The role of mast cell and airway smooth muscle interactions in the pathophysiology of asthma. (360G-Wellcome-082265_Z_07_Z)

We hypothesise that airway hyperresponsiveness and persistent airflow obstruction in asthma result from a) intrinsic abnormalities in airway smooth muscle (ASM) from asthmatics and b) interactions between ASM-mast cells. We aim i) to examine the differences between ASM from asthmatics and non-asthmatics and to determine the mechanisms underlying these differences in terms of calcium homeostasis using single cell and FLIPR analysis; contractility using collagen gel contraction assays and single c ell measurements of maximal contraction and velocity; synthetic capacity using a variety of approaches including ELISA, immunoblotting, proteomics, qPCR and gene array; migration using 2D assays; proliferation by MTS assay and thymidine incorporation; and survival by Annexin V and PI staining, ii) to investigate in vitro the effect of ASM-mast cell interactions on ASM and mast cell function respectively and to confirm effects, as appropriate, in tissue and iii) to confirm that localisation of ac tivated mast cells in the ASM-bundle is a feature of asthma across severity of disease and to determine whether this is affected by corticosteroids or experimental viral infection. For the in vitro experiments we shall use freshly isolated and primary ASM cultures from bronchial biopsies from asthmatics and controls and primary mast cells from lung resection material.