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Background & Aims: Mechanisms and clinical impact of portal microthrombosis featuring severe COVID-19 are unknown. Intrapulmonary vascular dilation (IPVD)-related hypoxia has been described in severe liver diseases. We hypothesized that portal microthrombosis is associated with IPVD and fatal respiratory failure in COVID-19., Methods: Ninety-three patients who died from COVID-19, were analysed for portal microvascular damage (histology), IPVD (histology and chest-computed tomography, CT), and hypoxemia (arterial blood gas). Seventeen patients who died from COVID-19-unrelated pneumonia served as controls. Vascular lesions and microthrombi were phenotyped for endothelial (vWF) and pericyte (αSMA/PDGFR-β) markers, tissue factor (TF), viral spike-protein and nucleoprotein (SP, NP), fibrinogen, platelets (CD41a). Viral particles in vascular cells were assessed by transmission electron microscopy (TEM). Cultured pericytes were infected with SARS-CoV-2 to measure TF expression and tubulisation of human pulmonary microvascular endothelial cells (HPMEC) was assessed upon vWF treatment., Results: IPVD was present in 16/66 COVID-19 patients with both liver and lung histology, with a younger age (62 vs 78yo), longer illness (25 vs 14 days), worsening hypoxemia (PaO 2 /FiO 2 from 209 to 89), and more ventilatory support (63 vs 22%) compared to COVID-19/Non-IPVD. IPVD, absent in controls, were confirmed by chest-CT. COVID-19/IPVD liver histology showed portal microthrombosis in >82.5% of portal areas, with a thicker wall of αSMA/PDGFR-β + / SP + /NP + pericytes compared with COVID-19/Non-IPVD. Thrombosed portal venules correlated with αSMA + area, whereas infected SP + /NP + pericytes expressed TF. SARS-CoV-2 viral particles were observed in portal pericytes. In-vitro SARS-CoV-2 infection of pericytes up-regulated TF and induced endothelial cells to overexpress vWF, which expanded HPMEC tubules., Conclusions: SARS-CoV-2 infection of liver pericytes elicits a local procoagulant response associated with extensive portal microthrombosis, IPVD and worsening respiratory failure in fatal COVID-19., Impact and Implications: Vascular involvement of the liver represents a serious complication of COVID-19 infection that must be considered in the work-up of patients with long-lasting and progressively worsening respiratory failure, as it may associate with the development of intrapulmonary vascular dilations. This clinical picture is associated with a pro-coagulant phenotype of portal venule pericytes, which is induced by SARS-CoV-2 infection of pericytes. Both observations provide a model that may apply, at least in part, to other vascular disorders of the liver, featuring obliterative portal venopathy, similarly characterized at the clinical level by development of hypoxemia and at the histological level, by phlebosclerosis and reduced caliber of the portal vein branches in the absence of cirrhosis. Moreover, our findings bring light to an as yet overlooked player of thrombosis pathophysiology, i.e. pericytes, which may provide novel therapeutic tools to halt prothrombotic mechanisms., Competing Interests: Declaration of Competing Interest Authors have nothing to disclosure., (Copyright © 2024. Published by Elsevier B.V.)

Metabolic dysfunction-associated fatty liver disease (MAFLD), the hepatic component of the metabolic syndrome caused by insulin resistance, is a major public health problem, affecting about the 25 % of the general population in Western countries. Morbidity and mortality of MAFLD patients is increased primarily due to cardiovascular disease (CVD). Liver fibrosis, the byproduct of hepatic repair, is the main determinant of MAFLD progression and the strongest predictor for overall mortality. Since the mechanistic relationship between MAFLD, fibrosis, insulin resistance and the cardiometabolic risk is far to be clear, deciphering the functional link of hepatic fibrogenesis with genetic factors and hypercoagulability in MAFLD-associated CVD may hold translational potential for risk profiling and innovative therapeutic targeting., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Biliary tract cancers are an uncommon set of gastrointestinal malignancies that are associated with high morbidity and mortality rates. Most patients present with incurable locally advanced or metastatic disease. The pathophysiology of biliary tract cancer can be exploited for direct therapeutic benefit, and indeed, chemotherapy, precision medicine, immunotherapy and combination treatments are now applied as both standard-of-care and investigational therapies. In the first-line setting, the immune-based chemotherapy combination of durvalumab plus gemcitabine and cisplatin has recently been shown to improve survival compared to chemotherapy alone. In the second-line, precision medicine can be employed in those with select genetic alterations in IDH1/2 (isocitrate dehydrogenase 1/2), FGFR2 (fibroblast growth factor receptor 2), KRAS, BRAF, ERBB2, NTRK (neurotrophic receptor tyrosine kinase), ROS, RET, and/or deficiencies in mismatch repair enzymes. In those patients without targetable genetic alterations, fluoropyridine doublets lead to