Due to donor liver shortage, donation after circulatory death (DCD) livers are increasingly used for liver transplantation (LT). However, non-anastomotic biliary strictures (NAS) are more often observed after DCD LT, which can cause serious morbidity. To provide early adequate NAS-treatment, reliable NAS classification is pivotal. Therefore, the current study determined the clinical applicability of two radiological NAS-classification systems, namely the system of Croome and the Groningen system. Patients included in the dual hypothermic oxygenated perfusion (DHOPE)-DCD trial (NCT02584283) who underwent LT between January 2016 and July 2019, and in whom per-protocol biliary imaging studies 6 months post-LT were available, were included in the study. NAS severity according to both scoring systems was scored by two independent radiologists, and the correlation for each system with clinical outcomes was made. In total, 133 patients were included. In our study population, both systems showed good correlation with clinical outcomes, as the highest rates of NAS-related cholangitis and biliary interventions were observed in patients with diffuse necrosis or multifocal progressive disease according to the Croome classification, and likewise in the moderate and severe NAS subgroups in the Groningen classification. Worst 5-year graft and patient survival rates were observed in the case of diffuse necrosis (77

Biliary atresia (BA) is a cholangiopathy affecting the extrahepatic bile duct (EHBD) of newborns. The etiology and pathophysiology of BA are not fully understood; however, multiple causes of damage and obstruction of the neonatal EHBD have been identified. Initial damage to the EHBD likely occurs before birth. We discuss how different developmental stages in utero and birth itself could influence the susceptibility of the fetal EHBD to damage and a damaging wound-healing response. We propose that a damage-repair response of the fetal and neonatal EHBD involving redox stress and a program of fetal wound healing could-regardless of the cause of the initial damage-lead to either obstruction and BA or repair of the duct and recovery. This overarching concept should guide future research targeted toward identification of factors that contribute to recovery as opposed to progression of injury and fibrosis. Viewing BA through the lens of an in utero damage-repair response could open up new avenues for research and suggests exciting new therapeutic targets.

Biliary atresia is a neonatal disease characterized by damage, inflammation, and fibrosis of the liver and bile ducts and by abnormal bile metabolism. It likely results from a prenatal environmental exposure that spares the mother and affects the fetus. Our aim was to develop a model of fetal injury by exposing pregnant mice to low-dose biliatresone, a plant toxin implicated in biliary atresia in livestock, and then to determine whether there was a hepatobiliary phenotype in their pups. Pregnant mice were treated orally with 15 mg/kg/d biliatresone for 2 days. Histology of the liver and bile ducts, serum bile acids, and liver immune cells of pups from treated mothers were analyzed at P5 and P21. Pups had no evidence of histological liver or bile duct injury or fibrosis at either timepoint. In addition, growth was normal. However, serum levels of glycocholic acid were elevated at P5, suggesting altered bile metabolism, and the serum bile acid profile became increasingly abnormal through P21, with enhanced glycine conjugation of bile acids. There was also immune cell activation observed in the liver at P21. These results suggest that prenatal exposure to low doses of an environmental toxin can cause subclinical disease including liver inflammation and aberrant bile metabolism even in the absence of histological changes. This finding suggests a wide potential spectrum of disease after fetal biliary injury.

Exploring the pathogenesis of and developing therapies for cholestatic liver diseases such as primary sclerosing cholangitis (PSC) remains challenging, partly due to a paucity of in vitro models that capture the complex environments contributing to disease progression and partly due to difficulty in obtaining cholangiocytes. Here we report the development of a human vascularized bile duct-on-a-chip (VBDOC) that uses cholangiocyte organoids derived from normal bile duct tissue and human vascular endothelial cells to model bile ducts and blood vessels structurally and functionally in three dimensions. Cholangiocytes in the duct polarized, formed mature tight junctions and had permeability properties comparable to those measured in ex vivo systems. The flow of blood and bile was modeled by perfusion of the cell-lined channels, and cholangiocytes and endothelial cells displayed differential responses to flow. We also showed that the device can be constructed with biliary organoids from cells isolated from both bile duct tissue and the bile of PSC patients. Cholangiocytes in the duct became more inflammatory under the stimulation of IL-17A, which induced peripheral blood mononuclear cells and differentiated Th17 cells to transmigrate across the vascular channel. In sum, this human VBDOC recapitulated the vascular-biliary interface structurally and functionally and represents a novel multicellular platform to study inflammatory and fibrotic cholestatic liver diseases.

