Innate immune defence to Campylobacter jejuni.

Campylobacter jejuni is the most prevalent cause of bacterial diarrhoea worldwide and is frequently associated with severe post-infectious complications such as the Guillain-Barre syndrome. Despite the serious health burden caused by the bacterium disease pathogenesis remains ill defined. Human (3-defensins (hBDs), a family of epithelial antimicrobial peptides, are a major component of host innate defence at mucosal surfaces. In the present study we investigated the effect of C. jejuni on intestinal epithelial innate responses. Up-regulation of IL-8, hBD-2 and hBD-3 gene and peptide expression was observed in Caco-2 and HT-29 cell-lines in response to C. jejuni strains 11168H and 81-176. Furthermore, recombinant hBDs were found to exhibit potent bactericidal activity against C. jejuni suggesting a major role for these peptides in disease pathogenesis. Secondly, we aimed to identify host receptor(s) involved in sensing of C. jejuni and initiating innate defence. Given the invasive nature of infection, we investigated the potential role of cytoplasmic nucleotide-binding oligomerisation domain (NOD) proteins. Using small interfering (si) RNA targeting NODI and transfection of NOD2 overexpression plasmids, we identified NODI as a major pattern recognition receptor involved in mediating innate host defence to C. jejuni while NOD2 was found to play a minor role. Additionally, reduced NODI expression resulted in an increased number of intracellular C. jejuni thus highlighting a critical role for NODI mediated antimicrobial defence in limiting infection. In the final part of the study an ex-vivo model of C. jejuni infection using human intestinal biopsies was developed. Additionally, a vertical diffusion chamber system was utilised to improve culture conditions in C. jejuni infection models. In conclusion, this study highlights the important role of intestinal innate host defence to C. jejuni. The development of new and improved models of infections has the potential to provide previously unavailable opportunities to study C. jejuni disease pathogenesis

The value of blood derived DNA methylation signatures in advancing our understanding of Crohn's Disease pathogenesis.

DNA methylation is one of the main epigenetic mechanisms, known to be operative in mammals. Extensive evidence has highlighted its pivotal role in several fundamental biological processes including organogenesis, X-chromosome inactivation and genomic imprinting (1). Although our understanding of how DNA methylation impacts on gene transcription and cellular function remains incomplete, it is clear that DNA methylation plays an important role in defining cell and tissue specific cellular function (2,3). Increasing evidence suggests that altered DNA methylation can contribute to either the development and/or persistence of many human diseases. Importantly, what makes epigenetic mechanisms a particularly attractive concept when it comes to investigating disease pathogenesis of modern, multi-factorial/complex diseases is their responsiveness to environmental factors (4)

Effect of cell separation on gene expression and DNA methylation profiles in intestinal epithelial cells

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Biobanking of human gut organoids for translational research.

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

Clinical course and outcomes of diagnosing Inflammatory Bowel Disease in children 10 years and under: retrospective cohort study from two tertiary centres in the United Kingdom and in Italy.

BACKGROUND: Most children with Inflammatory Bowel Disease (IBD) are diagnosed between 11 and 16 years of age, commonly presenting with features of typical IBD. Children with onset of gut inflammation under 5 years of age often have a different underlying pathophysiology, one that is genetically and phenotypically distinct from other children with IBD. We therefore set out to assess whether children diagnosed after the age of 5 years, but before the age of 11, have a different clinical presentation and outcome when compared to those presenting later. METHODS: Retrospective cohort study conducted at two European Paediatric Gastroenterology Units. Two cohorts of children with IBD (total number = 160) were compared: 80 children diagnosed between 5 and 10 years (Group A), versus 80 children diagnosed between 11 and 16 (Group B). Statistical analysis included multiple logistic regression. RESULTS: Group A presented with a greater disease activity (p = 0.05 for Crohn's disease (CD), p = 0.03 for Ulcerative Colitis (UC); Odds Ratio 1.09, 95 % Confidence Interval: 1.02-1.1), and disease extent (L2 location more frequent amongst Group A children with CD (p = 0.05)). No significant differences were observed between age groups in terms of gastro-intestinal and extra-intestinal signs and symptoms at disease presentation, nor was there a difference in the number of hospitalisations due to relapsing IBD during follow-up. However, children in Group A were treated earlier with immunosuppressants and had more frequent endoscopic assessments. CONCLUSION: While clinicians feel children between 5 and 10 years of age have a more severe disease course than adolescents, our analysis also suggests a greater disease burden in this age group. Nevertheless, randomized trials to document longer-term clinical outcomes are urgently needed, in order to address the question whether a younger age at disease onset should prompt per se a more "aggressive" treatment. We speculate that non-clinical factors (e.g. genetics, epigenetics) may have more potential to predict longer term outcome than simple clinical measures such as age at diagnosis

Intestinal Epithelial Organoids as Tools to Study Epigenetics in Gut Health and Disease.

The intestinal epithelium forms the inner layer of the human intestine and serves a wide range of diverse functions. Its constant exposure to a vast amount of complex microbiota highlights the critical interface that this single-cell layer forms between the host and our environment. Importantly, the well-documented contribution of environmental factors towards the functional development of the human intestinal epithelium directly implies epigenetic mechanisms in orchestrating this complex interplay. The development of intestinal epithelial organoid culture systems that can be generated from human tissue provides researchers with unpresented opportunities to study functional aspects of human intestinal epithelial pathophysiology. In this brief review, we summarise existing evidence for the role of epigenetics in regulating intestinal epithelial cell function and highlight the great potential for human gut organoids as translational research tools to investigate these mechanisms in vitro.Peer Reviewe

Guidance on the interpretation of faecal calprotectin levels in children.

BACKGROUND: Faecal calprotectin (FCP) is a powerful tool to predict inflammatory bowel disease (IBD) in patients with gastrointestinal symptoms. In the paediatric patient population, the reference value of 50 μg/g, 15% were ≥ 250 μg/g. Children 50 μg/g) was the sole reason for being referred for suspected IBD did not have IBD. CONCLUSION: Children with an FCP < 600 μg/g and without matching symptoms suggestive of IBD are unlikely to have IBD. A higher FCP reference value may provide cost-effective improvement that could avoid redundant investigations and specialist referrals. A guideline for specialist referrals is proposed

A functional genetic toolbox for human tissue-derived organoids.

Funder: Alzheimers Research UK Stem Cell Research CentreHuman 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 knock-down 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 on-going descriptive studies, such as the Human Cell Atlas Project

Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease.

DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.This study was supported by funds obtained from The Evelyn Trust, Crohn’s in Childhood Research Association (CICRA) and Crohn’s and Colitis in Childhood (3Cs) charity. J.K. was funded by a PhD studentship from CICRA. Funding for E.C. was provided by the Deutsche Forschungsgemeinschaft (Grant CA226/4-3) and Interne Forschungsförderung Essen (IFORES).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/mi.2015.8