CEACAM6 as a Novel Therapeutic Target to Boost HO-1-mediated Antioxidant Defense in COPD.

Rationale: Tobacco smoking and air pollution are primary causes of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop COPD. The mechanisms underlying the defense against nitrosative/oxidative stress in nonsusceptible smokers to COPD remain largely unresolved. Objectives: To investigate the defense mechanisms against nitrosative/oxidative stress that possibly prevent COPD development or progression. Methods: Four cohorts were investigated: 1) sputum samples (healthy, n = 4; COPD, n = 37), 2) lung tissue samples (healthy, n = 13; smokers without COPD, n = 10; smoker+COPD, n = 17), 3) pulmonary lobectomy tissue samples (no/mild emphysema, n = 6), and 4) blood samples (healthy, n = 6; COPD, n = 18). We screened 3-nitrotyrosine (3-NT) levels, as indication of nitrosative/oxidative stress, in human samples. We established a novel in vitro model of a cigarette smoke extract (CSE)-resistant cell line and studied 3-NT formation, antioxidant capacity, and transcriptomic profiles. Results were validated in lung tissue, isolated primary cells, and an ex vivo model using adeno-associated virus-mediated gene transduction and human precision-cut lung slices. Measurements and Main Results: 3-NT levels correlate with COPD severity of patients. In CSE-resistant cells, nitrosative/oxidative stress upon CSE treatment was attenuated, paralleled by profound upregulation of heme oxygenase-1 (HO-1). We identified carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) as a negative regulator of HO-1-mediated nitrosative/oxidative stress defense in human alveolar type 2 epithelial cells (hAEC2s). Consistently, inhibition of HO-1 activity in hAEC2s increased the susceptibility toward CSE-induced damage. Epithelium-specific CEACAM6 overexpression increased nitrosative/oxidative stress and cell death in human precision-cut lung slices on CSE treatment. Conclusions: CEACAM6 expression determines the hAEC2 sensitivity to nitrosative/oxidative stress triggering emphysema development/progression in susceptible smokers.

Identification of extracellular vesicle microRNA signatures specifically linked to inflammatory and metabolic mechanisms in obesity-associated low type-2 asthma.

RATIONALE AND OBJECTIVE: Plasma extracellular vesicles (EVs) represent a vital source of molecular information about health and disease states. Due to their heterogenous cellular sources, EVs and their cargo may predict specific pathomechanisms behind disease phenotypes. Here we aimed to utilize EV microRNA (miRNA) signatures to gain new insights into underlying molecular mechanisms of obesity-associated low type-2 asthma. METHODS: Obese low type-2 asthma (OA) and non-obese low type-2 asthma (NOA) patients were selected from an asthma cohort conjointly with healthy controls. Plasma EVs were isolated and characterised by nanoparticle tracking analysis. EV-associated small RNAs were extracted, sequenced and bioinformatically analysed. RESULTS: Based on EV miRNA expression profiles, a clear distinction between the three study groups could be established using a principal component analysis. Integrative pathway analysis of potential target genes of the differentially expressed miRNAs revealed inflammatory cytokines (e.g., interleukin-6, transforming growth factor-beta, interferons) and metabolic factors (e.g., insulin, leptin) signalling pathways to be specifically associated with OA. The miR-17-92 and miR-106a-363 clusters were significantly enriched only in OA. These miRNA clusters exhibited discrete bivariate correlations with several key laboratory (e.g., C-reactive protein) and lung function parameters. Plasma EV miRNA signatures mirrored blood-derived CD4+ T-cell transcriptome data, but achieved an even higher sensitivity in identifying specifically affected biological pathways. CONCLUSION: The identified plasma EV miRNA signatures and particularly the miR-17-92 and -106a-363 clusters were capable to disentangle specific mechanisms of the obesity-associated low type-2 asthma phenotype, which may serve as basis for stratified treatment development.

IgA+ memory B-cells are significantly increased in patients with asthma and small airway dysfunction.

