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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that spread around the world during the past 2 years, has infected more than 260 million people worldwide and has imposed an important burden on the healthcare system. Several risk factors associated with unfavorable outcome were identified, including elderly age, selected comorbidities, immune suppression as well as laboratory markers. The role of immune system in the pathophysiology of SARS-CoV-2 infection is indisputable: while an appropriate function of the immune system is important for a rapid clearance of the virus, progression to the severe and critical phases of the disease is related to an exaggerated immune response associated with a cytokine storm. We analyzed differences and longitudinal changes in selected immune parameters in 823 adult COVID-19 patients hospitalized in the Martin University Hospital, Martin, Slovakia. Examined parameters included the differential blood cell counts, various parameters of cellular and humoral immunity (serum concentration of immunoglobulins, C4 and C3), lymphocyte subsets (CD3+, CD4+, CD8+, CD19+, NK cells, CD4+CD45RO+), expression of activation (HLA-DR, CD38) and inhibition markers (CD159/NKG2A). Besides already known changes in the differential blood cell counts and basic lymphocyte subsets, we found significantly higher proportion of CD8+CD38+ cells and significantly lower proportion of CD8+NKG2A+ and NK NKG2A+ cells on admission in non-survivors, compared to survivors; recovery in survivors was associated with a significant increase in the expression of HLA-DR and with a significant decrease of the proportion of CD8+CD38+cells. Furthermore, patients with fatal outcome had significantly lower concentrations of C3 and IgM on admission. However, none of the examined parameters had sufficient sensitivity or specificity to be considered a biomarker of fatal outcome. Understanding the dynamic changes in immune profile of COVID-19 patients may help us to better understand the pathophysiology of the disease, potentially improve management of hospitalized patients and enable proper timing and selection of immunomodulator drugs. [ABSTRACT FROM AUTHOR]

The velocity of the COVID-19 pandemic spread and the variable severity of the disease course has forced scientists to search for potential predictors of the disease outcome. We examined various immune parameters including the markers of immune cells exhaustion and activation in 21 patients with COVID-19 disease hospitalised in our hospital during the first wave of the COVID-19 pandemic in Slovakia. The results showed significant progressive lymphopenia and depletion of lymphocyte subsets (CD3+, CD4+, CD8+ and CD19+) in correlation to the disease severity. Clinical recovery was associated with significant increase in CD3+ and CD3+CD4+ T-cells. Most of our patients had eosinopenia on admission, although no significant differences were seen among groups with different disease severity. Non-survivors, when compared to survivors, had significantly increased expression of PD-1 on CD4+ and CD8+ cells, but no significant difference in Tim-3 expression was observed, what suggests possible reversibility of immune paralysis in the most severe group of patients. During recovery, the expression of Tim-3 on both CD3+CD4+ and CD3+CD8+ cells significantly decreased. Moreover, patients with fatal outcome had significantly higher proportion of CD38+CD8+ cells and lower proportion of CD38+HLA-DR+CD8+ cells on admission. Clinical recovery was associated with significant decrease of proportion of CD38+CD8+ cells. The highest AUC values within univariate and multivariate logistic regression were achieved for expression of CD38 on CD8+ cells and expression of PD1 on CD4+ cells alone or combined, what suggests, that these parameters could be used as potential biomarkers of poor outcome. The assessment of immune markers could help in predicting outcome and disease severity in COVID-19 patients. Our observations suggest, that apart from the degree of depletion of total lymphocytes and lymphocytes subsets, increased expression of CD38 on CD3+CD8+ cells alone or combined with increased expression of PD-1 on CD3+CD4+ cells, should be regarded as a risk factor of an unfavourable outcome in COVID-19 patients. Increased expression of PD-1 in the absence of an increased expression of Tim-3 on CD3+CD4+ and CD3+CD8+ cells suggests potential reversibility of ongoing immune paralysis in patients with the most severe course of COVID-19. [ABSTRACT FROM AUTHOR]

Introduction: SYNAPSE, a Phase III, randomised, doubleblind trial, assessed the efficacy of mepolizumab in severe chronic rhinosinusitis with nasal polyps (CRSwNP) (Han JK, et al. Lancet Respir Med 2021; S2213-2600(21)00097-7). Aim: To assess the clinical response to mepolizumab in severe CRSwNP. Method: In SYNAPSE, patients received standard of care plus mepolizumab (100 mg subcutaneous) or placebo for 52 weeks. This post hoc, descriptive analysis, classified patients in the intent-totreat (ITT) population without sinus surgery during the study period as responders, using 5 modified European Forum for Research and Education in Allergy and Airway Diseases (EUFOREA) criteria (Bachert C, et al. JACI, 2021;147:29). Results: In the ITT population (n=407), 381 and 343 patients had not had sinus surgery by Weeks 24 and 52, respectively. For each EUFOREA criterion (reduced nasal polyp size and nasal obstruction, improved sense of smell, improved quality of life, improved overall symptoms) proportion of patients classified as responders was higher with mepolizumab than placebo at Weeks 24 and 52. The proportion of patients who met =3 criteria was 55% with mepolizumab vs 35% with placebo at Week 24, and 67% vs 49% at Week 52. Conclusion: Clinical response, as defined by the modified EUFOREA criteria, was greater with mepolizumab than placebo and increased from Week 24 to Week 52, suggesting a sustained, progressive response in patients with severe CRSwNP without sinus surgery during the study. [ABSTRACT FROM AUTHOR]

Summary: Background: Nitric oxide (NO) measurements in exhaled air and hypertonic saline-induced sputum are commonly used biomarker sampling methods of the lower airways. Both sampling methods have been validated in asthmatic patients and healthy controls, however, data from chronic smokers are scarce. Objectives: To evaluate the reproducibility and differences in fractional exhaled NO (FeNO) values in asymptomatic chronic smokers and healthy, non-smoking controls. Furthermore, to test the effect of hypertonic saline sputum induction (SI) on FeNO levels in both study groups. Methods: 16 asymptomatic chronic smokers and 16 non-smokers participated in this study. Baseline FeNO and forced expiratory volume in 1 s (FEV1) were recorded pre- and 30 min post NaCl 4.5% SI (3 × 5 min) on 2 study days (±2 h; 4–10 days apart). Mixed ANOVA was used to estimate the intra-subject Coefficient of Variation (CV) % over days; changes in FeNO and FEV1 values before and after SI, were analyzed by a Student''s paired t-test.The difference between smokers and non-smokers was estimated by a Student''s t-test. Results: On day 1, FeNO values in smokers were significantly lower than in non-smokers, 10.6 ppb, and 18.4 ppb, respectively, (42% difference, p = 0.0028, 95% CI: −59%, −19%). In both study groups, FeNO measurements were reproducible, with an intra-subject CV of 27.2% and 19.2%, for smokers and non-smokers, respectively. SI significantly decreased FeNO levels in both study groups on day 1. In smokers, there was a mean reduction in FeNO of almost 37% (p = 0.0045, 95% CI: −53%, −14%), and in non-smokers a mean decrease of almost 37% (95% CI : −53%, −14%; p = 0.0045). In both study groups SI did not affect FEV1 (p > 0.94). Conclusions: Our data extend previous findings in asthmatics and healthy controls to asymptomatic chronic smokers: 1. FeNO measurements are reproducible in both smokers and non-smokers; 2. baseline FeNO levels in chronic smokers are lower than in non-smokers and 3. sputum induction by hypertonic saline reduces FeNO levels in both study groups, without affecting lung function. [Copyright &y& Elsevier]

