Abstracts from the 3rd International Severe Asthma Forum (ISAF)

s from the 3rd International Severe Asthma Forum (ISAF) Manchester, United Kingdom. 17 November 2016–19 November 2016 © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. ORAL ABSTRACT SESSION 1—Asthma: from mechanisms to management O01 Serum IL‐1RL1‐A levels predict an eosinophilic subtype of asthma in preschool wheezing children M. E. Ketelaar, K. Van De Kant, F. N. Dijk, E. M. M. Klaassen, N. Grotenboer, M. C. Nawijn, E. Dompeling, G. H. Koppelman University Medical Center Groningen, Beatrix Children’s Hospital, Groningen Research Institute for Asthma and COPD (GRIAC), Groningen, The Netherlands; Department of Pediatric Pulmonology, School for Public Health and Primary Care (CAPHRI), Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands; Department of General Practice, School for Public Health and Primary Care (CAPHRI), Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands; University Medical Center Groningen, Department of Pathology and Medical Biology, Laboratory of Experimental Pulmonology and Inflammation Research (EXPIRE), Groningen Research Institute for Asthma and COPD (GRIAC), Groningen, The Netherlands Correspondence: Maria Elizabeth Ketelaar m.e.ketelaar@student.rug.nl Clinical and Translational Allergy 2017, 7(Suppl 2):O0

Introduction: Respiratory symptoms are common in preschool children. However, which of these wheezers will develop asthma at school age, and what phenotype they will develop remains difficult to predict. Current models such as the asthma prediction index (API) are based on clinical parameters and have only modest predictive accuracy. Expression levels of well replicated asthma genes could potentially form novel biomarkers for asthma prediction. IL1RL1 is an asthma susceptibility gene, and has also been linked to eosinophilia. Therefore, we hypothesized that expression levels of IL1RL1 in the form of soluble IL-1RL1-a measured in serum from wheezing preschool children contribute to the prediction of asthma at school age. Moreover, since IL1RL1 was previously associated with blood eosinophilia, our second aim was to determine whether serum IL-1RL1-a levels predict eosinophilic asthma. Method: We used logistic predictive modeling in a prospective Dutch birth cohort (n = 202 wheezers), and calculated the area under the curve (AUC) of the sensitivity/1-specificity curves of potential models. Results: Neither IL-1RL1-a serum levels at age 2-3 years alone nor its combination with the API had predictive value for doctors' diagnosed asthma at age 6y (IL-1RL1-a alone: AUC = 0.50 [95 CI 0.41-0.59, P = 0.98], API + IL-1RL1-a: AUC = 0.57 [95 CI 0.49-0.66, P = 0.12]). However, IL-1RL1-a serum levels at age 2-3 years correlated with the severity of airway eosinophilia (determined by levels of exhaled fraction of NO, [FeNO]) in children who had developed asthma at age 6y (Pearson's R = −0.24, P = 0.046, N = 59). Logistic predictive modeling of eosinophilic asthma at age 6y (asthma with FeNO ≥ 20 ppb) showed that IL-1RL1-a serum levels itself and in combination with the API could predict this eosinophilic subphenotype of asthma Conclusion: Our study shows that serum IL-1RL1-a levels measured in wheezing children at age 2-3 years do not predict doctors' diagnosed asthma as general phenotype at age 6 years, but negatively predict an eosinophilic subphenotype of asthma. This suggests that IL-1RL1 might play a protective role in the development of eosinophilia in children who experience asthma at school age and implies that IL-1RL1 targeted therapy could rather be further explored in the subphenotype of asthmatic children with predominant eosinophilic inflammation. Keywords: Childhood Asthma, Eosinophilic Asthma, Prediction, IL-1RL1, Serum Introduction: We have previously demonstrated significant molecular changes in the blood between mild allergic asthmatic individuals who develop isolated early responses (early responders, ERs) compared to those who also develop late-phase responses (dual responders, DRs) after allergen inhalation challenge. Identifying individuals likely to develop dual responses may aid in the screening of subjects for clinical trials that test drugs for the attenuation of the late-phase asthmatic response, which shares hallmark features of chronic disease. The objective of this study was to develop blood-based biomarker panels that could identify asthmatic individuals with high probability of developing a late-phase response. Method: The discovery cohort consisted of 36 mild asthmatic subjects (15 ERs and 21 DRs) and a validation cohort consisted of 45 mild asthmatic subjects (9 ERs and 36 DRs). Blood samples were collected prior to allergen challenge. Following RNA extraction, total RNA (globin-/ ribo-depleted) was sequenced using an Illumina HiSeq 2000 as 100 base paired-end reads. Both genome-guided datasets (UCSC genes, UCSC gene-isoforms, and Ensembl) and de novo assembled transcripts using the Trinity software were constructed. Top-ranked biomarker candidates were transferred to the high-precision and clinicallyapproved NanoString nCounter platform. Final biomarker panels were identified, and statistical algorithms were locked down prior to testing in the validated cohort. Results: Predictive biomarker panels had classification performance (based on the area under the receiver operating curve, AUC) ranging between 60 and 70% in the discovery cohort. 87 transcripts identified on the RNA-Seq platform were transferred to NanoString Elements assay chemistry. The transcripts were split with respect to their dataset of origin and tested in the validation cohort. The UCSC gene-isoforms and Trinity biomarker panels had AUCs of 68% and 72%, respectively. The biomarker transcripts were enriched for biological pathways such as NF-ĸB signaling (up-regulated in DRs) and apoptosis, decoy receptors and formyl peptide receptors (down-regulated in DRs). Conclusion: RNA transcript biomarker panels in the blood were successful at predicting asthmatic subjects likely to develop dual responses upon allergen inhalation challenge. Pathways and networks represented by these biomarker panels may reveal new avenues for therapeutic targeting of the pathobiology involved in chronic asthma. Keywords: Predictive Biomarkers, RNA-Seq, NanoString Elements, Statistical Algorithms Introduction: Asthma is an inflammatory disease with a strong circadian signature. Symptoms of asthma are worse overnight and in the early hours of the morning. Measurements of peak expiratory flow rate and forced expiratory volume in 1 s, are lower in early morning compared to afternoon. It is likely that circadian biology plays a role in the pathogenesis of asthma. A better understanding of the circadian nature of asthma may lead to better treatments through chronotherapy (taking medication at the most beneficial time of day). Clock genes control circadian rhythms within every cell in the body. The clock gene, REV-ERB alpha (NR1D1), connects the 'core' clock and the immune system. We employed a REV-ERB alpha global knockout mouse (rev-erb alpha −/− , these mice retain circadian rhythmicity, but show loss of temporal variation of innate immunity) to investigate the effect of REV-ERB alpha on airway hyper-responsiveness (AHR). AHR is a key pathophysiological feature of asthma. Method: All experimental procedures were carried out in accordance with the Animals (Scientific Procedures) Act,1986. Rev-erb alpha −/− mice were provided by Ueli Schibler (University of Geneva).
Measurements of dynamic resistance were performed using a Flexivent system (Scireq, Montreal, Canada). After induction of anaesthesia with an intrperitoneal injection of 'Hypnorm' (0.315 mg fentanyl; 10 mg fluanisone) and midazolam (5 mg) in water, at a dose of 0.1 ml/10 g, mice were tracheostomized and connected to the flexivent ventilator via an 18-guage needle. Mice were ventilated. Changes in resistance were measured in response to increasing concentrations of nebulized methacholine from 0 to 300 mg/ml. All measurements were made in the morning. Results: Rev-erb alpha −/− mice exhibit significantly (p < 0.01) greater AHR to increasing concentrations of methacholine compared to their wildtype and heterozygote litter-mate controls ( Figure 1). Conclusion: Rev-erb alpha −/− mice retain circadian rhythmicity yet demonstrate increased AHR to methacholine (as is seen in human asthma). REV-ERB alpha may play an important role in the pathogenesis of asthma. Next steps: Introduction: In a previous study (submitted to publish) we defined specific genes related with asthma and allergic diseases, by studying the gene-expression of 94 genes in a population composed by 4 groups of subjects: healthy control, nonallergic asthmatic, asthmatic allergic and nonasthmatic allergic patients. The analysis of differential gene-expression between control and patients with nonallergic asthma revealed a set of statistically relevant genes mainly associated with the disease's severity: IL10, MSR1, PHLDA1, SERPINB2, CHI3L1, IL8, and PI3. In this project we analyzed if methylation takes part in the regulation of the gene-expression of these potential asthma biomarkers and their correlation with protein expression and functional parameters (%FVE1, %FVC and %PBD).