BACKGROUND: Comprehensive studies investigated the role of T-cells in asthma which led to personalised treatment options targeting severe eosinophilic asthma. However, little is known about the contribution of B-cells to this chronic inflammatory disease. In this study we investigated the contribution of various B-cell populations to specific clinical features in asthma. METHODS: In the All Age Asthma Cohort (ALLIANCE), a subgroup of 154 adult asthma patients and 28 healthy controls were included for B-cell characterisation by flow cytometry. Questionnaires, lung function measurements, blood differential counts and allergy testing of participants were analysed together with comprehensive data on B-cells using association studies and multivariate linear models. RESULTS: Patients with severe asthma showed decreased immature B-cell populations while memory B-cells were significantly increased compared with both mild-moderate asthma patients and healthy controls. Furthermore, increased frequencies of IgA+ memory B-cells were associated with impaired lung function and specifically with parameters indicative for augmented resistance in the peripheral airways. Accordingly, asthma patients with small airway dysfunction (SAD) defined by impulse oscillometry showed increased frequencies of IgA+ memory B-cells, particularly in patients with mild-moderate asthma. Additionally, IgA+ memory B-cells significantly correlated with clinical features of SAD such as exacerbations. CONCLUSIONS: With this study we demonstrate for the first time a significant association of increased IgA+ memory B-cells with asthma and SAD, pointing towards future options for B-cell-directed strategies in preventing and treating asthma.

T2-high asthma phenotypes across lifespan.

RATIONALE: In adults, personalised asthma treatment targets patients with type 2 (T2)-high and eosinophilic asthma phenotypes. It is unclear whether such classification is achievable in children. OBJECTIVES: To define T2-high asthma with easily accessible biomarkers and compare resulting phenotypes across all ages. METHODS: In the multicentre clinical All Age Asthma Cohort (ALLIANCE), 1125 participants (n=776 asthmatics, n=349 controls) were recruited and followed for 2 years (1 year in adults). Extensive clinical characterisation (questionnaires, blood differential count, allergy testing, lung function and sputum induction (in adults)) was performed at baseline and follow-ups. Interleukin (IL)-4, IL-5 and IL-13 were measured after stimulation of whole blood with lipopolysaccharide (LPS) or anti-CD3/CD28. MEASUREMENTS AND MAIN RESULTS: Based on blood eosinophil counts and allergen-specific serum IgE antibodies, patients were categorised into four mutually exclusive phenotypes: "atopy-only", "eosinophils-only", "T2-high" (eosinophilia + atopy) and "T2-low" (neither eosinophilia nor atopy). The T2-high phenotype was found across all ages, even in very young children in whom it persisted to a large degree even after 2 years of follow-up. T2-high asthma in adults was associated with childhood onset, suggesting early origins of this asthma phenotype. In both children and adults, the T2-high phenotype was characterised by excessive production of specific IgE to allergens (p<0.0001) and, from school age onwards, by increased production of IL-5 after anti-CD3/CD28 stimulation of whole blood. CONCLUSIONS: Using easily accessible biomarkers, patients with T2-high asthma can be identified across all ages delineating a distinct phenotype. These patients may benefit from therapy with biologicals even at a younger age.

Raised sputum extracellular DNA confers lung function impairment and poor symptom control in an exacerbation-susceptible phenotype of neutrophilic asthma.