Summary: In recent years, it has become increasingly important to get as much as possible information on clinical efficacy already in the early phases of drug development. For proof of concept (POC) studies testing novel anti-inflammatory drugs in asthma, there are several validated exacerbation models, inducing various aspects of the airway inflammation and airway responsiveness. The choice of the appropriate asthma model depends on the drug''s targets within the inflammatory process. For adequate assessment of the drug''s anti-inflammatory potential, it is crucial to choose adequate (surrogate) biomarkers. Ideally, these should include measures of airway response, central and peripheral airway inflammation and airway hyperresponsiveness. Overall, there are validated non-invasive sampling techniques for the measurement of inflammatory markers in asthma that can be applied as outcome parameters in early clinical trials. If adequately implemented, these measurements can provide early indication of proof of pharmacological and potential therapeutic efficacy—even in first administration to humans. [Copyright &y& Elsevier]

Summary: In this review, we aim to lead the readers through the historical highlights of pathophysiological concepts and treatment of asthma. Understanding the nature and links of asthma has modeled our diagnostic, pathophysiological and therapeutic thinking and acting. The recognition of its heterogeneous nature in combination with several refined and sophisticated technologies will mark a new era of phenotype-specific approach and treatment of asthma. [Copyright &y& Elsevier]

Summary: Asthma is a chronic disorder, characterized by airway hyperresponsiveness (AHR), airway inflammation and airway remodelling. Evidence has been provided for a relationship between pathophysiology, airway inflammation and remodelling. Moreover, these asthma features have been shown to respond to anti-inflammatory therapy. According to current guidelines, monitoring of asthma is predominantly based on symptoms and lung function data. However, these parameters appeared as poor indices for asthma control. Alternatively, asthma control relates well to exacerbations and (anamnestic) surrogate biomarkers of airway inflammation. Hence, appropriate treatment of asthma should primarily target the airway inflammation. According to current guidelines for asthma management, anti-inflammatory therapy with inhaled corticosteroids (ICS) is the cornerstone in the treatment of persistent asthma. To further optimize asthma control, add-on therapy with long-acting β 2-agonists (LABA) or leukotriene receptor antagonists (LTRA) should be combined with low to high doses of ICS. While the first combination focuses on optimal control of symptoms and lung function, the second provides a more complete suppression of the airway inflammation. In this paper we discuss treatment of asthma according to current guidelines versus new insights, addressing practical issues. [Copyright &y& Elsevier]

Dynamic heterogeneity in lung ventilation is an important measure of pulmonary function and may be characteristic of early pulmonary disease. While standard indices like spirometry, body plethysmography, and blood gases have been utilized to assess lung function, they do not provide adequate information on regional ventilatory distribution nor function assessments of ventilation during the respiratory cycle. Emerging technologies such as xenon CT, volumetric CT, functional MRI and X-ray velocimetry can assess regional ventilation using non-invasive radiographic methods that may complement current methods of assessing lung function. As a supplement to current modalities of pulmonary function assessment, functional lung imaging has the potential to identify respiratory disease phenotypes with distinct natural histories. Moreover, these novel technologies may offer an optimal strategy to evaluate the effectiveness of novel therapies and therapies targeting localized small airways disease in preclinical and clinical research. In this review, we aim to discuss the features of functional lung imaging, as well as its potential application and limitations to adoption in research., Competing Interests: NE, TO, AF, and JK, are employed by 4D Medical. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Karmali, Sowho, Bose, Pearce, Tejwani, Diamant, Yarlagadda, Ponce, Eikelis, Otvos, Khan, Lester, Fouras, Kirkness and Siddharthan.)

