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Glucagon-like peptide-1 (GLP-1) receptor agonists and the dual GLP-1- and glucose-dependent insulinotropic polypeptide receptor co-agonist tirzepatide (referred to here collectively as "GLP-1-based therapy") are incretin-based therapies being used increasingly in the management of both type 2 diabetes and obesity. They are now recognized to have beneficial effects beyond improved glycemic control and weight loss, including cardiovascular and renal protection. GLP-1-based therapy also slows gastric emptying, which has benefits (lowering postprandial glucose), but also potential risks (eg, hypoglycemia in individuals on insulin or sulphonylurea therapy). Their effects on the gallbladder may also be beneficial, contributing to reducing postprandial triglycerides, but they also potentially increase the risk of biliary disease. In this review, we summarize the effects of GLP-1 and incretin-based therapeutics on gastric, biliary and small intestinal function. An improved understanding of these effects will optimize the use of these drugs., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Cancers evolve not only through the acquisition and clonal transmission of somatic mutations but also by epigenetic mechanisms that modify cell phenotype. Here, we use histology-guided and spatial transcriptomics to characterize hepatoblastoma, a childhood liver cancer that exhibits significant histologic and proliferative heterogeneity despite clonal activating mutations in the Wnt/β-catenin pathway. Highly proliferative regions with embryonal histology show high expression of Wnt target genes, the embryonic biliary transcription factor SOX4, and striking focal expression of the growth factor FGF19. In patient-derived tumoroids with constitutive Wnt activation, FGF19 is a required growth signal for FGF19-negative cells. Indeed, some tumoroids contain subsets of cells that endogenously express FGF19, downstream of Wnt/β-catenin and SOX4. Thus, the embryonic biliary lineage program cooperates with stabilized nuclear β-catenin, inducing FGF19 as a paracrine growth signal that promotes tumor cell proliferation, together with active Wnt signaling. In this pediatric cancer presumed to originate from a multipotent hepatobiliary progenitor, lineage-driven heterogeneity results in a functional growth advantage, a non-genetic mechanism whereby developmental lineage programs influence tumor evolution., Competing Interests: Competing interests: R.N. is a board member of Bio-Techne and a member of the Scientific Advisory Board of Surrozen Inc. The authors declare no other competing interests., (© 2024. The Author(s).)

Aquaporins (AQPs) are transmembrane proteins permeable to water and a series of small solutes. AQPs play a key role in pathways of hepatobiliary secretion at the level of the liver, bile ducts, and gallbladder. AQP8 and -9 are pivotal in facilitating the osmotic water movement of hepatic bile, which is composed of 95% water. In the biliary tract, AQP1 and -4 are involved in the rearrangement of bile composition by mechanisms of reabsorption/secretion of water. In the gallbladder, AQP1 and -8 are also involved in trans-epithelial bidirectional water flow with the ultimate goal of bile concentration. Pathophysiologically, AQPs have been indicated as players in several hepatobiliary disorders, including cholestatic diseases and cholesterol cholelithiasis. Research on AQP function and the modulation of AQP expression is in progress, with the identification of potent and homolog-specific compounds modulating the expression or inhibiting these membrane channels with promising pharmacological developments. This review summarizes the contribution of AQPs in physiological and pathophysiological stages related to hepatobiliary function.

Background: The relationship between non-alcoholic fatty liver disease (NAFLD) and cholelithiasis is intricate, with alterations in the microenvironment potentially mediating this interplay. Thus, this study aimed to explore the biliary microbiota and metabolites of patients with cholelithiasis and detect changes induced by comorbid NAFLD., Methods: In this study, 16S rRNA gene sequencing and metabolome analysis were performed on biliary samples collected from 35 subjects. Then, patients were stratified into two groups: the comorbidity group (n = 18), consisting of cholelithiasis patients with NAFLD, and the non-comorbidity group (n = 17), comprising cholelithiasis patients without NAFLD., Results: Comorbid NAFLD did not significantly increase α-diversity but affected β-diversity. A statistically significant difference was observed in the abundance of biliary metabolites between the two groups. Specifically, differences in the abundance of 4 phyla, 19 genera, and 28 metabolites were significant between the two groups. Correlation analysis demonstrated positive associations among 12α-hydroxylated bile acid levels, Pyramidobacter and Fusobacterium abundance, AST levels, and the fibrosis-4 index (p < 0.05, r > 0.3), all of which were increased in patients with cholelithiasis and comorbid NAFLD., Conclusions: The relationship between cholelithiasis and NAFLD influences the biliary microbial and metabolic profile, creating a detrimental microenvironment that promotes the disease progression., 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 © 2024. Published by Elsevier Masson SAS.)

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested these 24 mutations affect 24 different genes. We applied unsupervised clustering algorithms to unbiasedly classify the recovered mutants into three classes. Further computational analysis revealed that each of the recovered mutations in these three classes has a unique phenotype on node-subtype composition and distribution within the intrahepatic biliary network. In addition, we found most of the recovered mutations are viable. In those mutant fish, which are already good animal models to study chronic cholestatic liver diseases, the biliary network phenotypes persist into adulthood. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: In the developing liver, bipotent epithelial progenitor cells undergo lineage segregation to form hepatocytes, which constitute the bulk of the liver parenchyma, and biliary epithelial cells (cholangiocytes), which comprise the bile duct (a complex tubular network that is critical for normal liver function). Notch and TGFβ signalling promote the formation of a sheet of biliary epithelial cells, the ductal plate, that organises into discontinuous tubular structures. How these structures elongate and connect to form a continuous duct remains undefined. We aimed to define the mechanisms by which the ductal plate transitions from a simple sheet of epithelial cells into a complex and connected bile duct., Methods: By combining single-cell RNA sequencing of embryonic mouse livers with genetic tools and organoid models we functionally dissected the role of planar cell polarity in duct patterning., Results: We show that the planar cell polarity protein VANGL2 is expressed late in intrahepatic bile duct development and patterns the formation of cell-cell contacts between biliary cells. The patterning of these cell contacts regulates the normal polarisation of the actin cytoskeleton within biliary cells and loss of Vangl2 function results in the abnormal distribution of cortical actin remodelling, leading to the failure of bile duct formation., Conclusions: Planar cell polarity is a critical step in the post-specification sculpture of the bile duct and is essential for establishing normal tissue architecture., Impact and Implications: Like other branched tissues, such as the lung and kidney, the bile ducts use planar cell polarity signalling to coordinate cell movements; however, how these biochemical signals are linked to ductular patterning remains unclear. Here we show that the core planar cell polarity protein VANGL2 patterns how cell-cell contacts form in the mammalian bile duct and how ductular cells transmit confluent mechanical changes along the length of a duct. This work sheds light on how biological tubes are patterned across mammalian tissues (including within the liver) and will be important in how we promote ductular growth in patients where the duct is mis-patterned or poorly formed., (Copyright © 2024. Published by Elsevier B.V.)

The progress of research focused on cholangiocytes and the biliary tree during development and following injury is hindered by limited available quantitative methodologies. Current techniques include two-dimensional standard histological cell-counting approaches, which are rapidly performed, error prone, and lack architectural context or three-dimensional analysis of the biliary tree in opacified livers, which introduce technical issues along with minimal quantitation. The present study aims to fill these quantitative gaps with a supervised machine-learning model (BiliQML) able to quantify biliary forms in the liver of anti-keratin 19 antibody-stained whole slide images. Training utilized 5,019 researcher-labeled biliary forms, which following feature selection, and algorithm optimization, generated an F score of 0.87. Application of BiliQML on seven separate cholangiopathy models [genetic ( Afp -CRE; Pkd1l1 null/Fl , Alb- CRE; Rbp-jk fl/fl , and Albumin- CRE; ROSA NICD ), surgical (bile duct ligation), toxicological (3,5-diethoxycarbonyl-1,4-dihydrocollidine), and therapeutic ( Cyp2c70 -/- with ileal bile acid transporter inhibition)] allowed for a means to validate the capabilities and utility of this platform. The results from BiliQML quantification revealed biological and pathological differences across these seven diverse models, indicating a highly sensitive, robust, and scalable methodology for the quantification of distinct biliary forms. BiliQML is the first comprehensive machine-learning platform for biliary form analysis, adding much-needed morphologic context to standard immunofluorescence-based histology, and provides clinical and basic science researchers with a novel tool for the characterization of cholangiopathies. NEW & NOTEWORTHY BiliQML is the first comprehensive machine-learning platform for biliary form analysis in whole slide histopathological images. This platform provides clinical and basic science researchers with a novel tool for the improved quantification and characterization of biliary tract disorders.

