Showing papers by "Donna E. Davies published in 2010"

A new look at the pathogenesis of asthma

Abstract: Asthma is an inflammatory disorder of the conducting airways that has strong association with allergic sensitization. The disease is characterized by a polarized Th-2 (T-helper-2)-type T-cell response, but in general targeting this component of the disease with selective therapies has been disappointing and most therapy still relies on bronchodilators and corticosteroids rather than treating underlying disease mechanisms. With the disappointing outcomes of targeting individual Th-2 cytokines or manipulating T-cells, the time has come to re-evaluate the direction of research in this disease. A case is made that asthma has its origins in the airways themselves involving defective structural and functional behaviour of the epithelium in relation to environmental insults. Specifically, a defect in barrier function and an impaired innate immune response to viral infection may provide the substrate upon which allergic sensitization takes place. Once sensitized, the repeated allergen exposure will lead to disease persistence. These mechanisms could also be used to explain airway wall remodelling and the susceptibility of the asthmatic lung to exacerbations provoked by respiratory viruses, air pollution episodes and exposure to biologically active allergens. Variable activation of this epithelial-mesenchymal trophic unit could also lead to the emergence of different asthma phenotypes and a more targeted approach to the treatment of these. It also raises the possibility of developing treatments that increase the lung's resistance to the inhaled environment rather than concentrating all efforts on trying to suppress inflammation once it has become established.

Double-stranded RNA induces disproportionate expression of thymic stromal lymphopoietin versus interferon-beta in bronchial epithelial cells from donors with asthma.

Abstract: Background Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that strongly activates dendritic cells and can initiate allergic inflammation. Since exposure to rhinovirus or double-stranded (ds) RNA (a surrogate of viral infection) induces TSLP expression in bronchial epithelial cells (BECs), this cytokine may link innate antiviral responses and the type 2 adaptive immune response. Objective As BECs from donors with asthma have a deficient interferon (IFN) response to rhinovirus infection, a study was undertaken to test the hypothesis that their antiviral response shows a bias towards TSLP production. Methods Primary BECs were grown from subjects with asthma and healthy volunteers. After exposure to dsRNA, interleukin (IL)-8, IFN β and TSLP mRNA and protein expression were measured by RT-qPCR and ELISA, respectively. Results dsRNA dose-dependently increased IL-8 expression in BECs with no significant difference between the groups. However, BECs from subjects with asthma expressed less IFN β and more TSLP mRNA and protein in response to dsRNA than BECs from those without asthma (median (IQR) 57 (38–82) pg/ml vs 106 (57–214) pg/ml for IFN β (p Conclusion BECs from subjects with asthma are biased towards higher TSLP and lower IFN β production in response to dsRNA, suggesting that viral infection in asthma may lead to an altered mediator profile that biases towards a Th2 immune response.

Identification of Lipocalin and Apolipoprotein A1 as Biomarkers of Chronic Obstructive Pulmonary Disease

Abstract: Rationale: Much effort is being made to discover noninvasive biomarkers of chronic airway disease that might enable better management, predict prognosis, and provide new therapeutic targets. Objectives: To undertake a comprehensive, unbiased proteomic analysis of induced sputum and identify novel noninvasive biomarkers for chronic obstructive pulmonary disease (COPD). Methods: Induced sputum was obtained from patients with COPD with a spectrum of disease severity and from control subjects. Two-dimensional gel electrophoresis and mass spectrometric identification of differentially expressed proteins were first applied to induced sputum from patients with GOLD stage 2 COPD and healthy smoker control subjects. Initial results thus obtained were validated by a combination of immunoassays (Western blotting and ELISA) applied to a large subject cohort. The biomarkers were localized to bronchial mucosa by immunohistochemistry. Measurements and Main Results: Of 1,325 individual protein spots identified, 37 were quantitatively and 3 qualitatively different between the two groups (P < 0.05%). Forty protein spots were subjected to tandem mass spectrometry, which identified 15 separate protein species. Seven of these were further quantified in induced sputum from 97 individuals. Using this sequential approach, two of these potential biomarkers (apolipoprotein A1 and lipocalin-1) were found to be significantly reduced in patients with COPD when compared with healthy smokers. Their levels correlated with FEV1/FVC, indicating their relationship to disease severity. Conclusions: A potential role for apolipoprotein A1 and lipocalin-1 in innate defense has been postulated previously; our discovery of their reduction in COPD indicates a deficient innate defense system in airway disease that could explain increased susceptibility to infectious exacerbations.

