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Background: The anti-interleukin 13 (IL-13) monoclonal antibody lebrikizumab improves lung function in patients with moderate-to-severe uncontrolled asthma, but its effects on airway inflammation and remodelling are unknown. CLAVIER was designed to assess lebrikizumab's effect on eosinophilic inflammation and remodelling., Objective: To report safety and efficacy results from enrolled participants with available data from CLAVIER., Methods: We performed bronchoscopy on patients with uncontrolled asthma before and after 12 weeks of randomized double-blinded treatment with lebrikizumab (n = 31) or placebo (n = 33). The pre-specified primary end-point was relative change in airway subepithelial eosinophils per mm 2 of basement membrane (cells/mm 2 ). Pre-specified secondary and exploratory outcomes included change in IL-13-associated biomarkers and measures of airway remodelling., Results: There was a baseline imbalance in tissue eosinophils and high variability between treatment groups. There was no discernible change in adjusted mean subepithelial eosinophils/mm 2 in response to lebrikizumab (95% CI, -82.5%, 97.5%). As previously observed, FEV 1 increased after lebrikizumab treatment. Moreover, subepithelial collagen thickness decreased 21.5% after lebrikizumab treatment (95% CI, -32.9%, -10.2%), and fractional exhaled nitric oxide, CCL26 and SERPINB2 mRNA expression in bronchial tissues also reduced. Lebrikizumab was well tolerated, with a safety profile consistent with other lebrikizumab asthma studies., Conclusions & Clinical Relevance: We did not observe reduced tissue eosinophil numbers in association with lebrikizumab treatment. However, in pre-specified exploratory analyses, lebrikizumab treatment was associated with reduced degree of subepithelial fibrosis, a feature of airway remodelling, as well as improved lung function and reduced key pharmacodynamic biomarkers in bronchial tissues. These results reinforce the importance of IL-13 in airway pathobiology and suggest that neutralization of IL-13 may reduce asthmatic airway remodelling., Clinical Trial Registration: NCT02099656., (© 2020 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Background: Immunological biomarkers are the key to the diagnosis of allergic bronchopulmonary aspergillosis (ABPA) and fungal sensitisation, but how these relate to clinically relevant outcomes is unclear., Objectives: To assess how fungal immunological biomarkers are related to fixed airflow obstruction and radiological abnormalities in moderate to severe asthma., Methods: Cross-sectional study of 431 asthmatics. Inflammatory biomarkers, lung function and an IgE fungal panel to colonising filamentous fungi, yeasts and fungal aeroallergens were measured. CT scans were scored for the presence of radiological abnormalities. Factor analysis informed the variables used in a k-means cluster analysis. Fixed airflow obstruction and radiological abnormalities were then mapped to these immunological variables in the cluster analysis., Results: 329 (76.3%) subjects were sensitised to ≥ 1 fungi. Sensitisation to Aspergillus fumigatus and/or Penicillium chrysogenum was associated with a lower post-bronchodilator FEV 1 compared with those not sensitised to fungi ((73.0 (95% CI 70.2-76) vs. 82.8 (95% CI 78.5-87.2)% predicted, P < 0.001), independent of atopic status (P = 0.005)), and an increased frequency of bronchiectasis (54.5%, P < 0.001), tree-in-bud (18.7%, P < 0.001) and collapse/consolidation (37.5%, P = 0.002). Cluster analysis identified three clusters: (i) hypereosinophilic (n = 71, 16.5%), (ii) high immunological biomarker load and high frequency of radiological abnormalities (n = 34, 7.9%) and (iii) low levels of fungal immunological biomarkers (n = 326, 75.6%)., Conclusions and Clinical Relevance: IgE sensitisation to thermotolerant filamentous fungi, in particular A. fumigatus but not total IgE, is associated with fixed airflow obstruction and a number of radiological abnormalities in moderate to severe asthma. All patients with IgE sensitisation to A. fumigatus are at risk of lung damage irrespective of whether they meet the criteria for ABPA., (© 2016 John Wiley & Sons Ltd.)

Mast cells (MCs) play a central role in tissue homoeostasis, sensing the local environment through numerous innate cell surface receptors. This enables them to respond rapidly to perceived tissue insults with a view to initiating a co-ordinated programme of inflammation and repair. However, when the tissue insult is chronic, the ongoing release of multiple pro-inflammatory mediators, proteases, cytokines and chemokines leads to tissue damage and remodelling. In asthma, there is strong evidence of ongoing MC activation, and their mediators and cell-cell signals are capable of regulating many facets of asthma pathophysiology. This article reviews the evidence behind this., (© 2015 John Wiley & Sons Ltd.)

