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Copyright © 2015 Cutler et al.Background: The inflammatory bowel diseases (IBD) are common, complex disorders in which genetic and environmental factors are believed to interact leading to chronic inflammatory responses against the gut microbiota. Earlier

Background Food allergies are a growing health problem, and the development of therapies that prevent disease onset is limited by the lack of adjuvant-free experimental animal models. We compared allergic sensitization in patients with food allergy or Wiskott-Aldrich syndrome ( WAS) and defined whether spontaneous disease in Was −/− mice recapitulates the pathology of a conventional disease model and/or human food allergy. Methods Comparative Immuno CAP ISAC microarray was performed in patients with food allergy or WAS. Spontaneous food allergy in Was −/− mice was compared to an adjuvant-based model in wild-type mice ( WT- OVA/alum). Intestinal and systemic anaphylaxis was assessed, and the role of the high-affinity IgE Fc receptor (Fcε RI) in allergic sensitization was evaluated using Was −/− Fcer1a −/− mice. Results Polysensitization to food was detected in both WAS and food-allergic patients which was recapitulated in the Was −/− model. Oral administration of ovalbumin (OVA) in Was −/− mice induced low titers of OVA-specific IgE compared to the WT- OVA/alum model. Irrespectively, 79% of Was −/− mice developed allergic diarrhea following oral OVA challenge. Systemic anaphylaxis occurred in Was −/− mice (95%) with a mortality rate >50%. Spontaneous sensitization and intestinal allergy occurred independent of Fcε RI expression on mast cells (MCs) and basophils. Conclusions Was −/− mice provide a model of food allergy with the advantage of mimicking polysensitization and low food-antigen IgE titers as observed in humans with clinical food allergy. This model will facilitate studies on aberrant immune responses during spontaneous disease development. Our results imply that therapeutic targeting of the IgE/Fcε RI activation cascade will not affect sensitization to food. [ABSTRACT FROM AUTHOR]

Background Cysteinyl leukotrienes contribute to Th2-type inflammatory immune responses. Their levels in oesophageal tissue, however, do not distinguish patients with eosinophilic oesophagitis (EoE) from controls. Objective We asked whether mRNA levels of leukotriene C4 synthase (LTC4S), a key regulator of leukotriene production, could serve as a marker for EoE. Methods Digital mRNA expression profiling (nCounter® Technology) was performed on proximal and distal oesophageal biopsies of 30 paediatric EoE patients and 40 non-EoE controls. Expression data were confirmed with RT- qPCR. LTC4S mRNA levels were quantified in whole blood samples. Leukotriene E4 was measured in urine. Results LTC4S mRNA levels were elevated in proximal (2.6-fold, P < 0.001) and distal (2.9-fold, P < 0.001) oesophageal biopsies from EoE patients. Importantly, increased LTC4S mRNA transcripts identified a subpopulation of EoE patients (28%). This patient subgroup had higher serum IgE levels (669 U/mL vs. 106 U/mL, P = 0.01), higher mRNA transcript numbers of thymic stromal lymphopoietin (TSLP) (1.6-fold, P = 0.009) and CD4 (1.4-fold, P = 0.04) but lower IL-23 mRNA levels (0.5-fold, P = 0.04). In contrast, elevated levels of IL-23 mRNA were found in oesophageal biopsies of patients with reflux oesophagitis. LTC4S mRNA transcripts in whole blood and urinary excretion of leukotriene E4 were similar in EoE patient subgroups and non-EoE patients. Conclusion & Clinical Relevance Elevated oesophageal expression of LTC4S mRNA is found in a subgroup of EoE patients, concomitant with higher serum IgE levels and an oesophageal transcriptome indicative of a more-pronounced allergic phenotype. Together with TSLP and IL-23 mRNA levels, oesophageal LTC4S mRNA may facilitate diagnosis of an EoE subpopulation for personalized therapy. [ABSTRACT FROM AUTHOR]

Leprosy is of interest to immunologists because the varied clinical manifestations of the disease correlate closely with the immunological spectrum. Resistance to infection is dependent on appropriate cell-mediated immunity, but patients with the lepromatous form fail to respond to antigens of M. leprae. In vitro studies have revealed the existence of T-suppressor cells of the phenotype CD8+, CD3+, HLA-DR+, FcR+, 9·3-, which are restricted by major histocompatibility complex (MHC) class II antigens. Several new candidate vaccines against leprosy have been effective in breaking immunological unresponsiveness and engendering cell-mediated immunity in leproma- tous leprosy patients, including the combination of BCG + killed M. leprae. Because BCG has unique adjuvant properties, we have begun to use molecular genetic approaches to develop BCG into a multivaccine vehicle capable of immunizing simultaneously against several pathogens. Both phage- based and plasmid-based strategies have been successfully developed for introducing selectable markers into BCG for the first time. [ABSTRACT FROM AUTHOR]

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 104 to 105 times greater than those of the parent strain, yielding more than 105 transformants per μg of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria. [ABSTRACT FROM AUTHOR]

Background & Aims: The evolution of complicated pediatric Crohn's disease (CD) in the era of anti-tumor necrosis factor (aTNF) therapy continues to be described. Because CD progresses from inflammatory to stricturing (B2) and penetrating (B3) disease behaviors in a subset of patients, we aimed to understand the risk of developing complicated disease behavior or undergoing surgery in relation to aTNF timing and body mass index z-score (BMIz) normalization., Methods: Multicenter, 5-year longitudinal data from 1075 newly diagnosed CD patients were analyzed. Descriptive statistics, univariate and stepwise multivariate Cox proportional hazard regression (CPHR), and log-rank analyses were performed for risk of surgery and complicated disease behaviors. Differential gene expression from ileal bulk RNA sequencing was correlated with outcomes., Results: Stricturing complications had the largest increase: from 2.98% to 10.60% over 5 years. Multivariate CPHR showed aTNF exposure within 3 months from diagnosis (hazard ratio [HR], 0.33; 95% CI, 0.15-0.71) and baseline L2 disease (HR, 0.29; 95% CI, 0.09-0.92) to be associated with reduced B1 to B2 progression. For children with a low BMIz at diagnosis (n = 294), multivariate CPHR showed BMIz normalization within 6 months of diagnosis (HR, 0.47; 95% CI, 0.26-0.85) and 5-aminosalicyclic acid exposure (HR, 0.32; 95% CI, 0.13-0.81) were associated with a decreased risk for surgery while B2 (HR, 4.20; 95% CI, 1.66-10.65) and B2+B3 (HR, 8.24; 95% CI, 1.08-62.83) at diagnosis increased surgery risk. Patients without BMIz normalization were enriched for genes in cytokine production and inflammation., Conclusions: aTNF exposure up to 3 months from diagnosis may reduce B2 progression. In addition, lack of BMIz normalization within 6 months of diagnosis is associated with increased surgery risk and a proinflammatory transcriptomic profile., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Chronic granulomatous disease (CGD) is caused by defects in any 1 of the 6 subunits forming the nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2), leading to severely reduced or absent phagocyte-derived reactive oxygen species production. Almost 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). While conventional IBD therapies can treat CGD-IBD, their benefits must be weighed against the risk of infection. Understanding the impact of NOX2 defects on the intestinal microbiota may lead to the identification of novel CGD-IBD treatments., Objective: We sought to identify microbiome and metabolome signatures that can distinguish individuals with CGD and CGD-IBD., Methods: We conducted a cross-sectional observational study of 79 patients with CGD, 8 pathogenic variant carriers, and 19 healthy controls followed at the National Institutes of Health Clinical Center. We profiled the intestinal microbiome (amplicon sequencing) and stool metabolome, and validated our findings in a second cohort of 36 patients with CGD recruited through the Primary Immune Deficiency Treatment Consortium., Results: We identified distinct intestinal microbiome and metabolome profiles in patients with CGD compared to healthy individuals. We observed enrichment for Erysipelatoclostridium spp, Sellimonas spp, and Lachnoclostridium spp in CGD stool samples. Despite differences in bacterial alpha and beta diversity between the 2 cohorts, several taxa correlated significantly between both cohorts. We further demonstrated that patients with CGD-IBD have a distinct microbiome and metabolome profile compared to patients without CGD-IBD., Conclusion: Intestinal microbiome and metabolome signatures distinguished patients with CGD and CGD-IBD, and identified potential biomarkers and therapeutic targets., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Inflammatory bowel disease (IBD) is a group of chronic digestive tract inflammatory conditions whose genetic etiology is still poorly understood. The incidence of IBD is particularly high among Ashkenazi Jews. Here, we identify 8 novel and plausible IBD-causing genes from the exomes of 4453 genetically identified Ashkenazi Jewish IBD cases (1734) and controls (2719). Various biological pathway analyses are performed, along with bulk and single-cell RNA sequencing, to demonstrate the likely physiological relatedness of the novel genes to IBD. Importantly, we demonstrate that the rare and high impact genetic architecture of Ashkenazi Jewish adult IBD displays significant overlap with very early onset-IBD genetics. Moreover, by performing biobank phenome-wide analyses, we find that IBD genes have pleiotropic effects that involve other immune responses. Finally, we show that polygenic risk score analyses based on genome-wide high impact variants have high power to predict IBD susceptibility., (© 2023. The Author(s).)

