Co-Development of Three Dietary Indices to Facilitate Dietary Intake Assessment of Pediatric Crohn's Disease Patients.

Literature on dietary behaviours of the pediatric Crohn's Disease (CD) population and the relationship between dietary intake and CD activity is limited. Three dietary indices were developed and tested to conduct dietary pattern analysis in pediatric patients with CD consuming a free diet following remission induction via exclusive enteral nutrition (n = 11). Index scores underwent descriptive and inferential analysis. The mean adjusted scores (out of 100) for the Pediatric Western Diet Index, Pediatric Prudent Diet Index, and Pediatric-Adapted 2010 Alternate Healthy Eating Index (PA2010-AHEI) were 29.82 ± 15.22, 34.25 ± 15.18, and 51.50 ± 11.69, respectively. The mean Western-to-Prudent ratio was 0.94 ± 0.55. A significant correlation (r = -0.71) and relationship (F[1, 9] = 9.04, P < 0.05, R2 = 0.501) between the Western-to-Prudent ratio and PA2010-AHEI was found. The results suggest participants were not following a Western or Prudent diet, and were consuming foods not captured by the indices. More research is needed to describe dietary intake of individuals with CD, validate dietary indices in diverse samples, and explore the utility of these indices in CD assessment and treatment. The co-authors hope this work will stimulate/inspire subsequent interprofessional, dietitian-led research on this topic.

Gut bacterial gene changes following pegaspargase treatment in pediatric patients with acute lymphoblastic leukemia.

Treatment of pediatric acute lymphoblastic leukemia (ALL) with pegaspargase exploits ALL cells dependency on asparagine. Pegaspargase depletes asparagine, consequentially affecting aspartate, glutamine and glutamate. The gut as a confounding source of these amino acids (AAs) and the role of gut microbiome metabolism of AAs has not been examined. We examined asparagine, aspartate, glutamine and glutamate in stool samples from patients over pegaspargase treatment. Microbial gene-products, which interact with these AAs were identified. Stool asparagine declined significantly, and 31 microbial genes changed over treatment. Changes were complex, and included genes involved in AA metabolism, nutrient sensing, and pathways increased in cancers. While we identified changes in a gene (iaaA) with limited asparaginase activity, it lacked significance after correction leaving open other mechanisms for asparagine decline, possibly including loss from gut to blood. Understanding pathways that change AA availability, including by microbes in the gut, could be useful in optimizing pegaspargase therapy.

Investigating the gut microbial community and genes in children with differing levels of change in serum asparaginase activity during pegaspargase treatment for acute lymphoblastic leukemia.

Asparaginase (ASNase) is an effective treatment of pediatric acute lymphoblastic leukemia (ALL). Changes in ASNase activity may lead to suboptimal treatment and poorer outcomes. The gut microbiome produces metabolites that could impact ASNase therapy, however, remains uninvestigated. We examined gut-microbial community and microbial-ASNase and asparagine synthetase (ASNS) genes using 16SrRNA and metagenomic sequence data from stool samples of pediatric ALL patients. Comparing ASNase activity between consecutive ASNase-doses, we found microbial communities differed between decreased- and increased-activity samples. Escherichia predominated in the decreased-activity community while Bacteroides and Streptococcus predominated in the increased-activity community. In addition microbial ASNS was significantly (p=.004) negatively correlated with change in serum ASNase activity. These preliminary findings suggest microbial communities prior to treatment could affect serum ASNase levels, although the mechanism is unknown. Replication in an independent cohort is needed, and future research on manipulation of these communities and genes could prove useful in optimizing ASNase therapy.

Bacterial Taxa and Functions Are Predictive of Sustained Remission Following Exclusive Enteral Nutrition in Pediatric Crohn's Disease.

