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The U-BIOPRED study is a multicentre European study aimed at a better understanding of severe asthma. It included three steroid-treated adult asthma groups (severe nonsmokers (SAn group), severe current/ex-smokers (SAs/ex group) and those with mild–moderate disease (MMA group)) and healthy controls (HC group). The aim of this cross-sectional, bronchoscopy substudy was to compare bronchial immunopathology between these groups.In 158 participants, bronchial biopsies and bronchial epithelial brushings were collected for immunopathologic and transcriptomic analysis. Immunohistochemical analysis of glycol methacrylate resin-embedded biopsies showed there were more mast cells in submucosa of the HC group (33.6 mm−2) compared with both severe asthma groups (SAn: 17.4 mm−2, p−2, p=0.01) and with the MMA group (21.2 mm−2, p=0.01). The number of CD4+lymphocytes was decreased in the SAs/ex group (4.7 mm−2) compared with the SAn (11.6 mm−2, p=0.002), MMA (10.1 mm−2, p=0.008) and HC (10.6 mm−2, pAffymetrix microarray analysis identified seven probe sets in the bronchial brushing samples that had a positive relationship with submucosal eosinophils. These mapped toCOX-2(cyclo-oxygenase-2),ADAM-7(disintegrin and metalloproteinase domain-containing protein 7),SLCO1A2(solute carrier organic anion transporter family member 1A2),TMEFF2(transmembrane protein with epidermal growth factor like and two follistatin like domains 2) andTRPM-1(transient receptor potential cation channel subfamily M member 1); the remaining two are unnamed.We conclude that in nonsmoking and smoking patients on currently recommended therapy, severe asthma exists despite suppressed tissue inflammation within the proximal airway wall.

BACKGROUND Systematic assessment of severe asthma can be used to confirm the diagnosis identify comorbidities, and address adherence to therapy. However, the prospective usefulness of this approach is yet to be established. The objective of this study was to determine whether the systematic assessment of severe asthma is associated with improved quality of life (QoL) and health-care use and, using prospective data collection, to compare relevant outcomes in patients referred with severe asthma to specialist centers across the United Kingdom. METHODS Data from the National Registry for dedicated UK Difficult Asthma Services were used to compare patient demographics, disease characteristics, and health-care use between initial assessment and a median follow-up of 286 days. RESULTS The study population consisted of 346 patients with severe asthma. At follow-up, there were significant reductions in health-care use in terms of primary care or ED visits (66.4% vs 87.8%, P .0001) and hospital admissions (38% vs 48%, P = .0004). Although no difference was noted in terms of those requiring maintenance oral corticosteroids, there was a reduction in steroid dose (10 mg [8-20 mg] vs 15 mg [10-20 mg], P = .003), and fewer subjects required short-burst steroids (77.4% vs 90.8%, P = .01). Significant improvements were seen in QoL and control using the Asthma Quality of Life Questionnaire and the Asthma Control Questionnaire. CONCLUSIONS To our knowledge, this is the first time that a prospective study has shown that a systematic assessment at a dedicated severe asthma center is associated with improved QoL and asthma control and a reduction in health-care use and oral steroid burden.

Chronic cough is associated with airway inflammation and remodelling. Abnormal airway smooth muscle cell (ASMC) function may underlie mechanisms of chronic cough. Our objective was to examine the transcriptome and focused secretome of ASMCs from chronic cough patients and healthy non-cough volunteers. ASMC gene expression profiling was performed at baseline and/or after stimulation with polyinosinic:polycytidylic acid (poly(I:C)) to mimic viral infection. Supernatants were collected for multiplex analysis. Our results showed no significant differentially expressed genes (DEGs, false discovery rate (FDR) 1.5 fold-change, FDR

