Rantes

Extra blood left after the above tests was used to detect several inflammatory or anti-inflammatory cytokines before intervention, 6 months after intervention initiation, and 3 months after stopping the intervention. After centrifugation, the serum was refrigerated at −80°C until use for ELISA. The inflammatory cytokines used for ELISA test were as follows: TNF-α (Thermo Scientific, Carlsbad, CA, USA), IL-8 (Thermo Scientific, Carlsbad, CA, USA), IL-17 (R&D Systems, Minneapolis, Minnesota, USA), MIP-1β (PeproTech, Cranbury, NJ, USA), and RANTES (R&D Systems, Minneapolis, Minnesota, USA). The anti-inflammatory cytokine used was TGF-β1 (Thermo Scientific, Carlsbad, CA, USA). All samples were measured by the commercial protocol at least in triplicate.

In the Boyden chamber (BD Biosciences) migration assay, resting PBL or HUVECs (5 × 10⁴ or 2 × 10³, resp.) were plated onto the apical side of 50 μg/mL matrigel-coated filters (8.2 mm diameter and 0.3 μm or 0.5 μm pore size; Neuro Probe, Inc.; BIOMAP snc, Milan, Italy) in RPMI or M200 serum-free medium, with or without OPN-FL (10 μg/mL), OPN-N (5 μg/mL), or OPN-C (5 μg/mL). Mediums containing 1 ng/mL RANTES (R&D System) or 10 ng/mL vascular endothelial growth factor (VEGF-α, R&D System) were placed in the basolateral chamber as a positive chemoattractant stimuli for PBL and HUVECs, respectively. The chamber was incubated at 37°C under 5% CO₂. After 20 h, the cells on the apical side were wiped off with Q-tips. The cells on the bottom of the filter were stained with crystal violet, and all were counted (fourfold filter) with an inverted microscope (magnification 40x). Data are shown as percentages of the treated cells migration versus the control migration measured for untreated cells. Control migration is (mean ± SEM) 263 ± 45 cells for HUVECs (n = 5) and 155 ± 25 for lymphocytes (n = 5).

Brain tissue in the cerebral cortex around the injured area was collected and homogenized. The homogenates were centrifuged at 4°C at 12,000 × g for 15 minutes, and supernatants were collected carefully and evaluated in duplicate using IL-1β, IL-6, TNF-α, MCP (monocyte chemoattractant protein)-1 and RANTES (regulated upon activation, normal T cell expressed and secreted) assay kits (R&D Systems, Minneapolis, MN, US), in accordance with the manufacturer’s guidelines. Tissue cytokine concentrations are expressed as picograms per milligram of protein.
Cell culture supernatants were carefully collected at 24 hours after stimulation with LPS and centrifuged at 4°C at 12,000 × g for 15 minutes. Cytokine concentrations were evaluated using protein assay kits (R&D Systems, Minneapolis, MN, US), in accordance with the manufacturer’s guidelines. Cell cytokine concentrations are expressed as picograms per milliliter.