BACKGROUND: The gut microbiome is extensively involved in induction of remission in pediatric Crohn's disease (CD) patients by exclusive enteral nutrition (EEN). In this follow-up study of pediatric CD patients undergoing treatment with EEN, we employ machine learning models trained on baseline gut microbiome data to distinguish patients who achieved and sustained remission (SR) from those who did not achieve remission nor relapse (non-SR) by 24 weeks. METHODS: A total of 139 fecal samples were obtained from 22 patients (8-15 years of age) for up to 96 weeks. Gut microbiome taxonomy was assessed by 16S rRNA gene sequencing, and functional capacity was assessed by metagenomic sequencing. We used standard metrics of diversity and taxonomy to quantify differences between SR and non-SR patients and to associate gut microbial shifts with fecal calprotectin (FCP), and disease severity as defined by weighted Pediatric Crohn's Disease Activity Index. We used microbial data sets in addition to clinical metadata in random forests (RFs) models to classify treatment response and predict FCP levels. RESULTS: Microbial diversity did not change after EEN, but species richness was lower in low-FCP samples (<250 µg/g). An RF model using microbial abundances, species richness, and Paris disease classification was the best at classifying treatment response (area under the curve [AUC] = 0.9). KEGG Pathways also significantly classified treatment response with the addition of the same clinical data (AUC = 0.8). Top features of the RF model are consistent with previously identified IBD taxa, such as Ruminococcaceae and Ruminococcus gnavus. CONCLUSIONS: Our machine learning approach is able to distinguish SR and non-SR samples using baseline microbiome and clinical data.

The relationship between fecal bile acids and microbiome community structure in pediatric Crohn's disease.

Gut microbiome community structure is associated with Crohn's disease (CD) development and response to therapy. Bile acids (BAs) play a central role in modulating intestinal immune responses, and changes in gut bacterial communities can profoundly alter the intestinal BA pool. The liver synthesizes and conjugates primary bile acids (priBAs) that are then deconjugated, epimerized, and dehydroxylated by gut bacteria to produce secondary bile acids (secBAs). We investigated the relationship between the gut microbiome and the fecal BA pool in stool samples obtained from a well-characterized cohort of pediatric CD patients undergoing nutritional therapy to induce disease remission. We found that fecal BA composition was altered in a sub-group of CD patients who did not sustain remission. The microbial community structures associated with priBA and secBA-dominant profiles were distinct. In addition, the fecal BA concentrations were correlated with the abundance of distinct bacterial taxonomic groups. Finally, priBA dominant samples were associated with community-level decreases in enzymes for dehydroxylation but not deconjugation.

Opinion: Are Organoids the End of Model Evolution for Studying Host Intestinal Epithelium/Microbe Interactions?

In the pursuit to understand intestinal epithelial cell biology in health and disease, researchers have established various model systems, from whole animals (typically rodents) with experimentally induced disease to transformed human carcinomas. The obvious limitation to the ex vivo or in vitro cell systems was enriching, maintaining, and expanding differentiated intestinal epithelial cell types. The popular concession was human and rodent transformed cells of mainly undifferentiated cells, with a few select lines differentiating along the path to becoming goblet cells. Paneth cells, in particular, remained unculturable. The breakthrough came in the last decade with the report of conditions to grow mouse intestinal organoids. Organoids are 3-dimensional ex vivo "mini-organs" of the organ from which the stem cells were derived. Intestinal organoids contain fully differentiated epithelial cells in the same spatial organization as in the native epithelium. The cells are suitably polarized and produce and secrete mucus onto the apical surface. This review introduces intestinal organoids and provide some thoughts on strengths and weaknesses in the application of organoids to further advance our understanding of the intestinal epithelial-microbe relationship.

Infectious Complications Are Associated With Alterations in the Gut Microbiome in Pediatric Patients With Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia is the most common pediatric cancer. Fortunately, survival rates exceed 90%, however, infectious complications remain a significant issue that can cause reductions in the quality of life and prognosis of patients. Recently, numerous studies have linked shifts in the gut microbiome composition to infection events in various hematological malignances including acute lymphoblastic leukemia (ALL). These studies have been limited to observing broad taxonomic changes using 16S rRNA gene profiling, while missing possible differences within microbial functions encoded by individual species. In this study we present the first combined 16S rRNA gene and metagenomic shotgun sequencing study on the gut microbiome of an independent pediatric ALL cohort during treatment. In this study we found distinctive differences in alpha diversity and beta diversity in samples from patients with infectious complications in the first 6 months of therapy. We were also able to find specific species and functional pathways that were significantly different in relative abundance between samples that came from patients with infectious complications. Finally, machine learning models based on patient metadata and bacterial species were able to classify samples with high accuracy (84.09%), with bacterial species being the most important classifying features. This study strengthens our understanding of the association between infection and pediatric acute lymphoblastic leukemia treatment and warrants further investigation in the future.

