17q21 Variants Disturb Mucosal Host Defense in Childhood Asthma.

Rationale: The strongest genetic risk factor for childhood-onset asthma, the 17q21 locus, is associated with increased viral susceptibility and disease-promoting processes.Objectives: To identify biological targets underlying the escalated viral susceptibility associated with the clinical phenotype mediated by the 17q21 locus.Methods: Genome-wide transcriptome analysis of nasal brush samples from 261 children (78 healthy, 79 with wheezing at preschool age, 104 asthmatic) within the ALLIANCE (All-Age-Asthma) cohort, with a median age of 10.0 (range, 1.0-20.0) years, was conducted to explore the impact of their 17q21 genotype (SNP rs72163891). Concurrently, nasal secretions from the same patients and visits were collected, and high-sensitivity mesoscale technology was employed to measure IFN protein levels.Measurements and Main Results: This study revealed that the 17q21 risk allele induces a genotype- and asthma/wheeze phenotype-dependent enhancement of mucosal GSDMB expression as the only relevant 17q21-encoded gene in children with preschool wheeze. Increased GSDMB expression correlated with the activation of a type-1 proinflammatory, cell-lytic immune, and natural killer signature, encompassing key genes linked to an IFN type-2-signature (IFNG, CXCL9, CXCL10, KLRC1, CD8A, GZMA). Conversely, there was a reduction in IFN type 1 and type 3 expression signatures at the mRNA and protein levels.Conclusions: This study demonstrates a novel disease-driving mechanism induced by the 17q21 risk allele. Increased mucosal GSDMB expression is associated with a cell-lytic immune response coupled with compromised airway immunocompetence. These findings suggest that GSDMB-related airway cell death and perturbations in the mucosal IFN signature account for the increased vulnerability of 17q21 risk allele carriers to respiratory viral infections during early life, opening new options for future biological interventions.The All-Age-Asthma (ALLIANCE) cohort is registered at www.clinicaltrials.gov (pediatric arm, NCT02496468).

Urinary eosinophil-derived neurotoxin is associated with reduced lung function in pediatric asthma.

INTRODUCTION: Eosinophil-derived neurotoxin (EDN) is a biomarker for eosinophilic activation. Urinary (u) EDN may allow non-invasive monitoring of asthma, but clinical recommendations are lacking. We assessed the potential of uEDN as a marker of disease activity in pediatric asthma. METHODS: We assessed urine samples of 371 children from the German ALLIANCE study cohort, from which we had: 169 preschool wheezers (<6 years), 80 asthmatics (≥6 years), and 122 healthy controls using the ImmunoCAP™ EDN Assay. Creatinine (Cr)-adjusted uEDN values were analyzed using correlations, association tests, (non) parametric statistics, multiple linear, and multivariable regression. RESULTS: uEDN/uCr values were higher in atopic versus non-atopic preschool-aged subjects (p = .035) and associated with the sum of allergen-specific IgE in younger (r = 0.24, p = .003), and older subjects (r = 0.23, p = .043). uEDN/uCr was marginally a good determinant for atopy (p = .078, for subjects aged <6 years, and p = .058 for subjects ≥6 years). Children with the T2-high phenotype had higher uEDN/uCr (p < .001) versus T2-low-irrespective of using uEDN/uCr or blood eosinophils in combination to allergen sIgE for disease phenotyping. uEDN/uCr significantly correlated with reduced lung function among asthmatics (FEV1 z-scores: r = -0.30, p = .007, and FEV1/FVC z-scores: r = -0.24, p = .038). Using multivariable modeling, uEDN/uCr was an independent determinant of FEV1 (p = .038), and to a lesser extent, FEV1/FVC (p = .080). CONCLUSIONS: uEDN/uCr may serve as a non-invasive biomarker for clinical features such as lung function in pediatric asthma. We highlight the utility of uEDN/uCr as a biomarker that can be easily assessed using widely available robust diagnostic immunoassays.

Impact of Heated Tobacco Products, E-Cigarettes, and Cigarettes on Inflammation and Endothelial Dysfunction.

