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Supplementary Methods

Supplementary Figures 1-11. Supplementary Figure 1. DNA-PK expression and tumour specific PRKDC locus amplification Supplementary Figure 2. DDR gene expression in HCC cells Supplementary Figure 3: DDR protein expression and activity in HCC cells Supplementary Figure 4: Effects of DNA-PKcs knockdown on NHEJ activity Supplementary Figure 5: Clonogenic survival of HCC cells exposed to IR- and doxorubicin-induced cytotoxicity in HCC cells Supplementary Figure 6: DNA-PK inhibition potentiated IR- and doxorubicin-induced cytotoxicity in HCC cells Supplementary Figure 7: ATM inhibition potentiated IR- and doxorubicin-induced cytotoxicity in the majority of HCC cells Supplementary Figure 8: Combination DNA-PK and ATM inhibition suppressed clonogenic survival in the absence of cyctotoxic injury in HCC cells Supplementary Figure 9: Combined ATM and DNA-PK inhibition further potentiated IR and doxorubicin cytotoxicity in HCC cells Supplementary Figure 10: Neither NU7441 nor KU55933 effect nuclear doxorubicin accumulation Supplementary Figure 11: In vivo effects of NU7441 and doxorubicin treatment

Data Supplement from DNA-PK—A Candidate Driver of Hepatocarcinogenesis and Tissue Biomarker That Predicts Response to Treatment and Survival

Purpose: Therapy resistance and associated liver disease make hepatocellular carcinomas (HCC) difficult to treat with traditional cytotoxic therapies, whereas newer targeted approaches offer only modest survival benefit. We focused on DNA-dependent protein kinase, DNA-PKcs, encoded by PRKDC and central to DNA damage repair by nonhomologous end joining. Our aim was to explore its roles in hepatocarcinogenesis and as a novel therapeutic candidate.Experimental Design: PRKDC was characterized in liver tissues from of 132 patients [normal liver (n = 10), cirrhotic liver (n = 13), dysplastic nodules (n = 18), HCC (n = 91)] using Affymetrix U133 Plus 2.0 and 500 K Human Mapping SNP arrays (cohort 1). In addition, we studied a case series of 45 patients with HCC undergoing diagnostic biopsy (cohort 2). Histological grading, response to treatment, and survival were correlated with DNA-PKcs quantified immunohistochemically. Parallel in vitro studies determined the impact of DNA-PK on DNA repair and response to cytotoxic therapy.Results: Increased PRKDC expression in HCC was associated with amplification of its genetic locus in cohort 1. In cohort 2, elevated DNA-PKcs identified patients with treatment-resistant HCC, progressing at a median of 4.5 months compared with 16.9 months, whereas elevation of activated pDNA-PK independently predicted poorer survival. DNA-PKcs was high in HCC cell lines, where its inhibition with NU7441 potentiated irradiation and doxorubicin-induced cytotoxicity, whereas the combination suppressed HCC growth in vitro and in vivo.Conclusions: These data identify PRKDC/DNA-PKcs as a candidate driver of hepatocarcinogenesis, whose biopsy characterization at diagnosis may impact stratification of current therapies, and whose specific future targeting may overcome resistance. Clin Cancer Res; 21(4); 925–33. ©2014 AACR.

Supplementary Tables 1-2. Supplementary Table 1. Clinical features of the subset of patients receiving palliative TACE as first line therapy Supplementary Table 2 - Tumour Factors associated with survival in cohort two

Supplementary Figure Legends

A 39-year-old female presented with a one-week history of jaundice and nausea after taking an over-the-counter herbal supplement containing ashwagandha root extract. Initial investigations revealed a hepatocellular pattern of liver enzyme abnormality with jaundice. Investigations, including viral serology, liver specific autoantibodies and an ultrasound scan of the abdomen, were unremarkable. Liver biopsy showed an acute cholestatic hepatitis with confluent necrosis but no features of chronicity. These histopathological findings differ to that of a previously reported case. Review of recent literature revealed that some clinical features and the time course of liver injury were similar to previous reports of ashwagandha drug-induced liver injury (DILI). The patient received treatment with ursodeoxycholic acid. We compare this case to previous reported cases of ashwagandha DILI and discuss the biochemical and histopathological features of ashwagandha DILI, therapeutic strategies and the importance of recognising herbal supplements as a possible cause of DILI.

Professional societies play a major role in medicine and science. The societies tend to be large with well-developed administrative structures. An additional model, however, is based on small groups of experts who meet regularly in an egalitarian model in order to discuss disease-specific scientific and medical problems. In order to illustrate the effectiveness of this model, the history and practices are examined of a long-standing successful example, the International Liver Pathology Group, better known as the Gnomes. The history shows that groups such as the Gnomes offer a number of important benefits not available in larger societies and nurturing such groups advances science and medicine in meaningful ways. The success of the Gnomes’ approach provides a road map for future small scientific groups. Electronic supplementary material The online version of this article (10.1007/s00428-020-02879-5) contains supplementary material, which is available to authorized users.

[Objective] The full phenotypic expression of non-alcoholic fatty liver disease (NAFLD) in lean subjects is incompletely characterised. We aimed to investigate prevalence, characteristics and long-term prognosis of Caucasian lean subjects with NAFLD., [Design] The study cohort comprises 1339 biopsy-proven NAFLD subjects from four countries (Italy, UK, Spain and Australia), stratified into lean and non-lean (body mass index (BMI), [Results] Lean patients represented 14.4% of the cohort and were predominantly of Italian origin (89%). They had less severe histological disease (lean vs non-lean: non-alcoholic steatohepatitis 54.1% vs 71.2% p10 483 person-years), 4.7% of lean vs 7.7% of non-lean patients reported liver-related events (p=0.37). No difference in survival was observed compared with non-lean NAFLD (p=0.069)., [Conclusions] Caucasian lean subjects with NAFLD may progress to advanced liver disease, develop metabolic comorbidities and experience cardiovascular disease (CVD) as well as liver-related mortality, independent of longitudinal progression to obesity and PNPLA3 genotype. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD where the disease manifests at lower overall BMI thresholds. [Lay summary] NAFLD may affect and progress in both obese and lean individuals. Lean subjects are predominantly males, have a younger age at diagnosis and are more prevalent in some geographic areas. During the follow-up, lean subjects can develop hepatic and extrahepatic disease, including metabolic comorbidities, in the absence of weight gain. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD., This study has been supported by the EPoS (Elucidating Pathways of Steatohepatitis) consortium funded by the Horizon 2020 Framework Program of the European Union under Grant Agreement 634413 and the Newcastle NIHR Biomedical Research Centre. The authors are contributing members of The European NAFLD Registry. The study was also supported by the Italian Ministry of Health, grant RF-2016-02364358 (Ricerca Finalizzata, Ministero della Salute). ME and JG are supported by the Robert W. Storr Bequest to the Sydney Medical Foundation, University of Sydney; a National Health and Medical Research Council of Australia (NHMRC) Program Grant (APP1053206, APP1149976) and Project grants (APP1107178 and APP1108422).