Background: Emerging data showed patients with chronic inflammatory disorders, including inflammatory bowel disease, are more likely to develop atherosclerotic cardiovascular diseases, heart failure, and atrial fibrillation. This article aims to review the evidence of those associations., Methods: PubMed was searched from inception to January 2022 using the keywords, including inflammatory bowel diseases, Crohn disease, ulcerative colitis, atherosclerotic cardiovascular disease, coronary artery disease, cardiovascular disease, atrial fibrillation, heart failure, and premature coronary artery disease. Relevant literature, including retrospective/prospective cohort studies, clinical trials, meta-analyses, and guidelines, were reviewed and summarized., Results: Both ulcerative colitis and Crohn disease are associated with an increased risk of atherosclerotic cardiovascular diseases, cerebrovascular accidents, premature coronary artery disease, and atrial fibrillation. Ulcerative colitis is associated with an increased risk of heart failure. The increased atrial fibrillation occurred during inflammatory bowel disease flares and persistent activity but not during periods of remission. Hypotheses for the mechanism underlying the association of inflammatory bowel disease and atherosclerotic cardiovascular diseases include shared risk factors (ie, obesity, diabetes, smoking, diet) and pathophysiology (gut microbiome dysfunction) or adverse effects from inflammatory bowel disease itself or its treatment (ie, chronic inflammation, dyslipidemia, thrombocytosis, steroids)., Conclusion: Inflammatory bowel disease is associated with an increased risk of atherosclerotic cardiovascular diseases, heart failure, and atrial fibrillation. A multidisciplinary team with gastroenterologists and cardiologists is needed to optimize the care for patients with inflammatory bowel disease and associated cardiac diseases., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Research on the utilization and effectiveness of antitumor necrosis factor (TNF) biologics in children with very early onset inflammatory bowel disease (VEOIBD) is urgently needed. Here we describe anti-TNF use and durability in a multicenter cohort., Methods: We performed a retrospective cohort study of patients diagnosed with VEOIBD (<6 years) between 2008 and 2013 at 25 North American centers. We performed chart abstraction at diagnosis and 1, 3, and 5 years after diagnosis. We examined the rate of initiation and durability of infliximab and adalimumab and evaluated associations between treatment durability and the following covariates with multivariate Cox proportional hazard regression: age at diagnosis, sex, disease duration, disease classification, and presence of combined immunomodulatory treatment versus monotherapy., Results: Of 294 children with VEOIBD, 120 initiated treatment with anti-TNF therapy and 101 had follow-up data recorded [50% Crohn disease (CD), 31% ulcerative colitis (UC), and 19% IBD unclassified (IBD-U)]. The cumulative probability of anti-TNF treatment was 15% at 1 year, 30% at 3 years, and 45% at 5 years from diagnosis; 56 (55%) were treated between 0 and 6 years old. Anti-TNF durability was 90% at 1 year, 75% at 3 years, and 55% at 5 years. The most common reason for discontinuation of anti-TNF were loss of response in 24 (57%) children. Children with UC/IBD-U had lower durability than those with CD (hazard ratio [HR] 0.17; 95% confidence interval [CI], 0.06-0.51; P = 0.001)., Conclusions: Utilization and durability of anti-TNF in VEOIBD is relatively high and comparable with older children. Having Crohn disease (compared with UC/IBD-U) is associated with greater durability., (Copyright © 2022 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Mutations in the tetratricopeptide repeat domain 7A (TTC7A) gene are a rare cause of congenital enteropathy that can result in significant morbidity. TTC7A deficiency leads to disruption of the intestinal epithelium. The histopathology of this condition has been partly described in case reports and clinical studies. This manuscript describes an in-depth investigation of the pediatric gastrointestinal pathology of the largest histologically examined cohort with confirmed TTC7A mutations reported to date and, for the first time, compared the findings to age-matched and sex-matched control patients with intestinal atresia not thought to be associated with TTC7A mutations. Hematoxylin and eosin-stained slides of endoscopically obtained mucosal biopsies and surgical resection specimens from 7 patients with known TTC7A mutations were examined retrospectively. The microscopic findings were found to be on a spectrum from atresia-predominant to those with predominantly epithelial abnormalities. Several unique histopathologic characteristics were observed when compared with controls. These included neutrophilic colitis and prominent lamina propria eosinophilia throughout the gastrointestinal tract. Striking architectural abnormalities of the epithelium were observed in 4 of the 7 patients. The 5 patients with intestinal atresia demonstrated hypertrophy and disorganization of the colonic muscularis mucosae accompanied by bland spindle cell nodules within the intestinal wall. The components of the latter were further elucidated using immunohistochemistry, and we subsequently hypothesize that they represent obliterated mucosa with remnants of the muscularis mucosae. Finally, atrophic gastritis was noted in 4 patients. In conclusion, the unique histopathologic characteristics of TTC7A mutation-associated enteropathy described herein more fully describe this novel disease entity in infants who present with congenital enteropathy or enterocolitis., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background & Aims: Monogenic forms of inflammatory bowel disease (IBD) illustrate the essential roles of individual genes in pathways and networks safeguarding immune tolerance and gut homeostasis., Methods: To build a taxonomy model, we assessed 165 disorders. Genes were prioritized based on penetrance of IBD and disease phenotypes were integrated with multi-omics datasets. Monogenic IBD genes were classified by (1) overlapping syndromic features, (2) response to hematopoietic stem cell transplantation, (3) bulk RNA-sequencing of 32 tissues, (4) single-cell RNA-sequencing of >50 cell subsets from the intestine of healthy individuals and patients with IBD (pediatric and adult), and (5) proteomes of 43 immune subsets. The model was validated by addition of newly identified monogenic IBD defects. As a proof-of-concept, we explore the intersection between immunometabolism and antimicrobial activity for a group of disorders (G6PC3/SLC37A4)., Results: Our quantitative integrated taxonomy defines the cellular landscape of monogenic IBD gene expression across 102 genes with high and moderate penetrance (81 in the model set and 21 genes in the validation set). We illustrate distinct cellular networks, highlight expression profiles across understudied cell types (e.g., CD8 + T cells, neutrophils, epithelial subsets, and endothelial cells) and define genotype-phenotype associations (perianal disease and defective antimicrobial activity). We illustrate processes and pathways shared across cellular compartments and phenotypic groups and highlight cellular immunometabolism with mammalian target of rapamycin activation as one of the converging pathways. There is an overlap of genes and enriched cell-specific expression between monogenic and polygenic IBD., Conclusion: Our taxonomy integrates genetic, clinical and multi-omic data; providing a basis for genomic diagnostics and testable hypotheses for disease functions and treatment responses., (Copyright © 2022 AGA Institute. All rights reserved.)

