanced collagen-I synthesis by HSC. Lastly, recombinant TGF-β (rTGFβ) and rOPN promoted BEC proliferation and neutralisation of OPN and TGF-β reduced collagen-I expression in co-cultured HSC.OPN emerges as a key matricellular protein driving DR and contributing to scarring and liver fibrosis via TGF-β.

Introduction: The management of hepatocellular carcinoma (HCC) is a well-known public health problem, accounting for increasing mortality and high costs. Quality parameters able to measure clinical outcomes are still lacking. Aim of our study (Value Based Medicine in Hepatology, VBMH)was to identify and test outcome indicators (OIs) in HCC, in light of their potential use in policy decision models. Methods: A panel of experts identified a list of OIs using a modified Delphi method, according to experience and published evidence; four OIs with the highest RAND/UCLA score were tested in a prospective multicenter observational study. During 18 months, 711 HCC patients were enrolled and prospectively followed. 677 patients (95%) had at least one follow-up consultation. Median follow-up time was 14 months. Results: The first OI was 1–3–5 years survival stratified for BCLC stage or treatment (OI#1). One-year survival for BCLC stage 0/A–D was 93%, 86%, 50%, 26% respectively. One-year survival of 288 patients treated for the first time during the study was 88% for liver transplantation, 97% surgical resection, 100% ablation and 89% for TACE. The other three OIs evaluated the appropriateness of treatments: worsening of BCLC and/or CPT score after three months from a loco-regional therapy or surgical resection (OI#2), occurred in 16% of cases (76% after TACE). Recurrence of HCC within 6 months after curative treatments (OI#3) happened in 15% of patients treated, mainly after ablation (20%). Presence of severe morbidity three months after loco-regional procedure or resection, evaluated as grade≥3 according to Clavien-Dindo classification (OI#3), was found in 3% of patients treated. Conclusions: The outcome indicators identified in the VBMH study proved their feasibility in a large cohort of patients and could stand as a benchmark for healthcare providers to move towards a value-based approach in the management of HCC.

Introduction: Liver Cirrhosis (LC) is responsible for high morbidity, mortality and costs, with increasing need to improve quality of care. Aim of our study (Value Based Medicine in Hepatology Study, VBMH) was to identify outcome indicators (OIs) able to measure quality in liver diseases, including compensated (CC) and decompensated (DC) LC. Methods: A panel of hepatologists identified a list of 7 OIs for LC according to experience and published evidence, using a modified Delphi method and a standard 9-pointRANDappropriateness scale. Then, these 7 OIs were tested in clinical practice in a prospective multicenter observational study involving three tertiary centers in Italy, using a web-based electronic medical record. 1751 LC patients were enrolled in 18 months: 1004 CC and 747 DC. 92% of patients had at least two consultations in 17 months median follow-up. Results: Annual rate of decompensation in CC was 12% (OI#1). Annual incidence of 1st variceal bleeding (VB) was 2% for low-risk and 3% for high-risk varices (OI#2) indicating a significant success of primary profilaxis. Annual incidence of HCC in CC was 4.3%, with 81% patients found at early stage (OI#3), underlining the accuracy of oncologic surveillance. One-year survival after the first decompensation episode (ascites in 74% of cases) was 96%, 81% and 59% stratified for CPT score A, B, C respectively (OI#4), and 94%, 56% stratified for MELD below or above 15 (OI#5). 4% of DC patients had an episode of VB with 90% survival after 6 weeks, and 33% recurrence (OI#6). Similarly, 3% ofDCpatients had spontaneous bacterial peritonitis with 6 weeks survival of 86% and 13% recurrence (OI#7). Conclusions: The value-based OIs generated in this study performed well in a large cohort of consecutive patients, and could represent a reference tool for healthcare providers to improve care in LC.