For many adult human organs, tissue regeneration during chronic disease remains a controversial subject. Regenerative processes are easily observed in animal models, and their underlying mechanisms are becoming well characterized 1-4 , but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases., (© 2024. The Author(s).)

Purpose: Human epidermal growth factor receptor 2 (HER2) overexpression is seen in 4%-16% of biliary tract cancers (BTCs). We aimed to evaluate the clinical activity of gemcitabine-cisplatin (GC) plus anti-HER2 antibody trastuzumab as initial treatment in HER2-positive BTCs., Methods: This study was an investigator-initiated, open-label, single-arm, multi-institutional, phase II trial in adult patients with HER2-positive (defined as immunohistochemistry [IHC] 3+ or IHC 2+ and fluorescent in situ hybridization-positive), treatment-naïve BTCs. The primary end point of the study was 6-month progression-free survival (PFS). Next-generation sequencing was performed on tissue samples to evaluate mutational status., Results: From March 2020 to August 2022, of the 876 screened patients, 118 (13.4%) were found to have HER2-positive status, of whom 90 were enrolled in the study. Most patients had GBC (n = 96; 96%) with two or more sites of metastatic disease (n = 70; 78%). With a median follow-up of 17.3 (95% CI, 15.22 to 19.32) months, 72 patients had disease progression with a median PFS of 7 (95% CI, 6.2 to 7.8) months. The diagnosis to event 6-month PFS rate was 75.6% (95% CI, 66.6 to 84.6). A complete or partial response was seen in 50 (55.5%) patients and 22 (24.4%) patients had stable disease as the best response to treatment, for an overall disease control rate of 80%. The presence of isolated TP53 mutations was associated with inferior PFS compared with other mutations (TERT promoter, HER2, PIK3CA, etc) or no detected mutations (6.51 v 12.02 v 10.58 months; P < .001)., Conclusion: The combination of GC and trastuzumab achieved its primary end point of improving PFS compared with historical data in the treatment-naïve HER2-positive BTC. Evaluating additional mutations such as TP53 and PIK3CA along with HER2 testing may help to preferentially select patients for anti-HER2 therapy in the future (Clinical Trial Registry India number: CTRI/2019/11/021955).

Background: Individual host susceptibility is believed to be a risk factor in the interaction between the host and the parasite. Since studying time series in humans is limited, animal models are replaced., Aim: This study aims to explore and compare the pattern of inflammatory cell types along the biliary tract and their association with proliferative lesions in the early development of cholangiocarcinoma from susceptible and nonsusceptible animal models., Methods: Thirty male Syrian golden hamsters and 30 BALB/c mice, serving as the susceptible and nonsusceptible animal models, were used in this comparative study. The animals were infected with 50 Opisthorchis viverrini metacercariae via gastric intubation. At days 1, 2, 7, 14, 28, and 56 postinfection (p.i.), five animals were randomly selected from each group and humanely sacrificed. The hepatobiliary tissues were collected and processed for histopathological study. Histochemical and immunohistochemical staining were applied to differentiate the inflammatory cell types. Kruskal-Wallis and Mann-Whitney tests were applied to assess all semi-quantitative and quantitative variables. The correlation between each variable was also analyzed using Spearman rank at a p -value < 0.05., Results: The results demonstrated that mice had different patterns of infiltrating cell types when compared to hamsters. This suggested that the cellular response to the infection in mice occurred earlier than that in hamsters. The response in mice reached its peak at D7 to D14 and then rapidly declined at D28. In contrast, although the inflammatory response in hamsters started slowly, the response reached the peak at D28 and maintained a high level until D56. Significant differences in the number of inflammatory cells between mice and hamsters were seen at D1 ( p = 0.047), D7 ( p = 0.049), D28 ( p = 0.040), and D56 ( p < 0.040)., Conclusion: The inflammatory responses to O. viverrini infection in the nonsusceptible animal model occurred and declined earlier while the response in the susceptible animal model occurred later in a gradual manner. Both rodents are suitable animal models for the studies of opisthorchiasis susceptibility., Competing Interests: The authors declare there are no conflicts of interest.

Cholangiocarcinoma (CCA), a highly heterogeneous cancer, is the second most common type of primary liver cancer. It is characterized by resistance to therapy and poor prognosis, with a 5-year survival rate lower than 20%. The pathogenesis of CCA is complex and multifactorial, and in recent years, bile acids (BAs) have been implicated in CCA development and prognosis. BAs belong to a category of amphipathic compounds that hold significant importance as signaling molecules and inflammatory agents. They possess the ability to activate transcriptional factors and cellular signaling pathways, thereby governing the regulation of lipid, glucose, and energy metabolism in diverse human disorders. These disorders encompass chronic liver diseases among other conditions. In this review, we provided an update on the current knowledge on the molecular mechanisms involving BAs in cholangiocarcinogenesis. Additionally, we analyzed the role of gut and biliary microbiota in CCA pathogenesis. Future research is required to better understand how to modulate BA activity and, possibly, identify new therapeutic strategies.

Interleukin 35 (IL-35) mediates immunosuppression of T cells in autoimmune diseases. T cells play an important role in primary biliary cholangitis (PBC) with incompletely elucidated pathogenesis. Thus, we aimed to investigate the role of IL-35 regulation on T cells in PBC patients. Fifty-one PBC patients and 28 controls were enrolled in this study. Plasma IL-35 level was measured. Purified peripheral CD4+ and CD8+ T cells were stimulated with exogenous IL-35 to investigate their functional phenotypes. IL-35-treated CD8+ T cells were cultured with human intrahepatic biliary epithelial cell line to determine the cytotoxicity of CD8+ T cells from PBC patients. Plasma IL-35 concentration was lower in PBC patients and negatively correlated with alkaline phosphatase. CD4+ T cells from PBC patients exhibited elevated transcription factor expressions and cytokine secretion, whereas CD8+ T cells produced increased cytotoxic molecules and cytokines. In vitro IL-35 stimulation suppressed the production of IL-17 and IL-22 by CD4+ T cells from PBC patients. CD8+ T cells treated with IL-35 mediated reduced target cell death in the direct contact co-culture system in PBC patients. This process was accompanied by reduced production of cytotoxic molecules and cytokines and increased expressions of immune checkpoint receptors in CD8+ T cells. Reduced circulating IL-35 might be insufficient to suppress T cell function, leading to the immune dysregulation in PBC patients.

The development of surgical techniques, immunosuppressive strategies and new organ preservation methods have meant that transplant centers have to face the problem of an insufficient number of organs for transplantation concerning the constantly growing demand. Therefore, using organs from expanded criteria donors and developing new analytical solutions to find parameters or compounds that would allow a more efficient assessment of organ quality before transplantation are options for meeting this challenge. This study proposed bile metabolomic analysis to evaluate liver metabolism and biliary tract function depending on the organ preservation method and degree of warm ischemia time. The analyses were performed on solid-phase microextraction-prepared bile samples from porcine model donors with mild (heart beating donor [HBD]) and moderate warm ischemia (donation after circulatory death [DCD]) grafts subjected to static cold storage (SCS) or normothermic ex vivo liver perfusion (NEVLP) before transplantation. Bile produced in the SCS-preserved livers was characterized by increased levels of metabolites such as chenodeoxycholic acid, arachidonic acid and 5S-hydroxyeicosatetraeonic acid, as well as saturated and monounsaturated lysophosphatidylcholines (LPC). Such changes may be associated with differences in the bile acid synthesis pathways and organ inflammation. Moreover, it has been shown that NEVLP reduced the negative effect of ischemia on organ function. A linear relationship was observed between levels of lipids from the LPC group and the time of organ ischemia. This study identified metabolites worth considering as potential markers of changes occurring in preserved grafts.

Pancreatic ductal adenocarcinoma and cholangiocarcinoma constitute two aggressive tumor types that originate from the epithelial lining of the excretory ducts of the pancreatobiliary tract. Given their close histomorphological resemblance, a correct diagnosis can be challenging and almost impossible without clinical information. In this study, we investigated whether mass spectrometric peptide features could be employed to distinguish pancreatic ductal adenocarcinoma from cholangiocarcinoma. Three tissue microarrays of formalin-fixed and paraffin-embedded material (FFPE) comprising 41 cases of pancreatic ductal adenocarcinoma and 41 cases of cholangiocarcinoma were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The derived peptide features and respective intensities were used to build different supervised classification algorithms: gradient boosting (GB), support vector machine (SVM), and k-nearest neighbors (KNN). On a pixel-by-pixel level, a classification accuracy of up to 95% could be achieved. The tentative identification of discriminative tryptic peptide signatures revealed proteins that are involved in the epigenetic regulation of the genome and tumor microenvironment. Despite their histomorphological similarities, mass spectrometry imaging represents an efficient and reliable approach for the distinction of PDAC from CC, offering a promising complementary or alternative approach to the existing tools used in diagnostics such as immunohistochemistry.

