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Objectives: Alcohol-related liver disease (ALD) is among the most common indications for liver transplantation (LTX) in Europe and North America, with good five-year survival rates post-LTX. Here we evaluated survival up to and beyond 20 years after LTX for patients with ALD compared to a comparison group., Methods: Patients with ALD and a comparison group transplanted in the Nordic countries between 1982 and 2020 were included. Data were analyzed using descriptive statistics, Kaplan-Meier curves and predictors of survival were assessed with Cox-regressions., Results: 831 patients with ALD and 2979 patients in the comparison group were included in the study. Patients with ALD were older at the time of LTX ( p  < .001) and more likely to be male ( p  < .001). The estimated median follow-up time was 9.1 years for the ALD-group and 11.1 years for the comparison group. 333 (40.1%) patients with ALD and 1010 (33.9%) patients in the comparison group died during follow-up. The overall survival was impaired for patients with ALD compared to the comparison group ( p  < .001) and was evident for male and female patients, patients transplanted before and after 2005, and observed in all age-groups except patients over 60 years. Age at transplant, waiting time, year of LTX and country of LTX were associated with decreased survival after LTX for patients with ALD., Conclusions: Patients with ALD have a decreased long-term survival following LTX. This difference was evident in most sub-groups of patients and warrants close follow-up of liver transplanted patients with ALD with focus on risk reduction.

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2) 1 , could represent a new chemoprophylactic approach for COVID-19 that complements vaccination 2,3 . However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials., (© 2022. The Author(s).)

We developed a novel, pump-less directional flow recirculating organ-on-a-chip (rOoC) platform that creates controlled unidirectional gravity-driven flow by a combination of a 3D-tilting system and an optimized microfluidic layout. The rOoC platform was assembled utilizing a layer-to-layer fabrication technology based on thermoplastic materials. It features two organoid compartments supported by two independent perfusion channels and separated by a hydrogel barrier. We developed a computational model to predict wall shear stress values and then measured the flow rate in the microfluidic channels with micro-Particle-Image-Velocimetry (μPIV). The suitability of the rOoC for functional culture of endothelial cells was tested using HUVECs seeded in the perfusion channels. HUVECs aligned in response to the directional flow, formed a barrier and were able to sprout into the organoid compartments. Next, we demonstrated the viability of human stem-cell derived liver organoids in the organoid compartments. Finally, we show the possibility to circulate immune cells in the microfluidic channels that retain viability without being trapped or activated. The rOoC platform allows growing and connecting of two or more tissue or organ representations on-chip with the possibility of applying gradients, endothelial barriers, microvasculature and circulating cells independent of external tubing and support systems.

Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 serum samples. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring., (© 2022. The Author(s).)

Liver fibrosis and cirrhosis have limited therapeutic options and represent a serious unmet patient need. Recent use of single-cell RNA sequencing (scRNAseq) has identified enriched cell types infiltrating cirrhotic livers but without defining the microanatomical location of these lineages thoroughly. To assess whether fibrotic liver regions specifically harbor enriched cell types, we explored whether whole-tissue spatial transcriptomics combined with scRNAseq and gene deconvolution analysis could be used to localize cell types in cirrhotic explants of patients with end-stage liver disease (total n = 8; primary sclerosing cholangitis, n = 4; primary biliary cholangitis, n = 2, alcohol-related liver disease, n = 2). Spatial transcriptomics clearly identified tissue areas of distinct gene expression that strongly correlated with the total area (Spearman r = 0.97, p = 0.0004) and precise location (parenchyma, 87.9% mean congruency; range, 73.1%-97.1%; fibrosis, 68.5% mean congruency; range, 41.0%-91.7%) of liver regions classified as parenchymal or fibrotic by conventional histology. Deconvolution and enumeration of parenchymal and fibrotic gene content as measured by spatial transcriptomics into distinct cell states revealed significantly higher frequencies of ACTA2+ FABP4+ and COL3A1+ mesenchymal cells, IL17RA+ S100A8+ and FCER1G+ tissue monocytes, VCAM1+ SDC3+ Kupffer cells, CCL4+ CCL5+ KLRB1+ and GZMA+ IL17RA+ T cells and HLA-DR+, CD37+ CXCR4+ and IGHM+ IGHG+ B cells in fibrotic liver regions compared with parenchymal areas of cirrhotic explants. Conclusion: Our findings indicate that spatial transcriptomes of parenchymal and fibrotic liver regions express unique gene content within cirrhotic liver and demonstrate proof of concept that spatial transcriptomes combined with additional RNA sequencing methodologies can refine the localization of gene content and cell lineages in the search for antifibrotic targets., (© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background and Aims: Cholangiocarcinoma (CCA) is a malignancy arising from biliary epithelial cells of intra- and extrahepatic bile ducts with dismal prognosis and few nonsurgical treatments available. Despite recent success in the immunotherapy-based treatment of many tumor types, this has not been successfully translated to CCA. Mucosal-associated invariant T (MAIT) cells are cytotoxic innate-like T cells highly enriched in the human liver, where they are located in close proximity to the biliary epithelium. Here, we aimed to comprehensively characterize MAIT cells in intrahepatic (iCCA) and perihilar CCA (pCCA)., Approach and Results: Liver tissue from patients with CCA was used to study immune cells, including MAIT cells, in tumor-affected and surrounding tissue by immunohistochemistry, RNA-sequencing, and multicolor flow cytometry. The iCCA and pCCA tumor microenvironment was characterized by the presence of both cytotoxic T cells and high numbers of regulatory T cells. In contrast, MAIT cells were heterogenously lost from tumors compared to the surrounding liver tissue. This loss possibly occurred in response to increased bacterial burden within tumors. The residual intratumoral MAIT cell population exhibited phenotypic and transcriptomic alterations, but a preserved receptor repertoire for interaction with tumor cells. Finally, the high presence of MAIT cells in livers of iCCA patients predicted long-term survival in two independent cohorts and was associated with a favorable antitumor immune signature., Conclusions: MAIT cell tumor infiltration associates with favorable immunological fitness and predicts survival in CCA., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Primary sclerosing cholangitis (PSC) is associated with altered microbiota of the gut and bile. Mucosal-associated invariant T (MAIT) cells, enriched in human liver, uniquely recognize microbial-derived metabolites. This study aimed to determine whether bile from patients with PSC contains antigens activating MAIT cells. Bile was collected at the time of liver transplantation from patients with PSC (n = 28). The bile samples were either directly incubated with peripheral blood mononuclear cells from healthy donors or with antigen-presenting cells followed by co-culture with peripheral blood mononuclear cells. MAIT cell activation was assessed by flow cytometry. An anti-MR1 antibody was used to determine whether the activation was major histocompatibility complex class I-related protein (MR1) restricted. Biliary microbiota profiles were generated using 16S rRNA amplicon sequencing, and the abundance of the bacterial gene ribD was predicted. Eight of 28 bile samples could activate MAIT cells. This activation was partly MR1-dependent in five of eight bile samples. Microbial DNA was detected in 15 of 28 bile samples, including the five bile samples leading to MR1-dependent activation. A higher abundance of the ribD gene expression in the group of bile samples that could activate MAIT cells was predicted on the basis of the 16S sequencing. In co-culture experiments, cholangiocytes could take up and present biliary antigens to MAIT cells. These findings suggest a pathophysiological pathway in PSC connecting the immune system and the microbiome., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: There is continued uncertainty regarding the risks of hepato-pancreato-biliary cancers in patients with inflammatory bowel disease (IBD) with or without concomitant primary sclerosing cholangitis (PSC)., Objective: To give updated estimates on risk of hepato-pancreato-biliary cancers in patients with IBD, including pancreatic cancer, hepatocellular carcinoma, gall bladder cancer, and intra - and extrahepatic cholangiocarcinoma., Methods: In a population-based cohort study, we included all patients diagnosed with IBD in Norway and Sweden from 1987 to 2016. The cohort comprised of 141,960 patients, identified through hospital databases and the National Patient Register. Participants were followed through linkage to national cancer, cause of death, and population registries. We calculated absolute risk and standardized incidence ratios (SIRs) of hepato-pancreato-biliary cancers by PSC and other clinical characteristics., Results: Of the 141,960 IBD patients, 3.2% were diagnosed with PSC. During a median follow-up of 10.0 years, we identified 443 biliary tract cancers (SIR 5.2, 95% confidence interval [CI] 4.8-5.7), 161 hepatocellular carcinomas (SIR 2.4, 95% CI 2.0-2.7) and 282 pancreatic cancers (SIR 1.3, 95% CI 1.2-1.5). The relative risks were considerably higher in PSC-IBD patients, with SIR of 140 (95% CI 123-159) for biliary tract, 38.6 (95% CI 29.2-50.0) for hepatocellular, and 9.0 (95% CI 6.3-12.6) for pancreatic cancer. The SIRs were still slightly increased in non-PSC-IBD patients, compared to the general population. For biliary tract cancer, the cumulative probability at 25 years was 15.6% in PSC-IBD patients, and 0.4% in non-PSC-IBD patients., Conclusions: The dramatically increased risks of hepato-pancreato-biliary cancers in PSC-IBD patients support periodic surveillance for these malignancies. While much lower, the excess relative risks in non-PSC-IBD patients were not trivial compared to non-IBD related risk factors., (© 2022 The Authors. United European Gastroenterology Journal published by Wiley Periodicals LLC on behalf of United European Gastroenterology.)

