Inhibition of exocytotic activity in human mast cell line 1 by clostridial neurotoxin light chains

The main object of this work is to deliver the catalytically active portion of neurotoxins (light chain) into the cytosol of mast cells to inhibit the specific release of inflammatory mediators. Mast cells play a significant role in the pathophysiology of many different diseases. In most of these diseases there is increased recruitment of mast cells and sustained secretion of many proinflammatory mediators, including autacoids, cytokines and proteases. We use the human mast cell line 1 (HMC-1), a leukaemia line expressing several features of mature human mast cells that has proved a valuable model for studying human mast cell biology. In order to stimulate fusion among secretory granules, we use intracellular application of GTPgammaS and extracellular stimulation with concanavalin A. By patch-clamp technique, we determine the changes in cell membrane capacitance, thereby studying exocytosis. The whole-cell configuration of patch-clamp is used. By electroporation, the light chains of TeNT and BoNT/C & D will be delivered into the cytoplasm of mast cells to estimate their effects on mast cell activity. Cells treated with neurotoxins will be subjected to stimulation tests “in vitro”, recording of membrane capacitance by patch-clamp and detection of cytokine release to estimate effects on mast cell activity.