modest improvements in outcomes. Next-generation sequencing is critical for direct patient care and to help elucidate genomic mechanisms of resistance in a research context. Currently, multiple clinical trials are ongoing - hence, this review seeks to provide an update on evolving standards of care and ongoing investigational agents, limitations to current treatments, and a framework for effective combination drug development for the future., Competing Interests: Conflict of interest James J. Harding reports grant research support from BI, Bristol Myers Squibb, Calithera, CytomX, Eli Lilly, Genoscience, Incyte, Novartis, Polaris, Yiviam and Zymeworks; consulting supprot from Adaptimmune, Bristol Myers Squibb, Eisai, Elevar, Exelixis, Hepion, Medivir, and Merck; and honoraria from HCC Connect, and NCCN. Danny N. Khalil reports royalty for intellectual property rights associated with Merck Sharp & Dohme; consulting fees from AbbVie and Psioxus; and patents EP3331612A1, CA3042867A1 for which he holds intellectual property interests related to CD40, nanoparticle therapeutics, and in situ vaccination. Luca Fabris reports consulting fees from L.E.K. Consulting and MedPanel; spport for attending European Association for the Study of the Liver (EASL) Liver Cancer Summit (2020), Prague, CZE, and European Network for the Study of Cholangiocarcinoma (ENS-CCA) Biennal Meeting (2022), Edinburgh, Scotland, ledership fiudicary role in International PSC Study Group, Working Group Malignancy (ongoing) (unpaid), Member Expert Panel, National Science Centre, Poland - Narodowe Centrum Nauki (NCN), Life Science section NZ5, Krakow (2019 e 2020), and external Reviewer of Doctoral Thesis for International Assessment. Invited by the Rabdoud University, The Netherlands (2022). Ghassan K. Abou-Alfa reports grant research support from Arcus, Astra Zeneca, BioNtech, BMS, Celgene, Flatiron, Genentech/Roche, Genoscience, Incyte, Polaris, Puma, QED, Silenseed, Yiviva, and consulting supprot from Adicet, Alnylam, Astra Zeneca, Autem, Beigene, Berry Genomics, Boehringer Ingelheim, Celgene, Cend, CytomX, Eisai, Eli Lilly, Exelixis, Flatiron, Genentech/Roche, Genoscience, Helio, Helsinn, Incyte, Ipsen, Merck, Nerviano, Newbridge, Novartis, QED, Redhill, Rafael, Servier, Silenseed, Sobi, Vector, Yiviva. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression., Methods: Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated in vitro, in vivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1 +/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry., Results: The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation in vitro. NEDDylation depletion (pevonedistat or Nae1 +/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA in vivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells in vitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration., Conclusion: Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA., Lay Summary: Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors., Competing Interests: Conflict of interest Authors disclose no conflict of interest related to this manuscript. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: 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., Methods: 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., Results: 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., Conclusion: 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., Lay Summary: 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 11 th 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., Competing Interests: Conflict of interest AF declares lecture fees (from Bayer HealthCare, Gilead, Roche, MSD and Boston Scientific), and consultancy fees (from Bayer HealthCare, AstraZeneca, Roche, Guerbert, SIRTEX and Exact Science). AL reported travel and educational support (from Ipsen, Pfizer, Bayer, Advanced Acceletaro Applications, SirtEx, Novartis, Mylan and Delcath), speaker honoraria (from Merck, Pfizer, Ipsen, Incyte and AAA), advisory honoraria (from EISAI, Nutricia Ipsen, QED and Roche), and she is Member of the Knowledge Network and NETConnect Initiatives funded by Ipsen. ALl declares lecture fees (from Intercept Pharma, Abbvie, Gilead, AlfaSigma, and MSD) and consultancy fees (from Intercept and Alfa Sigma). AL-M declares travel and educational support (from Pfizer, Roche, Sanofi, Rovi, Pharma Mar and Merck). CB declares consultancy honoraria (from Incyte). GV declares consultancy fee (from Bayer HealthCare). JBA declares consulting role (for QED Therapeutics and SEALD). JMB declares research grants (from Incyte), personal fees for lecturer (from Bayer and Intercept), and consulting role (for QED Therapeutics, Albireo Pharma and OWL Metabolomics). JWV reports personal fees (from Agios, AstraZeneca, Baxter, Genoscience Pharma, Hutchison Medipharma, Imaging Equipment Ltd (AAA), Incyte, Ipsen, Mundipharma EDO, Mylan, QED, Servier, Sirtex, Zymeworks), and grants, personal fees and non-financial support (from NuCana). LF declares research grants (from Progetto di Ricerca Dipartimentale), honoraria (from Obesity 4C Complex Clinical Cases Contest), leadership role (for HCERES, NCN), and travel expenses (from AASLD, AISF and EASL). MP-S declares