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Although there is much circumstantial evidence implicating eosinophils as major orchestrators in the pathophysiology of asthma, recent studies have cast doubt on their importance. Not only does anti-interleukin-5 treatment not alter the course of the disease, but some patients with asthma do not have eosinophils in their airways, whereas patients with eosinophilic bronchitis exhibit a florid tissue eosinophilia but do not have asthma. In contrast, mast cells are found in all airways and localize specifically to key tissue structures such as the submucosal glands and airway smooth muscle within asthmatic bronchi, irrespective of disease severity or phenotype. Here they are activated and interact exclusively with these structural cells via adhesive pathways and through the release of soluble mediators acting across the distance of only a few microns. The location of mast cells within the airway smooth muscle bundles seems particularly important for the development and propagation of asthma, perhaps occurring in response to, and then serving to aggravate, an underlying abnormality in asthmatic airway smooth muscle function. Targeting this mast cell-airway smooth muscle interaction in asthma offers exciting prospects for the treatment of this common disease.

Abstract Background The role of TNFα in cancer is complex with both pro-tumourigenic and anti-tumourigenic roles proposed. We hypothesised that anatomical microlocalisation is critical for its function. Methods This study used immunohistochemistry to investigate the expression of TNFα in the tumour islets and stroma with respect to survival in 133 patients with surgically resected NSCLC. Results TNFα expression was increased in the tumour islets of patients with above median survival (AMS) compared to those with below median survival (BMS)(p = 0.006), but similar in the stroma of both groups. Increasing tumour islet TNFα density was a favorable independent prognostic indicator (p = 0.048) while stromal TNFα density was an independent predictor of reduced survival (p = 0.007). Patients with high TNFα expression (upper tertile) had a significantly higher 5-year survival compared to patients in the lower tertile (43% versus 22%, p = 0.01). In patients with AMS, 100% of TNFα+ cells were macrophages and mast cells, compared to only 28% in the islets and 50% in the stroma of BMS patients (p < 0.001). Conclusions The expression of TNFα in the tumour islets of patients with NSCLC is associated with improved survival suggesting a role in the host anti-tumour immunological response. The expression of TNFα by macrophages and mast cells is critical for this relationship.

Abstract Background We have previously demonstrated that tumour islet infiltration by macrophages is associated with extended survival (ES) in NSCLC. We therefore hypothesised that patients with improved survival would have high tumour islet expression of chemokine receptors known to be associated with favourable prognosis in cancer. This study investigated chemokine receptor expression in the tumour islets and stroma in NSCLC. Methods We used immunohistochemistry to identify cells expressing CXCR1, CXCR2, CXCR3, CXCR4, CXCR5 and CCR1 in the tumour islets and stroma in 20 patients with surgically resected NSCLC. Correlations were made with macrophage and mast cell expression. Results There was increased expression of CXCR2, CXCR3, and CCR1 in the tumour islets of ES compared with poor survival (PS) patients (p = 0.007, 0.01, and 0.002, respectively). There was an association between 5 year survival and tumour islet CXCR2, CXCR3 and CCR1 density (p = 0.02, 0.003 and s = 0.520, p = 0.02) and between mast cell density and CXCR3 expression (rs = 0.499, p = 0.03) in the tumour islets. Conclusion Above median expression of CXCR2, CXCR3 and CCR1 in the tumour islets is associated with increased survival in NSCLC, and expression of CXCR3 correlates with increased macrophage and mast cell infiltration in the tumour islets.

