Tensin1 expression and function in chronic obstructive pulmonary disease

open access articleChronic obstructive pulmonary disease (COPD) constitutes a major cause of morbidity and mortality. Genome wide association studies have shown significant associations between airflow obstruction or COPD with a non-synonymous SNP in the TNS1 gene, which encodes tensin1. However, the expression, cellular distribution and function of tensin1 in human airway tissue and cells are unknown. We therefore examined these characteristics in tissue and cells from controls and people with COPD or asthma. Airway tissue was immunostained for tensin1. Tensin1 expression in cultured human airway smooth muscle cells (HASMCs) was evaluated using qRT-PCR, western blotting and immunofluorescent staining. siRNAs were used to downregulate tensin1 expression. Tensin1 expression was increased in the airway smooth muscle and lamina propria in COPD tissue, but not asthma, when compared to controls. Tensin1 was expressed in HASMCs and upregulated by TGFβ1. TGFβ1 and fibronectin increased the localisation of tensin1 to fibrillar adhesions. Tensin1 and α-smooth muscle actin (αSMA) were strongly co-localised, and tensin1 depletion in HASMCs attenuated both αSMA expression and contraction of collagen gels. In summary, tensin1 expression is increased in COPD airways, and may promote airway obstruction by enhancing the expression of contractile proteins and their localisation to stress fibres in HASMCs

Evidence for a novel Kit adhesion domain mediating human mast cell adhesion to structural airway cells

Background: Human lung mast cells (HLMCs) infiltrate the airway epithelium and airway smooth muscle (ASM) in asthmatic airways. The mechanism of HLMC adhesion to both cell types is only partly defined, and adhesion is not inhibited by function-blocking anti-Kit and anti-stem cell factor (SCF) antibodies. Our aim was to identify adhesion molecules expressed by human mast cells that mediate adhesion to human ASM cells (HASMCs) and human airway epithelial cells. Methods: We used phage-display to isolate single chain Fv (scFv) antibodies with adhesion-blocking properties from rabbits immunised with HLMC and HMC-1 membrane proteins. Results: Post-immune rabbit serum labelled HLMCs in flow cytometry and inhibited their adhesion to human BEAS-2B epithelial cells. Mast cell-specific scFvs were identified which labelled mast cells but not Jurkat cells by flow cytometry. Of these, one scFv (A1) consistently inhibited mast cell adhesion to HASMCs and BEAS-2B epithelial cells by about 30 %. A1 immunoprecipitated Kit (CD117) from HMC-1 lysates and bound to a human Kit-expressing mouse mast cell line, but did not interfere with SCF-dependent Kit signalling. Conclusion: Kit contributes to human mast cell adhesion to human airway epithelial cells and HASMCs, but may utilise a previously unidentified adhesion domain that lies outside the SCF binding site. Targeting this adhesion pathway might offer a novel approach for the inhibition of mast cell interactions with structural airway cells, without detrimental effects on Kit signalling in other tissues

An Experimental Study of Effects of Step Roughness in Skimming Flows on Stepped Chutes

On a spillway chute, a stepped design increases the rate of energy dissipation on the chute itself and reduces the size of a downstream energy dissipator. Up to date, the effects of step roughness on the flow properties remain unknown despite the practical relevance to damaged concrete steps, rock chutes and gabions weirs. New measurements were conducted in a large-size laboratory facility with two step conditions (smooth and rough) and three types of step roughness. Detailed air-water flow properties were measured systematically for several flow rates. The results showed faster flow motion on rough step chutes. Although the finding is counter-intuitive, it is linked with the location of the inception point of free-surface aeration being located further downstream than for a smooth stepped chute for an identical flow rate. In the aerated flow region, the velocities on rough-step chutes were larger than those of smooth chute flows for a given flow rate and dimensionless location from the inception point of free-surface aeration both at step edges and between step edges. The results suggest that design guidelines for smooth (concrete) stepped spillway may not be suitable to rough stepped chutes including gabion stepped weirs, and older stepped chutes with damaged steps

Image based machine learning for identification of macrophage subsets

Macrophages play a crucial rule in orchestrating immune responses against pathogens and foreign materials. Macrophages have remarkable plasticity in response to environmental cues and are able to acquire a spectrum of activation status, best exemplified by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at the two ends of the spectrum. Characterisation of M1 and M2 subsets is usually carried out by quantification of multiple cell surface markers, transcription factors and cytokine profiles. These approaches are time consuming, require large numbers of cells and are resource intensive. In this study, we used machine learning algorithms to develop a simple and fast imaging-based approach that enables automated identification of different macrophage functional phenotypes using their cell size and morphology. Fluorescent microscopy was used to assess cell morphology of different cell types which were stained for nucleus and actin distribution using DAPI and phalloidin respectively. By only analysing their morphology we were able to identify M1 and M2 phenotypes effectively and could distinguish them from naïve macrophages and monocytes with an average accuracy of 90%. Thus we suggest high-content and automated image analysis can be used for fast phenotyping of functionally diverse cell populations with reasonable accuracy and without the need for using multiple markers

