Sicam 1

Dulbecco’s modified Eagle’s medium (DMEM), Calcein-AM (Calcein O, O’-diacetate tetrakis (acetoxymethyl) ester, and RPMI-1640 were purchased from Life Technologies (Grand Island, NY, USA). Enzyme-linked immunosorbent assay (ELISA) kits for human and mouse adhesion molecules ICAM-1 (sICAM-1) and VCAM-1 (sVCAM-1) and mouse chemokines MCP-1/JE and CXCL1/KC were obtained from R&D Systems (Minneapolis, MN, USA). Goat antirabbit IgG, DyLight™ 488 conjugated secondary antibody and goat antirabbit horseradish peroxidase (HRP) IgG secondary antibody were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Primary antibodies for immunohistochemistry were acquired from Cell Signaling Technology, Inc. for NF-κB p65 and IκB-α (Danvers, MA, USA), from Santa Cruz Biotechnology for VCAM-1 (Santa Cruz, CA, USA), and from BMA Biomedicals for F4/80 (Augst, Switzerland). Resveratrol (≥98%, HPLC) was from the Stanford Chemicals Company (Irvine, CA, USA), and other general chemicals, including DAPI, were procured from Sigma-Aldrich (St. Louis, MO, USA)

Serum or plasma samples were analyzed for biomarkers of intestinal injury in Uganda on all cryopreserved samples. Children were considered to have intestinal injury if they had either elevated TFF3 or I-FABP compared to the population reference levels (>99th percentile for the population was used as the upper limit of normal). Markers were assessed using a custom Luminex MagPix panel including plasma trefoil factor 3 (TFF3), angiopoietin 2 (Angpt-2), soluble Fms-related receptor tyrosine kinase 1 (sFlt-1), P-selectin, sE-selectin, soluble vascular cell adhesion molecule 1 (sVCAM-1), chitinase-3-like protein 1 (CHI3L1), and procalcitonin (R&D Systems, Minneapolis, MN). A modified cytokine Luminex panel was used to measure serum levels of selected cytokines, including tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-8 (IL-8), interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-4 (IL-4), vascular endothelial growth factor (VEGF), granzyme B, Fractalkine, and sFlt-3 (R&D Systems, Minneapolis, MN). Intestinal fatty acid binding protein (I-FABP), soluble CD14 (sCD14), LPS binding protein (LBP), C-reactive protein (CRP), and soluble intercellular adhesion molecule 1 (sICAM-1) were measured by enzyme-linked immunosorbent assay (ELISA) using DuoSets by R&D Systems.

ELISA kits for IFN-γ (cat #DIF50) and sICAM-1 (cat #P161289) were purchased from R&D (Minneapolis, MN, USA). Cells were placed in 0% FBS containing media, treated as described in individual experiments, and the resulting supernatant was collected. Supernatants were spun at 1000 rpm at 4 degrees C for 5 minutes in order to eliminate any cell debris from the samples.