Associations between Cellular Energy and Pediatric Inflammatory Bowel Disease Patient Response to Treatment.

Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis, are chronic diseases of the gastrointestinal tract, with an unknown etiology, that affect over 6.8 million people worldwide. To characterize disease pathogenesis, proteomic and bioinformatic analyses were performed on colon biopsies collected during diagnostic endoscopy from 119 treatment-naïve pediatric patients, including from 78 IBD patients and 41 non-IBD patients who served as controls. Due to the presence of noninflamed and/or inflamed regions in IBD patients, up to two biopsies were obtained from IBD patients as compared to a single noninflamed biopsy from non-IBD pediatric control patients. Additional biopsies were obtained and analyzed from 33 of the IBD patients after IBD-directed therapeutic intervention for comparison of pre- and post-treatment proteomes. SuperSILAC was utilized to perform quantitative analysis of homogenized tissues, which were processed by filter-aided sample preparation. Hierarchical clustering and principal component analyses revealed proteomic patterns that distinguished inflamed from noninflamed tissues independent of therapy. Gene ontology revealed that proteins downregulated in inflammation are associated with metabolism, whereas upregulated proteins contribute to protein processing. A comparison of pre- and post-treatment proteomes from CD patients identified over 100 proteins that are significantly different between patients who responded and those who did not respond to therapy, including creatine kinase B and basigin.

Factors contributing to fidelity in a pilot trial of individualized resistant starches for pediatric inflammatory bowel disease: a fidelity study protocol.

BACKGROUND: The consumption of resistant starches is a promising adjuvant therapy for patients with inflammatory bowel disease. Rigorous evaluation of resistant starches in this setting depends on the intervention being delivered, received, and enacted as intended, that is, with fidelity. As part of a planned pilot trial, participants will be randomized to ingest resistant starches or a placebo. They will also be asked to collect stool samples and keep symptom and dose diaries to inform trial outcomes. We aim to identify potential factors impacting fidelity to the receipt and enactment of trial intervention and data collection activities from the perspective of patients and caregivers in the trial. Identifying fidelity barriers and enablers at the pilot trial phase of a clinical intervention may help to determine optimization processes when expanding to multiple sites in future trials. METHODS: We will conduct 15-30 semi-structured interviews with pilot trial participants (aged 8-17) and their caregivers. Trial participants will be approached for interviews approximately 6 months after the start of their trial participation. Personal projects analysis, a tool for understanding how individuals manage competing demands in their daily lives, will guide an in-depth exploration of how trial participants engage in activities related to intervention and data collection fidelity (ingesting resistant starches or placebo, collecting stool samples, keeping a symptom and dose diary) amidst the complexities of daily living. DISCUSSION: The present study will seek to explore and demonstrate how theory-informed fidelity assessments can be conducted alongside pilot trials to inform future multisite trials. Study results will clarify what factors may affect fidelity to trial intervention and data collection activities. Results may suggest what to modify to optimize the design and conduct, and ensure the integrity, of future multisite trials. Conducting process evaluations alongside clinical trials has the potential to improve our understanding of trial participant experiences. Results will provide a better understanding of how trial participants manage to engage in necessary trial activities along with other priorities.

Metaproteomics reveals associations between microbiome and intestinal extracellular vesicle proteins in pediatric inflammatory bowel disease.

Alterations in gut microbiota have been implicated in the pathogenesis of inflammatory bowel disease (IBD), however factors that mediate the host-microbiota interactions remain largely unknown. Here we collected mucosal-luminal interface samples from a pediatric IBD inception cohort and characterized both the human and microbiota proteins using metaproteomics. We show that microbial proteins related to oxidative stress responses are upregulated in IBD cases compared to controls. In particular, we demonstrate that the expression of human proteins related to oxidative antimicrobial activities is increased in IBD cases and correlates with the alteration of microbial functions. Additionally, we reveal that many of these human proteins are present and show altered abundance in isolated free extracellular vesicles (EVs). Therefore, our study suggests that the alteration of intestinal EV proteomes is associated with the aberrant host-microbiota interactions in IBD.

Mucosal-luminal interface proteomics reveals biomarkers of pediatric inflammatory bowel disease-associated colitis.

