Gamal Mahdi

Microbiota of De-Novo Pediatric IBD: Increased Faecalibacterium Prausnitzii and Reduced Bacterial Diversity in Crohn ' s But Not in Ulcerative Colitis

OBJECTIVES:The gastrointestinal microbiota is considered important in inflammatory bowel disease (IBD) pathogenesis. Discoveries from established disease cohorts report reduced bacterial diversity, changes in bacterial composition, and a protective role for Faecalibacterium prausnitzii in Crohns disease (CD). The majority of studies to date are however potentially confounded by the effect of treatment and a reliance on established rather than de-novo disease.METHODS:Microbial changes at diagnosis were examined by biopsying the colonic mucosa of 37 children: 25 with newly presenting, untreated IBD with active colitis (13 CD and 12 ulcerative colitis (UC)), and 12 pediatric controls with a macroscopically and microscopically normal colon. We utilized a dual-methodology approach with pyrosequencing (threshold >10,000 reads) and confirmatory real-time PCR (RT-PCR).RESULTS:Threshold pyrosequencing output was obtained on 34 subjects (11 CD, 11 UC, 12 controls). No significant changes were noted at phylum level among the Bacteroidetes, Firmicutes, or Proteobacteria. A significant reduction in bacterial α-diversity was noted in CD vs. controls by three methods (Shannon, Simpson, and phylogenetic diversity) but not in UC vs. controls. An increase in Faecalibacterium was observed in CD compared with controls by pyrosequencing (mean 16.7% vs. 9.1% of reads, P=0.02) and replicated by specific F. prausnitzii RT-PCR (36.0% vs. 19.0% of total bacteria, P=0.02). No disease-specific clustering was evident on principal components analysis.CONCLUSIONS:Our results offer a comprehensive examination of the IBD mucosal microbiota at diagnosis, unaffected by therapeutic confounders or changes over time. Our results challenge the current model of a protective role for F. prausnitzii in CD, suggesting a more dynamic role for this organism than previously described.

Genotype-phenotype analysis in childhood-onset Crohn's disease: NOD2/CARD15 variants consistently predict phenotypic characteristics of severe disease.

Introduction: The incidence of early‐onset CD in Scotland is among the highest worldwide. Three single nucleotide polymorphisms (SNPs) R702W, G908R and Leu1007finsC in the NOD2/CARD15 gene predispose to adult CD. We investigated the contribution of these variants to disease susceptibility and phenotype in the Scottish early‐onset IBD population. Patients and Methods: 906 individuals including 247 Scottish IBD patients aged <16 years at diagnosis, 414 parents and 245 controls were genotyped. Transmission disequilibrium testing (TDT), case‐control analysis and detailed genotype‐phenotype analysis were performed. Results: The Leu1007finsC variant was associated with susceptibility to CD by case‐control (4.2% versus. 1.4%, P = 0.01) and TDT analysis (P = 0.006). The Population Attributable Risk (PAR) for the 3 NOD2/CARD15 mutations was 7.9%. Carriage of NOD2/CARD15 variants was associated with, at diagnosis: decreased albumin (31.0% versus. 9.0%, P = 0.001) and raised CRP (25% versus. 9.5%, P = 0.04) and at follow up: need for surgery (39.5% versus. 12.8%, P = 0.0002) jejunal involvement (50% versus. 18.4%, P = 0.01) jejunal and ileal involvement (50% versus. 10.7%, P = 0.009), raised CRP (57.1% and 12.8%, P = 0.0009), lower weight/height centile (75.0% versus. 20.2%, P = 0.03, 50.0% versus. 16.0%, P = 0.001 respectively) and stricturing disease (45.5% versus. 19.4%, P < 0.05). Multifactorial analysis demonstrated carriage was associated with need for surgery (P = 0.004, OR 4.9 [1.5‐14.7]). Conclusions: These NOD2/CARD 15 variants in the Scottish early onset CD population have a definite, albeit relatively small contribution to CD susceptibility (PAR 7.9%) but a major impact on phenotype. In particular NOD2/CARD15 variants are strongly associated with several markers of disease severity in pediatric CD, notably need for surgery.