BACKGROUND: Extracellular DNA (e-DNA) and neutrophil extracellular traps (NETs) are linked to asthmatics airway inflammation. However, data demonstrating the characterization of airway inflammation associated with excessive e-DNA production and its impact on asthma outcomes are limited. OBJECTIVE: To characterize the airway inflammation associated with excessive e-DNA production and its association with asthma control, severe exacerbations and pulmonary function, particularly, air trapping and small airway dysfunction. METHODS: We measured e-DNA concentrations in induced sputum from 134 asthma patients and 28 healthy controls. We studied the correlation of e-DNA concentrations with sputum neutrophils, eosinophils and macrophages and the fractional exhaled nitric oxide (FeNO). Lung function was evaluated using spirometry, body plethysmography, impulse oscillometry and inert gas multiple breath washout. We stratified patients with asthma into low-DNA and high-DNA to compare lung function impairments and asthma outcomes. RESULTS: Patients with severe asthma had higher e-DNA concentration (54.2 ± 42.4 ng/µl) than patients with mild-moderate asthma (41.0 ± 44.1 ng/µl) or healthy controls (26.1 ± 16.5 ng/µl), (all p values < 0.05). E-DNA concentrations correlated directly with sputum neutrophils (R = 0.49, p < 0.0001) and negatively with sputum macrophages (R = - 0.36, p < 0.0001), but neither with sputum eosinophils (R = 0.10, p = 0.26), nor with FeNO (R = - 0.10, p = 0.22). We found that 29% of asthma patients (n = 39) had high e-DNA concentrations above the upper 95th percentile value in healthy controls (55.6 ng /µl). High-DNA was associated with broad lung function impairments including: airflow obstruction of the large (FEV1) and small airways (FEF50%, FEF25-75), increased air trapping (RV, RV/TLC), increased small airway resistance (R5-20, sReff), decreased lung elasticity (X5Hz) and increased ventilation heterogeneity (LCI), (all P values < 0.05). We also found that high e-DNA was associated with nearly three-fold greater risk of severe exacerbations (OR 2·93 [95% CI 1.2-7.5]; p = 0·012), worse asthma control test (p = 0.03), worse asthma control questionnaire scores (p = 0.01) and higher doses of inhaled corticosteroids (p = 0.026). CONCLUSION: Increased production of extracellular DNA in the airway characterizes a subset of neutrophilic asthma patients who have broad lung function impairments, poor symptom control and increased risk of severe exacerbations.

Differential Regulation of Interferon Signaling Pathways in CD4+ T Cells of the Low Type-2 Obesity-Associated Asthma Phenotype.

In the era of personalized medicine, insights into the molecular mechanisms that differentially contribute to disease phenotypes, such as asthma phenotypes including obesity-associated asthma, are urgently needed. Peripheral blood was drawn from 10 obese, non-atopic asthmatic adults with a high body mass index (BMI; 36.67 ± 6.90); 10 non-obese, non-atopic asthmatic adults with normal BMI (23.88 ± 2.73); and 10 healthy controls with normal BMI (23.62 ± 3.74). All asthmatic patients were considered to represent a low type-2 asthma phenotype according to selective clinical parameters. RNA sequencing (RNA-Seq) was conducted on peripheral blood CD4+ T cells. Thousands of differentially expressed genes were identified in both asthma groups compared with heathy controls. The expression of interferon (IFN)-stimulated genes associated with IFN-related signaling pathways was specifically affected in obese asthmatics, while the gap junction and G protein-coupled receptor (GPCR) ligand binding pathways were enriched in both asthma groups. Furthermore, obesity gene markers were also upregulated in CD4+ T cells from obese asthmatics compared with the two other groups. Additionally, the enriched genes of the three abovementioned pathways showed a unique correlation pattern with various laboratory and clinical parameters. The specific activation of IFN-related signaling and viral infection pathways might provide a novel view of the molecular mechanisms associated with the development of the low type-2 obesity-associated asthma phenotype, which is a step ahead in the development of new stratified therapeutic approaches.

Exploration of the sputum methylome and omics deconvolution by quadratic programming in molecular profiling of asthma and COPD: the road to sputum omics 2.0.