Aims: Patients with rheumatoid arthritis (RA) have increased risk of heart failure (HF). The mechanisms and cardiac prerequisites explaining this association remain unresolved. In this study, we sought to determine the potential cardiac impact of an experimental model of RA in mice subjected to HF by constriction of the ascending aorta., Methods: Aorta was constricted via thoracotomy and placement of o-rings with inner diameter 0.55 mm or 0.66 mm, or sham operated. RA-like phenotype was instigated by delayed-type hypersensitivity arthritis (DTHA) two weeks after surgery and re-iterated after additional 18 days. Cardiac magnetic resonance imaging (MRI) was performed before surgery and at successive time points throughout the study. Six weeks after surgery the mice were euthanized, blood and tissue were collected, organ weights were documented, and expression levels of cardiac foetal genes were analysed. In a supplemental study, DTHA-mice were euthanized throughout 14 days after induction of arthritis, and blood was analysed for important markers and mediators of RA (SAP, TNF-α and IL-6). In order to put the latter findings into clinical context, the same molecules were analysed in serum from untreated RA patients and compared to healthy controls., Results: Significant elevations of inflammatory markers were found in both patient- and murine blood. Furthermore, the DTHA model appeared clinically relevant when compared to the inflammatory responses observed in three prespecified RA severity disease states. Two distinct trajectories of cardiac dysfunction and HF development were found using the two o-ring sizes. These differences were consistent by both MRI, organ weights and cardiac foetal gene expression levels. Still, no difference within the HF groups, nor within the sham groups, could be found when DTHA was induced., Conclusion: DTHA mediated systemic inflammation did not cause, nor modify HF caused by aortic constriction. This indicates other prerequisites for RA-induced cardiac dysfunction., Competing Interests: The authors have declared that no competing interests exist.

Recurrent attacks of acute intermittent porphyria (AIP) result in poor quality of life and significant risks of morbidity and mortality. Liver transplantation (LT) offers a cure, but published data on outcomes after LT are limited. We assessed the pretransplant characteristics, complications, and outcomes for patients with AIP who received a transplant. Data were collected retrospectively from the European Liver Transplant Registry and from questionnaires sent to identified transplant and porphyria centers. We studied 38 patients who received transplants in 12 countries from 2002 to 2019. Median age at LT was 37 years (range, 18-58), and 34 (89%) of the patients were women. A total of 9 patients died during follow-up, and 2 patients were retransplanted. The 1-year and 5-year overall survival rates were 92% and 82%, which are comparable with other metabolic diseases transplanted during the same period. Advanced pretransplant neurological impairment was associated with increased mortality. The 5-year survival rate was 94% among 19 patients with moderate or no neuropathy at LT and 83% among 10 patients with severe neuropathy (P = 0.04). Pretransplant renal impairment was common. A total of 19 (51%) patients had a GFR < 60 mL/minute. Although few patients improved their renal function after LT, neurological impairments improved, and no worsening of neurological symptoms was recorded. No patient had AIP attacks after LT, except for a patient who received an auxiliary graft. LT is a curative treatment option for patients with recurrent attacks of AIP. Severe neuropathy and impaired renal function are common and increase the risk for poor outcomes. If other treatment options fail, an evaluation for LT should be performed early., (Copyright © 2020 The Authors. Liver Transplantation published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease without clear etiology or effective treatment. Genetic factors contribute to PSC pathogenesis, but so far, no causative mutation has been found. We performed whole-exome sequencing in a family with autosomal dominant inheritance of PSC and identified a heterozygous germline missense mutation in SEMA4D , encoding a K849T variant of CD100. The mutation was located in an evolutionarily conserved, unstructured cytosolic region of CD100 affecting downstream signaling. It was found to alter the function of CD100-expressing cells with a bias toward the T cell compartment that caused increased proliferation and impaired interferon-γ (IFN-γ) production after stimulation. Homologous mutation knock-in mice developed similar IFN-γ impairment in T cells and were more prone to develop severe cholangitis when exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Transfer of wild-type T cells to knock-in mice before and during DDC exposure attenuated cholangitis. Taken together, we identified an inherited mutation in the disordered cytosolic region of CD100 resulting in T cell functional defects. Our findings suggest a protective role for T cells in PSC that might be used therapeutically., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Thoracic epidural analgesia (TEA) is not widely used for postoperative pain management in liver transplantation due to hepatic coagulopathy-related increased risk of inducing an epidural hematoma. However, an increasing number of patients are transplanted for other indications than the end-stage liver disease and without coagulopathy allowing insertion of an epidural catheter., Methods: This study is a retrospective observational single-center study of all adult patients undergoing first-time liver transplantation at Oslo University Hospital between January 1, 2008, and December 31, 2017. Data regarding patient characteristics were obtained from the Nordic liver transplant registry, medical records, and pain registration forms. Patients without coagulopathy (international normalized ratio <1.5 and platelets >100 × 10 9 /L) were eligible for TEA., Results: Out of 685 first-time liver transplantations in a 10-year period, 327 received TEA, and 358 did not. The median Model of End-stage Liver Disease score was lower in the TEA group than in the non-TEA-group (9 versus 17, P < 0.001), and fewer patients were hospitalized preoperatively (16 versus 127, P < 0.001). The median international normalized ratio (1.1 versus 1.6, P < 0.001) and platelet count (190 versus 78, P < 0.001) were different between the TEA and non-TEA groups. There were no serious complications related to insertion or removal of the TEA catheters. Patients in the TEA group had less pain with a mean numeric rating scale at postoperative days 0-5 of 1.4 versus 1.8 ( P = 0.008). Nearly 50% of the patients were prescribed opioids when discharged from hospital (non-TEA 154 versus TEA 158, P = 0.23), and there was no difference after 1 year ( P = 0.718)., Conclusions: Our report revealed very good pain control with both TEA and the non-TEA modality. TEA was without any serious complications like epidural hematoma or infection/abscess in selected liver transplant recipients without severe coagulopathy. Opioid prescription at hospital discharge and by 1-year follow-up did not differ between the groups., Competing Interests: The authors declare no funding or conflicts of interest., (Copyright © 2020 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