BACKGROUND: Asthma is a heterogeneous disease in which there is a differential response to asthma treatments. This heterogeneity needs to be evaluated so that a personalised management approach can be provided.OBJECTIVES: We stratified patients with moderate-to-severe asthma based on clinico-physiological parameters and performed an -omics analysis of sputum.METHODS: Partition-around-medoid clustering was applied to a training set of 266 asthma participants from the European U-BIOPRED adult cohort using 8 pre-specified clinic-physiological variables. This was repeated in a separate validation set of 152 asthmatics. The clusters were compared based on sputum proteomic and transcriptomic data.RESULTS: Four reproducible and stable clusters of asthmatics were identified. The training set cluster T1 consists of well-controlled moderate-to-severe asthmatics, while cluster T2 is a group of late-onset severe asthmatics with history of smoking and chronic airflow obstruction. Cluster T3 is similar to cluster T2 in terms of chronic airflow obstruction but is composed of non-smokers. Cluster T4 is predominantly composed of obese female uncontrolled severe asthmatics with increased exacerbations, but with normal lung function. The validation set exhibited similar clusters, demonstrating reproducibility of the classification. There were significant differences in sputum proteomics and transcriptomics between the clusters. The severe asthma clusters, T2, T3 and T4, had higher sputum eosinophilia than T1 with no differences in sputum neutrophil counts, exhaled nitric oxide and serum IgE levels.CONCLUSION: Clustering based on clinico-physiological parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.CLINICAL IMPLICATIONS: The definition of four distinct clusters of asthma linked to different pathobiological pathways provides a better template for the phenotyping and personalised treatment of severe asthma, where high unmet needs remain.CAPSULE SUMMARY: Unsupervised clustering of asthma on clinical features alone has led to the definition of four phenotypes. Sputum 'omics' analysis has revealed different biological pathways pointing towards potential new treatments.

A 54-year-old female presented with a 2-week history of increasing shortness of breath and fever. She had a history of a poorly differentiated sigmoid adenocarcinoma for which she underwent an anterior resection 6 months prior to admission, followed by 12 cycles of adjuvant FOLFOX chemotherapy. The patient was treated for a severe community-acquired pneumonia; however, she remained hypoxic. A chest CT revealed extensive right-sided fibrotic changes, tractional dilatation of the airways and ground glass density, which had developed since a staging CT scan performed 2 months previously. Although her symptoms improved with steroid therapy, repeat imaging revealed that right hydropneumothorax had developed, and this required the insertion of a chest drain. Following its successful removal, the patient continues to improve clinically and radiographically. The rapid onset and nature of these changes is consistent with a drug-induced fibrotic lung disease secondary to FOLFOX chemotherapy. The phenomenon is underreported and yet, it is relatively common: it occurs in approximately 10% of patients who are treated with antineoplastic agents, although information specifically relating to FOLFOX-induced pulmonary toxicity is limited. It is associated with significant morbidity and mortality, but is often hard to differentiate from other lung conditions, making the diagnosis a challenge. Pulmonary toxicity is an important complication associated with antineoplastic agents. It should be considered in any patient on a chemotherapeutic regimen who presents with dyspnoea and hypoxia in order to try to reduce the associated morbidity and mortality.

SummaryBackgroundThe presence of lipids in alveolar macrophages (AMs) may impair their phagocytic response, and determine airway inflammation and obstruction.ObjectiveTo determine the factors such as severity of asthma, chronic cough, airway inflammation and obesity that may influence the presence of lipids in lung macrophages.MethodsBronchoalveolar lavage fluid (BALF) was obtained from 38 asthmatics (21 severe and 17 mild/moderate), 16 subjects with chronic cough and 11 healthy control subjects. The presence of lipids in macrophages was detected using an Oil-red-O stain and an index of lipid-laden macrophages (LLMI) was obtained.ResultsLLMI scores were higher in healthy subjects (median 48 [IQR 10–61]) and the severe asthma group (37 [11.5–61]) compared to mild/moderate asthmatics (7 [0.5–37]; p

