Soluble cd163

Fasting blood samples were collected and separated into RBCs, serum, and plasma. Samples were stored at −80 °C until being analyzed. RBC deformability and aggregation were measured by a microfluidic ektacytometer (RheoScan-AnD 300, RheoMeditech, Seoul, Korea). RBC rheology was defined as either the critical shear stress (CSS) or shear stress required for one-half maximal elongation (SS^1/2). Greater CSS values reflect higher RBC aggregability [38 (link)], and higher SS^1/2 values indicate a decrease in RBC deformability [39 (link)]. Free Hb (ICL; Mountainside, NJ, USA) and soluble CD163 (R&D Systems, Shanghai, China) levels in serum were determined by enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer’s instructions. Hb levels were assessed by a colorimetric method (Fortress Diagnostics, Northern Ireland, UK). Serum total bilirubin was quantitated with a Bilirubin Assay Kit (Cell Biolabs, San Diego, CA, USA) based on the Jendrassik-Frof method. Serum and liver total iron (40 mg liver sample/test) were quantitated by an iron assay kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferease (ALT) were measured using a colorimetric method by the Beckman DxC 800 (Beckman Coulter, Brea, CA, USA).

Soluble-CRs were quantified by Luminex following manufacturer’s instructions, using a MAGPix instrument with xPonent v4.2 software (LuminexCorp, ’s-Hertogenbosch, Netherlands). Soluble-CRs included TIM3, CD80, LAG3, HVEM, BTLA, CD27, CD28, CD137, CD152, GITR, IDO, PDL1, PDL2, and PD1) (Thermo Fisher Scientific, United Kingdom). Using the same platform, we also measured TIM3 soluble ligands Galectin-9 and soluble-CEACAM1, 10 pro-/anti-inflammatory cytokines (IL-1a, IL-1b, IL-1Ra, IL-6, IL-8, IL-10, IL-18, IL-33, IFNg, and TNFa), and the gut bacterial translocation marker soluble-CD163 (R&D-Systems/Bio-Techne, Abingdon-Oxford, United Kingdom). Another gut bacterial translocation marker, D-lactate, was measured by colorimetric assay (AbCam, Cambridge, United Kingdom).

Sequential blood samples from a total of 120 participants were analyzed. Plasma βDG was measured by the Fungitell Limulus Amebocyte Lysate (LAL) assay (Associates of Cape Cod, Inc, East Falmouth, MA, USA) according to the manufacturer's instruction. Enzyme-linked immunosorbent assays (ELISAs) were performed to quantify plasma LPS (CUSABIO, Wuhan, Hubei, China), LPS binding protein (LBP, Hycultbiotech, Uden, Netherlands), soluble LPS receptor CD14 (sCD14), intestinal fatty acid binding protein (IFABP), regenerating islet-derived protein 3α (REG3α), and soluble CD163 (R&D Systems, Minneapolis, MN, USA).