Background and Aim Endoscopic submucosal dissection (ESD) is a challenging procedure. A dissection speed of ≥9 cm2/h has been acknowledged as a mark for expertise, alongside a complication rate of ≤5% and en bloc resection rate of ≥90%. However, there is lack of objective information on whether the three measures correlate with each other. This study aims to evaluate the dissection speed, safety, and efficacy of colorectal ESDs performed by experts and trainees. Methods Consecutive patients undergoing colorectal ESD at a Japanese hospital (2006–2017) were included in a prospectively collected database. Information on patient demographics, proceduralist, and intra‐/postprocedure data was retrieved. The primary outcome was the comparison in dissection speed. The secondary outcomes included differences in safety and efficacy. Log‐linear regression models adjusted for confounders (e.g. R0 resection) were used to assess the differences in dissection speed. Results Five hundred ninety procedures (514 patients) performed by 26 endoscopists were analyzed. Experts performed a higher number of difficult lesions (e.g. F2 fibrosis) but achieved higher dissection speed (10.3 vs 6.7 cm2/h). The difference was statistically significant for both unadjusted and adjusted models (P, Endoscopic submucosal dissection (ESD) expertise is commonly measured through dissection speed, en bloc resection, and complication rates. However, it is unclear whether the slower pace during ESD training is necessarily accompanied by worst outcomes regarding efficacy and safety. This study found that colorectal ESD can be safely and efficaciously performed by ESD trainees.

The tumour microenvironment, also termed the tumour stroma or tumour mesenchyme, includes fibroblasts, immune cells, blood vessels and the extracellular matrix and substantially influences the initiation, growth and dissemination of gastrointestinal cancer. Cancer-associated fibroblasts (CAFs) are one of the critical components of the tumour mesenchyme and not only provide physical support for epithelial cells but also are key functional regulators in cancer, promoting and retarding tumorigenesis in a context-dependent manner. In this Review, we outline the emerging understanding of gastrointestinal CAFs with a particular emphasis on their origin and heterogeneity, as well as their function in cancer cell proliferation, tumour immunity, angiogenesis, extracellular matrix remodelling and drug resistance. Moreover, we discuss the clinical implications of CAFs as biomarkers and potential targets for prevention and treatment of patients with gastrointestinal cancer., ファイル公開：2019-11-01

Background Alcohol is the main cause of chronic liver disease. The Enhanced Liver Fibrosis (ELF) test is a serological biomarker for fibrosis staging in chronic liver disease, however its utility in alcohol-related liver disease warrants further validation. We assessed the diagnostic and prognostic performance of ELF in alcohol-related liver disease. Methods Observational cohort study assessing paired ELF and histology from 786 tertiary care patients with chronic liver disease due to alcohol (n = 81) and non-alcohol aetiologies (n = 705). Prognostic data were available for 64 alcohol patients for a median of 6.4 years. Multiple ELF cut-offs were assessed to determine diagnostic utility in moderate fibrosis and cirrhosis. Survival data were assessed to determine the ability of ELF to predict liver related events and all-cause mortality. Results ELF identified cirrhosis and moderate fibrosis in alcohol-related liver disease independently of aminotransferase levels with areas under receiver operating characteristic curves of 0.895 (95% CI 0.823–0.968) and 0.923 (95% CI 0.866–0.981) respectively, which were non-inferior to non-alcohol aetiologies. The overall performance of ELF was assessed using the Obuchowski method: in alcohol = 0.934 (95% CI 0.908–0.960); non-alcohol = 0.907 (95% CI 0.895–0.919). Using ELF Conclusions ELF accurately stages liver fibrosis independently of transaminase elevations as a marker of inflammation and has superior prognostic performance to biopsy in alcohol-related liver disease.

Background & Aims: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. Methods: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting–purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. Results: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor β was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB–IL34/CCL8 signaling that promotes macrophage chemotaxis. Conclusions: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.

[Background & Aims] Non-invasive scoring systems (NSS) are used to identify patients with non-alcoholic fatty liver disease (NAFLD) who are at risk of advanced fibrosis, but their reliability in predicting long-term outcomes for hepatic/extrahepatic complications or death and their concordance in cross-sectional and longitudinal risk stratification remain uncertain., [Methods] The most common NSS (NFS, FIB-4, BARD, APRI) and the Hepamet fibrosis score (HFS) were assessed in 1,173 European patients with NAFLD from tertiary centres. Performance for fibrosis risk stratification and for the prediction of long-term hepatic/extrahepatic events, hepatocarcinoma (HCC) and overall mortality were evaluated in terms of AUC and Harrell’s c-index. For longitudinal data, NSS-based Cox proportional hazard models were trained on the whole cohort with repeated 5-fold cross-validation, sampling for testing from the 607 patients with all NSS available., [Results] Cross-sectional analysis revealed HFS as the best performer for the identification of significant (F0-1 vs. F2-4, AUC = 0.758) and advanced (F0-2 vs. F3-4, AUC = 0.805) fibrosis, while NFS and FIB-4 showed the best performance for detecting histological cirrhosis (range AUCs 0.85-0.88). Considering longitudinal data (follow-up between 62 and 110 months), NFS and FIB-4 were the best at predicting liver-related events (c-indices>0.7), NFS for HCC (c-index = 0.9 on average), and FIB-4 and HFS for overall mortality (c-indices >0.8). All NSS showed limited performance (c-indices, [Conclusions] Overall, NFS, HFS and FIB-4 outperformed APRI and BARD for both cross-sectional identification of fibrosis and prediction of long-term outcomes, confirming that they are useful tools for the clinical management of patients with NAFLD at increased risk of fibrosis and liver-related complications or death., [Lay summary] Non-invasive scoring systems are increasingly being used in patients with non-alcoholic fatty liver disease to identify those at risk of advanced fibrosis and hence clinical complications. Herein, we compared various non-invasive scoring systems and identified those that were best at identifying risk, as well as those that were best for the prediction of long-term outcomes, such as liver-related events, liver cancer and death., This study has been supported by the EPoS (Elucidating Pathways of Steatohepatitis) consortium funded by the Horizon 2020 Framework Program of the European Union under Grant Agreement 634413 and the Newcastle NIHR Biomedical Research Centre. The authors are contributing members of The European NAFLD Registry. The study was also supported by the Italian Ministry of Health, grant RF-2016-02364358 (Ricerca Finalizzata, Ministero della Salute), and the Italian Ministry for Education, University and Research (Ministero dell’Istruzione, dell’Università e della Ricerca - MIUR) under the programme “Dipartimenti di Eccellenza 2018 – 2022” Project code D15D18000410001.

