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Background Asthma prevalence is 339 million globally. ‘Severe asthma’ (SA) comprises subjects with uncontrolled asthma despite proper management. Objectives To compare asthma from diverse ethnicities and environments. Methods A cross-sectional analysis of two adult cohorts, a Brazilian (ProAR) and a European (U-BIOPRED). U-BIOPRED comprised of 311 non-smoking with Severe Asthma (SAn), 110 smokers or ex-smokers with SA (SAs) and 88 mild to moderate asthmatics (MMA) while ProAR included 544 SA and 452 MMA. Although these projects were independent, there were similarities in objectives and methodology, with ProAR adopting operating procedures of U-BIOPRED. Results Among SA subjects, age, weight, proportion of former smokers and FEV1 pre-bronchodilator were similar. The proportion of SA with a positive skin prick tests (SPT) to aeroallergens, the scores of sino-nasal symptoms and quality of life were comparable. In addition, blood eosinophil counts (EOS) and the % of subjects with EOS > 300 cells/μl were not different. The Europeans with SA however, were more severe with a greater proportion of continuous oral corticosteroids (OCS), worse symptoms and more frequent exacerbations. FEV1/FVC pre- and post-bronchodilator were lower among the Europeans. The MMA cohorts were less comparable in control and treatment, but similar in the proportion of allergic rhinitis, gastroesophageal reflux disease and EOS >3%. Conclusions ProAR and U-BIOPRED cohorts, with varying severity, ethnicity and environment have similarities, which provide the basis for global external validation of asthma phenotypes. This should stimulate collaboration between asthma consortia with the aim of understanding SA, which will lead to better management.

Background The role of IL-17 immunity is well established in patients with inflammatory diseases, such as psoriasis and inflammatory bowel disease, but not in asthmatic patients, in whom further study is required. Objective We sought to undertake a deep phenotyping study of asthmatic patients with upregulated IL-17 immunity. Methods Whole-genome transcriptomic analysis was performed by using epithelial brushings, bronchial biopsy specimens (91 asthmatic patients and 46 healthy control subjects), and whole blood samples (n = 498) from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. Gene signatures induced in vitro by IL-17 and IL-13 in bronchial epithelial cells were used to identify patients with IL-17–high and IL-13–high asthma phenotypes. Results Twenty-two of 91 patients were identified with IL-17, and 9 patients were identified with IL-13 gene signatures. The patients with IL-17–high asthma were characterized by risk of frequent exacerbations, airway (sputum and mucosal) neutrophilia, decreased lung microbiota diversity, and urinary biomarker evidence of activation of the thromboxane B2 pathway. In pathway analysis the differentially expressed genes in patients with IL-17-high asthma were shared with those reported as altered in psoriasis lesions and included genes regulating epithelial barrier function and defense mechanisms, such as IL1B, IL6, IL8, and β-defensin. Conclusion The IL-17–high asthma phenotype, characterized by bronchial epithelial dysfunction and upregulated antimicrobial and inflammatory response, resembles the immunophenotype of psoriasis, including activation of the thromboxane B2 pathway, which should be considered a biomarker for this phenotype in further studies, including clinical trials targeting IL-17.

Background The mechanisms by which mast cells in patients with unexplained anaphylaxis (UEA) are triggered remain elusive. Onset of episodes is unpredictable and often recurrent. The substantial overlap between the clinical manifestations of UEA and clonal mast cell disorders (CMD) suggests an association between these rare disorders. The two forms of CMD characterized to date are systemic mastocytosis (SM) and monoclonal mast cell activation syndrome (MMAS). Objective To examine the hypothesis that the pathogenesis of UEA reflects the presence of aberrant subpopulations of mast cells. Methods Thirty (14 men, 16 women) patients (≥ 18 years) suffering from UEA and with no signs of cutaneous mastocytosis were recruited. Patients underwent an initial complete allergy work-up to confirm the diagnosis of UEA. Level of baseline serum tryptase (sBT) and total IgE were determined. In addition, a bone marrow examination was performed on all 30 patients to investigate possible underlying CMD. Results Fourteen (47%) of our cases (nine men, five women) were diagnosed with CMD: 10 with SM and four with MMAS. Four of the 10 patients with SM had mast cell aggregates in their bone marrow. All patients with SM exhibited a sBT level > 11.4 ng/mL, whereas this level was elevated in only two of those with MMAS and four with UAE but not diagnosed with CMD. Total IgE levels were lower in the group of patients with CMD (P Conclusion and clinical relevance The pathogenic mechanism underlying UEA could be explained by the presence of immunophenotypically aberrant mast cells with clonal markers in 47% of our subjects, indicating that clonal mast cell disorders are present in a substantial subset of these patients. Thus, the presence of CMD should be considered in patients with UEA if they have an elevated level of sBT (≥ 11.4 ng/mL) and cardiovascular symptoms such as syncope.

SummaryBackground Patients with systemic mastocytosis (SM) have clinical signs of mast cell (MC) activation and increased levels of MC mediators. It is unclear whether the increased mediator levels are caused by increased numbers of tissue MCs, or whether these cells in affected individuals have a hyperactive phenotype. Objective To determine reactivity of the skin and the airways to directly acting mediators and indirectly acting mast cell secretagogues in subjects with SM. Methods Skin reactivity to morphine and histamine, and airway responsiveness to mannitol and methacholine, was assessed in 15 patients with SM, 11 patients with allergic asthma (A) and 13 healthy controls (HC). Serum tryptase and urinary metabolites of the MC mediators histamine and prostaglandin D2 were measured, as well as ex vivo basophil histamine release. Results Mast cell mediators in the blood and urine were significantly higher in patients with SM than in HC and A controls. Responsiveness to local activation of skin MCs (by morphine) and airway MCs (by mannitol) was similar in SM and HC groups. Likewise, end-organ responsiveness in the skin to histamine, and in the airways to methacholine, was similar in all three subject groups. There was no evidence of increased basophil reactivity in SM patients. Conclusions and Clinical Relevance Mast cells in the skin and airways of subjects with SM do not exhibit hyper-reactivity towards the MC-activating stimuli morphine and mannitol, respectively. Therefore, the highly elevated baseline levels of MC mediators in SM are most likely due to increased MC numbers, rather than altered MC responsiveness. The underlying mechanisms could involve leakage of MC mediators, or dysfunctions in mediator synthesis, storage and release. One clinical implication of our study is that there is no contraindication to perform skin tests using morphine in subjects with mastocytosis.

