Position paper: The potential role of optical biopsy in the study and diagnosis of environmental enteric dysfunction

Environmental enteric dysfunction (EED) is a disease of the small intestine affecting children and adults in low and middle income countries. Arising as a consequence of repeated infections, gut inflammation results in impaired intestinal absorptive and barrier function, leading to poor nutrient uptake and ultimately to stunting and other developmental limitations. Progress towards new biomarkers and interventions for EED is hampered by the practical and ethical difficulties of cross-validation with the gold standard of biopsy and histology. Optical biopsy techniques — which can provide minimally invasive or noninvasive alternatives to biopsy — could offer other routes to validation and could potentially be used as point-of-care tests among the general population. This Consensus Statement identifies and reviews the most promising candidate optical biopsy technologies for applications in EED, critically assesses them against criteria identified for successful deployment in developing world settings, and proposes further lines of enquiry. Importantly, many of the techniques discussed could also be adapted to monitor the impaired intestinal barrier in other settings such as IBD, autoimmune enteropathies, coeliac disease, graft-versus-host disease, small intestinal transplantation or critical care

Perianal magnetic resonance imaging findings and their potential impact on outcome in children with perianal fistulizing Crohn disease

Background: Children with perianal fistulizing Crohn disease require intensive medical management but also have a higher risk for subsequent surgical interventions.Objective: We performed a retrospective study to identify patient factors and perianal anatomical features by pelvic MR that are associated with surgical interventions in these children.Materials and methods: We included children with Crohn disease and perianal fistula who underwent pelvic MR with available, archived images and collected demographic, clinical and laboratory data. Radiologists reviewed pelvic MR exams and identified Park classification and additional anatomical features of perianal fistulas, including fistula branching, horseshoe ramifications, abscess, inflammatory mass, supralevator extension, anal sphincter damage, proctitis and posterior anal space involvement. We performed univariate and subsequent multivariate analysis to determine features associated with subsequent surgical intervention.Results: Ninety-nine children with Crohn disease underwent pelvic MR. In this cohort, 69 children had no surgical interventions prior to baseline MRI, with subsequent median clinical follow-up of 5.5 years. Univariate analysis demonstrated that branching (P=0.009), supralevator extension (P=0.015) and anal sphincter damage (P=0.031) were significantly associated with subsequent surgical intervention. Age at baseline MRI was also associated with intervention (hazard ratio [HR] every 5 years: 2.13; 95% confidence interval [CI]: 1.18-3.83; P=0.012). A multivariable model identified only fistula branching (HR: 2.31; 95% CI: 1.28-4.15; P=0.005) and age (HR: 5.18; CI: 1.57-17.14; P=0.007) as independent predictors of subsequent surgery. No demographic, clinical or laboratory parameter predicted subsequent surgical intervention.Conclusion: Age and anatomical MR features indicating fistula complexity (branching, supralevator extension) and sphincter damage confer a higher risk of subsequent surgical intervention in children with perianal Crohn disease

Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal InflammationSummary

Background & Aims: Forkhead box protein 3 (FOXP3)+ regulatory T cell (Treg) dysfunction is associated with autoimmune diseases; however, the mechanisms responsible for inflammatory bowel disease pathophysiology are poorly understood. Here, we tested the hypothesis that a physical interaction between transcription factor FOXP3 and the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is essential for gene co-repressive function. Methods: Human FOXP3 mutations clinically relevant to intestinal inflammation were generated by site-directed mutagenesis. T lymphocytes were isolated from mice, human blood, and lamina propria of Crohn’s disease (CD) patients and non-CD controls. We performed proximity ligation or a co-immunoprecipitation assay in FOXP3-mutant+, interleukin 6 (IL6)-treated or CD-CD4+ T cells to assess FOXP3–EZH2 protein interaction. We studied IL2 promoter activity and chromatin state of the interferon γ locus via luciferase reporter and chromatin-immunoprecipitation assays, respectively, in cells expressing FOXP3 mutants. Results: EZH2 binding was abrogated by inflammatory bowel disease–associated FOXP3 cysteine 232 (C232) mutation. The C232 mutant showed impaired repression of IL2 and diminished EZH2-mediated trimethylation of histone 3 at lysine 27 on interferon γ, indicative of compromised Treg physiologic function. Generalizing this mechanism, IL6 impaired FOXP3–EZH2 interaction. IL6-induced effects were reversed by Janus kinase 1/2 inhibition. In lamina propria–derived CD4+T cells from CD patients, we observed decreased FOXP3–EZH2 interaction. Conclusions: FOXP3–C232 mutation disrupts EZH2 recruitment and gene co-repressive function. The proinflammatory cytokine IL6 abrogates FOXP3–EZH2 interaction. Studies in lesion-derived CD4+ T cells have shown that reduced FOXP3–EZH2 interaction is a molecular feature of CD patients. Destabilized FOXP3–EZH2 protein interaction via diverse mechanisms and consequent Treg abnormality may drive gastrointestinal inflammation. Keywords: Proinflammatory Cytokine, Epigenetics, Regulatory T Cells, Crohn’s Diseas