Autophagy Gene ATG16L1 Influences Susceptibility and Disease Location but Not Childhood-Onset in Crohn's Disease in Northern Europe

Background: The rs2241880A/G variant of the ATG16L1 gene has been associated with susceptibility to ileal Crohns disease (CD) in adults. Our aim was to assess whether germline variation of ATG16L1 acts as an independent determinant of susceptibility to childhood‐onset CD in the high‐incidence Scottish population. Methods: In all, 2195 subjects (361 children (inflammatory bowel disease [IBD] diagnosis <17 years), their parents (n = 634), 855 adult IBD patients, and 345 controls were genotyped. Case‐control analysis was powered to detect effect sizes with an odds ratio (OR) >1.39 in pediatric CD. Case‐control analysis, transmission disequilibrium testing (TDT), analysis of variance (ANOVA) of growth parameter z‐scores, Kruskal–Wallis test (age at diagnosis), and multifactorial genotype–phenotype analysis (Montreal classification) were performed. 7.8% of pediatric CD patients and 37.2% of adult CD patients had pure ileal disease. Results: We confirmed the association of the rs2241880G‐allele with adult‐onset CD (60.7% versus controls 53.9%, P = 0.01, OR 1.32, 95% confidence interval [CI] 1.07–1.63) in contrast to childhood‐onset CD (54.1% versus controls, P = 0.95, OR 1.01, 95% CI 0.80–1.26). TDT analysis was negative. Genotype–phenotype analysis demonstrated an association of pure ileal disease with the rs2241880G‐allele (P = 0.02, OR 1.34, 95% CI 1.03–1.74). Using binary logistic regression analysis we confirmed the effect of rs2241880 genotype (GG) on ileal disease versus colonic disease (P = 0.03, OR 2.43, 95% CI 1.05–5.65). ATG16L1 genotype did not influence age at CD diagnosis. ANOVA of z‐scores of height, weight, and body mass index (BMI) at CD diagnosis in children showed no association with genotype. Conclusions: The ATG16L1 variant is associated with susceptibility to adult CD in Scotland, but not early‐onset disease. These contrasting effects are primarily driven by differences in disease location between early‐onset and adult‐onset disease.

The Microaerophilic Microbiota of De-Novo Paediatric Inflammatory Bowel Disease: The BISCUIT Study

Introduction Children presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study aetiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. We set out to study these and other microaerophilic bacteria in de-novo paediatric IBD. Patients and Methods 100 children undergoing colonoscopy were recruited including 44 treatment naïve de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis. Results 129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312). Conclusions This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon. Trial Registration This study is publically registered on the United Kingdom Clinical Research Network Portfolio (9633).

Contribution of the NOD1/CARD4 insertion/deletion polymorphism +32656 to inflammatory bowel disease in Northern Europe

Background: NOD1/CARD4 and NOD2/CARD15 are both intracellular pattern‐recognition receptors. The NOD1/CARD4 gene lies within a previously described inflammatory bowel disease (IBD) locus (7p14). An association has been suggested between the NOD1/CARD4+32656 deletion*1 variant of a complex deletion*1/insertion*2 polymorphism and IBD in 1 recent study in Europe. Our aim was to assess the influence of NOD1/CARD4+32656 on disease susceptibility and phenotype in the Scottish and Swedish IBD populations. Methods: A total of 3,962 individuals (1,791 IBD patients, 522 parents, 1,649 healthy controls) from 2 independent populations (Scotland and Sweden) were genotyped for NOD1/CARD4+32656 A/C by TaqMan and direct sequencing. Case‐control, Transmission Disequilibrium Testing (TDT) and detailed genotype–phenotype (Montreal) analyses were performed. The case‐control analysis had 80% power to detect an effect size of odds ratio (OR) 1.21 for IBD. Results: In case‐control analyses in Scottish and Swedish patients, none of the genotypes studied in IBD, Crohns disease (CD) or ulcerative colitis (UC), differed significantly from controls (deletion*1 allelic frequency 73.9%, 73.6%, 73.9%, and 73.6%, respectively: all P > 0.8). No epistatic interaction with NOD2/CARD15 was seen for CD susceptibility. TDT analysis in our Scottish early onset cohort was negative. Conclusions: This variant allele of NOD1/CARD4+32656 is not associated with a strong effect on susceptibility to IBD in children and adults in Northern Europe. A gene‐wide haplotype‐based approach may be preferable to analysis of individual variants to assess the contribution of the NOD1/CARD4 gene to IBD. (Inflamm Bowel Dis 2007)

Filaggrin loss-of-function variants are associated with atopic comorbidity in pediatric inflammatory bowel disease

