Achieving pan-microbiome biological insights via the dbBact knowledge base.

16S rRNA amplicon sequencing provides a relatively inexpensive culture-independent method for studying microbial communities. Although thousands of such studies have examined diverse habitats, it is difficult for researchers to use this vast trove of experiments when interpreting their own findings in a broader context. To bridge this gap, we introduce dbBact - a novel pan-microbiome resource. dbBact combines manually curated information from studies across diverse habitats, creating a collaborative central repository of 16S rRNA amplicon sequence variants (ASVs), which are assigned multiple ontology-based terms. To date dbBact contains information from more than 1000 studies, which include 1500000 associations between 360000 ASVs and 6500 ontology terms. Importantly, dbBact offers a set of computational tools allowing users to easily query their own datasets against the database. To demonstrate how dbBact augments standard microbiome analysis we selected 16 published papers, and reanalyzed their data via dbBact. We uncovered novel inter-host similarities, potential intra-host sources of bacteria, commonalities across different diseases and lower host-specificity in disease-associated bacteria. We also demonstrate the ability to detect environmental sources, reagent-borne contaminants, and identify potential cross-sample contaminations. These analyses demonstrate how combining information across multiple studies and over diverse habitats leads to better understanding of underlying biological processes.

Stratification of risk of progression to colectomy in ulcerative colitis via measured and predicted gene expression.

An important goal of clinical genomics is to be able to estimate the risk of adverse disease outcomes. Between 5% and 10% of individuals with ulcerative colitis (UC) require colectomy within 5 years of diagnosis, but polygenic risk scores (PRSs) utilizing findings from genome-wide association studies (GWASs) are unable to provide meaningful prediction of this adverse status. By contrast, in Crohn disease, gene expression profiling of GWAS-significant genes does provide some stratification of risk of progression to complicated disease in the form of a transcriptional risk score (TRS). Here, we demonstrate that a measured TRS based on bulk rectal gene expression in the PROTECT inception cohort study has a positive predictive value approaching 50% for colectomy. Single-cell profiling demonstrates that the genes are active in multiple diverse cell types from both the epithelial and immune compartments. Expression quantitative trait locus (QTL) analysis identifies genes with differential effects at baseline and week 52 follow-up, but for the most part, differential expression associated with colectomy risk is independent of local genetic regulation. Nevertheless, a predicted polygenic transcriptional risk score (PPTRS) derived by summation of transcriptome-wide association study (TWAS) effects identifies UC-affected individuals at 5-fold elevated risk of colectomy with data from the UK Biobank population cohort studies, independently replicated in an NIDDK-IBDGC dataset. Prediction of gene expression from relatively small transcriptome datasets can thus be used in conjunction with TWASs for stratification of risk of disease complications.

Multicenter Cohort Study of Infliximab Pharmacokinetics and Therapy Response in Pediatric Acute Severe Ulcerative Colitis.

BACKGROUND & AIMS: We aimed to model infliximab (IFX) pharmacokinetics (PK) in pediatric acute severe ulcerative colitis (ASUC) and assess the association between PK parameters, including drug exposure, and clinical response. METHODS: We studied a multicenter prospective cohort of hospitalized children initiating IFX for ASUC or IBD-unclassified. Serial IFX serum concentrations over 26 weeks were used to develop a PK model. We tested the association of PK parameter estimates with day 7 clinical response, week 8 clinical remission, week 26 corticosteroid-free clinical remission (CSF-CR) (using the Pediatric Ulcerative Colitis Activity Index), and colectomy-free survival. RESULTS: Thirty-eight participants received IFX (median initial dose, 9.9 mg/kg). Day 7 clinical response, week 8 clinical remission, and week 26 CSF-CR occurred in 71%, 55%, and 43%, respectively. Albumin, C-reactive protein, white blood cell count, platelets, weight, and antibodies to IFX were significant covariates incorporated into a PK model. Week 26 non-remitters exhibited faster IFX clearance than remitters (P = .013). However, cumulative IFX exposure did not differ between clinical response groups. One (2.7%) and 4 (10.8%) participants underwent colectomy by week 26 and 2 years, respectively. Day 3 IFX clearance >0.02 L/h was associated with colectomy (hazard ratio, 58.2; 95% confidence interval, 6.0-568.6; P < .001). CONCLUSIONS: At median higher-than-label IFX dosing for pediatric ASUC, baseline faster IFX CL was associated with colectomy and at week 26 with lack of CSF-CR. IFX exposure was not predictive of clinical outcomes. Higher IFX dosing may sufficiently optimize early outcomes in pediatric ASUC. Larger studies are warranted to determine whether sustained intensification can overcome rapid clearance and improve later outcomes. CLINICALTRIALS: gov identifier: NCT02799615.

