025, 1.055, and 1.21g/mL with NaBr before each following run, and fractions corresponding

respectively to IDL, LDL, and HDL were collected. Each fraction was then dialyzed at 4°C against 150 mM NaCl-0.24 mM ethylene diamine tetra-acetic acid (pH 7.4) buffer and filtered through 0.22-μm pore size filters (Millipore, Saint Quentin, France) before lipid extraction as described below. Plasma was adjusted to 1.055 g/mL with NaBr and centrifuged as described above. After collection, the upper low-density fraction (LDF) was dialyzed as described for lipoprotein fractions and stored at 4°C in the dark, in the presence of 2% protease inhibitor cocktail (P8340; Sigma-Aldrich). LVP purification Protease Inhibitor Library from normal lipoproteins contained in LDF was performed via protein A immunoprecipitation of the immune complexes that are only found in the LDFs of infected patients as described.4 Briefly, protein A–coated magnetic beads (Miltenyi Biotec, Paris, France) were incubated with LDFs in phosphate-buffered saline (PBS) with gentle rocking for 30 minutes (20 μL of beads per 1

mL LDF). A total of 2 mL sample were then passed through one magnetic column (Miltenyi Biotec), washed with PBS, and collected in 500 μL PBS. For experiments with larger LDF volume, multiple columns were used. Immunocaptured particles (purified LVPs) were Selleck Pritelivir subjected to lipid extraction as described below or stored at 4°C in the dark in the presence of 2% protease inhibitor cocktail for biochemical characterization. Protein concentration was determined using Coomassie Plus (Bradford)

Protein assay (ThermoScientific, Brebières, France). ApoB concentration in low-density fractions and sera was determined by immunochemical assay (SFRI Diagnostics, Saint-Jean d’Illiac, France). Total cholesterol (TChol), phospholipid, and triacylglycerol concentrations in sera were calculated with Cholesterol RTU, Phospholipid Enzymatic PAP150, and Triacylglycerol Enzymatic PAP150 kits (BioMérieux, Marcy l’étoile, France). ApoB concentration in purified LVPs was determined MCE公司 via enzyme-linked immunosorbent assay (ELISA) as described.20 Mock-prepared LVPs from healthy donors displayed a maximal background of <1% of lipids or apoB detected in LVPs prepared from patients. Lipid extraction of mock LVPs, as described below, has not allowed the detection of any fatty acid by gas chromatography (GC) in the final lipid extract. RNA was extracted from 150 μL serum, 10 μL lipoproteins, LDFs, or purified LVPs with a NucleoSpin RNA virus kit (Macherey-Nagel, Hoerdt, France) and stored at −80°C. HCV RNA quantification was performed using quantitative real-time polymerase chain reaction of the 5′ HCV noncoding region as described.22 HCV genotyping was performed using the INNO LiPA HCV assay (Innogenetics, Zwijnaarde, Belgium).