In addition to the market launch of heated tobacco products (HTPs) and the JUUL as well as the EVALI, they caused a widespread discussion on the risk reduction compared to a combustible cigarette. Furthermore, first data showed harmful effects on the cardiovascular system. We, therefore, conducted investigations including a control group with a nicotine-free liquid. Forty active smokers were studied in two different approaches during and after consuming an HTP, a cigarette, a JUUL, or a typical electronic cigarette with or without nicotine in a partly double-blinded randomised, cross-over trial. Inflammation, endothelial dysfunction, and blood samples (full blood count, ELISA, multiplex immunoassay) were analysed, and arterial stiffness was measured. In addition to the cigarette, an increase in the white blood cell count but also in proinflammatory cytokines was shown for the various nicotine delivery systems. These correlated with the parameters of arterial vascular stiffness as a clinical parameter of endothelial dysfunction. It can be shown that even a single consumption of the different nicotine delivery system or cigarette leads to a significant inflammatory reaction followed by endothelial dysfunction and increased arterial stiffness causing cardiovascular disease. Inflammation, endothelial dysfunction, and arterial stiffness should be addressed in long-term observational studies.

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.

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.

Two Beer(s) or Not Two Beer(s): The eNose as an Instrument to Pacify the World.

BACKGROUND: Science prizes that are not meant to be very serious, stand-up evenings, science slams or publications with a scientific twist: science comedy comes in very different forms. But all variants have one thing in common: humour. It can be used to hide the seriousness of life or, in this case, everyday scientific life for a brief moment. Moreover, serious social or ethical questions are also met. The GPP, a group of German, Austrian and Swiss Pediatric Pulmonologists (GPP) is a scientific society with regular annual meetings. Unsystematic observations and preliminary data suggest that beer consumption increased by some of the participants during this event. Recently, electronic nose (eNose) devices have been developed as a technology for disease screening using exhaled-breath analysis. Here we addressed the issue, if the eNose can be used to differentiate between real beer and fake beer. METHODS: In this single-centre experimental study, 12 different "real beer" types and one "fake beer" were analyzed with regard to their emittance of volatile organic compounds (VOCs) with the eNose as an electronic VOC-sensing technology. RESULTS: Every single beer type can be identified by a characteristic VOC-smell print using the eNose. Distinct clusters exist for bottom- and top-fermented ales. Intriguingly, "Sylter Hopfen", which is marketed as a "champagne-beer" and tested as representative of a "fake beer", can be clearly differentiated from all other genuine beer types. CONCLUSION: Our study provides the first objective data of beer flavor. In the long term perspective the eNose might help to overcome the agonizing controversy about beer flavors and, consequently, pacify the World. In the short run, however, our results give support to more targeted and reserved beer consumption during our annual meeting, especially since one specific beer shows a very similar pattern to indoor air. HINTERGRUND UND FRAGESTELLUNG: Sogenannte Stand-up-Abende, Science Slams oder Publikationen wie in der «Christmas-Edition¼ des "British Medical Journals" haben eines gemeinsam: Humor. Humor kann dabei helfen, der Ernsthaftigkeit des Alltags - auch der des Wissenschaftlers - für einen kurzen Moment zu entfliehen. Die wissenschaftliche Gesellschaft Pädiatrische Pneumologie (GPP e. V.) ist eine Gruppe deutscher, österreichischer und schweizer Kinderpneumolog:innen, die sich regelmässig zu ihrer Jahrestagung treffen. Nicht-systematisch erhobene Daten und persönliche Beobachtungen deuten darauf hin, dass der Bierkonsum von einigen der Teilnehmer:innen während dieser Veranstaltung signifikant ansteigt. Vor kurzem wurde mit der «eNose¼ eine sogenannte «elektronische Nase¼ entwickelt, die als Screening-Instrument zur Atemgasanalyse eingesetzt werden kann. Hier haben wir die Frage gestellt, ob die eNose verwendet werden kann, um unterschiedliche Biersorten zu unterscheiden und echte Biere von sogenannten «Fake-Bieren¼ zu diskriminieren. METHODEN: In dieser monozentrischen, experimentellen Studie wurden 12 verschiedene "echte Biersorten" und ein "Fake-Bier" hinsichtlich ihrer Emission flüchtiger organischer Verbindungen (VOCs) mit der eNose als elektronische VOC-Sensortechnologie analysiert. ERGEBNISSE: Jede einzelne Biersorte lässt sich anhand eines charakteristischen, reproduzierbaren VOC-Profils mit der eNose identifizieren. Dabei clustern sogenannte unter- und obergärige Biere mit einem spezifischen Muster. "Sylter Hopfen", das als "Champagner-Bier" vermarktet und als «Fake-Bier¼ getestet wurde, unterscheidet sich in seinem VOC-Profil von allen anderen «echten¼ Biersorten. SCHLUSSFOLGERUNG: Unsere Studie liefert die ersten objektiven Daten zu spezifischen VOC-Mustern von verschiedenen Biersorten. Langfristig könnte die eNose dabei helfen, die emotionale Kontroverse um Bieraromen zu überwinden und damit die Welt zu befrieden. Kurzfristig stützen unsere Ergebnisse die Empfehlung nach einem zurückhaltenden Bierkonsum während unserer Jahrestagung, zumal spezifischen VOC-Mustern einiger Biere ein sehr ähnliches Muster wie Raumluft zeigen.