Portal venous branches as an anatomic railroad for a gut-bile duct-axisJournal of HepatologyVol. 79Issue 2PreviewAffecting primarily the large extrahepatic bile ducts, primary sclerosing cholangitis (PSC) is a chronic, inflammatory, and fibro-obliterative cholangiopathy with an unknown trigger.1 In PSC, concomitant inflammatory bowel disease (IBD) is prevalent, with ulcerative colitis (UC) diagnosed in the majority of such cases. However, UC in patients with PSC differs from classical UC2 with (i) a predominantly right-sided and often mild-to-moderate colitis, (ii) a significantly higher risk for colon cancer in the ascending colon, and (iii) different responses to pharmacological therapies. Full-Text PDF We read with great interest the recent Letter to the Editor from Fickert and Lin et al. wherein they describe studies in search of an entero-biliary connection to explain disease processes in ulcerative colitis (UC) and primary sclerosing cholangitis (PSC).[1]Fickert P. Lin A.C. Ritschl H. Hammer N Denk H. Portal venous branches as an anatomic railroad for a gut-bile duct-axis.J Hepatol. 2023; 79: e82-e84Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar They injected the portal veins and hepatic arteries with blue- and red-colored acrylic, respectively, and demonstrated the existence of a connection between branches of the portal vein entering the liver and vessels traveling through the interstitial spaces of the large bile ducts. Importantly, the authors found such a connection in the healthy livers of both mice and humans. As they note, this finding has important implications as a potential explanation for the clinical link between UC and PSC, suggesting an anatomic route for inflammatory signals from the gut to reach the bile ducts via the portal vein and biliary interstitial spaces and then trigger an inflammatory response. We suggest an additional perspective: that this work is the human extension of groundbreaking studies from the great American anatomist Franklin P. Mall, who used a similar injection approach in cat livers to identify a perilobular space – now known as the "space of Mall" – connecting the portal vein and portal tract reticular tissues (Fig. 1).[2]Mall F.P. A study of the structural unit of the liver.Am J Anat. 1906; V: 227-308Crossref Scopus (136) Google Scholar Mall found that after an injection of Prussian-blue gelatin into the portal vein, a "zone of blue" appeared in the portal unit, especially in the larger portal tracts where the connective tissue is more extensive. He concluded that: "The blue extravasates from the capillaries at the center of the portal unit and invades the connective tissue to reach the beginning of the lymphatics, when of course it is carried rapidly from the liver"(2). Mall's studies demonstrated that materials flowing into the liver through the hepatic artery and the portal vein find their way to mixing in the pre-lymphatic, interstitial spaces within the portal tract stroma. Of relevance to the studies of Fickert and Lin et al., our own recent work suggests that the adventitial spaces surrounding portal veins are also continuous with the peribiliary interstitial spaces and thus are an additional, parallel channel between the gut and liver.[3]Cenaj O. Allison D.H.R. Imam R. Zeck B. Drohan L.M. Chiriboga L. et al.Evidence for continuity of interstitial spaces across tissue and organ boundaries in humans.Commun Biol. 2021; 4: 436Crossref PubMed Scopus (23) Google Scholar We congratulate the authors on their beautiful work, which extends the early efforts of Mall to human and rodent livers. These findings, in both cases from healthy livers, add to our understanding of PSC pathogenesis by demonstrating a channel in healthy livers that connects the gut and liver/biliary tree via the vasculature, and could serve as a conduit for inflammatory cells and signals (recognized by cholangiocytes, bile duct-associated macrophages, or other immune cells) that contribute to PSC. Notably, Fickert and Lin et al. also demonstrate the continued importance of anatomical studies and the relevance of even centuries-old work to modern scientific discovery. We look forward to further details as the authors investigate their hypothesis further, in particular as they explore possible relationships between the portal vein and small vs. large ducts in the pathogenesis of PSC. The authors did not receive any financial support to produce this manuscript. The authors of this study declare that they do not have any conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details. IEMdJ created Fig. 1B. All authors discussed the manuscript concept, participated in writing and editing all drafts of the manuscript, and approved the final version. The following are the supplementary data to this article: Download .pdf (.22 MB) Help with pdf files Multimedia component 1

