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Significance was evaluated according to Pfaffl et?al. (45). MCP-2 levels were measured from cell supernatants derived from poly-(I:C)-treated HaCaT cells, with or without the TRPV1 antagonist, I-RTX, in presence or in absence of PEA using the human MCP-2 ELISA kit protocol and http://www.selleckchem.com/products/VX-770.html according to the manufacture��s instructions (Ray Biotech, Inc. provided by Tebu-Bio, Magenta, Milan, Italy). Human keratinocytes cells were plated into six-well culture plates and sensitized with poly-(I:C). After 24?h, cells and supernatants were homogenized in chloroform/methanol/Tris�CHCl 50?mM pH 7.4 (2?:?1?:?1, v/v) containing 10?pmol of [2H]8-AEA, and 50?pmol of [2H]5-2AG, [2H]4-PEA and [2H]2-OEA as internal standards (41). The extraction and purification phases and the LC�CAPCI�CMS analysis of PEA were conducted as described earlier. LC�CAPCI�CMS analysis of AEA, 2-AG and OEA analysis were carried out using m/z values of 356 and 348 (molecular ions +1 for deuterated and undeuterated AEA), 384.35 and 379.35 (molecular ions http://www.selleck.cn/products/Verteporfin(Visudyne).html +1 for deuterated and undeuterated 2-AG), 328 and 326 (molecular ions +1 for deuterated and undeuterated OEA). AEA, 2-AG and OEA levels were calculated by isotopic dilution, as described for earlier for PEA. Palmitoylethanolamide (5 and 10?mg/kg), cpz (0.5 and 2?mg/kg) and the association of PEA, 5?mg/kg, and cpz, 0.5?mg/kg, or the association of PEA, 10?mg/kg, and cpz, 2?mg/kg, were administered intraperitoneally (i.p.) on day 5, 6, 7 (the days of the first challenge with DNFB), or on day 12, 13, 14 (the days of the second challenge with DNFB) after the initial sensitization with DNFB. N?=?11 mice per group were used for these experiments. For these experiments, we employed exactly the same mouse ear extracts previously shown to contain elevated AEA and 2-AG levels following DNFB treatment and subsequent second http://www.selleckchem.com/products/ch5424802.html challenge, and in which measures of ear thickness (oedema) had been already carried out (38). PEA amounts were measured by LC-MS in the ear skin of DNFB-sensitized mice after the second challenge with DNFB and induction of ear swelling. DNFB challenge caused in both WT and, particularly, double CB1/CB2 KO mice a significant increase in both ear thickness and ear MCP-2 mRNA levels assessed by q-PCR (see ref. 38 and Table?1). PEA levels were 29.9?��?1.5 vs 36.5?��?5.4?pmol/mg of lipid extract in vehicle-treated WT mice (WT/CTRL) vs DNFB-treated WT mice (WT/DNFB), and 33.5?��?4.2 vs 97.8?��?4.9?pmol/mg lipid extract in vehicle-treated CB1?/?/CB2?/? KO mice (CB1/CB2 KO/CTRL) vs DNFB-treated CB1?/?/CB2?/? mice (CB1/CB2 KO/DNFB) (Fig.?1). Thus, the higher the degree of inflammation (WT/CTRL?��?CB1/CB2 KO/CTRL?