Background: Pediatric inflammatory bowel disease (IBD) has a high prevalence of coexistent atopy. Filaggrin (FLG) loss‐of‐function variants (null‐alleles) are associated with eczema and asthma in association with eczema. The aim was to assess the contribution of FLG null‐alleles to pediatric IBD susceptibility and to coexistent atopy (eczema, asthma, allergic rhinitis, or food allergy). Methods: FLG variants (R501X and 2282del4) were genotyped in 403 children with IBD, 683 parents, and 996 population controls. Results: In all, 11% of IBD patients carried at least 1 FLG null‐allele compared to 11% of population controls (P > 0.4). Carriage of 1 or more null‐alleles in patients with atopy (present in 52% of IBD patients) differed from IBD patients without atopy (14% versus 6%, P = 0.01; odds ratio [OR] 2.4, 95% confidence interval [CI] 1.2–5.1). The effect of FLG null‐alleles was strongest for eczema (19% versus 7%, P = 0.0003; OR 3.3, 95% CI 1.7–6.6) and food allergy (28% versus 8%, P = 0.0001; OR 4.5, 95% CI 2.0–10.0). The presence of more than 1 atopic disease tended to increase the associated OR: eczema + asthma (23% versus 7%, P = 0.001; OR 3.9, 95% CI 1.6–9.1), eczema + asthma + allergic rhinitis (29% versus 7%, P = 0.0006; OR 5.4, 95% CI 1.9–15.4) and eczema + asthma + allergic rhinitis + food allergy (45% versus 6%, P < 10−4; OR 12.2, 95% CI 3.2–46.3). Logistic regression analysis of IBD cases confirmed the association of carriage of an FLG null‐allele with atopy (P = 0.01; OR 2.4, 95% CI 1.2–5.1) and co‐occurrence of different forms of atopy (P = 0.003; OR 3.5, 95% CI 1.5–8.1). Conclusions: Filaggrin null‐alleles have no effect on IBD susceptibility but contribute to coexistent eczema and food allergy. (Inflamm Bowel Dis 2009)

Sa1164 Exclusive Enteral Nutrition Optimizes Growth Outcomes Versus Corticosteroid Therapy for Pediatric Crohn's Disease

Background: The pathogenesis of IBD involves a genetically-determined dysregulation of mucosal immunity in response to environmental factors such as the gut microbiota. Multiple studies have shown the bacterial microbiota to be dysbiotic with an increase in Proteobacteria and a decrease in taxonomic richness. Serologic evidence in patients with IBD as well as mechanistic studies in murine models suggests that gut fungi, collectively referred to as the mycobiome, may also play a role in the pathogenesis of IBD. In this study, we utilized recently described analytic platforms to compare members of the gut microbiota from all three domains of life, Bacteria, Archaea, and Eukaryota in patients with IBD to healthy human subjects. Methods: A single stool sample was collected from pediatric patients with either Crohns disease (CD) or ulcerative colitis (UC). DNA was extracted using the PSP DNA extraction kit. Pyrosequencing was carried out using barcoded primers. Domainspecific16S rRNA gene primers were used to analyze bacteria and archaea and ITS primers were used to analyze fungi. Sequences obtained were decoded using the QIIME pipeline. The results were compared to a cohort of pediatric control subjects from a previously published study (Hoffmann C. et al. PloS ONE 2013 8(6):e66019). In the IBD cohort, clinical records were obtained and disease activity was measured by the Pediatric Crohn Disease Activity Index (PCDAI). Results: Stool samples were collected from 32 pediatric IBD patients. The majority of these patients (81%) had CD. Four patients had UC and 2 patients had indeterminate colitis. Similar to previously published studies, IBD patients demonstrated decreased bacterial diversity. Although ITS sequencing showed that fecal DNA from patients with IBD and healthy subjects had a similar number of fungal reads, the two groups clustered separately on a principal coordinate analysis (Figure 1) suggesting differences in the mycobiome. Both Pichia jadinii and Candida parapsilosis were significantly more abundant in IBD patients (p = 0.0034 and P=0.00038, respectively). By contrast, an unclassified Candida taxon (OTU EU490138) was more abundant in healthy subjects (p=0.0025). There were no statistically significant differences in the archaeal microbiota. Within the CD patients, age, race, current medications, and PCDAI were not correlated with bacterial microbiota composition. Conclusions: Pediatric IBD is associated with an alteration in the gut mycobiota with increased proportions of both Pichia jadinii and Candida parapsilosis. Although some species within the Pichia genus are known to be pathogenic in immunocompromised hosts, additional studies will be needed to determine whether or not they play a role in the pathogenesis of IBD in humans.