Normothermic machine perfusion (NMP) after static cold storage is increasingly used for preservation and assessment of human donor livers prior to transplantation. Biliary viability assessment during NMP reduces the risk of post-transplant biliary complications. However, understanding of molecular changes in the biliary system during NMP remains incomplete. We performed an in-depth, unbiased proteomics analysis of bile collected during sequential hypothermic machine perfusion, rewarming and NMP of 55 human donor livers. Longitudinal analysis during NMP reveals proteins reflective of cellular damage at early stages, followed by upregulation of secretory and immune response processes. Livers with bile chemistry acceptable for transplantation reveal protein patterns implicated in regenerative processes, including cellular proliferation, compared to livers with inadequate bile chemistry. These findings are reinforced by detection of regenerative gene transcripts in liver tissue before machine perfusion. Our comprehensive bile proteomics and liver transcriptomics data sets provide the potential to further evaluate molecular mechanisms during NMP and refine viability assessment criteria.

BACKGROUND:Cholestasis characterised by conjugated hyperbilirubinemia is a marker of hepatobiliary dysfunction following neonatal cardiac surgery. We aimed to characterise the incidence of conjugated hyperbilirubinemia following neonatal heart surgery and examine the effect of conjugated hyperbilirubinemia on post-operative morbidity and mortality. METHODS:This was a retrospective study of all neonates who underwent surgery for congenital heart disease (CHD) at our institution between 1/1/2010 and 12/31/2020. Patient- and surgery-specific data were abstracted from local registry data and review of the medical record. Conjugated hyperbilirubinemia was defined as perioperative maximum conjugated bilirubin level > 1 mg/dL. The primary outcome was in-hospital mortality. Survival analysis was conducted using the Kaplan-Meier survival function. RESULTS:Conjugated hyperbilirubinemia occurred in 8.5% of patients during the study period. Neonates with conjugated hyperbilirubinemia were more likely to be of younger gestational age, lower birth weight, and non-Caucasian race (all p < 0.001). Patients with conjugated hyperbilirubinemia were more likely to have chromosomal and non-cardiac anomalies and require ECMO pre-operatively. In-hospital mortality among patients with conjugated hyperbilirubinemia was increased compared to those without (odds ratio 5.4). Post-operative complications including mechanical circulatory support, reoperation, prolonged ventilator dependence, and multi-system organ failure were more common with conjugated hyperbilirubinemia (all p < 0.04). Patients with higher levels of conjugated bilirubin had worst intermediate-term survival, with patients in the highest conjugated bilirubin group (>10 mg/dL) having a 1-year survival of only 6%. CONCLUSIONS:Conjugated hyperbilirubinemia is associated with post-operative complications and worse survival following neonatal heart surgery. Cholestasis is more common in patients with chromosomal abnormalities and non-cardiac anomalies, but the underlying mechanisms have not been delineated.

BACKGROUND:End-ischemic ex situ normothermic machine perfusion (NMP) enables assessment of donor livers prior to transplantation. The objective of this study was to provide support for bile composition as a marker of biliary viability and to investigate whether bile ducts of high-risk human donor livers already undergo repair during NMP.METHODS:Forty-two livers that were initially declined for transplantation were included in our NMP clinical trial. After NMP, livers were either secondary declined (n = 17) or accepted for transplantation (n = 25) based on the chemical composition of bile and perfusate samples. Bile duct biopsies were taken before and after NMP and assessed using an established histological injury severity scoring system and a comprehensive immunohistochemical assessment focusing on peribiliary glands (PBGs), vascular damage, and regeneration.RESULTS:Bile ducts of livers that were transplanted after viability testing during NMP showed better preservation of PBGs, (micro)vasculature, and increased cholangiocyte proliferation, compared with declined livers. Biliary bicarbonate, glucose, and pH were confirmed as accurate biomarkers of bile duct vitality. In addition, we found evidence of PBG-based progenitor cell differentiation toward mature cholangiocytes during NMP.CONCLUSIONS:Favorable bile chemistry during NMP correlates well with better-preserved biliary microvasculature and PBGs, with a preserved capacity for biliary regeneration. During NMP, biliary tree progenitor cells start to differentiate toward mature cholangiocytes, facilitating restoration of the ischemically damaged surface epithelium.

