During red tide events aerosolized polyether brevetoxins (PbTxs) have been linked to upper and lower airway symptoms in both normal individuals and in "susceptible populations", i.e. those individuals with pre-existing airway disease. During the first grant cycle we showed that sheep inhaling PbTxs environmentally relevant concentrations of PbTx developed adverse pulmonary events including acute bronchoconstriction, airway hyperresponsiveness (AHR), airway inflammation and decreased mucociliary clearance. Both normal and allergic sheep, which serve as a surrogate for a "susceptible population", responded to toxin challenge, but the severity of the response was, in part, dependent on the underlying inflammatory status of the airways. The most striking findings, however, were that the airway effects of the inhaled PbTxs differed in their physiological actions and their response to pharmacologic intervention. The differences in responses were related to changes in the chemical structure of the PbTx tested. To further complicate the problem, we found that a natural congener of PbTx and chemically modified PbTxs not only could block the adverse pulmonary effects of PbTx, but in some instances, improve pulmonary function on their own. These findings suggest that synthetic and/or natural modifications of the PbTx molecules can result in the generation of compounds that have increased toxicity, or depending on the structural modification, beneficial effects in the airway. Therefore, in this proposal we will test the hypothesis that the severity of the airway effects of inhaled toxins, i.e. effects on bronchial tone, airway responsiveness, mucociliary clearance, inflammatory cell recruitment and airway cell function are dependent on the chemical structure(s) of the individual PbTxs that are aerosolized. Furthermore, we postulate that the PbTx congeners and analogs can stimulate different pulmonary cells/receptors and that the PbTx-induced effects (whether beneficial or harmful) will be dependent on cells/receptors on various pulmonary cells stimulated by the toxins. Three Specific Aims will be used to test this hypothesis. Specific Aim 1: A) To compare the effects of selected PbTxs (both natural and synthetic toxins), PbTx congeners and toxin complexes identified in field studies on measures of bronchial tone (pulmonary airflow resistance, RL) and mucociliary clearance (tracheal mucus velocity, TMV and whole lung mucociliary clearance, MCC) to identify the chemical structure(s) responsible for the observed effects whether beneficial or harmful. And B) To use in vivo pharmacology to identify airway cells/receptors responsible for toxin-induced effects. Specific Aim 2: To determine the mechanisms responsible for the airway inflammation and airway hyperresponsiveness (AHR) that result from 4-day PbTx exposure, including the regulation of nuclear factor kappa beta (NFkB) activation, subsequent cytokine release and the role of adhesion molecule activation. Specific Aim 3: To determine if the immunosuppressive effects of PbTx on macrophages and the PbTx-induced depressions in TMV seen after toxin exposures affect bacterial clearance in vivo. We will continue to utilize the sheep model for these studies because this model responds to inhaled concentrations of toxin that are present in the environment and cause respiratory symptoms in humans and comparisons between normal and allergic sheep allow us to model airway effects in "normal" and "susceptible populations". Furthermore, as demonstrated in the Progress Report / Preliminary Studies, the physiologic endpoints measured in this model have adequate sensitivity to differentiate amongst the PbTx congeners that differ by a single atom or functional group, thereby providing confidence that the proposed Specific Aims can be accomplished. The data generated in this proposal are novel in that they provide an approach to understanding how structure and composition of aerosolized toxin affects the airways. The experiments are being conducted at environmentally relevant concentrations. Because of our extensive experience with delineating inflammatory cascades in this model, we are well positioned to understand mechanisms involved in toxin-induced inflammation in the airways and the potential of toxin exposure to impair host defense mechanisms. With the current paucity of data addressing these issues, the proposed studies should provide needed information on the pathophysiological consequences of PbTx exposure.