Background: Vascular endothelial growth factor inhibitors (VEGFi) are compromised by a lack of validated biomarkers. Previously we showed that changes in the concentration of plasma Tie2 (pTie2) was a response biomarker for bevacizumab. Here, we investigated whether pTie2 can predict response and progression cross-tumour for generic VEGFi treatment., Patients and Methods: Patients (n = 124) with advanced biliary tract cancer (ABC) received cisplatin/gemcitabine with cediranib or placebo (ABC-03 trial). Concentrations of pTie2 were measured longitudinally from before treatment until disease progression. Data from patients with ovarian cancer (n = 92, ICON7 trial) and patients with colorectal cancer (CRC) (n = 70, Travastin trial) were also included., Results: Cediranib-treated ABC patients were deconvoluted into distinct groups where in one group pTie2 trajectories resembled those seen in placebo-treated patients and in another pTie2 significantly reduced (t-test P = 2.7 × 10 -14 ). Using the 95% confidence interval for these two groups, we defined a vascular complete response (vCR) as a 24% reduction in pTie2 within 9 weeks; vascular no response (vNR) as a 7% increase in pTie2, and a vascular partial response (between these limits). vCR cediranib-treated patients had significantly improved progression-free survival (8.8 versus 7.5 months, restricted mean ratio 0.73, P = 0.012) and overall survival (18.8 versus 12.1 months, hazard ratio 0.49, P = 0.02). By integrating data across ovarian cancer, CRC and ABC, we show that (i) patients with vNR do not benefit from VEGFi and (ii) Tie2-defined vascular progression occurs sufficiently in advance of radiological progressive disease that changes in treatment could be offered to prevent clinical deterioration., Conclusion: pTie2 is the first cross-tumour, generic VEGFi, vascular response biomarker to guide optimum use of VEGFi in clinical practice., Competing Interests: Disclosure JWV reports personal fees from Agios, AstraZeneca, Baxter, Genoscience Pharma, Hutchison Medipharma, Imaging Equipment Ltd (AAA), Incyte, Ipsen, Mundipharma EDO, Mylan, QED, Servier, Sirtex and Zymerworks; and grants, personal fees and non-financial support from NuCana, outside the submitted work. GCJ reports research funding from AstraZeneca for clinical trials outside of the submitted work. Research funding has been received from: AstraZeneca, Astex Pharmaceuticals, Bioven, Amgen, Carrick Therapeutics, Merck AG, Taiho Oncology, GSK, Bayer, Boehringer Ingelheim, Roche, BMS, Novartis, Celgene, Epigene Therapeutics Inc, Angle PLC, Menarini, Clearbridge Biomedics, Thermo Fisher Scientific and Neomed Therapeutics. CD has received honoraria for consultancy/advisory boards from Biocartis, Merck and AstraZeneca. All other authors have declared no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Patients with Alagille syndrome carry monogenic mutations in the Notch signaling pathway and face complications such as jaundice and cholestasis. Given the presence of intrahepatic ductopenia in these patients, Notch2 receptor signaling is implicated in driving normal biliary development and downstream branching morphogenesis. As a result, in vitro model systems of liver epithelium are needed to further mechanistic insight of biliary tissue assembly. Here, primary human intrahepatic cholangiocytes as a candidate population for such a platform are systematically evaluated, and conditions that direct their branching morphogenesis are described. It is found that extracellular matrix presentation, coupled with mitogen stimulation, promotes biliary branching in a Notch-dependent manner. These results demonstrate the utility of using 3D scaffolds for mechanistic investigation of cholangiocyte branching and provide a gateway to integrate biliary architecture in additional in vitro models of liver tissue., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The liver has a remarkable regenerative capacity with different modes of regeneration according to the type and extent of an injury. It has been reported that mature hepatocytes could transdifferentiate into a cholangiocyte phenotype. Sry HMG box protein 9 (SOX9) is one of the earliest biliary markers that regulate bile duct development. We have found that SOX9-positive biphenotypic hepatocytes appear in severe acute liver injury patients' liver specimens accompanied by an elevation in plasma interleukin-8 levels. In vitro assays revealed that interleukin-8 homologs induce the expression of SOX9 in mature mouse hepatocytes. Here, we describe the methods used to detect SOX9-positive hepatocytes in human liver specimens and to induce SOX9-positive hepatocytes in mature mouse hepatocytes., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Primary biliary cholangitis (PBC) is a classical autoimmune disease, which is highly influenced by genetic determinants. Many genome-wide association studies (GWAS) have reported that numerous genetic loci were significantly associated with PBC susceptibility. However, the effects of genetic determinants on liver cells and its immune microenvironment for PBC remain unclear., Results: We constructed a powerful computational framework to integrate GWAS summary statistics with scRNA-seq data to uncover genetics-modulated liver cell subpopulations for PBC. Based on our multi-omics integrative analysis, 29 risk genes including ORMDL3, GSNK2B, and DDAH2 were significantly associated with PBC susceptibility. By combining GWAS summary statistics with scRNA-seq data, we found that cholangiocytes exhibited a notable enrichment by PBC-related genetic association signals (Permuted P < 0.05). The risk gene of ORMDL3 showed the highest expression proportion in cholangiocytes than other liver cells (22.38%). The ORMDL3 + cholangiocytes have prominently higher metabolism activity score than ORMDL3 - cholangiocytes (P = 1.38 × 10 -15 ). Compared with ORMDL3 - cholangiocytes, there were 77 significantly differentially expressed genes among ORMDL3 + cholangiocytes (FDR < 0.05), and these significant genes were associated with autoimmune diseases-related functional terms or pathways. The ORMDL3 + cholangiocytes exhibited relatively high communications with macrophage and monocyte. Compared with ORMDL3 - cholangiocytes, the VEGF signaling pathway is specific for ORMDL3 + cholangiocytes to interact with other cell populations., Conclusions: To the best of our knowledge, this is the first study to integrate genetic information with single cell sequencing data for parsing genetics-influenced liver cells for PBC risk. We identified that ORMDL3 + cholangiocytes with higher metabolism activity play important immune-modulatory roles in the etiology of PBC., (© 2021. The Author(s).)

The mechanism of damage of the biliary epithelium remains partially unexplored. However, recently many works have offered new evidence regarding the cholangiocytes' damage process, which is the main target in a broad spectrum of pathologies ranging from acute cholestasis, cholangiopathies to cholangiocarcinoma. This is encouraging since some works addressed this epithelium's relevance in health and disease until a few years ago. The biliary tree in the liver, comprised of cholangiocytes, is a pipeline for bile flow and regulates key hepatic processes such as proliferation, regeneration, immune response, and signaling. This review aimed to compile the most recent advances on the mechanisms of cholangiocellular damage during cholestasis, which, although it is present in many cholangiopathies, is not necessarily a common or conserved process in all of them, having a relevant role cAMP and PKA during obstructive cholestasis, as well as Ca 2+ -dependent PKC in functional cholestasis. Cholangiocellular damage could vary according to the type of cholestasis, the aggressor, or the bile ducts' location where it develops and what kind of damage can favor cholangiocellular carcinoma development., Competing Interests: Conflicts of interest The authors declare that they have no conflict of interest., (Copyright © 2021 Fundación Clínica Médica Sur, A.C. Published by Elsevier España, S.L.U. All rights reserved.)

