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  • Conference paper
    Finney LJ, Belchamber KBR, Mallia P, Johnston SL, Donnelly LE, Wedzicha JAet al., 2016,

    HUMAN RHINOVIRUS IMPAIRS THE INNATE IMMUNE RESPONSE TO BACTERIA IN MONOCYTE DERIVED MACROPHAGES FROM PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE

    , British Thoracic Society Winter Meeting 2016, Publisher: BMJ PUBLISHING GROUP, Pages: A2-A2, ISSN: 0040-6376
  • Journal article
    Telcian AG, Zdrenghea MT, Edwards MR, Laza-Stanca V, Mallia P, Johnston SL, Stanciu LAet al., 2016,

    Vitamin D increases the antiviral activity of bronchial epithelial cells in vitro

    , Antiviral Research, Vol: 137, Pages: 93-101, ISSN: 1872-9096

    BACKGROUND: By modulating the antiviral immune response via vitamin D receptor, the active form of vitamin D (1,25-dihydroxyvitamin D, calcitriol) could play a central role in protection against respiratory virus infections. This in vitro study tested the hypothesis that respiratory viruses modulate vitamin D receptor expression in human bronchial epithelial cells and this modulation affects the antiviral response to exogenous vitamin D. METHODS: Human primary bronchial epithelial cells were infected with rhinoviruses and respiratory syncytial virus in the presence or absence of vitamin D. Expression of vitamin D receptor, 1α-hydroxylase (1α(OH)ase), 24-hydroxylase (24(OH)ase), innate interferons, interferon stimulated genes and cathelicidin were measured by quantitative polymerase chain reaction. The antiviral effect of vitamin D on rhinovirus replication was determined by measurement of virus load. A direct inactivation assay was used to determine the antiviral activity of cathelicidin. RESULTS: Both RV and RSV decreased vitamin D receptor and 24(OH)ase and, in addition, RSV increased 1α(OH)ase expression in epithelial cells. Vitamin D decreased rhinovirus replication and release, and increased rhinovirus-induced interferon stimulated genes and cathelicidin. Furthermore, cathelicidin had direct anti-rhinovirus activity. CONCLUSIONS: Despite lower vitamin D receptor levels in rhinovirus-infected epithelial cells, exogenous vitamin D increased antiviral defences most likely via cathelicidin and innate interferon pathways.

  • Journal article
    Carugo D, Aron M, Sezgin E, Bernardino de la Serna J, Kuimova MK, Eggeling C, Stride Eet al., 2016,

    Modulation of the molecular arrangement in artificial and biological membranes by phospholipid-shelled microbubbles

    , Biomaterials, Vol: 113, Pages: 105-117, ISSN: 1878-5905

    The transfer of material from phospholipid-coated microbubbles to cell membranes has been hypothesized to play a role in ultrasound-mediated drug delivery. In this study, we employed quantitative fluorescence microscopy techniques to investigate this phenomenon in both artificial and biological membrane bilayers in an acoustofluidic system. The results of the present study provide strong evidence for the transfer of material from microbubble coatings into cell membranes. Our results indicate that transfer of phospholipids alters the organization of molecules in cell membranes, specifically the lipid ordering or packing, which is known to be a key determinant of membrane mechanical properties, protein dynamics, and permeability. We further show that polyethylene-glycol, used in many clinical microbubble formulations, also has a major impact on both membrane lipid ordering and the extent of lipid transfer, and that this occurs even in the absence of ultrasound exposure.

  • Journal article
    Baker JR, Vuppusetty C, Colley T, Papaioannou AI, Fenwick P, Donnelly L, Ito K, Barnes PJet al., 2016,

    Oxidative stress dependent microRNA-34a activation via PI3Kα reduces the expression of sirtuin-1 and sirtuin-6 in epithelial cells