honoraria (from Roche and Servier), consulting or advisory role (for AstraZeneca) and travel expenses (from Roche, BMS and Incyte). KU declares fees for advisory role (BMS). ALC, AS, ASc, AS-L, AV, BGK, DA, FL-L, GC, GLG, HJK, JA, JI, JIE, JJGM, JK, JPJ, KE, LB, LI-S, LK, MK, MM, NJ, RB, RIRM, SB, TF, VC, VS, WH, and ZS declare no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Recent evidence has consistently suggested a role for HCV in the etiology of head and neck squamous cell carcinoma (HNSCC), but the conclusions of these studies have often been limited by small sample size. Therefore, a meta-analysis was performed to summarize present evidence on the association between HCV infection and HNSCC. After screening citations from literature search, eight observational studies investigating the association between HCV and cancer(s) of either oral cavity, oropharynx, hypopharynx or larynx were included. For each cancer site, risk ratios from individual studies were displayed in forest plots; pooled risk ratios (RR) and corresponding confidence intervals (CI) were calculated. A significant association was found between HCV infection and cancers of the oral cavity (RR = 2.13; 95%: 1.61-2.83), oropharynx (RR = 1.81; 95% CI: 1.21-2.72), and larynx (RR = 2.57; 95% CI: 1.11-5.94). A similar picture emerged for hypopharyngeal cancer, though this result did not fully reach statistical significance because of the small number of available studies (RR = 2.15; 95% CI: 0.73-6.31). These findings remained similar after exclusion of patients with HIV co-infection. Our results highlighted the importance of surveillance of the upper aerodigestive tract in patients with known chronic HCV infections in order to enable HNSCC early diagnosis. In addition, they could be a reminder of the possibility of undiagnosed HCV infection to the clinicians treating HNSCC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Melatonin, a neuroendocrine hormone synthesized by the pineal gland and cholangiocytes, decreases biliary hyperplasia and liver fibrosis during cholestasis-induced biliary injury via melatonin-dependent autocrine signaling through increased biliary arylalkylamine N-acetyltransferase (AANAT) expression and melatonin secretion, downregulation of miR-200b and specific circadian clock genes. Melatonin synthesis is decreased by pinealectomy (PINX) or chronic exposure to light. We evaluated the effect of PINX or prolonged light exposure on melatonin-dependent modulation of biliary damage/ductular reaction/liver fibrosis. Studies were performed in male rats with/without BDL for 1 week with 12:12 h dark/light cycles, continuous light or after 1 week of PINX. The expression of AANAT and melatonin levels in serum and cholangiocyte supernatant were increased in BDL rats, while decreased in BDL rats following PINX or continuous light exposure. BDL-induced increase in serum chemistry, ductular reaction, liver fibrosis, inflammation, angiogenesis and ROS generation were significantly enhanced by PINX or light exposure. Concomitant with enhanced liver fibrosis, we observed increased biliary senescence and enhanced clock genes and miR-200b expression in total liver and cholangiocytes. In vitro, the expression of AANAT, clock genes and miR-200b was increased in PSC human cholangiocyte cell lines (hPSCL). The proliferation and activation of HHStecs (human hepatic stellate cell lines) were increased after stimulating with BDL cholangiocyte supernatant and further enhanced when stimulated with BDL rats following PINX or continuous light exposure cholangiocyte supernatant via intracellular ROS generation. Conclusion: Melatonin plays an important role in the protection of liver against cholestasis-induced damage and ductular reaction., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Liver diseases negatively impact the quality of life and survival of patients, and often require liver transplantation in cases that progress to organ failure. Understanding the cellular and molecular mechanisms of liver development and pathogenesis has been a challenging task, in part for the lack of adequate cellular models directly relevant to the human diseases. Recent technological advances in the stem cell field have shown the potentiality of induced pluripotent stem cells (iPSC) and liver organoids as the next generation tool to model in vitro liver diseases. Hepatocyte-like cells and cholangiocyte are currently being generated from skin fibroblasts and mononuclear blood cells reprogrammed into iPSC and have been successfully used for disease modeling, drug testing and gene editing, with the hope to be able to find application also in regenerative medicine. Protocols to generate other liver cell types are still under development, but the field is advancing rapidly. On the other end, liver cells can now be isolated from liver specimens (liver explants or liver biopsies) and cultured in specific conditions to form polarized 3D organoids. The purpose of this review is to summarize all these recent technological advances and their potential applications but also to analyze the current issues to be addressed before the technology can reach its full potential., (Copyright © 2018. Published by Elsevier B.V.)

