Comparison of the Lipidomic Signature of Fatty Liver in Children and Adults: A Cross-Sectional Study.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition in children characterised 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 9500 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.JPM is supported by a Wellcome Trust fellowship (216329/Z/19/Z), a European Society for Paediatric Research (ESPR) Young Investigator Award, and a Children’s Liver Disease Foundation Grant. EU-PNAFLD Registry is supported by a European Association for Study of the Liver (EASL) Registry Grant. MZ is supported by a New Investigator Research Grant from the MRC (MR/T001917/1). BK is supported by grants from Van den Broek Lohman Foundation, Virtutis Opus Foundation and For Wishdom Foundation. SF, SGS & AK are supported by the BBSRC (BB/M027252/1 & BB/P028195/1), BJJ & AK are supported by the National Institute for Health Research (NIHR146281)

Expression of Human Beta-Defensins in Children with Chronic Inflammatory Bowel Disease

Background: Human beta-defensins (hBDs) are antimicrobial peptides known to play a major role in intestinal innate host defence. Altered mucosal expression of hBDs has been suggested to be implicated in chronic inflammatory bowel disease pathogenesis. However, little is known about expression of these peptides in children. Methods: Intestinal biopsies were obtained from the duodenum (n = 88), terminal ileum (n = 90) and ascending colon (n = 105) of children with Crohn’s disease (n = 26), ulcerative colitis (n = 11) and healthy controls (n = 16). Quantitative realtime (RT) PCR was performed and absolute mRNA copy numbers analyzed for hBD1-3 as well as inflammatory cytokines IL-8 and TNF-alpha. Results: Significant induction of hBD2 and hBD3 was observed in the inflamed terminal ileum and ascending colon of IBD children. In the ascending colon induction of hBD2 was found to be significantly lower in children with Crohn’s disease compared to ulcerative colitis. A strong correlation was found between inducible defensins hBD2 and 3 and the inflammatory cytokines IL-8 and TNF-alpha, both in the terminal ileum and ascending colon. Conclusion: Our study demonstrates distinct changes in hBD expression throughout the intestinal tract of children with IBD

DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development.

OBJECTIVE: Human intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited. DESIGN: We performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology. RESULTS: We discovered stable epigenetic signatures which define regional differences in gut epithelial function, including induction of segment-specific genes during cellular differentiation. Established DNA methylation profiles were independent of cellular environment since organoids retained their regional DNA methylation over prolonged culture periods. In contrast to paediatric and adult organoids, fetal gut-derived organoids showed distinct dynamic changes of DNA methylation and gene expression in culture, indicative of an in vitro maturation. By applying CRISPR/Cas9 genome editing to fetal organoids, we demonstrate that this process is partly regulated by TET1, an enzyme involved in the DNA demethylation process. Lastly, generating IEOs from a child diagnosed with gastric heterotopia revealed persistent and distinct disease-associated DNA methylation differences, highlighting the use of organoids as disease-specific research models. CONCLUSIONS: Our study demonstrates striking similarities of epigenetic signatures in mucosa-derived IEOs with matching primary epithelium. Moreover, these results suggest that intestinal stem cell-intrinsic DNA methylation patterns establish and maintain regional gut specification and are involved in early epithelial development and disease.This work was supported by funding from the following charitable organizations: Crohn’s in Childhood Research Association (CICRA), the Evelyn Trust, Crohn’s and Colitis in Childhood (“3Cs”), Addenbrooke’s Charitable Trust (ACT), and the Newlife Foundation for Disabled Children. J.K. was funded by a CICRA PhD studentship, K.H. was funded by an EBPOD EMBL-EBI/Cambridge Computational Biomedical Postdoctoral Fellowship. B-K.K. was supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [101241/Z/13/Z] and received core support from the Wellcome Trust and MRC to the WT - MRC Cambridge Stem Cell Institute. GD and JF received support from the Wellcome Trust. J.F. was supported by a studentship from the MRC. P.R was supported by the Deutsche Forschungsgemeinschaft EXC306 Cluster “Inflammation at Interfaces” and BMBF IHEC DEEP TP5.2

Matrix expansion and syncytial aggregation of syndecan-1+ cells underpin villous atrophy in coeliac disease