Background: Heterozygous germline mutations in cytotoxic T lymphocyte-associated antigen-4 (CTLA4) impair the immunomodulatory function of regulatory T cells. Affected individuals are prone to life-threatening autoimmune and lymphoproliferative complications. A number of therapeutic options are currently being used with variable effectiveness., Objective: Our aim was to characterize the responsiveness of patients with CTLA-4 insufficiency to specific therapies and provide recommendations for the diagnostic workup and therapy at an organ-specific level., Methods: Clinical features, laboratory findings, and response to treatment were reviewed retrospectively in an international cohort of 173 carriers of CTLA4 mutation. Patients were followed between 2014 and 2020 for a total of 2624 months from diagnosis. Clinical manifestations were grouped on the basis of organ-specific involvement. Medication use and response were recorded and evaluated., Results: Among the 173 CTLA4 mutation carriers, 123 (71%) had been treated for immune complications. Abatacept, rituximab, sirolimus, and corticosteroids ameliorated disease severity, especially in cases of cytopenias and lymphocytic organ infiltration of the gut, lungs, and central nervous system. Immunoglobulin replacement was effective in prevention of infection. Only 4 of 16 patients (25%) with cytopenia who underwent splenectomy had a sustained clinical response. Cure was achieved with stem cell transplantation in 13 of 18 patients (72%). As a result of the aforementioned methods, organ-specific treatment pathways were developed., Conclusion: Systemic immunosuppressants and abatacept may provide partial control but require ongoing administration. Allogeneic hematopoietic stem cell transplantation offers a possible cure for patients with CTLA-4 insufficiency., (Copyright © 2021. Published by Elsevier Inc.)

Background: Clinical and molecular subcategories of inflammatory bowel disease (IBD) are needed to discover mechanisms of disease and predictors of response and disease relapse. We aimed to develop a study of a prospective adult research cohort with IBD (SPARC IBD) including longitudinal clinical and patient-reported data and biosamples., Methods: We established a cohort of adults with IBD from a geographically diverse sample of patients across the United States with standardized data and biosample collection methods and sample processing techniques. At enrollment and at time of lower endoscopy, patient-reported outcomes (PRO), clinical data, and endoscopy scoring indices are captured. Patient-reported outcomes are collected quarterly. The quality of clinical data entry after the first year of the study was assessed., Results: Through January 2020, 3029 patients were enrolled in SPARC, of whom 66.1% have Crohn's disease (CD), 32.2% have ulcerative colitis (UC), and 1.7% have IBD-unclassified. Among patients enrolled, 990 underwent colonoscopy. Remission rates were 63.9% in the CD group and 80.6% in the UC group. In the quality study of the cohort, there was 96% agreement on year of diagnosis and 97% agreement on IBD subtype. There was 91% overall agreement describing UC extent as left-sided vs extensive or pancolitis. The overall agreement for CD behavior was 83%., Conclusion: The SPARC IBD is an ongoing large prospective cohort with longitudinal standardized collection of clinical data, biosamples, and PROs representing a unique resource aimed to drive discovery of clinical and molecular markers that will meet the needs of precision medicine in IBD., (© 2021 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Transmural healing (TH) is associated with better long-term outcomes in Crohn disease (CD), whereas pretreatment ileal gene signatures encoding myeloid inflammatory responses and extracellular matrix production are associated with stricturing. We aimed to develop a predictive model for ileal TH and to identify ileal genes and microbes associated with baseline luminal narrowing (LN), a precursor to strictures., Materials and Methods: Baseline small bowel imaging obtained in the RISK pediatric CD cohort study was graded for LN. Ileal gene expression was determined by RNASeq, and the ileal microbial community composition was characterized using 16S rRNA amplicon sequencing. Clinical, demographic, radiologic, and genomic variables were tested for association with baseline LN and future TH., Results: After controlling for ileal location, baseline ileal LN (odds ratio [OR], 0.3; 95% confidence interval [CI], 0.1-0.8), increasing serum albumin (OR, 4; 95% CI, 1.3-12.3), and anti-Saccharomyces cerevisiae antibodies IgG serology (OR, 0.97; 95% CI, 0.95-1) were associated with subsequent TH. A multivariable regression model including these factors had excellent discriminant power for TH (area under the curve, 0.86; positive predictive value, 80%; negative predictive value, 87%). Patients with baseline LN exhibited increased Enterobacteriaceae and inflammatory and extracellular matrix gene signatures, coupled with reduced levels of butyrate-producing commensals and a respiratory electron transport gene signature. Taxa including Lachnospiraceae and the genus Roseburia were associated with increased respiratory and decreased inflammatory gene signatures, and Aggregatibacter and Blautia bacteria were associated with reduced extracellular matrix gene expression., Conclusions: Pediatric patients with CD with LN at diagnosis are less likely to achieve TH. The association between specific microbiota, wound healing gene programs, and LN may suggest future therapeutic targets., (© 2021 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Abstract BACKGROUND: Members of the public will soon be taking commercial suborbital spaceflights with significant G x (chest-to-back) acceleration potentially reaching up to 6 G x . Pulmonary physiology is gravity-dependent and is likely to be affected, which may have clinical implications for medically susceptible individuals. METHODS: During 2-min centrifuge exposures ranging up to 6 G x , 11 healthy subjects were studied using advanced respiratory techniques. These sustained exposures were intended to allow characterization of the underlying pulmonary response and did not replicate actual suborbital G profiles. Regional distribution of ventilation in the lungs was determined using electrical impedance tomography. Neural respiratory drive (from diaphragm electromyography) and work of breathing (from transdiaphragmatic pressures) were obtained via nasoesophageal catheters. Arterial blood gases were measured in a subset of subjects. Measurements were conducted while breathing air and breathing 15 oxygen to simulate anticipated cabin pressurization conditions. RESULTS: Acceleration caused hypoxemia that worsened with increasing magnitude and duration of G x . Minimum arterial oxygen saturation at 6 G x was 86 1 breathing air and 79 1 breathing 15 oxygen. With increasing G x the alveolar-arterial (A-a) oxygen gradient widened progressively and the relative distribution of ventilation reversed from posterior to anterior lung regions with substantial gas-trapping anteriorly. Severe breathlessness accompanied large progressive increases in work of breathing and neural respiratory drive. DISCUSSION: Sustained high-G acceleration at magnitudes relevant to suborbital flight profoundly affects respiratory physiology. These effects may become clinically important in the most medically susceptible passengers, in whom the potential role of centrifuge-based preflight evaluation requires further investigation. Pollock RD, Jolley CJ, Abid N, Couper JH, Estrada-Petrocelli L, Hodkinson PD, Leonhardt S, Mago-Elliott S, Menden T, Rafferty G, Richmond G, Robbins PA, Ritchie GAD, Segal MJ, Stevenson AT, Tank HD, Smith TG. Pulmonary effects of sustained periods of high-G acceleration relevant to suborbital spaceflight . Aerosp Med Hum Perform. 2021; 92(7):633641.