    , Scientific Reports, Vol: 6, ISSN: 2045-2322

    Sirtuin-1 (SIRT1) and SIRT6, NAD(+)-dependent Class III protein deacetylases, are putative anti-aging enzymes, down-regulated in patients with chronic obstructive pulmonary disease (COPD), which is characterized by the accelerated ageing of the lung and associated with increased oxidative stress. Here, we show that oxidative stress (hydrogen peroxide) selectively elevates microRNA-34a (miR-34a) but not the related miR-34b/c, with concomitant reduction of SIRT1/-6 in bronchial epithelial cells (BEAS2B), which was also observed in peripheral lung samples from patients with COPD. Over-expression of a miR-34a mimic caused a significant reduction in both mRNA and protein of SIRT1/-6, whereas inhibition of miR-34a (antagomir) increased these sirtuins. Induction of miR-34a expression with H2O2 was phosphoinositide-3-kinase (PI3K) dependent as it was associated with PI3Kα activation as well as phosphatase and tensin homolog (PTEN) reduction. Importantly, miR-34a antagomirs increased SIRT1/-6 mRNA levels, whilst decreasing markers of cellular senescence in airway epithelial cells from COPD patients, suggesting that this process is reversible. Other sirtuin isoforms were not affected by miR-34a. Our data indicate that miR-34a is induced by oxidative stress via PI3K signaling, and orchestrates ageing responses under oxidative stress, therefore highlighting miR-34a as a new therapeutic target and biomarker in COPD and other oxidative stress-driven aging diseases.

  • Conference paper
    Jarrett R, Ogg G, Salio M, Lloyd-Lavery A, Subramaniam S, Bourgeois E, Archer C, Cheung A, Hardman C, Chandler D, Salimi M, Gutowska-Owsiak D, Bernardino de la Serna J, Fallon PG, Jolin H, Mckenzie A, Dziembowski A, Podobas EI, Bal W, Johnson D, Moody DB, Cerundolo Vet al., 2016,

    Filaggrin inhibits house dust mite phospholipase generation of CD1a lipid antigens for recognition by T cells

    , 20th Anniversary Conference of the British-Skin-Foundation on Skin Deep - 20 Years of Research, Publisher: WILEY-BLACKWELL, Pages: 50-50, ISSN: 0007-0963
  • Journal article
    Bernardino de la Serna J, Schuetz GJ, Eggeling C, Cebecauer Met al., 2016,

    There is no simple model of the plasma membrane organizationyy

    , Frontiers in Cell and Developmental Biology, Vol: 4, ISSN: 2296-634X

    Ever since technologies enabled the characterization of eukaryotic plasma membranes,heterogeneities in the distributions of its constituents were observed. Over the years thisled to the proposal of various models describing the plasma membrane organizationsuch as lipid shells, picket-and-fences, lipid rafts, or protein islands, as addressed innumerous publications and reviews. Instead of emphasizing on one model we in thisreview give a brief overview over current models and highlight how current experimentalwork in one or the other way do not support the existence of a single overarching model.Instead, we highlight the vast variety of membrane properties and components, theirinfluences and impacts. We believe that highlighting such controversial discoveries willstimulate unbiased research on plasma membrane organization and functionality, leadingto a better understanding of this essential cellular structure.

  • Journal article
    Stanly TA, Fritzsche M, Banerji S, Garcia E, Bernardino de la Serna J, Jackson DG, Eggeling Cet al., 2016,

    Critical importance of appropriate fixation conditions for faithful imaging of receptor microclusters

    , Biology Open, Vol: 5, Pages: 1343-1350, ISSN: 2046-6390

    Receptor clustering is known to trigger signalling events that contributeto critical changes in cellular functions. Faithful imaging of suchclusters by means of fluorescence microscopy relies on the applicationof adequate cell fixation methods prior to immunolabelling in order toavoid artefactual redistribution by the antibodies themselves. Previouswork has highlighted the inadequacy of fixation with paraformaldehyde(PFA) alone for efficient immobilisation of membrane-associatedmolecules, and the advantages of fixation with PFA in combinationwith glutaraldehyde (GA). Using fluorescence microscopy, we herehighlight how inadequate fixation can lead to the formation ofartefactual clustering of receptors in lymphatic endothelial cells,focussing on the transmembrane hyaluronan receptors LYVE-1 andCD44, and the homotypic adhesion molecule CD31, each of whichdisplays their native diffuse surface distribution pattern only whenvisualised with the right fixation techniques, i.e. PFA/GA incombination. Fluorescence recovery after photobleaching (FRAP)confirms that the artefactual receptor clusters are indeed introduced byresidual mobility. In contrast, we observed full immobilisation ofmembrane proteins in cells that were fixed and then subsequentlypermeabilised, irrespective of whether the fixative was PFA or PFA/GAin combination. Our study underlines the importance of choosingappropriate sample preparation protocols for preserving authenticreceptor organisation in advanced fluorescence microscopy

