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The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.

The global incidence and prevalence of paediatric inflammatory bowel disease (IBD) is increasing, with a notable emergence in developing countries with historically low rates. This suggests that environmental and epigenetic factors may play an important role in the pathogenesis and progression of IBD. Epigenetics refers to the study of biological mechanisms that result in a change of phenotype, without an change in the underlying DNA sequence. Epigenetic mechanisms drive many biological processes that occur in health, such as development and ageing, and are also implicated in disease, including cancer and other inflammatory diseases. Importantly, identification of cell-type-specific epigenetic mechanisms could lead to the identification of molecular disease subtypes allowing a personalised treatment approach. In this short review, we provide a summary of epigenetic mechanisms operative in mammals, and their potential involvement in IBD pathogenesis. Furthermore, we discuss key challenges associated with investigating epigenetics in IBD and provide potential strategies to overcome these, such as through the use of ‘omics’ and organoid technologies.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition in children characterized by insulin resistance and altered lipid metabolism. Affected patients are at increased risk of cardiovascular disease (CVD) and children with NAFLD are likely to be at risk of premature cardiac events. Evaluation of the plasma lipid profile of children with NAFLD offers the opportunity to investigate these perturbations and understand how closely they mimic the changes seen in adults with cardiometabolic disease.METHODS: We performed untargeted liquid chromatography mass spectrometry (LC-MS) plasma lipidomics on 287 children: 19 lean controls, 146 from an obese cohort, and 122 NAFLD cases who had undergone liver biopsy. Associations between lipid species and liver histology were assessed using regression adjusted for age and sex. Results were then replicated using data from 9,500 adults with metabolic phenotyping.RESULTS: More severe paediatric NAFLD was associated with lower levels of long chain, polyunsaturated phosphatidylcholines (PC) and triglycerides (TG). Similar trends in PC and TG chain length and saturation were seen in adults with hepatic steatosis. However, many of the specific lipids associated with NAFLD differed between children and adults. Five lipids replicated in adults (including PC(36:4)) have been directly linked to death and cardiometabolic disease, as well as indirectly via genetic variants.CONCLUSION: These findings suggest that, whilst similar pathways of lipid metabolism are perturbed in paediatric NAFLD as in cardiometabolic disease in adults, the specific lipid signature in children is different.

Peer reviewed: True, Funder: King’s College London, Funder: National Institute for Health Research (NIHR), Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.

Funder: MRC New Investigator Research Grant (MZ) European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN), The development of human organoid culture models has led to unprecedented opportunities to generate self-organizing, three-dimensional miniature organs that closely mimic in vivo conditions. The ability to expand, culture, and bank such organoids now provide researchers with the opportunity to generate next-generation living biobanks, which will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine as well as the development of a personalized treatment approach. However, compared to traditional tissue repositories, the generation of a living organoid biobank requires a much higher level of coordination, additional resources, and scientific expertise. In this short review, we discuss the opportunities and challenges associated with the generation of a living organoid biobank. Focusing on human intestinal organoids, we highlight some of the key aspects that need to be considered and provide an outlook for future development in this exciting field.

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[

Introduction: Inflammatory bowel diseases (IBDs) are lifelong conditions causing relapsing inflammation of the intestine. In the absence of a cure, clinical management of IBDs is extremely challeng...

Background & AimsHuman intestinal epithelial organoids (IEO) are a powerful tool to model major aspects of intestinal development, health and diseases, as patient derived cultures retain many features found in-vivo. A necessary aspect of the organoid model is the requirement to expand cultures in-vitro through several rounds of passaging. This is of concern, as the passaging of cells has been shown to affect cell morphology, ploidy, and function. In this study, we address concerns around long term passaging of IEO to better characterise and define effects on cell morphology and function.MethodsHere we have analysed 173 human IEO from two sampling sites, terminal ileum and sigmoid colon and examined the effect of culture duration on DNA methylation (DNAm), gene expression and cellular function including their response to proinflammatory cytokines and in-vitro cell differentiation.ResultsOur analyses revealed a major effect of culture duration on DNAm, leading to significant changes at 61,337 loci representing approximately 8% of all CpGs tested. Although global cellular functions such as gut segment-specific gene expression remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokine exposure and in-vitro differentiation. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication indicating similarities to malignant transformation.ConclusionsOur study reveals culture-associated epigenetic, transcriptomic and functional changes in human mucosa derived IEO and highlights the importance of considering passage number as a potentially confounding factor.SynopsisThis work describes cell culture induced changes to DNA methylation, gene expression and cellular function in human IEO. Globally organoids lost DNA methylation with time in culture while DNA methylation also became generally more variable. This work suggests a shifted epigenetic profile in organoids cultured long-term.