BACKGROUND: To date, most studies involving high-throughput analyses of sputum in asthma and COPD have focused on identifying transcriptomic signatures of disease. No whole-genome methylation analysis of sputum cells has been performed yet. In this context, the highly variable cellular composition of sputum has potential to confound the molecular analyses. METHODS: Whole-genome transcription (Agilent Human 4 × 44 k array) and methylation (Illumina 450 k BeadChip) analyses were performed on sputum samples of 9 asthmatics, 10 healthy and 10 COPD subjects. RNA integrity was checked by capillary electrophoresis and used to correct in silico for bias conferred by RNA degradation during biobank sample storage. Estimates of cell type-specific molecular profiles were derived via regression by quadratic programming based on sputum differential cell counts. All analyses were conducted using the open-source R/Bioconductor software framework. RESULTS: A linear regression step was found to perform well in removing RNA degradation-related bias among the main principal components of the gene expression data, increasing the number of genes detectable as differentially expressed in asthma and COPD sputa (compared to controls). We observed a strong influence of the cellular composition on the results of mixed-cell sputum analyses. Exemplarily, upregulated genes derived from mixed-cell data in asthma were dominated by genes predominantly expressed in eosinophils after deconvolution. The deconvolution, however, allowed to perform differential expression and methylation analyses on the level of individual cell types and, though we only analyzed a limited number of biological replicates, was found to provide good estimates compared to previously published data about gene expression in lung eosinophils in asthma. Analysis of the sputum methylome indicated presence of differential methylation in genomic regions of interest, e.g. mapping to a number of human leukocyte antigen (HLA) genes related to both major histocompatibility complex (MHC) class I and II molecules in asthma and COPD macrophages. Furthermore, we found the SMAD3 (SMAD family member 3) gene, among others, to lie within differentially methylated regions which has been previously reported in the context of asthma. CONCLUSIONS: In this methodology-oriented study, we show that methylation profiling can be easily integrated into sputum analysis workflows and exhibits a strong potential to contribute to the profiling and understanding of pulmonary inflammation. Wherever RNA degradation is of concern, in silico correction can be effective in improving both sensitivity and specificity of downstream analyses. We suggest that deconvolution methods should be integrated in sputum omics analysis workflows whenever possible in order to facilitate the unbiased discovery and interpretation of molecular patterns of inflammation.

Small airway dysfunction as predictor and marker for clinical response to biological therapy in severe eosinophilic asthma: a longitudinal observational study.

BACKGROUND: Anti-T2 biological therapies have proven to effectively reduce acute exacerbations and daily doses of oral steroids in severe eosinophilic asthma. Despite the remarkable clinical efficacy, there are usually only moderate improvements in airflow limitation, suggesting that other measures of lung function like small airway dysfunction (SAD) might better reflect the clinical response. We aimed to investigate if measures of small airway function would predict and correlate with the clinical response to anti-T2 therapy. METHODS: We studied data of patients who were previously included in the German prospective longitudinal All Age Asthma Cohort (ALLIANCE) that recruits asthma patients of all severity grades and inflammatory phenotypes. The selection criteria for this analysis were adult patients with severe eosinophilic asthma under treatment with anti-T2 biological agents. Asthma control was assessed by asthma control test (ACT) and number of severe exacerbations. Small airway function was assessed by the frequency dependence of resistance (FDR, R5-20)) derived from impulse oscillometry (IOS) and the mean forced expiratory flow between 25 and 75% of the forced vital capacity (FEF25-75). We also studied air trapping (RV and RV/TLC), blood eosinophils and FeNO. Patients were classified into responders and partial or non-responders. Clinical response was defined as at least 50% reduction in annualized severe exacerbations and daily oral steroid doses accompanied with a minimum increase of 3 points in the ACT score. We used a Receiver Operator Characteristic (ROC) to study the capacity of FDR in predicting clinical response compared to other clinical variable like blood eosinophils. We studied the correlation between FDR measures and clinical response, represented by the ACT score and number of exacerbations, using linear regressions. RESULTS: 20 patients were included (mean age, 59 ± 9 years; 60% female; mean body mass index (BMI), 27.6 ± 5.4 kg/m2; mean absolute blood eosinophils, 570 ± 389/µl; mean number of severe exacerbations 12 months prior to initiating the biological therapy, 5.0 ± 3; mean predicted FEV1, 76 ± 21%; mean predicted FDR, 224 ± 140%; mean daily prednisolone dose, 6.4 ± 4.9 mg; mean ACT score, 15 ± 5). Responders had significantly higher baseline FDR compared to partial or non-responders but similar FEV1, FEF25-75, RV and RV/TLC. ROC analysis showed that the combination of FDR and blood eosinophils had the best predictive capacity of the clinical response among all tested clinical markers (FeNO, FEV1, FDR, blood eosinophils) with an AUC of 85% [67-100%], (CI = 0.95, p = 0.01). Linear regressions indicated better associations between improvements in FDR and ACT score (R2 = 0.42, p = 0.001) than with FEV1 and ACT score (R2 = 0.25, p = 0.013). Likewise, we observed better associations between improvements in FDR and reduction of exacerbations (R2 = 0.41, p = 0.001) than with FEV1 (R2 = 0.20, p = 0.025). CONCLUSION: Our data suggest that severe SAD may represent a distinct phenotype of eosinophilic asthma that substantially improves under anti-T2 biological therapy. Measures of small airway function might be useful in selecting appropriate patients qualifying for anti-T2 biological therapy in addition to blood eosinophil count.