Background & Aims Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. Methods Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. Results We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor β and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)–mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. Conclusions Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

Introduction: Quality measures for colonoscopy such as adenoma detection rate (ADR) have been proposed to be surveilled for ensuring minimum standards. However, its direct measurement is time consuming and often neglected. Extrapolating ADR and other quality measures from polyp detection rate (PDR) can be a pragmatic alternative. Objective: To determine quotients for estimating ADR and sessile serrated adenoma/polyp detection rate (SSA/P-DR) from PDR in an Australian cohort. Methods: Consecutive adult patient colonoscopies during a 1-year period were retrospectively assessed in a single Australian tertiary endoscopy center. Adenoma detection quotient (ADQ) and SSA/P detection quotient (SSA/P-DQ) were defined as the division of ADR and SSA/P-DR by PDR, respectively. The primary outcome was the number of procedures to achieve a stable cumulative ADQ and SSA/P-DQ. Secondary outcomes included evaluation of ADQ and SSA/P-DQ in different subsets. Results: In total, 2,657 colonoscopies were performed by 15 endoscopists in 2016. The ADR, SSA/P-DR, and PDR found were 32.2, 6.7, and 47.3%, respectively. The ADQ and SSA/P-DQ values found were 0.68 and 0.14, respectively. After approximately 500 procedures, both ADQ and SSA/P-DQ became stable. Interclass correlation coefficient (ICC) for the prediction of ADR from ADQ was excellent for all endoscopists that performed >177 procedures in that year (ICC 0.84). Conclusions: ADQ and SSA/P-DQ values were consistent when over 500 procedures were analyzed. ADQ had an excellent correlation with ADR when >177 procedures per endoscopist were evaluated.

Introduction We investigated the longitudinal impact of antinuclear antibody (ANA) on clinical outcomes and survival in nonalcoholic fatty liver disease (NAFLD). Methods ANA were found in 16.9% of 923 biopsy-proven NAFLD patients, but none of them had histologic autoimmune hepatitis (AIH) or developed AIH after a mean follow-up of 106±50 months. Results Although ANA-positive cases had a higher prevalence of nonalcoholic steatohepatitis at baseline, the occurrence of liver-related events, hepatocellula carcinoma, cardiovascular events, extrahepatic malignancy, and overall survival were similar to ANA-negative. Discussion Once AIH has been ruled out, the long-term outcomes and survival are unaffected by the presence of ANA in patients with NAFLD.

Background & Aims: Genetic factors associated with nonalcoholic fatty liver disease (NAFLD) remain incompletely understood. To date, most genome-wide association studies (GWASs) have adopted radiologically assessed hepatic triglyceride content as the reference phenotype and so cannot address steatohepatitis or fibrosis. We describe a GWAS encompassing the full spectrum of histologically characterised NAFLD. Methods: The GWAS involved 1,483 European NAFLD cases and 17,781 genetically matched controls. A replication cohort of 559 NAFLD cases and 945 controls was genotyped to confirm signals showing genome-wide or close to genome-wide significance. Results: Case-control analysis identified signals showing p values = F3), the signals on chr2, chr19 and chr22 maintained their genome-wide significance. Except for GCKR/C2ORF16, the genome-wide significance signals were replicated. Conclusions: This study confirms PNPLA3 as a risk factor for the full histological spectrum of NAFLD at genome-wide significance levels, with important contributions from TM6SF2 and HSD17B13. PYGO1 is a novel steatosis modifier, suggesting that Wnt signalling pathways may be relevant in NAFLD pathogenesis. Lay summary: Non-alcoholic fatty liver disease is a common disease where excessive fat accumulates in the liver and may result in cirrhosis. To understand who is at risk of developing this disease and suffering liver damage, we undertook a genetic study to compare the genetic profiles of people suffering from fatty liver disease with genetic profiles seen in the general population. We found that particular sequences in 4 different areas of the human genome were seen at different frequencies in the fatty liver disease cases. These sequences may help predict an individuals risk of developing advanced disease. Some genes where these sequences are located may also be good targets for future drug treatments. (C) 2020 European Association for the Study of the Liver. Published by Elsevier B.V. Funding Agencies|EPoS (Elucidating Pathways of Steatohepatitis) consortium - Horizon 2020 Framework Program of the European Union [634413]; FLIP consortium(EuropeanUnion FP7 grant) [241762]; Newcastle NIHR Biomedical Research Centre

OBJECTIVES Non-invasive fibrosis scores are widely used to identify/exclude advanced fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). However, these scores were principally developed and validated in patients aged between 35 and 65 years of age. The objective of this study was to assess the effect of age on the performance of non-invasive fibrosis tests in NAFLD. METHODS Patients were recruited from European specialist hepatology clinics. The cohort was divided into five age-based groups: ≤35 (n=74), 36-45 (n=96), 46-55 (n=197), 56-64 (n=191), and ≥65 years (n=76), and the performance of the aspartate aminotransferase (AST)/alanine transaminase (ALT) ratio, fibrosis 4 (FIB-4), and NAFLD fibrosis score (NFS) for advanced fibrosis (stage F3-F4) for each group was assessed using liver biopsy as the standard. RESULTS Six hundred and thirty-four patients were included. The diagnostic accuracy of the AST/ALT ratio was lower than NFS and FIB-4 in all the age groups. The AST/ALT ratio, NFS, and FIB-4 score performed poorly for a diagnosis of advanced fibrosis in those aged ≤35 years (area under the receiver operating characteristic curves (AUROCs 0.52, 0.52, and 0.60, respectively). For all groups >35 years, AUROCs for advanced fibrosis were similar for the NFS and FIB-4 score (range 0.77-0.84). However, the specificity for advanced fibrosis using the FIB-4 and NFS declined with age, becoming unacceptably low in those aged ≥65 years (35% for FIB-4 and 20% for NFS). New cutoffs were derived (and validated) for those aged ≥65 years, which improved specificity to 70% without adversely affecting sensitivity (FIB-4 2.0, sensitivity 77%; NFS 0.12, sensitivity 80%). CONCLUSIONS The NFS and FIB-4 scores have similar accuracy for advanced fibrosis in patients aged >35 years. However, the specificity for advanced fibrosis is unacceptably low in patients aged ≥65 years, resulting in a high false positive rate. New thresholds for use in patients aged ≥65 years are proposed to address this issue.Am J Gastroenterol advance online publication, 11 October 2016; doi:10.1038/ajg.2016.453.