Background & Aims: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury.Methods: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. Results: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/ neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. Conclusion: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD.(c) 2023 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The human interstitium is a continuous, body-wide network of spaces with connectivity across tissue and organ boundaries. Continuity of the interstitium across all layers of the colon and into the attached mesenteric fascia has been demonstrated histologically, as has continuity between the interstitia of both the vascular adventitia and perineurium and the connective tissue interstitium of the organs through which they pass. Thus, there are interconnected physical spaces across all layers of the bowel wall, from lamina propria to the mesenteric fascia. The interstitial spaces of the adventitia of the vascular tree and the perineurium of the peripheral nervous system including in the mesentery provide additional connections to distant sites throughout the body. These findings indicate possibilities for communication between the mesentery and both adjoining and distant parts of the body. Mesenteric continuity is central to new perspectives on the mesentery, and understanding the role of the interstitium offers the potential for significant insight into mesenteric function. The interstitium, with the potential to regulate the mesentery itself and to communicate near and far through solutes, fluid flow mechanics, cell migration, and electrical signaling, positions the mesentery as a central organ in whole body physiology.

Background & Aims Biliary atresia is a neonatal disease characterized by bile duct and liver damage, fibrosis, inflammation and abnormal bile metabolism. It appears to result from a prenatal exposure that spares the mother and affects the fetus. Our aim was to define the phenotype in neonatal mice after maternal exposure to low-dose biliatresone, a plant toxin implicated in biliary atresia in livestock. Methods Pregnant mice were treated orally with low-doses of biliatresone. Histological changes, bile acid profiles and immune profiles were analyzed in postnatal day 5 and 21 pups born to treated mothers. Results The pups of mothers treated with this dose of biliatresone had no evidence of significant liver or ductular injury or fibrosis at postnatal day 5 or 21 and they grew normally. However, serum levels of glycocholic acid were elevated at postnatal day 5, suggesting altered bile metabolism, and bile metabolism became increasingly abnormal through postnatal day 21, with enhanced glycine conjugation of bile acids. There was also immune cell activation observed in the liver at postnatal day 21. Conclusion Prenatal exposure to low doses of an environmental toxin can cause liver inflammation and aberrant bile metabolism even in the absence of histological changes. Lay summary Prenatal exposure to low doses of an environmental toxin can cause changes in bile metabolism in neonatal mice.

ABSTRACT Introduction Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause; however, the response of the fetal EHBD to injury remains unknown. The objective of this study was to define the fetal response to EHBD injury and to determine whether it follows a fetal wound healing paradigm. Methods Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. Results A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland (PBG) hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. Conclusion The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with high levels of HA typical of the regenerative, scarless program termed fetal wound healing. Although generally beneficial, the expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD.

Ex situ normothermic machine perfusion (NMP) is increasingly used for viability assessment of high-risk donor livers, whereas dual hypothermic oxygenated machine perfusion (DHOPE) reduces ischemia-reperfusion injury. We aimed to resuscitate and test the viability of initially-discarded, high-risk donor livers using sequential DHOPE and NMP with two different oxygen carriers: an artificial hemoglobin-based oxygen carrier (HBOC) or red blood cells (RBC). In a prospective observational cohort study of 54 livers that underwent DHOPE-NMP, the first 18 procedures were performed with a HBOC-based perfusion solution and the subsequent 36 procedures were performed with an RBC-based perfusion solution for the NMP phase. All but one livers were derived from extended criteria donation after circulatory death donors, with a median donor risk index of 2.84 (IQR 2.52-3.11). After functional assessment during NMP, 34 livers (63% utilization), met the viability criteria and were transplanted. One-year graft and patient survival were 94% and 100%, respectively. Post-transplant cholangiopathy occurred in 1 patient (3%). There were no significant differences in utilization rate and post-transplant outcomes between the HBOC and RBC group. Ex situ machine perfusion using sequential DHOPE-NMP for resuscitation and viability assessment of high-risk donor livers results in excellent transplant outcomes, irrespective of the oxygen carrier used.