An inflammatory response is required for tissue healing after a myocardial infarction (MI), but the process must be balanced to prevent maladaptive remodeling. This study shows that improved survival and cardiac function following MI, in mice deficient for the NLRP3 inflammasome, can be recapitulated in wild-type mice receiving bone marrow from Nlrp3 -/- mice. This suggests that NLRP3 activation in hematopoietic cells infiltrating in the myocardium increases mortality and late ventricular remodeling. Our data should encourage performing clinical trials directly targeting NLRP3 inflammasome and their inflammatory cytokines (interleukin-1β and -18) in MI patients., Competing Interests: This work was supported by grants from the K.G. Jebsen Inflammation Research Centre, South-Eastern Norway Regional Health Authority, and Norwegian Research Council. Dr. Yndestad is currently an employee of Pfizer Norway; the current work was conducted when he was employed by Oslo University Hospital. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2020 The Authors.)

Invariant natural killer T (iNKT) cells recognize activating self and microbial lipids presented by CD1d. CD1d can also bind non-activating lipids, such as sphingomyelin. We hypothesized that these serve as endogenous regulators and investigated humans and mice deficient in acid sphingomyelinase (ASM), an enzyme that degrades sphingomyelin. We show that ASM absence in mice leads to diminished CD1d-restricted antigen presentation and iNKT cell selection in the thymus, resulting in decreased iNKT cell levels and resistance to iNKT cell-mediated inflammatory conditions. Defective antigen presentation and decreased iNKT cells are also observed in ASM-deficient humans with Niemann-Pick disease, and ASM activity in healthy humans correlates with iNKT cell phenotype. Pharmacological ASM administration facilitates antigen presentation and restores the levels of iNKT cells in ASM-deficient mice. Together, these results demonstrate that control of non-agonistic CD1d-associated lipids is critical for iNKT cell development and function in vivo and represents a tight link between cellular sphingolipid metabolism and immunity.

Disorders of the biliary tree develop and progress differently according to patient age. It is currently not known whether the aging process affects the response to injury of cholangiocytes. The aim of this study was to identify molecular pathways associated with cholangiocyte aging and to determine their effects in the biological response to injury of biliary cells. A panel of microRNAs (miRs) involved in aging processes was evaluated in cholangiocytes of young and old mice (2 months and 22 months of age, respectively) and subjected to a model of sclerosing cholangitis. Intracellular pathways that are common to elevated miRs were identified by in silico analysis. Cell proliferation and senescence were evaluated in Twinfilin-1 (Twf1) knocked-down cells. In vivo, senescence-accelerated prone mice (Samp8, a model for accelerated aging), Twf1 -/- , or their respective controls were subjected to DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine). Cholangiocytes from DDC-treated mice showed up-regulation of a panel of aging-related miRs. Twf1 was identified by in silico analysis as a common target of the up-regulated miRs. Twf1 expression was increased both in aged and diseased cholangiocytes, and in human cholangiopathies. Knock-down of Twf1 in cholangiocytes reduced cell proliferation. Senescence and senescence-associated secretory phenotype marker expression increased in Twf1 knocked-down cholangiocytes following pro-proliferative and pro-senescent (10-day lipopolysaccharide) stimulation. In vivo, Samp8 mice showed increased biliary proliferation, fibrosis, and Twf1 protein expression level, whereas Twf1 -/- had a tendency toward lower biliary proliferation and fibrosis following DDC administration compared with control animals. Conclusion: We identified Twf1 as an important mediator of both cholangiocyte adaptation to aging processes and response to injury. Our data suggest that disease and aging might share common intracellular pathways., (© 2018 by the American Association for the Study of Liver Diseases.)

Pediatric liver transplantation is often required as a consequence of biliary disorders because of the lack of alternative treatments for repairing or replacing damaged bile ducts. To address the lack of availability of pediatric livers suitable for transplantation, we developed a protocol for generating bioengineered biliary tissue suitable for biliary reconstruction. Our platform allows the derivation of cholangiocyte organoids (COs) expressing key biliary markers and retaining functions of primary extra- or intrahepatic duct cholangiocytes within 2 weeks of isolation. COs are subsequently seeded on polyglycolic acid (PGA) scaffolds or densified collagen constructs for 4 weeks to generate bioengineered tissue retaining biliary characteristics. Expertise in organoid culture and tissue engineering is desirable for optimal results. COs correspond to mature functional cholangiocytes, differentiating our method from alternative organoid systems currently available that propagate adult stem cells. Consequently, COs provide a unique platform for studies in biliary physiology and pathophysiology, and the resulting bioengineered tissue has broad applications for regenerative medicine and cholangiopathies.