It is currently known that colorectal cancers (CRC) arise from 3 different pathways: the adenoma to carcinoma chromosomal instability pathway (50%-70%); the mutator "Lynch syndrome" route (3%-5%); and the serrated pathway (30%-35%). The World Health Organization has classified serrated polyps into three types of lesions: hyperplastic polyps (HP), sessile serrated adenomas/polyps (SSA/P) and traditional serrated adenomas (TSA), the latter two strongly associated with development of CRCs. HPs do not cause cancer and TSAs are rare. SSA/P appear to be the responsible precursor lesion for the development of cancers through the serrated pathway. Both HPs and SSA/Ps appear morphologically similar. SSA/P are difficult to detect. The margins are normally inconspicuous. En bloc resection of these polyps can hence be troublesome. A careful examination of borders, submucosal injection of a dye solution (for larger lesions) and resection of a rim of normal tissue around the lesion may ensure total eradication of these lesions.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fifth most common cause of cancer death in the UK. Its poor prognosis is attributed to late detection and limited therapeutic options. Expression of SULF2, an endosulfatase that modulates heparan sulfate proteoglycan 6-O-sulfation and is reportedly tumourigenic in different types of cancer, was investigated. Methods: SULF2 expression was determined immunohistochemically in archival surgical resection tissue sections from 93 patients with a confirmed histological diagnosis of PDAC between 2002 and 2008 followed for a median of 9 years. Relationships with clinico-pathological parameters and patient survival were explored. Results: The majority of PDACs showed positive SULF2 staining in tumour cells and intratumoural or tumour-adjacent stroma. Greater than 25% SULF2-positive tumour cells was present in 60% of cancers and correlated with tumour stage (P=0.002) and perineural invasion (P=0.024). SULF2 intensity was scored moderate or strong in 81% of cancers and positively correlated with vascular invasion (P=0.015). High SULF2 expression, defined as >50% SULF2-positive tumour cells and strong SULF2 staining, was associated with shorter time to radiological progression (P=0.018, HR 1.98, CI 1.13–3.47). Similarly, by multivariate analysis, high SULF2 expression was independently associated with poorer survival (P=0.004, HR 2.10, CI 1.26–3.54), with a median survival of 11 months vs 21 months for lower PDAC SULF2. Conclusions: Elevated SULF2 in PDAC was associated with advanced tumour stage, vascular invasion, shorter interval to radiological progression and shorter overall survival. SULF2 may have roles as a prognostic biomarker and as a therapeutic target for patients with PDAC.

Obesity and non-alcoholic fatty liver disease (NAFLD) are contributing to the global rise in deaths from hepatocellular carcinoma (HCC). The pathogenesis of NAFLD-HCC is not well understood. The severity of hepatic steatosis, steatohepatitis and fibrosis are key pathogenic mechanisms, but animal studies suggest altered immune responses are also involved. Genetic studies have so far highlighted a major role of gene variants promoting fat deposition in the liver (PNPLA3 rs738409, TM6SF2 rs58542926). Here, we have considered single-nucleotide polymorphisms (SNPs) in candidate immunoregulatory genes (MICA rs2596542, CD44 rs187115, PDCD1 rs7421861 and rs10204525), in 594 patients with NAFLD and 391 with NAFLD-HCC, from three European centres. Associations between age, body mass index, diabetes, cirrhosis and SNPs with HCC development were explored. PNPLA3 and TM6SF2 SNPs were associated with both progression to cirrhosis and NAFLD-HCC development, while PDCD1 SNPs were specifically associated with NAFLD-HCC risk, regardless of cirrhosis. PDCD1 rs7421861 was independently associated with NAFLD-HCC development, while PDCD1 rs10204525 acquired significance after adjusting for other risks, being most notable in the smaller numbers of women with NAFLD-HCC. The study highlights the potential impact of inter individual variation in immune tolerance induction in patients with NAFLD, both in the presence and absence of cirrhosis.

Non-alcoholic fatty liver disease (NAFLD) is often associated with obesity and type 2 diabetes. To disentangle etiological relationships between these conditions and identify genetically-determined metabolites involved in NAFLD processes, we mapped 1H nuclear magnetic resonance (NMR) metabolomic and disease-related phenotypes in a mouse F2 cross derived from strains showing resistance (BALB/c) and increased susceptibility (129S6) to these diseases. Quantitative trait locus (QTL) analysis based on single nucleotide polymorphism (SNP) genotypes identified diet responsive QTLs in F2 mice fed control or high fat diet (HFD). In HFD fed F2 mice we mapped on chromosome 18 a QTL regulating liver micro- and macrovesicular steatosis and inflammation, independently from glucose intolerance and adiposity, which was linked to chromosome 4. Linkage analysis of liver metabolomic profiling data identified a QTL for octopamine, which co-localised with the QTL for liver histopathology in the cross. Functional relationship between these two QTLs was validated in vivo in mice chronically treated with octopamine, which exhibited reduction in liver histopathology and metabolic benefits, underlining its role as a mechanistic biomarker of fatty liver with potential therapeutic applications.

Surveillance post-endoscopic resection (ER) currently warrants biopsy samples from the resection site scar in most cases, although clinical practice is variable. A classification with standard criteria for scars has not yet been established. We aimed to create and validate a novel classification for post-ER scars by using specific criteria based on advanced imaging.Key endoscopic features for scars with and without recurrence were (1) dark brown color, elongated/branched pit pattern, and dense capillary pattern and (2) whitish, pale appearance, round/slightly large pits, and irregular sparse vessels. Scars were first assessed with high-definition white-light endoscopy (HD-WLE) followed by interrogation with narrow-band imaging (NBI). Scars with at least 2 concordant characteristics were diagnosed with "high confidence" for NBI for scar (NBI-SCAR) classification. The final endoscopic predictions were correlated with histopathology. The primary outcome was the difference in sensitivity between NBI-SCAR and HD-WLE predictions. Secondary outcomes included the validation of our findings in 6 different endoscopy settings (Australia, United States, Japan, Brazil, Singapore, and Malaysia). The validation took place in 2 sessions separated by 2 to 3 weeks, each with 10 one-minute videos of post-ER scars on underwater NBI with dual focus. Inter-rater and intrarater reliability were calculated with Fleiss' free-marginal kappa and Bennett et al. S score, respectively.One hundred scars from 82 patients were included. Ninety-five scars were accurately predicted with high confidence by NBI-SCAR in the exploratory phase. NBI-SCAR sensitivity was significantly higher compared with HD-WLE (100% vs 73.7%, P .05). In the validation phase, similar results were found for endoscopists who routinely perform colonoscopies and use NBI (sensitivity of 96.4%). The inter-rater and intrarater reliability throughout all centers were, respectively, substantial (κ = .61) and moderate (average S = .52) for this subset.NBI-SCAR has a high sensitivity and negative predictive value for excluding recurrence for endoscopists experienced in colonoscopy and NBI. In this setting, this approach may help to accurately evaluate or resect scars and potentially mitigate the burden of unnecessary biopsy samples.