Figure 3 Airway Hypersensitivity to Methacholine
Introduction: Inducible nitric oxide synthase (iNOS) and arginase-2 (ARG2) share a common substrate arginine. We recently showed that both enzymes are highly expressed in asthmatic airway epithelial cells where ARG2 plays a critical role in the regulation of mitochondrial bioenergetic driven Th2 response, as shown by the development of severe eosinophilic airway inflammation in mice deficient for ARG-2 (ARG-2 KO). The role of iNOS and ARG2 in a combined Th2 and Th17 response is unknown. Here we hypothesize that Th17 airway inflammation is also regulated by ARG2 driven metabolism. Method: Commercially available Wildtype (WT) and iNOS knockout (KO) were used. ARG2 KO was donated to us and ARG2iNOS double KO (dKO) strain was generated in house. Standard house dust mite extract (HDME) mouse model of Th2/Th17 asthma endotype was used. Airway inflammation was quantified by analysis of inflammatory cells in bronchoalveolar lavage fluid (BALF). Airway angiogenic remodeling was measured by quantification of lung microvessel density on tissue sections stained for the endothelial marker von Willebrand Factor. Epithelial and immune cell cytokines were measured by ELISA or flow cytometric multiplex assay. Results: HDME induced airway inflammation in all genotypes, but was the highest in ARG2 KO as showed by increased number inflammatory cells in the BALF. Both eosinophilic (Th2) and neutrophilic (Th17) inflammation peaked in ARG2 KO mice but were attenuated in ARG2iNOS dKO animals. Airway inflammation was similar among HDME exposed WT, iNOS KO and ARG2iNOS dKO mice. Angiogenic airway remodeling was also highest in ARG2 KO mice and comparable among WT, iNOS KO and ARG2iNOS dKO genotypes. Airway epithelial or immune cell-derived Th2 cytokines IL-5 and eotaxin-2 and, Th17 cytokine IL-17 were maximal in lungs of HDME exposed ARG2 KO mice, but decreased in ARG2iNOS dKO. Conclusion: The data show that ARG2 deficiency induces a severe combined Th2 and Th17 airway inflammation. Deletion of iNOS in addition to ARG2 inhibited disease severity, but iNOS deletion alone, had no effect, indicating that ARG2 is the most critical of the two arginine consuming enzymes in the regulation of inflammation and angiogenesis in asthma. Overall, the results expand our previous findings by demonstrating that in addition to Th2, Th17 endotype of airway inflammation is also critically regulated by arginine metabolism Keywords: Arginine, Metabolism, Th2, Th17, Mitochondria

O09 Hyaluronan-induced lipid mediators influence antiviral and antibacterial immunity in severe asthmatics
Milena Sokolowska 1 , Li-Yuan Chen 2 , Yueqin Liu 2 , Asuncion Martinez-Anton 2 , Carolea Logun 2 , Sara Alsaaty 2 , Rosemarie Cuento 3 , Rongman Cai 2 , Junfeng Sun 2 , Oswald Quehenberger 4 , Aaron Armando 5 , Edward Dennis 5 , Stewart Levine 3 , James Shelhamer 2 1 Critical Care Medicine Department, Clinical Center, NIH; Swiss Institute of Allergy and Asthma Research, University of Zurich; CK-CARE, Davos, Introduction: Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix involved in the pathogenesis of asthma and other chronic inflammatory diseases. During inflammation in asthma there is an increased breakdown of HA, resulting in the local and systemic accumulation of low molecular weight (LMW) HA. Eicosanoids, derived from cytosolic phospholipase A 2 group IVA (cPLA 2 alpha) activation, are potent lipid mediators also attributed to acute and chronic inflammation. Method: We investigated the effect of LMW HA on the lipidomic profile and global gene expression and their functional interactions in peripheral blood mononuclear cells of patients with mild-to-moderate (n = 7) and severe asthma (n = 6) as compared to controls (n = 6). Results: We found that LMW HA increased production of 68 unique lipid species, among which PGE 2 , PGB 2 , PGD 2 , 15-HETE, TxB 2 , 11(12)-EET, 14(15)-EET, 13-HOTrE(y) and 16(17)-EpDPE were significantly upregulated only in severe asthmatics. We also performed a genomewide expression analysis of LMW HA signaling, confirming its highly immunostimulatory potential. However, in severe asthmatics the LMW HA-induced global gene expression profile showed a comprehensive impairment in interferon signaling, cell apoptosis and cell movement, leading to diminished antiviral and antibacterial responses. We confirmed these findings at the protein level, finding that LMW HAinduced production of IL-12 p40, CXCL10, CXCL11 and CCL8 in severe asthmatics was markedly reduced. Importantly, upon cPLA 2 alpha inhibition, there was a significant decrease in lipid mediator production accompanied by a significant increase in IL-12 p40 and CXCL9 protein expressions in each phenotype.