Building a best-in-class automated de-identification tool for electronic health records through ensemble learning

Summary: The presence of personally identifiable information (PII) in natural language portions of electronic health records (EHRs) constrains their broad reuse. Despite continuous improvements in automated detection of PII, residual identifiers require manual validation and correction. Here, we describe an automated de-identification system that employs an ensemble architecture, incorporating attention-based deep-learning models and rule-based methods, supported by heuristics for detecting PII in EHR data. Detected identifiers are then transformed into plausible, though fictional, surrogates to further obfuscate any leaked identifier. Our approach outperforms existing tools, with a recall of 0.992 and precision of 0.979 on the i2b2 2014 dataset and a recall of 0.994 and precision of 0.967 on a dataset of 10,000 notes from the Mayo Clinic. The de-identification system presented here enables the generation of de-identified patient data at the scale required for modern machine-learning applications to help accelerate medical discoveries. The bigger picture: Clinical notes in electronic health records convey rich historical information regarding disease and treatment progression. However, this unstructured text often contains personally identifiable information such as names, phone numbers, or residential addresses of patients, thereby limiting its dissemination for research purposes. The removal of patient identifiers, through the process of de-identification, enables sharing of clinical data while preserving patient privacy. Here, we present a best-in-class approach to de-identification, which automatically detects identifiers and substitutes them with fabricated ones. Our approach enables de-identification of patient data at the scale required to harness the unstructured, context-rich information in electronic health records to aid in medical research and advancement

Functional Consequences of the Macrophage Stimulating Protein 689C Inflammatory Bowel Disease Risk Allele

<div><p>Background</p><p>Macrophage stimulating protein (MSP) is a serum growth factor that binds to and activates the receptor tyrosine kinase, Recepteur d'Origine Nantais (RON). A non-synonymous coding variant in MSP (689C) has been associated with genetic susceptibility to both Crohn's disease and ulcerative colitis, two major types of inflammatory bowel disease (IBD) characterized by chronic inflammation of the digestive tract. We investigated the consequences of this polymorphism for MSP-RON pathway activity and IBD pathogenesis.</p><p>Methods</p><p>RON expression patterns were examined on mouse and human cells and tissues under normal and disease conditions to identify cell types regulated by MSP-RON. Recombinant MSP variants were tested for their ability to bind and stimulate RON and undergo proteolytic activation. MSP concentrations were quantified in the serum of individuals carrying the MSP 689R and 689C alleles.</p><p>Results</p><p>In intestinal tissue, RON was primarily expressed by epithelial cells under normal and disease conditions. The 689C polymorphism had no impact on the ability of MSP to bind to or signal through RON. In a cohort of normal individuals and IBD patients, carriers of the 689C polymorphism had lower concentrations of MSP in their serum.</p><p>Conclusions</p><p>By reducing the quantities of circulating MSP, the 689C polymorphism, or a variant in linkage disequilibrium with this polymorphism, may impact RON ligand availability and thus receptor activity. Given the known functions of RON in regulating wound healing and our analysis of RON expression patterns in human intestinal tissue, these data suggest that decreased RON activity may impact the efficiency of epithelial repair and thus underlie the increased IBD susceptibility associated with the MSP 689C allele.</p></div

The 689C polymorphism does not affect MSP signaling through RON.