Background: The role of small airway obstruction in the clinical expression of asthma is incompletely understood., Objective: We tested the hypotheses that markers of small airway obstruction are associated with (i) increased asthma severity, (ii) impaired asthma control and quality of life and (iii) frequent exacerbations., Methods: Seventy-four adults with asthma and 18 healthy control subjects underwent impulse oscillometry (IOS), multiple breath inert gas washout (MBW), body plethysmography, single-breath determination of carbon monoxide uptake and spirometry. Patients completed the six-point Asthma Control Questionnaire (ACQ-6) and standardized Asthma Quality of Life Questionnaire [AQLQ(S)]. Asthma severity was classified according to the Global Initiative for Asthma (GINA) treatment steps., Results: The putative small airway obstruction markers Sacin , resistance at 5 Hz minus resistance at 20 Hz (R5-R20) and reactance area (AX) were not independently associated with asthma severity, control, quality of life or exacerbations. In contrast, markers of total (R5) and mean airway resistance of large and small airways (R20) were significantly higher in the severe asthma group compared with the mild-moderate group (0.47 vs. 0.37, P < 0.05 for R5; 0.39 vs. 0.31, P < 0.01 for R20). The strongest independent contributors to ACQ-6 score were R20 and forced expiratory volume in one second (% pred.), and the strongest independent contributors to AQLQ(S) score were R20 and forced vital capacity (% pred.). A history of one or more exacerbations within the previous year was independently associated with R20., Conclusions and Clinical Relevance: Previously reported markers of small airway obstruction do not appear to be independently associated with asthma disease expression. In contrast, the IOS parameter R20, a marker of mean airway resistance of both large and small airways, appears to have independent clinical significance. These observations require confirmation in prospective longitudinal studies., (© 2013 John Wiley & Sons Ltd.)

Mast cells play a central role in the pathophysiology of asthma and related allergic conditions. Mast cell activation leads to the degranulation of preformed mediators such as histamine and the secretion of newly synthesised proinflammatory mediators such as leukotrienes and cytokines. Excess release of these mediators contributes to allergic disease states. An influx of extracellular Ca2+ is essential for mast cell mediator release. From the Ca2+ channels that mediate this influx, to the K+ , Cl- and transient receptor potential channels that set the cell membrane potential and regulate Ca2+ influx, ion channels play a critical role in mast cell biology. In this review we provide an overview of our current knowledge of ion channel expression and function in mast cells with an emphasis on how channels interact to regulate Ca2+ signalling., (© 2012 Blackwell Publishing Ltd.)

Background: Fungal sensitization is common in severe asthma, but the clinical relevance of this and the relationship with airway colonization by fungi remain unclear. The range of fungi that may colonize the airways in asthma is unknown., Objective: To provide a comprehensive analysis on the range of filamentous fungi isolated in sputum from people with asthma and report the relationship with their clinico-immunological features of their disease., Methods: We recruited 126 subjects with a diagnosis of asthma, 94% with moderate-severe disease, and 18 healthy volunteers. At a single stable visit, subjects underwent spirometry; sputum fungal culture and a sputum cell differential count; skin prick testing to both common aeroallergens and an extended fungal panel; specific IgE to Aspergillus fumigatus. Fungi were identified by morphology and species identity was confirmed by sequencing. Four patients had allergic bronchopulmonary aspergillosis., Results: Forty-eight percent of asthma subjects were IgE-sensitized to one fungal allergen and 22% to ≥ 2. Twenty-seven different taxa of filamentous fungi were isolated from 54% of their sputa, more than one species being detected in 17%. This compared with 3 (17%) healthy controls culturing any fungus (P < 0.01). Aspergillus species were most frequently cultured in isolation followed by Penicillium species. Post-bronchodilator FEV (1) (% predicted) in the subjects with asthma was 71(± 25) in those with a positive fungal culture vs. 83 (± 25) in those culture-negative, (P < 0.01)., Conclusion and Clinical Relevance: Numerous thermotolerant fungi other than A. fumigatus can be cultured from sputum of people with moderate-to-severe asthma; a positive culture is associated with an impaired post-bronchodilator FEV (1) , which might be partly responsible for the development of fixed airflow obstruction in asthma. Sensitization to these fungi is also common., (© 2012 Blackwell Publishing Ltd.)