The Role of Succinate in the Regulation of Intestinal Inflammation.

Succinate is a metabolic intermediate of the tricarboxylic acid (TCA) cycle within host cells. Succinate is also produced in large amounts during bacterial fermentation of dietary fiber. Elevated succinate levels within the gut lumen have been reported in association with microbiome disturbances (dysbiosis), as well as in patients with inflammatory bowel disease (IBD) and animal models of intestinal inflammation. Recent studies indicate that succinate can activate immune cells via its specific surface receptor, succinate receptor 1(SUCNR1), and enhance inflammation. However, the role of succinate in inflammatory processes within the gut mucosal immune system is unclear. This review includes current literature on the association of succinate with intestinal inflammation and the potential role of succinate⁻SUCNR1 signaling in gut immune functions.

Exclusive Enteral Nutrition Therapy in Paediatric Crohn's Disease Results in Long-term Avoidance of Corticosteroids: Results of a Propensity-score Matched Cohort Analysis.

BACKGROUND AND AIMS: Exclusive enteral nutrition [EEN] is recommended as a first-line induction therapy for paediatric Crohn's disease [CD] although corticosteroids [CS] are still used commonly. Our aim was to compare short- and long-term disease outcomes of paediatric CD patients initially managed with either EEN or CS. METHODS: Medical records of newly diagnosed paediatric CD patients treated with EEN or CS as induction therapy were retrospectively reviewed. To minimise selection bias inherent in observational cohort studies, propensity analysis was carried out. Data on anthropometrics, medical history, and presenting phenotype were collected at time of diagnosis [baseline]; outcomes of interest, including medication use, hospitalisation, surgical procedures, and disease progression were assessed up to 6 years following diagnosis. RESULTS: Of 127 patients reviewed, a total of 111 propensity-score matched CD patients receiving EEN [n = 76] or CS [n = 35] were analysed. By 4-12 weeks of induction therapy, 86.6% of EEN-treated patients achieved remission (Paediatric Crohn's Disease Activity Index [PCDAI] ≤ 7.5) compared with 58.1% of patients in the CS-treated group [p < 0.01]. Choice of EEN over CS for induction was associated with avoidance of corticosteroids over a 6-year follow-up period. Analysis of long-term linear growth, hospitalisation, need for biologic therapy, or surgical intervention did not reveal any significant differences. CONCLUSIONS: These findings suggest that EEN induction therapy is more effective in achieving early remission and is associated with long-term steroid avoidance without increased use of biologics or need for surgery.

The Impact of Exclusive Enteral Nutrition (EEN) on the Gut Microbiome in Crohn's Disease: A Review.

Crohn's disease (CD), a form of inflammatory bowel disease (IBD), is thought to arise from a complex interaction of genetics, the gut microbiome, and environmental factors, such as diet. There is clear evidence that dietary intervention is successful in the treatment of CD-exclusive enteral nutrition (EEN) is able to induce remission in up to 80% of CD patients. While the mechanism of action of EEN is not clear, EEN is known to cause profound changes in the gut microbiome. Understanding how EEN modifies the gut microbiome to induce remission could provide insight into CD etiopathogenesis and aid the development of microbiome-targeted interventions to guide ongoing dietary therapy to sustain remission. This review includes current literature on changes in composition and function of the gut microbiome associated with EEN treatment in CD patients.

Early Changes in Microbial Community Structure Are Associated with Sustained Remission After Nutritional Treatment of Pediatric Crohn's Disease.