Background: The incidence of very early onset inflammatory bowel disease (VEOIBD) is increasing, yet the phenotype and natural history of VEOIBD are not well described., Methods: We performed a retrospective cohort study of patients diagnosed with VEOIBD (6 years of age and younger) between 2008 and 2013 at 25 North American centers. Eligible patients at each center were randomly selected for chart review. We abstracted data at diagnosis and at 1, 3, and 5 years after diagnosis. We compared the clinical features and outcomes with VEOIBD diagnosed younger than 3 years of age with children diagnosed with VEOIBD at age 3 to 6 years., Results: The study population included 269 children (105 [39%] Crohn's disease, 106 [39%] ulcerative colitis, and 58 [22%] IBD unclassified). The median age of diagnosis was 4.2 years (interquartile range 2.9-5.2). Most (94%) Crohn's disease patients had inflammatory disease behavior (B1). Isolated colitis (L2) was the most common disease location (70% of children diagnosed younger than 3 years vs 43% of children diagnosed 3 years and older; P = 0.10). By the end of follow-up, stricturing/penetrating occurred in 7 (6.6%) children. The risk of any bowel surgery in Crohn's disease was 3% by 1 year, 12% by 3 years, and 15% by 5 years and did not differ by age at diagnosis. Most ulcerative colitis patients had pancolitis (57% of children diagnosed younger than 3 years vs 45% of children diagnosed 3 years and older; P = 0.18). The risk of colectomy in ulcerative colitis/IBD unclassified was 0% by 1 year, 3% by 3 years, and 14% by 5 years and did not differ by age of diagnosis., Conclusions: Very early onset inflammatory bowel disease has a distinct phenotype with predominantly colonic involvement and infrequent stricturing/penetrating disease. The cumulative risk of bowel surgery in children with VEOIBD was approximately 14%-15% by 5 years. These data can be used to provide anticipatory guidance in this emerging patient population., (© 2020 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Advances in genetic technologies provide opportunities for patient care and ethical challenges. Clinical care of patients with rare Mendelian disorders is often at the forefront of those developments. Whereas in classical polygenic inflammatory bowel disease (IBD), the predictive value of genetic variants is very low, predictive prenatal genetic diagnosis can inform families at high risk of severe genetic disorders. Patients with IL-10 signalling defects because of pathogenic variants in IL10RA, Il10RB, and IL10 develop severe infantile onset inflammatory bowel disease that is completely penetrant and has a high morbidity and substantial mortality despite treatment., Methods: We performed a survey among tertiary specialist paediatric centers of 10 countries on the utilization of predictive prenatal genetic diagnosis in IL-10 signalling defects. We retrospectively report prenatal genetics in a series of 8 families., Results: International variation in legislation, guidelines, expert opinion, as well as cultural and religious background of families and clinicians results in variable utilization of preimplantation and prenatal genetic testing for IL-10 signalling defects. Eleven referrals for prenatal diagnosis for IL-10 signalling defects were identified across 4 countries. We report on 8 families who underwent prenatal preimplantation monogenic testing after in vitro fertilization (n = 2) and/or by amniocentesis/chorion villus sampling (n = 6). A genetic diagnosis was established in 1 foetus and excluded in 7 foetuses (all IL10RA variants)., Conclusions: Prenatal genetic testing for IL10R-defects is feasible, yet the legal and ethical considerations are complex and controversial. In some countries, predictive genetics for IL-10-related signalling defects is entering clinical practice., Competing Interests: The authors report no conflict of interest., (Copyright © 2020 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Background and Aims: Ileal strictures are the major indication for resective surgery in Crohn's disease (CD). We aimed to define ileal gene programs present at diagnosis linked with future stricturing behavior during five year follow-up, and to identify potential small molecules to reverse these gene signatures., Methods: Antimicrobial serologies and pre-treatment ileal gene expression were assessed in a representative subset of 249 CD patients within the RISK multicenter pediatric CD inception cohort study, including 113 that are unique to this report. These data were used to define genes associated with stricturing behavior and for model testing to predict stricturing behavior. A bioinformatics approach to define small molecules which may reverse the stricturing gene signature was applied., Results: 19 of the 249 patients developed isolated B2 stricturing behavior during follow-up, while 218 remained B1 inflammatory. Using deeper RNA sequencing than in our prior report, we have now defined an inflammatory gene signature including an oncostatin M co-expression signature, tightly associated with extra-cellular matrix (ECM) gene expression in those who developed stricturing complications. We further computationally prioritize small molecules targeting macrophage and fibroblast activation and angiogenesis which may reverse the stricturing gene signature. A model containing ASCA and CBir1 serologies and a refined eight ECM gene set was significantly associated with stricturing development by year five after diagnosis (AUC (95th CI) = 0.82 (0.7-0.94))., Conclusion: An ileal gene program for macrophage and fibroblast activation is linked to stricturing complications in treatment naïve pediatric CD, and may inform novel small molecule therapeutic approaches., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The rate of pediatric inflammatory bowel disease (IBD) has been increasing over the last decade and this increase has occurred most rapidly in the youngest children diagnosed <6 years, known as very early-onset inflammatory bowel disease (VEO-IBD). These children can present with more extensive and severe disease than older children and adults. The contribution of host genetics in this population is underscored by the young age of onset and the distinct, aggressive phenotype. In fact, monogenic defects, often involving primary immunodeficiency genes, have been identified in children with VEO-IBD and have led to targeted and life-saving therapy. This position paper will discuss the phenotype of VEO-IBD and outline the approach and evaluation for these children and what factors should trigger concern for an underlying immunodeficiency. We will then review the immunological assays and genetic studies that can facilitate the identification of the underlying diagnosis in patients with VEO-IBD and how this evaluation may lead to directed therapies. The position paper will also aid the pediatric gastroenterologist in recognizing when a patient should be referred to a center specializing in the care of these patients. These guidelines are intended for pediatricians, allied health professionals caring for children, pediatric gastroenterologists, pediatric pathologists, and immunologists.