Background Obesity has emerged as a risk factor for the development of asthma and it may also influence asthma control and airway inflammation. However, the role of obesity in severe asthma remains unclear. Thus, our objective was to explore the association between obesity (defined by BMI) and severe asthma. Methods Data from the British Thoracic Society Difficult Asthma Registry were used to compare patient demographics, disease characteristics, and health-care utilization among three BMI categories (normal weight: 18.5-24.99; overweight: 25-29.99; obese: ≥ 30) in a well-characterized group of adults with severe asthma. Results The study population consisted of 666 patients with severe asthma; the group had a median BMI of 29.8 (interquartile range, 22.5-34.0). The obese group exhibited greater asthma medication requirements in terms of maintenance corticosteroid therapy (48.9% vs 40.4% and 34.5% in the overweight and normal-weight groups, respectively), steroid burst therapy, and short-acting β 2 -agonist use per day. Significant differences were seen with gastroesophageal reflux disease (53.9% vs 48.1% and 39.7% in the overweight and normal weight groups, respectively) and proton pump inhibitor use. Bone density scores were higher in the obese group, while pulmonary function testing revealed a reduced FVC and elevated carbon monoxide transfer coefficient. Serum IgE levels decreased with increasing BMI and the obese group was more likely to report eczema, but less likely to have a history of nasal polyps. Conclusions Patients with severe asthma display particular characteristics according to BMI that support the view that obesity-associated severe asthma may represent a distinct clinical phenotype.

Severe asthma is thought to be a heterogeneous disease with different phenotypes predicated primarily on the nature of the inflammatory cell infiltrate and response to corticosteroid therapy. This group of patients often has refractory disease with an associated increase in morbidity and mortality, and there remains a need for better therapies for severe asthmatics. Inflammatory changes in asthma are driven by immune mechanisms, within which interleukins play an integral role. Interleukins are cell-signaling cytokines that are produced by a variety of cells, predominantly T cells. Knowledge about their actions has improved the understanding of the pathogenesis of asthma and provided potential targets for novel therapies. To date, this has not translated into clinical use. However, there are ongoing clinical trials that use monoclonal antibodies for various interleukins, some of which have shown to be promising in Phase II studies.

Background: Although previous studies have implicated tissue CD4 + T cells in the development and maintenance of the inflammatory response in asthmatic patients, little is known about the role of CD8 + T cells. There is now accumulating evidence that microRNAs and other noncoding RNAs are important regulators of T-cell function. Objectives: We sought to use transcriptomics to determine the activation state of circulating CD4 + and CD8 + T cells in patients with nonsevere and severe asthma. Methods: mRNA and noncoding RNA expression in circulating T cells was measured by means of microarray, quantitative real-time PCR, or both. Results: Comparison of mRNA expression showed widespread changes in the circulating CD8 + but not CD4 + T cells from patients with severe asthma. No changes were observed in the CD4 + and CD8 + T cells in patients with nonsevere asthma versus those in healthy control subjects. Bioinformatics analysis showed that the changes in CD8 + T-cell mRNA expression were associated with multiple pathways involved in T-cell activation. As with mRNAs, we also observed widespread changes in expression of noncoding RNA species, including natural antisense, pseudogenes, intronic long noncoding RNAs (lncRNAs), and intergenic lncRNAs in CD8 + T cells from patients with severe asthma. Measurement of the microRNA expression profile showed selective downregulation of miR-28-5p in CD8 + T cells and reduction of miR-146a and miR-146b in both CD4 + and CD8 + T cells. Conclusions: Severe asthma is associated with the activation of circulating CD8 + T cells but not CD4 + T cells. This response is correlated with the downregulation of miR-146a/b and miR-28-5p, as well as changes in the expression of multiple species of lncRNA that might regulate CD8 + T-cell function. © 2011 American Academy of Allergy, Asthma & Immunology.