In this Article, the sentence: "After 7 months of HFD, MUP-uPA mice developed HCC15, which contained numerous (usually 50-100 per tumour) non-recurrent coding mutations in pathways that are mutated in human HCC (Fig. 2d and Extended Data Fig. 6a).", should have read: "After 7 months of HFD, MUP-uPA mice developed HCC15, which contained numerous (usually 50-100 per tumour) non-recurrent mutations in pathways that are mutated in human HCC (Fig. 2d and Extended Data Fig. 6a).". This has been corrected online. In Extended Data Fig. 6a and b, which show the number of point mutations identified per sample and the mutational signatures, all sequence variants (including non-coding mutations) are shown. Fig. 2d also presents all variants compared to human mutations. In the Supplementary Information to this Amendment, we now provide the comparisons of all variants and coding variants to human mutations.

Nonalcoholic fatty liver disease (NAFLD) comprises a spectrum of clinical and histopathological changes including "simple" steatosis, steatosis with inflammation, steatohepatitis, cirrhosis, and hepatocellular carcinoma. It was initially described in the context of drug-induced liver injury and acute liver disease following jejunoileal bypass surgery, but since the early 1980s it has been widely acknowledged as the hepatic manifestation of metabolic syndrome. It now represents a burgeoning public health crisis and is fast becoming the main indication for liver transplantation in some parts of the world. Its true incidence and prevalence are unknown, although estimates have been made from large imaging studies. Liver biopsy interpretation is still regarded as the gold standard for making accurate diagnoses in NAFLD, but sampling limitations are recognized. Furthermore, clear definitions for key histopathological components have been lacking, resulting in significant interobserver variations in making a diagnosis of steatohepatitis. In this review the authors consider some aspects of classification and variant forms of NAFLD such as that occurring in children. They provide an update on grading and staging systems and histopathological prognostic factors, and address the role of liver biopsy in contemporary clinical care of patients with NAFLD.

Background and Aims There remains uncertainty about the natural history of non-alcoholic fatty liver disease (NAFLD). The spectrum of NAFLD includes non-alcoholic fatty liver (NAFL; steatosis without hepatocellular injury) and steatohepatitis (NASH; steatosis with hepatocyte ballooning degeneration±fibrosis). Our aim was to assess the histological severity of NAFLD in a cohort with serial biopsy data, and determine factors predicting progression. Methods Patients with two liver biopsies more than a year apart were identified. Clinical and laboratory data were collected from the time of liver biopsy. Results 108 patients had serial biopsies (median interval 6.6years, range 1.3–22.6). 81 (75%) patients had NASH and 27 had NAFL. Overall, 45 (42%) patients had fibrosis progression, 43 (40%) had no change in fibrosis, while 20 (18%) had fibrosis regression. Importantly, no significant difference in the proportion exhibiting fibrosis progression was found between those with NAFL or NASH at index biopsy (37% vs. 43%, p =0.65). Progression to NASH was seen in 44% of patients with baseline NAFL. Of 10 patients with NAFL who had fibrosis progression, 3 progressed by 1 stage, 5 by 2 stages and 2 by 3 stages; all had NASH on follow-up biopsy. Of concern, 6 of 27 (22%) patients with baseline NAFL, reached stage 3 fibrosis at follow-up biopsy. Among the patients with NAFL, 80% of those having fibrosis progression were diabetic at the follow-up liver biopsy compared with 25% of non-progressors ( p =0.005). Conclusions Contrary to current dogma, this study suggests that steatosis can progress to NASH and clinically significant fibrosis.

Optimal patient management benefits from comprehensive and accurate pathology reports that contribute to cancer staging and prognostication. Proforma reports are used in many countries, but these vary in their structure and implementation. The International Collaboration on Cancer Reporting (ICCR) is an alliance formed by the Royal College of Pathologists of Australasia, the Royal College of Pathologists of the United Kingdom, the College of American Pathologists, the Canadian Partnership Against Cancer the European Society of Pathology and the American Society of Clinical Pathology (ASCP), with the aim of developing an evidence-based reporting data set for each cancer site. It is argued that this should reduce the global burden of cancer data set development and reduplication of effort by different international institutions that commission, publish and maintain standardised cancer reporting data sets. The resultant standardisation of cancer reporting will benefit not only those countries directly involved in the collaboration but also others not in a position to develop their own data sets. We describe the development of a cancer data set by the ICCR expert panel for the reporting of the main malignant liver tumours: intrahepatic cholangiocarcinoma, perihilar cholangiocarcinoma and hepatocellular carcinoma and present the 'required' and 'recommended' elements to be included in the report with an explanatory commentary. This data set incorporates definitions and classifications in the most recent World Health Organisation (WHO) publication on hepatic malignancies (4th edition) and the recently published tumour-node-metastasis (TNM)8 staging system. Widespread adoption and implementation of this data set will enable consistent and accurate data collection, comparison of epidemiological and pathological parameters between different populations, facilitate research and ultimately result in better patient outcomes.

Purpose: Therapy resistance and associated liver disease make hepatocellular carcinomas (HCC) difficult to treat with traditional cytotoxic therapies, whereas newer targeted approaches offer only modest survival benefit. We focused on DNA-dependent protein kinase, DNA-PKcs, encoded by PRKDC and central to DNA damage repair by nonhomologous end joining. Our aim was to explore its roles in hepatocarcinogenesis and as a novel therapeutic candidate. Experimental Design: PRKDC was characterized in liver tissues from of 132 patients [normal liver (n = 10), cirrhotic liver (n = 13), dysplastic nodules (n = 18), HCC (n = 91)] using Affymetrix U133 Plus 2.0 and 500 K Human Mapping SNP arrays (cohort 1). In addition, we studied a case series of 45 patients with HCC undergoing diagnostic biopsy (cohort 2). Histological grading, response to treatment, and survival were correlated with DNA-PKcs quantified immunohistochemically. Parallel in vitro studies determined the impact of DNA-PK on DNA repair and response to cytotoxic therapy. Results: Increased PRKDC expression in HCC was associated with amplification of its genetic locus in cohort 1. In cohort 2, elevated DNA-PKcs identified patients with treatment-resistant HCC, progressing at a median of 4.5 months compared with 16.9 months, whereas elevation of activated pDNA-PK independently predicted poorer survival. DNA-PKcs was high in HCC cell lines, where its inhibition with NU7441 potentiated irradiation and doxorubicin-induced cytotoxicity, whereas the combination suppressed HCC growth in vitro and in vivo. Conclusions: These data identify PRKDC/DNA-PKcs as a candidate driver of hepatocarcinogenesis, whose biopsy characterization at diagnosis may impact stratification of current therapies, and whose specific future targeting may overcome resistance. Clin Cancer Res; 21(4); 925–33. ©2014 AACR.

Nature 551, 340–345 (2017); doi:10.1038/nature24302 In this Article, owing to an error during the production process, the text ‘(Iga is also known as Cd79a)’ was erroneously added to the sentence describing the generation of MUP-uPA/Iga−/− and STAM/Iga−/− mice; this should have stated ‘(Iga is also known as Igha)’.