Background and Aims: Stratified therapy has entered clinical practice in primary biliary cholangitis (PBC), with routine use of second-line therapy in nonresponders to first-line therapy with ursodeoxycholic acid (UDCA). The mechanism for nonresponse to UDCA remains, however, unclear and we lack mechanistic serum markers. The UK-PBC study was established to explore the biological basis of UDCA nonresponse in PBC and identify markers to enhance treatment., Approach and Results: Discovery serum proteomics (Olink) with targeted multiplex validation were carried out in 526 subjects from the UK-PBC cohort and 97 healthy controls. In the discovery phase, untreated PBC patients (n = 68) exhibited an inflammatory proteome that is typically reduced in scale, but not resolved, with UDCA therapy (n = 416 treated patients). Nineteen proteins remained at a significant expression level (defined using stringent criteria) in UDCA-treated patients, six of them representing a tightly linked profile of chemokines (including CCL20, known to be released by biliary epithelial cells (BECs) undergoing senescence in PBC). All showed significant differential expression between UDCA responders and nonresponders in both the discovery and validation cohorts. A linear discriminant analysis, using serum levels of C-X-C motif chemokine ligand 11 and C-C motif chemokine ligand 20 as markers of responder status, indicated a high level of discrimination with an AUC of 0.91 (CI, 0.83-0.91)., Conclusions: UDCA under-response in PBC is characterized by elevation of serum chemokines potentially related to cellular senescence and was previously shown to be released by BECs in PBC, suggesting a potential role in the pathogenesis of high-risk disease. These also have potential for development as biomarkers for identification of high-risk disease, and their clinical utility as biomarkers should be evaluated further in prospective studies., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Studies based on single cells have revealed vast cellular heterogeneity in stem cell and progenitor compartments, suggesting continuous differentiation trajectories with intermixing of cells at various states of lineage commitment and notable degrees of plasticity during organogenesis 1-5 . The hepato-pancreato-biliary organ system relies on a small endoderm progenitor compartment that gives rise to a variety of different adult tissues, including the liver, pancreas, gall bladder and extra-hepatic bile ducts 6,7 . Experimental manipulation of various developmental signals in the mouse embryo has underscored important cellular plasticity in this embryonic territory 6 . This is reflected in the existence of human genetic syndromes as well as congenital malformations featuring multi-organ phenotypes in liver, pancreas and gall bladder 6 . Nevertheless, the precise lineage hierarchy and succession of events leading to the segregation of an endoderm progenitor compartment into hepatic, biliary and pancreatic structures have not yet been established. Here we combine computational modelling approaches with genetic lineage tracing to accurately reconstruct the hepato-pancreato-biliary lineage tree. We show that a multipotent progenitor subpopulation persists in the pancreato-biliary organ rudiment, contributing cells not only to the pancreas and gall bladder but also to the liver. Moreover, using single-cell RNA sequencing and functional experiments we define a specialized niche that supports this subpopulation in a multipotent state for an extended time during development. Together these findings indicate sustained plasticity underlying hepato-pancreato-biliary development that might also explain the rapid expansion of the liver while attenuating pancreato-biliary growth., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background & Aims: Primary sclerosing cholangitis (PSC) is characterized by chronic inflammation and progressive fibrosis of the biliary tree. The bile acid receptor TGR5 (GPBAR1) is found on biliary epithelial cells (BECs), where it promotes secretion, proliferation and tight junction integrity. Thus, we speculated that changes in TGR5-expression in BECs may contribute to PSC pathogenesis., Methods: TGR5-expression and -localization were analyzed in PSC livers and liver tissue, isolated bile ducts and BECs from Abcb4 -/- , Abcb4 -/- /Tgr5 Tg and ursodeoxycholic acid (UDCA)- or 24-norursodeoxycholic acid (norUDCA)-fed Abcb4 -/- mice. The effects of IL8/IL8 homologues on TGR5 mRNA and protein levels were studied. BEC gene expression was analyzed by single-cell transcriptomics (scRNA-seq) from distinct mouse models., Results: TGR5 mRNA expression and immunofluorescence staining intensity were reduced in BECs of PSC and Abcb4 -/- livers, in Abcb4 -/- extrahepatic bile ducts, but not in intrahepatic macrophages. No changes in TGR5 BEC fluorescence intensity were detected in liver tissue of other liver diseases, including primary biliary cholangitis. Incubation of BECs with IL8/IL8 homologues, but not with other cytokines, reduced TGR5 mRNA and protein levels. BECs from Abcb4 -/- mice had lower levels of phosphorylated Erk and higher expression levels of Icam1, Vcam1 and Tgfβ2. Overexpression of Tgr5 abolished the activated inflammatory phenotype characteristic of Abcb4 -/- BECs. NorUDCA-feeding restored TGR5-expression levels in BECs in Abcb4 -/- livers., Conclusions: Reduced TGR5 levels in BECs from patients with PSC and Abcb4 -/- mice promote development of a reactive BEC phenotype, aggravate biliary injury and thus contribute to the pathogenesis of sclerosing cholangitis. Restoration of biliary TGR5-expression levels represents a previously unknown mechanism of action of norUDCA., Lay Summary: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease-associated with progressive inflammation of the bile duct, leading to fibrosis and end-stage liver disease. Bile acid (BA) toxicity may contribute to the development and disease progression of PSC. TGR5 is a membrane-bound receptor for BAs, which is found on bile ducts and protects bile ducts from BA toxicity. In this study, we show that TGR5 levels were reduced in bile ducts from PSC livers and in bile ducts from a genetic mouse model of PSC. Our investigations indicate that lower levels of TGR5 in bile ducts may contribute to PSC development and progression. Furthermore, treatment with norUDCA, a drug currently being tested in a phase III trial for PSC, restored TGR5 levels in biliary epithelial cells., Competing Interests: Conflict of interest MR, LS, CK, KF, JS, KD, JH, EL, JV, SFG, GH, SJF, IE, DN, CDF, MGB, GG, SV, JPM, MV, KS, TL, KK, DH, TL, MH have nothing to disclose.VK has served as speaker for Falk Foundation, Albireo and Abbvie. JRH has served on the advisory board for Novartis and Orkla Health. JRH has received research support from Biogen unrelated to the current work. THK has received consulting/speaker reimbursement from Gilead and Intercept unrelated to the current work. CS has served as speaker for Falk Foundation. MT has served as speaker for Falk Foundation, Gilead, Intercept and MSD; he has advised for Albireo, BiomX, Boehringer Ingelheim, Falk Pharma GmbH, Genfit, Gilead, Intercept, Jannsen, MSD, Novartis, Phenex, Regulus and Shire. MT further received travel grants from Abbvie, Falk, Gilead and Intercept and research grants from Albireo, Cymabay, Falk, Gilead, Intercept, MSD and Takeda. MT is also co-inventor of patents on the medical use of NorUDCA filed by the Medical Universities of Graz and Vienna (WO2006119803, WO20099013334). PF received norUDCA, an unrestricted research grant, travel grants and advisory board fees from Dr. Falk Pharma GmbH. PF is listed as co-inventor of patents for the medical use of norUDCA filed by the Medical University of Graz (WO2006119803, WO20099013334). UB received grants for investigator-initiated studies by Dr Falk GmbH, Freiburg, and Intercept, San Diego., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

HER2 amplification, which results in overexpression of the receptor tyrosine kinase HER2, has been described in a wide variety of malignancies. HER2-targeting agents have been incorporated into the treatment paradigms for HER2-overexpressing breast and gastric cancer. More recently, these agents have shown promise in other gastrointestinal malignancies, such as colon cancer and biliary tract tumors. This study discusses two patients with gallbladder carcinoma and a third with ampullary carcinoma who were able to achieve marked responses to HER2-directed therapy. These cases underscore the importance of molecular analysis for HER2 amplification/HER2 overexpression, irrespective of tumor histology, and highlight a need for further investigation of HER2-directed therapy beyond breast and gastroesophageal cancers. KEY POINTS: Current guidelines recommend molecular assessment for HER2 overexpression exclusively in breast and gastric adenocarcinoma. The focus of this report is on three cases (two biliary tract and one ampullary carcinoma) in which amplification of HER2 or overexpression of HER2 was detected and treatment with HER2-directed therapy resulted in robust responses. These cases exemplify responsiveness of non-breast/gastric histologies to HER2-directed therapies, highlighting several promising new settings for these agents. Testing for amplification of HER2 or overexpression of HER2 should be considered especially in rare diseases with limited treatment options., (© 2021 AlphaMed Press.)

The integration of a bile drainage structure into engineered liver tissues is an important issue in the advancement of liver regenerative medicine. Primary biliary cells, which play a vital role in bile metabolite accumulation, are challenging to obtain in vitro because of their low density in the liver. In contrast, large amounts of purified hepatocytes can be easily acquired from rodents. The in vitro chemically induced liver progenitors (CLiPs) from primary mature hepatocytes offer a platform to produce biliary cells abundantly. Here, we generated a functional CLiP-derived tubular bile duct-like structure using the chemical conversion technology. We obtained an integrated tubule-hepatocyte tissue via the direct coculture of hepatocytes on the established tubular biliary-duct-like structure. This integrated tubule-hepatocyte tissue was able to transport the bile, as quantified by the cholyl-lysyl-fluorescein assay, which was not observed in the un-cocultured structure or in the biliary cell monolayer. Furthermore, this in vitro integrated tubule-hepatocyte tissue exhibited an upregulation of hepatic marker genes. Together, these findings demonstrated the efficiency of the CLiP-derived tubular biliary-duct-like structures regarding the accumulation and transport of bile., (© 2021 Wiley Periodicals LLC.)

