Calprotectin

Stomach tissue collection was performed as described previously (10 (link)). Briefly, the stomachs were opened along the greater curvature. For histology, gastric strips from both the lesser and greater curvatures were fixed in formalin for paraffin sections. For DNA, gastric tissue was snap frozen in liquid nitrogen and extracted using the DNEasy Blood and Tissue Kit (Qiagen, Valencia, CA). For flow cytometry, gastric cells were digested using a modified version of the protocol described by Geem et al. (11 (link)), which utilizes 17.9 μg/ml Liberase TM (Cat #05401119001, Roche Diagnostics Corporation, Indianapolis, IN) instead of Type VIII collagenase. Immunohistochemistry and immunofluorescence were performed as described previously (10 (link)), using antibodies against the following targets: calprotectin (#ab22506, Abcam); CD11b-AF594 (clone M1/70, #101254, BioLegend); Cxcr2-AF488 (#FAB2164G, R&D Systems); GSII-FITC lectin (#FL-1211, Vector Labs, Burlingame); H⁺/K⁺-ATPase-β (#D032-3, Medical and Biological Laboratories, Woburn, MA); intrinsic factor (gift from David Alpers, Washington University, St. Louis, MO); E-cadherin-FITC (#612130, BD Biosciences, San Jose, CA); E-cadherin-AF647 (#560062, BD Biosciences); and 4 Hydroxynonenal antibody (#ab46545, Abcam).

In the derivation cohort, six biomarkers were measured by ELISA: IL-1R2, CRP, PCT, sTREM-1, calprotectin (Abcam) as well as S100A9 (Biolegend). In the external validation cohort, plasma concentrations of IL-1R2 (Abcam) and sTREM-1 (R&D Systems) were measured by ELISA. Samples were tested and analyzed per the manufacturers’ instructions.

Venous blood samples were drawn from all patients immediately after patients' visiting before the initiation of any treatment. These blood samples were centrifuged at 1,200 × g for 10 min, 4°C immediately after collection, and stored at −80°C until use. Citrated plasma samples were thawed on ice and thoroughly vortexed before the test of calprotectin concentrations (Abcam, USA). All samples were tested in duplicates and the means of each measurement were used for analysis. For samples with extreme numbers which distribute beyond means ± 3 SD, the analysis would be repeated with three previously validated samples as references. These experiments were performed in a blinded manner following the manufacturer's instructions.