View Large Image Figure ViewerDownload Hi-res image Download (PPT) The biliary tree and the liver are inseparably linked. They complement each other and are generally considered 1 organ. However, the biliary tree has a well-defined identity of its own. Moreover, if you look closely, an astonishing number of structural, functional, and embryological variation is evident within this ductular network. Grossly, the heterogeneity of the biliary tree can be appreciated along both the proximal-to-distal axis and the radial axis.[1]Lanzoni G. Cardinale V. Carpino G. The hepatic, biliary, and pancreatic network of stem/progenitor cell niches in humans: a new reference frame for disease and regeneration.Hepatology. 2016; 64: 277-286Crossref PubMed Scopus (99) Google Scholar Hepatocyte bile canaliculi are located at the most proximal end of the biliary tree and transition into the canals of Hering (CoH), which contains progenitor cells and is where cholangiocytes are juxtaposed to hepatocytes. From the CoH, bile flows into the ductules (<15 μm diameter) and through the merging network of ducts (increasing in size from 15 μm to several mm in diameter) into the duodenum (sizes apply to the human bile duct). The cholangiocyte population changes from proximal to distal: generally, the interlobular and septal ducts are lined with small cholangiocytes while the large intrahepatic, segmental, and hepatic ducts and the common bile duct are lined with large cholangiocytes.[2]Maroni L. Haibo B. Ray D. Zhou T. Wan Y. Meng F. et al.Functional and structural features of cholangiocytes in health and disease.Cell Mol Gastroenterol Hepatol. 2015; 1: 368-380Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar As bile duct diameter increases, the cytoplasm-to-nucleus ratio increases, cholangiocytes become hormone-responsive, and the involvement of cholangiocytes in bile modification intensifies. The total number of peribiliary glands (PBGs) increases as well as the number of mucus producing PBGs.[3]Terada T. Nakanuma Y. Morphological examination of intrahepatic bile ducts in hepatolithiasis.Virch Arch A Pathol Anat Histopathol. 1988; 413: 167-176Crossref PubMed Scopus (63) Google Scholar Distally located cholangiocytes play an increasing role in secretion and absorption. At the same time, the vascular and neural networks merge into a complex network of multiple layers in line with the growing role of the bile ducts in bile modification[4]Nakanuma Y. Miyata N. Vascular supply of the bile duct and ischemic cholangiopathy. Pathology of the bile duct Singapore. Springer Singapore, 2017: 55-70Google Scholar,[5]Balemba O.B. Salter M.J. Mawe G.M. Innervation of the extrahepatic biliary tract.Anat Rec A Discov Mol Cell Evol Biol. 2004; 280: 836-847Crossref PubMed Scopus (42) Google Scholar such that the distal end of the bile duct harbors a 3-layered peribiliary vascular plexus surrounding the PBGs. In addition, the bile duct is extrinsically and intrinsically innervated, resembling the gut. Biliary-nerve contacts are rare in the CoH, are more common in the ductules and interlobular ducts, and are part of multiple plexuses moving toward the common bile duct.[5]Balemba O.B. Salter M.J. Mawe G.M. Innervation of the extrahepatic biliary tract.Anat Rec A Discov Mol Cell Evol Biol. 2004; 280: 836-847Crossref PubMed Scopus (42) Google Scholar Radial axis heterogeneity refers to the epithelial diversity from the lumen toward the deeper PBGs in the bile duct wall. This axis parallels the transition from mucus-producing cells toward serous acini and from mature cholangiocytes toward progenitor/stem cells.[1]Lanzoni G. Cardinale V. Carpino G. The hepatic, biliary, and pancreatic network of stem/progenitor cell niches in humans: a new reference frame for disease and regeneration.Hepatology. 2016; 64: 277-286Crossref PubMed Scopus (99) Google Scholar,[3]Terada T. Nakanuma Y. Morphological examination of intrahepatic bile ducts in hepatolithiasis.Virch Arch A Pathol Anat Histopathol. 1988; 413: 167-176Crossref PubMed Scopus (63) Google Scholar The structural diversity in both the proximal-to-distal and radial directions reflects the functional heterogeneity along the biliary tree and its ability to withstand injury. Small cholangiocytes are more resistant to severe damage than large cholangiocytes and are able to proliferate, differentiate, and ultimately replace the large cholangiocytes, as has been shown after bile duct ligation of the rat bile duct.[2]Maroni L. Haibo B. Ray D. Zhou T. Wan Y. Meng F. et al.Functional and structural features of cholangiocytes in health and disease.Cell Mol Gastroenterol Hepatol. 2015; 1: 368-380Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar In the radial direction, PBGs harbor endoderm progenitor cells and stem cells that are less susceptible to damage than mature cholangiocytes. This epithelial cell compartment is able to survive in hypoxic conditions, ensuring the regeneration of injured epithelia.[6]de Jong I.E.M. Matton A.P.M. van Praagh J.B. van Haaften W.T. Wiersema-Buist J. van Wijk L.A. et al.Peribiliary glands are key in regeneration of the human biliary epithelium after severe bile duct injury.Hepatology. 2019; 69: 1719-1734Crossref PubMed Scopus (27) Google Scholar Participation of PBGs in bile duct regeneration has been demonstrated in a tissue culture using human extrahepatic bile duct and a mouse model in which lineage tracing was used after chemically induced biliary injury.[6]de Jong I.E.M. Matton A.P.M. van Praagh J.B. van Haaften W.T. Wiersema-Buist J. van Wijk L.A. et al.Peribiliary glands are key in regeneration of the human biliary epithelium after severe bile duct injury.Hepatology. 2019; 69: 1719-1734Crossref PubMed Scopus (27) Google Scholar,[7]Carpino G. Nevi L. Overi D. Cardinale V. Lu W.Y. Di Matteo S. et al.Peribiliary gland niche participates in biliary tree regeneration in mouse and human primary sclerosing cholangitis.Hepatology. 2020; 71: 972-989Crossref PubMed Scopus (22) Google Scholar Finally, the heterogeneity of the biliary tree is underlined by its embryological origins and the existence of distinct small and large duct cholangiopathies. The intrahepatic and extrahepatic bile duct develop from different parts of the ventral foregut at different gestational stages, merging at the level of the hepatic hilum.[8]Strazzabosco M. Fabris L. Development of the bile ducts: essentials for the clinical hepatologist.J Hepatol. 2012; 56: 1159-1170Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar Considering the embryological, structural, and biological heterogeneity of the bile duct, it should be no surprise that cholangiopathies manifest at specific sites of the biliary tree. For example, primary sclerosing cholangitis affects large ducts whereas primary biliary cholangitis targets small ducts;[9]Cheung A.C. Lorenzo Pisarello M.J. LaRusso N.F. Pathobiology of biliary epithelia.Biochim Biophys Acta Mol Basis Dis. 2018; 1864: 1220-1231Crossref PubMed Scopus (25) Google Scholar ischemic cholangiopathies, as would be expected from their exclusively arterial blood supply, are restricted to large bile ducts. Biliary atresia begins in the extrahepatic duct whereas Alagille Syndrome is characterized by paucity of the interlobular ducts.[10]Desmet V.J. Congenital diseases of intrahepatic bile ducts: variations on the theme "ductal plate malformation.Hepatology. 1992; 16: 1069-1083Crossref PubMed Scopus (477) Google Scholar Thus, it is clear that the biliary tree is highly complex and can by no means be considered a "simple" conduit for bile. Travel Grant from the Fulbright Foundation (to IEMdJ). Travel Grant from the International Liver Transplantation Society (to IEMdJ). National Institutes of Health grant DK119290 (to RGW). Fred and Suzanne Biesecker Pediatric Liver Center at The Children's Hospital of Philadelphia (to RGW). IEMdJ designed and executed the artwork and wrote the text. MCvdH, RGW, and RJP edited the figure and wrote and edited the text. The authors of this study have no conflicts of interest to declare. Please refer to the accompanying ICMJE disclosure forms for further details. The following is the supplementary data to this article: Download .pdf (.48 MB) Help with pdf files Multimedia component 1

