Assessment and Mechanism of the Refractory Period in Asthma

Hypothesis: The primary hypothesis of this thesis is that the refractory period in asthma is due to release of bronchoprotective mediator(s) within the airway and that these may be one or more of the pro-resolving lipid mediators such as lipoxins or resolvins. Methods: 1. Development of a LC-MS assay for the detection of pro-resolving mediators in sputum. 2. Development of an in vitro airway model using porcine and human bronchial rings. 3. A clinical study of mannitol-induced refractoriness with collection of urine and sputum to assess changes in prostaglandin and pro-resolving mediator levels. Key Results: 1. Approximate LC-MS limits of detection were 0.002, 0.01 and 0.04 pg/μl for PGE2, LXA4 and RvD1 respectively. These LOD compare favourably with ELISA. 2. Serial LC-MS analysis of spiked sputum samples showed that LXA4 appeared stable, while PGE2 and RvD1 showed some degradation over 5 months. 3. LXA4, RvD1 and MaR1 had no significant effect on the histamine response of porcine bronchial rings. 4. Bronchoconstriction with high concentration mannitol rendered an airway refractory to further contraction with direct ASM agonists via an unknown mechanism. 5. In human bronchial rings, neither 10 nM LXA4 nor 10 nM MaR1 had any effect on bronchoconstriction induced by mast cell FcμR1 activation. 6. In the clinical study, with bolus dosing the mean refractory index for mannitol was 24%. The degree of refractoriness to mannitol did not correlate with markers of disease severity. 7. Urinary PGD2 and PGE2 metabolite levels measured by ELISA showed no significant change in response to mannitol challenge. Sputum LXA4 and RvD1 levels measured by ELISA showed no consistent change in response to mannitol challenge. Conclusions: This study has found no evidence of a role for pro-resolving mediators in the mechanism of refractoriness.