Background: Nasal polyposis is a disease known to be associated with asthma. The management is anti-inflammatory, with topical and oral corticosteroids as the first-line treatment. The effect of surgical treatment on lower airway inflammation has not been sufficiently studied. Aim: The aim of this study is to investigate the effects of functional endoscopic sinus surgery (FESS) as well as fluticasone proprionate nasal drops (FPND) 400 lg b.i.d. on nasal and lower airway parameters in asthmatics with nasal polyposis. Methods: This was a prospective 21-week study of 68 patients with asthma and nasal polyposis, on the benefits of FESS on nasal (butanol test, subjective olfaction, peak nasal inspiratory flow, congestion, rhinorrhoea, and polyp score), and on the lower airway parameters (dyspnea, cough, mean daily peak expiratory flow rate (PEFR), and lung function tests). It also included a randomized, double-blind, placebo-controlled 14 weeks phase on FPND. Results: Functional endoscopic sinus surgery significantly improved mean asthma symptom scores and daily PEFR and all nasal parameters including subjective and objective olfaction tests. This is the first study that shows the benefits of FESS on butanol tests in patients with nasal polyposis. We found no significant difference between topical treatment with FPND or placebo in the nasal or lower airway variables. Conclusion: Functional endoscopic sinus surgery improved nasal and asthma symptoms in patients with nasal polyposis. Functional endoscopic sinus surgery could be considered early in the natural course of nasal polyposis with concomitant asthma, as well as a second-line treatment in nasal polyposis patients with a reduced sense of smell. The potential benefits of FPND 400 lg b.i.d. were probably overshadowed by FESS.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most prevalent drugs inducing hypersensitivity reactions. The aim of this analysis was to estimate the prevalence of NSAID-induced respiratory symptoms in population across Europe and to assess its association with upper and lower respiratory tract disorders.METHODS: The GA(2) LEN survey was conducted in 22 centers in 15 European countries. Each of 19 centers selected random samples of 5000 adults aged 15-74 from their general population, and in three centers (Athens, Munich, Oslo), a younger population was sampled. Questionnaires including questions about age, gender, presence of symptoms of asthma, allergic rhinitis, chronic rhinosinusitis, smoking status, and history of NSAID-induced hypersensitivity reactions were sent to participants by mail. Totally, 62 737 participants completed the questionnaires.RESULTS: The mean prevalence of NSAID-induced dyspnea was 1.9% and was highest in the three Polish centers [Katowice (4.9%), Krakow (4.8%), and Lodz (4.4%)] and lowest in Skopje, (0.9%), Amsterdam (1.1%), and Umea (1.2%). In multivariate analysis, the prevalence of respiratory reactions to NSAIDs was higher in participants with chronic rhinosinusitis symptoms (Odds Ratio 2.12; 95%CI 1.78-2.74), asthma symptoms in last 12 months (2.7; 2.18-3.35), hospitalization due to asthma (1.53; 1.22-1.99), and adults vs children (1.53; 1.24-1.89), but was not associated with allergic rhinitis.CONCLUSION: Our study documented significant variation between European countries in the prevalence of NSAID-induced respiratory hypersensitivity reactions, and association with chronic airway diseases, but also with environmental factors.

This study provides the first in vivo evidence for a mast cell stabilizing effect of the short-acting inhaled β2-adrenoceptor agonist terbutaline, when administered prophylactically at a clinically recommended dose (0.5 mg) before bronchial provocation with dry air. Our data therefore support the proposal that β2-adrenoceptor agonist-mediated mast cell stabilization is a major contributor to bronchoprotection in individuals with exercise-induced bronchoconstriction., Release of bronchoactive mediators from mast cells during exercise hyperpnea is a key factor in the pathophysiology of exercise-induced bronchoconstriction (EIB). Our aim was to investigate the effect of a standard, single dose of an inhaled β2-adrenoceptor agonist on mast cell activation in response to dry air hyperpnea in athletes with EIB. Twenty-seven athletes with EIB completed a randomized, double-blind, placebo-controlled, crossover study. Terbutaline (0.5 mg) or placebo was inhaled 15 min prior to 8 min of eucapnic voluntary hyperpnea (EVH) with dry air. Pre- and postbronchial challenge, urine samples were analyzed by enzyme immunoassay for 11β-prostaglandin F2α (11β-PGF2α). The maximum fall in forced expiratory volume in 1 s of 14 (12–20)% (median and interquartile range) following placebo was attenuated to 7 (5–9)% with the administration of terbutaline (P < 0.001). EVH caused a significant increase in 11β-PGF2α from 41 (27–57) ng/mmol creatinine at baseline to 58 (43–72) ng/mmol creatinine at its peak post-EVH following placebo (P = 0.002). The rise in 11β-PGF2α was inhibited with administration of terbutaline: 39 (28–44) ng/mmol creatinine at baseline vs. 40 (33–58) ng/mmol creatinine at its peak post-EVH (P = 0.118). These data provide novel in vivo evidence of mast cell stabilization following inhalation of a standard dose of terbutaline prior to bronchial provocation with EVH in athletes with EIB.

