Deenaz Zaidi

Mucosal Barrier Depletion And Loss Of Bacterial Diversity Are Primary Abnormalities In Paediatric Ulcerative Colitis

BACKGROUND AND AIMS Ulcerative colitis [UC] is associated with colonic mucosa barrier defects and bacterial dysbiosis, but these features may simply be the result of inflammation. Therefore, we sought to assess whether these features are inherently abrogated in the terminal ileum [TI] of UC patients, where inflammation is absent. METHODS TI biopsies from paediatric inflammatory bowel disease [IBD] subsets [Crohns disease [CD; n = 13] and UC [n = 10]], and non-IBD disease controls [n = 12] were histologically graded, and alcian blue/periodic acid-Schiff stained biopsies were quantified. The mucosal barrier was assessed for mucin [MUC2], immunoglobulin [Ig]A, IgG, and total bacteria (fluorescence in-situ hybridisation [FISH probe EUB338]) by immunofluorescence. The regulation of mucin secretion was investigated by NLRP6 gene expression and immunofluorescence. The composition of the active mucosa-associated microbiota was explored by sequencing the 16S rRNA amplicon generated from total RNA. RESULTS Despite the absence of ileitis, UC patients displayed ileal barrier depletion illustrated by reductions in mucin-containing goblet cells and mucin production and altered epithelial NLRP6 expression. In both CD patients with ileitis and UC patients with normal histology, bacteria coated with IgA and IgG penetrated the TI mucin layer. Biopsy 16S rRNA sequencing revealed a reduction in α-diversity by three methods [Shannon, Simpson, and Equitability indices] between UC and non-IBD paediatric patients. CONCLUSIONS These findings suggest an underlying defect in the UC-afflicted intestinal tract even in the absence of inflammation, implicating barrier and microbial changes as primary abnormalities in UC that may play a causative role in disease development.

Increased Epithelial Gap Density in the Noninflamed Duodenum of Children With Inflammatory Bowel Diseases

Objectives: Inflammatory bowel diseases (IBD) present commonly in childhood, with unknown etiology, but an important role for the epithelial lining is suggested. Epithelial cell extrusion, measured by counting gaps between epithelial cells, is higher in adult patients with Crohn disease (CD) than in controls. Our objectives were to compare epithelial gaps in the duodenum of IBD and non-IBD pediatric patients, to study the correlation between epithelial gaps, inflammation, and disease activity, and identify potential mechanisms. Methods: Epithelial gap density of the duodenum was evaluated using probe-based confocal laser endomicroscopy in 26 pediatric patients with IBD (16 CD, 10 ulcerative colitis [UC]) and 17 non-IBD controls during endoscopy. Epithelial gaps were correlated with serum inflammatory markers, disease activity indices, and intraepithelial lymphocytes. A panel of 10 inflammatory cytokines and expression of TNFAIP3 (A20; inhibits NF–&kgr;&bgr;-induced inflammation) were analyzed in duodenal and ileal biopsies. Results: Confocal imaging showed significantly higher epithelial gap density in patients with IBD, including UC. Interleukin (IL)-2 and IL-8 were higher in duodenal but not ileal biopsies of patients with UC. No significant correlation was present between C-reactive protein, erythrocyte sedimentation rate, disease activity indices, and epithelial gaps in patients with UC. In patients with CD, C-reactive protein positively correlated with epithelial gaps. A20 expression in the duodenum was unchanged among non-IBD and IBD cases. Conclusions: Duodenal epithelial gaps are increased in pediatric patients with IBD (including UC) but are unrelated to inflammation. This suggests that altered epithelial barrier is an important systemic feature of pediatric IBD and is not only secondary to inflammation.

An update on travelers' diarrhea.

Purpose of review Travelers’ diarrhea, affecting millions of travelers every year globally, continues to be a leading cause of morbidity despite advances in vaccination, prevention, and treatment. Complications of travelers’ diarrhea often present to gastroenterologists and some patients followed by gastroenterologists are at higher risk of developing travelers’ diarrhea. This review will provide an update on recent progress made in the epidemiology, pathogenesis, diagnosis, prevention, and treatment of travelers’ diarrhea. Recent findings Most causes of travelers’ diarrhea remain bacterial, but newly recognized pathogens are emerging. Patient-related and travel-related factors affect disease development risk and should guide prophylaxis and treatment. Although specific vaccines are being developed, they have not yet had a major impact on travelers’ diarrhea, and understanding their roles and limitations is especially important. Prophylaxis and treatment of populations at risk (children, chronically ill patients, and those on immunosuppressive medications) remain challenging and require a tailored approach. Summary Travelers’ diarrhea will continue to challenge patients and physicians despite the use of sanitation advice, prophylactic vaccines, and treatment with antibiotics. Effects may extend beyond the time of travel, such as postinfectious complications and exacerbation of preexisting disease. Future research should focus on novel strategies for reducing exposure to pathogens, vaccine development, early detection, and targeted treatments.