U-BIOPRED is a European Union consortium of 20 academic institutions, 11 pharmaceutical companies and six patient organisations with the objective of improving the understanding of asthma disease mechanisms using a systems biology approach.This cross-sectional assessment of adults with severe asthma, mild/moderate asthma and healthy controls from 11 European countries consisted of analyses of patient-reported outcomes, lung function, blood and airway inflammatory measurements.Patients with severe asthma (nonsmokers, n=311; smokers/ex-smokers, n=110) had more symptoms and exacerbations compared to patients with mild/moderate disease (n=88) (2.5 exacerbations versus 0.4 in the preceding 12 months; pversus 1.05%; p=0.004) despite treatment with higher doses of inhaled and/or oral corticosteroids.Consistent with other severe asthma cohorts, U-BIOPRED is characterised by poor symptom control, increased comorbidity and airway inflammation, despite high levels of treatment. It is well suited to identify asthma phenotypes using the array of “omic” datasets that are at the core of this systems medicine approach.

Hydrogen sulfide (H₂S) is a gas produced by respiratory cells including smooth muscle cells and may play a role as a cellular gasotransmitter. We evaluated whether H₂S levels in serum or sputum could represent a new biomarker of COPD in a cross-sectional study.H₂S levels in sputum and serum samples were measured using a sulfide-sensitive electrode in 64 patients with stable COPD (S-COPD), 29 COPD subjects during acute exacerbation (AE-COPD), 14 healthy smokers and 21 healthy non-smokers.Sputum H₂S levels in AE-COPD subjects were higher than those in S-COPD, healthy smoking and non-smoking subjects (p0.001), but serum H₂S levels in AE-COPD were lower than those in S-COPD (p0.001). Thus, the sputum-to-serum ratio of H₂S (H₂S ratio) in AE-COPD subjects were higher than those in stable COPD, healthy smoking and non-smoking subjects (p0.001). In 14 COPD subjects whose H₂S ratios were measured during and after an exacerbation, the mean ratio was increased during exacerbation (p0.05). H₂S ratio was positively correlated with St. George's Respiratory Questionnaire score, sputum neutrophils and IL-6 and IL-8 levels in sputum and serum (p0.01) but inversely correlated with sputum macrophages (%), FEV₁%predicted and FEV₁/FVC (p0.01). The cut-off level of H₂S ratio to indicate an exacerbation was ≥0.44 (sensitivity of 93.1% and specificity of 84.5%).The ratio of sputum-to-serum levels of H₂S may provide a useful marker of COPD indicative of obstructive neutrophilic inflammation and of potential ongoing exacerbation.

Asthma is a heterogeneous condition with multiple phenotypes that respond to treatments in different ways. Allergic asthma is an important phenotype and although currently available treatments are effective, about 5% of affected patients have severe, treatment-refractory disease. Despite advances in our understanding of the disease, there remains an unmet need in this group of patients. The most recent and significant advance in treatment has been anti--immunoglobulin E (IgE) therapy, which improves symptoms and quality of life in patients with severe allergic asthma. Clinical trials are ongoing with novel biologic agents that demonstrate potential efficacy; determining the subsets of patients for which they are suitable will be crucial to ensure cost effectiveness. Personalised medicine and targeted therapies may hold the key to long-term control in this group of patients.

Increased airway smooth muscle (ASM) mass is a feature of asthmatic airways, and could result from augmented proliferation. We determined whether proliferation and IL-6 release are abnormal in ASM cells (ASMCs) from patients with severe asthma, and whether these features could be mediated by microRNA-221 and microRNA-222, through modulation of the cyclin-dependent kinase inhibitors, p21(WAF1) and p27(kip1). ASMCs cultured from bronchial biopsies of healthy subjects and patients with nonsevere or severe asthma were studied. Proliferation was measured by the incorporation of bromodeoxyuridine and IL-6 by ELISA. FCS and transforming growth factor (TGF)-β caused greater proliferation and IL-6 release in patients with severe compared with nonsevere asthma and normal subjects. FCS + TGF-β inhibited p21(WAF1) and p27(kip1) expression, and increased microRNA-221 (miR-221) expression in ASMCs from individuals with severe asthma. miR-221, and not miR-222, mimics the increased proliferation and IL-6 release induced by FCS + TGF in healthy ASM, whereas in patients with severe asthma, the inhibition of miR-221, but not miR-222, inhibited proliferation and IL-6 release. miR-221 inhibition led to the increased expression of FCS + TGF-β-induced p21(WAF1) and p27(kip1). Dexamethasone suppressed proliferation in healthy subjects, but not in subjects with asthma. IL-6 was less suppressible by dexamethasone in patients with nonsevere and severe asthma, compared with healthy subjects. miR-221 did not influence the effects of dexamethasone. ASM from patients with severe asthma shows greater proliferation and IL-6 release than in patients with nonsevere asthma, but both groups show corticosteroid insensitivity. miR-221 regulates p21(WAF1) and p27(kip1) expression levels. Furthermore, miR-221 regulates the hyperproliferation and IL-6 release of ASMCs from patients with severe asthma, but does not regulate corticosteroid insensitivity.

