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To determine Th1/Th2 Alternaria-specific T cells, Alternaria-stimulated cultures were performed as previously described (26�C30). In 1?ml volume of Rosewell Park Memorial Institute (RPMI) supplemented with 10% fetal calf serum (FCS), 1?��?106 peripheral blood lymphocytes (PBL) were cultured for 1?week in a humidified 5% CO2 atmosphere at 37��C. Cultures were stimulated in media alone, 25?mcg/ml of A.?alternata extract, 25?mcg/ml of Alt a1, 25?mcg/ml of Asp f3, and 25?mcg of Der p1. Tetanus toxoid 4?LF/ml (Connaught Laboratories, Toronto, Canada) was used as a Th1 control antigen. The culture supernatants were obtained and frozen at ?70��C until analyzed. Alternaria extract and Alt diglyceride a1 were obtained from Dr Hari Vijay, and Asp f3 was obtained from Dr Viswanath P. Kurup. Der p1 was obtained from Greer Laboratories. Measurement of Th1/Th2 culture supernatant cytokines and chemokines was performed by Flex Cytometric Bead Assay from BD Pharmingen (San Diego, CA USA) to measure Th1/Th2 cytokines, IL-4, IL-5, IL-10, IL-13, and IFN-g synthesis, as previously described (27, 30). To examine HLA class II restriction, inhibition of Alternaria-stimulated lymphoproliferative responses was assessed with blocking anti-HLA-DR, -DP, and -DQ selleck compound monoclonal antibodies as previously described (31). In 0.2?ml volume of RPMI supplemented with 10% FCS, 2.5?��?105?PBL was cultured for 7?days in a humidified 5% CO2 atmosphere at 37��C. Cultures were stimulated in media alone or 25?mcg/ml of A.?alternata extract and Alt a1. In parallel cultures, blocking anti-DR, -DP-, and DQ mAbs 20?mcg/ml were added (Leinco Technologies, selleck screening library Inc., St Louis, MO, USA). Cultures were then pulsed for 16?h with 1.0?mcC tritiated thymidine (3H TdR). 3H TdR which was incorporated into DNA during lymphoproliferation was harvested onto glass filter paper. Proliferation was measured as 3H TdR incorporation and expressed as mean counts per minute and stimulation index. The data for pulmonary function tests (PFT) and cytokine levels were expressed as the mean?��?SD and for IgE geometric mean?��/��?SD. Statistical analysis using two-tailed Mann�CWhitney U-test was used comparing mold-sensitive moderate-severe asthma to other groups. For multiple comparisons, the Kruskal�CWallace test (nonparametric anova) was performed. Post hoc analysis was performed using Dunn��s multiple comparisons test; P-value