  • Journal article
    Kuo CS, Pavlidis S, Loza M, Baribaud F, Rowe A, Pandis I, Hoda U, Rossios C, Sousa A, Wilson SJ, Howarth P, Dahlen B, Dahlen SE, Chanez P, Shaw D, Krug N, Sandström T, De Meulder B, Lefaudeux D, Fowler S, Fleming L, Corfield J, Auffray C, Sterk PJ, Djukanovic R, Guo Y, Adcock IM, Chung KF, U-BIOPRED Project Teamet al., 2016,

    A Transcriptome-driven Analysis of Epithelial Brushings and Bronchial Biopsies to Define Asthma Phenotypes in U-BIOPRED

    , American Journal of Respiratory and Critical Care Medicine, Vol: 195, Pages: 443-455, ISSN: 1535-4970

    RATIONALE AND OBJECTIVES: Asthma is a heterogeneous disease driven by diverse immunologic and inflammatory mechanisms. We used transcriptomic profiling of airway tissues to help define asthma phenotypes. METHODS: The transcriptome from bronchial biopsies and epithelial brushings of 107 moderate-to-severe asthmatics were annotated by gene-set variation analysis (GSVA) using 42 gene-signatures relevant to asthma, inflammation and immune function. Topological data analysis (TDA) of clinical and histological data was used to derive clusters and the nearest shrunken centroid algorithm used for signature refinement. RESULTS: 9 GSVA signatures expressed in bronchial biopsies and airway epithelial brushings distinguished two distinct asthma subtypes associated with high expression of T-helper type 2 (Th-2) cytokines and lack of corticosteroid response (Group 1 and Group 3). Group 1 had the highest submucosal eosinophils, high exhaled nitric oxide (FeNO) levels, exacerbation rates and oral corticosteroid (OCS) use whilst Group 3 patients showed the highest levels of sputum eosinophils and had a high BMI. In contrast, Group 2 and Group 4 patients had an 86% and 64% probability of having non-eosinophilic inflammation. Using machine-learning tools, we describe an inference scheme using the currently-available inflammatory biomarkers sputum eosinophilia and exhaled nitric oxide levels along with OCS use that could predict the subtypes of gene expression within bronchial biopsies and epithelial cells with good sensitivity and specificity. CONCLUSION: This analysis demonstrates the usefulness of a transcriptomic-driven approach to phenotyping that segments patients who may benefit the most from specific agents that target Th2-mediated inflammation and/or corticosteroid insensitivity.

  • Journal article
    Barnes PJ, 2016,

    Kinases as Novel Therapeutic Targets in Asthma and Chronic Obstructive Pulmonary Disease

    , Pharmacological Reviews, Vol: 68, Pages: 788-815, ISSN: 1521-0081

    Multiple kinases play a critical role in orchestrating the chronic inflammation and structural changes in the respiratory tract of patients with asthma and chronic obstructive pulmonary disease (COPD). Kinases activate signaling pathways that lead to contraction of airway smooth muscle and release of inflammatory mediators (such as cytokines, chemokines, growth factors) as well as cell migration, activation, and proliferation. For this reason there has been great interest in the development of kinase inhibitors as anti-inflammatory therapies, particular where corticosteroids are less effective, as in severe asthma and COPD. However, it has proven difficult to develop selective kinase inhibitors that are both effective and safe after oral administration and this has led to a search for inhaled kinase inhibitors, which would reduce systemic exposure. Although many kinases have been implicated in inflammation and remodeling of airway disease, very few classes of drug have reached the stage of clinical studies in these diseases. The most promising drugs are p38 MAP kinases, isoenzyme-selective PI3-kinases, Janus-activated kinases, and Syk-kinases, and inhaled formulations of these drugs are now in development. There has also been interest in developing inhibitors that block more than one kinase, because these drugs may be more effective and with less risk of losing efficacy with time. No kinase inhibitors are yet on the market for the treatment of airway diseases, but as kinase inhibitors are improved from other therapeutic areas there is hope that these drugs may eventually prove useful in treating refractory asthma and COPD.