There is a clear genetic contribution to the risk of cardiovascular diseases, and a composite genetic risk score (GRS) based on 27 single nucleotide polymorphisms (SNPs) was reported to predict risk of cardiovascular events in the general population. We aimed to evaluate this risk score in renal transplant recipients, a population with heightened cardiovascular risk, with a yet unknown genetic contribution. A total of 1640 participants from the ALERT trial (Assessment of Lescol in Renal Transplantation), a study comparing fluvastatin with placebo in stable renal transplant recipients, were genotyped for all SNPs making up the GRS. Risk alleles were weighted by the log of odds ratios reported in genome wide association studies and summed. Associations between GRS and time from study inclusion to first major cardiovascular event (MACE) were analyzed by Cox regression. In analyses adjusted for cardiovascular risk factors, GRS was significantly associated with MACE (hazard ratio [HR] 1.81, P = .006) when comparing genetic high-risk patients (quartile 4) with genetic low-risk participants (quartile 1). A 27-SNP GRS, which predicted cardiovascular events in the nontransplant population, appears to have predictive value also in kidney allograft recipients. Refining the score to better fit the transplant population seems feasible., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background & Aims: Nonalcoholic fatty liver disease(NAFLD) is the second most common cause of liver transplantation in the US. Data on NAFLD as a liver transplantation indication from countries with lower prevalences of obesity are lacking. We studied the temporal trends of NAFLD as an indication for liver transplantation in the Nordic countries, and compared outcomes for patients with NAFLD to patients with other indications for liver transplantation., Method: Population-based cohort study using data from the Nordic Liver Transplant Registry on adults listed for liver transplantation between 1994 and 2015. NAFLD as the underlying indication for liver transplantation was defined as a listing diagnosis of NAFLD/nonalcoholic steatohepatitis, or cryptogenic cirrhosis with a body mass index ≥25 kg/m 2 and absence of other liver diseases. Waiting time for liver transplantation, mortality and withdrawal from the transplant waiting list were registered. Survival after liver transplantation was calculated using multivariable Cox regression, adjusted for age, sex, body mass index and model for end-stage liver disease., Results: A total of 4609 patients listed for liver transplantation were included. NAFLD as the underlying indication for liver transplantation increased from 2.0% in 1994-1995 to 6.2% in 2011-2015 (P = .01) and was the second most rapidly increasing indication. NAFLD patients had higher age, model for end-stage liver disease and body mass index when listed for liver transplantation, but overall survival after liver transplantation was comparable to non--NAFLD patients (aHR 1.03, 95% CI 0.70-1.53 P = .87)., Conclusion: NAFLD is an increasing indication for liver transplantation in the Nordic countries. Despite more advanced liver disease, NAFLD patients have a comparable survival to other patients listed for liver transplantation., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Diagnostic work-ups in transplanted immunosuppressed patients are a challenge as non-specific findings may be interpreted as transplant-related complications. If the disease in question is rare and slowly developing like pseudomyxoma peritonei (PMP), it is even more difficult. Cytoreductive surgery (CRS) and subsequent hyperthermic intraperitoneal chemotherapy (HIPEC) is the recommended treatment for PMP even with extensive peritoneal spread. CRS-HIPEC for PMP after liver transplantation (LTX) has not been described before., Case Presentation: A 48-year-old female patient with end-stage primary sclerosing cholangitis (PSC) underwent orthotopic LTX and subsequent pancreaticoduodenectomy after the finding of cholangiocarcinoma in situ in the native common bile duct. Ten years after the transplantation, she developed symptoms and signs suspected to represent graft-related complications. An extensive work-up revealed PMP. Upon reassessment, a cystic mass near the coecum could be seen on computed tomography scan 1 year after transplantation. The multidisiplinary team was hesitant to accept the patient for CRS-HIPEC because of extensive PMP and possible risk to the graft. However, she was eventually accepted and underwent the procedure. The Peritoneal Cancer Index (PCI) was 28 of 39, and surgical debulking was performed followed by HIPEC. The transplant control 2 months after surgery showed no harm to the graft., Conclusions: Previous LTX should not exclude the possibility for CRS-HIPEC in PMP, even with extensive burden of disease.

Innate lymphocytes are selectively enriched in the liver where they have important roles in liver immunology. Murine studies have shown that type I NKT cells can promote liver inflammation, whereas type II NKT cells have an anti-inflammatory role. In humans, type II NKT cells were found to accumulate in the gut during inflammation and IL13Rα2 was proposed as a marker for these cells. In the human liver, less is known about type I and II NKT cells. Here, we studied the phenotype and function of human liver T cells expressing IL13Rα2. We found that IL13Rα2 was expressed by around 1% of liver-resident memory T cells but not on circulating T cells. In support of their innate-like T-cell character, the IL13Rα2 + T cells had higher expression of promyelocytic leukaemia zinc finger (PLZF) compared to IL13Rα2 - T cells and possessed the capacity to produce IL-22. However, only a minority of human liver sulfatide-reactive type II NKT cells expressed IL13Rα2. Collectively, these findings suggest that IL13Rα2 identifies tissue-resident intrahepatic T cells with innate characteristics and the capacity to produce IL-22., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Primary sclerosing cholangitis (PSC) is a genetically complex, inflammatory bile duct disease of largely unknown aetiology often leading to liver transplantation or death. Little is known about the genetic contribution to the severity and progression of PSC. The aim of this study is to identify genetic variants associated with PSC disease progression and development of complications., Design: We collected standardised PSC subphenotypes in a large cohort of 3402 patients with PSC. After quality control, we combined 130 422 single nucleotide polymorphisms of all patients-obtained using the Illumina immunochip-with their disease subphenotypes. Using logistic regression and Cox proportional hazards models, we identified genetic variants associated with binary and time-to-event PSC subphenotypes., Results: We identified genetic variant rs853974 to be associated with liver transplant-free survival (p=6.07×10 -9 ). Kaplan-Meier survival analysis showed a 50.9% (95% CI 41.5% to 59.5%) transplant-free survival for homozygous AA allele carriers of rs853974 compared with 72.8% (95% CI 69.6% to 75.7%) for GG carriers at 10 years after PSC diagnosis. For the candidate gene in the region, RSPO3 , we demonstrated expression in key liver-resident effector cells, such as human and murine cholangiocytes and human hepatic stellate cells., Conclusion: We present a large international PSC cohort, and report genetic loci associated with PSC disease progression. For liver transplant-free survival, we identified a genome-wide significant signal and demonstrated expression of the candidate gene RSPO3 in key liver-resident effector cells. This warrants further assessments of the role of this potential key PSC modifier gene., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Cholangiopathies are progressive disorders with largely unknown pathoetiology and limited treatment options. We aimed to develop a novel surgical technique with direct access to the bile ducts that would complement existing mouse models of cholestasis, biliary inflammation, and fibrosis and present a new route of administration for testing of potential treatment strategies. We developed a surgical technique to access the murine biliary tree by injection of different solvents through catheterization of the gall bladder with simultaneous clamping of the common bile duct. To demonstrate the applicability of the technique, we injected either phosphate-buffered saline or dimethyl sulfoxide in concentrations of 50 or 65% and compared these groups with sham-operated mice. The surgery was optimized to achieve a mortality rate close to 0. There were no significant changes in pain, activity level, or mortality from the day of the surgery until euthanization for any groups. Injection of phosphate-buffered saline or 50% dimethyl sulfoxide was generally well-tolerated, whereas 65% dimethyl sulfoxide led to higher weight loss, an increase of serum alanine transaminase, and histological portal inflammation. There were no signs of inflammation in the gut. We have developed a bile duct injection technique that is well-tolerated, easily reproducible, and that may complement existing models of cholangiopathies. Direct access to the bile ducts without causing harm to the hepatobiliary or intestinal tissue may be valuable in future studies of normal biliary physiology and different pathophysiological mechanisms of disease and to test novel therapeutic strategies. NEW & NOTEWORTHY To evaluate tolerability of the bile duct to injection of both polar and nonpolar compounds, we established a novel biliary injection technique. This technique is well-tolerated, easily reproducible, and with direct access to the bile ducts for studies of the murine biliary tree. The bile duct injection technique may complement existing animal models and be a valuable tool in future studies of normal biliary physiology or pathophysiology and to test novel therapeutic strategies.