Evaluating Eosinophilic Colitis as a Unique Disease Using Colonic Molecular Profiles: A Multi-Site Study.

BACKGROUND & AIMS: Colonic eosinophilia, an enigmatic finding often referred to as eosinophilic colitis (EoC), is a poorly understood condition. Whether EoC is a distinct disease or a colonic manifestation of eosinophilic gastrointestinal diseases (EGIDs) or inflammatory bowel disease (IBD) is undetermined. METHODS: Subjects with EoC (n = 27) and controls (normal [NL, n = 20], Crohn's disease [CD, n = 14]) were enrolled across sites associated with the Consortium of Eosinophilic Gastrointestinal Disease Researchers. EoC was diagnosed as colonic eosinophilia (ascending ≥100, descending ≥85, sigmoid ≥65 eosinophils/high-power field) with related symptoms. Colon biopsies were subjected to RNA sequencing. Associations between gene expression and histologic features were analyzed with Spearman correlation; operational pathways and cellular constituents were computationally derived. RESULTS: We identified 987 differentially expressed genes (EoC transcriptome) between EoC and NL (>1.5-fold change, P < .05). Colonic eosinophil count correlated with 31% of EoC transcriptome, most notably with CCL11 and CLC (r = 0.78 and 0.77, P < .0001). Among EoC and other EGIDs, there was minimal transcriptomic overlap and minimal evidence of a strong allergic type 2 immune response in EoC compared with other EGIDs. Decreased cell cycle and increased apoptosis in EoC compared with NL were identified by functional enrichment analysis and immunostaining using Ki-67 and cleaved caspase-3. Pericryptal circumferential eosinophil collars were associated with the EoC transcriptome (P < .001). EoC transcriptome-based scores were reversible with disease remission and differentiated EoC from IBD, even after controlling for colonic eosinophil levels (P < .0001). CONCLUSIONS: We established EoC transcriptomic profiles, identified mechanistic pathways, and integrated findings with parallel IBD and EGID data. These findings establish EoC as a distinct disease compared with other EGIDs and IBD, thereby providing a basis for improving diagnosis and treatment.

Transcriptome analysis in acute gastrointestinal graft-versus host disease reveals a unique signature in children and shared biology with pediatric inflammatory bowel disease.

We performed transcriptomic analyses on freshly frozen (n=21) and paraffin-embedded (n=35) gastrointestinal (GI) biopsies from children with and without acute acute GI graft-versus-host disease (GvHD) to study differential gene expressions. We identified 164 significant genes, 141 upregulated and 23 downregulated, in acute GvHD from freshy frozen biopsies. CHI3L1 was the top differentially expressed gene in acute GvHD, involved in macrophage recruitment and bacterial adhesion. Mitochondrial genes were among the top downregulated genes. Immune deconvolution identified a macrophage cellular signature. Weighted gene co-expression network analysis showed enrichment of genes in the ERK1/2 cascade. Transcriptome data from 206 ulcerative colitis (UC) patients were included to uncover genes and pathways shared between GvHD and UC. Comparison with the UC transcriptome showed both shared and distinct pathways. Both UC and GvHD transcriptomes shared an innate antimicrobial signature and FCγ1RA/CD64 was upregulated in both acute GvHD (log-fold increase 1.7, P=0.001) and UC. Upregulation of the ERK1/2 cascade pathway was specific to GvHD. We performed additional experiments to confirm transcriptomics. Firstly, we examined phosphorylation of ERK (pERK) by immunohistochemistry on GI biopsies (acute GvHD n=10, no GvHD n=10). pERK staining was increased in acute GvHD biopsies compared to biopsies without acute GvHD (P=0.001). Secondly, plasma CD64, measured by enzyme-linked immunsorbant assay (n=85) was elevated in acute GI GvHD (P<0.001) compared with those without and was elevated in GVHD compared with inflammatory bowel disease (n=47) (P<0.001), confirming the upregulated expression seen in the transcriptome.

Mucosal Genomics Implicate Lymphocyte Activation and Lipid Metabolism in Refractory Environmental Enteric Dysfunction.

