Histamine challenge was performed to assess bronchial and laryngeal thresholds, as previously described.12 Histamine was delivered in doubling concentrations, starting from 0.5 mg/mL up to 32 mg/mL by using a compressed air nebulizer controlled by a breath-actuated dosimeter (MEFAR MB3; Markos, Monza, Italy). Each concentration was inhaled by taking 5 slow vital capacity breaths from the nebulizer. After each set of inhalations, FEV1 and flow-volume loops were recorded, and the best of 3 trials was selected. MIF50 was used as the index of laryngeal narrowing because we previously found that it reflects the decrease in cross-sectional glottic area during histamine challenge.11 The challenge was stopped when a 20% FEV1 decrease was obtained or the highest histamine concentration was reached. The histamine concentration provoking a 20% decrease in FEV1 was the bronchial threshold (PC20FEV1) and that provoking a 25% decrease in MIF50 was the laryngeal threshold (PC25MIF50). BHR and LHR were defined when the thresholds were equal or less than the 8 mg/mL histamine concentration.