Background: Airway smooth muscle (ASM) hyperplasia and mast cell localization within the ASM bundle are important features of asthma. The cause of this increased ASM mass is uncertain and whether it is a consequence of ASM-mast cell interactions is unknown., Objective: We sought to investigate ASM proliferation and survival in asthma and the effects of co-culture with mast cells., Methods: Primary ASM cultures were derived from 11 subjects with asthma and 12 non-asthmatic controls. ASM cells were cultured for up to 10 days in the presence or absence of serum either alone or in co-culture with the human mast cell line-1, unstimulated human lung mast cells (HLMC) or IgE/anti-IgE-activated HLMC. Proliferation was assessed by cell counts, CFSE assay and thymidine incorporation. Apoptosis and necrosis were analysed by Annexin V/propidium iodide staining using flow cytometry and by assessment of nuclear morphology using immunofluorescence. Mast cell activation was confirmed by the measurement of histamine release., Results: Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma. Co-culture with mast cells did not affect the rate of proliferation or survival of ASM cells., Conclusion: Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma.

Background: Chemokine receptors play an important role in cell migration and wound repair. In asthma, CCR3 and 7 are expressed by airway smooth muscle (ASM) and CCR7 has been implicated in the development of ASM hyperplasia. The expression profile of other chemokine receptors by ASM and their function needs to be further explored., Objective: We sought to investigate ASM chemokine receptor expression and function in asthma., Methods: ASM cells were derived from 17 subjects with asthma and 36 non-asthmatic controls. ASM chemokine receptor expression was assessed by flow cytometry and immunofluorescence. The function of chemokine receptors expressed by more than 10% of ASM cells was investigated by intracellular calcium measurements, chemotaxis, wound healing, proliferation and survival assays., Results: In addition to CCR3 and 7, CXCR1, 3 and 4 were highly expressed by ASM. These CXC chemokine receptors were functional with an increase in intracellular calcium following ligand activation and promotion of wound healing [CXCL10 (100 ng/mL) 34 +/- 2 cells/high-powered field (hpf) vs. control 29 +/- 1; P=0.03; n=8]. Spontaneous wound healing was inhibited by CXCR3 neutralizing antibody (mean difference 7 +/- 3 cells/hpf; P=0.03; n=3). CXC chemokine receptor activation did not modulate ASM chemotaxis, proliferation or survival. No differences in chemokine receptor expression or function were observed between ASM cells derived from asthmatic or non-asthmatic donors., Conclusions: Our findings suggest that the chemokine receptors CXCR1, 3 and 4 modulate some aspects of ASM function but their importance in asthma is uncertain.

In 2008, many thousands of articles were published on the subject of allergic disease with over 200 reviews, editorials and original papers in Clinical & Experimental Allergy alone. These represent a considerable amount of data and even the most avid reader could only hope to assimilate a small fraction of this knowledge. There is therefore a pressing need for the key messages that emerge from a journal such as Clinical & Experimental Allergy to be summarized by experts in the field in a form that highlights the significance of the developments and sets them in the context of important findings in the field published in other journals. This also has the advantage of making connections between new data in conditions such as asthma, where articles often appear in different sections of the journal. As can be seen from this review, the body of work is diverse both in terms of the disease of interest and the discipline that has been used to investigate it. However, taken as a whole, we hope that the reader will gain a flavour of where the field is mature, where there remain controversies and where the cutting edge is leading.

Background: Mast cells contribute to the pathogenesis of asthma and allergy through the release of a plethora of pro-inflammatory mediators and cytokines. Their study is hampered by the difficult access to human tissue samples. Human mast cells have been cultured from CD34+ progenitors in the bone marrow of normal volunteers following iliac crest bone marrow biopsy but this is invasive. Hip bone marrow could provide a more convenient less invasive source of mast cell progenitors., Objective: To characterize mast cells cultured from human bone marrow obtained at routine hip surgery., Methods: Mononuclear cells were isolated from the bone marrow reamings of patients undergoing routine hip replacement surgery and were cultured with recombinant stem cell factor (SCF), IL-6 and IL-10. Cell surface markers were examined using flow cytometry, protease expression monitored using immunohistochemistry, histamine measured by radioenzymic assay, Ca2+ responses analysed using ratiometric Ca2+ imaging, and ion currents recorded via the patch-clamp technique., Results: Mast cells were absent at baseline, but accounted for 65 +/- 7% of cells after 8-12 weeks of culture, equating to a mean 0.6 +/- 0.14 x 10(6) mast cells per culture. Fifty-three percent of tryptase+ cells also contained chymase. The remaining cells comprised a population of large CD1a+ HLA-DR+ and Fc epsilon RI alpha+ cells, most likely dendritic cells. All mast cells expressed CD117 and the high-affinity IgE receptor alpha-chain (Fc epsilon RI alpha) constitutively, and developed a Ca2+ response following IgE-dependent activation. These cells exhibited 7.8 +/- 2.9% net IgE-dependent histamine release, and demonstrated a similar ion channel profile to human lung mast cells. In particular, the intermediate conductance Ca(2+)-activated K+ channel opened following IgE-dependent activation., Conclusions: Mast cells grown from human hip marrow provide a rich non-invasive source of functionally mature mast cells. In addition, this culture system may be useful for the generation of Fc epsilon RI alpha+ dendritic cells.

