267 colon (from splenic flexure to rectosigmoid junction) and rectum. All 5 segments were evaluated for 4 different endoscopic variables scored from 0 to 3: size of ulcers, ulcerated surface, affected surface and the presence of narrowings. Ultimately, SES-CD scores were defined as previously described: endoscopic remission 0 – 3 points (category 0), mild disease activity 4 – 10 points (category 1), moderate disease activity 11 – 19 points (category 2) and severe disease activity ≥ 20 points (category 3).26 For UC, the Mayo endoscopic subscore for endoscopic disease activity was obtained from endoscopy reports written by certified gastroenterologists. Here, Mayo 0 was defined as endoscopic remission (normal mucosa), Mayo 1 as mild disease activity (erythema, decreased vascular pattern, mild friability), Mayo 2 as moderate disease activity (marked erythema, lack of vascular pattern, friability, erosions) and Mayo 3 as severe disease activity (spontaneous bleeding and ulceration). For the purpose of analysis, categories from both endoscopy scores of CD (SES-CD) and UC (Mayo endoscopic subscore) were merged on categorical level of mucosal damage (0 – 3) to finally create an IBD composite endoscopy score.27 Measurement of inflammatory biomarkers A selection of 10 inflammatory biomarkers were measured based on a previously performed study and available literature.22 In short, serum samples from all subjects were collected and stored in 1 mL aliquots at -80°C. After thawing and prior to analysis, samples were centrifuged for 3 minutes at 2,000 xg to remove remaining debris. Measurement of serum levels of C-reactive protein (CRP), serum amyloid A (SAA), IFN-γ, TNF-α, IL-6, IL-8, IL-10, IL-17A, Eotaxin-1 and Eotaxin-3 was implemented using a customized electrochemiluminescence (ECL) multiplex assay (Meso Scale Discovery (MSD®), Meso Scale Diagnostics, Rockville, MD). ECL signals were fitted to a 4- parameter logistic model with 1/y2 weighting, ensuring a broad and dynamic range of molecule detection. Serum concentrations of all detected molecules were determined using calibration curves to which the ECL signals were back-fitted. Final concentrations were calculated using the MSD DiscoveryWorkbench analysis software®. Of all detected biomarker concentrations, 94.0% of values were within the detection range and remaining values (6.0%) were excluded from further analysis. Statistical analysis Baseline demographic and clinical characteristics were presented as means ± standards errors (SEM) or proportions with corresponding percentages (n, %). Serum concentrations of inflammatory biomarkers were presented as median ± interquartile ranges (IQR). Assessment of normality of continuous variables was performed using normal Q-Q plots. Continuous variables were compared using Student’s t-tests or Mann-Whitney U-tests according to normality. Categorical variables were compared using chi-square tests or Fisher’s exact test, as appropriate. All consecutive analyses were performed in the subset of 71 IBD patients with available endoscopic results within 3 months prior to serum analysis. Simple correlations between inflammatory biomarkers and measures of disease activity were established using the nonparametric Spearman’s correlation coefficient (ρ). To evaluate predictive performance of all detected inflammatory biomarkers regarding composite IBD endoscopic disease activity, receiver operating characteristics (ROC) curves were established with associated areas under the ROC Predicting endoscopic disease activity in IBD
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