Liver disease is a severe complication in patients with Cystic Fibrosis (CF), a genetic disease caused by mutations in the gene encoding for cystic fibrosis transmembrane conductance regulator (CFTR) channel. The sequence of events leading to CFLD is still unclear and has limited the development of more specific treatments other than the bile acid UDCA. However, in the last twenty years, several gaps have been filled, which have mainly been possible due to the availability of different animal models that mimic CF. CF mice, although they lack a spontaneous liver manifestation, have been essential to better understand the multiple functions of CFTR expression on the apical membrane of cholangiocytes, from chloride channel to regulator of epithelial innate immunity. Additionally, we have learned that the gut microbiota might be a pathogenetic factor for the development of liver disease. The recent creation of novel CF animal models (i.e. pig and ferret) that better reproduce the human disease, will allow for comparative studies with species that spontaneously develop the liver disease and will hopefully lead to novel therapeutic treatments. In this review, we have compared and summarized the main features of the current available CF animal models and their applicability for the study of the liver phenotype., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cholestasis is a frequent clinical condition initiating or complicating chronic liver diseases, particularly cholangiopathies, where the biliary epithelium is the primary target of the pathogenetic sequence. Until a few decades ago, understanding of cholestasis relied mostly on the experimental model of bile duct ligation in rodents. However, a simple model of biliary obstruction cannot reproduce the complex mechanisms and networks leading to cholestasis in cholangiopathies. These networks are underpinned by an intricate dysregulation of pro-inflammatory and pro-fibrotic signals involving besides cholangiocytes, multiple cell elements of both innate and adaptive immunity. Therefore, in the last years, a wide range of animal models of biliary injury have been developed, mostly in mice, following three main approaches, chemical induction, immunization and genetic manipulation. In this review, we will give an update of the animal models of the two main cholangiopathies, primary sclerosing cholangitis and primary biliary cholangitis, which have provided us with the most relevant insights into the pathogenesis of these still controversial diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background & Aims: In cholangiocarcinoma, early metastatic spread via lymphatic vessels often precludes curative therapies. Cholangiocarcinoma invasiveness is fostered by an extensive stromal reaction, enriched in cancer-associated fibroblasts (CAFs) and lymphatic endothelial cells (LECs). Cholangiocarcinoma cells recruit and activate CAFs by secreting PDGF-D. Herein, we investigated the role of PDGF-D and liver myofibroblasts in promoting lymphangiogenesis in cholangiocarcinoma., Methods: Human cholangiocarcinoma specimens were immunostained for podoplanin (LEC marker), α-SMA (CAF marker), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants. Using human LECs incubated with conditioned medium from PDGF-D-stimulated fibroblasts we assessed migration, 3D vascular assembly, transendothelial electric resistance and transendothelial migration of cholangiocarcinoma cells (EGI-1). We then studied the effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymph node metastases in vivo., Results: In cholangiocarcinoma specimens, CAFs and LECs were closely adjacent. CAFs expressed VEGF-A and VEGF-C, while LECs expressed VEGFR2 and VEGFR3. Upon PDGF-D stimulation, fibroblasts secreted increased levels of VEGF-C and VEGF-A. Fibroblasts, stimulated by PDGF-D induced LEC recruitment and 3D assembly, increased LEC monolayer permeability, and promoted transendothelial EGI-1 migration. These effects were all suppressed by the PDGFRβ inhibitor, imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion, markedly reduced lymphatic vascularization and reduced lymph node metastases., Conclusion: PDGF-D stimulates VEGF-C and VEGF-A production by fibroblasts, resulting in expansion of the lymphatic vasculature and tumor cell intravasation. This critical process in the early metastasis of cholangiocarcinoma may be blocked by inducing CAF apoptosis or by inhibiting the PDGF-D-induced axis., Lay Summary: Cholangiocarcinoma is a highly malignant cancer affecting the biliary tree, which is characterized by a rich stromal reaction involving a dense population of cancer-associated fibroblasts that promote early metastatic spread. Herein, we show that cholangiocarcinoma-derived PDGF-D stimulates fibroblasts to secrete vascular growth factors. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce the invasiveness of cholangiocarcinoma., (Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The most studied physiological function of biliary epithelial cells (cholangiocytes) is to regulate bile flow and composition, in particular the hydration and alkalinity of the primary bile secreted by hepatocytes. After almost three decades of studies it is now become clear that cholangiocytes are also involved in epithelial innate immunity, in inflammation, and in the reparative processes in response to liver damage. An increasing number of evidence highlights the ability of cholangiocyte to undergo changes in phenotype and function in response to liver damage. By participating actively to the immune and inflammatory responses, cholangiocytes represent a first defense line against liver injury from different causes. Indeed, cholangiocytes express a number of receptors able to recognize pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), such as Toll-like receptors (TLR), which modulate their pro-inflammatory behavior. Cholangiocytes can be both the targets and the initiators of the inflammatory process. Derangements of the signals controlling these mechanisms are at the basis of the pathogenesis of different cholangiopathies, both hereditary and acquired, such as cystic fibrosis-related liver disease and sclerosing cholangitis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 54months 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., (Copyright © 2017. Published by Elsevier B.V.)