Background: Lack of Hepatitis C virus (HCV) incidence data in (Norwegian) high-risk groups impedes the ability to make informed decisions on prevention measures. Thus we rely on modelling to estimate the incidence and burden of HCV infections., Methods: We constructed a compartmental model for HCV infections in Norway among active and former people who inject drugs (PWIDs). We based yearly transition rates on literature. The model was fitted to absolute numbers of hepatitis C associated cirrhosis, hepatocellular carcinoma (HCC) and death from national data sources (2000-2013). We estimated the number (95%CI) of HCV infections, cirrhosis, HCC and death and disability adjusted life years (DALYs) due to HCV infections in Norway, 1973-2030. We assumed treatment rates in the projected period were similar to those in 2013., Results: The estimated proportion of chronic HCV (including those with cirrhosis and HCC) among PWIDs was stable from 2000 (49%; 4441/9108) to 2013 (43%; 3667/8587). We estimated that the incidence of HCV among PWIDs was 381 new infections in 2015. The estimated number of people with cirrhosis, HCC, and liver transplant was predicted to increase until 2022 (1537 people). DALYs among active PWIDs estimated to peak in 2006 (3480 DALYs) and decrease to 1870 DALYs in 2030. Chronic HCV infection contributes most to the total burden of HCV infection, and peaks at 1917 DALYs (52%) in 2007. The burden of HCV related to PWID increased until 2006 with 81/100,000 DALYs inhabitants and decreased to 68/100,000 DALYs in 2015., Conclusion: The burden of HCV associated with injecting drug use is considerable, with chronic HCV infection contributing most to the total burden. This model can be used to estimate the impact of different interventions on the HCV burden in Norway and to perform cost-benefit analyses of various public health measures.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

The pan lymphocyte marker CD45 exists in various isoforms arising from alternative splicing of the exons 4, 5 and 6. While naïve T cells express CD45RA translated from an mRNA containing exon 4, exons 4-6 are spliced out to encode the shorter CD45R0 in antigen-experienced effector/memory T cells. The SNP C77G (rs17612648) is located in exon 4 and blocks the exon's differential splicing from the pre-mRNA, enforcing expression of CD45RA. Several studies have linked C77G to autoimmune diseases but lack of validation in other cohorts has left its role elusive. An incidental finding in an ovarian cancer patient cohort from West Norway (Bergen region, n = 312), suggested that the frequency of C77G was higher among ovarian cancer patients than in healthy Norwegians (n = 1,357) (3.0% vs. 1.8% allele frequency). However, this finding could not be validated in a larger patient cohort from South-East Norway (Oslo region, n = 1,198) with 1.2% allele frequency. Hence, C77G is not associated with ovarian cancer in the Norwegian population. However, its frequency was increased in patients with FIGO stage II, endometrioid histology or an age at diagnosis of 60 years or older indicating a possible association with a less aggressive cancer type.

Targeting CD100 by antibody blockade is a potential therapeutic strategy for cancers, but the functional effects on T cells following blockade of this immune activating molecule are rarely considered. Indeed, CD100 is highly expressed in T cells and anti-CD100 antibodies play a role during T cell proliferation; however, the outcome varies from different studies and the underlying mechanism is still unclear. To address this, monoclonal antibody clones directed against CD100 were evaluated. In their soluble form, four of these antibodies significantly reduced the expansion of T cells in the presence of bead-bound anti-CD3/CD28, either in total peripheral blood mononuclear cell or purified T cell culture systems. Similar inhibition was seen when blocking CD100-CD72 interaction by soluble anti-CD72 instead of anti-CD100 antibodies. Conversely, restoring the interaction by CD72-Fc eliminated the soluble anti-CD100-induced inhibitory effect. Taken together, these results reveal that T cell proliferation is regulated by CD100 via interaction with CD72. They further establish an in vitro system to evaluate the inhibitory effect of anti-CD100 antibodies on T cells, to which attention should be paid in clinical trials in order to avoid potential side effects.

Background & Aims: Prognostic biomarkers are lacking in primary sclerosing cholangitis, hampering patient care and the development of therapy. We aimed to identify novel protein biomarkers of disease severity and prognosis in primary sclerosing cholangitis (PSC)., Methods: Using a bead-based array targeting 63 proteins, we profiled a derivation panel of Norwegian endoscopic retrograde cholangiography bile samples (55 PSC, 20 disease controls) and a Finnish validation panel (34 PSC, 10 disease controls). Selected identified proteins were measured in serum from two Norwegian PSC cohorts (n=167 [1992-2006] and n=138 [2008-2012]), inflammatory bowel disease (n=96) and healthy controls (n=100)., Results: In the bile derivation panel, the levels of 14 proteins were different between PSC patients and controls (p<0.05); all were confirmed in the validation panel. Twenty-four proteins in the bile derivation panel were significantly (p<0.05) different between PSC patients with mild compared to severe cholangiographic changes (modified Amsterdam criteria); this was replicated for 18 proteins in the validation panel. Interleukin (IL)-8, matrix metallopeptidase (MMP)9/lipocalin (LCN)2-complex, S100A8/9, S100A12 and tryptophan hydroxylase (TPH)2 in the bile were associated with both a PSC diagnosis and grade of cholangiographic changes. Stratifying PSC patients according to tertiles of serum IL-8, but not MMP9/LCN2 and S100A12, provided excellent discrimination for transplant-free survival both in the serum derivation and validation cohort. Furthermore, IL-8 was associated with transplant-free survival in multivariable analyses in both serum panels independently of age and disease duration, indicating an independent influence on PSC progression. However, the Enhanced Liver Fibrosis (ELF®) test and Mayo risk score proved to be stronger predictors of transplant-free survival., Conclusions: Based on assaying of biliary proteins, we have identified novel biliary and serum biomarkers as indicators of severity and prognosis in PSC., Lay Summary: Prognostic biomarkers are lacking in primary sclerosing cholangitis, hampering patient care and the development of therapy. We have identified inflammatory proteins including calprotectin and IL-8 as important indicators of disease severity and prognosis in bile and serum from patients with primary sclerosing cholangitis., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