OBJECTIVE: Improved biomarkers are an unmet clinical need for suspected inflammatory bowel disease (IBD). Need is greatest for children, since current biomarkers suffers from low specificity, particularly in this population; thus, invasive testing methods, with the accompanying risk of complications, are necessary. Additionally, current biomarkers do not delineate disease extent assessment for ulcerative colitis (UC), a factor involved in therapeutic decisions. METHODS: Intestinal mucosal-luminal interface (MLI) aspirates from the ascending colon (AC) and descending colon (DC) were collected during diagnostic colonoscopy from treatment-naïve children. The MLI proteomes of 18 non-IBD and 42 IBD patients were analyzed by liquid chromatography mass spectrometry. Analyses of proteomic data generated protein panels distinguishing IBD from non-IBD and pancolitis from non-pancolitis (UC disease extent). Select protein biomarkers were evaluated in stool samples by enzyme-linked immunosorbent assay (n = 24). RESULTS: A panel of four proteins discriminated active IBD from non-IBD (discovery cohort) with a sensitivity of 0.954 (95% confidence interval (CI): 0.772-0.999) and >0.999 (95% CI: 0.824-1.00) for the AC and DC, respectively, and a specificity of >0.999 (AC, 95% CI: 0.815-1.00; DC, 95% CI:0.692-1.00) for both the AC and DC. A separate panel of four proteins distinguished pancolitis from non-pancolitis in UC patients with sensitivity >0.999 (95% CI: 0.590-1.00) and specificity >0.999 (95% CI: 0.715-1.00). Catalase (p < 0.0001) and LTA4H (p = 0.0002) were elevated in IBD stool samples compared to non-IBD stool samples. CONCLUSION: This study identified panels of proteins that have significantly different expression levels and contribute to accurate IBD diagnosis and disease extent characterization in children with UC. Biomarkers identified from the MLI demonstrate transferable results in stool samples.

Proteomic analysis of ascending colon biopsies from a paediatric inflammatory bowel disease inception cohort identifies protein biomarkers that differentiate Crohn's disease from UC.

OBJECTIVE: Accurate differentiation between Crohn's disease (CD) and UC is important to ensure early and appropriate therapeutic intervention. We sought to identify proteins that enable differentiation between CD and UC in children with new onset IBD. DESIGN: Mucosal biopsies were obtained from children undergoing baseline diagnostic endoscopy prior to therapeutic interventions. Using a super-stable isotope labeling with amino acids in cell culture (SILAC)-based approach, the proteomes of 99 paediatric control and biopsies of patients with CD and UC were compared. Multivariate analysis of a subset of these (n=50) was applied to identify novel biomarkers, which were validated in a second subset (n=49). RESULTS: In the discovery cohort, a panel of five proteins was sufficient to distinguish control from IBD-affected tissue biopsies with an AUC of 1.0 (95% CI 0.99 to 1.0); a second panel of 12 proteins segregated inflamed CD from UC within an AUC of 0.95 (95% CI 0.86 to 1.0). Application of the two panels to the validation cohort resulted in accurate classification of 95.9% (IBD from control) and 80% (CD from UC) of patients. 116 proteins were identified to have correlation with the severity of disease, four of which were components of the two panels, including visfatin and metallothionein-2. CONCLUSIONS: This study has identified two panels of candidate biomarkers for the diagnosis of IBD and the differentiation of IBD subtypes to guide appropriate therapeutic interventions in paediatric patients.

Altered intestinal microbiota-host mitochondria crosstalk in new onset Crohn's disease.

Intestinal microbial dysbiosis is associated with Crohn's disease (CD). However, the mechanisms leading to the chronic mucosal inflammation that characterizes this disease remain unclear. In this report, we use systems-level approaches to study the interactions between the gut microbiota and host in new-onset paediatric patients to evaluate causality and mechanisms of disease. We report an altered host proteome in CD patients indicative of impaired mitochondrial functions. In particular, mitochondrial proteins implicated in H2S detoxification are downregulated, while the relative abundance of H2S microbial producers is increased. Network correlation analysis reveals that Atopobium parvulum controls the central hub of H2S producers. A. parvulum induces pancolitis in colitis-susceptible interleukin-10-deficient mice and this phenotype requires the presence of the intestinal microbiota. Administrating the H2S scavenger bismuth mitigates A. parvulum-induced colitis in vivo. This study reveals that host-microbiota interactions are disturbed in CD and thus provides mechanistic insights into CD pathogenesis.

In Vitro Metabolic Labeling of Intestinal Microbiota for Quantitative Metaproteomics.

Intestinal microbiota is emerging as one of the key environmental factors influencing or causing the development of numerous human diseases. Metaproteomics can provide invaluable information on the functional activities of intestinal microbiota and on host-microbe interactions as well. However, the application of metaproteomics in human microbiota studies is still largely limited, in part due to the lack of accurate quantitative intestinal metaproteomic methods. Most current metaproteomic microbiota studies are based on label-free quantification, which may suffer from variability during the separate sample processing and mass spectrometry runs. In this study, we describe a quantitative metaproteomic strategy, using in vitro stable isotopically ((15)N) labeled microbiota as a spike-in reference, to study the intestinal metaproteomes. We showed that the human microbiota were efficiently labeled (>95% (15)N enrichment) within 3 days under in vitro conditions, and accurate light-to-heavy protein/peptide ratio measurements were obtained using a high-resolution mass spectrometer and the quantitative proteomic software tool Census. We subsequently employed our approach to study the in vitro modulating effects of fructo-oligosaccharide and five different monosaccharides on the microbiota. Our methodology improves the accuracy of quantitative intestinal metaproteomics, which would promote the application of proteomics for functional studies of intestinal microbiota.