BACKGROUND & AIMS: Human intestinal epithelial organoids (IEOs) are a powerful tool to model major aspects of intestinal development, health, and diseases because patient-derived cultures retain many features found in vivo. A necessary aspect of the organoid model is the requirement to expand cultures in vitro through several rounds of passaging. This is of concern because the passaging of cells has been shown to affect cell morphology, ploidy, and function. METHODS: Here, we analyzed 173 human IEO lines derived from the small and large bowel and examined the effect of culture duration on DNA methylation (DNAm). Furthermore, we tested the potential impact of DNAm changes on gene expression and cellular function. RESULTS: Our analyses show a reproducible effect of culture duration on DNAm in a large discovery cohort as well as 2 publicly available validation cohorts generated in different laboratories. Although methylation changes were seen in only approximately 8% of tested cytosine-phosphate-guanine dinucleotides (CpGs) and global cellular function remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokine exposure and withdrawal of Wnt agonists. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication, indicating similarities to malignant transformation. CONCLUSIONS: Our study shows distinct culture-associated epigenetic changes in mucosa-derived human IEOs, some of which appear to impact gene transcriptomic and cellular function. These findings highlight the need for future studies in this area and the importance of considering passage number as a potentially confounding factor.

Clinical psychology intervention in paediatric gastroenterology is vital given the biopsychosocial aetiology of paediatric functional gastrointestinal disorders, and the psychological impact of chronic conditions. The aim was to assess the availability and benefit of clinical psychology in paediatric gastroenterology across the UK and Germany. A retrospective assessment of referrals (n = 936 referrals) to clinical psychology was performed at our tertiary paediatric gastroenterology centre between 2010 and 2018. The availability of clinical psychologists and outcome of psychology intervention for children with functional abdominal pain were also assessed. Access to clinical psychology across the UK and Germany was assessed using an online questionnaire. We observed a substantial rise in the number of clinical psychology referrals between 2010 and 2018. Increasing demand was not matched by sufficient increase in availability of clinical psychology, leading to longer waiting times. A major benefit of clinical psychology intervention was highlighted with 95% of patients (n = 20) reporting a significant reduction in symptoms. Of the 12 centres who responded, 11 centres have direct access to clinical psychology with a mean of 13% of patients requiring psychology referrals annually.Conclusion: Despite evidence of its benefit and increasing demand, there is insufficient access to clinical psychological services, highlighting the urgent need to address this important issue. What is known:• The biopsychosocial pathophysiology of functional gastrointestinal disorders involves a disordered brain-gut interaction, which emphasizes the close link between psychological factors and altered gut function.• Psychological intervention, as an adjunct to medical treatment, improves outcomes in paediatric patients with gastrointestinal (GI) disease such as functional gastrointestinal disorders and inflammatory bowel diseasesWhat is new:• There is a rising number of referrals from paediatric gastroenterology to clinical psychology in our centre which is not met by a sufficient increase in the availability of clinical psychologists. Similarly, access to clinical psychological services is lacking in several paediatric gastroenterology centres in the UK and Germany.• Strategic action is required to address this important gap in the care of children suffering from GI diseases.

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ stimulated cells guanylate binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, whereas GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP-Caspase-4 pathway in anti-bacterial defense.

