Specific enzyme immunoassays

Concentrations of VEGF in cultured HCSMCs that had been treated with different kinds of reagents in medium supplemented with 0.5% FBS and without growth supplement for 18 h in a humidified at 37 °C were determined as described previously [11 (link)] in duplicate by specific enzyme immunoassays (R&D Systems) according to the manufacturer’s instructions.

Native BAL fluids were collected on ice and BAL leukocyte counts were immediately determined on an automated cell counter (XE‐5000, Sysmex Corporation, Kobe, Japan). BAL differential cell counts were done by morphological examination of the cytospin preparation (Shandon cytospin 3 centrifuge, Cheshire, UK) stained with modified Wright's stain. Lavage samples were than centrifuged and the supernatant was collected and stored at −80 °C until assays were performed. Blood and BAL concentrations of IL‐6, IL‐8, TNF‐α and surfactant protein D (SP‐D) were measured using specific enzyme immunoassays (R&D Systems, Minneapolis, MN, USA). The lower limits of quantification are 0.156 pg·ml⁻¹ for IL‐6, 1.6 pg·ml⁻¹ for IL‐8, 0.5 pg·ml⁻¹ for TNF‐α and 0.63 ng·ml⁻¹ for SP‐D, and allow the detection of even very low normal values in healthy volunteers 14, 15. C‐reactive protein (CRP), blood differential, BAL total protein and immunoglobulin (Ig) G were determined in an accredited routine laboratory.