Repeated low-dose allergen inhalation challenge mimics natural allergen exposure, providing a model for early mechanisms in the triggering of asthma. The current authors performed a controlled study to evaluate the time course of changes in exhaled nitric oxide fraction (F(e,NO)) and urinary biomarkers of airway inflammation. Eight subjects with mild allergic asthma completed two 7-day repeated low-dose challenge periods, with diluent and allergen, respectively. Subjects were symptom free at inclusion and were investigated when not exposed to specific allergen. Pulmonary function and symptoms were followed, and F(e,NO) and urinary mediators were correlated to changes in airway responsiveness to histamine and adenosine. Despite no change in pulmonary function (forced expiratory volume in one second mean+/-sem fall 0.3+/-0.7 versus 0.6+/-1.0%, for diluent and allergen, respectively) and no asthma symptoms, repeated allergen exposure, in contrast to diluent, caused significant increases in histamine responsiveness (2.3 doubling doses), an early and gradual increase in F(e,NO) (up to a doubling from baseline) and a small increase in the mast cell marker 9alpha11beta-prostaglandin F(2) after adenosine challenge. In conclusion, serial measurements of exhaled nitric oxide fraction have the potential to provide a very sensitive strategy for early detection of emerging airway inflammation and subsequent changes in airway hyperresponsiveness to histamine.

Since severe asthma is a poorly understood, major health problem, 12 clinical specialist centres in nine European countries formed a European Network For Understanding Mechanisms Of Severe Asthma (ENFUMOSA).In a cross-sectional observational study, a total of 163 subjects with severe asthma were compared with 158 subjects whose asthma was controlled by low doses of inhaled corticosteroids (median dose of beclomethasone equivalents 666 mug). Despite being treated with higher doses of inhaled corticosteroids; (median dose 1773 mug) and for a third of the severe asthmatics also being treated with regular, oral-steroid therapy (median daily dose 19 mg), the subjects with severe asthma met the inclusion criteria. The criteria required subjects to have undergone at least one asthma exacerbation in the past year requiring oral steroid treatment. Females dominated the severe asthma group (female/male ratio 4.4:1 versus 1.6:1 in the controlled asthmatics), and compared with controlled asthmatics, they had a predominantly neutrophilic inflammation (sputum neutrophils, 36 versus 28%) and evidence of ongoing mediator release but less atopy.From these findings and other physiological and clinical data reported in this paper, it is suggested that severe asthma might be a different form of asthma rather than an increase in asthma symptoms. The findings prompt for longitudinal studies and interventions to define the mechanisms in severe asthma.

Indirect challenges act by causing the release of endogenous mediators that cause the airway smooth muscle to contract. This is in contrast to the direct challenges where agonists such as methacholine or histamine cause airflow limitation predominantly via a direct effect on airway smooth muscle. Direct airway challenges have been used widely and are well standardised. They are highly sensitive, but not specific to asthma and can be used to exclude current asthma in a clinic population. Indirect bronchial stimuli, in particular exercise, hyperventilation, hypertonic aerosols, as well as adenosine, may reflect more directly the ongoing airway inflammation and are therefore more specific to identify active asthma. They are increasingly used to evaluate the prevalence of bronchial hyperresponsiveness and to assess specific problems in patients with known asthma, e.g. exercise-induced bronchoconstriction, evaluation before scuba diving. Direct bronchial responsiveness is only slowly and to a modest extent, influenced by repeated administration of inhaled steroids. Indirect challenges may reflect more closely acute changes in airway inflammation and a change in responsiveness to an indirect stimulus may be a clinically relevant marker to assess the clinical course of asthma. Moreover, some of the indirect challenges, e.g. hypertonic saline and mannitol, can be combined with the assessment of inflammatory cells by induction of sputum.

Background Allergen provocation is a very useful way to study the inflammatory response in asthmatic patients. Although cumulative dose regimens are most often applied, another provocation model with repeated inhalations of low doses of allergens has recently come into use. Objective We were interested to compare these two allergen provocation models. To evaluate the inflammation induced by either model, we examined the mRNA expression of several cytokines that are implicated in the orchestration of the inflammatory response observed in asthma. Methods Interleukin (IL)-4, IL-5, IL-13 and interferon (IFN)-γ mRNA expression was analysed in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) CD4+ and PB CD8+ T cells following any of the two provocation regimens. IL-4 and IFN-γ mRNA expression was analysed by a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) method, while IL-5 and IL-13 were analysed semiquantitatively, before and after allergen provocation with either model. Results After low dose provocations none of the cell populations studied showed a clear change in the pattern of IL-4 or IFN-γ gene expression. In contrast, after cumulative dose provocation there was a clear tendency towards an increased IL-4 mRNA expression in BAL cells, correlating with a significant increase in IL-4 mRNA in PB CD4+ as well as in CD8+ T cells (P = 0.005 and P = 0.04, respectively). Regardless of the allergen provocation method used, in PB IL-4 mRNA was preferentially expressed by CD4+ cells while IFN-γ was expressed more by CD8+ cells. IL-5 transcripts increased after low dose provocations in PB CD4+ T cells in six of eight patients, while after cumulative dose provocation IL-5 mRNA increased in BAL cells in six out of nine patients but decreased especially in PB CD8+ T cells in six out of eleven patients, suggesting an accumulation of IL-5 expressing cells to the lungs. Conclusion Thus, the cumulative dose provocation regimen can induce a more pronounced Th2-like immune response in asthmatic patients than the low dose provocation model.

Background Cysteinyl-leukotrienes are central mediators in asthma and urinary leukotriene E 4 (LTE 4 ) is a reliable marker of their endogenous formation. Objective This study tested the hypothesis that the procedure used for allergen bronchoprovocation influences the bronchoconstrictor response and the amount of LTE 4 excreted following allergen challenge. Methods Seven atopic asthmatic men underwent two allergen bronchoprovocations 4 weeks apart. The same total dose of allergen was given at both sessions, cumulatively on one occasion and as a single dose at the other session. Urine was collected in hourly samples before and after challenge and LTE 4 was measured with previously validated methodology. Results The mean (± SE) drop in FEV 1 was not significantly different between the cumulative (29 ± 2.4%) and the single dose challenge (25 ± 2.8%). There was a significant increase in post-challenge levels of urinary LTE 4 after both sessions. The peak excretion of LTE 4 occurred 1 h following the maximal drop in FEV 1 for both challenges. However, the post-challenge increase in urinary LTE 4 was significantly larger at the cumulative session. In fact, the net increase (post-challenge minus prechallenge) of urinary LTE 4 was more than twofold higher after the cumulative session (AUC 0-3 h post-challenge: 46.7 ± 8.2 vs 22.1 ± 9.8, P