Background: We studied the expression of sulphated glycosaminoglycans (GAGs) in coeliac disease (CD) mucosa, as they are critical determinants of tissue volume, which increases in active disease. We also examined mucosal expression of IL-6, which stimulates excess GAG synthesis in disorders such as Grave's ophthalmopathy. Methods: We stained archival jejunal biopsies from 5 children with CD at diagnosis, on gluten-free diet and challenge for sulphated GAGs. We then examined duodenal biopsies from 9 children with CD compared to 9 histological normal controls, staining for sulphated GAGs, heparan sulphate proteoglycans (HSPG), short-chain HSPG (Δ-HSPG) and the proteoglycan syndecan-1 (CD138), which is expressed on epithelium and plasma cells. We confirmed findings with a second monoclonal in another 12 coeliac children. We determined mucosal IL-6 expression by immunohistochemistry and PCR in 9 further cases and controls, and used quantitative real time PCR for other Th17 pathway cytokines in an additional 10 cases and controls. Results: In CD, HSPG expression was lost in the epithelial compartment but contrastingly maintained within an expanded lamina propria. Within the upper lamina propria, clusters of syndecan-1+ plasma cells formed extensive syncytial sheets, comprising adherent plasma cells, lysed cells with punctate cytoplasmic staining and shed syndecan ectodomains. A dense infiltrate of IL-6+ mononuclear cells was detected in active coeliac disease, also localised to the upper lamina propria, with significantly increased mRNA expression of IL-6 and IL-17A but not IL-23 p19. Conclusions: Matrix expansion, through syndecan-1+ cell recruitment and lamina propria GAG increase, underpins villous atrophy in coeliac disease. The syndecan-1+ cell syncytia and excess GAG production recapitulate elements of the invertebrate encapsulation reaction, itself dependent on insect transglutaminase and glutaminated early response proteins. As in other matrix expansion disorders, IL-6 is upregulated and represents a logical target for immunotherapy in patients with coeliac disease refractory to gluten-free diet

IL-18 and VEGF-A trigger type 2 innate lymphoid cell accumulation and pro-tumoral function in chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematologic malignancy associated to an unregulated growth of myeloid cells in Bone Marrow (BM) and Peripheral Blood (PB), characterized by the BCR-ABL1 translocation. Given the known cytokine impairment in the leukemic niche of CML, we investigated the impact of this microenvironmental dysregulation on Innate Lymphoid Cells (ILCs), whose role in cancer has recently emerged. Three ILC subsets are identified based on transcriptional profiles and cytokine secretion. We observed that IL-18 and VEGF-A are increased in CML patients' sera and that ILC2s are enriched in CML PB and BM. We found that IL-18 drives ILC2 proliferation and that CML ILC2s highly express CXCR4 and CXCR7 BM-homing receptors, potentially explaining their enrichment in PB and BM, respectively. Next, we showed that ILC2s are hyper-activated through a tumor-derived VEGF-A-dependent mechanism, which leads to higher IL-13 secretion. In response to IL-13, leukemic cells increase their clonogenic capacity. Finally, we discovered that the pro-tumoral axis involving VEGF-A, IL-18 and ILC2s was disrupted upon Tyrosine Kinase Inhibitors' (TKIs) treatment, normalizing the levels of all these players in CML patients responding to therapy. Overall, our study uncovers the involvement of ILC2s in CML progression, mediated by VEGF-A and IL-18

Guidelines for the management of growth failure in childhood inflammatory bowel disease