Conclusion:
In summary, we demonstrated here that fragmented hyaluronan increased production of several unique lipid species in severe asthmatics. Moreover, through analysis of the whole genome profile, captured simultaneously with the lipidomic profile, we observed decreases in antiviral gene and protein expression upon LMW HA treatment in severe asthmatics, which was partially reversed upon inhibition of lipid production. Therefore, our current findings provide new evidence on the connection between extracellular matrix, global lipid mediator production and decreased antiviral responses in severe asthma. Keywords: Severe Asthma, Eicosanoid, Virus, Lipidomics, Transcriptomics  Introduction: Asthma is a complex heterogeneous disease that likely comprises several distinct disease phenotypes. Usually atopy presence or any allergic diseases could interfere and obstacle the control of asthma. Clustering approach has been used to classify heterogeneous asthma population into distinct phenotypes. Method: For a period of 1 year we evaluated medical history data of 110 children with asthma aged 3 to 17 years. For all children we performed pulmonary function tests, nasal smears for eosinophil counts, drew blood for IgE against inhalation and food allergies antibodies detection and ACQ. IgE were detected with the predesigned kit Euro-linePediatric and total IgE with Immunocap. Differences in clinical indices including ΔFEV1(pre-and post-bronchodilator) and nasal and blood biomarkers at enrollment among clusters were analyzed. Results: Five distinct phenotypes were determined. Cluster 1 (n = 13): (non-atopic) the lowest IgE level, very low ACQ, median age of diagnosis and IgE levels. Cluster 2 (n = 45): (mixed) the highest BMI with the latest age of diagnosis and high ACQ and BDR levels. Cluster 3 (n = 33) (atopic) early diagnosis, highest BDR, highest ACQ score, highest total and specific IgE levels among the clusters. Cluster 4 (n = 12): (atopic) the highest Immunocap result, relatedly high BMI and IgE with median ACQ score among clusters. Cluster 5 (n = 7): (non-atopic) the earliest age for diagnosis, with the lowest BMI, the lowest ACQ score, and Immunocap result, with high BDR and median level of IgE among clusters.
The patients with severe asthma were over 65% of the patients in cluster 3. Only one severe asthma patient was in cluster 2, and none in the other three clusters. Introduction: Pulmonary function testing (PFT) is of great importance in the evaluation of lung function. Spirometry is simple, noninvasive, and has been the most commonly used technique in children. Scoliosis is the most common abnormality of the spine with direct effects on the thoracic cage. Scoliosis has generally been associated with the development of restrictive lung disease. Objectives: To evaluate the PFT data of children with scoliosis and to compare them to children with asthma. Method: After obtaining signed informed consent from the parents we performed PFT in 50 children aged 5 to 17 years. The children were divided into four groups -10 children with scoliosis without asthma (Sc),10 children with scoliosis and asthma (AS), 10 children with moderate asthma (BA), 10 children with severe asthma (SA) and 10 healthy children (HC Introduction: For consideration and discussion of data obtained from a survey on asthma, two official statistical reports are available in Japan. The fundamental data in these reports are statistically analyzed and compared to clarify the tendency of asthma in Japan.

