We investigated the clinical and pathologic features of fungal sensitization in children with STRA and delineated mechanistic differences underlying chronic fungal and HDM exposure in a neonatal mouse model of allergic airways disease. Methods Subjects Children aged 6 to 16 years with STRA were recruited Bifemelane HCl from the Royal Brompton Hospital (London, United Kingdom). exposure. Lung IL-33 levels, IL-13+ ILC numbers, TH2 cell numbers, IL-13 levels, and AHR remained increased with inhaled budesonide during exposure, but all features were significantly reduced in ST2?/? mice lacking a functional receptor for IL-33. Conclusion Pediatric SAFS was associated with more oral steroid therapy and higher IL-33 levels. exposure resulted in increased IL-33Cmediated ILC2 numbers, TH2 cell numbers, and steroid-resistant AHR. IL-33 might be a novel therapeutic target for SAFS. did not show any benefit.15 Recently, IL-33 has been shown to contribute to the development of fungal exacerbation of allergic airways disease in an adult murine model after chronic house dust mite (HDM) exposure.16 However, mechanisms underlying chronic fungal exposure and sensitization remain unknown. We hypothesized that fungal sensitization in children with severe therapy-resistant asthma (STRA) is associated with more severe disease and is mediated by the innate cytokine IL-33. We investigated the Bifemelane HCl clinical and pathologic features of fungal sensitization in children with STRA and delineated mechanistic differences underlying chronic fungal and HDM exposure in a neonatal mouse model of allergic airways disease. Methods Subjects Children aged 6 to 16 years with STRA were recruited from the Royal Brompton Hospital (London, United Kingdom). They had already undergone a detailed assessment to optimize adherence and address underlying modifiable factors as much as possible.17 STRA was defined as previously described,1 as persistent chronic symptoms, exacerbations, or both despite high-dose inhaled?corticosteroids (beclomethasone equivalent 800 g/d), long-acting -agonists, and either current or a previous failed trial of leukotriene receptor antagonists. Two groups were defined: (1) patients with SAFS with sIgE or positive SPT responses to any of and (2) nonCfungus-sensitized patients (non-SAFS) with negative sIgE levels and SPT responses to all these fungal allergens. Sensitivity to other fungi is not routinely tested in our department. The study was approved by the local research ethics committee, and informed parental consent and child assent were obtained. Clinical assessment Age at onset of symptoms, medications, and symptom scores (Asthma Control Test)18 were recorded. Spirometry with bronchodilator reversibility was performed according to American Thoracic Society/European Respiratory Society guidelines.19 Atopy Atopy was assessed based on total serum IgE levels, sIgE levels, and SPT responses to was administered intranasally 3 times per week (5 g for the first 2 weeks, followed by 10 g in the third week). Airway hyperresponsiveness (AHR) to methacholine was determined by using the forced oscillation technique in anaesthetized and tracheostomized mice 4 hours after final challenge with HDM or 18 hours after the final challenge, as previously described.22 In experiments to assess the effects of steroid therapy, mice were treated with 0.6 mg/kg intranasal budesonide (Pulmicort Respules; AstraZeneca, London, United Kingdom) or PBS (10 L) daily during the period of allergen exposure. All?experiments were performed in accordance with UK Home Office guidelines. Tissue processing and analysis Serum, BAL fluid, and lung tissue were collected and analyzed, as previously described.22 Paired antibodies for murine IgE, (BD Biosciences, Oxford, United Kingdom), IL-13, IL-33 (R&D Systems), IL-4, and IL-5 (PharMingen, Oxford, United Kingdom), were used in standardized sandwich ELISAs, according to the manufacturer’s protocol. Serum HDM- and valueand test: *test: Bifemelane HCl **exposure to HDM exposure in neonatal mice Because most children with STRA are polysensitized to several aeroallergens, it is difficult to disentangle mechanisms attributable to fungal sensitization alone. exposure was PLS3 compared with HDM exposure in neonatal mice (Fig 2, exposure compared with that seen after HDM exposure. Open in a separate window Fig 2 Fungal exposure in neonatal mice resulted in more severe atopy and inflammation than HDM exposure, but AHR was similar with both allergens. Neonatal BALB/c mice were challenged with intranasal HDM (20 g for the first 2 weeks.