The role of adaptive immunity in early cancer development is controversial. Here we show that chronic inflammation and fibrosis in humans and mice with non-alcoholic fatty liver disease is accompanied by accumulation of liver-resident immunoglobulin-A-producing (IgA+) cells. These cells also express programmed death ligand 1 (PD-L1) and interleukin-10, and directly suppress liver cytotoxic CD8+ T lymphocytes, which prevent emergence of hepatocellular carcinoma and express a limited repertoire of T-cell receptors against tumour-associated antigens. Whereas CD8+ T-cell ablation accelerates hepatocellular carcinoma, genetic or pharmacological interference with IgA+ cell generation attenuates liver carcinogenesis and induces cytotoxic T-lymphocyte-mediated regression of established hepatocellular carcinoma. These findings establish the importance of inflammation-induced suppression of cytotoxic CD8+ T-lymphocyte activation as a tumour-promoting mechanism.

A third of the population are estimated to have NAFLD of varying severity. Serum immunoglobulins are frequently elevated in patients with chronic liver disease, but little is known about serum immunoglobulin levels in patients with NAFLD. Aim of this study was to evaluate serum immunoglobulin levels (IgA, IgG, and IgM) in a large cohort of patients with biopsy-proven NAFLD and determine if immunoglobulin levels are associated with clinical or histological features.Patients seen in a tertiary fatty liver clinic between 1999 and 2009 were included. Liver biopsies were assessed using the Kleiner score. Immunoglobulin levels and other blood tests were taken at time of biopsy.285 patients (110 simple steatosis and 175 NASH) had serum immunoglobulins measured within 6months of liver biopsy. 130 (46%) patients had elevated (1× upper limit of normal) serum IgA levels, 28 (10%) patients had elevated IgG and 22 (8%) raised IgM. Serum IgA levels were elevated more frequently in patients with NASH compared with subjects with simple steatosis (55% vs. 31%, p0.001). Overall, 55 (19%) patients had advanced liver fibrosis (Kleiner stage 3-4). There was a significant positive association between serum IgA levels and the stage of fibrosis (p0.001). Serum IgA, age, platelets, AST/ALT ratio and BMI were all independently with advanced fibrosis following multivariate analysis. A model constructed from these independent predictors accurately predicted advanced fibrosis (AUROC 0.87).The serum IgA level was frequently elevated in patients with NAFLD and was an independent predictor of advanced fibrosis.

Bile duct ligation (BDL) is commonly employed as a model for hepatic periportal fibrosis. However, BDL is limited in that it is a severe procedure; is irreversible in practice; the severity of injury cannot be modulated and the procedure is associated with high mortality. Methapyrilene (MP) administration has therefore been investigated as an alternative model. Male rats were subjected to BDL or orally dosed with 150 mg MP per kg body weight 3 times per week for up to 6 weeks. Both procedures resulted in increases in serum alkaline phosphatase enzyme levels and periportal liver injury as judged by marked inflammatory cell recruitment to the periportal regions of the liver lobule. Injury in both models was associated with an accumulation of pro-fibrogenic myofibroblast and fibroblast populations – and to fibrosis – in the periportal regions of the liver lobule. Both procedures also caused an expansion in the number of biliary epithelial cells and a ductular reaction. However, the studies with MP resulted in no rat mortality, in contrast to the BDL procedure. Injury, inflammation, fibrosis and the ductular reaction that occurred in response to MP treatment all reversed within 3 weeks after MP treatment withdrawal. The MP model of periportal fibrosis is therefore a superior – more refined – model of periportal fibrosis than BDL since the degree of injury may be modulated (through alterations in injuring MP dose); the procedure is less severe; is subject to minimal mortality and is reversible (and therefore a more sensitive model to identify effective anti-fibrogenic drugs).

Biliary epithelial cells (BEC) are important targets in some liver diseases, including acute allograft rejection. Although some injured BEC die, many can survive in function compromised states of senescence or phenotypic de-differentiation. This study was performed to examine changes in the phenotype of BEC during acute liver allograft rejection and the mechanism driving these changes. Liver allograft sections showed a positive correlation (p < 0.0013) between increasing T cell mediated acute rejection and the number of BEC expressing the senescence marker p21(WAF1/Cip) or the mesenchymal marker S100A4. This was modeled in vitro by examination of primary or immortalized BEC after acute oxidative stress. During the first 48 h, the expression of p21(WAF1/Cip) was increased transiently before returning to baseline. After this time BEC showed increased expression of mesenchymal proteins with a decrease in epithelial markers. Analysis of TGF-β expression at mRNA and protein levels also showed a rapid increase in TGF-β2 (p < 0.006) following oxidative stress. The epithelial de-differentiation observed in vitro was abrogated by pharmacological blockade of the ALK-5 component of the TGF-β receptor. These data suggest that stress induced production of TGF-β2 by BEC can modify liver allograft function by enhancing the de-differentiation of local epithelial cells.

Liver fibrosis is characterized by excessive deposition of collagenous scar tissue after persistent liver damage. The primary liver-scar–forming cell is the hepatic myofibroblast (HM), which is derived from quiescent hepatic stellate cells (qHSCs).1 Liver injury instructs qHSCs to undergo a transdifferentiation program from retinoid-storing cells into HMs, which produce collagen I and enzymes, which prevent collagen degradation, the tissue inhibitors of matrix metalloproteinase.1 Continued damage causes perpetuation of the HM phenotype and an imbalance in the deposition and breakdown of fibrotic matrix and, ultimately, the progression of liver fibrosis. Fibrosis in both rodents and humans is a dynamic process that can reverse, as well as progress,2 and removal of the injury stimulus or promoting HM apoptosis is associated with fibrolysis.3,4 Nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) is a transcription factor that contains five subunits, RelA (p65), p50, c-Rel, RelB, and p52, which form either homo-or heterodimers to bind DNA. NF-κB is a master regulator of essential cellular functions, including cell cycle, survival, and immunity.5,6 The pathway is activated by canonical (RelA, p50, and c-Rel subunits) or noncanonical (RelB and p52 subunit) signaling.5 In unstimulated cells, canonical NF-κB is predominantly bound to its inhibitor (IκBα) and retained in the cytoplasm. Stimulation by an appropriate signal, for example, tumor necrosis factor alpha (TNF-α) or lipopolysaccharide (LPS), promotes IκB kinase (IKK)-dependent degradation of IκBα, nuclear translocation of NF-κB, and subsequent DNA binding. In the noncanonical pathway, RelB is bound to p100, the precursor of p52. Stimulation by CD40 or lymphotoxin activates NF-κB-inducing kinase, which then activates IKKα. This promotes the phosphorylation and polyubiquitination of p100, which is then degraded by the proteasome, releasing active p52/RelB. Canonical NF-κB signaling influences liver fibrosis and promotes HM survival. In models of liver damage, mice lacking nfkb1 (p105/p50) develop severe inflammation and fibrosis,7 whereas crel knockout mice develop less fibrosis, but have impaired liver regeneration, compared to wild-type controls.8 Pharmacological blockade of NF-κB in HM promotes their apoptosis and enhanced reversal of liver fibrosis.9,10 However, long-term global NF-κB blockade may alter immune responses or cause cancer.11,12 In HM, NF-κB is constitutively active. Deregulation of healthy NF-κB activity is controlled by at least two reprogramming events that occur during HSC transdifferentiation. The first is persistent down-regulation in IκBα levels, which is mediated by epigenetic changes,13 and the second is phosphorylation of RelA at serine 536 (RelA-P-Ser536).9 This post-translational modification promotes RelA nuclear translocation and increases the ability of RelA-containing dimers to interact with coactivators as well as the transcriptional machinery to enhance the transactivation potential of RelA in multiple cells.14–16 RelA-P-Ser536 is a feature of HM in culture and diseased human liver and can be controlled by autocrine renin-angiotensin system (RAS) signaling.9 However, angiotensin blockade does not completely inhibit RelA-P-Ser536 in cultured HM, suggesting that other stimuli or signaling pathways regulate RelA-P-Ser536. Here, we report that in human HM, as with other cells, this modification can be induced by TNF-α stimulation.15,17 We show that a cell-permeable RelA-P-Ser536 competing peptide (P6) inhibits RelA-P-Ser536 in HM, both in vitro and in vivo, and has antifibrotic, but not anti-inflammatory, effects in multiple models of liver injury.