BACKGROUND & AIMS: Environmental enteric dysfunction (EED) limits the Sustainable Development Goals of improved childhood growth and survival. We applied mucosal genomics to advance our understanding of EED. METHODS: The Study of Environmental Enteropathy and Malnutrition (SEEM) followed 416 children from birth to 24 months in a rural district in Pakistan. Biomarkers were measured at 9 months and tested for association with growth at 24 months. The duodenal methylome and transcriptome were determined in 52 undernourished SEEM participants and 42 North American controls and patients with celiac disease. RESULTS: After accounting for growth at study entry, circulating insulin-like growth factor-1 (IGF-1) and ferritin predicted linear growth, whereas leptin correlated with future weight gain. The EED transcriptome exhibited suppression of antioxidant, detoxification, and lipid metabolism genes, and induction of anti-microbial response, interferon, and lymphocyte activation genes. Relative to celiac disease, suppression of antioxidant and detoxification genes and induction of antimicrobial response genes were EED-specific. At the epigenetic level, EED showed hyper-methylation of epithelial metabolism and barrier function genes, and hypo-methylation of immune response and cell proliferation genes. Duodenal coexpression modules showed association between lymphocyte proliferation and epithelial metabolic genes and histologic severity, fecal energy loss, and wasting (weight-for-length/height Z < -2.0). Leptin was associated with expression of epithelial carbohydrate metabolism and stem cell renewal genes. Immune response genes were attenuated by giardia colonization. CONCLUSIONS: Children with reduced circulating IGF-1 are more likely to experience stunting. Leptin and a gene signature for lymphocyte activation and dysregulated lipid metabolism are implicated in wasting, suggesting new approaches for EED refractory to nutritional intervention. ClinicalTrials.gov, Number: NCT03588013. (https://clinicaltrials.gov/ct2/show/NCT03588013).

Colonic Epithelial-Derived Selenoprotein P Is the Source for Antioxidant-Mediated Protection in Colitis-Associated Cancer.

BACKGROUND & AIMS: Patients with inflammatory bowel disease (IBD) demonstrate nutritional selenium deficiencies and are at greater risk of developing colon cancer. Previously, we determined that global reduction of the secreted antioxidant selenium-containing protein, selenoprotein P (SELENOP), substantially increased tumor development in an experimental colitis-associated cancer (CAC) model. We next sought to delineate tissue-specific contributions of SELENOP to intestinal inflammatory carcinogenesis and define clinical context. METHODS: Selenop floxed mice crossed with Cre driver lines to delete Selenop from the liver, myeloid lineages, or intestinal epithelium were placed on an azoxymethane/dextran sodium sulfate experimental CAC protocol. SELENOP loss was assessed in human ulcerative colitis (UC) organoids, and expression was queried in human and adult UC samples. RESULTS: Although large sources of SELENOP, both liver- and myeloid-specific Selenop deletion failed to modify azoxymethane/dextran sodium sulfate-mediated tumorigenesis. Instead, epithelial-specific deletion increased CAC tumorigenesis, likely due to elevated oxidative stress with a resulting increase in genomic instability and augmented tumor initiation. SELENOP was down-regulated in UC colon biopsies and levels were inversely correlated with endoscopic disease severity and tissue S100A8 (calprotectin) gene expression. CONCLUSIONS: Although global selenium status is typically assessed by measuring liver-derived plasma SELENOP levels, our results indicate that the peripheral SELENOP pool is dispensable for CAC. Colonic epithelial SELENOP is the main contributor to local antioxidant capabilities. Thus, colonic SELENOP is the most informative means to assess selenium levels and activity in IBD patients and may serve as a novel biomarker for UC disease severity and identify patients most predisposed to CAC development.

Practice Variations in the Management of Infants With Non-IgE-Mediated Cow's Milk Protein Allergy.

OBJECTIVES: Infants with non-IgE-mediated food allergies are often referred to gastroenterologists or immunologists. We hypothesized that there are practice variations between these disciplines in the diagnosis and management of such infants. METHODS: A computerized questionnaire was distributed between pediatric gastroenterologists and immunologists. The questions addressed diagnosis, management, and follow-up in 3 scenarios of infants with concern for food protein-induced allergic proctocolitis (FPIAP) due to non-IgE-mediated responses to cow's milk. RESULTS: Three cases of infants with suspected FPIAP were presented: milk-based formula-fed (case 1) or breast-fed (case 2) infants that are well appearing and thriving, and a breast-fed infant who is not growing appropriately along with a personal and family history of atopy (case 3). Fifty-eight pediatric gastroenterologists and 32 immunologists completed the questionnaire. Significant differences between gastroenterologists and immunologists were noted regarding the recommended dietary changes in these scenarios. Moreover, despite available guidelines generated by both societies, most physicians confirm the diagnosis based on resolution of symptoms after the dietary change, without re-exposure to the the suspected trigger. In addition, time for recommended re-exposure in infants with FPIAP was also different; most gastroenterologists recommended waiting until 12 months of age, while immunologists suggested reintroduction earlier, up to 6 months of age. CONCLUSIONS: We identified significant practice variations in diagnosis and management of FPIAP between pediatric gastroenterologists and immunologists, with lack of adherence to society guidelines. Joint task forces of primary care pediatricians, gastroenterologists, and immunologists should provide uniform guidelines to standardize care.