Method:
One is annual report of school health statistics (SHS) from Ministry of Education, Culture, Sports, Science and Technology which has competence to survey the school age population including diseases and related health problems. The incidence of asthma of SHS from 1995 to 2015 in 5 to 17 years old are used in this report. The other is the patient survey by Ministry of health, Labour and Welfare. The patient survey covers all generations and diseases but the survey carried on every third year and numbers of patients are summarized by 5 years old. Numbers of asthma case from 1996 to 2014 are extracted and adjusted to the corresponding age population as per 1,000 capita in this report. Results: According to SHS, the incidence of asthma was most frequent in 6 years old and statistically significant positive correlations were observed between the incidence and a lapse of years in all ages. Average slope of regression lines in the 5 to 9 years old was 0.10 in female and 0.16 in male. This analysis means that the incidence may be keep increase over 0.1% in annual in the near future. From the patient survey, asthma cases were most numerous in the 0 to 4 years old group and the 5 to 9 years old group was next. Asthma cases increased in elderly and the level of the 70 to 74 years old group was almost same level of the 10 to 14 years old group. Contrary to SHS, statistically significant negative correlations were observed between the asthma cases and a lapse of years in the age groups, 15 to 19 (p < 0.01), 20 to 24 (p < 0.05) and 25 to 29 (p < 0.05) years old in male. The analysis of the patient survey suggests that the asthma has decreased in these two decades. Conclusion: Contradictory results in younger generation are obtained by the analysis of the two official statistical reports. The patient survey is based on actual number of persons had a medical examination in medical facilities, on the other hand, SHS is carried out using the questionnaire. The data of SHS is seemed to be influenced by the anamnesis. Especially in lower age children, SHS data are also possibly influenced by the levels of interests and anxieties for asthma in their protectors. Keywords: Annual Report Of School Health Statistics, Patient Survey, Statistical Analysis Clin Transl Allergy 2017, 7(Suppl 2):14 Introduction: Patients with severe asthma experience unpredictable daily symptoms and an intense treatment regimen that can impact upon physical, emotional well-being and quality of life. Sexual function is a basic human requirement, contributing to quality of life; yet there is a dearth of research exploring relationships and intimacy in people with severe asthma. The overall aim was to explore the impact of severe asthma on patient's experiences of intimacy and relationships, establish their information needs and the role of the healthcare professional.
Method: A qualitative study using semi-structured interviews with patients receiving treatment for severe asthma at a dedicated severe asthma clinic was undertaken. Interviews were audio recorded and transcribed verbatim. Transcripts were verified then analysed for emergent themes which were then categorised into super-ordinate themes and sub themes. Results: All nine participants had a proven diagnosis of severe asthma. A higher proportion were female n = 6 (66.6%) compared to male n = 3(33.3%). The mean age was 45 years, range 34-59 years.
The majority of participants 8(88.9%) were married, with 1(11.1%) co-habiting. The analysis of interviews provided complex, detailed and novel insights into patients' perspectives on how living with severe asthma impacts upon intimacy and spousal relationships. Four super-ordinate themes emerged: (i) 'Physical intimacy' including disclosure of physical limitations of severe asthma upon intimacy and sexual activity. (ii) 'Emotional intimacy' the cyclical impact of the often negative emotional struggle of living with severe asthma upon both relationships and intimacy. (iii) 'Image of self ' as participants divulged their battle with body image and confusion in changing relationship roles. (iv) 'The role of the healthcare professional' patient identified information requirements within this area.

Conclusion:
The participants in this study have provided invaluable and novel insights into the implications of living with severe asthma upon a sensitive and rarely discussed topic. It is anticipated that these findings will serve to increase understanding and assist practitioners to help patients to adopt positive strategies to improve their quality of life within this area. Introduction: The use of unsupervised clustering has identified different subtypes of asthma. Choosing the variables to input into the clustering algorithm is one of the important considerations. The majority of previous studies selected variables based on expert advice, whilst others used dimension reduction techniques such as principal component analysis (PCA). We aimed to compare the results of unsupervised clustering when using raw variables, or variables transformed using dimensionality reduction techniques. Method: We performed our analysis on 613 asthmatics aged 6-23 years from Ankara, Turkey. We conducted extensive phenotyping and recorded 49 variables including demographic data, sensitization, lung function, medication, peripheral eosinophilia, and markers of asthma severity. We performed hierarchical clustering (HC) using: (1) all variables; and (2) variables transformed using dimensionality reduction techniques. Results: PCA revealed 5 components describing atopy and variations in asthma severity, which were then used to infer cluster assignment. The optimal HC solution in both PCA-transformed and raw untransformed data identified five clusters. However, these clusters were not identical. Both identified mild asthma with good lung function, severe atopic asthma and late-onset mild atopic asthma. However, the overlap between children assigned to these three clusters in two HC analyses was modest. Clustering without PCA identified early-onset severe atopic asthma and late-onset atopic asthma with high BMI, whilst early onset non-atopic mild asthma in females was identified in HC with PCA. Conclusion: Different methodologies applied to the same dataset identified differing clusters of asthma. Despite cluster instability, both methodologies provided meaningful clinical insights for understanding asthma heterogeneity.