Background and aims Monogenic forms of inflammatory bowel disease (IBD) illustrate the essential roles of individual genes in pathways and networks safeguarding immune tolerance and gut homeostasis. Methods To build a taxonomy model we assessed 165 disorders. Genes were prioritized based on penetrance of IBD and disease phenotypes were integrated with multi-omics datasets. Monogenic IBD genes were classified by: (1) overlapping syndromic features; (2) response to hematopoietic stem cell transplantation; (3) bulk RNA-seq of 32 tissues; (4) single-cell RNA-seq of >50 cell subsets from the intestine of healthy individuals and IBD patients (pediatric and adult), and (5) proteomes of 43 immune subsets. The model was validated by addition of newly identified monogenic IBD defects. As a proof-of-concept, we explore the intersection between immunometabolism and antimicrobial activity for a group of disorders (G6PC3/SLC37A4). Results Our quantitative integrated taxonomy defines the cellular landscape of monogenic IBD gene expression across 102 genes with high and moderate penetrance (81 in the model set and 21 genes in the validation set). We illustrate distinct cellular networks, highlight expression profiles across understudied cell types (e.g., CD8+ T cells, neutrophils, epithelial subsets and endothelial cells) and define genotype-phenotype associations (perianal disease and defective antimicrobial activity). We illustrate processes and pathways shared across cellular compartments and phenotypic groups and highlight cellular immunometabolism with mTOR activation as one of the converging pathways. There is an overlap of genes and enriched cell-specific expression between monogenic and polygenic IBD. Conclusion Our taxonomy integrates genetic, clinical and multi-omic data; providing a basis for genomic diagnostics and testable hypotheses for disease functions and treatment responses.

Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example, we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes ‘Organoid Easytag’, our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly targeted cells. Our toolbox also incorporates conditional gene knockdown or overexpression using tightly inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many ongoing descriptive studies, such as the Human Cell Atlas Project.

Funder: Medical Research Council, The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Recent research breakthroughs have emerged from applied basic research throughout all scientific areas, including adult and paediatric gastroenterology, hepatology and nutrition (PGHAN). The research landscape within the European Society of Paediatric Gastroenterology and Nutrition (ESPGHAN) is also inevitably changing from clinical research to studies involving applied laboratory research. This position paper aims to depict the current status quo of basic science and translational research within ESPGHAN, and to delineate how the society could invest in research in the present and future time. The paper also explores which research areas in the field of PGHAN represent the current and future priorities, and what type of support is needed across the ESPGHAN working groups (WGs) and special interest groups (SIGs) to fulfil their research goals.

Inflammatory bowel diseases [IBD] such as Crohn’s disease [CD] and ulcerative colitis [UC] are complex conditions presenting with a wide range of phenotypes. Given major variation in disease severity and outcomes as well as response to existing therapies, a personalised treatment approach stands the chance of improving the overall disease outcome as well as minimising potentially harmful side effects. However, disease activity or distribution at the point of diagnosis are poor predictors of future disease outcome. Hence, the urgent need to develop biomarkers that could either predict the overall disease course [i.e., disease prognostic biomarkers] or the response to individual therapies [i.e., disease predictive biomarkers]. Despite the widely accepted need for such biomarkers to improve the management of IBD patients, their development has proven to be challenging for a number of reasons. Based on our own experience in this field, we perform a reality check on existing evidence, discuss main challenges, and outline future perspectives.

Background Intestinal failure (IF) is associated with significant morbidity and mortality, yet specific parameters that determine medium- and long-term outcomes remain ill defined. Objective The aim of this study was to determine the long-term outcomes in childhood IF and identify patient characteristics associated with clinical endpoints. Design MEDLINE and EMBASE were searched for cohorts of >10 pediatric-onset IF patients with >12 mo follow-up. Random-effects meta-analysis and meta-regression weighted by follow-up duration were used to calculate clinical outcome rates and patient factors associated with outcomes. Primary outcome was mortality rate; secondary outcomes included neurodevelopmental status, transplantation, IF-associated liver disease (IFALD), enteral autonomy, and sepsis. Results In total, 175 cohorts (9318 patients and 34,549 y follow-up) were included in the meta-analysis. Overall mortality was 5.2% per y (95% CI: 4.3, 6.0) and was associated with sepsis and IFALD on meta-regression. Mortality rate improved with time from 5.9% per y pre-2000 to 4.5% per y post-2005. Sepsis rate was also predictive of IFALD and liver failure. Enteral autonomy was associated with small bowel length but not presence of ileo-cecal valve. There was a relative lack of data on neurodevelopmental outcomes. Conclusions Sepsis is the primary modifiable factor associated with mortality and liver failure, whereas enteral autonomy correlates with small-bowel length. No clear parameters have been identified that accurately predict neurodevelopmental outcomes, and hence further research is needed. Together, our findings are helpful for parental counseling and resource planning, and support targeting reduction in sepsis.

