Paraic McGrogan

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.

The use of exclusive enteral nutrition for induction of remission in children with Crohn’s disease demonstrates that disease phenotype does not influence clinical remission

Background  Exclusive enteral nutrition (EEN) achieves variable remission rates in patients with Crohn’s disease (CD).

Analysis of the influence of OCTN1/2 variants within the IBD5 locus on disease susceptibility and growth indices in early onset inflammatory bowel disease

Background and aims: The OCTN1 (SLC22A4 1672C→T) and OCTN2 (SLC22A5 −207G→C) variants within the IBD5 locus have been associated with susceptibility to adult onset Crohn’s disease (CD), but their contribution in children has not been examined. Methods: These OCTN1/2 variants and IBD5 marker single nucleotide polymorphisms (SNPs) (IGR2096a_1, IGR2198a_1, and IGR2230a_1) were examined in 299 Scottish children (200 with CD, 74 with ulcerative colitis (UC), and 25 with indeterminate colitis (IC)), together with 502 parents (for transmission disequilibrium testing) and 256 controls. Results: All SNPs were in strong linkage disequilibrium (D′ >0.94). TDT analysis showed association of the OCTN1 variant with inflammatory bowel disease (IBD) (p = 0.01) and CD (p = 0.04). Allele frequencies of the OCTN1/2 variants were significantly higher in IBD/CD cases (p<0.04). The homozygous mutant OCTN1/2 haplotype was increased in IBD (24.3% v 16.1%, p = 0.02) and UC (28.2% v 16.1%, p = 0.02) compared with controls. The OCTN1/2 variants were not independent of the background IBD5 risk haplotype in conferring disease susceptibility. Unifactorial analysis in CD patients showed that carriage of the TC haplotype was associated with lower weight, height, and BMI centile (<9th centile) at diagnosis (weight: 87.9% v 67.3% (p = 0.002), odds ratio (OR) = 3.52 (95% confidence interval, 1.51 to 8.22); height: 84.1% v 68.4% (p<0.05), OR = 2.44 (1.00 to 5.99); BMI: 79.6% v 61.1% (p = 0.02), OR = 2.49 (1.14 to 5.44)), and lower weight centile at follow up (87.5% v 64.6% (p = 0.03), OR = 3.83 (1.03 to 14.24)). Multifactorial binary logistic regression analysis confirmed association of the TC haplotype with lower weight centile at diagnosis (p = 0.02, OR = 3.41 (1.20 to 9.66)). Conclusions: These data implicate variants within the IBD5 haplotype, as determinants of disease susceptibility and growth indices in early onset IBD. The OCTN1/2 variants remain potential positional candidate genes, but require further analysis.

IL23R Arg381Gln is associated with childhood onset inflammatory bowel disease in Scotland

The discovery of NOD2/CARD15 as the first susceptibility gene in Crohn’s disease has contributed significantly to a fundamental change in the direction of basic research in inflammatory bowel disease (IBD), triggering renewed interest in the integrity of the innate immune response in IBD and appropriate orchestration of a subsequent adaptive immune response.1,2 More widely, in all complex diseases this finding in 2001 provided a much welcomed and needed proof of principle for non-parametric linkage analysis. Another study with major implications for the pathogenesis of Crohn’s disease as well as for investigation of all complex disorders has recently been published.3 The North American consortium performed an association study testing 308 332 markers spanning the entire genome in 567 patients with ileal Crohn’s disease and 571 controls of non-Jewish European ancestry. Of the three markers reported to retain significance after stringent Bonferroni correction, two were located in the NOD2/CARD15 gene. The third marker (rs11209026) was a non-synonymous variant in the interleukin-23 receptor (IL23R) gene on chromosome 1p31. Replication was obtained in the index paper in a Jewish ancestry case-control analysis of patients with Crohn’s disease by transmission disequilibrium testing in 883 families with offspring affected by IBD and in a combined case-control analysis …

Decline in Presumptively Protective Gut Bacterial Species and Metabolites Are Paradoxically Associated with Disease Improvement in Pediatric Crohn’s Disease During Enteral Nutrition