Abstract Background Mast cells play a key role in asthma and recent evidence indicates that their ongoing activation in this disease is mediated, in part, via IgE in the absence of antigen. In this study we have examined whether IgE alone enhances human lung mast cell (HLMC) survival. Methods Purified HLMC were cultured for 4 weeks and survival assays then performed over 10 days following cytokine withdrawal in the presence or absence of human myeloma IgE. Quantitative real time RT-PCR was carried out to examine IL-6 mRNA expression and IL-6 protein was measured in HLMC supernatants by ELISA. Results IgE alone promoted the survival of HLMC in a dose-dependent manner following cytokine withdrawal. IgE-induced survival was eliminated with the addition of neutralising anti-IL-6 antibody but not by the addition of neutralising anti-stem cell factor. IgE sensitisation initiated profound upregulation of IL-6 mRNA in HLMC, and IL-6 concentrations were also raised in the culture supernatants of IgE-exposed cells. Conclusion These data taken together suggest that IgE in the absence of antigen promotes HLMC survival through the autocrine production of IL-6. This provides a further mechanism through which IL-6 and IgE contribute to the pathogenesis of asthma, and through which anti-IgE therapy might achieve its therapeutic effect.

Abstract Background Analysis of cell-selective gene expression for families of proteins of therapeutic interest is crucial when deducing the influence of genes upon complex traits and disease susceptibility. Presently, there is no convenient tool for examining isoform-selective expression for large gene families. A multigene isoform profiling strategy was developed and used to investigate the inwardly rectifying K+ (Kir) channel family in human leukocytes. Comprised of seven subfamilies, Kir channels have important roles in setting the resting membrane potential in excitable and non-excitable cells. Results Gene sequence alignment allowed determination of "islands" of amino acid homology, and sub-family "centred" priming permitted simultaneous co-amplification of each family member. Validation and cross-priming analysis was performed against a panel of cognate Kir channel clones. Radiolabelling and diagnostic restriction digestion of pooled PCR products enabled determination of distinct Kir gene expression profiles in pure populations of human neutrophils, eosinophils and lung mast cells, with conservation of Kir2.0 isoforms amongst the leukocyte subsets. We also identified a Kir2.0 channel product, which may potentially represent a novel family member. Conclusions We have developed a novel, rapid and flexible strategy for the determination of gene family isoform composition in any cell type with the additional capacity to detect hitherto unidentified family members and verified its application in a study of Kir channel isoform expression in human leukocytes.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease with a poor prognosis and increasing incidence. Pirfenidone and nintedanib are the only approved treatments for IPF but have limited efficacy and their mechanisms of action are poorly understood. Here we have examined the effects of pirfenidone and nintedanib in a human model of lung fibrogenesis, and compared these with the putative anti-fibrotic compounds Lipoxin A4 (LXA4), and senicapoc, a KCa3.1 ion channel blocker.Methods: Early fibrosis was induced in cultured human lung parenchyma using TGFβ1 for 7 days, ± pirfenidone, nintedanib, or LXA4. Pro-fibrotic responses were examined by RT-PCR, immunohistochemistry and soluble collagen secretion.Results: Thirty six out of eighty four IPF and fibrosis-associated genes tested were significantly upregulated by TGFβ1 in human lung parenchyma with a ≥0.5 log2FC (n = 32). Nintedanib (n = 13) reduced the mRNA expression of 14 fibrosis-associated genes including MMPs (MMP1,−4,−13,−14), integrin α2, CXCR4 and PDGFB, but upregulated α-smooth muscle actin (αSMA). Pirfenidone only reduced mRNA expression for MMP3 and −13. Senicapoc (n = 11) previously attenuated the expression of 28 fibrosis-associated genes, including αSMA, several growth factors, collagen type III, and αV/β6 integrins. Pirfenidone and nintedanib significantly inhibited TGFβ1-induced fibroblast proliferation within the tissue, but unlike senicapoc, neither pirfenidone nor nintedanib prevented increases in tissue αSMA expression. LXA4 was ineffective.Conclusions: Pirfenidone and nintedanib demonstrate modest anti-fibrotic effects and provide a benchmark for anti-fibrotic activity of new drugs in human lung tissue. Based on these data, we predict that the KCa3.1 blocker senicapoc will show greater benefit than either of these licensed drugs in IPF.