COL4A3 is degraded in allergic asthma and degradation predicts response to anti-IgE therapy.

BACKGROUND: Asthma is a heterogeneous syndrome substantiating the urgent requirement for endotype-specific biomarkers. Dysbalance of fibrosis and fibrolysis in asthmatic lung tissue leads to reduced levels of the inflammation-protective collagen 4 (COL4A3). OBJECTIVE: To delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response. METHODS: The serological COL4A3 degradation marker C4Ma3 (Nordic Bioscience, Denmark) and serum cytokines were measured in the ALLIANCE cohort (paediatric cases/controls: n=134/n=35; adult cases/controls: n=149/n=31). Exacerbation of allergic airway disease in mice was induced by sensitising to ovalbumin (OVA), challenge with OVA aerosol and instillation of poly(cytidylic-inosinic). Fulacimstat (chymase inhibitor; Bayer) was used to determine the role of mast cell chymase in COL4A3 degradation. Patients with cystic fibrosis (n=14) and cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA; n=9) as well as patients with severe allergic uncontrolled asthma (n=19) were tested for COL4A3 degradation. Omalizumab (anti-IgE) treatment was assessed using the Asthma Control Test. RESULTS: Serum levels of C4Ma3 were increased in asthma in adults and children alike and linked to a more severe, exacerbating allergic asthma phenotype. In an experimental asthma mouse model, C4Ma3 was dependent on mast cell chymase. Serum C4Ma3 was significantly elevated in cystic fibrosis plus ABPA and at baseline predicted the success of the anti-IgE therapy in allergic, uncontrolled asthmatics (diagnostic OR 31.5). CONCLUSION: C4Ma3 levels depend on lung mast cell chymase and are increased in a severe, exacerbating allergic asthma phenotype. C4Ma3 may serve as a novel biomarker to predict anti-IgE therapy response.

Allergen extract- and component-based diagnostics in children of the ALLIANCE asthma cohort.