P06
Introduction: An important unexplored source of interpersonal variation in asthma presentation is the complex and dynamic community of microorganisms located on different sites of the respiratory tract. Our aim was to identify and explore the bacterial and viral diversity in a small pilot study of asthmatic children using metagenomics. Method: Nasopharyngeal samples were obtained from five asthmatic children (2 males) at a baseline asymptomatic visit. Mean age was 5.2 years (range 4.6-5.8). Communities of viruses (DNA and RNA genomes) and bacteria were evaluated using high depth pairedend shotgun sequencing. We performed taxonomic classification of sequences and calculated environmental indexes (alpha and beta) to estimate the microbial diversity both within (Shannon, Chao1, Jack1) and between (Jaccard) patients. Rarefaction curves were produced to study the relationship between microbial diversity and the number of generated sequences and/or number of samples. A correlation matrix was used to identify positive and negative correlations between bacterial and viral families and the results were superimposed in a network graph. Introduction: Asthma is closely associated with the endogenous circadian rhythm of the lungs; in severe and uncontrolled asthma nocturnal wakening is common, as is a dip in early morning peak expiratory flow. This variability makes asthma a potential target for chronotherapy to improve the efficacy of treatment. To personalise our approach to chronotherapy in asthma, it is essential to have an easy-to-measure biomarker that reflects an individual patient's chronotype. This would allow chronotherapy to be targeted to the right time of day for each patient. Breath samples are easy to collect and non-invasive, and exhaled volatile organic compounds (VOCs) have been related to airway inflammatory patterns in asthma. We hypothesise that the exhaled VOC profile contains potential biomarkers that track the circadian rhythm in asthma. Samples are analysed using thermal desorption-gas chromatography-mass spectrometry which is currently the gold standard for offline VOC detection, and allows high sensitivity and reliable identification of detected compounds. Method: Twenty non-smoking volunteers (10 healthy, 10 with moderate asthma) will be recruited. So far nine individuals have been recruited; the study design is detailed in the figure. Breath samples are taken at 15:00 on the first visit; 15:00, 21:00, 03:00 and 09:00 on the second and 09:00 on the final visit. This gives six samples per participant at four time points allowing investigation of both the changes in an individual's VOC profiles over the course of a day and longitudinal variation over the course of several weeks. All samples are analysed using thermal desorption-gas chromatography-mass spectrometry Introduction: The aim of the study is to determine the relationship between serum periostin (SP) levels and clinical characteristics in adult patients with moderate to severe asthma (AP). Method: SP levels were measured by ELISA method in 42 AP out of exacerbations and 10 healthy controls (HC). AP were divided into two groups according asthma control test (ACT) score: controlled AP (CA, n = 19) and uncontrolled AP (UA, n = 23). Atopic status was confirmed by skin prick testing and measurement of total and specific serum IgE levels. Lung functional parameters were assessed by spirometry. Results: We have observed significantly higher levels of SP in AP (1.68 ± 0.52 ng/ml) compared to HC (1.08 ± 0.12 ng/ml) (p < 0.001).