Safety and efficacy of the human neutrophil elastase inhibitor BAY 85-8501 for the treatment of non-cystic fibrosis bronchiectasis: A randomized controlled trial.

BACKGROUND: There are only limited treatment options for patients with non-cystic fibrosis bronchiectasis (non-CF BE). Human neutrophil elastase (HNE) is a mediator of tissue destruction in non-CF BE. BAY 85-8501, a selective and reversible HNE inhibitor, could represent a new treatment option for this disease. METHODS: This was a phase 2a, randomized, placebo-controlled, double-blind, parallel-group study. The primary objective was to assess the safety and tolerability of 1 mg BAY 85-8501 once daily (OD) for 28 days compared with placebo in patients with non-CF BE. Secondary objectives were to investigate the effects of 4 weeks of treatment with BAY 85-8501 on health-related quality of life, pulmonary function, and inflammatory and tissue damage biomarkers in sputum, blood and/or urine, and to evaluate the pharmacokinetics of BAY 85-8501. RESULTS: Overall, 94 patients (mean age, 66 years; 53% male) were randomized (n = 47 per group), and 82 completed the study (BAY 85-8501, n = 37; placebo, n = 45). Treatment-emergent adverse events (TEAEs) occurred in 31 patients (66%) taking BAY 85-8501 and in 36 patients (77%) taking placebo, and were mostly mild or moderate. The serious TEAEs (BAY 85-8501, n = 3; placebo, n = 1) were not considered to be study-drug related. There were no changes in pulmonary function parameters from baseline to end of treatment, and health-related quality of life did not improve in any group. HNE activity in blood after zymosan challenge decreased significantly with BAY 85-8501 treatment (P = 0.0250 versus placebo). There were no significant differences in other biomarkers between treatment groups, with the exception of a small increase in interleukin-8 levels in sputum in the BAY 85-8501 group. Trough plasma concentrations of BAY 85-8501 plateaued after 2 weeks. CONCLUSIONS: 1 mg BAY 85-8501 OD had a favourable safety and tolerability profile when administered for 28 days to patients with non-CF BE. Further studies with a longer treatment duration are needed to evaluate the potential clinical efficacy in this study population.

A GATA3-specific DNAzyme attenuates sputum eosinophilia in eosinophilic COPD patients: a feasibility randomized clinical trial.

BACKGROUND: A subset of COPD-patients presents with eosinophilic airway inflammation. While treatment of asthmatic patients with the GATA3-specific DNAzyme SB010 attenuated sputum eosinophilia after allergen challenge, this specific treatment has not been evaluated in patients with COPD. Our objective was to evaluate the feasibility and safety of inhaled SB010 in COPD patients with sputum eosinophilia. METHODS: We conducted a randomized, double-blind, placebo-controlled, multicentre clinical trial in COPD-patients with sputum eosinophilia (≥2.5% non-squamous cells). Patients inhaled 10 mg SB010 bid or matching placebo via the controlled inhalation system AKITA2 APIXNEB for 28 days. Endpoints included the feasibility of the study (primary), patient's safety, sputum eosinophils, FENO, lung function, symptoms, and biomarkers. The study was registered in the German Clinical Trials Register: DRKS00006087. RESULTS: One hundred thirty patients were screened, 23 patients were randomized (FEV1 49.4 ± 11.5%; sputum eosinophils 8.0 ± 8.4%) and 19 patients completed the study (10 placebo, 9 SB010. After 28 days, SB010 decreased the relative sputum eosinophil count (p = 0.004) as compared to no changes in placebo-treated patients. FENO, lung function, and symptoms were not affected significantly. We found an increase in blood IFN-γ (p = 0.02) and a trend to lower IL-5 levels in patients treated with SB010. SB010 was safe and well tolerated. Thirty five AEs (22 SB010, 13 placebo including 1 SAE) were observed with 3 AEs in each group judged to be possibly treatment-related. CONCLUSION: In patients with eosinophilic COPD, sputum eosinophils could be reduced by inhalation of SB010. Long-term studies are needed to demonstrate clinical efficacy.