CD8 + T cells that express retinoic acid-related orphan receptor (ROR)γt (T C 17 cells) have been shown to promote procarcinogenic inflammation and contribute to a tolerogenic microenvironment in tumors. We investigated their phenotype and functional properties in relationship to the pathogenesis of human distal bile duct cancer (DBDC). DBDC patients had an elevated level of type 17 immune responses and the frequency of CD8 + RORγt + T cells (T C 17 cells) was increased in peripheral blood. The CD8 + RORγt + T cells represented a highly activated subset and produced IL-17A in equal amount as CD4 + RORγt + T cells (T H 17 cells). Most CD8 + RORγt + T cells coexpressed T-bet, a lineage transcription factor for T H 1 and T C 1 development, suggesting that CD8 + RORγt + T cells undergo plasticity toward a T C 17/1-like phenotype with coproduction of IL-17A and INF-γ. In comparison with CD8 + RORγt - T cells, the CD8 + RORγt + T cells had a higher level of TCR signaling and were terminally differentiated and exhausted. These cells also had impaired ability to re-express perforin after degranulation and reduced cytotoxic immune function. A subset of CD8 + RORγt + T cells expressing a low level of programmed cell death protein 1 and a high level of OX40 were associated with reduced patient survival. In conclusion, CD8 + RORγt + T cells are proinflammatory and functionally impaired and may contribute to the pathogenesis of DBDC., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Natural killer T (NKT) cells are activated by lipid antigens presented by CD1d molecules and represent a major lymphocyte subset of the liver. NODc3c4 mice spontaneously develop biliary inflammation in extra- and intrahepatic bile ducts. We demonstrated by flow cytometry that invariant NKT (iNKT) cells were more abundant in the thymus, spleen, and liver of NODc3c4 mice compared to NOD mice. iNKT cells in NODc3c4 mice displayed an activated phenotype. Further, NOD and NOD Cd1d -/- mice were irradiated and injected with NODc3c4 bone marrow, and injection of NODc3c4 bone marrow resulted in biliary infiltrates independently of CD1d expression in recipient mice. Activation or blocking of NKT cells with α -galactosylceramide or anti-CD1d antibody injections did not affect the biliary phenotype of NODc3c4 mice. NODc3c4. Cd1d -/- mice were generated by crossing NOD Cd1d -/- mice onto a NODc3c4 background. NODc3c4. Cd1d -/- and NODc3c4 mice developed the same extent of biliary disease. This study demonstrates that iNKT cells are more abundant and activated in the NODc3c4 model. The portal inflammation of NODc3c4 mice can be transferred to irradiated recipients, which suggests an immune-driven disease. Our findings imply that NKT cells can potentially participate in the biliary inflammation, but are not the primary drivers of disease in NODc3c4 mice., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Primary sclerosing cholangitis (PSC) is a rare progressive disorder leading to bile duct destruction; ∼75% of patients have comorbid inflammatory bowel disease (IBD). We undertook the largest genome-wide association study of PSC (4,796 cases and 19,955 population controls) and identified four new genome-wide significant loci. The most associated SNP at one locus affects splicing and expression of UBASH3A, with the protective allele (C) predicted to cause nonstop-mediated mRNA decay and lower expression of UBASH3A. Further analyses based on common variants suggested that the genome-wide genetic correlation (r G ) between PSC and ulcerative colitis (UC) (r G = 0.29) was significantly greater than that between PSC and Crohn's disease (CD) (r G = 0.04) (P = 2.55 × 10 -15 ). UC and CD were genetically more similar to each other (r G = 0.56) than either was to PSC (P < 1.0 × 10 -15 ). Our study represents a substantial advance in understanding of the genetics of PSC.

Microbial products are thought to influence the progression of cholangiopathies, in particular primary sclerosing cholangitis (PSC). Inflammasomes are molecular platforms that respond to microbial products through the synthesis of proinflammatory cytokines. We investigated the role of inflammasome activation in cholangiocyte response to injury. Nucleotide-binding oligomerization domain (NOD)-like receptor family, pyrin domain-containing protein 3 (Nlrp3) expression was tested in cholangiocytes of normal and cholestatic livers. Effects of Nlrp3 activation induced by incubation with lipopolysaccharide and ATP was studied in vitro in normal and siRNA-Nlrp3 knocked-down cholangiocytes. Wild-type and Nlrp3 knockout (Nlrp3 -/- ) mice were fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; a model of sclerosing cholangitis) for 4 weeks. Nlrp3 and its components were overexpressed in cholangiocytes of mice subjected to DDC and in patients affected by PSC. In vitro, Nlrp3 activation stimulated expression of Il-18 but not of Il-1β and Il-6. Nlrp3 activation had no effect on cholangiocyte proliferation but significantly decreased the expression of Zonulin-1 and E-cadherin, whereas Nlrp3 knockdown increased the permeability of cholangiocyte monolayers. In vivo, the DDC-stimulated number of cytokeratin-19-positive cells in the liver of wild-type animals was slightly reduced in Nlrp3 -/- mice, and expression of E-cadherin was reestablished. In conclusion, Nlrp3 is expressed in reactive cholangiocytes, in both murine models and patients with PSC. Activation of Nlrp3 leads to synthesis of proinflammatory cytokines and influences epithelial integrity of cholangiocytes., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Recruitment of gut-derived memory T-cells to the liver is believed to drive hepatic inflammation in primary sclerosing cholangitis (PSC). However, whether gut-infiltrating and liver-infiltrating T-cells share T cell receptors (TCRs) and antigenic specificities is unknown. We used paired gut and liver samples from PSC patients with concurrent inflammatory bowel disease (PSC-IBD), and normal tissue samples from colon cancer controls, to assess potential T cell clonotype overlap between the two compartments., Methods: High-throughput sequencing of TCRβ repertoires was applied on matched colon, liver and blood samples from patients with PSC-IBD (n=10), and on paired tumor-adjacent normal gut and liver tissue samples from colon cancer patients (n=10)., Results: An average of 9.7% (range: 4.7-19.9%) memory T cell clonotypes overlapped in paired PSC-IBD affected gut and liver samples, after excluding clonotypes present at similar frequencies in blood. Shared clonotypes constituted on average 16.0% (range: 8.7-32.6%) and 15.0% (range: 5.9-26.3%) of the liver and gut memory T-cells, respectively. A significantly higher overlap was observed between paired PSC-IBD affected samples (8.7%, p=0.0007) compared to paired normal gut and liver samples (3.6%), after downsampling to equal number of reads., Conclusion: Memory T-cells of common clonal origin were detected in paired gut and liver samples of patients with PSC-IBD. Our data indicate that this is related to PSC-IBD pathogenesis, suggesting that memory T-cells driven by shared antigens are present in the gut and liver of PSC-IBD patients. Our findings support efforts to therapeutically target memory T cell recruitment in PSC-IBD., Lay Summary: Primary sclerosing cholangitis (PSC) is a devastating liver disease strongly associated with inflammatory bowel disease (IBD). The cause of PSC is unknown, but it has been suggested that the immune reactions in the gut and the liver are connected. Our data demonstrate for the first time that a proportion of the T-cells in the gut and the liver react to similar triggers, and that this proportion is particularly high in patients with PSC and IBD., (Copyright © 2016 European Association for the Study of the Liver. All rights reserved.)

Unlabelled: Hepatic T-cell infiltrates and a strong genetic human leukocyte antigen association represent characteristic features of various immune-mediated liver diseases. Conceptually the presence of disease-associated antigens is predicted to be reflected in T-cell receptor (TCR) repertoires. Here, we aimed to determine if disease-associated TCRs could be identified in the nonviral chronic liver diseases primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and alcoholic liver disease (ALD). We performed high-throughput sequencing of the TCRβ chain complementarity-determining region 3 of liver-infiltrating T cells from PSC (n = 20), PBC (n = 10), and ALD (n = 10) patients, alongside genomic human leukocyte antigen typing. The frequency of TCRβ nucleotide sequences was significantly higher in PSC samples (2.53 ± 0.80, mean ± standard error of the mean) compared to PBC samples (1.13 ± 0.17, P < 0.0001) and ALD samples (0.62 ± 0.10, P < 0.0001). An average clonotype overlap of 0.85% was detected among PSC samples, significantly higher compared to the average overlap of 0.77% seen within the PBC (P = 0.024) and ALD groups (0.40%, P < 0.0001). From eight to 42 clonotypes were uniquely detected in each of the three disease groups (≥30% of the respective patient samples). Multiple, unique sequences using different variable family genes encoded the same amino acid clonotypes, providing additional support for antigen-driven selection. In PSC and PBC, disease-associated clonotypes were detected among patients with human leukocyte antigen susceptibility alleles., Conclusion: We demonstrate liver-infiltrating disease-associated clonotypes in all three diseases evaluated, and evidence for antigen-driven clonal expansions. Our findings indicate that differential TCR signatures, as determined by high-throughput sequencing, may represent an imprint of distinctive antigenic repertoires present in the different chronic liver diseases; this thereby opens up the prospect of studying disease-relevant T cells in order to better understand and treat liver disease., (© 2015 by the American Association for the Study of Liver Diseases.)