In the last 25years, a number of animal models, mainly rodents, have been generated with the goal to mimic cholestatic liver injuries and, thus, to provide in vivo tools to investigate the mechanisms of biliary repair and, eventually, to test the efficacy of innovative treatments. Despite fundamental limitations applying to these models, such as the distinct immune system and the different metabolism regulating liver homeostasis in rodents when compared to humans, multiple approaches, such as surgery (bile duct ligation), chemical-induced (3,5-diethoxycarbonyl-1,4-dihydrocollidine, DDC, α-naphthylisothiocyanate, ANIT), viral infections (Rhesus rotavirustype A, RRV-A), and genetic manipulation (Mdr2, Cftr, Pkd1, Pkd2, Prkcsh, Sec63, Pkhd1) have been developed. Overall, they have led to a range of liver phenotypes recapitulating the main features of biliary injury and altered bile acid metabolisms, such as ductular reaction, peribiliary inflammation and fibrosis, obstructive cholestasis and biliary dysgenesis. Although with a limited translability to the human setting, these mouse models have provided us with the ability to probe over time the fundamental mechanisms promoting cholestatic disease progression. Moreover, recent studies from genetically engineered mice have unveiled 'core' pathways that make the cholangiocyte a pivotal player in liver repair. In this review, we will highlight the main phenotypic features, the more interesting peculiarities and the different drawbacks of these mouse models. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We show how spatial variability in channel bed morphology affects the hydraulic characteristics of river reaches available to Atlantic salmon parr (Salmo salar) under different flow conditions in an upland stream. The study stream, the Girnock Burn, is a long-term monitoring site in the Scottish Highlands. Six site characterised by different bed geometry and morphology were investigated. Detailed site bathymetries were collected and combined with discharge time series in a 2D hydraulic model to obtain spatially distributed depth-averaged velocities under different flow conditions. Available habitat (AH) was estimated for each site. Stream discharge was used according to the critical displacement velocity (CDV) approach. CDV defines a velocity threshold above which salmon parr are not able to hold station and effective feeding opportunities or habitat utilization are reduced, depending on fish size and water temperature. An average value of the relative available habitat () for the most significant period for parr growth - April to May - was used for inter-site comparison and to analyse temporal variations over 40years. Results show that some sites are more able than others to maintain zones where salmon parr can forage unimpeded by high flow velocities under both wet and dry conditions. With lower flow velocities, dry years offer higher values of than wet years. Even though can change considerably across the sites as stream flow changes, the directions of change are consistent. Relative available habitat (RAH) shows a strong relationship with discharge per unit width, whilst channel slope and bed roughness either do not have relevant impact or compensate each other. The results show that significant parr habitat was available at all sites across all flows during this critical growth period, suggesting that hydrological variability is not a factor limiting growth in the Girnock., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Genetic defects in polycystin-1 or -2 (PC1 or PC2) cause polycystic liver disease associated with autosomal dominant polycystic kidney disease (PLD-ADPKD). Progressive cyst growth is sustained by a cAMP-dependent Ras/ERK/HIFα pathway, leading to increased vascular endothelial growth factor A (VEGF-A) signaling. In PC2-defective cholangiocytes, cAMP production in response to [Ca 2+ ] ER depletion is increased, while store-operated Ca 2+ entry (SOCE), intracellular and endoplasmic reticulum [Ca 2+ ] ER levels are reduced. We investigated whether the adenylyl cyclases, AC5 and AC6, which can be inhibited by Ca 2+ , are activated by the ER chaperone STIM1. This would result in cAMP/PKA-dependent Ras/ERK/HIFα pathway activation in PC2-defective cells, in response to [Ca 2+ ] ER depletion., Methods: PC2/AC6 double conditional knockout (KO) mice were generated (Pkd2/AC6 KO) and compared to Pkd2 KO mice. The AC5 inhibitor SQ22,536 or AC5 siRNA were used in isolated cholangiocytes while the inhibitor was used in biliary organoid and animals; liver tissues were harvested for histochemical analysis., Results: When comparing Pkd2/AC6 KO to Pkd2 KO mice, no decrease in liver cyst size was found, and cellular cAMP after [Ca 2+ ] ER depletion only decreased by 12%. Conversely, in PC2-defective cells, inhibition of AC5 significantly reduced cAMP production, pERK1/2 expression and VEGF-A secretion. AC5 inhibitors significantly reduced growth of biliary organoids derived from Pkd2 KO and Pkd2/AC6 KO mice. In vivo treatment with SQ22,536 significantly reduced liver cystic area and cell proliferation in PC2-defective mice. After [Ca 2+ ] ER depletion in PC2-defective cells, STIM1 interacts with AC5 but not with Orai1, the Ca 2+ channel that mediates SOCE., Conclusion: [Ca 2+ ] ER depletion in PC2-defective cells activates AC5 and results in stimulation of cAMP/ERK1-2 signaling, VEGF production and cyst