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Soluble CD40 ligand levels were measured in serum and homogenized tissues. All samples were stored at ?80��C until assayed. Assessment of serum concentrations of sCD40L was performed using a commercially available ELISA kit for quantitative detection of human sCD40L (Human sCD40L Platinum ELISA High Sensitive, Bioscience, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, plasma samples (20 ��l per well) or standards (100 ��l per well) were incubated for 2 h at room temperature. After washing, 100 ��l of HRP conjugate was added for 1 h at room temperature. Following incubation, unbound HRP-conjugated anti-human sCD40L antibody was removed during a wash step, and substrate solution (TMB ELISA substrate) was added to the all wells. A range of 0.08�C5.00 ng ml?1 selleck products of sCD40L was used to establish standard curves and to maximize the sensitivity. The sCD40L levels were determined using a spectrophotometer at a wavelength of 450 nm, and the data are reported in nanograms per millilitre. FR122047 [cyclo-oxygenase (COX)-1 inhibitor] selleck chemical and DuP 697 (COX-2 inhibitor), were purchased from Tocris Cookson Inc. (Menville, MO, USA), while atenolol (��1-adrenoceptor antagonist), isoprenaline (non-selective ��-adrenoceptor agonist), SC19220 (PGE2 receptor inhibitor), SP600125 [enzymatic c-Jun N-terminal kinase (JNK) inhibitor] and PGE2 were from Sigma Chemical Co. (St Louis, MO, USA). Stock solutions were freshly prepared in the corresponding buffers. The vehicles used were ethanol for PGE2 ALG1 and DMSO in distilled water for DuP 697, FR122047, SC19220 and SP600125. The drugs were diluted in the bath to achieve the final concentration stated in the text. The final concentration of DMSO was not more than 1 in 1000 and it lacked pharmacological action. Student's unpaired t test was used to determine the levels of significance. When multiple comparisons were necessary, after ANOVA the Student�CNewman�CKeuls test was applied. Differences between means were considered significant if P