<p>(A) Western blot analysis of total Akt and pAkt in A2780-hRON and BxPC3 cells treated as indicated. Blot was performed in triplicate and mean of the pAkt/total ratios is shown. (B) Quantitation of pAkt by MSD analysis in 3T3-hRON cells treated with scMSP or MSP variants. Mean +/− SD of triplicate wells is shown. Data are representative of three independent experiments. (C) Quantification of pAkt by MSD analysis in HCT15 cells treated with MSP variants. Mean +/− SD of triplicate wells is shown. Data are representative of two independent experiments. (D) Quantitation of pAkt by MSD analysis in human primary colon cells treated with MSP variants. Mean +/− SD of triplicate wells is shown. Data are representative of two independent experiments. (E) Images from scratch wound assay of 3T3-hRON cells treated with scMSP or MSP variants. Images are from the same cell culture at the indicated times after scratch wounding. Dashed lines represent position of initial scratch. (F) Quantitation of scratch wound assay from 3T3-hRON cells treated with medium-alone, scMSP, or MSP variants. Mean +/− SD of three treatments is shown. Data are representative of three independent experiments. ^#not significant, **p≤0.0003 for comparisons between medium and MSP variants.</p

RON is expressed by myeloid and epithelial cell populations in mice.

<p>(A) Single cell suspensions of the indicated cell populations or tissues were analyzed by flow cytometry for RON expression using a monoclonal antibody specific for murine RON (blue histogram) or an isotype control antibody (shaded histogram). Macrophage populations were gated as CD45⁺F4/80⁺CD11b⁺MHC class II⁺ for <i>ex vivo</i>-derived cells and F4/80⁺CD11b⁺ for bone marrow cultured cells. Colonic epithelial cells were gated as CD45⁻ECadherin⁺. B) Representative image from IHC analysis of RON expression (brown) on tissue sections from normal mouse colon. Scale bars  =  100 µm. Data are representative of at least three similar experiments.</p

Binding kinetics and affinities of MSP 689R and 689C proteins to RON.

<p>^aData are the average ± standard deviations of three or more independent determinations in general.</p><p>SPR capturing RON-Fc at 25°C,</p><p>SPR using immobilized RON Sema/PSI at 25°C,</p><p>BLI using RON-Fc at 30°C,</p><p>Radioligand binding to 3T3-hRON cells for 2 h at room temperature,</p><p>Radioligand binding to 3T3-hRON cells for 4 h on ice.</p

RON is preferentially expressed by epithelial cells in human tissues under steady-state and disease conditions.

<p>(A) Representative images from IHC analysis of RON protein expression (brown) on tissue sections from normal, UC, and CD colons. Scale bar  =  100 µm. (B) Representative images from ISH analysis of RON transcript expression (red) on tissue sections from normal, UC, and CD colons. Scale bar  =  20 µm. (C) Quantitative RT-PCR analysis of RON expression in human intestinal biopsies samples taken from normal individuals and uninflamed and inflamed regions of UC and CD patients. (D) Analysis of RON expression in single cell suspensions of resected intestinal tissues from an ulcerative colitis patient by flow cytometry using a monoclonal antibody specific for human RON (blue histogram) or an isotype control antibody (shaded histogram). Macrophages were gated as CD45⁺CD14⁺HLADR⁺ and epithelial cells were gated as CD45⁻EpCAM⁺ cells. (E) Quantification of RON expression by flow cytometry on macrophages and epithelial cells from resected intestinal tissue of multiple donors (macrophages: 1 non-IBD (N), 3 UC, 2 CD; epithelial cells: 2 UC, 1 CD). Patient numbers are indicated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083958#pone.0083958.s007" target="_blank">Table S1</a>). Data represents the mean fluorescence intensity (MFI) ratio of RON staining to isotype control staining. Dashed line indicates a ratio of 1∶1, <i>i.e.</i> lack of RON expression. N, non-IBD.</p