Objective: IBD is a group of complex, systemic disorders associated with intestinal inflammation and extraintestinal manifestations. Recent studies revealed Mendelian forms of IBD, which contributed significantly to our understanding of disease pathogenesis and the heritability of IBD., Design: We performed exome sequencing in a family with Crohn's disease (CD) and severe autoimmunity, analysed immune cell phenotype and function in affected and non-affected individuals, and performed in silico and in vitro analyses of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) structure and function., Results: A novel missense variant was identified in CTLA4 encoding CTLA-4, a coinhibitory protein expressed by T cells and required for regulation of T cell activation. The residue affected by the mutation, CTLA-4 Tyr60, is evolutionarily highly conserved, and the identified Y60C variant is predicted to affect protein folding and structural stability and demonstrated to cause impaired CTLA-4 dimerisation and CD80 binding. Intestinal inflammation and autoimmunity in carriers of CTLA-4 Y60C exhibit incomplete penetrance with a spectrum of clinical presentations ranging from asymptomatic carrier status to fatal autoimmunity and intestinal inflammation. In a clinically affected CTLA-4 Y60C carrier, T cell proliferation was increased in vitro and associated with an increased ratio of memory to naive T cells in vivo, consistent with impaired regulation of T cell activation., Conclusions: Our results support the concept that variants in CTLA4 provide the basis for a novel Mendelian form of early-onset CD associated with systemic autoimmunity. Incomplete penetrance of autoimmunity further indicates the presence of other genetic and/or environmental modifiers., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background: Primary sclerosing cholangitis (PSC) is 1 of the leading causes of liver transplantation (LTX) in Scandinavia, and an increasing number of PSC patients have been transplanted in Norway during the last 2 decades. This trend is partly attributable to the recently established practice in Norway of offering LTX to PSC patients with cholangiocellular dysplasia. Based on the controversy associated with this practice, we herein aimed to report the main features and outcomes of our LTX program in PSC., Methods: The primary indication for LTX (quality of life/end-stage liver disease or suspected neoplasia) was retrospectively determined for 222 patients undergoing LTX for PSC or other autoimmune liver diseases (primary biliary cirrhosis/autoimmune hepatitis) with at least 5 years of follow-up., Results: In PSC patients impaired quality of life (43.5%) and end-stage liver disease (38.4%) were the most frequent indications for LTX, whereas suspected neoplasia accounted for 18.1%. The proportion of PSC patients with manifest encephalopathy, variceal bleeding, or ascites declined over time. In patients with suspected neoplasia as the primary indication for LTX (n = 25), neoplasia was confirmed in the explanted liver in 20 patients (80%). Five-year survival rates for PSC patients transplanted between 2001 and 2009 were 91.9% for patients receiving LTX due to impaired quality of life or end-stage liver disease and 83.3% for suspected neoplasia., Conclusions: The PSC patients are increasingly listed for LTX at an earlier stage of their liver disease. In patients with suspected neoplasia before LTX, 5-year survival was acceptable, despite confirmation of neoplasia in 80% of the liver explants.

Unlabelled: Cholangiocytes express antigen-presenting molecules, but it has been unclear whether they can present antigens. Natural killer T (NKT) cells respond to lipid antigens presented by the major histocompatibility complex class I-like molecule CD1d and are abundant in the liver. We investigated whether cholangiocytes express CD1d and present lipid antigens to NKT cells and how CD1d expression varies in healthy and diseased bile ducts. Murine and human cholangiocyte cell lines as well as human primary cholangiocytes expressed CD1d as determined by flow cytometry and western blotting. Murine cholangiocyte cell lines were able to present both exogenous and endogenous lipid antigens to invariant and noninvariant NKT cell hybridomas and primary NKT cells in a CD1d-dependent manner. A human cholangiocyte cell line, cholangiocarcinoma cell lines, and human primary cholangiocytes also presented exogenous CD1d-restricted antigens to invariant NKT cell clones. CD1d expression was down-regulated in the biliary epithelium of patients with late primary sclerosing cholangitis, primary biliary cirrhosis, and alcoholic cirrhosis compared to healthy controls., Conclusions: Cholangiocytes express CD1d and present antigens to NKT cells and CD1d expression is down-regulated in diseased biliary epithelium, findings which show that the biliary epithelium can activate an important lymphocyte subset of the liver. This is a potentially important immune pathway in the biliary system, which may be capable of regulating inflammation in the context of biliary disease., (© 2015 by the American Association for the Study of Liver Diseases.)

The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening.

Aim and Background: The Nordic Liver Transplant Registry (NLTR) accounts for all liver transplants performed in the Nordic countries since the start of the transplant program in 1982. Due to short waiting times, donor liver allocation has been made without considerations of the model of end-stage liver disease (MELD) score. We aimed to summarize key outcome measures and developments for the activity up to December 2013., Materials and Methods: The registry is integrated with the operational waiting-list and liver allocation system of Scandiatransplant (www.scandiatransplant.org) and accounted at the end of 2013 for 6019 patients out of whom 5198 were transplanted. Data for recipient and donor characteristics and relevant end-points retransplantation and death are manually curated on an annual basis to allow for statistical analysis and the annual report., Results: Primary sclerosing cholangitis, acute hepatic failure, alcoholic liver disease, primary biliary cirrhosis and hepatocellular carcinoma are the five most frequent diagnoses (accounting for 15.3%, 10.8%, 10.6%, 9.3% and 9.0% of all transplants, respectively). Median waiting time for non-urgent liver transplantation during the last 10-year period was 39 days. Outcome has improved over time, and for patients transplanted during 2004-2013, overall one-, five- and 10-year survival rates were 91%, 80% and 71%, respectively. In an intention-to-treat analysis, corresponding numbers during the same time period were 87%, 75% and 66%, respectively., Conclusion: The liver transplant program in the Nordic countries provides comparable outcomes to programs with a MELD-based donor liver allocation system. Unique features comprise the diagnostic spectrum, waiting times and the availability of an integrated waiting list and transplant registry (NLTR).

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.