Clinical, neuroimaging, and molecular spectrum of TECPR2-associated hereditary sensory and autonomic neuropathy with intellectual disability.

Bi-allelic TECPR2 variants have been associated with a complex syndrome with features of both a neurodevelopmental and neurodegenerative disorder. Here, we provide a comprehensive clinical description and variant interpretation framework for this genetic locus. Through international collaboration, we identified 17 individuals from 15 families with bi-allelic TECPR2-variants. We systemically reviewed clinical and molecular data from this cohort and 11 cases previously reported. Phenotypes were standardized using Human Phenotype Ontology terms. A cross-sectional analysis revealed global developmental delay/intellectual disability, muscular hypotonia, ataxia, hyporeflexia, respiratory infections, and central/nocturnal hypopnea as core manifestations. A review of brain magnetic resonance imaging scans demonstrated a thin corpus callosum in 52%. We evaluated 17 distinct variants. Missense variants in TECPR2 are predominantly located in the N- and C-terminal regions containing ß-propeller repeats. Despite constituting nearly half of disease-associated TECPR2 variants, classifying missense variants as (likely) pathogenic according to ACMG criteria remains challenging. We estimate a pathogenic variant carrier frequency of 1/1221 in the general and 1/155 in the Jewish Ashkenazi populations. Based on clinical, neuroimaging, and genetic data, we provide recommendations for variant reporting, clinical assessment, and surveillance/treatment of individuals with TECPR2-associated disorder. This sets the stage for future prospective natural history studies.

Bile Acid Profiling Reveals Distinct Signatures in Undernourished Children with Environmental Enteric Dysfunction.

BACKGROUND: Intestinal inflammation and malabsorption in environmental enteric dysfunction (EED) are associated with early childhood growth faltering in impoverished settings worldwide. OBJECTIVES: The goal of this study was to identify candidate biomarkers associated with inflammation, EED histology, and as predictors of later growth outcomes by focusing on the liver-gut axis by investigating the bile acid metabolome. METHODS: Undernourished rural Pakistani infants (n = 365) with weight-for-height Z score (WHZ) < -2 were followed up to the age of 24 mo and monitored for growth, infections, and EED. Well-nourished local children (n = 51) were controls, based on consistent WHZ > 0 and height-for-age Z score (HAZ) > -1 on 2 consecutive visits at 3 and 6 mo. Serum bile acid (sBA) profiles were measured by tandem MS at the ages of 3-6 and 9 mo and before nutritional intervention. Biopsies and duodenal aspirates were obtained following upper gastrointestinal endoscopy from a subset of children (n = 63) that responded poorly to nutritional intervention. BA composition in paired plasma and duodenal aspirates was compared based on the severity of EED histopathological scores and correlated to clinical and growth outcomes. RESULTS: Remarkably, >70% of undernourished Pakistani infants displayed elevated sBA concentrations consistent with subclinical cholestasis. Serum glycocholic acid (GCA) correlated with linear growth faltering (HAZ, r = -0.252 and -0.295 at the age of 3-6 and 9 mo, respectively, P <0.001) and biomarkers of inflammation. The proportion of GCA positively correlated with EED severity for both plasma (rs = 0.324 P = 0.02) and duodenal aspirates (rs = 0.307 P = 0.06) in children with refractory wasting that underwent endoscopy, and the proportion of secondary BA was low in both undernourished and EED children. CONCLUSIONS: Dysregulated bile acid metabolism is associated with growth faltering and EED severity in undernourished children. Restoration of intestinal BA homeostasis may offer a novel therapeutic target for undernutrition in children with EED. This trial was registered at clinicaltrials.gov as NCT03588013.

Social isolation in mice: behavior, immunity, and tumor growth.