BACKGROUND: Current in vitro allergen-specific IgE (sIgE) detection assays measure IgE against allergen extracts or molecules in a single- or multiplex approach. Direct comparisons of the performance of such assays among young children with common presentations of allergic diseases regardless of sensitization status are largely missing. OBJECTIVES: The aim of this study was a comparison of the analytical and diagnostic performance for common clinical questions of three commonly used technologies which rely upon different laboratory methodologies among children of the All Age Asthma (ALLIANCE) cohort (clinicaltrials.gov: NCT02496468). METHODS: Sera from 106 paediatric study participants (mean age 4 years) were assessed for the presence of sIgE by means of the ImmunoCAP™ sx1 and fx5 mixes, the ImmunoCAP ISAC™ 112 microarray and a Euroline™ panel. RESULTS: Total and negative concordance was high (>82%->89%), while positive concordance varied considerably (0%-100%) but was also >50% for the most common sensitizations analysed (house dust mite and birch). All three test systems showed good sensitivity and specificity (AUC consistently > 0.7). However, no significant differences with regard to identifying sIgE sensitizations associated with symptoms in children with suspected pollen- or dust-triggered wheeze or presenting with symptoms of allergic rhinoconjunctivitis or food allergy were detected. Extending the number of allergens did not change the similar performance of the three assay systems. CONCLUSION AND CLINICAL RELEVANCE: Among young children, the three sIgE assays showed good analytical and diagnostic concordance. Our results caution that the identification of larger numbers of sensitizations by more comprehensive multiplex approaches may not improve the clinical utility of sIgE testing in this age group.

Differential DNA methylation in bronchial biopsies between persistent asthma and asthma in remission.

Approximately 40% of asthmatics experience remission of asthma symptoms. A better understanding of biological pathways leading to asthma remission may provide insight into new therapeutic targets for asthma. As an important mechanism of gene regulation, investigation of DNA methylation provides a promising approach. Our objective was to identify differences in epigenome wide DNA methylation levels in bronchial biopsies between subjects with asthma remission and subjects with persistent asthma or healthy controls.We analysed differential DNA methylation in bronchial biopsies from 26 subjects with persistent asthma, 39 remission subjects and 70 healthy controls, using the limma package. The comb-p tool was used to identify differentially methylated regions. DNA methylation of CpG-sites was associated to expression of nearby genes from the same biopsies to understand function.Four CpG-sites and 42 regions were differentially methylated between persistent asthma and remission. DNA methylation at two sites was correlated i n cis with gene expression at ACKR2 and DGKQ Between remission subjects and healthy controls 1163 CpG-sites and 328 regions were differentially methylated. DNA methylation was associated with expression of a set of genes expressed in ciliated epithelium.CpGs differentially methylated between remission and persistent asthma identify genetic loci associated with resolution of inflammation and airway responsiveness. Despite the absence of symptoms, remission subjects have a DNA methylation profile that is distinct from that of healthy controls, partly due to changes in cellular composition, with a higher gene expression signal related to ciliated epithelium in remission versus healthy controls.

Lost in transition: biomarkers of remodeling in patients with asthma.

PURPOSE OF REVIEW: 'Biomarkers of remodeling' represent a loose collection of features referring to several biological adaptations of the lung to cope with stressing factors. In addition, remodel-'ing' infers a dynamic process that would require a spatiotemporal resolution. This review focuses on different aspects of remodeling in pediatric and adult care. RECENT FINDINGS: This review will cover aspects of pediatric remodeling, adult remodeling and techniques and procedures to adequately assess remodeling across different age spectra. In pediatrics, the onset and first features of remodeling are discussed and the continuation into adolescence is addressed. For adults, this review addresses predominant features of remodeling throughout the adult life span and whether there are currently interventions available to treat or reverse remodeling. SUMMARY: The term 'remodeling' is often referred to via biomarkers that reflect the endstage of a process, although it rather reflects a continuous process starting in childhood and progressing to all age-levels in patients with asthma. Hence, only few biomarkers or surrogates are able to 'capture' its spatiotemporal component, and hardly any are ready for routine use in clinical practice. Given the clinical impact of the remodeling processes, new biomarkers are needed to adequately treat patients with asthma and objectively monitor treatment response beyond symptom control and lung function.

Novel therapeutic roles for surfactant-inositols and -phosphatidylglycerols in a neonatal piglet ARDS model: a translational study.