Obesity has emerged as a risk factor for the development of asthma and it may also influence asthma control and airway inflammation. However, the role of obesity in severe asthma remains unclear. Thus, our objective was to explore the association between obesity (defied by BMI) and severe asthma.Data from the British Thoracic Society Difficult Asthma Registry were used to compare patient demographics, disease characteristics, and health-care utilization among three BMI categories (normal weight: 18.5-24.99; overweight: 25-29.99; obese: 30) in a well-characterized group of adults with severe asthma.The study population consisted of 666 patients with severe asthma; the group had a median BMI of 29.8 (interquartile range, 22.5-34.0). The obese group exhibited greater asthma medication requirements in terms of maintenance corticosteroid therapy (48.9% vs 40.4% and 34.5% in the overweight and normal-weight groups, respectively), steroid burst therapy, and short-acting b 2 -agonist use per day. Significant differences were seen with gastroesophageal reflux disease (53.9% vs 48.1% and 39.7% in the overweight and normal weight groups, respectively) and proton pump inhibitor use. Bone density scores were higher in the obese group, while pulmonary function testing revealed a reduced FVC and elevated carbon monoxide transfer coefficient. Serum IgE levels decreased with increasing BMI and the obese group was more likely to report eczema, but less likely to have a history of nasal polyps.Patients with severe asthma display particular characteristics according to BMI that support the view that obesity-associated severe asthma may represent a distinct clinical phenotype.

Rationale: Asthma is a chronic inflammatory airway disease. Microbial colonization of the respiratory tract may have a role in the chronic inflammatory process. Methods : We recruited 23 non-smoking patients with stable asthma and 9 healthy subjects in Guangzhou, and obtained induced sputum samples for 16S ribosomal RNA analysis. The V3-V5 regions of bacterial 16S rRNA genes were amplified and sequenced by 454 pyrosequencing. Sequences were assigned to bacterial taxa by comparing them with 16s rRNA sequences in the Ribosomal Database Project (BGI, Shenzhen). Results : Asthmatics with an ACT score of 22.8±1.8 had lower %FEV1 predicted (73.4 vs 99.4; pl0.001) and higher sputum eosinophils (24.0 % vs 0.5%; pl0.001). The species richness was significantly decreased in asthmatic patients compared with control subjects (85.0±20.6 vs 107.9±15.2 taxa; p=0.005). 16S rRNA sequencing detected bacteria containing 6 major phyla: Bacteroidetes, Firmicutes, Proteobacteria,Fusobacterium, Actinobacteria and Spirochaetes, with the first 3 accounting for g 90%of all sequences. Actinobacteria (2.2% vs 0.6%, P=0.02) were found more frequently in asthmatic samples. Conclusions : Sputum from asthmatic patients has an altered microbial composition with lower species richness in the respiratory tract compared with non-asthmatics. The corresponding biological effect on airway inflammation remains to be determined. Supported by State Key Laboratory of Respiratory Diseases, Guangzhou Medical University.