growth. This mechanism may represent a novel therapeutic target., Lay Summary: Polycystic liver diseases are characterized by progressive cyst growth until their complications mandate surgery or liver transplantation. In this manuscript, we demonstrate that inhibiting cell proliferation, which is induced by increased levels of cAMP, may represent a novel therapeutic target to slow the progression of the disease., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Transcribed ultraconserved regions (T-UCRs) are genomic regions conserved across large evolutionary distances, which encode for noncoding RNAs that serve as regulators of gene expression. Although T-UCRs have been linked to multiple aspects of mammalian gene regulation, the roles of their extreme evolutionary conservation remain largely unexplained. Growing body of literature is now focusing on T-UCRs as potential cancer biomarkers or as new drug targets. Here we present an overview of the discoveries so far published about the role of T-UCR in cancer and disease., (© 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Multiplex analysis permits the detection of several analytical targets at the same time. This approach may permit to draw a rapid and accurate diagnostic about the health of an individual or an environment. Among the analytical techniques with potential for multiplexing surface-enhanced Raman scattering (SERS) offer unique advantages such as ultrasensitive detection down low the deconvolution times, a unique signature containing all the vibrational information of the target molecules, and the possibility of performing the experiments even in very demanding environments such as natural or biological fluids. Here we review the late advances in multiplex SERS including the direct methods, those aided by the surface functionalization of the plasmonic nanoparticles and the use of SERS encoded particles., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Several 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., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Very few studies assessed cholangiocarcinoma clinical characteristics., Aim: To evaluate the clinical characteristics of intra-hepatic (IH) and extra-hepatic (EH)-CCA., Methods: We performed a national survey based on a questionnaire., Results: 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., Conclusions: 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., (Copyright © 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: The HLA complex on chromosome 6p21 is firmly established as a risk locus for primary sclerosing cholangitis (PSC). We aimed to exploit genetic differences between Northern Europe and Italy in an attempt to define a causative locus in this genetic region., Methods: Seventy-eight North-Italian PSC patients and 79 controls were included. We performed sequencing-based genotyping of HLA-C, HLA-B, and HLA-DRB1. The major histocompatibility chain-related A (MICA) transmembrane microsatellite was analysed using PCR fragment length determination. The tumour necrosis factor-alpha (TNF-alpha)-308G-->A polymorphism was genotyped with TaqMan. Allele frequencies were compared with Chi-square tests. Uncorrected p-values <0.05 were considered statistically significant when replicating findings in previous studies. The p-values of novel associations were corrected for multiple comparisons (Bonferroni)., Results: The frequency of the strong inhibitory HLA-C2 killer-immunoglobulin receptor (KIR) ligand variant was significantly reduced in PSC vs. controls (0.39 vs. 0.58, p=0.0006). Consequently, HLA-C1 homozygosity was associated with an increased risk of PSC (OR 3.1; 95% CI 1.4-6.7, p=0.004). Importantly, there were no significant associations with the HLA-Bw4 KIR ligand variant, at the neighbouring MICA locus or with TNF-alpha-308G-->A. At HLA-DRB1, we confirmed positive and negative associations with DRB1*15 and DRB1*07, respectively, while there were no associations with the DRB1*03, *04 or *1301 alleles typically detected in PSC in Northern Europe., Conclusions: The strong inhibitory of the KIR ligand HLA-C2 protects against PSC development in all populations hitherto studied. Further studies on the role of natural killer cells and T-lymphocytes expressing KIRs in PSC pathogenesis are warranted., (Copyright (c) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Hyperbilirubinemia is a common laboratory finding in clinical practice, being found in several haematological and liver diseases as well as in familial conditions (5-10% in Western countries). Although most of the familial forms of hyperbilirubinemia are classically viewed as benign conditions, they have gained an increased interest in the last few years since recent data have indicated that subjects with an impaired bilirubin metabolism may have an increased susceptibility to drug toxicity. The authors briefly review the main steps of bilirubin metabolism, with a special emphasis on the emerging concepts on the molecular mechanisms of regulation by nuclear receptors (NRs) and genetic factors. Then the different forms of isolated hyperbilirubinemia occurring in both adults and paediatrics are systematically analysed, and a new categorisation is also proposed in light of the recent advances in bilirubin research. Finally, a diagnostic algorithm is discussed, along with a correct approach to its management, in order to avoid unnecessary medical investigations.