Background: Airway smooth muscle infiltration by mast cells is a feature of asthma and not eosinophilic bronchitis. In asthma, Th2 cytokines have been implicated as playing a critical role in the development of airway inflammation and hyper-responsiveness. Whether inflammatory cells within the airway smooth muscle release these cytokines is unknown., Methods: We have undertaken a comparative immunohistochemical study in bronchial biopsies from 14 subjects with asthma, 10 with eosinophilic bronchitis and eight normal controls recruited from two centres., Results: The median number of IL-4+ cells/mm2 smooth muscle was significantly higher in subjects with asthma than eosinophilic bronchitis and normal controls for both the anti-IL-4 mAb 3H4 (2.4, 0, 0, respectively; P=0.001) and anti-IL-4 mAb 4D9 (1.6, 0, 0, respectively; P=0.02). There were no group differences in the number of IL-5+ cells (P=0.31). In six subjects with asthma, IL-13 expression by cells within the airway smooth muscle was studied. The median (range) of IL-13+cells was 2 (0.9-2.7). Ninety-four percent of the cells expressing IL-4 (3H4), 92% of those expressing IL-4 (4D9) and 100% expressing IL-13 in the airway smooth muscle were mast cells. Fifty-five percent of the mast cells within the airway smooth muscle co-localized to IL-4 (3H4), 29% to IL-4 (4D9) and 17% to IL-13., Conclusions: In asthma, IL-4+ and IL-13+ cells were present within the airway smooth muscle and were expressed predominantly by mast cells, suggesting that IL-4 and IL-13 may play an important role in mast cell-airway smooth muscle interactions.

Background: Human mast cells synthesize and secrete many cytokines of relevance to the pathogenesis of allergic diseases such as asthma and rhinitis. In particular, interleukin (IL) -4 and IL-5 are likely to play key roles in the development of the inflammatory response that characterizes these diseases. Immunohistochemical studies on human nasal and bronchial mucosal biopsies suggest that IL-4 and IL-5 may be stored preformed in mast cells., Objective: To identify whether IL-4 and IL-5 are stored within mast cell secretory granules., Methods: We used immunogold electron microscopic analysis on bronchial mucosa and lung parenchyma from resected lung specimens, and a nasal mucosal biopsy from a patient with active allergic rhinitis. Samples were fixed in 4% paraformaldehyde plus 0.5% glutaraldehyde and processed into Lowicryl K4M resin by the 'Progressive Lowering of Temperature' technique. Ultrathin sections were stained immunohistochemically by an indirect immunogold method., Results: Immunoreactivity for IL-4, but not IL-5, was localized to the granules of mast cells in all tissue samples. IL-5 was localized to the matrix of eosinophil granules in these samples, but neither cytokine was detected in T cells. IL-4 immunoreactivity increased in the granules of mast cells 24 h after immunoglobulin (Ig) E-dependent activation (mean 17.5 +/- 1.4 gold particles per granule) compared with nonactivated mast cells (mean 6.8 +/- 0.8 gold particles per granule, P < 0.001), suggesting replenishment of stores by newly generated protein. Immunoreactive IL-5 remained undetectable in mast cells 24 h after activation, a time point at which they are known to secrete large quantities of this cytokine., Conclusion: Human mast cells store IL-4 within the matrix of their granules. Very few, if any, lung or nasal mast cells store IL-5. A store of preformed IL-4 within mast cell granules is likely to have an important influence during the initiation and maintenance of the allergic immunological response.

