Supplementary Material for: Clinician-Reported Barriers to Implementing Breast Cancer Chemoprevention in the UK: A Qualitative Investigation

<i>Aims:</i> The use of tamoxifen and raloxifene as preventive therapy for women at increased risk of breast cancer was approved by the National Institute for Health and Care Excellence (NICE) in 2013. We undertook a qualitative investigation to investigate the factors affecting the implementation of preventive therapy within the UK. <i>Methods:</i> We recruited general practitioners (GPs) (n = 10) and clinicians working in family history or clinical genetics settings (FHCG clinicians) (n = 15) to participate in semi-structured interviews. Data were coded thematically within the Consolidated Framework for Implementation Research. <i>Results:</i> FHCG clinicians focussed on the perceived lack of benefit of preventive therapy and difficulties interpreting the NICE guidelines. FHCG clinicians felt poorly informed about preventive therapy, and this discouraged patient discussions on the topic. GPs were unfamiliar with the concept of preventive therapy, and were not aware that they may be asked to prescribe it for high-risk women. GPs were reluctant to initiate therapy because it is not licensed, but were willing to continue a prescription if it had been started in secondary or tertiary care. <i>Conclusions:</i> Barriers to implementing preventive therapy within routine clinical practice are common and could be addressed by engaging all stakeholders during the development of policy documents

Supplementary Material for: IL-33 and Its Receptor ST2 after Inhaled Allergen Challenge in Allergic Asthmatics

<b><i>Background:</i></b> Previous murine models have demonstrated interleukin (IL)-33 to be an important mediator of type-2 inflammation and to promote airway hyperresponsiveness in allergic asthma. A number of inflammatory cells produce IL-33 and eosinophils express ST2 mRNA. The relationship between IL-33 and eosinophils in allergic asthma, however, remains unclear. <b><i>Objective:</i></b> The aim of this work was to evaluate in vitro the effect of allergen inhalation on IL-33 levels and expression of its receptor (ST2L) on eosinophils in allergic asthmatics, and the effect of IL-33 stimulation on eosinophil activity. <b><i>Methods:</i></b> Plasma and sputum IL-33, soluble ST2 (sST2) levels, and ST2L expression on eosinophils were measured in 10 healthy controls and 10 allergic asthmatics. Asthmatics underwent allergen and diluent inhalation challenges. Blood and sputum samples were collected to measure IL-33, sST2, and ST2L eosinophil expression before and 24 h after allergen inhalation. Purified blood eosinophils from allergic asthmatics were incubated overnight with IL-33 to assess ST2 and intracellular IL-5 expression. <b><i>Results:</i></b> Baseline levels of IL-33 in sputum and sST2 in plasma and sputum were similar in allergic asthmatics compared to healthy controls. In addition, there was no difference in blood or sputum eosinophil ST2L expression in healthy controls versus allergic asthmatics. Eosinophil ST2L expression was significantly increased 24 h postallergen inhalation in allergic asthmatics. In vitro stimulation of human eosinophils with IL-33 and LPS significantly increased eosinophil ST2L expression and IL-33 stimulation increased intracellular IL-5 expression, which was attenuated by treatment with sST2 and ST2 blockade. <b><i>Conclusion and Clinical Relevance:</i></b> In mild asthmatics, there was a significant upregulation of ST2 surface expression on eosinophils from blood and sputum following allergen inhalation challenge. In vitro, IL-33 stimulation of eosinophils increases both ST2 membrane expression and IL-5 production. These results support a role for IL-33 in causing allergen-induced eosinophilia. Blockade of IL-33 and ST2 signaling may present a novel therapeutic avenue for asthma treatment

Supplementary Material for: Allergen-Induced Increases in Interleukin-25 and Interleukin-25 Receptor Expression in Mature Eosinophils from Atopic Asthmatics

<p><b><i>Background:</i></b> Interleukin (IL)-25 plays a pivotal role in type 2 immune responses. In a baseline cross-sectional study, we previously showed that IL-25 plasma levels and IL-25 receptor (IL-25R: IL-17RA, IL-17RB, and IL-17RA/RB) expression on mature blood eosinophils are increased in atopic asthmatics compared to normal nonatopic controls. This study investigated allergen-induced changes in IL-25 and IL-25R expression in eosinophils from asthmatics. <b><i>Methods:</i></b> Dual responder atopic asthmatics (n = 14) were enrolled in this randomized diluent-controlled crossover allergen challenge study. Blood was collected before and 24 h after the challenge. The surface expression of IL-25R was evaluated by flow cytometry on eosinophils and Th2 memory cells. In addition, plasma levels of IL-25 were measured by ELISA, and functional responses to IL-25 including type 2 cytokine expression, degranulation, and the migrational responsiveness of eosinophils were evaluated in vitro. <b><i>Results:</i></b> Following the allergen but not the diluent inhalation challenge, significant increases in the expression of IL-17RB and IL-17RA/B were found on eosinophils but not on Th2 memory cells. IL-25 plasma levels and the number of eosinophils but not of Th2 memory cells expressing intracellular IL-25 increased significantly in response to the allergen but not the diluent challenge. Stimulation with physiologically relevant concentrations of IL-25 in vitro caused (i) degranulation of eosinophils (measured by eosinophil peroxidase release), (ii) enhanced intracellular expression of IL-5 and IL-13, and (iii) priming of eosinophil migration to eotaxin. IL-25 stimulated intracellular cytokine expression, and the migration of eosinophils was blocked in the presence of a neutralizing IL-25 antibody. <b><i>Conclusions:</i></b> Our findings suggest that the IL-25/IL-25R axis may play an important role in promoting the recruitment and proinflammatory function of eosinophils in allergic asthma.</p