2.7. Systemic and Intestinal Inflammation
INTESTINAL INFLAMMATION
Intestinal inflammation was assessed based on measures of fecal calprotectin (30-CALPHU-CH01) and myeloperoxidase (30-6630; Alpco). Assays were performed in accordance with the manufacturer’s instructions, and an easy stool extraction device (30-EZEX-100) was used to accurately measure stool mass prior to sample analysis. Intra- and inter-assay CVs of both proteins were <3.0% and <7.3%, respectively.Whole blood pro-inflammatory gene expression was measured by real-time quantitative polymerase chain reaction. Total RNA was extracted using a RiboPure-Blood Kit (ThermoFisher Scientific, AM 1928). RNA yield and purity were assessed using a BioSpec-nano spectrophotometer (Shimadzu, Tokyo, Japan), and integrity was determined following native agarose gel (2%) electrophoresis. cDNA was synthesized from high-quality RNA using an iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Primers (IL-8, MCP1, TNFα, IL6, TLR4, p65, β-actin) were purchased from Integrated DNA Technologies (