Objective: The molecular adsorbent recirculating system (MARS) is used to purify blood from albumin-bound toxins in patients with liver failure. However, the application of MARS has not demonstrated improved survival in randomized clinical trials and the clinical utility has not been finally established. In our department, the use of MARS is now restricted to the most critically ill patients with acute or acute on chronic liver failure., Material and Methods: Since 2005, we have treated 69 patients (30 males/39 females with median age of 49 years ranging from 1 months to 70 years) listed for liver transplantation (LT) with MARS. Median model of end-stage liver disease score in patients older than 12 years of age (n = 56) was 33 (interquartile range 26-39). The flow rate was 35-40 mL/kg/h and treatment kits were changed every 8-12 h. The patients were treated for a median of 27 h (range 1-144 h)., Results: Fifty-six patients (81%) were transplanted. Nine died before they could be transplanted, and four patients recovered without transplantation. Forty-six (82%) of the transplanted patients were alive 30 days after transplantation. Ammonium decreased modestly from a median of 148 to 124 µM (p = 0.03) during MARS treatment. We detected worsening of coagulopathy with significant decreases in platelet count and fibrinogen concentrations, and increase in International Normalized Ratio. Phosphate and magnesium decreased significantly during MARS treatment., Conclusion: Continuous MARS therapy may bridge liver failure patients to LT under close observation and treatment of coagulopathy and electrolyte disturbances.

Background: Genetic variants within the major histocompatibility complex (MHC) represent the strongest genetic susceptibility factors for primary sclerosing cholangitis (PSC). Identifying the causal variants within this genetic complex represents a major challenge due to strong linkage disequilibrium and an overall high physical density of candidate variants. We aimed to refine the MHC association in a geographically restricted PSC patient panel., Methodology/principal Findings: A total of 365 PSC cases and 368 healthy controls of Scandinavian ancestry were included in the study. We incorporated data from HLA typing (HLA-A, -B, -C, -DRB3, -DRB1, -DQB1) and single nucleotide polymorphisms across the MHC (n = 18,644; genotyped and imputed) alongside previously suggested PSC risk determinants in the MHC, i.e. amino acid variation of DRβ, a MICA microsatellite polymorphism and HLA-C and HLA-B according to their ligand properties for killer immunoglobulin-like receptors. Breakdowns of the association signal by unconditional and conditional logistic regression analyses demarcated multiple PSC associated MHC haplotypes, and for eight of these classical HLA class I and II alleles represented the strongest association. A novel independent risk locus was detected near NOTCH4 in the HLA class III region, tagged by rs116212904 (odds ratio [95% confidence interval] = 2.32 [1.80, 3.00], P = 1.35×10-11)., Conclusions/significance: Our study shows that classical HLA class I and II alleles, predominantly at HLA-B and HLA-DRB1, are the main risk factors for PSC in the MHC. In addition, the present assessments demonstrated for the first time an association near NOTCH4 in the HLA class III region.

Background and Aims: Curative treatment of hepatocellular carcinoma (HCC) is dependent on early diagnosis. Surveillance of patients at high risk for HCC is a key determinant to achieve this goal, but may be an underutilized tool. The aim of this study was to determine the rate of pre-diagnosis surveillance in patients with HCC in a large population-based cohort and to assess to what extent cirrhosis was known prior to the diagnosis of HCC., Methods: All patients diagnosed with HCC during 2000-2009 in The South-Eastern Regional Health Authority, representing 56% of the Norwegian population, were identified from The National Cancer Registry and the medical records were reviewed., Results: Fifteen out of 486 patients (3%) were diagnosed by surveillance. Potential curative treatment was offered to 58% of the patients who underwent surveillance as opposed to 15% in the non-surveillance group. Only age ≤ 65 years was an independent predictor of screening in a multivariate model. Almost two thirds of the patients with cirrhosis were unrecognized prior to the HCC diagnosis. Two hundred and fourteen patients (44%) were non-cirrhotics., Conclusion: Regular HCC surveillance in at-risk populations is virtually not applied in Norway and this may contribute to inferior overall survival. Failure to recognize cirrhosis and a high rate of HCC in non-cirrhotic patients will be limiting factors for the overall effectiveness of a potential surveillance program.

Background & Aims: Small duct primary sclerosing cholangitis (PSC) is phenotypically a mild version of large duct PSC, but it is unknown whether these phenotypes share aetiology. We aimed to characterize their relationship by investigating genetic associations in the human leucocyte antigen (HLA) complex, which represent the strongest genetic risk factors in large duct PSC., Methods: Four classical HLA loci (HLA-A, HLA-B, HLA-C and HLA-DRB1) were genotyped in 87 small duct PSC patients, 485 large duct PSC patients and 1117 controls across three geographical regions., Results: HLA-DRB1*13:01 (OR = 2.0, 95% CI 1.2-3.4, P = 0.01) and HLA-B*08 (OR = 1.6, 95% CI 1.1-2.4, P = 0.02) were significantly associated with small duct PSC compared with healthy controls. Based on the observed frequency of HLA-B*08 in small duct PSC, the strongest risk factor in large duct PSC, an estimated 32% (95% CI 4-65%) of this population can be hypothesized to represent early stages or mild variants of large duct PSC. This subgroup may be constituted by small duct PSC patients with inflammatory bowel disease (IBD), which greatly resembled large duct PSC in its HLA association. In contrast, small duct PSC without IBD was only associated with HLA-DRB1*13:01(P = 0.03) and was otherwise distinctly dissimilar from large duct PSC., Conclusions: Small duct PSC with IBD resembles large duct PSC in its HLA association and may represent early stages or mild variants of large duct disease. Different HLA associations in small duct PSC without IBD could indicate that this subgroup is a different entity. HLA-DRB1*13:01 may represent a specific risk factor for inflammatory bile duct disease., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Primary sclerosing cholangitis (PSC) is a chronic cholangiopathy characterized by biliary fibrosis, development of cholestasis and end stage liver disease, high risk of malignancy, and frequent need for liver transplantation. The poor understanding of its pathogenesis is also reflected in the lack of effective medical treatment. Well-characterized animal models are utterly needed to develop novel pathogenetic concepts and study new treatment strategies. Currently there is no consensus on how to evaluate and characterize potential PSC models, which makes direct comparison of experimental results and effective exchange of study material between research groups difficult. The International Primary Sclerosing Cholangitis Study Group (IPSCSG) has therefore summarized these key issues in a position paper proposing standard requirements for the study of animal models of PSC., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Purpose of Review: The pathogenesis of primary sclerosing cholangitis (PSC) involves heritable factors. This review summarizes the recent genetic studies and discusses the implications of identified risk loci., Recent Findings: A total of 16 PSC susceptibility loci have been identified in genome-wide association studies and related study designs. At least 33 additional loci are involved in what is increasingly acknowledged to represent a general pool of genetic risk loci for immune-mediated diseases. One important group of genes is part of well characterized immune pathways (e.g. interleukin 2 signaling), whereas for other loci the relationship to PSC pathophysiology is less evident. Importantly, the loci collectively account for only 7.3% of overall PSC liability, thus pointing to a large contribution from environmental factors to PSC development. The individual PSC risk genes cannot be interpreted within a simple cause-effect model used for monogenic traits, but need to be explored for their individual biological correlates, preferably in a disease context. To some extent, as exemplified for the human leukocyte antigen and FUT2 associations, genetic findings may guide the discovery of interacting and co-occuring environmental susceptibility factors., Summary: Multiple PSC susceptibility loci are now available for exploration in experimental model systems and patient-centered research.