The aim of this study was to investigate the behavioral, immunological, and neurological effects of long-term isolation in an animal model. Male C3H/eB mice wereraised in either social isolation or standard conditions for 6 weeks. At 10 weeks, each group was further divided into 3 sets. (A) Physical strength and behavior were evaluated with the grip strength, hot plate, staircase, and elevated plus-maze tests. Natural-killer cell activity and lymphocyte proliferation were measured. (B) Half the animals were subjected to electric shock with 3 reminders, and freezing time was evaluated at each reminder. Cortisone levels were evaluated after 16 weeks. (C)Mice were injected with 38 C-13 B lymphoma cells and followed for tumor size and survival. Strength evaluation yielded asignificantly lower body weight and grip strength in the socially isolated mice. Behavioral test results were similar in the two groups. The pattern of reactions to stress conditioning differed significantly, with the socially isolated mice showing an incline in freezing with each successive reminder, and the control mice showing a decline. The socially isolated mice had significantly attenuated tumor growth, with no significant difference in survival from control mice. There were no significant between-group differences in immunological parameters. In conclusion, social isolation serves as a model for chronic stress. It was associated with significant changes in stress conditioning reaction, resembling symptoms of post-traumatic stress disorder, and attenuated tumor development. No differences from controls were found in behavior tests, immune parameters, or survival after tumor cell inoculation.Lay summaryThis article explores biological and behavioral consequences of social isolation in a mice model. Our results show that social isolation leads to changes in the Hypothalamic-hypophyseal-adrenal axis, which in turn alter the response to stress. Additionally, social isolation was shown to impact tumor progression.

Trabecular Bone Score in Children and Adolescents With Inflammatory Bowel Diseases.

INTRODUCTION: Trabecular bone score (TBS) is a textural index that evaluates bone microarchitecture of the lumbar spine. Our aim was to assess TBS in children with inflammatory bowel diseases and to evaluate correlations with clinical, laboratory and densitometric variables. METHODS: A retrospective study of TBS and areal bone mineral density measurements by dual-energy X-ray absorptiometry (DXA) of children with either Crohn's disease (CD) or ulcerative colitis (UC). Bone mineral apparent density was calculated for size adjustment. TBS Z-score for each child were calculated based on data from a healthy population of similar age and gender distribution. Variables significantly associated with TBS were included in stepwise linear regression models to examine independent predictors of TBS. RESULTS: Fifty patients (age at DXA scan 13.8 ± 3.0 years, 29 males) were included. No significant differences were observed between the patients with CD and UC, in age at diagnosis, age at DXA scan and disease duration. The mean TBS of patients with CD (n = 35) was lower than of patients with UC (n = 15): 1.340 ± 0.080 vs 1.395 ± 0.092, p = 0.040. The mean TBS Z-score of patients with CD, -0.443 ± 0.788, was significantly lower than expected in healthy children (p = 0.002), while the mean TBS Z-score of patients with UC, 0.231 ± 0.685, was similar to that of healthy children (p = 0.212). In the stepwise linear regression analysis, BMI Z-score at diagnosis, phosphorus level at diagnosis and age at the time of the DXA scan were significant independent predictors of TBS (r²â€¯= 0.604; ß = 0.037, 95% confidence interval (CI) for ß 0.022-0.051, p < 0.001; ß = 0.045, 95% CI: 0.017-0.073, p = 0.002; and ß = 0.031, 95% CI: 0.005-0.021, p < 0.002, respectively). CONCLUSIONS: TBS is significantly reduced in pediatric patients with CD but not in patients with UC. This finding likely reflects the effect of CD on bone microarchitecture.

Clinical and biological predictors of response to standardised paediatric colitis therapy (PROTECT): a multicentre inception cohort study.