Background. Hypothermic oxygenated machine perfusion (HOPE) reduces ischemia-reperfusion injury of donor livers and is increasingly used in clinical transplantation. However, it remains unclear whether perfusion via the portal vein alone (HOPE) or via both the portal vein and hepatic artery (dual HOPE or DHOPE) is superior. Methods. Twelve porcine livers donated after circulatory death were randomized for 2 h of HOPE (n = 6) or DHOPE (n = 6), followed by 4 h of warm reperfusion with whole blood, to mimic transplantation. Hepatobiliary and endothelial cell function and injury markers were determined in perfusate and bile samples. Biopsies of bile ducts, hepatic arteries, and liver parenchyma were collected to assess histological damage and the expression of endothelial protective genes (KLF-2, eNOS, ET-1, CD31, VWF, VEGF-A). Results. There were no differences in hepatobiliary function and injury after warm reperfusion between the groups, apart from a 2-fold lower concentration of alanine aminotransferase in the perfusate (P = 0.045) and a lower peak lactate dehydrogenase in bile (P = 0.04) of livers preserved by DHOPE. Endothelial cell function and injury, as assessed by perfusate nitric oxide and von Willebrand factor antigen levels, as well as endothelial protective gene expressions, were similar between the groups. The hepatic arteries of both groups showed no microscopic evidence of injury. Conclusions. This study did not reveal major differences in hepatobiliary or endothelial function and injury after preservation by single or dual HOPE of porcine livers donated after circulatory death.