BACKGROUND: Clinical remission achieved by exclusive enteral nutrition (EEN) is associated with marked microbiome changes. In this prospective study of exclusive enteral nutrition, we employ a hierarchical model of microbial community structure to distinguish between pediatric Crohn's disease patients who achieved sustained remission (SR) and those who relapsed early (non-SR), after restarting a normal diet. METHODS: Fecal samples were obtained from 10 patients (age 10-16) and from 5 healthy controls (age 9-14). The microbiota was assessed via 16S rRNA sequencing. In addition to standard measures of microbial biodiversity, we employed Bayesian methods to characterize the hierarchical community structure. Community structure between patients who sustained remission (wPCDAI <12.5) up to their 24-week follow-up (SR) was compared with patients that had not sustained remission (non-SR). RESULTS: Microbial diversity was lower in Crohn's disease patients relative to controls and lowest in patients who did not achieve SR. SR patients differed from non-SR patients in terms of the structure and prevalence of their microbial communities. The SR prevalent community contained a number of strains of Akkermansia muciniphila and Bacteroides and was limited in Proteobacteria, whereas the non-SR prevalent community had a large Proteobacteria component. Their communities were so different that a model trained to discriminate SR and non-SR had 80% classification accuracy, already at baseline sampling. CONCLUSIONS: Microbial community structure differs between healthy controls, patients who have an enduring response to exclusive enteral nutrition, and those who relapse early on introduction of normal diet. Our novel Bayesian approach to these differences is able to predict sustained remission after exclusive enteral nutrition.

The Gut Microbiome of Pediatric Crohn's Disease Patients Differs from Healthy Controls in Genes That Can Influence the Balance Between a Healthy and Dysregulated Immune Response.

BACKGROUND: Exclusive enteral nutrition (EEN) is a first-line therapy in pediatric Crohn's disease (CD) thought to induce remission through changes in the gut microbiome. With microbiome assessment largely focused on microbial taxonomy and diversity, it remains unclear to what extent EEN induces functional changes that thereby contribute to its therapeutic effect. METHODS: Fecal samples were collected from 15 pediatric CD patients prior to and after EEN treatment, as well as from 5 healthy controls. Metagenomic data were obtained via next-generation sequencing, and nonhuman reads were mapped to KEGG pathways, where possible. Pathway abundance was compared between CD patients and controls, and between CD patients that sustained remission (SR) and those that did not sustain remission (NSR). RESULTS: Of 132 KEGG pathways identified, 8 pathways differed significantly between baseline CD patients and controls. Examination of these eight pathways showed SR patients had greater similarity to controls than NSR patients in all cases. Pathways fell into one of three groups: 1) no prior connection to IBD, 2) previously reported connection to IBD, and 3) known roles in innate immunity and immunoregulation. CONCLUSIONS: The microbiota of CD patients and controls represent alternative ecological states that have broad differences in functional capabilities, including xenobiotic and environmental pollutant degradation, succinate metavolism, and bacterial HtpG, all of which can affect barrier integrity and immune regulation. Moreover, our finding that SR patients were more similar to healthy controls suggests that community microbial function, as inferred from fecal microbiomes, could serve as a valuable diagnostic tool.

Novel Strategies for Applied Metagenomics.

Detailed analyses of the gut microbiome and its effect on human physiology and disease are emerging, thanks to advances in high-throughput DNA-sequencing technology and the burgeoning field of metagenomics. Metagenomics examines the structure and functional potential of microbial communities in their native habitats through the direct isolation and analysis of community DNA. In inflammatory bowel disease, gut microbiome studies have shown an association with perturbations in community composition and, especially, function. In this review, we discuss the application of next-generation sequencing to microbiome research and highlight the importance of modeling microbiome structure and function to the future of inflammatory bowel disease research and treatment.

CD4⁺CD25⁺Foxp3⁺ regulatory T cells promote Th17 responses and genital tract inflammation upon intracellular Chlamydia muridarum infection.

The functional role of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in host responses to intracellular bacterial infection was investigated in an in vitro coculturing system and a murine model of Chlamydia muridarum genital tract infection. Remarkably, C. muridarum infection subverted the immune suppressive role of CD4⁺CD25⁺Foxp3⁺ Tregs; instead of hampering immune responses, Tregs not only promoted Th17 differentiation from conventional CD4⁺ T cells but also themselves converted into proinflammatory Th17 cells in both in vitro and in vivo settings. Anti-CD25 mAb PC61 treatment to deplete ∼50% of pre-existing Tregs prior to C. muridarum genital tract infection markedly reduced the frequency and the total number of Th17 but not Th1 CD4⁺ cells at both immune induction and memory phases. Most importantly, Treg-depleted mice displayed significantly attenuated inflammation, neutrophil infiltration, and reduced severity of oviduct pathology upon C. muridarum genital infection. To our knowledge, this is the first report demonstrating that the level of pre-existing CD4⁺CD25⁺Foxp3⁺ Tregs in Chlamydia-infected hosts has a major impact on the development Chlamydia-associated diseases.