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

Background: Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the refractory period observed in patients with EIB. Objective: This study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction. Methods: We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP) antagonists for EP1-4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed. Results: Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP2 and EP4 receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone. Conclusions:Our data show a protective role for the PGE2 receptors EP2 and EP4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition

Temperature-controlled laminar airflow in severe asthma for exacerbation reduction (The LASER Trial): study protocol for a randomised controlled trial

BackgroundAsthma affects more than 5 million patients in the United Kingdom. Nearly 500,000 of these patients have severe asthma with severe symptoms and frequent exacerbations that are inadequately controlled with available treatments. The burden of severe asthma on the NHS is enormous, accounting for 80 % of the total asthma cost (£1 billion), with frequent exacerbations and expensive medications generating much of this cost.Of those patients with severe asthma, 70 % are sensitised to indoor aeroallergens, and the level of exposure to allergens determines the symptoms; patients exposed to high levels are therefore most at risk of exacerbations and hospital admissions.The LASER trial aims to assess whether a new treatment, temperature controlled laminar airflow (TLA) delivered by the Airsonett™ device, can reduce the frequency of exacerbations in patients with severe allergic asthma by reducing exposure to aeroallergens overnight.MethodsThis multicentre study is a placebo-controlled, blinded, randomised controlled, parallel group trial. A total of 222 patients with a new or current diagnosis of severe allergic asthma will be assigned with a random element in a 1:1 ratio to receive either an active device for one year or a placebo device. The primary outcome is the frequency of severe asthma exacerbations occurring over a 12-month period, defined in accordance with the American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines. Secondary outcomes include changes in asthma control, lung function, asthma-specific and global quality of life for participants and their carers, adherence to intervention, healthcare resource use and costs, and cost-effectiveness. Qualitative interviews will be conducted to elicit participant’s and their partner’s perceptions of the treatment.DiscussionEffective measures of allergen avoidance have, to date, proved elusive. The LASER trial aims to address this. The study will ascertain whether home-based nocturnal TLA usage over a 12-month period can reduce the frequency of exacerbations and improve asthma control and quality of life as compared to placebo, whilst being cost-effective and acceptable to adults with poorly controlled, severe allergic asthma. The results of this study will be widely applicable to the many patients with allergic asthma both in the UK and internationally.Trial registrationCurrent controlled trials ISRCTN46346208 (Date assigned 22 January 2014)

Effects of intranasal TNFα on granulocyte recruitment and activity in healthy subjects and patients with allergic rhinitis

<p>Abstract</p> <p>Background</p> <p>TNFα may contribute to the pathophysiology of airway inflammation. For example, we have recently shown that nasal administration of TNFα produces late phase co-appearance of granulocyte and plasma exudation markers on the mucosal surface. The objective of the present study was to examine indices of granulocyte presence and activity in response to intranasal TNFα challenge.</p> <p>Methods</p> <p>Healthy subjects and patients with allergic rhinitis (examined out of season) were subjected to nasal challenge with TNFα (10 μg) in a sham-controlled and crossover design. Nasal lavages were carried out prior to and 24 hours post challenge. Nasal biopsies were obtained post challenge. Nasal lavage fluid levels of myeloperoxidase (MPO) and eosinophil cationic protein (ECP) were analyzed as indices of neutrophil and eosinophil activity. Moreover, IL-8 and α₂-macroglobulin were analyzed as markers of pro-inflammatory cytokine production and plasma exudation. Nasal biopsy numbers of neutrophils and eosinophils were monitored.</p> <p>Results</p> <p>Nasal lavage fluid levels of MPO recorded 24 hours post TNFα challenge were increased in healthy subjects (p = 0.0081) and in patients with allergic rhinitis (p = 0.0081) (<it>c.f</it>. sham challenge). Similarly, α₂-macroglobulin was increased in healthy subjects (p = 0.014) and in patients with allergic rhinitis (p = 0.0034). Lavage fluid levels of ECP and IL-8 were not affected by TNFα challenge. TNFα increased the numbers of subepithelial neutrophils (p = 0.0021), but not the numbers of eosinophils.</p> <p>Conclusion</p> <p>TNFα produces a nasal inflammatory response in humans that is characterised by late phase (i.e., 24 hours post challenge) neutrophil activity and plasma exudation.</p