Inhaled corticosteroids (ICS) are the core component of asthma treatment and the only maintenance therapy known to prevent asthma death. There is currently no evidence that biologics prevent asthma death in people with asthma, and as such, biologics cannot be recommended as an alternative to ICS therapy. Taking the time to assess adherence and provide interventions and education to support patients in asthma self-management has been shown to improve patient outcomes. It is therefore our responsibility as healthcare professionals to ensure that patients are supported, educated and motivated to adhere to ICS therapy before progressing to biologic therapies.

The mechanisms underlying corticosteroid insensitivity in severe asthma have not been elucidated although some indirect clinical evidence points toward a role of mast cells. Here, we tested the hypothesis that mast cells can drive corticosteroid insensitivity in airway smooth muscle cells, a key player in asthma pathogenesis. Conditioned media from resting or FcεR1-activated human lung mast cells were incubated with serum-deprived ASM cells (1:4 dilution, 24 h) to determine their impact on the anti-inflammatory action of fluticasone on ASM cell chemokine expression induced by TNFα (10 ng/ml). Conditioned media from FcεR1-activated mast cells (but not that from non-activated mast cells or control media) significantly reduced the ability of 100 nM fluticasone to suppress ASM TNFα-dependent CCL5 and CXCL10 production at both mRNA and protein levels. In contrast, fluticasone inhibition of CXCL-8 production by TNFα was still preserved in the presence of activated mast cell conditioned media. Transcriptomic analysis validated by individual qPCR assays revealed that activated mast cell conditioned media dramatically reduced the number of anti-inflammatory genes induced by fluticasone in ASM cells. Our study demonstrates for the first time that conditioned media from FcεR1-activated mast cells blunt the anti-inflammatory action of corticosteroids in ASM cells by altering their transactivation properties. Because infiltration of mast cells within the ASM bundles is a defining feature of asthma, mast cell-derived mediators may contribute to the glucocorticoid insensitivity present in severe asthma.

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with limited therapeutic options. KCa3.1 ion channels play a critical role in TGFβ1-dependent pro-fibrotic responses in human lung myofibroblasts. We aimed to develop a human lung parenchymal model of fibrogenesis and test the efficacy of the selective KCa3.1 blocker senicapoc. 2 mm3 pieces of human lung parenchyma were cultured for 7 days in DMEM ± TGFβ1 (10 ng/ml) and pro-fibrotic pathways examined by RT-PCR, immunohistochemistry and collagen secretion. Following 7 days of culture with TGFβ1, 41 IPF- and fibrosis-associated genes were significantly upregulated. Immunohistochemical staining demonstrated increased expression of ECM proteins and fibroblast-specific protein after TGFβ1-stimulation. Collagen secretion was significantly increased following TGFβ1-stimulation. These pro-fibrotic responses were attenuated by senicapoc, but not by dexamethasone. This 7 day ex vivo model of human lung fibrogenesis recapitulates pro-fibrotic events evident in IPF and is sensitive to KCa3.1 channel inhibition. By maintaining the complex cell-cell and cell-matrix interactions of human tissue, and removing cross-species heterogeneity, this model may better predict drug efficacy in clinical trials and accelerate drug development in IPF. KCa3.1 channels are a promising target for the treatment of IPF.