Background It is generally accepted that the early asthmatic response to inhaled allergen is a result of IgE-mediated mast cell activation. In contrast, the underlying mechanism of the late asthmatic response is much less clear. Objective In order to investigate the pattern of mediator release during the early and late asthmatic responses to allergen, measurements of the urinary excretion of the mast cell markers 9α,11β-PGF2 and Nτ-methylhistamine were made. In addition, urinary levels of eosinophil protein X (EPX) and leukotriene E4 (LTE4) were measured. Methods Twelve mild atopic asthmatics participated in the study. On the study day, pulmonary function was recorded at baseline and for 12 h after inhalation of allergen. Urine was collected prior to challenge and thereafter at 1 h intervals. Measurements of 9α,11β-PGF2 and LTE4 were made with enzyme-immunoassay, and levels of Nτ-methylhistamine and EPX were analysed with radioimmunoassay. Results All subjects developed both an early and late phase airway response. Within 1 h of the early peak airway response, there was a significant increase in the urinary concentrations (AUC/h) of 9α,11β-PGF2 (49.3 ± 9.2 to 142.5 ± 49.2; P

SummaryBackground Exacerbations represent a major source of morbidity and mortality in asthma and are a prominent feature of poorly controlled, difficult-to-treat disease. Objective The goal of our study was to provide a detailed characterization of the frequent exacerbator phenotype and to identify risk factors associated with frequent and seasonal exacerbations. Methods Ninety-three severe asthmatics (SA) and 76 mild-to-moderate patients (MA) were screened and prospectively followed up for 1 year (NCT00555607). Medical history, baseline clinical data and biomarkers were used to assess risk factors for frequent exacerbations. Results During the study, 104 exacerbations were recorded in the SA group and 18 in the MA. Frequent exacerbators were characterized by use of higher doses of inhaled (1700 vs. 800 μg) and oral (6.7 vs. 1.7 mg) glucocorticosteroids, worse asthma control (ACQ score 2.3 vs. 1.4), lower quality of life (SGRQ score 48.5 vs. 33.3), higher sputum eosinophils (25.7% vs. 8.2%) and a more rapid decline in FEV1/FVC ratio (−0.07 vs. −0.01 ΔFEV1/FVC, frequent vs. non-frequent, respectively, P 45 p.p.b. and a history of smoking were associated with an increased risk of frequent exacerbations (odds ratios: 4.32 and 2.90 respectively). Conclusion and Clinical Relevance We were able to distinguish and characterize a subphenotype of asthma subjects – frequent exacerbators – who are significantly more prone to exacerbations. Patients with FeNO > 45 p.p.b. and a history of smoking are at increased risk of frequent exacerbations and require careful monitoring in clinical practice.

Background: Exacerbations represent a major source of morbidity and mortality in asthma and are a prominent feature of poorly controlled, difficult-to-treat disease. Objective: The goal of our study was to provide a detailed characterization of the frequent exacerbator phenotype and to identify risk factors associated with frequent and seasonal exacerbations. Methods: Ninety-three severe asthmatics (SA) and 76 mild-to-moderate patients (MA) were screened and prospectively followed up for 1 year (NCT00555607). Medical history, baseline clinical data and biomarkers were used to assess risk factors for frequent exacerbations. Results: During the study, 104 exacerbations were recorded in the SA group and 18 in the MA. Frequent exacerbators were characterized by use of higher doses of inhaled (1700 vs. 800 μg) and oral (6.7 vs. 1.7 mg) glucocorticosteroids, worse asthma control (ACQ score 2.3 vs. 1.4), lower quality of life (SGRQ score 48.5 vs. 33.3), higher sputum eosinophils (25.7% vs. 8.2%) and a more rapid decline in FEV1/FVC ratio (-0.07 vs. -0.01 ΔFEV1/FVC, frequent vs. non-frequent, respectively, P 45 p.p.b. and a history of smoking were associated with an increased risk of frequent exacerbations (odds ratios: 4.32 and 2.90 respectively). Conclusion and Clinical Relevance: We were able to distinguish and characterize a subphenotype of asthma subjects - frequent exacerbators - who are significantly more prone to exacerbations. Patients with FeNO > 45 p.p.b. and a history of smoking are at increased risk of frequent exacerbations and require careful monitoring in clinical practice. © 2013 John Wiley & Sons Ltd.

BACKGROUND: Although asthma is characterized by variable airways obstruction, most studies of asthma phenotypes are cross-sectional. The stability of phenotypes defined either by biomarkers or by physiological variables was assessed by repeated measures over 1�year in the Pan-European BIOAIR cohort of adult asthmatics. METHODS: A total of 169 patients, 93 with severe asthma (SA) and 76 with mild-to-moderate asthma (MA), were examined at six or more visits during 1�year. Asthma phenotype clusters were defined by physiological variables (lung function, reversibility and age of onset of the disease) or by biomarkers (eosinophils and neutrophils in induced sputum). RESULTS: After 1�year of follow-up, the allocation to clusters was changed in 23.6% of all asthma patients when defined by physiological phenotypes and, remarkably, in 42.3% of the patients when stratified according to sputum cellularity (P�=�0.034). In the SA cohort, 30% and 48.6% of the patients changed allocation according to physiological and biomarker clustering, respectively. Variability of phenotypes was not influenced by change in oral or inhaled corticosteroid dose, nor by the number of exacerbations. Lower stability of single and repeated measure was found for all evaluated biomarkers (eosinophils, neutrophils and FeNO) in contrast to good stability of physiological variables (FEV1 ), quality of life and asthma control. CONCLUSION: Phenotypes determined by biomarkers are less stable than those defined by physiological variables, especially in severe asthmatics. The data also imply that definition of asthma phenotypes is improved by repeated measures to account for fluctuations in lung function, biomarkers and asthma control.