Liver biopsy is the reference standard for the detection of nonalcoholic steatohepatitis (NASH) within nonalcoholic fatty liver disease (NAFLD). The aim of this study was to identify a biomarker of NASH in patients without significant fibrosis. In all, 172 patients from two centers with biopsy-proven NAFLD were included in this study. Eighty-four patients from a single center were included as a derivation cohort and 88 patients from a second center were included as a validation cohort. Serum samples were tested for candidate markers of fibrosis and inflammation alongside hematological and biochemical markers. Among patients without advanced fibrosis, terminal peptide of procollagen III (PIIINP) was the only marker found to be associated with a histological diagnosis of NASH in both cohorts. PIIINP also correlated with the total NAFLD activity score (NAS) and its constituent components (P < 0.001). Area under receiver operating characteristic curve (AUROC) for PIIINP in discriminating between NASH and simple steatosis (SS) was 0.77-0.82 in patients with F0-2 fibrosis and 0.82-0.84 in patients with F0-3 fibrosis. PIIINP was elevated in patients with advanced fibrosis, the overwhelming majority of whom had NASH. When incorporating patients with all degrees of fibrosis from both cohorts, PIIINP was able to discriminate between patients with SS and those with NASH or advanced fibrosis with AUROC 0.85-0.87. Conclusion: PIIINP discriminates between SS and NASH or advanced fibrosis. The use of a single biomarker in this context will be of clinical utility in detecting the minority of patients with NAFLD who have NASH or advanced fibrosis related to NASH. (HEPATOLOGY 2013)

Reduction in the use of animals in toxicology is an important goal despite the continued need to assess drug and chemical safety in man. However, a limitation to in vitro screening for drug and chemical toxicity is the lack of available human hepatocytes and the difficulties associated with generating fully functional hepatocytes from stem cells. Previously, we have shown that a rat pancreatic acinar cell line is capable of trans-differentiating into fully functional hepatocyte-like cells in response to glucocorticoid via a serine/threonine protein kinase mechanism alone. Here we demonstrated that differentiation only occurs with glucocorticoids, not other steroids. We also investigated the potential of human pancreatic cells to undergo the same process. Analysis of adult human pancreata at the level of mRNA, protein and by immunohistochemical staining demonstrated that long term systemic exposure to glucocorticoid therapy resulted in differentiation of exocrine tissue to hepatocyte-like tissue. Glucocorticoid treatment of human pancreatic acinar cells in culture also resulted in trans-differentiation to hepatocyte-like cells. Both in vivo and in vitro, trans-differentiation of pancreas cells to hepatocytes was associated with an induction of SGK1 variant transcripts that have been previously shown to drive B-13 differentiation to hepatocytes. Adult exocrine human pancreas therefore responds in a similar qualitative fashion to that previously observed in rodents exposed to elevated glucocorticoid – that of a differentiation into hepatocyte-like cells. Understanding the enhanced response of B-13 cells to glucocorticoid and engineering this response in a replicating human acinar cell could generate an unlimited supply of functional human hepatocytes in vitro that could be useful in a variety of applications, including screening drugs and chemicals for hepatic metabolism and toxicity.

Disturbed intestinal barrier function due to 'leaky' tight junctions may cause secondary sepsis via paracellular translocation across the gut wall. Our objective was to describe the effects of critical illness on duodenal morphology and ultrastructure.Prospective observational study of 12 mechanically ventilated critically ill patients in an intensive care unit and 15 control participants in an outpatient endoscopy suite.We took six endoscopic biopsy samples of the duodenum from each participant for analysis by electron and light microscopy.Our primary outcome was tight junction morphology, examined with electron microscopy. Secondary outcomes were microvillus length and density, vascular endothelium morphology and mitochondrial density and morphology, examined with electron microscopy, and morphology examined with light microscopy.We observed no abnormalities of tight junction ultrastructure in either group. There was a tendency towards shorter microvilli in the critically ill group: mean length in critically ill patients, 1.17 µm (interquartile range [IQR], 1.05-1.60 µm) v mean length in control patients, 1.58 µm (IQR, 1.30-1.72 µm); P = 0.07. There was a tendency towards less dense microvilli in the critically ill group: mean density in critically ill patients, 7.29 microvilli/µm (IQR, 6.83-8.05 microvilli/µm) v mean density in control patients, 8.23 microvilli/µm (IQR, 7.34-9.11 microvilli/µm); P = 0.07. Vascular endothelium appeared normal in all critically ill patients and abnormal in one control participant. Abnormal mitochondrial morphology was noted in one critically ill patient and one control participant, and no differences were seen in mitochondrial density. Using light microscopy, we saw more apoptotic cells in the critically ill patients (P = 0.018), but villus height, crypt depth and lymphocyte density were normal.We did not detect any morphological abnormalities of duodenal tight junctions in critically ill patients. Our results should be interpreted with caution because of the small sample population, but our observations challenge the concept that paracellular translocation facilitates secondary sepsis.