BACKGROUND: Lack of evidence-based outcomes data leads to uncertainty in developing treatment regimens in children who are newly diagnosed with ulcerative colitis. We hypothesised that pretreatment clinical, transcriptomic, and microbial factors predict disease course. METHODS: In this inception cohort study, we recruited paediatric patients aged 4-17 years with newly diagnosed ulcerative colitis from 29 centres in the USA and Canada. Patients initially received standardised mesalazine or corticosteroids, with pre-established criteria for escalation to immunomodulators (ie, thiopurines) or anti-tumor necrosis factor-α (TNFα) therapy. We used RNA sequencing to define rectal gene expression before treatment, and 16S sequencing to characterise rectal and faecal microbiota. The primary outcome was week 52 corticosteroid-free remission with no therapy beyond mesalazine. We assessed factors associated with the primary outcome using logistic regression models of the per-protocol population. This study is registered with ClinicalTrials.gov, number NCT01536535. FINDINGS: Between July 10, 2012, and April 21, 2015, of 467 patients recruited, 428 started medical therapy, of whom 400 (93%) were evaluable at 52 weeks and 386 (90%) completed the study period with no protocol violations. 150 (38%) of 400 participants achieved week 52 corticosteroid-free remission, of whom 147 (98%) were taking mesalazine and three (2%) were taking no medication. 74 (19%) of 400 were escalated to immunomodulators alone, 123 (31%) anti-TNFα therapy, and 25 (6%) colectomy. Low baseline clinical severity, high baseline haemoglobin, and week 4 clinical remission were associated with achieving week 52 corticosteroid-free remission (n=386, logistic model area under the curve [AUC] 0·70, 95% CI 0·65-0·75; specificity 77%, 95% CI 71-82). Baseline severity and remission by week 4 were validated in an independent cohort of 274 paediatric patients with newly diagnosed ulcerative colitis. After adjusting for clinical predictors, an antimicrobial peptide gene signature (odds ratio [OR] 0·57, 95% CI 0·39-0·81; p=0·002) and abundance of Ruminococcaceae (OR 1·43, 1·02-2·00; p=0·04), and Sutterella (OR 0·81, 0·65-1·00; p=0·05) were independently associated with week 52 corticosteroid-free remission. INTERPRETATION: Our findings support the utility of initial clinical activity and treatment response by 4 weeks to predict week 52 corticosteroid-free remission with mesalazine alone in children who are newly diagnosed with ulcerative colitis. The development of personalised clinical and biological signatures holds the promise of informing ulcerative colitis therapeutic decisions. FUNDING: US National Institutes of Health.

Longitudinal changes in bone mineral density in children with inflammatory bowel diseases.

AIM: Children with inflammatory bowel disease (IBD) are prone to low bone mineral density (BMD). Our aim was to assess longitudinal changes in BMD in this population. METHODS: A retrospective longitudinal study of children with IBD, treated at two tertiary centres in Israel, who underwent two BMD measurements by dual-energy X-ray absorptiometry (DXA). Changes in lumbar spine BMD (∆L1-4 z-scores) were examined for correlations with clinical characteristics. RESULTS: The cohort included 41 patients (age at diagnosis 12.1 ± 3.5 years, 23 females).The mean interval between the scans was 3.4 ± 2.0 years. There was a trend towards improvement in L1-4 z-scores (-1.64 ± 1.02 vs -1.45 ± 0.83, P = .12). ∆L1-4 z-scores correlated positively with ∆weight-standard deviation scores (SDS), ∆height-SDS and ∆BMI-SDS, and with age at the second scan (R = .55, P < .01; R = .42, P < .01; R = .42, P = .01; R = .35, P = .02, respectively); and negatively with L1-4 z-scores at the first scan (R = -.63, P < .01). Stepwise linear regression analysis identified the first scan L1-4 z-scores and ∆weight-SDS as independent predictors of ∆L1-4 z-scores. An L1-4 z-score ≤-2 at the first DXA scan was associated with significant improvement at the second scan. CONCLUSION: Improvement in BMD was more pronounced in children who gained weight or whose BMD was low at the first scan.

Genetic variants and pathways implicated in a pediatric inflammatory bowel disease cohort.

In the United States, approximately 5% of individuals with inflammatory bowel disease (IBD) are younger than 20 years old. Studies of pediatric cohorts can provide unique insights into genetic architecture of IBD, which includes Crohn's disease (CD) and ulcerative colitis (UC). Large genome-wide association studies have found more than 200 IBD-associated loci but explain a minority of disease variance for CD and UC. We sought to characterize the contribution of rare variants to disease development, comparing exome sequencing of 368 pediatric IBD patients to publicly available exome sequencing (dbGaP) and aggregate frequency data (ExAC). Using dbGaP data, we performed logistic regression for common variants and optimal unified association tests (SKAT-O) for rare, likely-deleterious variants. We further compared rare variants to ExAC counts with Fisher's exact tests. We did pathway enrichment analysis on the most significant genes from each comparison. Many variants overlapped with known IBD-associated genes (e.g. NOD2). Rare variants were enriched in CD-associated loci (p = 0.009) and showed suggestive enrichment in neutrophil function genes (p = 0.05). Pathway enrichment implicated immune-related pathways, especially cell killing and apoptosis. Variants in extracellular matrix genes also emerged as an important theme in our analysis.