The hypothesis that cysteinyl-leukotrienes (LTC4, LTD4 and LTE4) are mediators of allergen-induced airway obstruction in asthmatics was tested with the specific receptor antagonist ICI-204,219, in a double-blind, placebo-controlled, randomized, cross-over bronchoprovocation study. On three occasions, cumulative bronchial challenge with specific allergen was performed in 10 males with mild allergic asthma. The first control session established the baseline provocative dose of allergen producing a decrease in forced expiratory volume in one second (FEV1) by 20% (PD20FEV1). The two rechallenges were performed 2 h after oral administration of placebo or 20 mg of ICI-204,219. The allergen dose-response relations were highly reproducible, producing PD20 values at the control session and after placebo treatment which varied by no more than 0.7-1.3 fold (95% confidence interval (95% CI)). After ICI-204,219, the median cumulated allergen dose was 5.5 fold higher, and the group geometric mean PD20 was increased 2.5 times. Furthermore, the recovery time after the immediate bronchoconstriction was shorter (40 vs 60 min). The wheal and flare responses to intradermally injected LTD4 were somewhat inhibited by ICI-204,219, whereas responses to histamine were unaffected. However, the findings suggest that skin testing with LTD4 is unlikely to predict the degree of leukotriene-antagonism in the airways. The findings confirm and extend the indications that cysteinyl-leukotrienes are important mediators of allergen-induced airway obstruction, and that leukotriene-antagonists should be evaluated as a potential new therapy in allergic asthma.

Allergic asthma is IgE-mediated and the IgE-sensitisation is usually demonstrated by skin prick tests (SPT) and IgE antibody determinations in serum. The SPT and IgE-antibody values do not directly predict if the allergy clinically contributes to the asthma. There is therefore a need for new objective tests that may indicate the clinical importance of an IgE-sensitisation.To evaluate basophil allergen threshold sensitivity (CD-sens) as a measure of allergen sensitivity in allergic asthma.Twenty-six subjects with stable, intermittent allergic asthma were tested with SPT and spirometry, and methacholine and allergen inhalation challenges to determine methacholine PD(20) (provocative dose causing a 20% drop in forced expiratory volume in 1 s) and allergen PD(20) . The results were compared with CD-sens and serological parameters, i.e. IgE- and IgG4 antibodies to the relevant allergens.A significant correlation was found between CD-sens and allergen PD(20) (P = 0.01; r = 0.49; n = 26) as well as between CD-sens and the ratio of allergen PD(20) to methacholine PD(20) (P = 0.007; r = 0.52; n = 26). In patients with a moderate to low degree of bronchial hyperresponsiveness there was an excellent correlation (P = 0.0001; r = 0.88, n = 13) between CD-sens and allergen sensitivity. No relation to either allergen PD(20) or the ratio was found for basophil allergen reactivity measured as CD63 up-regulation at high concentrations of the respective allergen.CD-sens was found to be an objective marker of airway allergen sensitivity in stable allergic asthmatics, that may be used to predict airway responsiveness when bronchial challenge tests cannot be performed.

Prostaglandin (PG) D(2) is a pro-inflammatory and bronchoconstrictive mediator released from mast cells, and is currently evaluated as a new target for treatment of asthma and rhinitis. It is not known which cyclooxygenase (COX) isoenzyme catalyses its biosynthesis in subjects with asthma.Primarily, to assess whether treatment with the COX-2 selective inhibitor celecoxib inhibited biosynthesis of PGD(2) , monitored as urinary excretion of its major tetranor metabolite (PGDM). Secondarily, to determine the effects of the treatment on biosynthesis of PGE(2) , thromboxane A(2) and PGI(2) , also measured as major urinary metabolites.Eighteen subjects with asthma participated in a cross-over study where celecoxib 200mg or placebo were given b.i.d. on 3 consecutive days following 2 untreated baseline days. Six healthy controls received active treatment with the same protocol. Urinary excretion of the eicosanoid metabolites was determined by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Lung function was followed as FEV(1) and airway inflammation as fraction of exhaled nitric oxide (F(E) NO).Celecoxib treatment inhibited urinary excretion of PGEM by 50% or more in subjects with asthma and healthy controls, whereas there was no significant change in the excretion of PGDM. In comparison with the healthy controls, the subjects with asthma had higher baseline levels of urinary PGDM but not of PGEM. The 3-day treatment did not cause significant changes in FEV(1) or F(E) NO.Biosynthesis of PGD(2) was increased in subjects with asthma and its formation is catalysed predominantly by COX-1. By contrast, COX-2 contributes substantially to the biosynthesis of PGE(2) . The asymmetric impact of celecoxib on prostanoid formation raises the possibility of long-term adverse consequences of COX-2 inhibition on airway homeostasis by the decreased formation of bronchodilator PGs and maintained production of increased levels of bronchoconstrictor PGs in asthmatics.

According to the GA2LEN recommendations, nasal challenge test with lysine-aspirin should be performed only in patients with severe asthma, because the sensitivity of this test has been lower than in bronchial and oral challenge tests. The AIA patient group often have severe asthma with impaired lung function, and therefore improvement of the nasal challenge is warranted. The outcomes of this study clearly indicate that a prolonged detection time from two to three hours might improve the sensitivity of the nasal challenge as a method for diagnosing aspirin intolerance. Moreover, we found a different vascular response in the nasal mucosa in the subjects with AIA after local challenge with lysine-aspirin as compared to an ATA patient group. This puts RSM-LDF as a possible new method in addition to those previously recommended for this particular test.