The liver is beyond any doubt the most important metabolic organ of the human body. This function requires an intensive crosstalk within liver cellular structures, but also with other organs. Membrane transport proteins are therefore of upmost importance as they represent the sensors and mediators that shuttle signals from outside to the inside of liver cells and/or vice versa. In this review, we summarize the known literature of liver transport proteins with a clear emphasis on functional and structural information on ATP binding cassette (ABC) transporters, which are expressed in the human liver. These primary active membrane transporters form one of the largest families of membrane proteins. In the liver, they play an essential role in for example bile formation or xenobiotic export. Our review provides a state of the art and comprehensive summary of the current knowledge of hepatobiliary ABC transporters. Clearly, our knowledge has improved with a breath-taking speed over the last few years and will expand further. Thus, this review will provide the status quo and will lay the foundation for new and exciting avenues in liver membrane transporter research.

Cholangiopathies caused by biliary epithelial cell (BEC) injury represent a leading cause of liver failure. No effective pharmacologic therapies exist, and the underlying mechanisms remain obscure. We aimed to explore the mechanisms of bile duct repair after targeted BEC injury. Injection of intermedilysin into BEC-specific human CD59 (hCD59) transgenic mice induced acute and specific BEC death, representing a model to study the early signals that drive bile duct repair. Acute BEC injury induced cholestasis followed by CCR2+ monocyte recruitment and BEC proliferation. Using microdissection and next-generation RNA-Seq, we identified 5 genes, including Mapk8ip2, Cdkn1a, Itgb6, Rgs4, and Ccl2, that were most upregulated in proliferating BECs after acute injury. Immunohistochemical analyses confirmed robust upregulation of integrin αvβ6 (ITGβ6) expression in this BEC injury model, after bile duct ligation, and in patients with chronic cholangiopathies. Deletion of the Itgb6 gene attenuated BEC proliferation after acute bile duct injury. Macrophage depletion or Ccr2 deficiency impaired ITGβ6 expression and BEC proliferation. In vitro experiments revealed that bile acid-activated monocytes promoted BEC proliferation through ITGβ6. Our data suggest that BEC injury induces cholestasis, monocyte recruitment, and induction of ITGβ6, which work together to promote BEC proliferation and therefore represent potential therapeutic targets for cholangiopathies.

The alimentary limb has been proposed to be a key driver of the weight-loss-independent metabolic improvements that occur upon bariatric surgery. However, the one anastomosis gastric bypass (OAGB) procedure, consisting of one long biliary limb and a short common limb, induces similar beneficial metabolic effects compared to Roux-en-Y Gastric Bypass (RYGB) in humans, despite the lack of an alimentary limb. The aim of this study was to assess the role of the length of biliary and common limbs in the weight loss and metabolic effects that occur upon OAGB. OAGB and sham surgery, with or without modifications of the length of either the biliary limb or the common limb, were performed in Gottingen minipigs. Weight loss, metabolic changes, and the effects on plasma and intestinal bile acids (BAs) were assessed 15 days after surgery. OAGB significantly decreased body weight, improved glucose homeostasis, increased postprandial GLP-1 and fasting plasma BAs, and qualitatively changed the intestinal BA species composition. Resection of the biliary limb prevented the body weight loss effects of OAGB and attenuated the postprandial GLP-1 increase. Improvements in glucose homeostasis along with changes in plasma and intestinal BAs occurred after OAGB regardless of the biliary limb length. Resection of only the common limb reproduced the glucose homeostasis effects and the changes in intestinal BAs. Our results suggest that the changes in glucose metabolism and BAs after OAGB are mainly mediated by the length of the common limb, whereas the length of the biliary limb contributes to body weight loss. NEW & NOTEWORTHY Common limb mediates postprandial glucose metabolism change after gastric bypass whereas biliary limb contributes to weight loss.

E7766 represents a novel class of macrocycle-bridged dinucleotides and is under clinical development for immuno-oncology. In this report, we identified mechanism of systemic clearance E7766 and investigated the hepatobiliary transporters involved in the disposition of E7766 and potential drug interactions of E7766 as a victim of organic anion-transporting polypeptide (OATP) inhibitors. In bile-duct cannulated rats and dogs, E7766 was mainly excreted unchanged in bile (>80%) and to a lesser extent in urine (<20%). Sandwich-cultured human hepatocytes (SCHHs), transfected cells, and vesicles were used to phenotype the hepatobiliary transporters involved in the clearance of E7766. SCHH data showed temperature-dependent uptake of E7766 followed by active biliary secretion. In vitro transport assays using transfected cells and membrane vesicles confirmed that E7766 was a substrate of OATP1B1, OATP1B3, and multidrug resistance-associated protein 2. Phenotyping studies suggested predominant contribution of OATP1B3 over OATP1B1 in the hepatic uptake of E7766. Studies in OATP1B1/1B3 humanized mice showed that plasma exposure of E7766 increased 4.5-fold when coadministered with Rifampicin. Physiologically based pharmacokinetic models built upon two independent bottom-up approaches predicted elevation of E7766 plasma exposure when administered with Rifampicin, a clinical OATP inhibitor. In conclusion, we demonstrate that OATP-mediated hepatic uptake is the major contributor to the clearance of E7766, and inhibition of OATP1B may increase its systemic exposure. Predominant contribution of OATP1B3 in the hepatic uptake of E7766 was observed, suggesting polymorphisms in OATP1B1 would be unlikely to cause variability in the exposure of E7766. SIGNIFICANCE STATEMENT: Understanding the clearance mechanisms of new chemical entities is critical to predicting human pharmacokinetics and drug interactions. A physiologically based pharmacokinetic model that incorporated parameters from mechanistic in vitro and in vivo experiments was used to predict pharmacokinetics and drug interactions of E7766, a novel dinucleotide drug. The findings highlighted here may shed a light on the pharmacokinetic profile and transporter-mediated drug interaction propensity of other dinucleotide drugs., (Copyright © 2021 by The Author(s).)

There is a dearth of effective parameters for selecting potentially transplantable liver grafts from expanded-criteria donors. In this study, we used a nuclear magnetic resonance (NMR) relaxation analyzer-based assay to assess the viability of ex vivo livers obtained via porcine donation after circulatory death (DCD). Ex situ normothermic machine perfusion (NMP) was utilized as a platform for viability test of porcine DCD donor livers. A liver-targeted contrast agent, gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA), was injected into the perfusate during NMP, and the dynamic biliary excretion of the Gd-EOB-DTPA was monitored by measuring the longitudinal relaxation time (T1). The longitudinal relaxation rate (R1) of the bile was served as a parameter. The delay of increase in biliary R1 during early stage of NMP indicated the impaired function of liver grafts in both warm and cold ischemia injury, which was correlated with the change of alanine aminotransferase. The preservative superiority in cold ischemia of dual hypothermic oxygenated machine perfusion could also be verified by assessing biliary R1 and other biochemical parameters. This study allows for the dynamic assessment of the viability of porcine DCD donor livers by combined usage of ex situ NMP and NMR relaxation time based assay, which lays a foundation for further clinical application.

Multiplexed immunohistochemical techniques give insight into contextual cellular relationships by offering the ability to collect cell-specific data with spatial information from formalin-fixed, paraffin-embedded tissue sections. We established an automated sequential elution-stripping multiplex immunohistochemical assay to address two controversial scientific questions in the field of hepatopathology: 1) whether epithelial-to-mesenchymal transition or mesenchymal-to-epithelial transition occurs during liver injury and repair of a chronic liver disease and 2) if there is a stromal:epithelial relationship along the canals of Hering that would support the concept of this biliary structure being a stem/progenitor cell niche. Our 4-plex assay includes both epithelial and mesenchymal clinical immunohistochemical markers and was performed on clinical human liver specimens in patients with primary biliary cholangitis. The assay demonstrated that in each specimen, co-expression of epithelial and mesenchymal markers was observed in extraportal cholangiocytes. In regard to possible mesenchymal components in a stem cell niche, 82.3% ± 5.5% of extraportal cholangiocytes were intimately associated with a vimentin-positive cell. Co-expression of epithelial and mesenchymal markers by extraportal cholangiocytes is evidence for epithelial to mesenchymal transition in primary biliary cholangitis. Vimentin-positive stromal cells are frequently juxtaposed to extraportal cholangiocytes, supporting an epithelial:mesenchymal relationship within the hepatobiliary stem cell niche. Our automated sequential elution-stripping multiplex immunohistochemical assay is a cost-effective multiplexing technique that can be readily applied to a small series of clinical pathology samples in order to answer scientific questions involving cell:cell relationships and cellular antibody expression.