Background: Variation in care is common in medical practice. Reducing variation in care is shown to improve quality and increase favorable outcomes in chronic diseases. We sought to identify factors associated with variation in care in children with newly diagnosed Crohn's disease (CD)., Methods: Prospectively collected data from a 28-site multicenter inception CD cohort were analyzed for variations in diagnostic modalities, treatment, and follow-up monitoring practices, along with complicated disease outcomes over 3 years in 1046 children. Generalized linear mixed effects models were used to investigate the intercenter variations in each outcome variable., Results: The mean age at diagnosis was 12 years, and 25.9% were nonwhite. The number of participants ranged from 5 to 112 per site. No variation existed in the initial diagnostic approach. When medication exposure was analyzed, steroid exposure varied from 28.6% to 96.9% (P < 0.01) within 90 days, but variation was not significant over a 3-year period (P = 0.13). Early anti-tumor necrosis factor (anti-TNF) exposure (within 90 days) varied from 2.1% to 65.7% (P < 0.01), but variation was not significant over a 3-year period (P > 0.99). Use of immunomodulators (IMs) varied among centers both within 90 days (P < 0.01) and during 3 years of follow-up (P < 0.01). A significant variation was seen at the geographic level with follow-up small bowel imaging and colonoscopy surveillance after initial therapy., Conclusions: Intercenter variation in care was seen with the initial use of steroids and anti-TNF, but there was no difference in total 3-year exposure to these drugs. Variation in the initiation and long-term use of IMs was significant among sites, but further research with objective measures is needed to explain this variation of care. Small bowel imaging or repeat colonoscopy in CD patients was not uniformly performed across sites. As our data show the widespread existence of variation in care and disease monitoring at geographic levels among pediatric CD patients, future implementation of various practice strategies may help reduce the variation in care., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Children with oropharyngeal dysphagia have impaired airway protection mechanisms and are at higher risk for pneumonia and other pulmonary complications. Aspiration of gastric contents is often implicated as a cause for these pulmonary complications, despite being supported by little evidence. The goal of this study is to determine the relative contribution of oropharyngeal and gastric microbial communities to perturbations in the lung microbiome of children with and without oropharyngeal dysphagia and aspiration., Methods: We conducted a prospective cohort study of 220 patients consecutively recruited from a tertiary aerodigestive center undergoing simultaneous esophagogastroduodenoscopy and flexible bronchoscopy. Bronchoalveolar lavage, gastric and oropharyngeal samples were collected from all recruited patients and 16S sequencing was performed. A subset of 104 patients also underwent video fluoroscopic swallow studies to assess swallow function and were categorized as aspiration/no aspiration. To ensure the validity of the results, we compared the microbiome of these aerodigestive patients to the microbiome of pediatric patients recruited to a longitudinal cohort study of children with suspected GERD; patients recruited to this study had oropharyngeal, gastric and/or stool samples available. The relationships between microbial communities across the aerodigestive tract were described by analyzing within- and between-patient beta diversities and identifying taxa which are exchanged between aerodigestive sites within patients. These relationships were then compared in patients with and without aspiration to evaluate the effect of aspiration on the aerodigestive microbiome., Results: Within all patients, lung, oropharyngeal and gastric microbiomes overlap. The degree of similarity is the lowest between the oropharynx and lungs (median Jensen-Shannon distance (JSD) = 0.90), and as high between the stomach and lungs as between the oropharynx and stomach (median JSD = 0.56 for both; p = 0.6). Unlike the oropharyngeal microbiome, lung and gastric communities are highly variable across people and driven primarily by person rather than body site. In patients with aspiration, the lung microbiome more closely resembles oropharyngeal rather than gastric communities and there is greater prevalence of microbial exchange between the lung and oropharynx than between gastric and lung sites (p = 0.04 and 4x10-5, respectively)., Conclusions: The gastric and lung microbiomes display significant overlap in patients with intact airway protective mechanisms while the lung and oropharynx remain distinct. In patients with impaired swallow function and aspiration, the lung microbiome shifts towards oropharyngeal rather than gastric communities. This finding may explain why antireflux surgeries fail to show benefit in pediatric pulmonary outcomes., Competing Interests: The authors have declared that no competing interests exist.

Age-of-diagnosis associated variation in disease location and antimicrobial sero-reactivity has suggested fundamental differences in pediatric Crohn Disease (CD) pathogenesis. This variation may be related to pubertal peak incidence of ileal involvement and Peyer's patches maturation, represented by IFNγ-expressing Th1 cells. However, direct mucosal evidence is lacking. We characterize the global pattern of ileal gene expression and microbial communities in 525 treatment-naive pediatric CD patients and controls (Ctl), stratifying samples by their age-of-diagnosis. We show a robust ileal gene signature notable for higher expression of specific immune genes including GM-CSF and INFγ, and reduced expression of antimicrobial Paneth cell α-defensins, in older compared to younger patients. Reduced α-defensin expression in older patients was associated with higher IFNγ expression. By comparison, the CD-associated ileal dysbiosis, characterized by expansion of Enterobacteriaceae and contraction of Lachnospiraceae and Ruminococcaceae, was already established within the younger group and did not vary systematically with increasing age-of-diagnosis. Multivariate analysis considering individual taxa, however did demonstrate negative associations between Lachnospiraceae and IFNγ, and positive associations between Bacteroides and α-defensin expression. These data provide evidence for maturation of mucosal Th1 immune responses and loss of epithelial antimicrobial α-defensins which are associated with specific taxa with increasing age-of-diagnosis in pediatric CD.

Background: Granulocyte-macrophage colony-stimulating factor auto-antibodies (GMAbs) suppress neutrophil-extrinsic GM-CSF signaling and increase risk for stricturing behavior in Crohn's disease (CD). We aimed to define clinical, genomic, and functional associations with neutrophil-intrinsic GM-CSF signaling., Methods: Missense mutations in CSF2RA, CSF2RB, JAK2, STAT5A, and STAT5B were identified using whole-exome sequencing in 543 pediatric inflammatory bowel disease (IBD) patients. Neutrophil-intrinsic GM-CSF signaling was defined using the GM-CSF-induced STAT5 stimulation index (GMSI) in 180 pediatric IBD patients and 26 non-IBD controls. Reduced GM-CSF signaling (GMSI-Lo) was defined as the 20th percentile within the control group. Variation in neutrophil phospho-protein abundance, bacterial killing, and the global pattern of gene expression with the GMSI was determined., Results: We validated 18 potentially damaging missense mutations in CSF2RA and CSF2RB. CSF2RA A17G carriage increased from 10% in those with intact neutrophil GMSI to 32% in those with low GMSI (P = 0.02). The frequency of reduced Staphylococcus aureus killing increased from 17% in those with intact neutrophil GMSI to 35% in GMSI-Lo neutrophils (P = 0.043). Crohn's disease neutrophils with low GMSI exhibited specific alterations in phospho-protein networks and genes regulating cytokine production, wound healing, and cell survival and proliferation. Stricturing behavior increased from 7% in patients with both low GMAb and intact GMSI to 64% in patients with both elevated GMAb and low GMSI (P < 0.0001)., Conclusions: Low/normal neutrophil-intrinsic GM-CSF signaling is associated with CSF2RA missense mutations, alterations in gene expression networks, and higher rates of disease complications in pediatric CD., (© 2018 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