M1138 Epidemiological Risk Factors for Childhood Onset Inflammatory Bowel Disease in Scotland: A Case-Control Study

G A A b st ra ct s care provider, emergency rooms (ER) may represent a significant point of contact with the healthcare system, and may represent poor maintenance care for their illness . National trends in ER visits by IBD patients have not been explored previously. Methods: We used data from the National Hospital Ambulatory Medical Care Survey (NHAMCS), a complex survey maintained by the National Center for Health Statistics that includes information from hospital emergency departments maintained by the National Center for Health Statistics. IBD-related ER visits were identified using free text or ICD-9-CM codes for Crohns disease or ulcerative colitis among the reasons for visit for the encounter. Trends in number of annual IBD-related ER visits were analyzed during four time periods 1994-96, 1997-99, 2000-02 and 2003-05. Results: There were a total estimated 28,752 annual ER visits related to IBD during 1994-96 which increased to an estimated 76,374 annual visits during 200305 (+165 %). The increase was much greater for CD (16,575 to 61,598) than UC (12,178 to 14,776), though these estimates were derived from small numbers of patients. During the time period 2003-05, over half the ER visits were from female patients (60%), with over three-quarters being from patients younger than age 50 (81%). Uninsured patients comprised about 17% of visits and patients belonging to non-white races comprised about 26% of visits, a proportion than increased from 1994-96 (11.8%, p < 0.0001). Just fewer than half the patients were admitted to the hospital. Adjusting for US population estimates revealed an average of 26 IBD-related ER visits per 100,000 population. Comparing the ER visits to ambulatory IBD visits showed an increase in rate from 25.5 ER visits per 1000 ambulatory IBD encounters in 1994-96 to 44.6 ER visits per 1000 ambulatory IBD encounters in 200305 (p <0.0001). This proportion was higher for CD (58.1 / 1000) compared to UC (21.4 / 1000) during the period 2003-05. Conclusion: There is increasing utilization of emergency rooms among patients with IBD with ER visits representing a rising proportion of ambulatory IBD encounters. Reasons underlying this phenomenon warrant investigation.

W1740 Loss-of-Function Variants of the Epithelial Barrier Protein Filaggrin Predispose to Marked Atopic Co-Morbidity in Paediatric Inflammatory Bowel Disease

Background: The gastric epithelial cell layer is a pivotal physical defence mechanism necessary for the integrity of gastric mucosa. Disruption of cell junctions and cell cytoskeleton, key elements in the efficiency of this barrier, leads to the initiation of gastric mucosal injury. Isoflavones, selective estrogen receptor modulators, exert their effects by selectively binding to estrogen receptors (ER), particularly ERβ, and are involved in the control of gastric mucosal homeostasis. ERβ are expressed mainly in the human gastric antrum and their role in the gastric physiological functions is still unknown. Aim: In this study we evaluated the effects of isoflavones, administrated as soy germ pasta, on gastric mucosal gene expression through an extensive microarray analysis in healthy individuals. Materials and Methods: Biologically active isoflavones (aglycones) were administrated using a 2% soy-germ pasta (SP) (Aliveris srl, Perugia, Italy). Equivalent conventional pasta (CP) was used as a control. 5 healthy volunteers (3 F, age: 30±3 years) consumed a standard diet containing CP (80gr/ daily) for 1 week followed by SP (80gr/daily; approx. 33mg of isoflavones) for 1 week. An upper endoscopy with biopsy was performed at the end of each week. Gastric samples were collected for microarray analysis and qRT-PCR. Biotin-labeled cRNA was obtained and hybridized to an Affymetrix HG-U133 Plus2.0 GeneChipArray according to standard procedures. Results: Microarray analysis highlighted a dramatic difference in gene expression after consumption of SP compared to CP. The gene list obtained was clearly efficient in separating the two groups at the end of the two different diets. Functional gene classification was performed based on different sources (GeneOntology, KEGG, SOURCE) with integrated information from the literature and 58 genes were included in 8 different gene categories: cytoskeleton, organization and biogenesis, cell-cell communication, contractility, signal transduction, stress response, transcription and other/unknown. Interestingly, one of these genes, PGTDS, which encodes an enzyme involved in prostaglandin synthesis, was 5-fold upregulated after SP administration compared to CP. Of note, while the expression of ERα and ERβ was not affected, 10 out of the 58 modulated genes contained one estrogen responsive element. Conclusion: This is the first evidence that isoflavones modulate the expression of genes involved in cell-cell and cell-extra cellular matrix adhesion process in human gastric mucosa, suggesting a potential role of isoflavones in gastric mucosal defense.