  • Journal article
    Barnes PJ, 2016,

    Inflammatory mechanisms in patients with chronic obstructive pulmonary disease

    , Journal of Allergy and Clinical Immunology, Vol: 138, Pages: 16-27, ISSN: 1097-6825

    Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation affecting predominantly the lung parenchyma and peripheral airways that results in largely irreversible and progressive airflow limitation. This inflammation is characterized by increased numbers of alveolar macrophages, neutrophils, T lymphocytes (predominantly TC1, TH1, and TH17 cells), and innate lymphoid cells recruited from the circulation. These cells and structural cells, including epithelial and endothelial cells and fibroblasts, secrete a variety of proinflammatory mediators, including cytokines, chemokines, growth factors, and lipid mediators. Although most patients with COPD have a predominantly neutrophilic inflammation, some have an increase in eosinophil counts, which might be orchestrated by TH2 cells and type 2 innate lymphoid cells though release of IL-33 from epithelial cells. These patients might be more responsive to corticosteroids and bronchodilators. Oxidative stress plays a key role in driving COPD-related inflammation, even in ex-smokers, and might result in activation of the proinflammatory transcription factor nuclear factor κB (NF-κB), impaired antiprotease defenses, DNA damage, cellular senescence, autoantibody generation, and corticosteroid resistance though inactivation of histone deacetylase 2. Systemic inflammation is also found in patients with COPD and can worsen comorbidities, such as cardiovascular diseases, diabetes, and osteoporosis. Accelerated aging in the lungs of patients with COPD can also generate inflammatory protein release from senescent cells in the lung. In the future, it will be important to recognize phenotypes of patients with optimal responses to more specific therapies, and development of biomarkers that identify the therapeutic phenotypes will be important.

  • Conference paper
    Jarrett R, Salio M, Lloyd-Lavery A, Subramaniam S, Bourgeois E, Archer C, Cheung A, Hardman C, Chandler D, Salimi M, Gutowska-Owsiak D, Bernardino de la Serna J, Fallon P, Jolin H, Mckenzie A, Dziembowski A, Podobas E, Bal W, Johnson D, Moody DB, Cerundolo V, Ogg Get al., 2016,

    Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite-derived phospholipase

    , Annual Meeting of the British-Society-for-Investigative-Dermatology (BSID), Publisher: WILEY-BLACKWELL, Pages: E49-E50, ISSN: 0007-0963
  • Journal article
    Garcia E, Ragazzini C, Yu X, Cuesta-Garcia E, Bernardino de la Serna J, Zech T, Sarrio D, Machesky LM, Anton IMet al., 2016,

    WIP and WICH/WIRE co-ordinately control invadopodium formation and maturation in human breast cancer cell invasion

    , Scientific Reports, Vol: 6, ISSN: 2045-2322

    Cancer cells form actin-rich degradative protrusions (invasive pseudopods and invadopodia), whichallows their efficient dispersal during metastasis. Using biochemical and advanced imaging approaches,we demonstrate that the N-WASP-interactors WIP and WICH/WIRE play non-redundant roles incancer cell invasion. WIP interacts with N-WASP and cortactin and is essential for invadopodiumassembly, whereas WICH/WIRE regulates N-WASP activation to control invadopodium maturationand degradative activity. Our data also show that Nck interaction with WIP and WICH/WIRE modulatesinvadopodium maturation; changes in WIP and WICH/WIRE levels induce differential distribution ofNck. We show that WIP can replace WICH/WIRE functions and that elevated WIP levels correlate withhigh invasiveness. These findings identify a role for WICH/WIRE in invasiveness and highlight WIP asa hub for signaling molecule recruitment during invadopodium generation and cancer progression, aswell as a potential diagnostic biomarker and an optimal target for therapeutic approaches.

  • Conference paper
    Bernardino de la Serna J, Chang VT, Waithe D, Fernandes RA, Fritzsche M, Santos AM, Shrestha D, Felce JH, Assmann MC, Davis SJ, Eggeling Cet al., 2016,

    T-Cells in Suspension Do Not Show Pre-Clustered LCK

    , 60th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 570A-570A, ISSN: 0006-3495
  • Journal article
    Jarrett R, Salio M, Lloyd-Lavery A, Subramaniam S, Bourgeois E, Archer C, Cheung KL, Hardman C, Chandler D, Salimi M, Gutowska-Owsiak D, Bernardino de la Serna J, Fallon PG, Jolin H, Mckenzie A, Dziembowski A, Podobas EI, Bal W, Johnson D, Moody DB, Cerundolo V, Ogg Get al., 2016,

    Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite-derived phospholipase

    , SCIENCE TRANSLATIONAL MEDICINE, Vol: 8, ISSN: 1946-6234
  • Journal article
    Mitani A, Ito K, Vuppusetty C, Barnes PJ, Mercado Net al., 2016,

    Restoration of Corticosteroid Sensitivity in Chronic Obstructive Pulmonary Disease by Inhibition of Mammalian Target of Rapamycin

    , AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 193, Pages: 143-153, ISSN: 1073-449X
  • Journal article
    Clausen MP, Sezgin E, Bernardino de la Serna J, Waithe D, Lagerholm BC, Eggeling Cet al., 2015,

    A straightforward approach for gated STED-FCS to investigate lipid membrane dynamics

    , Methods, Vol: 88, Pages: 67-75, ISSN: 1046-2023

    Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity. A unique advantage of STED-FCS is that the observation spot for the FCS data recordings can be tuned to sub-diffraction scales, i.e. <200 nm in diameter, in a gradual manner to investigate fast diffusion of membrane-incorporated labelled entities. Unfortunately, so far the STED-FCS technology has mostly been applied on a few custom-built setups optimised for far-red fluorescent emitters. Here, we summarise the basics of the STED-FCS technology and highlight how it can give novel details into molecular diffusion modes. Most importantly, we present a straightforward way for performing STED-FCS measurements on an unmodified turnkey commercial system using a time-gated detection scheme. Further, we have evaluated the STED-FCS performance of different commonly used green emitting fluorescent dyes applying freely available, custom-written analysis software.

  • Journal article
    Barnes PJ, 2015,

    Therapeutic approaches to asthma-chronic obstructive pulmonary disease overlap syndromes

    , JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, Vol: 136, Pages: 531-545, ISSN: 0091-6749
  • Journal article
    Wiegman CH, Michaeloudes C, Haji G, Narang P, Clarke CJ, Russell KE, Bao W, Pavlidis S, Barnes PJ, Kanerva J, Bittner A, Rao N, Murphy MP, Kirkham PA, Chung KF, Adcock IMet al., 2015,

    Oxidative stress-induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease

    , Journal of Allergy and Clinical Immunology, Vol: 136, Pages: 769-780, ISSN: 1097-6825

    BackgroundInflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress–induced pathology.ObjectiveWe sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells.MethodsMice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ.ResultsMice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-β–induced ASM cell proliferation and CXCL8 release.ConclusionsMitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammat

  • Journal article
    Barnes PJ, 2015,

    Identifying Molecular Targets for New Drug Development for Chronic Obstructive Pulmonary Disease: What Does the Future Hold?

    , SEMINARS IN RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 36, Pages: 508-522, ISSN: 1069-3424
  • Journal article
    Tetley TD, Ruenraroengsak P, 2015,

    Differential bioreactivity of neutral, cationic and anionic polystyrene nanoparticles with cells from the human alveolar compartment: robust response of alveolar type 1 epithelial cells

    , Particle and Fibre Toxicology, Vol: 12, ISSN: 1743-8977

    BackgroundEngineered nanoparticles (NP) are being developed for inhaled drug delivery. This route is non-invasive and the major target; alveolar epithelium provides a large surface area for drug administration and absorption, without first pass metabolism. Understanding the interaction between NPs and target cells is crucial for safe and effective NP-based drug delivery. We explored the differential effect of neutral, cationic and anionic polystyrene latex NPs on the target cells of the human alveolus, using primary human alveolar macrophages (MAC) and primary human alveolar type 2 (AT2) epithelial cells and a unique human alveolar epithelial type I-like cell (TT1). We hypothesized that the bioreactivity of the NPs would relate to their surface chemistry, charge and size as well as the functional role of their interacting cells in vivo.MethodsAmine- (ANP) and carboxyl- surface modified (CNP) and unmodified (UNP) polystyrene NPs, 50 and 100 nm in diameter, were studied. Cells were exposed to 1–100 μg/ml (1.25-125 μg/cm 2 ; 0 μg/ml control) NP for 4 and 24 h at 37 °C with or without the antioxidant, N-acetyl cysteine (NAC). Cells were assessed for cell viability, reactive oxygen species (ROS), oxidised glutathione (GSSG/GSH ratio), mitochondrial integrity, cell morphology and particle uptake (using electron microscopy and laser scanning confocal microscopy).ResultsANP-induced cell death occurred in all cell types, inducing increased oxidative stress, mitochondrial disruption and release of cytochrome C, indicating apoptotic cell death. UNP and CNP exhibited little cytotoxicity or mitochondrial damage, although they induced ROS in AT2 and MACs. Addition of NAC reduced epithelial cell ROS, but not MAC ROS, for up to 4 h. TT1 and MAC cells internalised all NP formats, whereas only a small fraction of AT2 cells internalized ANP (not UNP or CNP). TT1 cells were the most resistant to the effects of UNP and CNP.ConclusionANP induced marked oxidative damage

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