Background: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a negative immune regulator. Heterozygous CTLA4 germline mutations can cause a complex immune dysregulation syndrome in human subjects., Objective: We sought to characterize the penetrance, clinical features, and best treatment options in 133 CTLA4 mutation carriers., Methods: Genetics, clinical features, laboratory values, and outcomes of treatment options were assessed in a worldwide cohort of CTLA4 mutation carriers., Results: We identified 133 subjects from 54 unrelated families carrying 45 different heterozygous CTLA4 mutations, including 28 previously undescribed mutations. Ninety mutation carriers were considered affected, suggesting a clinical penetrance of at least 67%; median age of onset was 11 years, and the mortality rate within affected mutation carriers was 16% (n = 15). Main clinical manifestations included hypogammaglobulinemia (84%), lymphoproliferation (73%), autoimmune cytopenia (62%), and respiratory (68%), gastrointestinal (59%), or neurological features (29%). Eight affected mutation carriers had lymphoma, and 3 had gastric cancer. An EBV association was found in 6 patients with malignancies. CTLA4 mutations were associated with lymphopenia and decreased T-, B-, and natural killer (NK) cell counts. Successful targeted therapies included application of CTLA-4 fusion proteins, mechanistic target of rapamycin inhibitors, and hematopoietic stem cell transplantation. EBV reactivation occurred in 2 affected mutation carriers after immunosuppression., Conclusions: Affected mutation carriers with CTLA-4 insufficiency can present in any medical specialty. Family members should be counseled because disease manifestation can occur as late as 50 years of age. EBV- and cytomegalovirus-associated complications must be closely monitored. Treatment interventions should be coordinated in clinical trials., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Objectives: Environmental factors play an important role in the pathogenesis of Crohn's Disease (CD). In particular, by virtue of the instability of the microbiome and development of immunologic tolerance, early life factors may exert the strongest influence on disease risk and phenotype., Methods: We used data from 1119 CD subjects recruited from RISK inception cohort to examine the impact of early life environment on disease progression. Our primary exposures of interest were breastfeeding in infancy and exposure to maternal, active, or passive smoke. Our primary outcomes were development of complicated (stricturing or penetrating) disease, and need for CD-related hospitalization, and surgery. Multivariable logistic regression models were used to define independent associations, adjusting for relevant covariates., Results: Our study cohort included 1119 patients with CD among whom 15% had stricturing (B2) or penetrating disease (B3) by 3 years. 331 patients (35%) and 95 patients (10.6%) required CD-related hospitalizations and surgery respectively. 74.5% were breastfed in infancy and 31% were exposed to smoking among whom 7% were exposed to maternal smoke. On multivariable analysis, a history of breastfeeding was inversely associated with complicated (B2/B3 disease) 0.65, CI 95% 0.44-96; P = 0.03) in pediatric CD. Maternal smoking during pregnancy was associated with increased risk of hospitalization during the 3-year follow-up period (OR 1.75, CI 95% 1.05-2.89; P = 0.03)., Conclusions: Early life environmental factors influence the eventual phenotypes and disease course in CD.

Background: Inflammatory bowel disease (IBD) mainly consists of Crohn's disease (CD) and ulcerative colitis (UC). About 10%-15% of patients with IBD cannot be firmly diagnosed with CD or UC; hence, they are initially diagnosed as inflammatory bowel disease unclassified (IBD-U). Having a firm diagnosis is clearly preferred to guide treatment choices, and better understanding of the nature of IBD-U is required., Methods: We performed an analysis of a subset of pediatric subjects from an inception IBD cohort of patients initially enrolled in a prospective multicenter study (the RISK study). Initial diagnosis and 2-year follow-up data from the subjects diagnosed with IBD-U were analyzed. An expert panel verified final diagnosis using predefined criteria as a guide. Serological and disease-relevant ileal and rectal tissue gene expression profiles were investigated. The use and the time to initiate anti-TNFα treatment was analyzed among the outcome groups., Results: A total of 1411 subjects were enrolled with initial diagnosis of IBD, and among them, 136 subjects were initially diagnosed as IBD-U at enrollment. And 26% were reclassified as UC and 14% as CD within 2 years of diagnosis, while 60% remained as IBD-U. Of those who were reclassified, there was a 2:1 ratio, UC (n = 35) to CD (n = 19). The molecular and serological features of IBD-U at the end of follow-up were very similar to UC and very different from CD. There was less likelihood of receiving anti-TNFα agents if the diagnosis was IBD-U compared with CD (P < 0.0001)., Conclusions: In our cohort, 60% of the IBD-U subjects remained as unclassified at 2 years; of those subsequently classified, a higher percentage followed a course more similar to UC. Most of the IBD-U subjects at diagnosis had serological and molecular signatures that are very similar to UC. Although the atypical presentations made the clinician to make an interim diagnosis of IBD-U, results of the molecular and serological factors performed at the time of diagnosis suggests that they were very similar to UC. However, long-term studies are needed to better understand the natural history and molecular characterization of pediatric onset IBD-U. 10.1093/ibd/izy136&#95;video1Video 1.Video 1. Watch now at https://academic.oup.com/ibd/article-lookup/doi/10.1093/ibd/izy136izy136.video15791389938001.

Background: Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is a negative immune regulator on the surface of T cells. In humans, heterozygous germline mutations in CTLA4 can cause an immune dysregulation syndrome. The phenotype comprises a broad spectrum of autoinflammatory, autoimmune, and immunodeficient features. An increased frequency of malignancies in primary immunodeficiencies is known, but their incidence in CTLA-4 insufficiency is unknown. Methods: Clinical manifestations and details of the clinical history were assessed in a worldwide cohort of 184 CTLA4 mutation carriers. Whenever a malignancy was reported, a malignancy-specific questionnaire was filled. Results: Among the 184 CTLA4 mutation carriers, 131 were considered affected, indicating a penetrance of 71.2%. We documented 17 malignancies, which amounts to a cancer prevalence of 12.9% in affected CTLA4 mutation carriers. There were ten lymphomas, five gastric cancers, one multiple myeloma, and one metastatic melanoma. Seven lymphomas and three gastric cancers were EBV-associated. Conclusion: Our findings demonstrate an elevated cancer risk for patients with CTLA-4 insufficiency. As more than half of the cancers were EBV-associated, the failure to control oncogenic viruses seems to be part of the CTLA-4-insufficient phenotype. Hence, lymphoproliferation and EBV viral load in blood should be carefully monitored, especially when immunosuppressing affected CTLA4 mutation carriers.

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., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Goal: The aim of this study was to evaluate the effect of combination therapy with methotrexate or 6-mercaptopurine on infliximab levels (IFXL) and antibodies to infliximab (ATI)., Background: Infliximab (IFX) is a highly effective therapy for inflammatory bowel disease (IBD). Unfortunately, 25%-50% of patients will lose response to IFX. Loss of response is correlated with low IFXL and ATI formation which accelerates drug clearance. Combination therapy is thought to decrease ATI formation., Methods: We performed a cross-sectional analysis of 223 pediatric and young adult patients with IBD on IFX. IFXL and ATI were measured and compared between subjects on current combination therapy, prior combination therapy, and IFX monotherapy., Results: Eighty-four (37.7%) patients were on combination therapy and 139 (62.3%) were on IFX monotherapy. Within the current monotherapy group, 112 (80.6%) had previously been on combination therapy, while 27 (19.4%) had never been on a concomitant immunomodulator. Patients currently on combination therapy had a higher IFXL (17.00 ± 1.33 μg/mL) than those currently on IFX monotherapy (13.18 ± 1.26 μg/mL), P < 0.01. IFXL was lowest in patients who had never been on combination therapy (11.53 ± 2.05 μg/mL) and highest in patients currently on combination therapy (17.00 ± 1.33 μg/mL). Patients currently on combination therapy had a lower rate of detectable ATI (9.5%) compared with those on monotherapy (20.0%) in multivariate analysis (odds ratio [OR]: 0.3; 95% confidence interval (CI), 0.1-0.7, P < 0.01)., Conclusions: Current or prior combination therapy is associated with higher IFXL and lower rates of ATI formation.