Abstract Background Patients with difficult-to-control asthma consume 50–60% of healthcare costs attributed to asthma and cost approximately five-times more than patients with mild stable disease. Recent evidence demonstrates that not all patients with asthma have a typical type 2 (T2)-driven eosinophilic inflammation. These asthmatics have been called ‘T2-low asthma’ and have a minimal response to corticosteroid therapy. Adjustment of corticosteroid treatment using sputum eosinophil counts from induced sputum has demonstrated reduced severe exacerbation rates and optimized corticosteroid dose. However, it has been challenging to move induced sputum into the clinical setting. There is therefore a need to examine novel algorithms to target appropriate levels of corticosteroid treatment in difficult asthma, particularly in T2-low asthmatics. This study examines whether a composite non-invasive biomarker algorithm predicts exacerbation risk in patients with asthma on high-dose inhaled corticosteroids (ICS) (± long-acting beta agonist) treatment, and evaluates the utility of this composite score to facilitate personalized biomarker-specific titration of corticosteroid therapy. Methods/design Patients recruited to this pragmatic, multi-centre, single-blinded randomised controlled trial are randomly allocated into either a biomarker controlled treatment advisory algorithm or usual care group in a ratio of 4:1. The primary outcome measure is the proportion of patients with any reduction in ICS or oral corticosteroid dose from baseline to week 48. Secondary outcomes include the rate of protocol-defined severe exacerbations per patient per year, time to first severe exacerbation from randomisation, dose of inhaled steroid at the end of the study, cumulative dose of inhaled corticosteroid during the study, proportion of patients on oral corticosteroids at the end of the study, proportion of patients who decline to progress to oral corticosteroids despite composite biomarker score of 2, frequency of hospital admission for asthma, change in the 7-item Asthma Control Questionnaire (ACQ-7), Asthma Quality of Life Questionnaire (AQLQ), forced expiratory volume in 1 s (FEV1), exhaled nitric oxide, blood eosinophil count, and periostin levels from baseline to week 48. Blood will also be taken for whole blood gene expression; serum, plasma, and urine will be stored for validation of additional biomarkers. Discussion Multi-centre trials present numerous logistical issues that have been addressed to ensure minimal bias and robustness of study conduct. Trial registration ClinicalTrials.gov, NCT02717689 . Registered on 16 March 2016.

Background: Severe asthma exacerbations are costly to patients and the NHS, and occur frequently in severely allergic patients. Objective: To ascertain whether or not nocturnal temperature-controlled laminar airflow (TLA) device usage over 12 months can reduce severe exacerbations and improve asthma control and quality of life compared with a placebo device, while being cost-effective and acceptable to adults with severe allergic asthma. Design: A pragmatic, multicentre, randomised, double-blind, placebo-controlled, parallel-group, superiority trial with qualitative interviews. The trial included an internal pilot with qualitative focus groups. Setting: Fourteen hospitals in the UK that manage patients with severe asthma. Participants: Adults (16–75 years) with severe, poorly controlled, exacerbation-prone asthma despite high-intensity treatment, and who are sensitised to a perennial indoor aeroallergen. Intervention: Nocturnal, home-based TLA treatment using an Airsonett® (Airsonett AB, Ängelholm, Sweden) device. The comparator was a placebo device that was identical to the active device except that it did not deliver the laminar airflow. Participants were allocated 1 : 1 to TLA therapy or placebo, minimised by site, origin of case, baseline severe exacerbation frequency, maintenance oral corticosteroid use and pre-bronchodilator forced expiratory volume in 1 second. Main outcome measures: Primary outcome – frequency of severe asthma exacerbations occurring within the 12-month follow-up period, defined as worsening of asthma requiring systemic corticosteroids [≥ 30 mg of prednisolone or equivalent daily (or ≥ 50% increase in dose if on maintenance dose of ≥ 30 mg of prednisolone)] for ≥ 3 days. Secondary outcomes – changes in asthma control, lung function, asthma-specific and global quality of life for participants, adherence to the intervention, device acceptability, health-care resource use and cost-effectiveness. Results: Between May 2014 and January 2016, 489 patients consented to participate in the trial, of whom 249 failed screening and 240 were randomised (n = 119 in the treatment group and n = 121 in the placebo group); all were analysed. In total, 202 participants (84%) reported use of the device for 9–12 months. Qualitative analyses showed high levels of acceptability. The mean [standard deviation (SD)] rate of severe exacerbations did not differ between groups [active 1.39 (1.57), placebo 1.48 (2.03); risk ratio 0.92, 95% CI 0.66 to 1.27; p = 0.616]. There were no significant differences in secondary outcomes for lung function, except for a reduction in mean daily peak expiratory flow [mean (SD) difference 14.7 l/minute (7.35 l/minute), 95% CI 0.32 to 29.1 l/minute; p = 0.045) for those in the active device group. There were no differences in asthma control or airway inflammation and no serious harms related to the device. No significant difference between the groups in quality-adjusted life-years gained over 1 year was observed. In addition, there was no difference in generic or disease-specific health-related quality of life overall, although statistically significant higher quality of life at month 6 was observed. Increases in quality of life were not sufficient to offset the annual costs associated with use of the TLA device. Limitations: Missing outcome data could have resulted in an underestimation of exacerbations and rendered the study inconclusive. Conclusions: Within the limits of the data, no consistent benefits of the active device were demonstrated, and the differences observed were not sufficient to make the device cost-effective. The types of patients who may benefit from the TLA device, and the reasons for large reductions in exacerbation frequency in severe asthma trials, which also incorporate other methods of recording exacerbations, need to be explored. Trial registration: Current Controlled Trials ISRCTN46346208. Funding: This project was funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 23, No. 29. See the NIHR Journals Library website for further project information.