The allergen provocation test is an established model of allergic airway diseases, including asthma and allergic rhinitis, allowing the study of allergen-induced changes in respiratory physiology and inflammatory mechanisms in sensitised individuals as well as their associations. In the upper airways, allergen challenge is focused on the clinical and pathophysiological sequelae of the early allergic response, and is applied both as a diagnostic tool and in research settings. In contrast, bronchial allergen challenge has almost exclusively served as a research tool in specialised research settings with a focus on the late asthmatic response and the underlying type 2 inflammation. The allergen-induced late asthmatic response is also characterised by prolonged airway narrowing, increased nonspecific airway hyperresponsiveness and features of airway remodelling including the small airways, and hence allows the study of several key mechanisms and features of asthma. In line with these characteristics, allergen challenge has served as a valued tool to study the cross-talk of the upper and lower airways and in proof-of-mechanism studies of drug development. In recent years, several new insights into respiratory phenotypes and endotypes including the involvement of the upper and small airways, innovative biomarker sampling methods and detection techniques, refined lung function testing as well as targeted treatment options further shaped the applicability of the allergen provocation test in precision medicine. These topics, along with descriptions of subject populations and safety, in line with the updated Global Initiative for Asthma 2021 document, will be addressed in this review., Competing Interests: G.M. Gauvreau declares grants from AstraZeneca, Biohaven, Novartis and Genentech; consulting fees from AstraZeneca, Biohaven, Novartis, Sterna Biologicals and Certior Consulting; and payment or honoraria from AstraZeneca, Genzyme and Genentech, all in the 36 months prior to manuscript submission. B.E. Davis declares no competing interests. G. Scadding declares support from ALK-Abelló (provision of active and placebo medication for the GRASS clinical trial referenced in the article) and the Immune Tolerance Network (NIAID, NIH, USA; sponsor of the GRASS clinical trial) related to the present manuscript; as well as payment for lectures from ALK-Abelló in 2020 and 2021; and support from GlaxoSmithKline for attendance at the 2021 European Academy of Allergy and Clinical Immunology meeting. L-P. Boulet declares research grants for participation in multicentre studies or research projects proposed by the investigator from Amgen, AstraZeneca, GlaxoSmithKline, Merck, Novartis and Sanofi-Regeneron; royalties from UptoDate and Taylor & Francis; lecture fees from AstraZeneca, Covis, GlaxoSmithKline, Novartis, Merck and Sanofi, all in the 36 months prior to manuscript submission; and the following unpaid contributions: Chair of the Global Initiative for Asthma (GINA) Board of Directors; President of the Global Asthma Organisation (Interasma); Member of the Canadian Thoracic Society Respiratory Guidelines Committee; and Laval University Chair on Knowledge Transfer, Prevention and Education in Respiratory and Cardiovascular Health. L. Bjermer declares no competing interests. A. Chaker declares grants or contracts, via Technical University Munich, from EIT Health (European Institute of Technology), BMBF (German Federal Ministry of Education and Research), AstraZeneca, Sanofi-Genzyme, Roche, GlaxoSmithKline and ALK-Abelló; payments or honoraria, via Technical University Munich, from ALK-Abelló, Allergopharma, AstraZeneca, GlaxoSmithKline, Immunotek, Novartis, Regeneron, Sanofi-Genzyme, Leti, Zeller and Bencard; travel reimbursement to attend European Academy of Allergy and Clinical Immunology meetings (2018 and 2021 from the European Academy of Allergy and Clinical Immunology; 2019 from ALK-Abelló); patents, via Technical University Munich, relating to allergen-specific immunotherapy and treatment of SARS-CoV-2 infection; participation, via Technical University Munich, on a data safety monitoring board or advisory board for ALK-Abelló, Allergopharma, AstraZeneca, GlaxoSmithKline, Immunotek, Novartis, Regeneron and Sanofi-Genzyme; and the following unpaid roles: Past Chair of the Allergy, Clinical Immunology and Environmental Medicine Section of the German ENT Society; board member at large of the German Allergy Society (DGAKI); scientific advisor/board member to the German Society for Applied Allergy (AeDA); board member of the Allergen Immuntherapy Interest Group, European Academy of Allergy and Clinical Immunology. D.W. Cockcroft reports the following research grants in the 36 months prior to manuscript submission: research grant Dept of Medicine University of Saskatchewan (Effect of tiotropium on airway response to allergen); Canadian Society of Allergy and Clinical Immunology (The effect of deep inhalation on mannitol responsiveness); AstraZeneca (A phase 2a double blind randomised parallel group placebo controlled multicentre study to evaluate the effect of AZD8154 administered via nebuliser once daily on allergen-induced inflammation in subjects with mild allergic asthma challenged with inhaled allergen); AllerGen NCE (Methacholine challenge comparison of a jet nebuliser and vibrating mesh nebuliser); Novartis (Inhaled CSJ117 in adult asthmatics with mild atopic asthma). B. Dahlén declares payment to their institution in 2004 for a clinical trial by AstraZeneca, related to the present work; and grants to support the Karolinska Severe Asthma Centre from GlaxoSmithKline and Novartis; consulting fees from Teva, AstraZeneca and Sanofi; and payment for lectures from AstraZeneca and Sanofi, all in the 36 months prior to manuscript submission. W. Fokkens declares grants to their institution from ALK, Mylan, Allergy Therapeutics, GlaxoSmithKline, Sanofi, Novartis and Chordate; consulting fees from Sanofi and Bioinspire; payment or honoraria from Sanofi, GlaxoSmithKline and Novartis; and participation on a data safety monitoring board or advisory board for Lyra, Sanofi and GlaxoSmithKline, all in the 36 months prior to manuscript submission; and that they are Secretary General of the European Rhinologic Society. P. Hellings declares no competing interests. N. Lazarinis declares no competing interests. P.M. O'Byrne reports grants from AstraZeneca, GlaxoSmithKline, Biohaven, Merck, Bayer and Novartis; consulting fees from AstraZeneca, Covis, GlaxoSmithKline, Amgen and Novartis; and payment or honoraria from AstraZeneca, Covis and Novartis, all in the 36 months prior to manuscript submission; and that they are a Section Editor of the European Respiratory Journal. E. Tufvesson declares no competing interests. S. Quirce declares consulting fees and payment or honoraria from GlaxoSmithKline, Sanofi and AstraZeneca; and payment or honoraria from Novartis, Chiesi, Mundipharma and Teva, all in the 36 months prior to manuscript submission. M. Van Maaren declares payment or honoraria from ALK-Abelló, Takeda and CSL Behring, in the 36 months prior to manuscript submission; and an unpaid role as Chairman of the Dutch Society of Allergology and Clinical Immunology since 2020. F. H. de Jongh declares an unpaid role as European Respiratory Society Assessment Director. Z. Diamant reports that until May 2020 they worked as Research Director Respiratory & Allergy at a CRO (QPS-NL), which received funding from biotech and several pharma companies for conduct of phase I–II clinical studies (drug development); and declares consulting fees from ALK-Abelló, AstraZeneca, Antabio, GlaxoSmithKline, HAL Allergy, QPS-NL and Sanofi-Genzyme; and payment or honoraria from BMR, Boehringer Ingelheim, European Forum for Research and Education in Allergy and Airway Diseases (EUFOREA), Merck Sharp & Dohme and Sanofi-Genzyme, all in the 36 months prior to manuscript submission; and unpaid roles as the European Academy of Allergy and Clinical Immunology Asthma Section Chair (2017–2019) and EUFOREA Asthma Expert Panel Chair (since 2020)., (Copyright ©The authors 2022.)