Multi-analyte profiling of inflammatory mediators in COPD sputum--the effects of processing.

Prior to using a new multi-analyte platform for the detection of markers in sputum it is advisable to assess whether sputum processing, especially mucus homogenization by dithiothreitol (DTT), affects the analysis. In this study we tested a novel Human Inflammation Multi Analyte Profiling® Kit (v1.0 Luminex platform; xMAP®). Induced sputum samples of 20 patients with stable COPD (mean FEV1, 59.2% pred.) were processed in parallel using standard processing (with DTT) and a more time consuming sputum dispersion method with phosphate buffered saline (PBS) only. A panel of 47 markers was analyzed in these sputum supernatants by the xMAP®. Twenty-five of 47 analytes have been detected in COPD sputum. Interestingly, 7 markers have been detected in sputum processed with DTT only, or significantly higher levels were observed following DTT treatment (VDBP, α-2-Macroglobulin, haptoglobin, α-1-antitrypsin, VCAM-1, and fibrinogen). However, standard DTT-processing resulted in lower detectable concentrations of ferritin, TIMP-1, MCP-1, MIP-1ß, ICAM-1, and complement C3. The correlation between processing methods for the different markers indicates that DTT processing does not introduce a bias by affecting individual sputum samples differently. In conclusion, our data demonstrates that the Luminex-based xMAP® panel can be used for multi-analyte profiling of COPD sputum using the routinely applied method of sputum processing with DTT. However, researchers need to be aware that the absolute concentration of selected inflammatory markers can be affected by DTT.

Inhaled pan-selectin antagonist Bimosiamose attenuates airway inflammation in COPD.

Selectins, a family of cell adhesion molecules, are involved in leukocyte extravasation to sites of inflammation. We investigated the safety and efficacy of the inhaled pan-selectin antagonist Bimosiamose in patients with chronic obstructive pulmonary disease (COPD). 77 COPD patients (mean forced expiratory volume in 1 s, 57% pred.) were enrolled in a cross-over, double-blind, randomized, Placebo-controlled, multi-center trial. Bimosiamose (10 mg) or Placebo was inhaled twice daily via the breath actuated nebulizer Akita2 Apixneb™ for 28 days on top of standard bronchodilator therapy. Efficacy was assessed by measurement of inflammatory parameters in induced sputum (differential cell count, interleukin-8, matrix-metalloproteinase-9, myeloperoxidase) and lung function at day 28 of both treatment periods. The total adverse event ratio of Bimosiamose compared to Placebo treatment was balanced. Compared to Placebo, treatment with Bimosiamose led to a decrease of the interleukin-8 concentration (-9.49 ng/mL, 95%CI -18.8 to -2.7 ng/mL, p = 0.008), for the neutrophil count a difference of -0.368 × 10(6) cells/mL (95%CI -1.256 to 0.407 × 10(6)/mL, p = 0.313) was found. The macrophage count decreased by -0.200 × 10(6) cells/mL (95%CI -0.365 to -0.044 × 10(6) cells/mL, p = 0.012). Most lung function parameters showed a small numeric increase. Inhalation of Bimosiamose for 28 days was safe and well tolerated in patients with COPD. It led to an attenuation of airway inflammation (EudraCT 2009-017257-35; NCT ID: NCT01108913).

Longitudinal Impact of Sputum Inflammatory Phenotypes on Small Airway Dysfunction and Disease Outcomes in Asthma.

