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?4). To exclude the possibility that basophils had died after allergen-induced activation and mediator release, we checked them by incubation with labeled Annexin V (a marker for early apoptosis) and the dye 7-AAD (a marker for cell death). After cultivation in the presence of Bet v 1, IL-3, and IFN-�� for 16?h, irradiation and incubation for another 6?h, a maximum of 11% of basophils were positive for Annexin V, and Endonuclease the observed nonreactivity of T cells resulted from toxic effects of the mediators released by Bet v 1-activated basophils, we stimulated two Bet v 1-specific TCC with synthetic 12mer peptides containing their respective epitopes in the presence of basophils preincubated with Bet v 1, IL-3 and IFN-�� for 16?h. Such 12mer peptides are loaded directly into functional MHC class II molecules expressed by activated CD4+ T cells and induce proliferative responses in TCC albeit less strongly than in the presence of professional APC. The presence of Bet v 1-activated basophils did not ABT-263 order reduce peptide-induced proliferation in the TCC, thereby demonstrating that basophil-derived mediators did not impair T-cell reactivity (Fig.?5B). To study the antigen-processing and antigen-presenting capacity of human basophils, we developed a sophisticated experimental set-up involving a clinically relevant and immunologically well-characterized allergen, highly purified basophils, and specific TCC with defined epitope reactivity and HLA-restriction. The major birch pollen allergen has high allergenic potential and is a major cause for IgE-mediated respiratory allergy in Northern and Central Europe as well as North America [22]. We have extensively characterized Bet v 1 with regard to antigen-processing and T-cell signaling pathway reactivity by employing various types of professional APC and Bet v 1-specific TCC [17, 21, 23]. Bet v 1 lacks the cysteine-protease activity that, in the case of the major house dust mite allergen Der p 1, has been shown to activate basophils in a nonspecific manner [24]. Collectively, these features make Bet v 1 an ideal candidate to study the potential role of human basophils as APC in allergic disorders. We took great care to obtain highly purified basophils. Human monocytes (CD14+) and pDC (BDCA-2+), two cell types with potent antigen-processing capacity, express CD123, a molecule widely used as basophil marker. Even trace amounts of these cell types in our preparations might lead to false-positive detection of molecules involved in antigen presentation and/or activation of Bet v 1-specific TCC. Using the recently identified basophil selection marker CCR3 [25], we sorted basophils from PBMC by flow cytometry as CD14?BDCA-2?CCR3+CD123+ cells. In contrast to negative selection by commercially available kits, our sorting strategy also prevented the loss of potential HLA-DR-positive basophils.