Biliary epithelial cells (cholangiocytes) modulate bile fluidity and alkalinity absorbing and/or secreting fluid and electrolytes, particularly HCO3- and Cl-. Mechanisms responsible for transepithelial H+/HCO3- secretion in human cholangiocytes are largely unknown. Human cholangiocytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatric liver transplantation were cultured in the presence of human hepatocyte growth factor. Maintenance of cholangiocyte phenotypic features was assessed using markers such as cytokeratin 19, gamma-glutamyltranspeptidase, vimentin, factor VIII-related antigen, desmin, epithelial membrane antigen (EMA), and human epithelial antigen (HEA) 125. Intracellular pH (pHi) transients were measured microfluorimetrically 2'7'-Bis(2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCO3-, pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na(+)-dependent and amiloride-inhibitable. No Na(+)-independent recovery was recorded even after stimulation with agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCO3-, recovery from an intracellular acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4-diisothiocyan atostilben-2,2-disulfonic acid (DIDS) and by intracellular Cl- depletion. Acute removal of extracellular Cl induced a pHi alkalinization that was inhibited by DIDS. pHi recovery from an intracellular alkaline load (isohydric CO2 changes) was Cl(-)-dependent and DIDS-inhibitable. Administration of agents raising intracellular cAMP concentrations increased both Na(+)-dependent and Na(+)-independent Cl-/HCO-3 exchange activity. Stimulation of Cl-/HCO3- exchange activity was not prevented by the Cl- channel inhibitor 5'-nitro-2(2)-phenylpropyl-amino-benzoate(NPPB). In conclusion, human cholangiocytes possess two acid extruders (Na+/H+exchanger and Na(+)-dependent Cl-/HCO3- exchange) and an acid loader (Cl-/HCO3- exchange), whereas no evidence was found for cAMP activated H(+)-ATPase. Bicarbonate influx is thus mainly mediated by Na-dependent Cl-/HCO3- exchange, whereas Na+:HCO-3 cotransport is not active in the physiological range of pHi. Stimulation of Na(+)-independent Cl-/HCO3- exchanger by cAMP does not require activation of Cl- conductances. These mechanisms may underlay hormone-regulated biliary HCO3- secretion in the human biliary tree.

Background & Aims Repair from biliary damages requires the biliary specification of hepatic progenitor cells and the remodeling of ductular reactive structures into branching biliary tubules. We hypothesized that the morphogenetic role of Notch signaling is maintained during the repair process and have addressed this hypothesis using pharmacologic and genetic models of defective Notch signaling. Methods Treatment with DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) or ANIT (alpha-naphthyl-isothiocyanate) was used to induce biliary damage in wild type mice and in mice with a liver specific defect in the Notch-2 receptor ( Notch-2 -cKO) or in RPB-Jk. Hepatic progenitor cells, ductular reaction, and mature ductules were quantified using K19 and SOX-9. Results In DDC treated wild type mice, pharmacologic Notch inhibition with dibenzazepine decreased the number of both ductular reaction and hepatic progenitor cells. Notch-2 -cKO mice treated with DDC or ANIT accumulated hepatic progenitor cells that failed to progress into mature ducts. In RBP-Jk -cKO mice, mature ducts and hepatic progenitor cells were both significantly reduced with respect to similarly treated wild type mice. The mouse progenitor cell line BMOL cultured on matrigel, formed a tubular network allowing the study of tubule formation in vitro ; γ-secretase inhibitor treatment and siRNAs silencing of Notch-1 , Notch-2 or Jagged-1 significantly reduced both the length and number of tubular branches. Conclusions These data demonstrate that Notch signaling plays an essential role in biliary repair. Lack of Notch-2 prevents biliary tubule formation, both in vivo and in vitro . Lack of RBP-Jk inhibits the generation of biliary-committed precursors and tubule formation.

Notch signalling is an evolutionarily conserved intercellular pathway involved in many aspects of development and tissue renewal in several organs. The importance of Notch signalling in liver development and morphogenesis is well established. However, the post-natal role of Notch in liver repair/regeneration is only now beginning to be unveiled. Despite the simplicity of the pathway activation, a fine spatial-temporal regulation of Notch signalling is required to avoid pathologic effects. This review highlights recent advances in the field indicating that Notch signalling is involved in the reparative morphogenesis of the biliary tree and in liver carcinogenesis. Defective Notch signalling leads to impaired ability of the liver to repair liver damage, while excessive activation may be involved in liver cancer. Even though much remains to be understood about these mechanisms, including the cross-talk between Notch signalling and other liver morphogens, current evidence suggests that the modulation of the Notch pathway may represent a therapeutic target in chronic liver disease.