Orbital complications of rhinosinusitis can be life-threatening. Accurate diagnosis by computerized tomography (CT) scanning, immediate administration of intravenous antibiotics and surgical drainage are the recommended management strategy. Faulty diagnosis by CT scan may lead to visual deterioration, intracranial complications and even fatality. The purpose of this study is to increase the awareness of the possibility of misdiagnosing orbital abscess (OA) as subperiosteal orbital abscess (SPOA) and propose a novel surgical technique to overcome this surgical circumstance.Three cases of OA and 29 of SPOA cases that were surgically managed between 1998 and 2008 were retrospectively reviewed.Three cases of OA diagnosed by CT scan as SPOA were primarily medically treated by intravenous antibiotics. Once the therapeutic management failed to resolve the condition, endonasal endoscopic surgery (EES) was carried out, upon which the diagnosis of OA was made and drainage successfully achieved.Whenever SPOA is diagnosed by CT scan and no purulent discharge is evidenced after removal of the lamina papyracea, OA should be suspected and incisions along the orbital periosteum should be made to release the pus from the orbit. The EES approach in cases of OS and SPOA can confirm an accurate diagnosis.

Non-steroidal anti-inflammatory drugs (NSAIDs) are analgesic and anti-inflammatory by virtue of inhibition of the cyclooxygenase (COX) reaction that initiates biosynthesis of prostaglandins. Findings in a pulmonary pharmacology project gave rise to the hypothesis that certain members of the NSAID class might also be antagonists of the thromboxane (TP) receptor.Functional responses due to activation of the TP receptor were studied in isolated airway and vascular smooth muscle preparations from guinea pigs and rats as well as in human platelets. Receptor binding and activation of the TP receptor was studied in HEK293 cells.Diclofenac concentration-dependently and selectively inhibited the contraction responses to TP receptor agonists such as prostaglandin D2 and U-46619 in the tested smooth muscle preparations and the aggregation of human platelets. The competitive antagonism of the TP receptor was confirmed by binding studies and at the level of signal transduction. The selective COX-2 inhibitor lumiracoxib shared this activity profile, whereas a number of standard NSAIDs and other selective COX-2 inhibitors did not.Diclofenac and lumiracoxib, in addition to being COX unselective and highly COX-2 selective inhibitors, respectively, displayed a previously unknown pharmacological activity, namely TP receptor antagonism. Development of COX-2 selective inhibitors with dual activity as potent TP antagonists may lead to coxibs with improved cardiovascular safety, as the TP receptor mediates cardiovascular effects of thromboxane A2 and isoprostanes.

The present approach to the diagnosis, management and follow-up of anaphylaxis during anaesthesia varies in the Scandinavian countries. The main purpose of these Scandinavian Clinical Practice Guidelines is to increase the awareness about anaphylaxis during anaesthesia amongst anaesthesiologists. It is hoped that increased focus on the subject will lead to prompt diagnosis, rapid and correct treatment, and standardised management of patients with anaphylactic reactions during anaesthesia across Scandinavia. The recommendations are based on the best available evidence in the literature, which, owing to the rare and unforeseeable nature of anaphylaxis, mainly includes case series and expert opinion (grade of evidence IV and V). These guidelines include an overview of the epidemiology of anaphylactic reactions during anaesthesia. A treatment algorithm is suggested, with emphasis on the incremental titration of adrenaline (epinephrine) and fluid therapy as first-line treatment. Recommendations for primary and secondary follow-up are given, bearing in mind that there are variations in geography and resources in the different countries. A list of National Centres from which anaesthesiologists can seek advice concerning follow-up procedures is provided. In addition, an algorithm is included with advice on how to manage patients with previous suspected anaphylaxis during anaesthesia. Lastly, Appendix 2 provides an overview of the incidence, mechanisms and possibilities for follow-up for some common drug groups.

The increased number of lymphocytes in airways during an asthmatic response is believed to be the result of increased recruitment of these cells. However, it is possible that a decreased apoptotic rate could also contribute to the increased number. The aim of the present study was to investigate whether allergen airway provocation influences the apoptotic phenotype of lung and peripheral blood lymphocytes (PBL) in subjects with atopic asthma. Bronchoalveolar lavage (BAL) lymphocytes and PBL from 12 asthmatic subjects previously challenged with allergen (n = 7) or saline (n = 5) were exposed to the apoptotic stimulus tributyltin (TBT) in vitro and assayed for apoptosis. Airway allergen provocation resulted in decreased sensitivity of BAL lymphocytes to TBT-induced apoptosis, with 42.2% (range 33.9–62.5%) apoptotic cells before challenge versus 23.5% (range 15.3–42.4%) after challenge, while PBL were unaffected. The increased apoptosis resistance correlated with higher numbers of Bcl-2-expressing lymphocytes. Interestingly, baseline caspase-3-like activity was significantly elevated in viable BAL lymphocytes compared with viable PBL, and was unaltered by allergen exposure. In conclusion, allergen inhalation renders bronchoalveolar lavage lymphocytes more resistant to apoptosis while peripheral blood lymphocytes were not influenced at all, indicating that the apoptotic phenotype of airway lymphocytes may play a role in asthmatic inflammation.

Allergen provocation is a very useful way to study the inflammatory response in asthmatic patients. Although cumulative dose regimens are most often applied, another provocation model with repeated inhalations of low doses of allergens has recently come into use.We were interested to compare these two allergen provocation models. To evaluate the inflammation induced by either model, we examined the mRNA expression of several cytokines that are implicated in the orchestration of the inflammatory response observed in asthma.Interleukin (IL)-4, IL-5, IL-13 and interferon (IFN)-gamma mRNA expression was analysed in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) CD4+ and PB CD8+ T cells following any of the two provocation regimens. IL-4 and IFN-gamma mRNA expression was analysed by a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) method, while IL-5 and IL-13 were analysed semiquantitatively, before and after allergen provocation with either model.After low dose provocations none of the cell populations studied showed a clear change in the pattern of IL-4 or IFN-gamma gene expression. In contrast, after cumulative dose provocation there was a clear tendency towards an increased IL-4 mRNA expression in BAL cells, correlating with a significant increase in IL-4 mRNA in PB CD4+ as well as in CD8+ T cells (P = 0.005 and P = 0.04, respectively). Regardless of the allergen provocation method used, in PB IL-4 mRNA was preferentially expressed by CD4+ cells while IFN-gamma was expressed more by CD8+ cells. IL-5 transcripts increased after low dose provocations in PB CD4+ T cells in six of eight patients, while after cumulative dose provocation IL-5 mRNA increased in BAL cells in six out of nine patients but decreased especially in PB CD8+ T cells in six out of eleven patients, suggesting an accumulation of IL-5 expressing cells to the lungs.Thus, the cumulative dose provocation regimen can induce a more pronounced Th2-like immune response in asthmatic patients than the low dose provocation model.