Patients with Alagille syndrome (AGS), a genetic disorder of Notch signaling, suffer from severe ductopenia and cholestasis, but progression to biliary cirrhosis is rare. Instead, in biliary atresia (BA) severe cholestasis is associated with a pronounced "ductular reaction" and rapid progression to biliary cirrhosis. Given the role of Notch in biliary development, we hypothesized that defective Notch signaling would influence the reparative mechanisms in cholestatic cholangiopathies. Thus we compared phenotype and relative abundance of the epithelial components of the hepatic reparative complex in AGS (n = 10) and BA (n = 30) using immunohistochemistry and computer-assisted morphometry. BA was characterized by an increase in reactive ductular and hepatic progenitor cells, whereas in AGS, a striking increase in intermediate hepatobiliary cells contrasted with the near absence of reactive ductular cells and hepatic progenitor cells. Hepatocellular mitoinhibition index (p21(waf1)/Ki67) was similar in AGS and BA. Fibrosis was more severe in BA, where portal septa thickness positively correlated with reactive ductular cells and hepatic progenitor cells. AGS hepatobiliary cells failed to express hepatic nuclear factor (HNF) 1beta, a biliary-specific transcription factor. These data indicate that Notch signaling plays a role in liver repair mechanisms in postnatal life: its defect results in absent reactive ductular cells and accumulation of hepatobiliary cells lacking HNF1beta, thus being unable to switch to a biliary phenotype.

Background and Aim: To clarify the precise mode of inheritance of Gilbert syndrome, an unconjugated familial hyperbilirubinemia, where impaired bilirubin conjugation is caused by reduced UGT1A1 activity determined by a defective function of the A(TA)6TAA promoter region of the UGT1A1 gene., Subjects and Methods: Serum bilirubin levels were measured in a large, homogeneous resident population from North-Eastern Italy, consisting of 1.639 males (age 44.5+/-13.9, range 18-89 years), and 1.420 females (age 45.1+/-15.0, range 18-85). In 112 nuclear families from hyperbilirubinemic probands living in the same area a complex segregation analysis was then performed. In both samples we carefully excluded potentially confounding factors of bilirubin levels (alcohol abuse, excessive cigarette smoking, drug consumption, overt haemolysis and liver disease)., Results: Mean serum bilirubin concentrations are higher in males than in females, showing fluctuations through the different age periods in males. Complex segregation results demonstrate that unconjugated hyperbilirubinemia exhibits a precise mode of inheritance in which a major recessive gene with a frequency of 0.45 is responsible for higher serum bilirubin values., Conclusions: This major recessive gene accounts only for a part of the serum bilirubin concentration, thus implying additional, environmental factors for the clinical appearance of GS.

Background: The natural history of hepatitis C virus infection in the elderly is poorly known., Objective: To assess the mortality rate, the progression of liver disease, the hepatitis C virus carrier state and the co-morbidity in a cohort of 35 out of 1,063 anti-hepatitis C virus positive elderly people prospectively followed-up from 1992 to 2002., Methods: Liver function tests, hepatitis C virus-RNA analysis, hepatitis C virus genotyping and abdominal ultrasonography were assessed at the beginning of the study, and then, liver function tests and ultrasonography were performed annually during the first 5 years of the follow-up. At the end of the 10-year period, causes of death were recorded, while surviving patients underwent again medical examination, liver function tests and abdominal ultrasonography., Results: Out of 35 patients with a 10-year follow-up, 12 patients died: only 2 (5.7%) from liver-related disease (hepatocellular carcinoma and liver failure), whilst 10 (28.5%) from extrahepatic causes. Out of the two patients dying from liver-related causes, one was hepatitis C virus-RNA positive and one hepatitis C virus-RNA negative. Among the 23 living patients, 13 were hepatitis C virus-RNA positive (56.5%), the majority being infected with genotype 2 (69%); of them, 6 (46.1%) had persistently normal alanine aminotransferase levels. None of the hepatitis C virus-RNA positive individuals had excessive alcohol intake., Conclusion: Despite the presumably long duration of infection in our cohort, the liver-related mortality was five-fold lower than that from extrahepatic causes (five-fold higher). Lack of hepatic co-morbidity factors, such as alcohol consumption, seems to be relevant for the limited severity of liver disease.

