14–17 cDNAs were normalized

on the basis of the expression of HPRT. The reaction mixture (20 μl) contained normalized cDNA, 200 mmol/l of each deoxyribonucleotide MK-8669 in vitro triphosphate (dNTP), 1·5 mmol/l of MgCl2, 25 pmol of each primer and 0·5 U of Thermus aquaticus (Taq) DNA polymerase in polymerase chain reaction (PCR) buffer (Invitrogen). PCR was performed and the products were analyzed as described previously.14–16,18 The PCR products were scanned using a gel documentation system (Alpha-Innotech Corporation, San Leandro, CA), and the intensity of PCR products present in each lane was measured densitometrically using AlphaImager (software version 5.5; Alpha-Innotech Corporation, Santa Clara, CA). Whole blood (1 ml) was collected into sterile tubes at pretreatment and post-treatment stages, and from healthy volunteers. Blood was allowed to coagulate for 2–3 hr at 4° before centrifugation. Sera were preserved at −70° until use. Sera were collected 2–4 weeks after the last dose of treatment in clinically cured patients. Cytokine levels in serum were determined by flow cytometry utilizing

the inflammatory AZD9291 in vivo cytokine bead array (CBA) kit (BD Biosciences, San Jose, CA). Briefly, 50 μl of bead populations with discrete fluorescence intensities, coated with cytokine-specific capture antibodies, were added to 50-μl samples of patient sera and 50 μl of phycoerythrin (PE)-conjugated anti-human inflammatory cytokine antibodies. Simultaneously, standards for each cytokine (0–5000 pg/ml) were mixed with cytokine capture beads. The vortexed mixtures were allowed to incubate for 1·5 hr in the dark. After washing the beads, 50 μl of the human inflammation PE detection reagent was added and incubated for 1·5 hr in dark. Beads were washed and analyzed using flow cytometry (FACS Calibur; BD Biosciences). The quantity (pg/ml) of respective cytokine was calculated using CBA software. Standard curves were derived from the cytokine standards supplied with the kit. The BD OptEIA™ (BD Biosciences)

human IL-8 and MCP-1 enzyme-linked immunosorbent GNA12 assay (ELISA) kit was used for quantitative determination, as per the manufacturer’s instructions. The absorbance was measured at 450 nm within 30 min of stopping the reaction. Nitric oxide (NO) is degraded quickly into nitrite and nitrate, and therefore the serum nitrite concentration was determined using the Griess reaction as an indicator of NO. The Griess reagent (Sigma, St Louis, MO) was dissolved in 250 ml of nitrite-free water, and then 50 μl of reagent and an equal volume of the sample was added in an ELISA plate (Griener, Monroe, NC) and mixed immediately. After 30 min of incubation at room temperature, the absorbance was read at 540 nm. The EnVision TM G/2 system/AP (DakoCytomation, Glostrup, Denmark) procedure for light microscopic immunohistochemistry (IHC) in dermal lesions and control tissues was performed.