The biological and immune-protective properties of surfactant-derived phospholipids and phospholipid subfractions in the context of neonatal inflammatory lung disease are widely unknown. Using a porcine neonatal triple-hit acute respiratory distress syndrome (ARDS) model (repeated airway lavage, overventilation, and LPS instillation into airways), we assessed whether the supplementation of surfactant (S; poractant alfa) with inositol derivatives [inositol 1,2,6-trisphosphate (IP3) or phosphatidylinositol 3,5-bisphosphate (PIP2)] or phosphatidylglycerol subfractions [16:0/18:1-palmitoyloleoyl-phosphatidylglycerol (POPG) or 18:1/18:1-dioleoyl-phosphatidylglycerol (DOPG)] would result in improved clinical parameters and sought to characterize changes in key inflammatory pathways behind these improvements. Within 72 h of mechanical ventilation, the oxygenation index (S+IP3, S+PIP2, and S+POPG), the ventilation efficiency index (S+IP3 and S+POPG), the compliance (S+IP3 and S+POPG) and resistance (S+POPG) of the respiratory system, and the extravascular lung water index (S+IP3 and S+POPG) significantly improved compared with S treatment alone. The inositol derivatives (mainly S+IP3) exerted their actions by suppressing acid sphingomyelinase activity and dependent ceramide production, linked with the suppression of the inflammasome nucleotide-binding domain, leucine-rich repeat-containing protein-3 (NLRP3)-apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-caspase-1 complex, and the profibrotic response represented by the cytokines transforming growth factor-ß1 and IFN-γ, matrix metalloproteinase (MMP)-1/8, and elastin. In addition, IκB kinase activity was significantly reduced. S+POPG and S+DOPG treatment inhibited polymorphonuclear leukocyte activity (MMP-8 and myeloperoxidase) and the production of interleukin-6, maintained alveolar-capillary barrier functions, and reduced alveolar epithelial cell apoptosis, all of which resulted in reduced pulmonary edema. S+DOPG also limited the profibrotic response. We conclude that highly concentrated inositol derivatives and phosphatidylglycerol subfractions in surfactant preparations mitigate key inflammatory pathways in inflammatory lung disease and that their clinical application may be of interest for future treatment of the acute exudative phase of neonatal ARDS.

Profiling of healthy and asthmatic airway smooth muscle cells following interleukin-1ß treatment: a novel role for CCL20 in chronic mucus hypersecretion.

Chronic mucus hypersecretion (CMH) contributes to the morbidity and mortality of asthma, and remains uncontrolled by current therapies in the subset of patients with severe, steroid-resistant disease. Altered cross-talk between airway epithelium and airway smooth muscle cells (ASMCs), driven by pro-inflammatory cytokines such as interleukin (IL)-1ß, provides a potential mechanism that influences CMH. This study investigated mechanisms underlying CMH by comparing IL-1ß-induced gene expression profiles between asthma and control-derived ASMCs and the subsequent paracrine influence on airway epithelial mucus production in vitroIL-1ß-treated ASMCs from asthmatic patients and healthy donors were profiled using microarray analysis and ELISA. Air-liquid interface (ALI)-cultured CALU-3 and primary airway epithelial cells were treated with identified candidates and mucus production assessed.The IL-1ß-induced CCL20 expression and protein release was increased in ASMCs from moderate compared with mild asthmatic patients and healthy controls. IL-1ß induced lower MIR146A expression in asthma-derived ASMCs compared with controls. Decreased MIR146A expression was validated in vivo in bronchial biopsies from 16 asthmatic patients versus 39 healthy donors. miR-146a-5p overexpression abrogated CCL20 release in ASMCs. CCL20 treatment of ALI-cultured CALU-3 and primary airway epithelial cells induced mucus production, while CCL20 levels in sputum were associated with increased levels of CMH in asthmatic patients.Elevated CCL20 production by ASMCs, possibly resulting from dysregulated expression of the anti-inflammatory miR-146a-5p, may contribute to enhanced mucus production in asthma.

Tumstatin fragment selectively inhibits neutrophil infiltration in experimental asthma exacerbation.