Funder: European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN), Eosinophilic esophagitis (EoE) is a leading cause of dysphagia and food impaction in children and adults. The diagnosis relies on histological examination of esophageal mucosal biopsies and requires the presence of > 15 eosinophils per high-powered field. Potential pitfalls include the impact of biopsy sectioning as well as regional variations of eosinophil density. We performed genome-wide DNA methylation analyses on 30 esophageal biopsies obtained from children diagnosed with EoE (n = 7) and matched controls (n = 13) at the time of diagnosis as well as following first-line treatment. Analyses revealed striking disease-associated differences in mucosal DNA methylation profiles in children diagnosed with EoE, highlighting the potential for these epigenetic signatures to be developed into clinically applicable biomarkers.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used singlecell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprung’s disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.

The incidence of chronic inflammatory bowel diseases (IBDs), such as Crohn's disease (CD) and ulcerative colitis (UC) have significantly increased in recent decades implicating environmental effects. The developmental origin of disease concept provides a theoretical framework by which the complex interplay between environmental factors and host cells, particularly during vulnerable time periods, ultimately cause disease, such as IBD. Epigenetics has been proposed as the underlying mechanism within this concept, turning environmental triggers into stable changes of cellular function. Adding further to the complexity of IBD is the gut microbiome, which is equally responsive to the environment, and can impact host cell function, where recent findings underscore the stochastic and individualized nature of such effects. We review the microbiome literature through a novel triple environmental hit concept (priming, modulation, and trigger) of IBD pathogenesis. We propose that there are at least 3 distinct stages during an individual's lifespan where random/stochastic events driven by environmental influences are necessary for ultimately developing IBD. By this means, we speculate that microbiome-directed therapeutics carry potential for individualized prevention and dynamic treatment of IBD.

Background & Aims Gene expression patterns of CD8+ T cells have been reported to correlate with clinical outcomes of adults with inflammatory bowel diseases (IBD). We aimed at validating these findings in independent patient cohorts. Methods We obtained peripheral blood samples from 112 children with a new diagnosis of IBD (71 with Crohn’s disease and 41 with ulcerative colitis) and 19 children without IBD (controls) and recorded medical information on disease activity and outcomes. CD8+ T cells were isolated from blood samples by magnetic bead sorting at the point of diagnosis and during the course of disease. Genome-wide transcription (n=192) and DNA methylation (n=66) profiles were generated using Affymetrix and Illumina arrays, respectively. Publicly available transcriptomes and DNA methylomes of CD8+ T cells from three adult patient cohorts with and without IBD were included in data analyses. Results Previously reported CD8+ T cell prognostic expression and exhaustion signatures were only found in the original adult IBD patient cohort. These signatures could not be detected either in a pediatric, or in a second adult IBD cohort. In contrast, an association between CD8+ T cell gene expression with age and sex was detected across all three cohorts. CD8+ gene transcription was clearly associated with IBD in the two cohorts that included non-IBD controls. Lastly, DNA methylation profiles of CD8+ T cells from children with Crohn’s disease correlated with age but not with disease outcome. Conclusions We were unable to validate previously reported findings of an association between CD8+ T cell gene transcription and disease outcome in IBD. Our findings reveal the challenges of developing prognostic biomarkers for patients with IBD and the importance of their validation in large, independent cohorts before clinical application., Graphical abstract

Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition in children and adults characterized by insulin resistance and altered lipid metabolism. Affected patients are at increased risk of cardiovascular disease (CVD) and children with NAFLD are likely to be at risk of premature cardiac events. Evaluation of the plasma lipid profile of children with NAFLD offers the opportunity to investigate these perturbations and understand how closely they mimic the changes seen in adults with cardiometabolic disease. We hypothesized that change in the concentration of lipid species in pediatric NAFLD would mimic the alterations known to be associated with CVD in adults (and be largely reflective of insulin resistance). Here, we performed untargeted liquid chromatography mass spectrometry (LC-MS) plasma lipidomics on 287 children: 19 lean controls, 146 from an obese cohort, and 122 NAFLD cases who had undergone liver biopsy. Associations between lipid species and liver histology were assessed using regression adjusted for age and sex. Results were then replicated using data from 9,500 adults with metabolic phenotyping. Phosphatidylcholine (PC) and triglyceride (TG) desaturation and chain length were inversely associated with histological severity of paediatric NAFLD. Nine lipids species (lyso-PC, PC, and TG classes) were also associated with hepatic steatosis and insulin resistance in an independent cohort of adults. Five of the 9 lipids replicated in the adults cohort (including PC(36:4)) have been directly linked to death and cardiometabolic disease in adults, as well as indirectly via genetic variants that influence the concentration of these species. Together, these findings suggest that lipid metabolism is altered in paediatric NAFLD in a similar way as in cardiometabolic disease in adults and it is therefore critical to alleviate insulin resistance in these children to mitigate their long-term morbidity.

Background & AimsCD8+ T-cell gene expression has been implicated in the pathogenesis of Inflammatory Bowel Diseases (IBD) and has been shown to correlate with disease outcome in adult patients. Moreover, CD8+ T-cell exhaustion was identified as a contributing mechanism that impacts on disease behaviour. We aimed to explore CD8+ T-cell gene expression and DNA methylation in children newly diagnosed with IBD and test their correlation with disease outcome.MethodsWe prospectively recruited 112 children with IBD at the point of diagnosis and 19 non-IBD controls. Follow-up samples were obtained from a subset of patients at 3-month intervals (n=62). CD8+ T-cells were purified from peripheral blood samples using magnetic bead sorting and genome-wide transcriptional (n=192) and DNA methylation (n=66) profiles were generated using Affymetrix and Illumina arrays respectively. Publicly available adult CD8+ T-cell transcriptomes and DNA methylomes were included in data analyses to investigate age dependant differences.ResultsVariation amongst CD8+ T-cell transcriptomes obtained from children showed association with disease, systemic inflammation, age and gender but lacked correlation with disease outcome in pediatric IBD. In contrast to CD8+ T-cell transcriptomes in adult Crohn’s Disease (CD), samples from pediatric patients did not show variation within genes forming part of the previously reported prognostic expression or T-cell exhaustion signatures. Pediatric CD patient derived DNA methylation profiles also lacked correlation with disease outcome but in comparison to adult CD8+ methylomes showed a higher predicted proportion of CD8+ naïve T-cells.ConclusionsOur findings indicate age-related differences in IBD pathogenesis and highlight the importance of validating adult clinical biomarkers in pediatric cohorts.

SummaryHuman gut development requires the orchestrated interaction of various differentiating cell types. Here we generate an in-depth single-cell map of the developing human intestine at 6–10 weeks post-conception, a period marked by crypt-villus formation. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells, which are distinct from LGR5-expressing cells. We use computational analyses to show that these cells contribute to differentiated cell subsets directly and indirectly via the generation of LGR5-expressing stem cells and receive signals from the surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes ofex vivotissues andin vitrofetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNAseq profiles from paediatric Crohn’s disease epithelium alongside matched healthy controls to reveal disease associated changes in epithelial composition. Contrasting these with the fetal profiles reveals re-activation of fetal transcription factors in Crohn’s disease epithelium. Our study provides a unique resource, available atwww.gutcellatlas.org, and underscores the importance of unravelling fetal development in understanding disease.