Biliary tract cancer (BTC) is typically lethal due to the difficulty of early stage diagnosis. Thus, novel biomarkers of BTC precursors are necessary. Biliary intraepithelial neoplasia (BilIN) is a major precursor of BTC and is classified as low or high grade based on cell atypia. In normal gastric mucosa, gastric gland mucin-specific O-glycans are unique in having α1,4-linked N-acetylglucosamine (αGlcNAc) attached to MUC6. Previously, we reported that αGlcNAc functions as a tumor suppressor of differentiated-type gastric adenocarcinoma and that decreased αGlcNAc glycosylation on MUC6 in gastric, pancreatic, and uterine cervical neoplasms occurs in cancer as well as in their precursor lesions. However, αGlcNAc and MUC6 expression patterns in biliary tract neoplasms have remained unclear. Here, we analyzed MUC5AC, MUC6, and αGlcNAc expression status in 51 BTC cases and compared the expression of each with progression from low-grade BilIN to invasive adenocarcinoma (IAC). The frequency of αGlcNAc-positive and MUC6-positive lesions decreased with tumor progression. When we compared each marker's expression level with tumor progression, we found that the MUC6 expression score in IAC was significantly lower than in low-grade or high-grade BilIN (P < 0.001 or P < 0.01, respectively). However, the αGlcNAc expression score was low irrespective of histological grade, and also lower than that of MUC6 across all histological grades (P < 0.001 for low-grade and high-grade BilIN, and P < 0.01 for IAC). These results suggest that decreased expression of αGlcNAc relative to MUC6 marks the initiation of BTC progression., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Biliary senescence and hepatic fibrosis are hallmarks of cholangiopathies including primary sclerosing cholangitis (PSC). Senescent cholangiocytes display senescence-associated secretory phenotypes [SASPs, e.g., transforming growth factor-1 (TGF-1)] that further increase biliary senescence (by an autocrine loop) and trigger liver fibrosis by paracrine mechanisms. The aim of this study was to determine the effect of p16 inhibition and role of the TGF-1/microRNA (miR)-34a/sirtuin 1 (SIRT1) axis in biliary damage and liver fibrosis in the Mdr2 / mouse model of PSC. We treated (i) in vivo male wild-type (WT) and Mdr2 / mice with p16 Vivo-Morpholino or controls before measuring biliary mass [intrahepatic bile duct mass (IBDM)] and senescence, biliary SASP levels, and liver fibrosis, and (ii) in vitro intrahepatic murine cholangiocyte lines (IMCLs) with small interfering RNA against p16 before measuring the mRNA expression of proliferation, senescence, and fibrosis markers. p16 and miR-34a increased but SIRT1 decreased in Mdr2 / mice and PSC human liver samples compared to controls. p16 immunoreactivity and biliary senescence and SASP levels increased in Mdr2 / mice but decreased in Mdr2 / mice treated with p16 Vivo-Morpholino. The increase in IBDM and hepatic fibrosis (observed in Mdr2 / mice) returned to normal values in Mdr2 / mice treated with p16 Vivo-Morpholino. TGF-1 immunoreactivity and biliary SASPs levels were higher in Mdr2 / compared to those of WT mice but returned to normal values in Mdr2 / mice treated with p16 Vivo-Morpholino. The expression of fibrosis/senescence markers decreased in cholangiocytes from Mdr2 / mice treated with p16 Vivo-Morpholino (compared to Mdr2 / mice) and in IMCLs (after p16 silencing) compared to controls. Modulation of the TGF-1/miR-34a/SIRT1 axis may be important in the management of PSC phenotypes.

Background and Aims: During liver development, bipotent progenitor cells differentiate into hepatocytes and biliary epithelial cells to ensure a functional liver required to maintain organismal homeostasis. The developmental cues controlling the differentiation of committed progenitors into these cell types, however, are incompletely understood. Here, we discover an essential role for estrogenic regulation in vertebrate liver development to affect hepatobiliary fate decisions., Approach and Results: Exposure of zebrafish embryos to 17β-estradiol (E2) during liver development significantly decreased hepatocyte-specific gene expression, liver size, and hepatocyte number. In contrast, pharmacological blockade of estrogen synthesis or nuclear estrogen receptor (ESR) signaling enhanced liver size and hepatocyte marker expression. Transgenic reporter fish demonstrated nuclear ESR activity in the developing liver. Chemical inhibition and morpholino knockdown of nuclear estrogen receptor 2b (esr2b) increased hepatocyte gene expression and blocked the effects of E2 exposure. esr2b -/- mutant zebrafish exhibited significantly increased expression of hepatocyte markers with no impact on liver progenitors, other endodermal lineages, or vasculature. Significantly, E2-stimulated Esr2b activity promoted biliary epithelial differentiation at the expense of hepatocyte fate, whereas loss of esr2b impaired biliary lineage commitment. Chemical and genetic epistasis studies identified bone morphogenetic protein (BMP) signaling as a mediator of the estrogen effects. The divergent impact of estrogen on hepatobiliary fate was confirmed in a human hepatoblast cell line, indicating the relevance of this pathway for human liver development., Conclusions: Our studies identify E2, esr2b, and downstream BMP activity as important regulators of hepatobiliary fate decisions during vertebrate liver development. These results have significant clinical implications for liver development in infants exposed to abnormal estrogen levels or estrogenic compounds during pregnancy., (© 2020 by the American Association for the Study of Liver Diseases.)

Background: We have previously shown that obeticholic acid (OCA) upregulates the biliary excretion of asymmetric dimethylarginine (ADMA), an inhibitor of iNOS regulating the activity of matrix metalloproteinases (MMPs). Here, the effects of OCA on MMP-2 and MMP-9 activity in liver, bile and serum were evaluated after hepatic ischemia/reperfusion (I/R) injury., Material and Methods: Male Wistar rats (n = 20) were orally administered 10 mg/kg/day of OCA (5 days) and subjected to a 60-min ischemia and 60-min reperfusion. Bile, serum and tissue were collected for MMP-2 and MMP-9 activity quantification. The MMP regulator tissue reversion-inducing cysteine rich protein with Kazal motifs (RECK), tissue inhibitor of metalloproteinases (TIMPs), iNOS and biliary levels of LDH, γGT, glucose and ADMA were quantified., Results: In the I/R group, OCA administration reduced MMP-2 and MMP-9 in liver, bile and serum. A downregulation of tissue RECK and TIMPs, observed under I/R, were recovered by OCA. Immunohistochemical staining of hepatic tissue demonstrated that RECK expression is mainly localized in both cholangiocytes and hepatocytes. Hepatic iNOS positively correlated with tissue MMP-2 and MMP-9 activity. Biliary levels of LDH, γGT and glucose were lower in I/R rats treated with OCA; in bile, MMP levels positively correlated with LDH and γGT., Conclusion: Thus, OCA administration confers protection to cholangiocytes via downregulation of biliary MMPs in livers submitted to I/R. This event is associated with hepatic RECK- and TIMP-mediated MMP decrease., Competing Interests: The authors declare that Luciano Adorini is a part-time consultant of Intercept Pharmaceuticals. The specific roles of this author are articulated in the ‘author contributions’ section. However, the Intercept Pharmaceuticals did not provide support in the form of salaries for authors and did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

The autonomic nervous system plays a role in a variety of liver regenerative and metabolic functions, including modulating bile secretion and cholangiocyte and hepatobiliary progenitors of the canals of Hering. However, the nature and location of nerves which link to the proximal biliary tree have remained uncertain. We investigate the anatomic relationship of nerves to the proximal biliary tree including the putative stem/progenitor cell niche of the canal of Hering. Using double immunostaining (fluorescence, histochemistry) to highlight markers of cholangiocytes (biliary-type keratins), nerves (S100, neurofilament protein, PGP9.5, tyrosine hydroxylase), and stellate cells (CRBP-1), we examined sections from normal adult livers from autopsy or surgical resections. There is extensive contact between nerves and interlobular bile ducts, bile ductules, and canals of Hering (CoH). In multiple serial sections from 4 normal livers, biliary-nerve contacts were seen in all of these structures and were more common in the interlobular bile ducts (78/137; 57%) than in the ductules and CoH (95/294; 33%) (p < 0.001). Contacts appear to consist of nerves in juxtaposition to the biliary basement membrane, though crossing through basement membrane to interface directly with cholangiocytes is also present. These nerves are positive for tyrosine hydroxylase and are, thus, predominately adrenergic. Electron microscopy confirms nerves closely approximating ductules. Nerve fiber-hepatic stellate cell juxtaposition is observed but without stellate cell approximation to cholangiocytes. We present novel findings of biliary innervation, perhaps mediated in part, by direct cholangiocyte-nerve interactions. The implications of these findings are protean for studies of neuromodulation of biliary physiology and hepatic stem/progenitor cells.