Cysteinyl-leukotrienes are central mediators in asthma and urinary leukotriene E4 (LTE4) is a reliable marker of their endogenous formation.This study tested the hypothesis that the procedure used for allergen bronchoprovocation influences the bronchoconstrictor response and the amount of LTE4 excreted following allergen challenge.Seven atopic asthmatic men underwent two allergen bronchoprovocations 4 weeks apart. The same total dose of allergen was given at both sessions, cumulatively on one occasion and as a single dose at the other session. Urine was collected in hourly samples before and after challenge and LTE4 was measured with previously validated methodology.The mean (+/- SE) drop in FEV1 was not significantly different between the cumulative (29 +/- 2.4%) and the single dose challenge (25 +/- 2.8%). There was a significant increase in post-challenge levels of urinary LTE4 after both sessions. The peak excretion of LTE4 occurred 1 h following the maximal drop in FEV1 for both challenges. However, the post-challenge increase in urinary LTE4 was significantly larger at the cumulative session. In fact, the net increase (post-challenge minus prechallenge) of urinary LTE4 was more than twofold higher after the cumulative session (AUC 0-3 h post-challenge: 46.7 +/- 8.2 vs 22.1 +/- 9.8, P0.05).The peak excretion of urinary LTE4 occurred within 2 h after the termination of either challenge but the magnitude of urinary excretion of LTE4 was larger when cumulative challenge was performed. The findings are important to consider when designing studies where allergen-induced urinary excretion of LTE4 is an outcome variable.

The alveolar macrophage (AM) constitutes an important link between pulmonary innate and adaptive immunity due to its antigen-presenting capacity and ability to express different immunomodulating mediators. The role of AMs in the pathogenesis of allergic inflammation has yet to be fully determined.To investigate clinical effects and any change in the AM phenotype pattern after inhalation of sub-clinical doses of allergen by asthmatic patients.Eight subjects with allergic asthma underwent repeated low-dose allergen provocations equivalent to 10% of PD20. AMs recovered with bronchoalveolar lavage (BAL) were characterized by flow cytometric analysis of adhesion molecules, co-stimulatory molecules and markers for AM population activation and heterogeneity.An allergic airway inflammation, sub-clinical in six out of eight subjects, was obtained after low-dose allergen provocations, as determined by increased airway methacholine reactivity, increased BAL fluid total cell and eosinophil counts and increased serum ECP levels. The AMs showed a post-challenge altered phenotype pattern with a decreased expression of CD11a, CD16, CD71 and HLA class I and an increased expression of CD11b and CD14. The AMs were positive for CD83 and a weak post-challenge increase in the CD83 expression was found.Repeated low-dose allergen exposure induces an allergic airway inflammation in asthmatic subjects. The inflammation is associated with an altered AM phenotype pattern, consistent with an influx of monocytes and a hypothetical increased accessory cell function in the airways, possibly contributing to the development and sustenance of airway inflammation in asthma.

It is generally accepted that the early asthmatic response to inhaled allergen is a result of IgE-mediated mast cell activation. In contrast, the underlying mechanism of the late asthmatic response is much less clear.In order to investigate the pattern of mediator release during the early and late asthmatic responses to allergen, measurements of the urinary excretion of the mast cell markers 9alpha,11beta-PGF2 and Ntau-methylhistamine were made. In addition, urinary levels of eosinophil protein X (EPX) and leukotriene E4 (LTE4) were measured.Twelve mild atopic asthmatics participated in the study. On the study day, pulmonary function was recorded at baseline and for 12 h after inhalation of allergen. Urine was collected prior to challenge and thereafter at 1 h intervals. Measurements of 9alpha, 11beta-PGF2 and LTE4 were made with enzyme-immunoassay, and levels of Ntau-methylhistamine and EPX were analysed with radioimmunoassay.All subjects developed both an early and late phase airway response. Within 1 h of the early peak airway response, there was a significant increase in the urinary concentrations (AUC/h) of 9alpha, 11beta-PGF2 (49.3 +/- 9.2 to 142.5 +/- 49.2; P0.001) Ntau-methylhistamine (10.4 +/- 1.4 to 19.5 +/- 1.4; P0.001) and LTE4 (43.7 +/- 5.9 to 105.9 +/- 21.3; P0.001). Levels of all three mediators were also significantly increased above baseline during the LAR to 79.4 +/- 9.5 (P0.01), 19.8 +/- 1.9 (P0.001) and 85.6 +/- 10.4 (P0.001), respectively. Levels of EPX remained unchanged during the early and late responses (39.2 +/- 10.2 to 37.5 +/- 18.5, 33.9 +/- 6.8).These results indicate that mast cell activation is a feature not only of the early but also the late asthmatic response. Finally, increased LTE4 supports the contribution of the leukotrienes to airway obstruction during both phases of the asthmatic response to allergen.