Background: Type 2 inflammation underlies the chronicity of disease in subgroups of patients with asthma, chronic rhinosinusitis with nasal polyps (CRSwNP) and atopic dermatitis (AD), that often co-exist. Although several studies have investigated the unmet needs of asthma, AD and CRSwNP as such, little is known about the similarities and differences in experiences and perspectives of the current management of patients with comorbid Type 2 inflammatory diseases., Aims: To improve insight into the common and organ-specific needs of patients with Type 2 inflammation and comorbidities, allowing the formulation of recommendations to better address these needs in the future., Methodology: This qualitative study was conducted between July 2021 and December 2021 using semi-structured face-to-face or telephone interviews with patients suffering from year-long severe chronic Type 2 inflammation and at least one co-morbid inflammatory condition. Seven participating academic centers in Europe interviewed asthma (Copenhagen and Leuven), CRSwNP (London, Amsterdam and Crete) and/or AD (Oldenburg and Zurich) patients on patient characteristics, disease severity, shortcomings of current care pathways and suggestions for improvement of care. Transcripts were analyzed using an inductive thematic analysis approach., Results: Eighty-one patients with severe Type 2 inflammation and comorbidities were interviewed. Similar needs were recognized by patients with Type 2 inflammation, with both a lack of coordination in care and a lack of a real cure reported as being most frustrating. However, several needs are specific to asthma, CRSwNP and AD. Suggestions for improvement of care were generic across diseases, such as the implementation of a multidisciplinary approach, the improved facilitation of access to better treatments, the increase of general awareness on disease burden, and better educational programs for healthcare providers and patients. Of note, patients with CRSwNP also stated the need for alternatives to sinus surgery, whereas patients with asthma requested better medical care to prevent exacerbations and patients with AD would warmly welcome the reimbursement of emollients., Conclusion: Patients with asthma, CRSwNP and AD have shared unmet needs that need to be addressed by physicians, the academic community and health policy makers. This survey provides unique recommendations made by patients for the implementation of better care., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De Prins, Raap, Mueller, Schmid-Grendelmeier, Haase, Backer, Fokkens, Benoist, Prokopakis, Hopkins, Claeys, Teeling, Cypers, Cools, Bjermer, Diamant, Wahn, Scadding, Bachert, Patel, Van Staeyen and Hellings.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that spread around the world during the past 2 years, has infected more than 260 million people worldwide and has imposed an important burden on the healthcare system. Several risk factors associated with unfavorable outcome were identified, including elderly age, selected comorbidities, immune suppression as well as laboratory markers. The role of immune system in the pathophysiology of SARS-CoV-2 infection is indisputable: while an appropriate function of the immune system is important for a rapid clearance of the virus, progression to the severe and critical phases of the disease is related to an exaggerated immune response associated with a cytokine storm. We analyzed differences and longitudinal changes in selected immune parameters in 823 adult COVID-19 patients hospitalized in the Martin University Hospital, Martin, Slovakia. Examined parameters included the differential blood cell counts, various parameters of cellular and humoral immunity (serum concentration of immunoglobulins, C4 and C3), lymphocyte subsets (CD3 + , CD4 + , CD8 + , CD19 + , NK cells, CD4 + CD45RO + ), expression of activation (HLA-DR, CD38) and inhibition markers (CD159/NKG2A). Besides already known changes in the differential blood cell counts and basic lymphocyte subsets, we found significantly higher proportion of CD8 + CD38 + cells and significantly lower proportion of CD8 + NKG2A + and NK NKG2A + cells on admission in non-survivors, compared to survivors; recovery in survivors was associated with a significant increase in the expression of HLA-DR and with a significant decrease of the proportion of CD8 + CD38 + cells. Furthermore, patients with fatal outcome had significantly lower concentrations of C3 and IgM on admission. However, none of the examined parameters had sufficient sensitivity or specificity to be considered a biomarker of fatal outcome. Understanding the dynamic changes in immune profile of COVID-19 patients may help us to better understand the pathophysiology of the disease, potentially improve management of hospitalized patients and enable proper timing and selection of immunomodulator drugs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bobcakova, Barnova, Vysehradsky, Petriskova, Kocan, Diamant and Jesenak.)

Exercise induced bronchial (EIB) constriction is a common and highly specific feature of pediatric asthma and should be diagnosed with an exercise challenge test (ECT). The impact of EIB in asthmatic children's daily lives is immense, considering the effects on both physical and psychosocial development. Monitoring childhood asthma by ECT's can provide insight into daily life disease burden and the control of asthma. Current guidelines for bronchoprovocation tests restrict both the use of reliever and maintenance asthma medication before an exercise challenge to prevent false-negative testing, as both have significant acute bronchoprotective properties. However, restricting maintenance medication before an ECT may be less appropiate to evaluate EIB symptoms in daily life when a diagnosis of asthma is well established. Rigorous of maintenance medication before an ECT according to guidelines may lead to overestimation of the real, daily life asthma burden and lead to an inappropiate step-up in therapy. The protection against EIB offered by the combined acute and chronic bronchoprotective effects of maintenance medication can be properly assessed whilst maintaining them. This may aid in achieving the goal of unrestricted participation of children in daily play and sports activities with their peers without escalation of therapy. When considering a step down in medication, a strategic wash-out of maintenance medication before an ECT aids in providing objective support of potential discontinuation of maintenance medication., Competing Interests: In the past 3 years, ZD acted as Research Director at QPS-NL, an institution which received research support from several bio-pharmaceutical companies, esp within respiratory: HAL Allergy, Foresee Pharmaceuticals, Patara Pharma (now Respivant), Novartis. Furthermore, ZD received honoraria or speaker fees serving on advisory boards or as a consultant from: ALK, Antabio, AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, HAL Allergy, Merck Sharp & Dohme, Sanofi-Genzyme-Regeneron, all outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hengeveld, Keijzer, Diamant and Thio.)

