250 Materials and Methods Study population Patients aged 19-67 years with an established diagnosis of Crohn’s disease (CD) were included from March 2016 to April 2017 at the University Medical Center Groningen (UMCG), the Netherlands. In total, 39 CD patients were included and divided into two groups according to inflammatory disease activity, as determined by fecal calprotectin levels. Patients having a calprotectin level below 200 mg/kg were defined as the ‘normal’ calprotectin group (indicative of remissive disease) and patients with calprotectin levels above 200 mg/kg were defined as the‘increased’calprotectin group (indicative of inflammatory active CD). Clinically relevant data were obtained frommedical records: age, gender, BMI, smoking history, maintenance medication, Montreal score, ileocecal resection and laboratory parameters. Fecal calprotectin levels were determined in the laboratory of the UMCG as a routine measurement. Serum samples were obtained after patients gave written informed consent (study approved by the Medical Ethical Committee of the UMCG). Measurement of cytokines, chemokines and markers for angiogenesis and vascular injury Serum samples from all patients were collected and stored in 1 mL aliquots in the freezer (-20°C). After thawing, serum samples were centrifuged for 3 minutes at 2000 xg to remove particulates prior to sample preparation and analysis. Measurement of serum levels of cytokines, chemokines andmarkers for angiogenesis andvascular injurywasperformedby theelectrochemiluminescence (ECL) multiplex assay (Meso Scale Discovery (MSD ®), Meso Scale Diagnostics, Rockville, MD). The MSDV-plex Pro-inflammatory panel 1 (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and TNF- α), Cytokine panel 1 (GM-CSF, IL-5, IL-7, IL-12/23p40, IL-15, IL-16, IL-17A and TNF-β), Chemokine panel 1 (Eotaxin, MIP-1β, Eotaxin-3, TARC, IP-10, MIP-1α, MCP-1 and MDC), Angiogenesis panel 1 (VEGF, VEGF-C, VEGF-D, Tie-2, Flt-1, PIGF and bFGF) and Vascular injury panel 1 (SAA, CRP, VCAM1 and ICAM-1) were used to detect a total of 37 molecules. Calibration curves were created in order to calculate serum biomarker concentrations. Calibrator signals were fitted to a 4-parameter logistic model with 1⁄y2 weighting, providing the assay with a wide dynamic range of detection. Biomarker concentrations were calculated by back-fitting ECL signals to the calibration curves. Determination of final concentrations was performed using the MSD Discovery Workbench® analysis software. Concentrations of all molecules were above the lower limit of detection (LLOD). For the V-plex Pro-inflammatory panel 1, Cytokine panel 1 and Angiogenesis panel 1 assays, standard volumes of 50 μL of each serum sample were added and diluted 2-fold. For the Chemokine panel 1, samples were diluted 4-fold. For the Vascular injury panel 1, a standard volume of 25 μL of each serum sample was added and 1000-fold diluted. Statistics Study population characteristics were presented as means ± standard errors (SEM) or proportions (n, %). Serum biomarker distributions were presented as median ± interquartile ranges (IQR) and shown in boxplots (10th-90th percentiles) grouped by inflammatory disease activity, as determined by the fecal calprotectin level. Correlations between fecal calprotectin levels and serumbiomarker levels were established using the nonparametric Spearman’s correlation coefficient (ρ). Data were analyzed using SPSS Statistics 23.0 software package for Windows. P-values ≤ 0.05 were considered as statistically significant. Chapter 7
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