The KCa3.1 K+ channel has been proposed as a novel target for pulmonary diseases such as asthma and pulmonary fibrosis. It is expressed in epithelia but its expression and function in primary human bronchial epithelial cells (HBECs) has not been described. Due to its proposed roles in the regulation of cell proliferation, migration, and epithelial fluid secretion, inhibiting this channel might have either beneficial or adverse effects on HBEC function. The aim of this study was to assess whether primary HBECs express the KCa3.1 channel and its role in HBEC function. Primary HBECs from the airways of healthy and asthmatic subjects, SV-transformed BEAS-2B cells and the neoplastic H292 epithelial cell line were studied. Primary HBECs, BEAS-2B and H292 cells expressed KCa3.1 mRNA and protein, and robust KCa3.1 ion currents. KCa3.1 protein expression was increased in asthmatic compared to healthy airway epithelium in situ, and KCa3.1 currents were larger in asthmatic compared to healthy HBECs cultured in vitro. Selective KCa3.1 blockers (TRAM-34, ICA-17043) had no effect on epithelial cell proliferation, wound closure, ciliary beat frequency, or mucus secretion. However, several features of TGFβ1-dependent epithelial-mesenchymal transition (EMT) were inhibited by KCa3.1 blockade. Treatment with KCa3.1 blockers is likely to be safe with respect to airway epithelial biology, and may potentially inhibit airway remodelling through the inhibition of EMT.

CADM1 is a major receptor for the adhesion of mast cells (MCs) to fibroblasts, human airway smooth muscle cells (HASMCs) and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM). Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion.

Mast cells (MCs) play a central role in the development of many diseases including asthma and pulmonary fibrosis. Interactions of human lung mast cells (HLMCs) with human airway smooth muscle cells (HASMCs) are partially dependent on adhesion mediated by cell adhesion molecule-1 (CADM1), but the adhesion mechanism through which HLMCs interact with human lung fibroblasts (HLFs) is not known. CADM1 is expressed as several isoforms (SP4, SP1, SP6) in HLMCs, with SP4 dominant. These isoforms differentially regulate HLMC homotypic adhesion and survival.In this study we have investigated the role of CADM1 isoforms in the adhesion of HLMCs and HMC-1 cells to primary HASMCs and HLFs.CADM1 overexpression or downregulation was achieved using adenoviral delivery of CADM1 short hairpin RNAs or isoform-specific cDNAs respectively.Downregulation of CADM1 attenuated both HLMC and HMC-1 adhesion to both primary HASMCs and HLFs. Overexpression of either SP1 or SP4 isoforms did not alter MC adhesion to HASMCs, whereas overexpression of SP4, but not SP1, significantly increased both HMC-1 cell and HLMC adhesion to HLFs. The expression level of CADM1 SP4 strongly predicted the extent of MC adhesion; linear regression indicated that CADM1 accounts for up to 67% and 32% of adhesion to HLFs for HMC-1 cells and HLMCs, respectively. HLFs supported HLMC proliferation and survival through a CADM1-dependent mechanism. With respect to CADM1 counter-receptor expression, HLFs expressed both CADM1 and nectin-3, whereas HASMCs expressed only nectin-3.Collectively these data indicate that the CADM1 SP4 isoform is a key receptor mediating human MC adhesion to HASMCs and HLFs. The differential expression of CADM1 counter-receptors on HLFs compared to HASMCs may allow the specific targeting of either HLMC-HLF or HLMC-HASMC interactions in the lung parenchyma and airways.