Introduction: Clinical recommendations for childhood asthma are often based on data extrapolated from studies conducted in adults, despite significant differences in mechanisms and response to treatments. The Paediatric Asthma in Real Life (PeARL) Think Tank aspires to develop recommendations based on the best available evidence from studies in children. An overview of systematic reviews (SRs) on paediatric asthma maintenance management and an SR of treatments for acute asthma attacks in children, requiring an emergency presentation with/without hospital admission will be conducted., Methods and Analysis: Standard methodology recommended by Cochrane will be followed. Maintenance pharmacotherapy of childhood asthma will be evaluated in an overview of SRs published after 2005 and including clinical trials or real-life studies. For evaluating pharmacotherapy of acute asthma attacks leading to an emergency presentation with/without hospital admission, we opted to conduct de novo synthesis in the absence of adequate up-to-date published SRs. For the SR of acute asthma pharmacotherapy, we will consider eligible SRs, clinical trials or real-life studies without time restrictions. Our evidence updates will be based on broad searches of Pubmed/Medline and the Cochrane Library. We will use A MeaSurement Tool to Assess systematic Reviews, V.2, Cochrane risk of bias 2 and REal Life EVidence AssessmeNt Tool to evaluate the methodological quality of SRs, controlled clinical trials and real-life studies, respectively.Next, we will further assess interventions for acute severe asthma attacks with positive clinical results in meta-analyses. We will include both controlled clinical trials and observational studies and will assess their quality using the previously mentioned tools. We will employ random effect models for conducting meta-analyses, and Grading of Recommendations Assessment, Development and Evaluation methodology to assess certainty in the body of evidence., Ethics and Dissemination: Ethics approval is not required for SRs. Our findings will be published in peer reviewed journals and will inform clinical recommendations being developed by the PeARL Think Tank., Prospero Registration Numbers: CRD42020132990, CRD42020171624., Competing Interests: Competing interests: AGM reports grants from Boehringer Ingelheim outside the submitted work. AC reports personal fees from Novartis, Regeneron / Sanofi, Thermo Fisher Scientific, Boehringer Ingelheim and Philips, outside the submitted work. LB reports personal fees from Aerocrine, GlaxoSmithKline, Genentech/Novartis, Merck, DBV Technologies, Teva, Boehringer Ingelheim, AstraZeneca, WebMD/Medscape, Sanofi/Regeneron, Vectura and Circassia outside the submitted work. TC reports grants and personal fees CSL Behring, Dyax, Takeda, BioCryst, Pharming, personal fees from Grifols, grants and non-financial support from GSK, Regeneron, Novartis/Genetech outside the submitted work. AD reports grants and personal fees from Stallergenes Greer, personal fees from Novartis, ALK, TEVA, GSK, MEDA-MYLAN, CHIESI, AImmune, DBV technologies and Astra Zeneca, outside the submitted work. ZD reports personal fees from academic affiliations, ZD acts as Executive and Scientific Medical Director at a phase I/II pharmacological unit (QPS-NL), which performs clinical studies for pharmaceutical companies. ZD reports personal fees from Astrazeneca, ALK, Aquilon, Boehringer Ingelheim, CSL, HAL Allergy, MSD, and Sanofi-Genzyme outside the submitted work. FMD reports grants from Thorasys; personal fees from Jean-Coutu Pharmaceuticals, unrestricted research funds from Novartis Canada, Teva and Trudell Medical, research grants from GlaxoSmithKline and MEDteq in partnership with Thorasys; honorarium for consultancy work from Covis Pharma and Teva; and honorarium as invited speaker from Covis Pharma, Pharmacy Brunet, outside the submitted work. JEG reports grants from NIH/NIAID, personal fees from Regeneron, Ena Theraputics and MedImmune outside the submitted work; personal fees and stock options from Meissa Vaccines Inc outside the submitted work. JG reports personal fees from GSK, Vifor Pharmaceuticals, Novartis, BV Pharma and AstraZeneca outside the submitted work. AK reports personal fees Astra Zeneca, Behring, Boehringer Ingelheim, Covis, GSK, NovoNordisk, Novartis, Griffols, Pfizer, Sanofi, Teva and Trudel, outside the submitted work. RFL reports grants from NIH, non-financial support from GlaxoSmithKline, Boehringer-Ingelheim, Merck, TEVA, American Academy of Allergy, Asthma and Immunology, grants from Clinical and Translational Science Award (NIH), Childhood Origins of ASThma (COAST) grant, AsthmaNet, personal fees from LSU, Elsevier, UpToDate, the University of Kentucky, ThermoFischer, and Food Allergy Research and Education (FARE) Network, outside the submitted work. CM reports personal fees from Novartis, GSK, Astra Zeneca, Thermo Fisher and Boehringer Ingelheim outside the submitted work. NGP reports personal fees from ALK, Novartis, Nutricia, HAL, Menarini/FAES Farma, Sanofi, Mylan/MEDA, Biomay, AstraZeneca, GSK, MSD, ASIT BIOTECH and Boehringer Ingelheim; grants from Gerolymatos International SA and Capricare outside the submitted work. WP reports grants from NIH; grants and personal fees from Genentech/Novartis, Sanofi/Rgeneron; personal fees GSK; non-financial support from Thermo Fisher, Lincoln Diagnostics, Alk Abello, and Monaghen, outside the submitted work. PP reports grants from Astra Zeneca, Chiesi and TEVA; personal fees from Astra Zeneca, TEVA, Novartis, Mundipharma, S&D Pharma, and GlaxoSmithKline outside the submitted work. DP reports grants from AKL Research and Development, British Lung Foundation, Respiratory Effectiveness Group and the UK National Health Service; grants and personal fees from Boehringer Ingelheim, Chiesi, Circassia, Mylan, Mundipharma, Napp, Novartis, Pfizer, Regeneron Pharmaceuticals, Sanofi Genzyme, TEVA, Theravance and Zentiva (Sanofi Generics); personal fees from Cipla, GlaxoSmithKline, Kyorin and Merck; non-financial support from Efficacy and Mechanism Evaluation programme, Health Technology Assessment, outside the submitted work; DP also reports stock/stock options from AKL Research and Development which produces phytopharmaceuticals; and owns 74% of the social enterprise Optimum Patient Care (Australia and UK) and 74% of Observational and Pragmatic Research Institute (Singapore), outside the submitted work. GR reports personal fees from ALK, Allergen Therapeutics, Meda Plus, Merck; and a patent for the use of sublingual immunotherapy to prevent the development of allergy in at-risk infants, outside the submitted work. IT reports personal fees from Novartis, GSK, Boehringer Ingelheim and Astra Zeneca; grants from GSK Hellas, outside the submitted work. PX reports personal fees from Nutricia, Nestle, Friesland, Uriach, Novartis Pharma AG, and GlaxoSmithkline outside the submitted work., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Once-daily asthma treatment should prevent night-time deterioration, irrespective of the time of dosing. IND/GLY/MF, a fixed-dose combination of inhaled indacaterol acetate (IND, long-acting β 2 -agonist (LABA)), glycopyrronium bromide (GLY, long-acting muscarinic antagonist) and mometasone furoate (MF, inhaled corticosteroid (ICS)) delivered by Breezhaler, is indicated in adult asthma patients inadequately controlled on LABA/ICS. A randomised, double-blind, placebo-controlled, three-period, crossover, phase II study was performed to investigate the bronchodilator effect of IND/GLY/MF (150/50/80 μg) dosed morning and evening versus placebo in patients with mild-moderate asthma. The primary end-point was weighted mean forced expiratory volume in 1 s (FEV 1 ) over 24 h following 14 days of IND/GLY/MF dosed a.m. and p.m. versus placebo. Secondary end-points included the effect of dosing time on peak expiratory flow (PEF) and safety/tolerability. Of 37 randomised patients (age 18-72 years; 21 male, 16 female) 34 completed all three treatment periods. At screening, median (range) pre-bronchodilator FEV 1 was 75.8% (60-96%). Patients were using stable low- (83.8%) or medium-dose (16.2%) ICS. Morning and evening dosing of IND/GLY/MF improved FEV 1 (area under the curve from 0 to 24 h) by 610 mL (90% CI 538-681 mL) and 615 mL (90% CI 544-687 mL), respectively, versus placebo. Mean PEF over 14 days increased by 70.7 L·min -1 (90% CI 60.5-80.9 L·min -1 ) following a.m. dosing, and by 59.7 L·min -1 (90% CI 49.5-69.9 L·min -1 ) following p.m. dosing of IND/GLY/MF versus placebo. IND/GLY/MF demonstrated a safety profile comparable with placebo. Once-daily inhaled IND/GLY/MF was well tolerated and provided sustained lung function improvements over 24 h, irrespective of a.m. or p.m. dosing, in patients with mild-moderate asthma., Competing Interests: Conflict of interest: J. Beier reports personal fees from Novartis during the conduct of the study, and personal fees from AstraZeneca, Berlin Chemie/Menarini and Pohl Boskamp outside the submitted work. Conflict of interest: H. Watz reports grants, personal fees and nonfinancial support from Novartis during the conduct of the study; and grants, personal fees and nonfinancial support from AZ, Berlin Chemie/Menarini, GSK, Chiesi, Bayer and Takeda, outside the submitted work. Conflict of interest: Z. Diamant reports grants from Novartis during the conduct of the study, and personal fees from Acucort, AstraZeneca, ALK, Aquilon, Boehringer Ingelheim, CSL, HAL Allergy, MSD and Sanofi Genzyme outside the submitted work. Conflict of interest: J.M. Hohlfeld reports grants paid to his institution from Novartis during the conduct of the study; and consultancy fees from Boehringer Ingelheim, grants paid to his institution from AstraZeneca AB, Novartis, Janssen Pharmaceutica NV, ALK, Boehringer Ingelheim, LETI, GlaxoSmithKline and grants from Sanofi-Aventis, consultancy fees from Merck & Co, Inc., lecture fees from Novartis, grants paid to his institution from Astellas Pharma and Allergopharma, and lectures fees from HAL, outside the submitted work. Conflict of interest: D. Singh reports personal fees from AstraZeneca, Boehringer Ingelheim, Chiesi, Cipla, Genentech, GlaxoSmithKline, Glenmark, Gossamerbio, Menarini, Mundipharma, Novartis, Peptinnovate, Pfizer, Pulmatrix, Theravance and Verona, outside the submitted work. Conflict of interest: P. Pinot is an employee of Novartis. Conflict of interest: I. Jones is an employee of Novartis. Conflict of interest: H-C. Tillmann is an employee of Novartis., (Copyright ©ERS 2021.)