Results
No statistically significant differences were found in SP levels between CA and UA, atopic (n = 14) and non-atopic AP (n = 28), current smokers (n = 13) and non-smokers (n = 29) (p = 0.85, p = 0.33, p = 0.19 respectively). We have found significantly higher SP levels in AP on medium (n = 22) and high doses ICS (n = 9) compared to those on low dose ICS (n = 11) therapy (p = 0.011, p = 0.007 respectively). Twenty AP (n = 5, CA and n = 15, UA) demonstrated impaired lung function (FEV 1 < 80%), which was associated with higher levels of SP (p = 0.04). Similarly, in UA was observed significantly negative correlation between SP and FEV 1 % (p = 0.006, r = − 0.56 Introduction: Serum periostin is an emerging biomarker for T2 asthma, and for the response to new biologicals such as anti IL-13 lebrikizumab. But a clear cut-off has not been established, and other pathologies beyond asthma are known to course with high periostin levels. The aim of this study was to establish reference values for serum periostin in a representative sample of non-asthmatic non-COPD healthy population in our geographical area. Method: A cross-sectional study was conducted between May-October 2015. After signing the informed consent, we collected sera samples from consecutive blood donors of the Blood Bank of Universitary Hospital 12 de Octubre, Madrid, Spain. By choosing this source of donors, all diseases and other conditions related to any periostin elevations described so far were excluded, as they constitute contraindications for donating blood: chronic illnesses (diabetes; heart, pulmonary, renal or hepatic diseases); any type of cancer; chronic or acute infectious diseases, including stomathologycal disordes; pregnancy; recent traumatic injury or surgery. Moreover, all participants completed a questionnaire aimed to exclude those with symptoms and/or a physician diagnosis of asthma and/or COPD. Sera were frozen at −80 °C until periostin was measured using an EISA commercial kit. We calculated the sample size to estimate a media, using a standard deviation (SD) of 15, with 3 ng/mL precision and 95% confidence interval (Anastasilakis et al. 2014). Results: 100 non-asthmatic non-COPD and otherwise healthy subjects, aged between 18 and 65 years, were included in the study. Results are expressed in media (SD) (min-max range  Introduction: Severe asthma affects 5% of the asthma population but drives the majority of the morbidity and cost. Despite cough being a major symptom and a marker of both severity and poor prognosis, current medications are not designed to treat cough directly. Bronchial thermoplasty (BT) is a novel therapy in severe asthma, delivering radiofrequency thermal energy to the large airways. There are provisional data suggesting BT reduces the number of airway nerves 1 .
If this is the case, then BT may affect cough in asthma. Inhaled capsaicin challenge has been safely used to evoke cough responses and to assess cough threshold in research. However, to date no study has adopted this method to assess cough in patients with severe asthma or following BT. We aim to assess the safety and feasibility of capsaicin challenge in people with severe asthma. We will also explore the effects of BT on 24 h cough frequency, capsaicin evoked cough responses and coughrelated quality of life.

Method:
The protocol for this two-visit observational study has been approved by the local Research Ethic Committee. All recruited patients (target n = 24, of whom half will have had BT) will be treated at British Thoracic Society steps 4 or 5. During visit 1 patients will be consented, the Leicester Cough and Asthma Control Questionnaires administered and baseline spirometry performed. They will undergo ambulatory 24-hour cough monitoring. A capsaicin challenge will be performed during the second visit. Spirometry will be performed before and immediately after the challenge to assess any change in FEV 1 .

Results:
The primary outcome measure will be the number of participants with a 20% or greater fall in FEV1 and/or requiring rescue bronchodilator therapy during the capsaicin challenge period. Secondary outcomes include: change in %FEV1 predicted during capsaicin challenge; tolerability of inhaled capsaicin; number of adverse events during and after capsaicin evoked cough challenge; differences in capsaicin dose response curves between patients who have had BT versus those have not.

Conclusion:
This pilot study will lead to a larger prospective study investigating the effects of BT on cough responses in patients with severe asthma.  Introduction: Cough is a common and troublesome symptom in asthma but little is known about the neuronal pathways that trigger cough. The mechanisms by which airway inflammation, bronchial hyper-responsiveness and variable airflow obstruction cause cough is unclear. We have previously shown that methacholine induced bronchoconstriction heightens cough responses to inhaled capsaicin.