BACKGROUND AND AIMS:Nonanastomotic biliary strictures (NAS) are a major cause of morbidity after orthotopic liver transplantation (OLT). Although ischemic injury of peribiliary glands (PBGs) and peribiliary vascular plexus during OLT has been associated with the later development of NAS, the exact underlying mechanisms remain unclear. We hypothesized that bile ducts of patients with NAS suffer from ongoing biliary hypoxia and lack of regeneration from PBG stem/progenitor cells.APPROACH AND RESULTS:Forty-two patients, requiring retransplantation for either NAS (n = 18), hepatic artery thrombosis (HAT; n = 13), or nonbiliary graft failure (controls; n = 11), were included in this study. Histomorphological analysis of perihilar bile ducts was performed to assess differences in markers of cell proliferation and differentiation in PBGs, microvascular density (MVD), and hypoxia. In addition, isolated human biliary tree stem cells (hBTSCs) were used to examine exo-metabolomics during in vitro differentiation toward mature cholangiocytes. Bile ducts of patients with NAS or HAT had significantly reduced indices of PBG mass, cellular proliferation and differentiation (mucus production, secretin receptor expression, and primary cilia), reduced MVD, and increased PBG apoptosis and hypoxia marker expression, compared to controls. Metabolomics of hBTSCs during in vitro differentiation toward cholangiocytes revealed a switch from a glycolytic to oxidative metabolism, indicating the need for oxygen.CONCLUSIONS:NAS are characterized by a microscopic phenotype of chronic biliary hypoxia attributed to loss of microvasculature, resulting in reduced proliferation and differentiation of PBG stem/progenitor cells into mature cholangiocytes. These findings suggest that persistent biliary hypoxia is a key mechanism underlying the development of NAS after OLT.

Introduction Chimerism after orthotopic liver transplantation (OLT) has largely been investigated in intrahepatic cellular constituents. However, little is known about chimerism in the extrahepatic and large intrahepatic bile ducts. Our aim was to evaluate the presence and extent of chimerism after OLT in the peribiliary glands (PBG) and the luminal epithelium of the large donor bile ducts. Methods For this study, we examined six extrahepatic and large intrahepatic bile ducts from livers that were re-transplanted. In all cases there was a sex-mismatch between donor and recipient (female donor organ and male recipient), which allowed to discriminate between donor- and recipient-derived cells. Specimens from female to female transplants were used as negative controls and male to male transplants as positive controls. Fluorescencein situhybridization (FISH) for Y and X chromosomes was performed and the percentage of XY positive cells was determined among biliary epithelial cells. Immunohistochemistry was used to correlate chimerism with histological features. Results Cholangiocellular chimerism in all studied specimens ranged from 14 to 52%. The degree of chimerism was not associated with biliary damage. Marked chimerism was present at 5 days post-OLT. Ki-67-positivity was detected in 1-8% of the epithelial cells at the time of liver re-transplantation, and this correlated inversely with the degree of chimerism. Conclusion Recipient-derived cholangiocytes are present in the large bile ducts of the donor liver after OLT. The presence of chimerism in the large bile ducts suggests that recipient-derived cells may play a role in biliary regeneration following ischemia-induced injury during OLT.

Necroptosis is a type of regulated cell death that is increasingly being recognized as a relevant pathway in different pathological conditions. Necroptosis can occur in response to multiple stimuli, is triggered by the activation of death receptors, and is regulated by receptor-interacting protein kinases 1 and 3 and mixed-lineage kinase domain-like, which form a regulatory complex called the necrosome. Accumulating evidence suggests that necroptosis plays a complex role in cancer, which is likely context-dependent and can vary among different types of neoplasms. Necroptosis serves as an alternative mode of programmed cell death overcoming apoptosis and, as a pro-inflammatory death type, it may inhibit tumor progression by releasing damage-associated molecular patterns to elicit robust cross-priming of anti-tumor CD8+ T cells. The development of therapeutic strategies triggering necroptosis shows great potential for anti-cancer therapy. In this review, we summarize the current knowledge on necroptosis and its role in liver biliary neoplasms, underlying the potential of targeting necroptosis components for cancer treatment.