Background and aims Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown. Approach and results Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine-rich repeat-containing G-protein-coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional-specific markers corresponding to their location of origin. Of particular interest, down-regulation of biliary markers and up-regulation of cell-cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions. Conclusions Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development.

The disease course of children with ulcerative colitis (UC) varies substantially. Published data on predictors of disease outcomes in children remain scarce. We validate clinical predictors of outcomes in 93 children with UC in a single centre (age range: 2-18 years, minimum follow-up: 18 months). We stratified children into 3 groups according to their disease course, that is, 1 = mild (38/93, 40.9%), 2 = moderate (38/93, 40.9%), 3 = severe (17, 18.2%). Comparison of clinical and biochemical parameters was performed between groups using Chi-square, Mann-Whitney, and log-rank tests. Predictors of a severe disease course included pancolitis (P 0.01), low albumin (P 0.005), low haemoglobin at diagnosis (P 0.04), paediatric ulcerative colitis activity index (PUCAI) at 3 months, and nonresponse to steroids at 3 months (P 0.0001). In our cohort, failure to achieve remission at 3 months implied an 80% likelihood to require biologics or major surgery within 18 months. A specific 3-month review point is recommended to guide future management.

Human noroviruses (HuNoV) are a leading cause of viral gastroenteritis worldwide and a significant cause of morbidity and mortality in all age groups. The recent finding that HuNoV can be propagated in B cells and mucosa derived intestinal epithelial organoids (IEOs), has transformed our capability to dissect the life cycle of noroviruses. Using RNA-Seq of HuNoV infected intestinal epithelial cells (IECs), we have found that replication of HuNoV in IECs results in interferon-induced transcriptional responses and that HuNoV replication in IECs is sensitive to IFN. This contrasts with previous studies that suggest that the innate immune response may play no role in the restriction of HuNoV replication in immortalised cells. We demonstrate that the inhibition of JAK1/JAK2 enhances HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular RNA polymerase II-mediated transcription was not detrimental to HuNoV replication, but enhanced replication to a greater degree compared to blocking of JAK signalling directly. Furthermore, we demonstrate for the first time that IECs generated from genetically modified intestinal organoids, engineered to be deficient in the interferon response, are more permissive to HuNoV infection. Together our work identifies the IFN-induced transcriptional responses restrict HuNoV replication in IECs and demonstrates that the inhibition of these responses by modifications to the culture conditions can greatly enhance the robustness of the norovirus culture system.ImportanceNoroviruses are a major cause of gastroenteritis worldwide yet the challenges associated with their growth culture has greatly hampered the development of therapeutic approaches and has limited our understanding of cellular pathways that control infection. Here we show that human intestinal epithelial cells, the first point of entry of human noroviruses into the host, limit virus replication by the induction of the innate responses. Furthermore we show that modulating the ability of intestinal epithelial cells to induce transcriptional responses to HuNoV infection can significantly enhance human norovirus replication in culture. Collectively our findings provide new insights into the biological pathways that control norovirus infection but also identify mechanisms to enhance the robustness of norovirus culture.

Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. Until now, all the enteroviral proteins were thought to derive from proteolytic processing of a polyprotein encoded in a single open reading frame (ORF). We report that many enterovirus genomes also harbor an upstream ORF (uORF) that is subject to strong purifying selection. Using echovirus 7 and poliovirus 1, we confirmed expression of uORF protein (UP) in infected cells. Using ribosome profiling (a technique for global footprinting of translating ribosomes), we also demonstrated translation of the uORF in representative members of the predominant human enterovirus species, namely Enterovirus A, B and C. In differentiated human intestinal organoids, UP-knockout echoviruses are attenuated compared to wild-type virus at late stages of infection where membrane-associated UP facilitates virus release. Thus we have identified a previously unknown enterovirus protein that facilitates virus growth in gut epithelial cells – the site of initial viral invasion into susceptible hosts. These findings overturn the 50-year-old dogma that enteroviruses use a single-polyprotein gene expression strategy, and have important implications for understanding enterovirus pathogenesis.