Background:The gut microbiota is implicated in the pathogenesis of Crohn’s disease (CD). Exclusive enteral nutrition (EEN) is a successful treatment, but its mode of action remains unknown. This study assessed serial changes in the fecal microbiota milieu during EEN. Methods:Five fecal samples were collected from CD children: 4 during EEN (start, 15, 30, end EEN approximately 60 days) and the fifth on habitual diet. Two samples were collected from healthy control subjects. Fecal pH, bacterial metabolites, global microbial diversity abundance, composition stability, and quantitative changes of total and 7 major bacterial groups previously implicated in CD were measured. Results:Overall, 68 samples were from 15 CD children and 40 from 21 control subjects. Fecal pH and total sulfide increased and butyric acid decreased during EEN (all P < 0.05). Global bacterial diversity abundance decreased (P < 0.05); a higher degree of microbiota composition stability was seen in control subjects than in CD children during EEN (at P ⩽ 0.008). Faecalibacterium prausnitzii spp concentration significantly decreased after 30 days on EEN (P < 0.05). In patients who responded to EEN, the magnitude of the observed changes was greater and the concentration of Bacteroides/Prevotella group decreased (P < 0.05). All these changes reverted to pretreatment levels on free diet, and EEN microbiota diversity increased when the children returned to their free diet. Conclusions:EEN impacts on gut microbiota composition and changes fecal metabolic activity. It is difficult to infer a causative association between such changes and disease improvement, but the results do challenge the current perception of a protective role for F. prausnitzii in CD.

A British Society of Paediatric Gastroenterology, Hepatology and Nutrition survey of the effectiveness and safety of adalimumab in children with inflammatory bowel disease

Aliment Pharmacol Ther 2011; 33: 946–953

The role of pro-inflammatory cytokines in inflammatory bowel disease growth retardation

ABSTRACT Childhood inflammatory bowel disease (IBD) especially those with Crohn disease is commonly complicated by faltering growth and pubertal delay. Pro-inflammatory cytokines are often elevated in IBD and may affect linear growth and puberty either systemically or at the level of the growth plate. Further study of the underlying mechanisms of the deleterious effects of cytokines on the growth plate may improve management of faltering growth in childhood IBD. Well-controlled clinical studies of the respective effect of nutritional support, immunomodulatory therapy, biological agents and growth and puberty promoting agents on managing faltering growth also require further attention.

Extensive modulation of the fecal metagenome in children With Crohn’s disease during exclusive enteral nutrition

OBJECTIVES:Exploring associations between the gut microbiota and colonic inflammation and assessing sequential changes during exclusive enteral nutrition (EEN) may offer clues into the microbial origins of Crohn’s disease (CD).METHODS:Fecal samples (n=117) were collected from 23 CD and 21 healthy children. From CD children fecal samples were collected before, during EEN, and when patients returned to their habitual diets. Microbiota composition and functional capacity were characterized using sequencing of the 16S rRNA gene and shotgun metagenomics.RESULTS:Microbial diversity was lower in CD than controls before EEN (P=0.006); differences were observed in 36 genera, 141 operational taxonomic units (OTUs), and 44 oligotypes. During EEN, the microbial diversity of CD children further decreased, and the community structure became even more dissimilar than that of controls. Every 10 days on EEN, 0.6 genus diversity equivalents were lost; 34 genera decreased and one increased during EEN. Fecal calprotectin correlated with 35 OTUs, 14 of which accounted for 78% of its variation. OTUs that correlated positively or negatively with calprotectin decreased during EEN. The microbiota of CD patients had a broader functional capacity than healthy controls, but diversity decreased with EEN. Genes involved in membrane transport, sulfur reduction, and nutrient biosynthesis differed between patients and controls. The abundance of genes involved in biotin (P=0.005) and thiamine biosynthesis decreased (P=0.017), whereas those involved in spermidine/putrescine biosynthesis (P=0.031), or the shikimate pathway (P=0.058), increased during EEN.CONCLUSIONS:Disease improvement following treatment with EEN is associated with extensive modulation of the gut microbiome.

The aetiology and impact of malnutrition in paediatric inflammatory bowel disease

Disease-associated undernutrition of all types is very common in paediatric inflammatory bowel disease (IBD). Recent weight loss remains one of the triad of clinical manifestations and a cornerstone for the diagnosis of Crohns disease (CD), although significantly fewer patients now present as being underweight. Recent evidence suggests that the introduction of medical treatment will quickly restore body weight, although this does not reflect concomitant changes in body composition. CD children present with features of nutritional cachexia with normal fat stores but depleted lean mass. Poor bone health, delayed puberty and growth failure are additional features that further complicate clinical management. Suboptimal nutritional intake is a main determinant of undernutrition, although activation of the immune system and secretion of pro-inflammatory cytokines exert additional independent effects. Biochemically low concentrations of plasma micronutrients are commonly reported in IBD patients, although their interpretation is difficult in the presence of an acute phase response and other indices of body stores adequacy are needed. Anaemia is a common extraintestinal manifestation of the IBD child. Iron-deficient anaemia is the predominant type, with anaemia of chronic disease second. Decreased dietary intake, as a result of decreased appetite and food aversion, is the major cause of undernutrition in paediatric IBD. Altered energy and nutrient requirements, malabsorption and increased gastrointestinal losses are additional factors, although their contribution to undernutrition in paediatric CD needs to be studied further.