Fig. (2) IL-8 secretion in endothelial cells treated with APS IgG and vitamin D Overnight pretreatment with vitamin D (1α,25-dihydroxy-vitamin D3, 1,25-OH-D3; SIGMA-Aldrich - Saint Louis, MI, USA) 80 nM resulted in a significant reduction of upregulated levels of interleukin (IL)-8 (R&D Systems – Minneapolis, MN, USA). No significant difference was observed after treatment with Healthy Donor (HD) IgG and culture medium alone. Human Umbilical Vein Endothelial Cells (HUVECs) cultured in E199 (Gibco-Life Technologies - Groningen, The Netherlands), supplemented with 20% heat-injected Fetal Bovine Serum (FBS, PAA-GE Healthcare - Buckinghamshire, United Kingdom) in the presence of LPS (100 ng/mL, SIGMA-Aldrich - Saint Louis, MI, USA); Poly (I:C) (20 μg/mL, InvivoGen - San Diego, CA, USA); polyclonal IgG directed against β2GPI isolated from APS patient sera and HD IgG (200 μg/mL) from a pool of HD sera respectively. IgG were isolated on a protein-G-Sefarose column (HiTrap Protein G, GE Healthcare Bio-Science AB, Uppsala, Sweden) as previously described by Tincani et al., (97). The choice of the doses and regimens was based on previous unpublished experimental data from our laboratoryanti-b2GPI: anti-b2glycoprotein I, HD: Healthy Donor, IL-8: Interleukin-8, LPS: Lipopolysaccharide, NT: No Treatment, Poly (I:C): Polyinosinic:polycytidylic acid. (*p <0.05).

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