Tuft cells are a rare population of chemosensory cells at the mucosal surface epithelia of hollow organs. Their name-giving morphological feature is an apical tuft of stiff microvilli. Accordingly, the actin-binding protein, villin, was identified as one of the first tuft cell markers in immunohistochemical analysis. Unfortunately, villin expression is not restricted to tuft cells, but is also prominent e.g. in enterocytes, which limits the use of this gene as a marker and as an experimental tool to genetically target tuft cells. Here, we report that the villin-related protein, advillin, is a specific tuft cell marker in the gastro-intestinal and biliary tract epithelia. In situ hybridization and immunohistochemistry revealed that advillin expression, unlike villin, was restricted to solitary cholinergic tuft cells in the mucosal linings of the small and large intestine, and in the gall bladder. In the glandular stomach, villin and advillin mRNA were present in all epithelial cells, while detectable protein levels were confined to solitary tuft cells. Advillin expression was no longer detectable in the mucosa of the intestinal and biliary tract from Pou2f3 deficient mice that lack tuft cells. Finally, crossing Avil-Cre transgenic mice with a double-fluorescent reporter mouse line resulted in specific targeting of gastro-intestinal and biliary tuft cells. Our analysis introduces advillin as a selective marker and tool in histological and functional analysis of the alimentary tract tuft cell system.

Normal liver tissue is highly vulnerable towards irradiation, which remains a challenge in radiotherapy of hepatic tumours. Here, we examined the effects of radiation-induced liver injury on two specific liver functions and hepatocellular regeneration in a minipig model. Five Göttingen minipigs were exposed to whole-liver stereotactic body radiation therapy (SBRT) in one fraction (14 Gy) and examined 4-5 weeks after; five pigs were used as controls. All pigs underwent in vivo positron emission tomography (PET) studies of the liver using the conjugated bile acid tracer [N-methyl- 11 C]cholylsarcosine ([ 11 C]CSar) and the galactose-analogue tracer [ 18 F]fluoro-2-deoxy-D-galactose ([ 18 F]FDGal). Liver tissue samples were evaluated histopathologically and by immunohistochemical assessment of hepatocellular mitosis, proliferation and apoptosis. Compared with controls, both the rate constant for secretion of [ 11 C]CSar from hepatocytes into intrahepatic bile ducts as well as back into blood were doubled in irradiated pigs, which resulted in reduced residence time of [ 11 C]CSar inside the hepatocytes. Also, the hepatic systemic clearance of [ 18 F]FDGal in irradiated pigs was slightly increased, and hepatocellular regeneration was increased by a threefold. In conclusion, parenchymal injury and increased regeneration after whole-liver irradiation was associated with enhanced hepatobiliary secretion of bile acids. Whole-liver SBRT in minipigs ultimately represents a potential large animal model of radiation-induced liver injury and for testing of normal tissue protection methods.

Background and Purpose: Irinotecan-induced diarrhea (IID) results from intestinal damages by its active metabolite SN-38. Alleviation of these damages has focused on lowering luminal SN-38 concentrations. However, it is unclear if the enteric bioavailability of SN-38 is mostly dependent on luminal SN-38 concentrations., Experimental Approach: Irinotecan (50 mg/kg, i.p. once daily for 6 days) was administered to female wildtype FVB, Mdr1a (-/-), Mrp2 (-/-) and Bcrp1 (-/-) mice for pharmacokinetic (PK), toxicokinetic (TK) and biodistribution studies. Plasma PK/TK profiles and tissues drug distribution were determined after first or sixth daily doses, along with activities of blood and gut esterases and intestinal Ugts. Caco-2 cells and bile-cannulate mice were used to further investigate intestinal and biliary disposition of irinotecan and its metabolites., Key Results: Significant differences in IID severity were observed with the susceptible rank of Bcrp1(-/-) > wildtype FVB > Mdr1a(-/-) > Mrp2(-/-). This rank order did not correlate with biliary excretion rates of SN-38/SN-38G. Rather, the severity was best correlated (R = 0.805) with the intestinal ratio of C ss SN-38/SN-38G, a measure of gut Ugt activity. On the contrary, IID was poorly correlated with plasma AUC ratio of SN-38/SN-38G (R = 0.227). Increased intestinal esterase activities due to repeated dosing and gut efflux transporter functionality are the other key factors that determine SN-38 enteric exposures., Conclusion and Implications: Intestinal SN-38 exposure is mainly affected by intestinal Ugt activities and blood esterase activities, and strongly correlated with severity of IID. Modulating intestinal SN-38 concentration and gut Ugt expression should be the focus of future studies to alleviate IID., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Biliary tract infections (BTIs), including cholangitis and cholecystitis, are common causes of bacteremia. Bacteremic BTIs are associated with a mortality rate of 9-12%. The extent to which antibiotics are excreted in the bile and the ratio of their exposure to the minimum inhibitory concentration of the infecting organism are among the important factors for the treatment of BTIs. This review updates health care professionals on the distribution of antibiotics in the common bile duct, gallbladder, and gallbladder wall. Antibiotic efficacy in treating BTIs based on the latest available clinical studies is also discussed. The efficacy and pharmacokinetics of 50 antibiotics are discussed. Overall, most antibiotic classes exhibit biliary penetration that translates into clinical efficacy. Only seven antibiotics (amoxicillin, cefadroxil, cefoxitin, ertapenem, gentamicin, amikacin, and trimethoprim/sulfamethoxazole) had poor biliary penetration profiles. Three antibiotics (ceftibuten, ceftolozane/tazobactam, and doripenem) had positive clinical outcomes despite the lack of pharmacokinetic studies on their penetration into the biliary tract. Conflicting efficacy data were reported for ampicillin despite adequate biliary penetration, whereas conflicting pharmacokinetic data were reported with cefaclor and moxifloxacin. Even in the absence of supportive clinical studies, antibiotics with good biliary penetration profiles may have a place in the treatment of BTIs., (© 2020 Pharmacotherapy Publications, Inc.)

The development of the biliary system is a complex yet poorly understood process, with relevance to multiple diseases, including biliary atresia, choledochal cysts and gallbladder agenesis. We present here a crucial role for Hippo-Yap/Taz signaling in this context. Analysis of sav1 mutant zebrafish revealed dysplastic morphology and expansion of both intrahepatic and extrahepatic biliary cells, and ultimately larval lethality. Biliary dysgenesis, but not larval lethality, is driven primarily by Yap signaling. Re-expression of Sav1 protein in sav1 -/- hepatocytes is able to overcome these initial deficits and allows sav1 -/- fish to survive, suggesting cell non-autonomous signaling from hepatocytes. Examination of sav1 -/- rescued adults reveals loss of gallbladder and formation of dysplastic cell masses expressing biliary markers, suggesting roles for Hippo signaling in extrahepatic biliary carcinomas. Deletion of stk3 revealed that the phenotypes observed in sav1 mutant fish function primarily through canonical Hippo signaling and supports a role for phosphatase PP2A, but also suggests Sav1 has functions in addition to facilitating Stk3 activity. Overall, this study defines a role for Hippo-Yap signaling in the maintenance of both intra- and extrahepatic biliary ducts., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by biliary inflammation and stricturing. Exploration of the pathogenesis of PSC in light of its association with inflammatory bowel disease (IBD) and the "gut-liver" axis is an emerging area of interest. A growing number of studies have begun to elucidate the role of the gut microbiota, its metabolites and its influence on host immune responses in the development of PSC and PSC-IBD. Studies of the fecal microbiota have highlighted enriched levels of certain species, including Veillonella , Streptococcus and Enterococcus , among others. A heightened immune response to enteric dysbiosis and bacterial translocation have also been implicated. For example, Klebsiella pneumoniae strains derived from gnotobiotic mice transplanted with PSC-IBD microbiota were found to induce pore formation in human intestinal epithelial cells and enhanced Th17 responses. Gut microbes have additionally been hypothesized to be implicated in PSC pathogenesis through their role in the synthesis of various metabolites, including bile acids (BAs), which function as signaling molecules with important gut and hepatic effects. An expanded knowledge of the gut microbiome as it relates to PSC offers critical insight into the development of microbe-altering therapeutic interventions, such as antibiotics, nutritional interventions and fecal microbial transplantation. Some of these have already shown some preliminary evidence of benefit. Despite exciting progress in the field, much work remains to be done; areas that are particularly lacking include functional characterization of the microbiome and examination of pediatric populations. In this review, we summarize studies that have investigated the microbiome in PSC and PSC-IBD as well as putative mechanisms, including the potential role of metabolites, such as BAs. We then briefly review the evidence for interventions with microbe-altering properties for treating PSC., Competing Interests: Conflict-of-interest statement: Dr. Kamath BM reports - Consultant Shire, Albireo and Mirum since they make ASBT inhibitors. All other authors no disclosures to report., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

The gut microbiota is part of the human body that is involved in body metabolism and the occurrence of various diseases. Detecting and analyzing their genetic information (microbiome) is as important as analyzing human genes. The core microbiome, the key functional genes shared by all humans, helps better understand the physiology of the human body. Information on the gut microbiome of a diseased person can help diagnose and treat disease. The pancreatobiliary system releases functional antimicrobial substances, such as bile acids and antimicrobial peptides, which affect the gut microbiota directly. In response, the gut microbiota influences pancreatobiliary secretion by controlling the generation and emission of substances through indirect signaling. This crosstalk maintains homeostasis of the pancreatobiliary system secretion and microbiota. Dysbiosis and disease can occur if this fails to work properly. Bile acid therapy has been used widely and may affect the microbial environment in the intestine. An association of the gut microbiota has been reported in many cases of pancreatobiliary diseases, including malignant tumors. Traditionally, most pancreatobiliary diseases are accompanied by infections from the gut microbiota, which is an important target for treatment. The pancreatobiliary system can control its function through physical and drug therapy. This may be a new pioneering field in the study or treatment of the gut microbiota.