BACKGROUND: Little is known about the relationship between airway inflammatory phenotypes and some important asthma features such as small airway dysfunction (SAD). OBJECTIVE: To describe the longitudinal impact of airway inflammatory phenotypes on SAD and asthma outcomes. METHODS: We measured eosinophil and neutrophil counts in induced sputum at baseline and 1 year later to stratify 197 adult patients with asthma into 4 inflammatory phenotypes. We conducted a comprehensive assessment of lung function using spirometry, body plethysmography, impulse oscillometry, and inert gas single and multiple breath washouts. We compared lung function, asthma severity, exacerbation frequency, and symptom control between the phenotypes. We studied the longitudinal impact of persistent sputum inflammatory phenotypes and the change of sputum cell counts on lung function. RESULTS: Patients were stratified into eosinophilic (23%, n = 45), neutrophilic (33%, n = 62), mixed granulocytic (22%, n = 43), and paucigranulocytic (24%, n = 47) phenotypes. Patients with eosinophilic and mixed granulocytic asthma had higher rates of airflow obstruction and severe exacerbation as well as poorer symptom control than patients with paucigranulocytic asthma. All SAD measures were worse in patients with eosinophilic and mixed asthma than in those with paucigranulocytic asthma (all P values <.05). Eosinophilic asthma also indicated worse distal airflow obstruction, increased ventilation inhomogeneity (all P values <.05), and higher tendency for severe exacerbation (P = .07) than neutrophilic asthma. Longitudinally, persistent mixed granulocytic asthma was associated with the worst follow-up measures of SAD compared with persistent neutrophilic, persistent paucigranulocytic, or nonpersistent asthma phenotypes. In patients with stable forced expiratory volume in 1 second (FEV1), the mean increase in small airway resistance (R5-20) was greater in patients with persistent mixed granulocytic asthma (+103%) than in patients with persistent neutrophilic (+26%), P = .040, or persistent paucigranulocytic asthma (-41%), P = .028. Multivariate models adjusted for confounders and treatment with inhaled or oral corticosteroids or antieosinophilic biologics indicated that the change of sputum eosinophil rather than neutrophil counts is an independent predictor for the longitudinal change in FEV1, forced expiratory flow at 25% to 75% of forced vital capacity, specific effective airway resistance, residual lung volume, and lung clearance index. CONCLUSIONS: In asthma, airway eosinophilic inflammation is the main driver of lung function impairment and poor disease outcomes, which might also be aggravated by the coexistence of airway neutrophilia to confer a severe mixed granulocytic asthma phenotype. Persistent airway eosinophilia might be associated with dynamic SAD even in patients with stable FEV1.

Efficacy of the pan-selectin antagonist Bimosiamose on ozone-induced airway inflammation in healthy subjects--a double blind, randomized, placebo-controlled, cross-over clinical trial.

Selectins, a family of cell adhesion molecules, are involved in the activation and extravasation of leukocytes in inflammatory diseases. Inhalation of ozone induces an inflammation of the airways, which is dominated by neutrophils. We investigated the effect of repeated inhalations of the pan-selectin antagonist Bimosiamose on ozone-induced airway inflammation in healthy volunteers. In a double-blind, placebo-controlled, randomized, cross-over study Bimosiamose (10 mg bid) was inhaled via a breath actuated nebulizer (AKITA2 APIXNEB(®)) for 4 days. Treatment was followed by inhalation of ozone (250 ppb) for 3 h with intermittent exercise. Induced sputum was collected 3 h post ozone challenge for analysis of cellular and non-cellular composition. 18 subjects were randomized and completed the study. All treatments were safe and well tolerated. Compared to placebo Bimosiamose reduced the numbers of sputum neutrophils by 40% (p = 0.068) and concentrations of interleukin-8 and matrix-metalloproteinase-9 in sputum supernatant by 35% (p = 0.004) and 46% (p = 0.022), respectively. Inhalation of Bimosiamose showed favourable anti-inflammatory effects on ozone-induced airway inflammation in healthy volunteers. Further studies have to proof and translate this anti-inflammatory effect of Bimosiamose into a clinical benefit in patients with chronic obstructive pulmonary disease. (ClinTrialgov Ident: NCT01108913).