Spring and Summer 2020 are unique in that the challenges of care for those suffering from pollen allergy coincide with the COVID-19 pandemic. Several considerations are important to allow optimal care of allergic rhinitis (AR) and asthma and hence prevention of coronavirus spread through sneezing, rhinorrhoea, and coughing. This compact overview of recommendations by the EUFOREA expert teams on allergic airway diseases and allergen-specific immunotherapy (AIT) is based on investigation of the current COVID-19 literature in association with the key words above and shared clinical experience of the experts involved. It deals with similarities and differences between AR and coronavirus infection, specific recommendations for allergic disease care in the COVID-19 era, including guidance on AIT., (Crown Copyright © 2020 Published by Elsevier Inc. on behalf of World Allergy Organization.)

Despite the use of effective medications to control asthma, severe exacerbations in asthma are still a major health risk and require urgent action on the part of the patient and physician to prevent serious outcomes such as hospitalisation or death. Moreover, severe exacerbations are associated with substantial healthcare costs and psychological burden, including anxiety and fear for patients and their families. The European Academy of Allergy and Clinical Immunology (EAACI) and the European Respiratory Society (ERS) set up a task force to search for a clear definition of severe exacerbations, and to also define research questions and priorities. The statement includes comments from patients who were members of the task force., Competing Interests: Conflict of interest: A. Bourdin reports personal and institutional fees for advisory board work from AstraZeneca, Novartis, GSK, Boehringher Ingelheim, Chiesi, Actelion, Pfizer and Teva, outside the submitted work. Conflict of interest: L. Bjermer has nothing to disclose. Conflict of interest: C. Brightling reports grants and personal fees for consultancy from GlaxoSmithKline, AstraZeneca/Medimmune, Novartis, Chiesi, Roche/Genentech and Boehringer Inglheim, personal fees for consultancy from Vectura, Theravance, PreP, Gilead, Sanofi/Regeneron, Teva, Gossamer and 4DPharma, grants from Pfizer and Mologic, outside the submitted work. Conflict of interest: G.G. Brusselle reports personal fees for advisory board work and lectures from AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Novartis and Teva, personal fees for advisory board work from Sanofi, outside the submitted work. Conflict of interest: P. Chanez reports research grants and personal fees for consultancy, advisory board work and lectures from ALK, Almirall, Boehringer Ingelheim, GSK, AstraZeneca, Novartis, TEVA and Chiesi, grants from AMU, outside the submitted work. Conflict of interest: K.F. Chung has received honoraria for participating in advisory board meetings of GSK, AZ, Novartis, Merck, BI and TEVA regarding treatments for asthma and COPD, and has also been renumerated for speaking engagements. Conflict of interest: A. Custovic reports personal fees for consultancy from Novartis, Regeneron/Sanofi, Boehringer Ingelheim and Philips, personal fees for lectures from Thermo Fisher Scientific and Novartis, outside the submitted work. Conflict of interest: Z. Diamant reports personal fees from AstraZeneca and Sanofi-Genzyme, during the conduct of the study; personal fees from Aquilon, ALK, Boehringer Ingelheim, Gilead, Hal Allergy and MSD, outside the submitted work; and in addiction to academic affiliations, also works at a phase I/II unit performing clinical studies for different biotech and pharma companies. Conflict of interest: S. Diver has nothing to disclose. Conflict of interest: R. Djukanovic reports receiving fees for lectures at symposia organised by Novartis, AstraZeneca and TEVA, consultation for TEVA and Novartis as member of advisory boards, and participation in a scientific discussion about asthma organised by GlaxoSmithKline; in addition, is a co-founder and current consultant, and has shares in, Synairgen, a University of Southampton spin out company. Conflict of interest: D. Hamerlijnck has nothing to disclose. Conflict of interest: I. Horvath reports personal fees from AstraZeneca, Berlin-Chemie, Boehringer Ingelheim, Chiesi, GSK, Novartis, CSL-Behring and Roche, outside the submitted work. Conflict of interest: S.L. Johnston reports personal fees for advisory board work from Therapeutic Frontiers and Virtus Respiratory Research, personal fees for consultancy from Myelo Therapeutics GmbH, Concert Pharmaceuticals, Bayer, Gerson Lehrman Group, resTORbio, Bioforce, Materia Medical Holdings, PrepBio Pharma, Pulmotect, Virion Health and Lallemand Pharma, personal and insititutional fees for consultancy from Synairgen, Novartis, Boehringer Ingelheim and Chiesi; and has received personal fees for the following patents planned, issued or pending: transgenic animal models of HRV with human ICAM-1 sequences (UK patent application number 02 167 29.4, and international patent application number PCT/EP2003/007939); anti-virus therapy for respiratory diseases (UK patent application number GB 0405634.7); interferon-beta for anti-virus therapy for respiratory diseases (international patent application number PCT/GB05/50031); interferon lambda therapy for the treatment of respiratory disease (UK patent application number 6779645.9, granted); induction of cross-reactive cellular response against rhinovirus antigens (European patent number 13305152), outside the submitted work. Conflict of interest: F. Kanniess reports personal fees for lectures and advisory board work from AstraZeneca, Novartis, Mundipharma and TEVA, outside the submitted work. Conflict of interest: N. Papadopoulos reports personal fees for advisory board work and lectures from Novartis, Nutricia, HAL, personal fees from Menarini/Faes Farma and Mylan/Meda, personal fees for lectures from Sanofi, Biomay, MSD, ASIT Biotech and Boehringer Ingelheim, personal fees for advisory board work from AstraZeneca and GSK, grants from Gerolymatos International SA and Capricare, outside the submitted work. Conflict of interest: A. Papi reports grants, personal fees for lectures, advisory board work and consultancy, and travel expenses reimbursement from AstraZeneca, Boehringer Ingelheim, Chiesi Farmaceutici, GlaxoSmithKline and Teva, personal fees for advisory board work and consultancy from Sanofi/Regeneron, personal fees for lectures and travel expenses reimbursement from Zambon and Novartis, personal fees for lectures, advisory board work and consultancy, and travel expenses reimbursement from Mundipharma, personal fees for lectures and advisory board work, and travel expenses reimbursement Almirall, grants, personal fees for lectures and travel expenses reimbursement from Menarini, grants from Fondazione Maugeri, grants from Fondazione Chiesi Farmaceutici, outside the submitted work. Conflict of interest: R.J. Russell has nothing to disclose. Conflict of interest: D. Ryan reports personal fees for advisory board work from GSK and Trudell Medical, personal fees for advisory board work and lectures from AZ, personal fees for lectures from Mylan and Chiesi, personal fees for consultancy from Optimum Patient Care, outside the submitted work. Conflict of interest: K. Samitas has nothing to disclose. Conflict of interest: T. Tonia acts as ERS methodologist. Conflict of interest: E. Zervas reports personal fees consultancy and lectures from Astra, Bristol-Myers Squibb, Chiesi, GSK, Elpen, Merck, MSD, Novartis, Menarini and Pfizer, non-financial support for travel, accommodation and meeting expenses from Astra, Bristol-Myers Squibb, Galenica, Chiesi, Elpen, Novartis, Menarini and Roche, outside the submitted work. Conflict of interest: M. Gaga reports grants and personal fees from AZ, grants from BI, Elpen, Novartis and Menarini, personal fees from BMS, MSD, Chiesi and Pharmaten, outside the submitted work., (Copyright ©ERS 2019.)