Tumor necrosis factor α-induced protein 3 (A20) is dysregulated in pediatric Crohn disease

Purpose A significant feature of pediatric inflammatory bowel diseases (IBD), which include Crohn disease (CD), and ulcerative colitis (UC), is failure to suppress inflammation. The inability to regulate inflammation renders a major challenge toward establishing effective treatments in IBD. Nuclear factor kappa-light-chain-enhancer of activated B-cells-induced inflammation is inhibited by A20 through interactions with TAX1BP1 (Tax1-binding protein 1) and A20-binding inhibitor of NF-κβ activation (ABIN)-1 (A20 binding and inhibitor of NF-κβ) and upon phosphorylation by inhibitor of nuclear factor kappa-β kinase subunit beta (IKKβ), which stabilizes it. We hypothesized that dysregulation of A20 is an important factor in uncontrolled inflammation in pediatric IBD. Patients and methods Gene expression of A20, IKKβ, ABIN-1, TAX1BP1, A20 protein, cytokine levels, and A20 phosphorylation was analyzed in the terminal ileum (TI) of 39 patients (14 non-IBD, 15 CD, and 10 UC). A20 expression and protein in T-84 cells and ex vivo biopsies of patients were measured after treatment with Escherichia coli strains or tumor necrosis factor (TNF)-α. Results TNF-α levels and A20 expression were increased in the TI of CD patients. A20 protein levels and ABIN-1 expression were low, TAX1BP1 expression was high, and IKKβ was unchanged. A20 expression positively correlated with biopsy TNF-α levels and inflammatory markers in CD patients. A20 phosphorylation appeared lower in CD patients. A20 expression in TI biopsies from CD patients and T84 cells was triggered with E. coli, strain LF82, while A20 protein levels remained unchanged. Conclusion We describe a potential mechanism related to failure of A20 to suppress inflammation in CD, characterized by high A20 expression and low A20 protein levels. The dysregulation of A20 is potentially due to alterations in ABIN-1, and infection with E. coli strain LF82 could affect the function and stability of A20. Our study signifies an important finding in A20 regulation in IBD, which prevents it from suppressing inflammation.

P-323 Immunoglobulin G Selectively Identifies Pathosymbionts in Paediatric Inflammatory Bowel Diseases

Background: Inflammatory bowel diseases (IBD) are a group of complex and multifactorial disorders with unknown etiology. Both environmental and genetic factors contribute to disease pathogenesis. This complexity is further exacerbated with an uncontrolled immune response to the gut microbiota. Given that immunoglobulin (Ig) G is formed in response to invasive microbes, we anticipated that bacteria bound by IgG are more likely to be virulent; their isolation could help identify such invasive strains and define mechanisms of immune activation. We hypothesized that host IgG immune response can be used to identify bacteria that may be involved in the pathogenesis of IBD. Methods: Aspirate washes from the terminal ileum of pediatric IBD (including Crohn disease [CD] and ulcerative colitis [UC]) and non-IBD controls were collected during endsocopy, stringently washed, and then fixed in paraformaldehyde. An aliquot was stored (presort) and the remaining washes were labeled with propidium iodide and anti-IgG fluorescent antibody to allow for identification of bacteria bound by IgG and those that are not. Using fluorescence-activated cell sorting (FACS), the samples were then fractioned for IgG bound (IgG+) and non-IgG bound (IgG−) bacteria. Some samples were also imaged by image cytometry to validate the FACS procedure. After sorting, DNA was extracted using beads and purified by phenol/chloroform. DNA was then analyzed by 16S sequencing using the Ilumina MiSeq platform. Results: A total of 36 washes from children without IBD (n = 10), and with CD (n = 17), and UC (n = 9), were suitable for analysis with DNA meeting quality control criteria. The ileal mucosa-associated microbiome composition of non-IBD and IBD groups did not differ significantly, with predominance of Firmicutes and Bacteroidetes. When sorted for IgG coated bacteria, the ratio of IgG+/IgG−bacteria (indicating taxa over-represented by IgG binding) were increased in CD and UC patients by 2- and 1.5-fold, respectively. Notably, the IgG+/IgG−ratio of Bacteroidetes and Proteobacteria taxa were increased in CD, Actinobacteria was increased in UC patients, and there was no changes in Firmicutes, the major phyla of the ileal mucosal microbiome. Although there was considerable inter-individual variation, at the family level, IgG binding favored Porphyromonadaceae (Bacteroidetes) and Enterobacteriaceae (Proteobacteria) in children with CD; Bifidobacteriaceae (Actinobacteria) and Barnesiellaceae (Bacteroidetes) in UC; and Clostridiaceae and Veillonellaceae (both Firmicutes) in non-IBD. Conclusions: IgG coating of mucosa associated bacteria in the ileum of IBD patients is elevated and is selective for unique phyla compared to non-IBD controls. Taxa identified by IgG include bacteria that have been associated with disease and virulence. Identification of individual microbes using this method could facilitate development of targeted diagnostic and therapeutic approaches for IBD.