Background: Eosinophil-bronchial epithelial cell interactions are thought to be central to the pathogenesis of asthma, both in terms of the epithelium as a source of pro-inflammatory mediators and as a target for eosinophil-mediated damage. We have therefore investigated adhesion interactions between these two cell types., Objectives: To determine the role of eosinophil and epithelial activation on eosinophil adhesion to bronchial epithelium and to characterize the adhesion receptors mediating eosinophil adhesion., Methods: Eosinophils were purified from human peripheral blood by immunomagnetic selection and adhesion to confluent cultures of the airway epithelial cell lines A549 and BEAS-2B was studied., Results: Stimulation of A549 cells with TNFalpha, IFNgamma or a combination of 50 ng/mL of TNFalpha, IFNgamma and IL-1 (cytomix) did not effect eosinophil binding despite an increase in ICAM-1 expression. Similarly stimulation of eosinophils with PAF or IL-5 had no effect on eosinophil binding to medium- or cytokine-treated A549 cells. In contrast stimulation of BEAS-2B cells with cytomix caused a significant increase in eosinophil adhesion. This was associated with an increase in expression of ICAM-1 and induced expression of VCAM-1. Treatment of eosinophils with Mn2+ and IL-5 but not eotaxin, RANTES or PAF also significantly enhanced eosinophil adhesion to medium-treated BEAS-2B cells. Using blocking mAbs we were able to demonstrate that the increased adhesion resulting from stimulation of eosinophils or BEAS-2B cells was in both cases mediated by a combination of CD18 and alpha4 integrins., Conclusions: This study demonstrates a selective role for IL-5 in mediating integrin-dependent eosinophil adhesion to airway epithelium and once again emphasizes the importance of this cytokine in controlling eosinophil activation in diseases such as asthma.

Background: Immunoglobulin E (IgE) plays an important role in asthma, with total serum IgE levels closely related to both clinical expression of the disease and airway hyperresponsiveness. IgE binds to a high affinity cell-surface receptor (Fc epsilon RI) which is present on mast cells and which has also recently been demonstrated on cutaneous dendritic cells. If pulmonary dendritic cells were also able to express this receptor, this would have important implications with regard to their potential role in asthma., Objectives: The aims of the study were to investigate the expression of the alpha subunit of the high affinity IgE receptor (Fc epsilon RI-alpha) in normal and asthmatic airways, and to analyse its cellular provenance with particular emphasis on the dendritic cell., Methods: Bronchial biopsy specimens were obtained using fibreoptic bronchoscopy from 10 atopic asthmatics and nine non-atopic non-asthmatic control subjects. Specimens were processed into glycolmethacrylate resin and analysed by immunohistochemistry using specific monoclonal antibodies against Fc epsilon RI-alpha, and against tryptase and CD1a, markers for mast cells and dendritic cells, respectively., Results: The numbers of dendritic cells were significantly higher in the airways of asthmatics compared with those of control subjects (P < 0.02). Analysis of sequential sections revealed that the alpha subunit of Fc epsilon RI was localized to both mast cells and dendritic cells. The proportion of dendritic cells expressing Fc epsilon RI-alpha was significantly increased in the asthmatic group (P < 0.003)., Conclusion: These results support the hypothesis that dendritic cells play an important role in allergic asthma although the functional significance of Fc epsilon RI-alpha expression needs further investigation.

Allergic mucosal inflammation is characterized by tissue infiltration with eosinophils, and associated activation of mast cells and T lymphocytes. Tumour necrosis factor (TNF) alpha/cachectin is a candidate cytokine relevant to the pathogenesis of these events through its capacity to upregulate the expression of endothelial cell adhesion molecules, mediate granulocyte chemoattraction, and activate eosinophils, mast cells and T cells. To investigate the presence and localization of TNF alpha in the nasal mucosa in allergic rhinitis, nasal biopsies from perennial rhinitic (n = 13) and non-rhinitic volunteers (n = 11) were embedded in glycol methacrylate and immunostained with a monoclonal antibody directed against TNF alpha, and adjacent 2 microns sections stained for tryptase, CD3 and eosinophil cationic protein. This identified positive immunostaining for TNF alpha in the submucosa of both the rhinitic and normal subjects (median cell counts 13 and 23 cells/mm2 respectively, P = 0.24) with cellular localization to mast cells but not to T-lymphocytes or eosinophils. In a subsequent study of seven atopic subjects, nasal allergen challenge produced increases in lavage levels of histamine and albumin, which was associated with significant release of TNF alpha as early as 2 min post-allergen when compared with the saline control day (P = 0.05). This difference was also apparent when studying the area under the curve both at 30 and 60 min post-challenge t-test (P = 0.015 and 0.02 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)