Disruption of Epithelial HDAC3 in Intestine Prevents Diet-Induced Obesity in Mice.

BACKGROUND & AIMS: Intestinal microbiota modulate metabolism and associate closely with epithelial cells in the intestine. In intestinal epithelial cells (IECs), histone deacetylase 3 (HDAC3) integrates microbiota-derived signals to control intestinal homeostasis. We investigated whether HDAC3 in IECs regulates metabolism and the development of obesity in mice. METHODS: Adult C57BL/6 (control) mice and mice with constitutive or inducible IEC-specific disruption of Hdac3 (HDAC3ΔIEC mice) were placed on a standard chow or high-fat diet (HFD, 60% kcal from fat). We measured body composition, weight, glucose tolerance, and energy expenditure. IECs were isolated from small intestine and gene expression, and lipid levels were analyzed. HDAC3 levels were determined in 43 pediatric patient ileal biopsy samples and compared with body weight. RESULTS: Control mice fed an HFD gained weight, became obese, and had reduced glucose tolerance with increased serum insulin, whereas HFD-fed HDAC3ΔIEC mice did not develop obesity. Serum levels of triglycerides were reduced in HDAC3ΔIEC mice, and these mice had less liver fat and smaller adipocytes, compared with HFD-fed control mice. HDAC3ΔIEC mice had similar food intake and activity as control mice, but higher energy expenditure because of increased catabolism. IECs from HDAC3ΔIEC mice had altered expression levels of genes that regulate metabolism in response to the microbiota (such as Chka, Mttp, Apoa1, and Pck1) and accumulated triglycerides compared with IECs from control mice. The microbiota-derived short-chain fatty acid butyrate was decreased in obese mice. Butyrate significantly reduced the activity of HDAC3 and increased Pck1 expression in only control IECs. Administration of butyrate to control mice with diet-induced obesity, but not HDAC3ΔIEC mice, led to significant weight loss. Disruption of HDAC3 in IECs of mice after they became obese led to weight loss and improved metabolic profile. Levels of HDAC3 in intestinal biopsy samples correlated with patient weight. CONCLUSIONS: We found that epithelial HDAC3 promotes development of diet-induced obesity in studies of mice and that butyrate reduces activity of HDAC3 in IECs to prevent diet-induced obesity. This pathway might be manipulated to prevent or reduce obesity-associated disease.

Clinical and Genomic Correlates of Neutrophil Reactive Oxygen Species Production in Pediatric Patients With Crohn's Disease.

BACKGROUND & AIMS: Individuals with monogenic disorders of phagocyte function develop chronic colitis that resembles Crohn's disease (CD). We tested for associations between mutations in genes encoding reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, neutrophil function, and phenotypes of CD in pediatric patients. METHODS: We performed whole-exome sequence analysis to identify mutations in genes encoding NADPH oxidases (such as CYBA, CYBB, NCF1, NCF2, NCF4, RAC1, and RAC2) using DNA from 543 pediatric patients with inflammatory bowel diseases. Blood samples were collected from an additional 129 pediatric patients with CD and 26 children without IBD (controls); we performed assays for neutrophil activation, reactive oxygen species (ROS) production, and bacteria uptake and killing. Whole-exome sequence analysis was performed using DNA from 46 of the children with CD to examine associations with NADPH gene mutations; RNA sequence analyses were performed using blood cells from 46 children with CD to test for variations in neutrophil gene expression associated with ROS production. RESULTS: We identified 26 missense mutations in CYBA, CYBB, NCF1, NCF2, and NCF4. Patients with CD who carried mutations in these genes were 3-fold more likely to have perianal disease (P = .0008) and stricturing complications (P = .002) than children with CD without these mutations. Among patients with CD with none of these mutations, 9% had undergone abdominal surgery; among patients with mutations in these NADPH oxidase genes, 31% had undergone abdominal surgery (P = .0004). A higher proportion of neutrophils from children with CD had low ROS production (47%) than from controls (15%) among the 129 patients tested for ROS (P = .002). Minor alleles of the NADPH genes were detected in 7% of children with CD whose neutrophils produced normal levels of ROS vs 38% of children whose neutrophils produced low levels of ROS (P = .009). Neutrophils that produced low levels of ROS had specific alterations in genes that regulate glucose metabolism and antimicrobial responses. CONCLUSIONS: We identified missense mutations in genes that encode NADPH oxidases in children with CD; these were associated with a more aggressive disease course and reduced ROS production by neutrophils from the patients.