Background: Long noncoding RNAs (lncRNA) are key regulators of gene transcription and many show tissue-specific expression. We previously defined a novel inflammatory and metabolic ileal gene signature in treatment-naive pediatric Crohn disease (CD). We now extend our analyses to include potential regulatory lncRNA., Methods: Using RNAseq, we systematically profiled lncRNAs and protein-coding gene expression in 177 ileal biopsies. Co-expression analysis was used to identify functions and tissue-specific expression. RNA in situ hybridization was used to validate expression. Real-time polymerase chain reaction was used to test lncRNA regulation by IL-1β in Caco-2 enterocytes., Results: We characterize widespread dysregulation of 459 lncRNAs in the ileum of CD patients. Using only the lncRNA in discovery and independent validation cohorts showed patient classification as accurate as the protein-coding genes, linking lncRNA to CD pathogenesis. Co-expression and functional annotation enrichment analyses across several tissues and cell types 1showed that the upregulated LINC01272 is associated with a myeloid pro-inflammatory signature, whereas the downregulated HNF4A-AS1 exhibits association with an epithelial metabolic signature. We confirmed tissue-specific expression in biopsies using in situ hybridization, and validated regulation of prioritized lncRNA upon IL-1β exposure in differentiated Caco-2 cells. Finally, we identified significant correlations between LINC01272 and HNF4A-AS1 expression and more severe mucosal injury., Conclusions: We systematically define differentially expressed lncRNA in the ileum of newly diagnosed pediatric CD. We show lncRNA utility to correctly classify disease or healthy states and demonstrate their regulation in response to an inflammatory signal. These lncRNAs, after mechanistic exploration, may serve as potential new tissue-specific targets for RNA-based interventions., (© 2018 Crohn’s & Colitis Foundation of America. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Background and aims: Currently there is considerable interest in ketone metabolism owing to recently reported benefits of ketosis for human health. Traditionally, ketosis has been achieved by following a high-fat, low-carbohydrate "ketogenic" diet, but adherence to such diets can be difficult. An alternative way to increase blood D-β-hydroxybutyrate (D-βHB) concentrations is ketone drinks, but the metabolic effects of exogenous ketones are relatively unknown. Here, healthy human volunteers took part in three randomized metabolic studies of drinks containing a ketone ester (KE); (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, or ketone salts (KS); sodium plus potassium βHB. Methods and Results: In the first study, 15 participants consumed KE or KS drinks that delivered ~12 or ~24 g of βHB. Both drinks elevated blood D-βHB concentrations (D-βHB C max : KE 2.8 mM, KS 1.0 mM, P < 0.001), which returned to baseline within 3-4 h. KS drinks were found to contain 50% of the L-βHB isoform, which remained elevated in blood for over 8 h, but was not detectable after 24 h. Urinary excretion of both D-βHB and L-βHB was <1.5% of the total βHB ingested and was in proportion to the blood AUC. D-βHB, but not L-βHB, was slowly converted to breath acetone. The KE drink decreased blood pH by 0.10 and the KS drink increased urinary pH from 5.7 to 8.5. In the second study, the effect of a meal before a KE drink on blood D-βHB concentrations was determined in 16 participants. Food lowered blood D-βHB C max by 33% (Fed 2.2 mM, Fasted 3.3 mM, P < 0.001), but did not alter acetoacetate or breath acetone concentrations. All ketone drinks lowered blood glucose, free fatty acid and triglyceride concentrations, and had similar effects on blood electrolytes, which remained normal. In the final study, participants were given KE over 9 h as three drinks ( n = 12) or a continuous nasogastric infusion ( n = 4) to maintain blood D-βHB concentrations greater than 1 mM. Both drinks and infusions gave identical D-βHB AUC of 1.3-1.4 moles.min. Conclusion: We conclude that exogenous ketone drinks are a practical, efficacious way to achieve ketosis.

Background: Inflammatory bowel disease (IBD) affects 1.6 million people in the United States. IBD is associated with an increased risk of thrombosis, which rises with disease activity. The pathogenesis of IBD and its increased thrombotic risk is not completely understood. Ultra large von Willebrand factor (ULVWF) multimers are secreted from activated endothelium, leading to recruitment of platelets and leukocytes. A disintegrin and metalloproteinase with thrombospondin type I repeats motif 13 (ADAMTS13) cleaves highly adhesive ULVWF into smaller, less bioactive, multimers, releasing them into circulation. Mice deficient in ADAMTS13 (ADAMTS13 -/- ) have heightened inflammatory and thrombotic responses., Objectives: We hypothesized that upon colitis induction, ADAMTS13 -/- mice would have more severe symptoms compared with wild-type (WT) mice, and rhADAMTS13 administration to mice with colitis would improve their condition., Results: Dextran sodium sulfate-induced colitis was worse in ADAMTS13 -/- mice than WT. ADAMTS13 -/- showed increased weight loss, worse anemia, and increased clinical and histologic colitis severity, compared with WT mice. ADAMTS13 -/- mice had increased VWF release, with accumulation at inflamed colonic sites. Also, the majority of mice showed one or more submucosal colonic thrombi. ADAMTS13 deficiency worsened colitis and propagated intestinal inflammation, most likely through increased platelet-leukocyte recruitment by VWF. Treatment of WT mice with rhA-DAMTS13 decreased colitis severity without worsening anemia. Additionally, several immune-mediated chronic murine colitis models, and inflamed colon tissue specimens from IBD patients, showed increased VWF release at inflamed sites, suggesting a generalizability of our findings., Conclusion: Measuring VWF/ADAMTS13 levels could have clinical utility. When applicable, the administration of ADAMTS13, in addition to primary treatment, may improve outcomes for IBD patients.

Interleukin 10 (IL-10) is an anti-inflammatory cytokine that plays a critical role in the control of immune responses. However, its mechanisms of action remain poorly understood. Here, we show that IL-10 opposes the switch to the metabolic program induced by inflammatory stimuli in macrophages. Specifically, we show that IL-10 inhibits lipopolysaccharide-induced glucose uptake and glycolysis and promotes oxidative phosphorylation. Furthermore, IL-10 suppresses mammalian target of rapamycin (mTOR) activity through the induction of an mTOR inhibitor, DDIT4. Consequently, IL-10 promotes mitophagy that eliminates dysfunctional mitochondria characterized by low membrane potential and a high level of reactive oxygen species. In the absence of IL-10 signaling, macrophages accumulate damaged mitochondria in a mouse model of colitis and inflammatory bowel disease patients, and this results in dysregulated activation of the NLRP3 inflammasome and production of IL-1β., (Copyright © 2017, American Association for the Advancement of Science.)