Recently, this international task force reported the general considerations for bronchial challenge testing and the performance of the methacholine challenge test, a "direct" airway challenge test. Here, the task force provides an updated description of the pathophysiology and the methods to conduct indirect challenge tests. Because indirect challenge tests trigger airway narrowing through the activation of endogenous pathways that are involved in asthma, indirect challenge tests tend to be specific for asthma and reveal much about the biology of asthma, but may be less sensitive than direct tests for the detection of airway hyperresponsiveness. We provide recommendations for the conduct and interpretation of hyperpnoea challenge tests such as dry air exercise challenge and eucapnic voluntary hyperpnoea that provide a single strong stimulus for airway narrowing. This technical standard expands the recommendations to additional indirect tests such as hypertonic saline, mannitol and adenosine challenge that are incremental tests, but still retain characteristics of other indirect challenges. Assessment of airway hyperresponsiveness, with direct and indirect tests, are valuable tools to understand and to monitor airway function and to characterise the underlying asthma phenotype to guide therapy. The tests should be interpreted within the context of the clinical features of asthma., Competing Interests: Conflict of interest: T.S. Hallstrand has nothing to disclose. Conflict of interest: J.D. Leuppi has nothing to disclose. Conflict of interest: G. Joos reports grants and personal fees for advisory board work and lecturing from AstraZeneca and Novartis, grants from Boehringer Ingelheim and Chiesi, grants and personal fees for advisory board work from GlaxoSmithKline, and personal fees for lecturing from Teva, outside the submitted work. Conflict of interest: G.L. Hall has nothing to disclose. Conflict of interest: K.H. Carlsen has nothing to disclose. Conflict of interest: D.A. Kaminsky has received honoraria for teaching at an annual Cardiorespiratory Diagnostics Course from MGC Diagnostics, Inc., outside the submitted work. Conflict of interest: A.L. Coates has nothing to disclose. Conflict of interest: D.W. Cockcroft is a member of a medical advisory board for Methapharm, as has received products for research purposes from Aerogen and Pharmaxis, outside the submitted work. Conflict of interest: B.H. Culver has nothing to disclose. Conflict of interest: Z. Diamant reports personal fees from Aerocrine, ALK, Aquilon, AstraZeneca, Boehringer Ingelheim, Gilead, HAL Allergy, MSD and Sanofi Genzyme-Regeneron, outside the submitted work. Conflict of interest: G.M. Gauvreau has nothing to disclose. Conflict of interest: I. Horvath reports personal fees and non-financial support from AstraZeneca, Berlin-Chemie, Chiesi, Boehringer Ingelheim, Novartis, CSL Behring and Roche, personal fees from Sandoz, GSK, Sager Pharma, Affidea, Orion Pharma and Teva, and non-financial support from MSD, outside the submitted work. Conflict of interest: F.H.C. de Jongh has nothing to disclose. Conflict of interest: B.L. Laube has nothing to disclose. Conflict of interest: P.J. Sterk has nothing to disclose. Conflict of interest: J. Wanger has nothing to disclose., (Copyright ©ERS 2018.)

This international task force report updates general considerations for bronchial challenge testing and the performance of the methacholine challenge test. There are notable changes from prior recommendations in order to accommodate newer delivery devices. Rather than basing the test result upon a methacholine concentration (provocative concentration (PC 20 ) causing a 20% fall in forced expiratory volume in 1 s (FEV 1 )), the new recommendations base the result upon the delivered dose of methacholine causing a 20% fall in FEV 1 (provocative dose (PD 20 )). This end-point allows comparable results from different devices or protocols, thus any suitable nebuliser or dosimeter may be used, so long as the delivery characteristics are known. Inhalation may be by tidal breathing using a breath-actuated or continuous nebuliser for 1 min (or more), or by a dosimeter with a suitable breath count. Tests requiring maximal inhalations to total lung capacity are not recommended because the bronchoprotective effect of a deep breath reduces the sensitivity of the test., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