Summary Severe asthmatics often exhibit poor control despite high doses of inhaled corticosteroids with or without systemic corticosteroids and suffer from persistent symptoms and/or recurrent exacerbations. Five to ten percentage of the asthmatic population falls within this category. Patients with severe asthma are a heterogeneous group and should be investigated to confirm the diagnosis, identify comorbidities, exclude alternative diagnoses, together with an evaluation of treatment adherence and side-effects from medications. Optimization of asthma medications and monitoring the control and pattern of asthma usually takes place over a period of 6 months. In patients with confirmed severe refractory asthma, further evaluation is needed in terms of detailed lung function, of airway and lung structure using high resolution computed tomographic scanning, and of airway inflammatory processes and biomarkers using induced sputum or bronchial biopsies. Patients with severe asthma are best investigated and managed with a multidisciplinary team. Severe asthma consists of different phenotypes that need defining. Investigation of severe asthma should bring into the open the various characteristics of the disease that could point to particular phenotype. Inclusion of investigations based on transcriptomics and proteomics should expand, improve classification and understanding of severe asthma, with the ultimate hope of finding more effective treatments and a step towards personalized medicine.

Background: The airway smooth muscle (ASM) cell maintains its own proliferative rate and contributes to the inflammatory response in the airways, effects that are inhibited by corticosteroids, used in the treatment of airways diseases.Objective: We determined the differential expression of mRNAs, microRNAs (miRNAs) and long noncoding RNA species (lncRNAs) in primary ASM cells following treatment with a corticosteroid, dexamethasone, and fetal calf serum (FCS).Methods: mRNA, miRNA and lncRNA expression was measured by microarray and quantitative real-time PCR.Results: A small number of miRNAs (including miR-150, -371-5p, -718, -940, -1181, -1207-5p, -1915, and -3663-3p) were decreased following exposure to dexamethasone and FCS. The mRNA targets of these miRNAs were increased in expression. The changes in mRNA expression were associated with regulation of ASM actin cytoskeleton. We also observed changes in expression of lncRNAs, including natural antisense, pseudogenes, intronic lncRNAs, and intergenic lncRNAs following dexamethasone and FCS. We confirmed the change in expression of three of these, LINC00882, LINC00883, PVT1, and its transcriptional activator, c-MYC. We propose that four of these lincRNAs (RP11-46A10.4, LINC00883, BCYRN1, and LINC00882) act as miRNA 'sponges' for 4 miRNAs (miR-150, -371-5p, -940, -1207-5p).Conclusion: This in-vitro model of primary ASM cell phenotype was associated with the regulation of several ncRNAs. Their identification allows for in-vitro functional experimentation to establish causality with the primary ASM phenotype, and in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD).

Background: Bacteria are frequently cultured from sputum samples of severe asthma patients suggesting a defect in bacterial clearance from the airway. We measured the capacity of macrophages from patients with asthma to phagocytose bacteria. Methods: Phagocytosis of fluorescently-labelled polystyrene beads, Haemophilus influenzae or Staphylococcus aureus by broncholaveolar lavage alveolar macrophages (AM) and by monocyte-derived macrophages (MDM) from non-asthmatics, mild-moderate and severe asthmatic patients was assessed using fluorimetry. Results: There were no differences in phagocytosis of polystyrene beads by AMs or MDMs from any of the subject groups. There was reduced phagocytosis of Haemophilus influenzae and Staphylococcus aureus in MDMs from patients with severe asthma compared to non-severe asthma (p < 0.05 and p < 0.01, respectively) and healthy subjects (p < 0.01and p < 0.001, respectively). Phagocytosis of Haemophilus influenzae and Staphylococcus aureus by AM was also reduced in severe asthma compared to normal subjects (p < 0.05). Dexamethasone and formoterol did not suppress phagocytosis of bacteria by MDMs from any of the groups. Conclusions: Persistence of bacteria in the lower airways may result partly from a reduced phagocytic capacity of macrophages for bacteria. This may contribute to increased exacerbations, airway colonization and persistence of inflammation.