Background: Stricturing and penetrating complications account for substantial morbidity and health-care costs in paediatric and adult onset Crohn's disease. Validated models to predict risk for complications are not available, and the effect of treatment on risk is unknown., Methods: We did a prospective inception cohort study of paediatric patients with newly diagnosed Crohn's disease at 28 sites in the USA and Canada. Genotypes, antimicrobial serologies, ileal gene expression, and ileal, rectal, and faecal microbiota were assessed. A competing-risk model for disease complications was derived and validated in independent groups. Propensity-score matching tested the effect of anti-tumour necrosis factor α (TNFα) therapy exposure within 90 days of diagnosis on complication risk., Findings: Between Nov 1, 2008, and June 30, 2012, we enrolled 913 patients, 78 (9%) of whom experienced Crohn's disease complications. The validated competing-risk model included age, race, disease location, and antimicrobial serologies and provided a sensitivity of 66% (95% CI 51-82) and specificity of 63% (55-71), with a negative predictive value of 95% (94-97). Patients who received early anti-TNFα therapy were less likely to have penetrating complications (hazard ratio [HR] 0·30, 95% CI 0·10-0·89; p=0·0296) but not stricturing complication (1·13, 0·51-2·51; 0·76) than were those who did not receive early anti-TNFα therapy. Ruminococcus was implicated in stricturing complications and Veillonella in penetrating complications. Ileal genes controlling extracellular matrix production were upregulated at diagnosis, and this gene signature was associated with stricturing in the risk model (HR 1·70, 95% CI 1·12-2·57; p=0·0120). When this gene signature was included, the model's specificity improved to 71%., Interpretation: Our findings support the usefulness of risk stratification of paediatric patients with Crohn's disease at diagnosis, and selection of anti-TNFα therapy., Funding: Crohn's and Colitis Foundation of America, Cincinnati Children's Hospital Research Foundation Digestive Health Center., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

TGF-β1 induces an increase in paracellular permeability and actin stress fiber formation in lung microvascular endothelial and alveolar epithelial cells via small Rho GTPase. The molecular mechanism involved is not fully understood. Neuronal Wiskott-Aldrich syndrome protein (N-WASP) has an essential role in actin structure dynamics. We hypothesized that N-WASP plays a critical role in these TGF-β1-induced responses. In these cell monolayers, we demonstrated that N-WASP down-regulation by short hairpin RNA prevented TGF-β1-mediated disruption of the cortical actin structure, actin stress filament formation, and increased permeability. Furthermore, N-WASP down-regulation blocked TGF-β1 activation mediated by IL-1β in alveolar epithelial cells, which requires actin stress fiber formation. Control short hairpin RNA had no effect on these TGF-β1-induced responses. TGF-β1-induced phosphorylation of Y256 of N-WASP via activation of small Rho GTPase and focal adhesion kinase mediates TGF-β1-induced paracellular permeability and actin cytoskeleton dynamics. In vivo, compared with controls, N-WASP down-regulation increases survival and prevents lung edema in mice induced by bleomycin exposure-a lung injury model in which TGF-β1 plays a critical role. Our data indicate that N-WASP plays a crucial role in the development of TGF-β1-mediated acute lung injury by promoting pulmonary edema via regulation of actin cytoskeleton dynamics.-Wagener, B. M., Hu, M., Zheng, A., Zhao, X., Che, P., Brandon, A., Anjum, N., Snapper, S., Creighton, J., Guan, J.-L., Han, Q., Cai, G.-Q., Han, X., Pittet, J.-F., Ding, Q. Neuronal Wiskott-Aldrich syndrome protein regulates TGF-β1-mediated lung vascular permeability., (© FASEB.)

Mutations of the Wiskott-Aldrich syndrome gene (WAS) are responsible for Wiskott-Aldrich syndrome (WAS), a disease characterized by thrombocytopenia, eczema, immunodeficiency, and autoimmunity. Mice with conditional deficiency of Was in B lymphocytes (B/WcKO) have revealed a critical role for WAS protein (WASP) expression in B lymphocytes in the maintenance of immune homeostasis. Neural WASP (N-WASP) is a broadly expressed homolog of WASP, and regulates B-cell signaling by modulating B-cell receptor (BCR) clustering and internalization. We have generated a double conditional mouse lacking both WASP and N-WASP selectively in B lymphocytes (B/DcKO). Compared with B/WcKO mice, B/DcKO mice showed defective B-lymphocyte proliferation and impaired antibody responses to T-cell-dependent antigens, associated with decreased autoantibody production and lack of autoimmune kidney disease. These results demonstrate that N-WASP expression in B lymphocytes is required for the development of autoimmunity of WAS and may represent a novel therapeutic target in WAS., (© 2016 by The American Society of Hematology.)

Alteration of cortical actin structures is the common final pathway leading to podocyte foot process effacement and proteinuria. The molecular mechanisms that safeguard podocyte foot process architecture and maintain the three-dimensional actin network remain elusive. Here, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which promotes actin nucleation, is required to stabilize podocyte foot processes. Mice lacking N-WASP specifically in podocytes were born with normal kidney function but developed significant proteinuria 3 weeks after birth, suggesting an important role for N-WASP in maintaining foot processes. In addition, inducing deletion of N-WASP in adult mice resulted in severe proteinuria and kidney failure. Electron microscopy showed an accumulation of electron-dense patches of actin and strikingly altered morphology of podocyte foot processes. Although basic actin-based processes such as cell migration were not affected, primary cultures of N-WASP-deficient podocytes revealed significant impairment of dynamic actin reorganization events, including the formation of circular dorsal ruffles. Taken together, our findings suggest that N-WASP-mediated actin nucleation of branched microfilament networks is specifically required for the maintenance of foot processes, presumably sustaining the mechanical resistance of the filtration barrier.

Chemotaxis promotes neutrophil participation in cellular defense by enabling neutrophil migration to infected tissue and is controlled by persistent cell polarization. One long-standing question of neutrophil polarity has been how the pseudopod and the uropod are coordinated. In our previous report, we suggested that Rho GTPase Cdc42 controls neutrophil polarity through CD11b signaling at the uropod, albeit through an unknown mechanism. Here, we show that Cdc42 controls polarity, unexpectedly, via its effector WASp. Cdc42 controls WASp activation and its distant localization to the uropod. At the uropod, WASp regulates the reorganization of CD11b integrin into detergent resistant membrane domains; in turn, CD11b recruits the microtubule end binding protein EB1 to capture and stabilize microtubules at the uropod. This organization is necessary to maintain neutrophil polarity during migration and is critical for neutrophil emigration into inflamed lungs. These results suggest unrecognized mechanism of neutrophil polarity in which WASp mediates long-distance control of the uropod by Cdc42 to maintain a proper balance between the pseudopod and the uropod. Our study reveals a new function for WASp in the control of neutrophil polarity via crosstalk between CD11b and microtubules.

Wiskott Aldrich syndrome (WAS) is caused by mutations in the WAS gene that encodes for a protein (WASp) involved in cytoskeleton organization in hematopoietic cells. Several distinctive abnormalities of T, B, and natural killer lymphocytes; dendritic cells; and phagocytes have been found in WASp-deficient patients and mice; however, the in vivo consequence of WASp deficiency within individual blood cell lineages has not been definitively evaluated. By conditional gene deletion we have generated mice with selective deficiency of WASp in the B-cell lineage (B/WcKO mice). We show that this is sufficient to cause a severe reduction of marginal zone B cells and inability to respond to type II T-independent Ags, thereby recapitulating phenotypic features of complete WASp deficiency. In addition, B/WcKO mice showed prominent signs of B-cell dysregulation, as indicated by an increase in serum IgM levels, expansion of germinal center B cells and plasma cells, and elevated autoantibody production. These findings are accompanied by hyperproliferation of WASp-deficient follicular and germinal center B cells in heterozygous B/WcKO mice in vivo and excessive differentiation of WASp-deficient B cells into class-switched plasmablasts in vitro, suggesting that WASp-dependent B cell-intrinsic mechanisms critically contribute to WAS-associated autoimmunity.