Background: Inhaled allergen challenge is a validated disease model of allergic asthma offering useful pharmacodynamic assessment of pharmacotherapeutic effects in a limited number of subjects., Objectives: To evaluate whether an RNA signature can be identified from induced sputum following an inhaled allergen challenge, whether a RNA signature could be modulated by limited doses of inhaled fluticasone, and whether these gene expression profiles would correlate with the clinical endpoints measured in this study., Methods: Thirteen non-smoking, allergic subjects with mild-to-moderate asthma participated in a randomised, placebo-controlled, 2-period cross-over study following a single-blind placebo run-in period. Each period consisted of three consecutive days, separated by a wash-out period of at least 3 weeks. Subjects randomly received inhaled fluticasone ((FP) MDI; 500 mcg BID×5 doses in total) or placebo. On day 2, house dust mite extract was inhaled and airway response was measured by FEV1 at predefined time points until 7 h post-allergen. Sputum was induced by NaCl 4.5%, processed and analysed at 24 h pre-allergen and 7 and 24 h post-allergen. RNA was isolated from eligible sputum cell pellets (<80% squamous of 500 cells), amplified according to NuGEN technology, and profiled on Affymetrix arrays. Gene expression changes from baseline and fluticasone treatment effects were evaluated using a mixed effects ANCOVA model at 7 and at 24 h post-allergen challenge., Results: Inhaled allergen-induced statistically significant gene expression changes in sputum, which were effectively blunted by fluticasone (adjusted p<0.025). Forty-seven RNA signatures were selected from these responses for correlation analyses and further validation. This included Th2 mRNA levels for cytokines, chemokines, high-affinity IgE receptor FCER1A, histamine receptor HRH4, and enzymes and receptors in the arachidonic pathway. Individual messengers from the 47 RNA signatures correlated significantly with lung function and sputum eosinophil counts., Conclusion: Our RNA extraction and profiling protocols allowed reproducible assessments of inflammatory signatures in sputum including quantification of drug effects on this response in allergic asthmatics. This approach offers novel possibilities for the development of pharmacodynamic (PD) biomarkers in asthma.

Fractional exhaled nitric oxide (FeNO) is a convenient, non-invasive method for the assessment of active, mainly Th2-driven, airway inflammation, which is sensitive to treatment with standard anti-inflammatory therapy. Consequently, FeNO serves as a valued tool to aid diagnosis and monitoring in several asthma phenotypes. More recently, FeNO has been evaluated in several other respiratory, infectious, and/or immunological conditions. In this short review, we provide an overview of several clinical studies and discuss the status of potential applications of NO measurements in clinical conditions beyond asthma.

Background: Allergen-induced late airway response offers important pharmacodynamic targets, including T helper 2 (TH2) biomarkers. However, detection of inflammatory markers has been limited in dithiothreitol-processed sputum., Objectives: To test whether allergen-induced TH2 inflammatory markers can be reproducibly quantified by sensitive detection techniques in ultracentrifuged sputum and the effect of fluticasone (FP) on these endpoints., Methods: Thirteen allergic asthmatics with dual allergen-induced airway responses, documented during a single-blind placebo run-in period, participated in a double-blind, two-period crossover study. Each period consisted of three consecutive days, separated by ≥3 weeks. Following randomization, subjects inhaled FP (500 µg bid, five doses total) or placebo. On Day 2 in each study period, allergen challenge was performed and airway response measured by forced expiratory volume in 1 sec (FEV1) until 7 h post-challenge. Sputum was induced 24 h pre-allergen and 7 and 24 h post-allergen. Sputum samples were split into two portions: TH2 biomarkers were quantified by Meso Scale multiplex platform following ultracentrifugation, and cell differentials were counted on Giemsa-May-Grünwald-stained cytospins. Allergen-induced changes in inflammatory endpoints were compared between FP and placebo using a mixed model ANCOVA., Results: Inhaled allergen induced dual airway responses in all subjects during both placebo periods with reproducible late asthmatic response (LAR) and increased sputum inflammatory biomarkers (IL-2, IL-4, IL-13, and eotaxin-1) and eosinophil counts. FP effectively blunted both the LAR and the inflammatory biomarkers., Conclusions: Combining novel, sensitive quantification methods with ultracentrifugation allows reproducible quantification of sputum biomarkers following allergen challenge, reversed by FP. This approach allows non-invasive identification of pharmacodynamic targets for anti-asthma therapies.

Good clinical practice (GCP) guidelines should always be implemented and obeyed in clinical interventional studies. In this mini-review, we will address several burning questions relating to GCP in a concise 'frequently asked questions' format. While compliance to current rules and regulations is our mission, we also wish to play devil's advocate attempting to translate the rules into sizeable chunks using a high dose of common sense.

Background: The current maintenance dose (10,000 AUeq/monthly) of a subcutaneous allergoid for house dust mite (HDM) immunotherapy has previously shown significant clinical efficacy in patients with HDM induced allergic rhinitis or rhinoconjunctivitis. In order to comply with the 2009 EMA guidelines on immunotherapy products, a study was conducted to evaluate the safety, tolerability and short-term treatment effects of up-dosing regimens with high doses (up to 40,000 AUeq) of allergoid HDM immunotherapy., Methods: In total 48 patients with HDM-allergic rhinitis or rhinoconjunctivitis (29 M/19 F; 18-53 years) were included and enrolled into one of three up-dosing regimens (1:4:4): 1) a regular regimen with up-dosing to 40,000 AUeq followed by two maintenance doses (total duration 17 weeks), 2) an intermediate regimen (14 weeks) or 3) a fast regimen (11 weeks). Safety and tolerability were evaluated by monitoring of early and late local reactions and systemic reactions. In addition, short-term effects were assessed by conjunctival provocation test (CPT) and levels of serum allergen-specific IgE, IgG and IgG4., Results: Thirty-nine patients completed the study according to protocol. No early local reactions occurred. Late local reactions (LLR) were observed in 12% of the injections. In total, 31 systemic reactions, all grade 1, were reported of which two needed oral antihistamine treatment. No grade 2 or higher systemic reactions were observed. Six patients (15%) did not reach the highest dose due to LLR and/or systemic reactions needing antihistamines (20% in the regular regimen, 16% in the intermediate regimen and 13% in the fast regimen). At the end of the study, an improvement in the CPT was observed in 82.1% of patients, indirectly indicating an early treatment effect at the current dose and higher doses. In addition, IgG4 immunoglobulin levels were significantly increased in all groups following treatment., Conclusions: In this open-label study, allergoid HDM immunotherapy in doses up to 40,000 AUeq was generally well tolerated and no clinically relevant safety issues were identified. In the safety aspects of the three up-dosing regimens no clinically relevant differences were encountered. Therefore, these dose ranges and up-dosing regimens can be safely included in future dose-finding efficacy studies.