BACKGROUND: Asthma is a chronic inflammatory disease with structural changes present. Burgess and colleagues recently found tumstatin markedly reduced in adult asthmatic lung tissue compared with nonasthmatics. ECM fragments such as tumstatin are named matrikines and act independently of the parent molecule. The role of Col IV matrikines in neutrophil inflammation (eg. exacerbation in asthma) has not been investigated to date. Severe adult asthma phenotypes are dominated by neutrophilic inflammation and show a high frequency of severe exacerbations. OBJECTIVE: This study sought to investigate the role of a novel active region within tumstatin (CP17) and its implication in neutrophil inflammatory responses related to asthma exacerbation. METHODS: For reactive oxygen production, isolated neutrophils were preincubated with peptides or vehicle for 1 hour and stimulated (PMA). Luminescence signal was recorded (integration over 10 seconds) for 1.5 hours. Neutrophil migration was performed according to the SiMA protocol. Mice were sensitized to OVA/Alumn by intraperitoneal (i.p.) injections. Mice were then treated with CP17, vehicle (PBS) or scrambled peptide (SP17) after OVA exposure (days 27 and 28, polyI:C stimulation). All animals were killed on day 29 with lung function measurement, histology and lavage. RESULTS: CP17 decreased total ROS production rate to 52.44% (0.5 µmol/L, P < 0.05 vs SP17), reduced the in vitro directionality (vs SP17, P = 1 × 10-6 ) and migration speed (5 µmol/L, P = 1 × 10-3 ). In vivo application of CP17 decreased neutrophil inflammation ~1.8-fold (P < 0.001 vs SP17) and reduced numbers of mucus-producing cells (-29%, P < 0.05). CONCLUSION: CP17 reduced the ROS production rate, migrational speed and selectively inhibited neutrophil accumulation in the lung interstitium and lumen. CLINICAL RELEVANCE: CP17 may serve as a potential precursor for drug development to combat overwhelming neutrophil inflammation.

The all age asthma cohort (ALLIANCE) - from early beginnings to chronic disease: a longitudinal cohort study.

BACKGROUND: Asthma and wheezing disorders in childhood and adulthood are clinically heterogeneous regarding disease presentation, natural course, and response to treatment. Deciphering common disease mechanisms in distinct subgroups requires harmonized molecular (endo-) phenotyping of both children and adult patients with asthma in a prospective, longitudinal setting. METHODS: The ALL Age Asthma Cohort (ALLIANCE) of the German Center for Lung Research (DZL) is a prospective, multi-center, observational cohort study with seven recruiting sites across Germany. Data are derived from four sources: (a) patient history from medical records, (b) standardized questionnaires and structured interviews, (c) telephone interviews, and (d) objective measurements. Objective measurements include amongst others lung function and quantitative assessment of airway inflammation and exhaled breath, peripheral blood, skin, nasal, pharyngeal, and nasopharyngeal swabs, nasal secretions, primary nasal epithelial cells, and induced sputum. In cases, objective measurements and biomaterial collection are performed regularly, while control subjects are only examined once at baseline. DISCUSSION: The standardized and detailed collection of epidemiological and physiological data, and the molecular deep phenotyping of a comprehensive range of biomaterials in a considerable number of study participants across all ages are the outstanding characteristics of this multi-center cohort. Despite extensive biomaterial sampling, and a recruitment strategy that also includes pre-school children as young as 6 months, attrition is low. In children 83.9%, and in adults 90.5% attended the 12-month follow-up. The earliest time-point to include cases, however, is disease manifestation. Therefore, unraveling mechanisms that drive disease onset is limited, as this question can only be answered in a population-based birth cohort. Nonetheless, ALLIANCE offers a unique, integrative and inter-disciplinary framework with a comprehensive molecular approach in a prospective and identical fashion across ages in order to identify biomarkers and predictors for distinct childhood wheeze and asthma trajectories as well as their further course during adulthood. Ultimately, this approach aims to translate its most significant findings into clinical practice, and to improve asthma transition from adolescence to adulthood. TRIAL REGISTRATION: NCT02496468 for pediatric arm, NCT02419274 for adult arm.