We investigated whether ancestral liver damage leads to heritable reprogramming of hepatic wound healing in male rats. We found that a history of liver damage corresponds with transmission of an epigenetic suppressive adaptation of the fibrogenic component of wound healing to the male F1 and F2 generations. Underlying this adaptation was less generation of liver myofibroblasts, higher hepatic expression of the antifibrogenic factor peroxisome proliferator-activated receptor γ (PPAR-γ) and lower expression of the profibrogenic factor transforming growth factor β1 (TGF-β1) compared to rats without this adaptation. Remodeling of DNA methylation and histone acetylation underpinned these alterations in gene expression. Sperm from rats with liver fibrosis were enriched for the histone variant H2A.Z and trimethylation of histone H3 at Lys27 (H3K27me3) at PPAR-γ chromatin. These modifications to the sperm chromatin were transmittable by adaptive serum transfer from fibrotic rats to naive rats and similar modifications were induced in mesenchymal stem cells exposed to conditioned media from cultured rat or human myofibroblasts. Thus, it is probable that a myofibroblast-secreted soluble factor stimulates heritable epigenetic signatures in sperm so that the resulting offspring better adapt to future fibrogenic hepatic insults. Adding possible relevance to humans, we found that people with mild liver fibrosis have hypomethylation of the PPARG promoter compared to others with severe fibrosis.

Transdifferentiation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype is the pivotal event in liver fibrosis. The dramatic change in phenotype associated with transdifferentiation is underpinned by a global change in gene expression. Orchestrated changes in gene expression take place at the level of chromatin packaging which is regulated by enzymatic activity of epigenetic regulators that in turn affect histone modifications. Using expression profiling of epigenetic regulators in quiescent and activated primary HSCs we found a number of histone methyltranferases including MLL1, MLL5, Set1 and ASH1 to be highly up-regulated during transdifferentiation of HSCs. All of these histone methyltranferases regulate methylation of lysine 4 of histone H3, which is a signature of actively transcribed genes. We therefore postulated that one or more of these enzymes may be involved in positively influencing expression of profibrogenic genes. Conclusion: We find that ASH1 directly binds to the regulatory regions of alpha smooth muscle actin (αSMA), collagen I, tissue inhibitor of metalloproteinase-1 (TIMP1) and transforming growth factor beta1 (TGFβ1) in activated HSCs while depletion of ASH1 caused broad suppression of fibrogenic gene expression. We also discovered that MeCP2 positively regulates ASH1 expression and therefore identify ASH1 as a key transcriptional activator component of the MeCP2 epigenetic relay pathway that orchestrates coordinated induction of multiple profibrogenic genes. (Hepatology 2012;56:1129–1139)

The polymorphism, KLF6-IVS1-27A, in the Kruppel-like factor 6 (KLF6) transcription factor gene enhances its splicing into antagonistic isoforms and is associated with delayed histological progression of nonalcoholic fatty liver disease (NAFLD). To explore a potential role for KLF6 in the development of insulin resistance, central to NAFLD pathogenesis, we genotyped KLF6-IVS1-27 in healthy subjects and assayed fasting plasma glucose (FPG) and insulin sensitivities. Furthermore, we quantified messenger RNA (mRNA) expression of KLF6 and glucokinase (GCK), as an important mediator of insulin sensitivity, in human livers and in liver tissues derived from a murine Klf6 knockdown model (DeltaKlf6). Klf6 overexpression studies in a mouse hepatocyte line were utilized to mechanistically link KLF6 with Gck promoter activity. KLF6-IVS1-27Gwt (i.e., less KLF6 splicing) was associated with stepwise increases in FPG and insulin and reduced hepatic insulin sensitivity. KLF6 binds to the liver-specific Gck promoter and activates a GCK promoter-reporter, identifying GCK as a KLF6 direct transcriptional target. Accordingly, in DeltaKlf6 hepatocytes Gck expression was reduced and stable transfection of Klf6 led to up-regulation of Gck. GCK and KLF6 mRNAs correlate directly in human NAFLD tissues and immunohistochemistry studies confirm falling levels of both KLF6 and GCK in fat-laden hepatocytes. In contrast to full-length KLF6, splice variant KLF6-SV1 increases in NAFLD hepatocytes and inversely correlates with glucokinase regulatory protein, which negatively regulates GCK activity. Conclusion: KLF6 regulation of GCK contributes to the development of hepatic insulin resistance. The KLF6-IVS1-27A polymorphism, which generates more KLF6-SV1, combats this, lowering hepatic insulin resistance and blood glucose. (HEPATOLOGY 2011)

Tissue homeostasis requires an effective, limited wound-healing response to injury. In chronic disease, failure to regenerate parenchymal tissue leads to the replacement of lost cellular mass with a fibrotic matrix. The mechanisms that dictate the balance of cell regeneration and fibrogenesis are not well understood. Here we report that fibrogenic hepatic stellate cells (HSCs) in the liver are negative regulators of hepatocyte regeneration. This negative regulatory function requires stimulation of the 5-hydroxytryptamine 2B receptor (5-HT(2B)) on HSCs by serotonin, which activates expression of transforming growth factor β1 (TGF-β1), a powerful suppressor of hepatocyte proliferation, through signaling by mitogen-activated protein kinase 1 (ERK) and the transcription factor JunD. Selective antagonism of 5-HT(2B) enhanced hepatocyte growth in models of acute and chronic liver injury. We also observed similar effects in mice lacking 5-HT(2B) or JunD or upon selective depletion of HSCs in wild-type mice. Antagonism of 5-HT(2B) attenuated fibrogenesis and improved liver function in disease models in which fibrosis was pre-established and progressive. Pharmacological targeting of 5-HT(2B) is clinically safe in humans and may be therapeutic in chronic liver disease.

Glucocorticoids are antiinflammatory therapeutics that have potent effects on cell differentiation. The aim of this study was to establish whether systemic glucocorticoid exposure significantly affects pancreatic differentiation in vivo because hepatocyte-like cells have been documented to occur in the diseased rodent pancreas. Expression of hepatic markers was examined in pancreata from mice genetically modified to secrete elevated circulating endogenous glucocorticoid [Tg(Crh)]. Tg(Crh) mice with elevated glucocorticoid appeared cushingoid and by 21 weeks of age were obese, insulin-resistant, and had extensive areas of hepatic gene expression in exocrine tissue. Acinar cells from Tg(Crh) mice costained for both amylase and cyp2e1, suggesting direct acinar-hepatic transdifferentiation. Hepatic expression increased with age in the pancreas to such an extent that malabsorption and rapid weight loss occurred in a subset of aging mice; this effect was reversed by dietary porcine pancreatic enzyme supplementation. Indeed, pancreatic expression of hepatic markers was prevented by adrenalectomy, establishing a direct role for glucocorticoid. Elevated levels of circulating glucocorticoid therefore promote a transdifferentiation of adult exocrine pancreas into hepatocyte-like cells, and chronic exposure results in pancreatic malfunction. Glucocorticoids are thus capable of modulating the differentiation of terminally differentiated adult cells.