Individualized Dynamics in the Gut Microbiota Precede Crohn's Disease Flares.

OBJECTIVES: Crohn's disease (CD) is a chronic relapsing-remitting gut inflammatory disorder with a heterogeneous unpredictable course. Dysbiosis occurs in CD; however, whether microbial dynamics in quiescent CD are instrumental in increasing the risk of a subsequent flare remains undefined. METHODS: We analyzed the long-term dynamics of microbial composition in a prospective observational cohort of patients with quiescent CD (45 cases, 217 samples) over 2 years or until clinical flare occurred, aiming to identify whether changes in the microbiome precede and predict clinical relapse. Machine learning was used to prioritize microbial and clinical factors that discriminate between relapsers and nonrelapsers in the quiescent phase. RESULTS: Patients with CD in clinical, biomarker, and mucosal remission showed significantly reduced microbial richness and increased dysbiosis index compared with healthy controls. Of the 45 patients with quiescent CD, 12 (27%) flared during follow-up. Samples in quiescent patients preceding flare showed significantly reduced abundance of Christensenellaceae and S24.7, and increased abundance of Gemellaceae compared with those in remission throughout. A composite flare index was associated with a subsequent flare. Notably, higher individualized microbial instability in the quiescent phase was associated with a higher risk of a subsequent flare (hazard ratio 11.32, 95% confidence interval 3-42, P = 0.0035) using two preflare samples. Importantly, machine learning prioritized the flare index and the intrapersonal instability over clinical factors to best discriminate between relapsers and nonrelapsers. DISCUSSION: Individualized microbial variations in quiescent CD significantly increase the risk of future exacerbation and may provide a model to guide personalized preemptive therapy intensification.

Study of Environmental Enteropathy and Malnutrition (SEEM) in Pakistan: protocols for biopsy based biomarker discovery and validation.

BACKGROUND: Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority. METHODS: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn's disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community. DISCUSSION: Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals. TRIAL REGISTRATION: Retrospectively registered; clinicaltrials.gov ID NCT03588013 .

Mucosal Expression of Type 2 and Type 17 Immune Response Genes Distinguishes Ulcerative Colitis From Colon-Only Crohn's Disease in Treatment-Naive Pediatric Patients.

BACKGROUND & AIMS: There is controversy regarding the role of the type 2 immune response in the pathogenesis of ulcerative colitis (UC)-few data are available from treatment-naive patients. We investigated whether genes associated with a type 2 immune response in the intestinal mucosa are up-regulated in treatment-naive pediatric patients with UC compared with patients with Crohn's disease (CD)-associated colitis or without inflammatory bowel disease (IBD), and whether expression levels are associated with clinical outcomes. METHODS: We used a real-time reverse-transcription quantitative polymerase chain reaction array to analyze messenger RNA (mRNA) expression patterns in rectal mucosal samples from 138 treatment-naive pediatric patients with IBD and macroscopic rectal disease, as well as those from 49 children without IBD (controls), enrolled in a multicenter prospective observational study from 2008 to 2012. Results were validated in real-time reverse-transcription quantitative polymerase chain reaction analyses of rectal RNA from an independent cohort of 34 pediatric patients with IBD and macroscopic rectal disease and 17 controls from Cincinnati Children's Hospital Medical Center. RESULTS: We measured significant increases in mRNAs associated with a type 2 immune response (interleukin [IL]5 gene, IL13, and IL13RA2) and a type 17 immune response (IL17A and IL23) in mucosal samples from patients with UC compared with patients with colon-only CD. In a regression model, increased expression of IL5 and IL17A mRNAs distinguished patients with UC from patients with colon-only CD (P = .001; area under the receiver operating characteristic curve, 0.72). We identified a gene expression pattern in rectal tissues of patients with UC, characterized by detection of IL13 mRNA, that predicted clinical response to therapy after 6 months (odds ratio [OR], 6.469; 95% confidence interval [CI], 1.553-26.94), clinical response after 12 months (OR, 6.125; 95% CI, 1.330-28.22), and remission after 12 months (OR, 5.333; 95% CI, 1.132-25.12). CONCLUSIONS: In an analysis of rectal tissues from treatment-naive pediatric patients with IBD, we observed activation of a type 2 immune response during the early course of UC. We were able to distinguish patients with UC from those with colon-only CD based on increased mucosal expression of genes that mediate type 2 and type 17 immune responses. Increased expression at diagnosis of genes that mediate a type 2 immune response is associated with response to therapy and remission in pediatric patients with UC.