To monitor endogenous production of cysteinyl-containing leukotrienes, the end-metabolite leukotriene E4 (LTE4) was analysed in urine. Results obtained with a sensitive enzyme immunoassay (EIA), performed on crude urine samples correlated well with data obtained from a previously reported radioimmunoassay. Enzyme immunoassay analysis of unextracted urine was justified by an excellent agreement between analyses in crude samples and measurements achieved after purification on solid phase extraction followed by separation on reversed-phase high performance liquid chromatography. Moreover, LTE4 was stable in urine samples stored at -20 degrees C, for months without the addition of preservatives. The stability of LTE4 in urine was not improved by addition of the antioxidant 4-hydroxy-TEMPO and pH adjustment to 9. As assessed by EIA analysis in crude urine samples, baseline values for urinary leukotriene E4 were not significantly different between atopic asthmatic subjects and non-asthmatic individuals, and there was no diurnal variation in urinary excretion of LTE4 in healthy subjects. However, we confirmed earlier data on significantly higher basal levels of urinary LTE4 in aspirin-intolerant asthmatics. In addition, a post-challenge increase in urinary LTE4 levels was detected in association with allergen-induced airway obstruction in atopic asthmatics. The per cent increase in urinary LTE4 was similar, irrespective of whether the samples were purified or not prior to EIA. Thus, combined with random validation by high performance liquid chromatography, the strategy of direct EIA of serially diluted urine samples was found to be a good index of in vivo production of leukotrienes. This was further reinforced by the demonstration that pretreatment with the leukotriene biosynthesis inhibitor Bay x 1005 inhibited the post allergen-challenge increase in urinary LTE4, as shown both with unpurified and purified samples.

The ability of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) to inhibit the cyclo-oxygenase which catalyzes formation of prostaglandins appears to be central to the mechanisms involved in aspirin sensitivity. We have investigated whether the plasma levels of acetylsalicylic acid (ASA) and its main metabolite salicylic acid (SA) at the time of intolerance reactions correspond with the concentrations required for enzyme inhibition in vitro. Twelve aspirin-sensitive and 15 aspirin-tolerant subjects were followed during provocation with aspirin. ASA and SA concentrations in plasma were determined by HPLC. After oral provocation (up to 460 mg cumulative dose), the levels of ASA and SA in plasma were equivalent in aspirin-sensitive and aspirin-tolerant subjects. For the aspirin-sensitive subjects, at the time of adverse reaction, the concentration range was 2.9-33.3 microM for ASA and 18.1-245 microM for SA. Oral provocation with sodium salicylate yielding 10-fold higher SA levels did not elicit intolerance reactions. Statistically significantly lower levels of ASA and SA (P < or = 0.01) evoked airway obstruction, as compared with merely extrapulmonary symptoms. Bronchial absorption of aspirin was found after inhalation of lysine-aspirin and was comparable in asthmatic and nonasthmatic subjects. In three aspirin-sensitive subjects who developed airway obstruction, the plasma levels for ASA and SA were 0.9-2.6 microM and 0.0-6.7 microM, respectively. In conclusion, the plasma levels of ASA reached at the time of a positive reaction are of the magnitude known to inhibit cyclo-oxygenases. Neither differences in bioavailability of ASA nor the formation of SA seems to contribute to the aspirin-elicited reactions.

Drugs which block the action or formation of the cysteinyl leukotrienes (LTC4, LTD4 and LTE4) inhibit asthmatic responses evoked by allergen, exercise and cold dry air. The purpose of this study was to determine whether the specific leukotriene-receptor antagonist MK-0679 could block the airway obstruction induced by aspirin (acetylsalicylic acid (ASA)) in aspirin-intolerant asthmatics. Eight asthmatics (mean age 45 yrs), with an average history of asthma and ASA-sensitivity of about 10 yrs duration, were subjected to bronchial provocation with lysine-ASA. Baseline ASA-sensitivity was first determined in an open prestudy session by inhalation of cumulative doses of lysine-ASA to establish the dose of ASA decreasing forced expiratory volume in one second (FEV1) by 20% (PD20). Rechallenge with lysine-ASA was performed on two different occasions, 1 h after oral administration of placebo, or 750 mg of MK-0679, under double-blind conditions, in a randomized, cross-over design. Leukotriene formation was estimated by the measurement of urinary LTE4. The lysine-ASA challenge was highly reproducible (geometric mean for group PD20 being identical for the open prestudy and the placebo session), and was associated with a post-challenge increase in urinary LTE4. In contrast, after MK-0679, there was a rightward shift in the dose response relationship for all eight subjects (median shift being 4.4 fold), with three of the subjects failing to produce a 20% decrease in FEV1 despite inhalation of the highest dose of lysine-ASA feasible to deliver.(ABSTRACT TRUNCATED AT 250 WORDS)

The phase behaviour of a ceramide species containing C18-phytosphingosine and C24-fatty acid was studied by X-ray diffraction methods, in order to elucidate the packing principles of lipids with unequally long hydrocarbon chains. Six solid phases were observed. In five of them, the ceramide molecules have an extended, V-shaped conformation and pack in single layer arrangements with the sphingosine and fatty acid chains forming separate matrices. Differences between these phases are mainly due to thermotropic changes in packing efficiency and thus in tilt of the hydrocarbon chains. The chain packing undergoes transitions from triclinic (T|) to monoclinic (M|) and hexagonal, and between orthorhombic (O⊥) and hexagonal subcell arrangements, respectively. Only one case was observed, in which the molecules pack with their two chains parallelly stacked in a double layer arrangement in which the long fatty acid tails deeply interdigitate between the two opposite layer halves. In a natural membrane containing different lipids, however, long fatty acid tails probably arrange randomly and contribute to the formation of a liquid hydrocarbon zone in the center of the bilayer.

2-DL-Hydroxytetradecanoic acid is triclinic (PT) with a= 5.170, b= 5"385, c= 32.307 A, ~= 79.97, fl= 101" 14 and 7 = 121.92 °. The molecules are linked together as hydrogen-bonded dimers in which the chain axes are tilted 53 ° to the end group contact planes. The 0c-OH group is close to the plane of the carboxyl group which leads to a short intramolecular contact of 2"68 .~. The shortest intermolecular oxygenoxygen distance involving the hydroxyl group is 2.95/~,. The hydroxyl group is thus considered to be a hydrogen donor to two weak hydrogen bonds. The carbon chain has a sharp bend at carbon C(2) and the plane through the carboxyl group is perpendicular to the carbon chain plane.