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 gammaGT 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 understanding of the physiology and function of human biliary epithelial cells (hBEC) has been improved by studies in monolayer culture systems. The aim was to develop a polarized model to elucidate the mechanisms of ductular morphogenesis and functional differentiation of hBEC., Methods: The morphological, phenotypic and functional properties of hBEC cultured as three-dimensional aggregates in collagen gel were assessed in medium supplemented with (or without) human hepatocyte growth factor (hHGF) and foetal bovine serum., Results: In the absence of added mitogens and serum, cells maintained as morphologically polarized aggregates, organized around a central lumen, were positive for phenotypic markers of biliary epithelium and negative for markers of other cell types. Functional markers, gamma-glutamyl-transferase, anion exchanger-2, responses to gamma interferon and forskolin induced secretion, were preserved. hHGF increased both the size and number of aggregates and induced hBEC to invade the gel and lumena forming anastomosing networks of cells., Conclusions: Collagen gel culture in the absence of added growth factors and serum provides a model for analysis of the polarized functions of hBEC. The formation of poorly organized cords of cells in response to hHGF suggests that collagen gel culture may provide a model for the investigation of atypical ductular morphogenesis of the human biliary tract.

It has recently been shown that reactive bile ductules display neuroendocrine features, including immunoreactivity for the neural cell adhesion molecule (NCAM). In this study we have compared the immunohistochemical expression of NCAM with that of HEA-125 (biliary specific) and LKM-1 (hepatocyte specific) and other markers relevant to morphogenesis (Bcl-2, EMA) and cell proliferation (Ki-67) in cryostat sections from different chronic liver diseases and from fetal livers at different gestational ages. In parallel, viable NCAM-positive ductular cells were purified from collagenase digests of cirrhotic livers by immunomagnetic separation and characterized by immunocytochemistry and transmission electron microscopy. We demonstrated that reactive ductules with atypical morphology coexpressed NCAM and Bcl-2 and were found mainly in congenital diseases associated with ductal plate malformation and in primary cholangiopathies. On the contrary, reactive ductules with typical morphology were negative for NCAM/Bcl-2 and positive for EMA. Reactive ductules coexpressing NCAM/Bcl-2 were negative for the proliferation marker Ki-67 and appeared to be directly connected with periportal hepatocytes. In fetal livers NCAM/Bcl-2 was transiently expressed during the early developmental stages of ductal plate (10-16 weeks) and started to disappear as the ductal plate began duplicating. NCAM-positive ductal plate cells were Ki-67 negative, becoming positive in duplicated segments. Thus the histogenesis of ductular reactive cells seems to recapitulate the early stages of biliary ontogenesis. In primary cholangiopathies and ductal plate malformations, these cells do not appear to maturate further, and thus abundant ductular structures coexist with vanishing mature ducts. These NCAM-positive ductular cells were immunopurified from patients with chronic cholestatic liver diseases and showed ultrastructural features consistent with a less differentiated phenotype than mature cholangiocytes. These isolated cells represent a useful model for in vitro studies.

The term oval cell describes small cells with oval nuclei that arise in the periphery of the portal tracts in rat models of hepatocarcinogenesis and injury and can differentiate into either hepatocytes or bile duct cells, ie, are bipotential. The presence of such cells in human liver is controversial. Here, immunolocalization of OV-6 and two biliary markers, cytokeratin 19 (CK-19) and human epithelial antigen 125 (HEA-125) is compared in normal adult human livers and in primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) liver sections. CK-19 and HEA-125 stained bile ducts and ductules in normal liver as well as proliferating ductular structures in diseased livers. OV-6 did not label ducts or ductules in normal liver, but in PBC and PSC stained numerous proliferating ductular and periductular cells and lobular hepatocytes. In PBC, discrete OV-6-positive cells with a mature biliary-cell-like morphology were seen integrated into some intact bile ducts as well as occasional small immature oval-like cells. In addition, in PSC, hepatocytes in regenerating lobules were also strongly stained with OV-6, and on close inspection, in both PBC and PSC, oval cells and small hepatocytes at the margins of the lobules were strongly labeled. In contrast to the rat liver, OV-6 and CK-19 staining did not always co-localize. It is proposed that the small OV-6-positive oval cells are analogous to those seen in rat models and may represent human liver progenitor cells that may differentiate into OV-6-positive ductal cells or lobular hepatocytes.