The aim of the current study was to investigate the effects of allergen exposure on cough reflex sensitivity. Method: We performed a 8 visit randomised, single-blind, placebo controlled, two-way cross-over study comparing cough responses to inhaled capsaicin in allergic asthma patients during and 24 h after exposure to allergen compared with diluent (saline) control. Mild atopic asthmatics who are dual responders will undergo full dose allergen/diluent (saline) challenge. Early asthmatic responses (EAR) (fall in FEV1 > 20% at 30 min) and late asthmatic responses (LAR) (fall in FEV1 > 15% at 3-7 h) will be documented. Full dose capsaicin challenge was performed at screening to determine the excitatory dose evoking half the maximum cough response (ED50 vs. diluent at 30 min 15.0 (4.9) vs. 9.6(3.5), at 24 h 12.2 (8.0) vs. 6 (2.6).  Introduction: During a near-fatal attack of asthma (NFA) alveolar hypoventilation underlies respiratory acidosis while severe hypoxemia indicates intrapulmonary shunt. Illustrate the differential role of lowflow extracorporeal CO 2 removal (ECCO 2 R) and high-flow veno-venous extracorporeal membrane oxygenation (VV-ECMO) as rescue therapies in NFA. Method: Patient 1 A 31-year-old male with early-onset asthma and epilepsy was admitted with acute respiratory failure (ARF) complicated by generalized tonic-clonic seizure. He presented severe acute respiratory acidosis without hypoxemia after intubation (pH-7.10 pCO 2 -82 mmHg P/F 556). Thoracic CT: diffuse bronchial wall thickness without atelectasis. On Day-5 dynamic hyperinflation (auto-PEEP 10 cmH 2 O) under deep sedation and neuromuscular blockade persisted, precluding weaning from invasive mechanical ventilation (IMV). ECCO 2 R was initiated. Extubation, active physical therapy and full patient mobilization were possible on Day-6. ECCO 2 R discontinuation, ICU and hospital discharges occurred on Day-8, -9 and -11, respectively. Results: Patient 2 A 34-year-old obese female with late-onset and poorly controlled asthma was admitted with ARF with severe hypoxemia (pH-7.45 pCO 2 -30 mmHg P/F 69). After a trial of non-invasive ventilation, IMV was initiated. On Day-1, despite medical therapy and IMV optimizations, severe dynamic hyperinflation with refractory hypoxemia persisted and VV-ECMO was initiated. Thoracic CT revealed almost complete bilateral lung collapse. Methicillin-sensitive S. aureus and S. pneumoniae were isolated in endotracheal aspirate. Normal lung function was progressively reestablished following considerable viscid mucus secretions elimination and antibiotherapy. ECMO-VV was discontinued on Day-18. Extubation, ICU and hospital discharges occurred on Day-24, -27 and -55, respectively. Conclusion: How this report contributes to current knowledge. During refractory NFA, low-flow ECCO 2 R allows correction of severe respiratory acidosis and facilitates extubation while high-flow VV-ECMO is needed when significant atelectasis complicates airway narrowing and severe hypoxemia ensues. Knowledge of respiratory failure pathophysiology in refractory NFA allows the correct use of different ECLS modalities as a bridge to recovery.

Mechanisms of cross-talk between pulmonary epithelial cells and dendritic cells during type 2 inflammation
Consent to publish: Consent to publish was received from the patients. Introduction: 69.9% of patients with severe asthma respond to omalizumab, when it is used according to International Guidelines of asthma. We describe clinical and functional characteristics of 4 patients with severe asthma treated with omalizumab and belong to our series of severe asthmatic patients treated with this drug. Method: We collected 24 patients with severe asthma treated with omalizumab from the Allergy Unit of the University Hospital Foundation Alcorcón, during the period 2007 to 2015. Of these, 4 patients did not respond to treatment. The "No responders" was defined by the responsible doctor, based on the clinical and functional assessment of each patient considering several clinical and functional characteristics: beta-adrenergic use, number of exacerbations and scores ACQ and ACT, other comorbidities, spirometric and lung volumes. These values were measured in the last year before the start of omalizumab and the final year of treatment with omalizumab. Results: In 4 patients it was withdrawn omalizumab because of no efficacy. Responders and non-responders were treated, at least, during 1 year. The non-responders had more non-respiratory comorbidities than responders (75%-25%,p = 0.07). However responders, seemed to have more severe asthma, with a greater number of exacerbations (responders 3.24 vs no responders 2.75), more use of beta-adrenergic   baseline and response after 4 months ( Table 3). The response rate remained stable over the years 2012-2015 (Table 4). Figure 1 shows that most of the responders had an improvement of both FEV1 and ACQ. It also shows that ACQ appears to be a better measurement for a response than FEV1. Conclusion: 63% of the 403 patients with inadequately controlled severe allergic asthma had a good or excellent response to omalizumab after 16 weeks. Overall the ACQ improved, FEV1 increased and there was lower use of OCS at 16 weeks. There was no relationship between patients with a FEV1 < 80 and ≥ 80% at baseline and the response rate. Improvement of ACQ appears to be a better measurement for response than improvement of FEV1. Keywords: Asthma, Omalizumab