The influx of extracellular Ca(2+) into mast cells is critical for the FcεR1-dependent release of preformed granule-derived mediators and newly synthesised autacoids and cytokines. The Orai(CRACM) ion channel family provide the major pathway through which this Ca(2+) influx occurs. However the individual role of each of the three members of the Orai channel family in Ca(2+) influx and mediator release has not been defined in human mast cells.To assess whether there might be value in targeting individual Orai family members for the inhibition of FcεRI-dependent human lung mast cells (HLMC) mediator release.We used an adenoviral delivery system to transduce HLMCs with shRNAs targeted against Orai1 and Orai2 or with cDNAs directing the expression of dominant-negative mutations of the three known Orai channels.shRNA-mediated knockdown of Orai1 resulted in a significant reduction of approximately 50% in Ca(2+) influx and in the release of β-hexosaminidase (a marker of degranulation) and newly synthesized LTC4 in activated HLMCs. In contrast shRNA knockdown of Orai2 resulted in only marginal reductions of Ca(2+) influx, degranulation and LTC4 release. Transduced dominant-negative mutants of Orai1, -2 and -3 markedly reduced Orai currents and completely inhibited HLMC degranulation suggesting that Orai channels form heteromultimers in HLMCs, and that Orai channels comprise the dominant Ca(2+) influx pathway following FceRI-dependent HLMC activation. Inhibition of Orai currents did not alter HLMC survival. In addition we observed a significant down-regulation of the level of CRACM3 mRNA transcripts together with a small increase in the level of CRACM1 and CRACM2 transcripts following a period of sustained HLMC activation.Orai1 plays an important role in Ca(2+) influx and mediator release from HLMCs. Strategies which target Orai1 will effectively inhibit FcεRI-dependent HLMC activation, but spare off-target inhibition of Orai2 in other cells and body systems.

Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that K(Ca)3.1 K(+) channel-dependent cell processes contribute to IPF pathophysiology.K(Ca)3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of K(Ca)3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct K(Ca)3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]).Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed K(Ca)3.1 channel mRNA and protein. K(Ca)3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. K(Ca)3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. K(Ca)3.1 was expressed strongly in IPF tissue. K(Ca)3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca(2+).K(Ca)3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking K(Ca)3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic.

The Ca(2+)-activated K(+) channel K(Ca)3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of K(Ca)3.1 modifies experimental asthma in sheep.Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the K(Ca)3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling.These findings suggest that K(Ca)3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of K(Ca)3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.

Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism.HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures.Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G(0)/G(i) receptor coupled mechanism. Solid phase extraction of lipids (

BACKGROUND: We have previously investigated the microlocalisation of M1 and M2 macrophages in NSCLC. This study investigated the non-macrophage (NM) expression of proteins associated with M1 and M2 macrophages in NSCLC. METHODS: Using immunohistochemistry, CD68(+) macrophages and proteins associated with either a cytotoxic M1 phenotype (HLA-DR, iNOS, and MRP 8/14), or a non-cytotoxic M2 phenotype (CD163 and VEGF) were identified. NM expression of the markers was analysed in the islets and stroma of surgically resected tumours from 20 patients with extended survival (ES) (median 92.7 months) and 20 patients with poor survival (PS) (median 7.7 months). RESULTS: The NM expression of NM-HLA-DR (p