323 0.036% Bronidox L5, 0.1% Tween-20) using a standardized ELISA plate washing machine (BioTek Instruments, Microplate washer, ELx405 Select CW, Winooski, VT, USA). For colorimetric assays, tetramethylbenzidine (TMB) (Kem-En-Tec, CAT no. 438 OH, Taastrup, Denmark) was added as 100 μL per well; the plates were incubated for 15 minutes at room temperature and shaken again at 300 rpm. After this, 1% H2SO4 stopping buffer was added to stop the TMB reaction. An ELISA reader (VersaMAX; Molecular Devices, Wokingham Berkshire, UK) was applied to read optical densities at 450 nm and 650 nm. For chemiluminescence assays, BM Chemiluminescence ELISA Substrate (Merck, CAT no. 11 582 950 001, Germany) was added as 100 μl per well. The plates were then shaken at 300 rpm while incubating for 3 minutes at 20°C. A fluorescence plate reader (Fluoroskan FL, Thermo Fisher) was applied to read light emission at 1000 ms with no filter. Finally, standard curves were created using 4-parameter logistic models. Detection limits and detection rates of biomarkers can be found in Table S1. Table 1 | Serological biomarkers of extracellular matrix turnover and intestinal inflammation. Abbreviations: BM, basement membrane; IM, interstitial matrix; MMP, matrix metalloproteinase. Statistical analysis Descriptive and general inferential statistics Baseline characteristics of the study population were presented as means ± standard deviations (SD), medians with interquartile ranges (IQR) or as proportions n with corresponding percentages (%). Assessment of normality of continuous variables was performed by visual inspection of normal probability (Q-Q) plots and histograms. Differences in demographic and clinical data were compared using independent sample t-tests, Mann-Whitney U-tests or chi-squared tests, depending on normality and type of variable. Serum biomarker levels were presented as median [IQR], and differences between groups were tested non-parametrically using Kruskal-Wallis tests and Mann-Whitney U-tests with post-hoc Bonferroni correction for multiple comparisons, as appropriate. Correlations between different biomarkers were calculated using Spearman’s rank correlation coefficients. Statistical analyses were performed using the Python programming Collagen biomarkers and Crohn’s disease behavior Table 1. Serological biomarkers of extracellular matrix turnover and intestinal inflammation. Protein Biomarker of degradation Biomarker of formation BM/IM References Type I collagen C1M: Specific fragment of MMP-2, -9, -13mediated degradation of type I collagen IM 13 Type III collagen C3M: Specific fragment of MMP-9-mediated degradation of type III collagen PRO-C3: Released N-terminal pro-peptide of type III collagen IM 14,15 Type IV collagen C4M: Neo-epitope generated by MMP-2, -9, - 12-mediated degradation of type IV collagen C4G: Neo-epitope generated by T-cell granzyme-B-mediated degradation of type IV collagen PRO-C4: Internal epitope in 7s domain of type IV collagen BM 16-18 Type VI collagen C6Ma3: MMP-2 and -9-degraded type VI collagen (alpha chain) BM/IM 19 Abbreviations: BM, basement membrane; IM, interstitial matrix; MMP, matrix metalloproteinase.
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