Influence of Cell Quality on Inflammatory Biomarkers in COPD Sputum Supernatant.

PURPOSE: We recently introduced a sputum cell quality score to rate how cell morphology, cellular debris and squamous cell contamination influence inflammatory cell identification during microscopic evaluation. However, sputum cell quality is generally not considered for the interpretation of sputum fluid phase biomarkers. Therefore, we compared the soluble protein concentrations between sputum samples with different cell quality. The impact of cell quality was compared to other factors potentially affecting soluble biomarker concentrations. METHODS: A comprehensive sputum dataset from 154 clinically stable COPD patients was used to analyse the differences and the variability of sputum supernatant concentrations for 23 proteins between low, medium, and high sputum cell quality samples. A model was developed and tested to compare the impact of different factors on sputum supernatant protein levels. RESULTS: Mean percentages of sputum macrophages, neutrophils, eosinophils, monocytes and lymphocytes showed no significant differences between low, medium and high cell quality levels. The mean percentage of squamous cells were lower, while total cell count/mL sputum and cell viability were significantly higher in sputum samples with higher cell quality. The concentrations of Interleukin-6, Interleukin-8 and Tumor Necrosis Factor Receptor 2 were significantly increased in sputum samples of higher cell quality. The variability of most protein concentrations declined with increasing cell quality levels. Sixteen proteins showed significantly negative correlations with the percentage of squamous cells. For 14 proteins we observed a positive correlation with cell number/mL sputum. Multiple regression analysis shows that generally less than 30% of the protein variability can be explained by the included factors. CONCLUSION: Sputum cell quality has a significant impact on some soluble biomarker concentrations in sputum supernatant. Sputum samples with low sputum cell quality show a higher variability of fluid phase proteins in comparison to medium and high sputum cell quality levels.

The Relevance of Small Airway Dysfunction in Asthma with Nocturnal Symptoms.

RATIONALE: Small airway dysfunction (SAD) is a frequent feature of asthma that has been linked to disease severity and poor symptom control. However, little is known about the role of SAD in nocturnal asthma. OBJECTIVE: To study the association between the severity of SAD and frequency of nocturnal symptoms compared to conventional lung function testing. METHODS: We assessed the frequency of self-reported nocturnal symptoms through the asthma control test. We studied the impact of nocturnal asthma using the Asthma Quality of Life Questionnaire (AQLQ) and the Multidimensional Fatigue Inventory (MFI-20). We assessed the lung function using spirometry, body plethysmography, impulse oscillometry, single and multiple inert gas washout and measured markers of T2-inflammation (blood and sputum eosinophils; fractional exhaled nitric oxide (FeNo)). We stratified the patients according to the presence and frequency of nocturnal asthma. RESULTS: A total of 166 asthma patients were enrolled in the analysis. Eighty-seven patients (52%) reported to have nocturnal symptoms at least once in the last four weeks. The odds ratio of nocturnal asthma correlated with the severity of all non-spirometric measures of SAD, yet neither with airflow obstruction (FEV1 and FEV/FVC) nor with large airway resistance (R20). Patients with frequent nocturnal asthma (n = 29) had a numerical increase of T2 markers and more severe SAD, as indicated by all non-spirometric measures of SAD (all p-values < 0.05), worse overall asthma control, increased fatigue and reduced quality of life (all p-values < 0.01) compared to patients with infrequent nocturnal asthma (n = 58) or patients without nocturnal asthma (n = 79). We identified 63 patients without airflow obstruction, nearly 43% of them (n = 27) had nocturnal asthma. In this subgroup, only markers of air trapping and ventilation heterogeneity were significantly elevated and correlated with the frequency of nocturnal symptoms: LCI (Spearman's coefficient = -0.42, p < 0.001), RV% (-0.32, p = 0.02). CONCLUSION: SAD is closely associated to asthma with nocturnal symptoms. Spirometry might underestimate the broad spectrum of distal lung function impairments in this population of patients.