Around 1 in 4 patients with inflammatory bowel disease (IBD) present in childhood, the majority around the time of their pubertal growth spurt. This presents challenges over and above those of managing IBD in adults as this period is a time of dramatic psychological and physical transition for a child. Growth and nutrition are key priorities in the management of adolescents and young adults with IBD. Growth failure in IBD is characterized by delayed skeletal maturation and a delayed onset of puberty, and is best described in terms of height-for-age standard deviation score (Z score) or by variations in growth velocity over a period of 3-4 months. Growth failure is common at presentation in Crohn's disease (CD), but less common in ulcerative colitis (UC). The etiology of growth failure is multifactorial. Principal determinants, however, include the inflammatory process per se, with proinflammatory cytokines (e.g., IL-1, IL-6) being directly implicated. Furthermore, poor nutrition and the consequences of prolonged corticosteroid use also contribute to the significant reduction in final adult height of almost 1 in 5 children. Initially a prompt, where possible steroid-free, induction of remission is indicated. The ideal is then to sustain a relapse-free remission until growth is complete, which is often not until early adulthood. These goals can often be achieved with a combination of exclusive enteral nutrition (EEN) and early use of immunosuppressants. The advent of potent and efficacious biological agents considerably improves the range of growth-sparing interventions available to children around puberty, although well-timed surgery remains another highly effective means of achieving remission and significant catch-up growth. We carried out a systematic review of publications to identify the best available evidence for managing growth failure in children with IBD. Despite the paucity of high-quality publications, sufficient data were available in the literature to allow practical, evidence-based where possible, management guidelines to be formulated. Although there is clear evidence that exclusive enteral nutrition achieves mucosal healing, its effect on growth has only been assessed at 6 months. In contrast to corticosteroids, EEN has no negative effect on growth. Corticosteroids remain the key therapy responsible for medication-induced growth impairment, although the use of budesonide in selected patients may minimize the steroid effect on dividing growth plates. Immunosuppressants have become a mainstay of treatment in children with IBD, and are being used earlier in the disease course than ever before. However, there are currently no long-term data reporting better growth outcome if these agents are introduced very soon after diagnosis. In comparison, recent data from a large prospective trial of infliximab in children with moderate to severe CD suggested significant catch-up growth during the first year of regular infusions. The only other intervention that has documented clear catch-up growth has been surgical resection. Resection of localized CD, in otherwise treatment-resistant children, early in the disease process achieves clear catch-up growth within the next 6 months. There are no data available that growth hormone improves final adult height in children with CD. In conjunction with expert endocrinological support, pubertal delay, more common in boys, may be treated with parenteral testosterone if causing significant psychological problems. The optimal management of children and adolescents requires a multidisciplinary approach frequently available within the pediatric healthcare setting. Dedicated dietetic support, along with nurse-specialist, child psychologist, and with closely linked medical and surgical care will likely achieve the best possible start for children facing a lifetime of chronic gut disease

Syndecan-1 (CD138) expression in normal and coeliac mucosa using monoclonal MCA2459GA.

<p><b>A</b>. Expression within normal intestine, showing syndecan-1 localises predominantly to the basolateral epithelial surface (original magnification ×10). <b>B</b>. Higher power view (x40) of normal duodenum, showing similar epithelial localisation plus the presence of scattered syndecan-1+ plasma cells within the lamina propria. <b>C,D</b>. Low power view (x10) of 2 cases of coeliac disease, showing retention of epithelial staining similar to controls, plus additionally a dense confluent expression within the upper lamina propria. <b>E,F</b>. Higher power view (x40) of the subepithelial region in these cases. The lamina propria aggregates consist of cells with dense membrane staining (plasma cells), cells with recognisable nuclei and punctate cytoplasmic staining and scattered syndecan-1+ debris (shed ectodomains). <b>G</b>–<b>J</b>. Findings within the same case in two biopsies showing contrasting areas with containing areas with preserved villous architecture (G,H) and villous atrophy (I,J). G,I are at low power (x10), H,J at high power (x 40). The biopsy with villous atrophy shows dense subepithelial syndecan-1 syncytial expansion in comparison to that with preserved villous architecture. Mucosal syndecan-1 expression from all normal controls and coeliac patients studied is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106005#pone.0106005.s004" target="_blank">Figure S4</a>.</p

Quantitative analysis of mRNA expression for IL-17α, IL-6, IL-23α and TGF-β1 in biopsies from 10 children with coeliac disease and 8 histologically normal controls.

<p>For each cytokine, the left column represents results for coeliac disease and the right column results from controls. Il-17A and IL-6 showed significant increase in coeliac disease. Original data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106005#pone.0106005.s007" target="_blank">Figure S7</a>.</p

Representative staining for sulphated GAGs, HSPG and Δ-HSPG stubs in control (top row) and coeliac cases (middle and bottom row).

<p>All views are at same magnification (original ×40). In the control biopsies, there is strong expression of heparan sulphate and sulphated GAGs within the subepithelial basement membrane and on the basolateral surface of epithelial cells, most marked towards the villous tip. The coeliac biopsies show loss of HSPG and sulphated GAG expression within the basement membrane and from the basolateral epithelial surface. By contrast there is maintained expression within the lamina propria. Additional images from each of the patients studied can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106005#pone.0106005.s001" target="_blank">Figures S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106005#pone.0106005.s002" target="_blank">S2</a>. Quantitative staining intensity data derived from colour deconvoluted images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106005#pone.0106005.s003" target="_blank">Figure S3</a>) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106005#pone-0106005-g002" target="_blank">Figure 2</a>.</p