Correction to: The all age asthma cohort (ALLIANCE) - from early beginnings to chronic disease: a longitudinal cohort study.

Following publication of the original article [1], the author flagged aspects of the article that affected readability of some of the article's scientific content.

Tumstatin regulates the angiogenic and inflammatory potential of airway smooth muscle extracellular matrix.

The extracellular matrix (ECM) creates the microenvironment of the tissue; an altered ECM in the asthmatic airway may be central in airway inflammation and remodelling. Tumstatin is a collagen IV-derived matrikine reduced in the asthmatic airway wall that reverses airway inflammation and remodelling in small and large animal models of asthma. This study hypothesized that the mechanisms underlying the broad asthma-resolving effects of tumstatin were due to autocrine remodelling of the ECM. Neutrophils and endothelial cells were seeded on decellularized ECM of non-asthmatic (NA) or asthmatic (A) airway smooth muscle (ASM) cells previously exposed to tumstatin in the presence or absence of a broad matrix metalloproteinase inhibitor, Marimastat. Gene expression in NA and A ASM induced by tumstatin was assessed using RT-PCR arrays. The presence of tumstatin during ECM deposition affected neutrophil and endothelial cell properties on both NA and A ASM-derived matrices and this was only partly due to MMP activity. Gene expression patterns in response to tumstatin in NA and A ASM cells were different. Tumstatin may foster an anti-inflammatory and anti-angiogenic microenvironment by modifying ASM-derived ECM. Further work is required to examine whether restoring tumstatin levels in the asthmatic airway represents a potential novel therapeutic approach.

SiMA: A simplified migration assay for analyzing neutrophil migration.

In lung inflammation, neutrophils are the first leukocytes migrating to an inflammatory site, eliminating pathogens by multiple mechanisms. The term "migration" describes several stages of neutrophil movement to reach the site of inflammation, of which the passage of the interstitium and basal membrane of the airway are necessary to reach the site of bronchial inflammation. Currently, several methods exist (e.g., Boyden Chamber, under-agarose assay, or microfluidic systems) to assess neutrophil mobility. However, these methods do not allow for parameterization on single cell level, that is, the individual neutrophil pathway analysis is still considered challenging. This study sought to develop a simplified yet flexible method to monitor and quantify neutrophil chemotaxis by utilizing commercially available tissue culture hardware, simple video microscopic equipment and highly standardized tracking. A chemotaxis 3D µ-slide (IBIDI) was used with different chemoattractants [interleukin-8 (IL-8), fMLP, and Leukotriene B4 (LTB4 )] to attract neutrophils in different matrices like Fibronectin (FN) or human placental matrix. Migration was recorded for 60 min using phase contrast microscopy with an EVOS® FL Cell Imaging System. The images were normalized and texture based image segmentation was used to generate neutrophil trajectories. Based on these spatio-temporal information a comprehensive parameter set is extracted from each time series describing the neutrophils motility, including velocity and directness and neutrophil chemotaxis. To characterize the latter one, a sector analysis was employed enabling the quantification of the neutrophils response to the chemoattractant. Using this hard- and software framework we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB4 , the effect of the matrices FN versus HEM as well as the response to different medications (Prednisolone). Additionally, a comparison of four asthmatic and three non-asthmatic patients gives a first hint to the capability of SiMA assay in the context of migration based diagnostics. Using SiMA we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB4 , the effect of the matrices FN versus HEM as well as the response to different medications, that is, Prednisolone induced a change of direction of migrating neutrophils in FN but no such effect was observed in human placental matrix. In addition, neutrophils of asthmatic individuals showed an increased proportion of cells migrating toward the vehicle. With the SiMA platform we presented a simplified but yet flexible platform for cost-effective tracking and quantification of neutrophil migration. The introduced method is based on a simple microscopic video stage, standardized, commercially available, µ-fluidic migration chambers and automated image analysis, and track validation software. © 2017 International Society for Advancement of Cytometry.