Despite over seven decades of production and hundreds of oil spills per year, there were no comprehensive baselines for petroleum contamination in the Gulf of Mexico (GoM) prior to this study. Subsequent to the 2010 Deepwater Horizon (DWH) spill, we implemented Gulf-wide fish surveys extending over seven years (2011-2018). A total of 2,503 fishes, comprised of 91 species, were sampled from 359 locations and evaluated for biliary polycyclic aromatic hydrocarbon (PAH) concentrations. The northern GoM had significantly higher total biliary PAH concentrations than the West Florida Shelf, and coastal regions off Mexico and Cuba. The highest concentrations of biliary PAH metabolites occurred in Yellowfin Tuna (Thunnus albacares), Golden Tilefish (Lopholatilus chamaeleonticeps), and Red Drum (Sciaenops ocellatus). Conversely, biliary PAH concentrations were relatively low for most other species including economically important snappers and groupers. While oil contamination in most demersal species in the north central GoM declined in the first few years following DWH, more recent increases in exposure to PAHs in some species suggest a complex interaction between multiple input sources and possible re-suspension or bioturbation of oil-contaminated sediments. This study provides the most comprehensive baselines of PAH exposure in fishes ever conducted for a large marine ecosystem.

Physical activity encompasses a series of overall benefits on cardiovascular health and metabolic disorders. Research has recently focused on the hepatobiliary tract, as an additional target of the health-related outcomes of different types of physical exercise. Here, we focus on the global features of physical activity with respect to exercise modality and intensity, and on studies linking physical activity to lipid metabolism, gallbladder diseases (gallstones, symptoms, complications and health-related quality of life), gallbladder motor-function, enterohepatic circulation of bile acids, and systemic metabolic inflammation. Additional studies need to unravel the pathophysiological mechanisms involved in both beneficial and harmful effects of physical activity in populations with different metabolic conditions.

Biliary brushing cytology has become the standard of practice for the investigation of strictures of the biliary and pancreatic duct systems. The methodology however has a limitation in that it has low diagnostic sensitivity when only cytologic evaluation is used. A number of testing methodologies have been applied to brushing specimens in an attempt to improve overall sensitivity without loss of specificity. These have included DNA ploidy analysis, immunocytochemistry, individual gene mutational analysis, fluorescence in-situ hybridization (FISH), and next generation sequencing (NGS). Currently, FISH coupled with routine cytology appears to be the method of choice for improving diagnostic sensitivity. NGS shows significant promise for improvement of diagnostic sensitivity., (© 2019 S. Karger AG, Basel.)

The aim of the present study was to investigate the potential effects of isotretinoin on the biliary system in patients with acne vulgaris receiving isotretinoin therapy. This was a preliminary retrospective study involving 40 patients with severe acne vulgaris who attended the dermatology clinic and were administered different doses (20 or 30 mg/day) of isotretinoin. Serum levels of AST, ALT, ALP, GGT, total bilirubin, direct bilirubin, and indirect bilirubin at the beginning and at the first month of therapy were scanned, recorded, and statistically analyzed. Total and indirect bilirubin levels at the first month of treatment in 30 patients, receiving isotretinoin at a dose of 20 mg/day, were significantly lower compared to the baseline values (p = .02 and p = .03, respectively), whereas AST and GGT serum levels were significantly higher (p = .003 and p = .006 respectively). No significant reduction in total and indirect bilirubin levels was detectable at the first month of treatment in 10 patients receiving isotretinoin at a dose of 30 mg/day; however, AST, ALP, and GGT levels were significantly elevated in these patients (p = .023; p = .004; and p = .001, respectively). To our knowledge, there is no previous study investigating the effects of isotretinoin on the biliary system, and, therefore, the present study is a preliminary one. Our findings implicate that low dose (20 mg/day) isotretinoin therapy can potentially reduce total and indirect bilirubin levels. Long-term, large-scale, prospective studies with patients receiving different doses of isotretinoin may provide more reliable information regarding the bilirubin lowering effects of isotretinoin and optimum dosing for achieving this clinical effect., (© 2019 Wiley Periodicals, Inc.)

Active secretion of bile salts into the canalicular lumen drives bile formation and promotes biliary cholesterol and phospholipid output. Disrupting hepatic bile salt uptake, by inhibition of sodium-taurocholate cotransporting polypetide (NTCP; Slc10a1) with Myrcludex B, is expected to limit bile salt flux through the liver and thereby to decrease biliary lipid excretion. Here, we show that Myrcludex B-mediated NTCP inhibition actually causes an increase in biliary cholesterol and phospholipid excretion whereas biliary bile salt output and bile salt composition remains unchanged. Increased lysosomal discharge into bile was excluded as a potential contributor to increased biliary lipid secretion. Induction of cholesterol secretion was not a consequence of increased ATP-binding cassette subfamily G member 5/8 activity given that NTCP inhibition still promoted cholesterol excretion in Abcg8 -/- mice. Stimulatory effects of NTCP inhibition were maintained in Sr-b1 -/- mice, eliminating the possibility that the increase in biliary lipids was derived from enhanced uptake of high-density lipoprotein-derived lipids. NTCP inhibition shifts bile salt uptake, which is generally more periportally restricted, toward pericentral hepatocytes, as was visualized using a fluorescently labeled conjugated bile salt. As a consequence, exposure of the canalicular membrane to bile salts was increased, allowing for more cholesterol and phospholipid molecules to be excreted per bile salt. Conclusion: NTCP inhibition increases biliary lipid secretion, which is independent of alterations in bile salt output, biliary bile salt hydrophobicity, or increased activity of dedicated cholesterol and phospholipid transporters. Instead, NTCP inhibition shifts hepatic bile salt uptake from mainly periportal hepatocytes toward pericentral hepatocytes, thereby increasing exposure of the canalicular membrane to bile salts linking to increased biliary cholesterol secretion. This process provides an additional level of control to biliary cholesterol and phospholipid secretion., (© 2019 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.)

Metabolism is a critical regulator of cell fate determination. Recently, the significance of metabolic reprogramming in environmental adaptation during tumorigenesis has attracted much attention in cancer research. Recurrent mutations in the isocitrate dehydrogenase (IDH) 1 or 2 genes have been identified in several cancers, including intrahepatic cholangiocarcinoma (ICC). Mutant IDHs convert α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), which affects the activity of multiple α-KG-dependent dioxygenases including histone lysine demethylases. Although mutant IDH can be detected even in the early stages of neoplasia, how IDH mutations function as oncogenic drivers remains unclear. In this study, we aimed to address the biological effects of IDH1 mutation using intrahepatic biliary organoids (IBOs). We demonstrated that mutant IDH1 increased the formation of IBOs as well as accelerated glucose metabolism. Gene expression analysis and ChIP results revealed the upregulation of platelet isoform of phosphofructokinase-1 (PFKP), which is a rate-limiting glycolytic enzyme, through the alteration of histone modification. Knockdown of the Pfkp gene alleviated the mutant IDH1-induced increase in IBO formation. Notably, the high expression of PFKP was observed more frequently in patients with IDH-mutant ICC compared to in those with wild-type IDH (p < 0.01, 80.9% vs. 42.5%, respectively). Furthermore, IBOs expressing mutant IDH1 survived the suppression of ATP production caused by growth factor depletion and matrix detachment by retaining high ATP levels through 5' adenosine monophosphate-activated protein kinase (AMPK) activation. Our findings provide a systematic understanding as to how mutant IDH induces tumorigenic preconditioning by metabolic rewiring in intrahepatic cholangiocytes.