On-chip epithelial barrier function assays using electrical impedance spectroscopy

Abstract: A bio-impedance chip has been developed for real-time monitoring of the kinetics of epithelial cell monolayers in vitro. The human bronchial epithelial cell line (16-HBE 14o-) was cultured in Transwells® creating a sustainable and interactive model of the airway epithelium. Conducting polymer polypyrrole (PPy) doped with polystyrene sulfonate (PSS) was electrochemically deposited onto the surface of gold-plated electrodes to reduce the influence of the electrical double layer on the impedance measurements. Finite element and equivalent circuit models were used to model and determine the electrical properties of the epithelial cell monolayer from the impedance spectra. Electrically tight, confluent monolayers of 16 HBE 14o- cells were treated with increasing concentrations of either Triton X-100 to solubilize cell membranes or ethylene glycol-bis(2-aminoethyl-ether)-N,N,N′N′-tetraacetic acid (EGTA) to disrupt cell–cell adhesion. Experimental impedance data showed that disruption of epithelial barrier function in response to Triton X-100 and EGTA can be successfully measured by the bio-impedance chip. The results were consistent with the conventional hand-held trans-epithelial electrical resistance measurements. Immunofluorescent staining of the ZO-1 tight junction protein in the untreated and treated 16HBEs was performed to verify the disruption of the tight junctions by EGTA.

Osteopontin is expressed and functional in human eosinophils.

Abstract: BACKGROUND: Eosinophils are critically involved in allergic inflammation and tissue remodeling. Osteopontin (OPN) is a glycoprotein molecule which exhibits pro-fibrogenic and pro-angiogenic properties and has recently also been implicated in allergic diseases. In this study, we investigated the expression and function of OPN in human eosinophils. METHODS: Osteopontin mRNA (RT-PCR) and protein (immunofluorescence) expression in peripheral blood eosinophils from atopic human subjects were evaluated. Soluble OPN release was determined in resting and activated eosinophils. The contribution of OPN to eosinophil-induced angiogenesis was determined using the chick embryo chorio- allantoic membrane (CAM) assay and OPN-induced eosinophil chemotaxis was determined (ChemoTx System microplate wells). Finally, OPN expression in bronchoalveolar lavage (BAL) fluids from mild asthmatic and normal control subjects was determined. RESULTS: Osteopontin is expressed in human eosinophils and is increased following GM-CSF and IL-5 activation. Eosinophil-derived OPN contributes to eosinophil-induced angiogenesis. Recombinant OPN promotes eosinophil chemotaxis in vitro and this effect is mediated by alpha(4)beta(1) integrin binding. Soluble OPN is increased in the bronchoalveolar lavage fluid from mild asthmatic subjects and correlates with eosinophil counts. CONCLUSIONS: We therefore conclude that OPN is likely to contribute to the process of angiogenesis observed in the airways in asthma

Frmd7 expression in developing mouse brain.

Abstract: Aims: Mutations in the FERM domain containing 7 (FRMD7) genes are known to cause a significant number of cases of congenital idiopathic nystagmus (CIN). Only limited expression data exist suggesting low levels of expression in all tissues. In this study, we assess the expression profile of the murine homologue of FRMD7(Frmd7) in tissue from three murine organs during development. Methods: cDNA was extracted from heart, lung, and brain tissues of MF-1 mice at 12 developmental time points, embryonic days 11–19, postnatal days 1 and 8, and from adult mice. Relative expression of Frmd7mRNA was calculated using quantitative real-time PCR techniques with two normalising genes (Gapdhand Actb). Results: Expression of Frmd7was low in all tissues consistent with earlier reports. In heart and lung tissues, expression remained very low with an increase only in adult samples. In brain tissue, expression levels were higher at all time points with a significant increase at embryonic day 18, with no gender-specific influence on Frmd7expression. Conclusions: Frmd7is expressed at low levels in all tissues studied suggesting a role in many tissue types. However, higher overall expression and a sharp increase at ED18 in the murine brain suggest a different role in this tissue. Earlier studies have shown that genes expressed in the murine brain during development exhibit temporal functional clustering. The temporal pattern of Frmd7 expression found in this study mirrors that of genes involved in synapse formation/function, and genes related to axon growth/guidance. This suggests a role for Frmd7 in these processes and should direct further expression studies.

Sensors for chemical detection based on top-down fabricated polycrystalline silicon nanowires

Abstract: Semiconducting Silicon (Si) nanowires (NWs) have been widely investigated for their potential to function as highly sensitive and selective sensors for both chemical and biological purposes. A key point of this sensing method is to be real-time and label-free. Several interesting sensing assays have been demonstrated such as sensing of ions, proteins, DNA and viruses[1-3]. The available approaches of silicon nanowire fabrication usually use some advanced lithographic techniques i.e., deep-UV, electron-beam or nanoimprint lithography to pattern silicon nanowires on SOI wafers. Recently, spacer nanowires patterned by a conventional anisotropic dry etch were used to form transistors. While this approach has the advantage of CMOS-compatibility, these techniques are extremely expensive and accessible only to large-scale integrated circuit manufacturers. While this approach delivers a cheap route for nanowire definition, nanowire volume control across the wafer remains challenging as the nanowire sidewall region generally receives unwanted etching.

Polycrystalline silicon nanowires patterned by top-down lithography for biosensor applications

Abstract: Recently, Si nanowires are receiving much attention for biosensing because they offer the prospect of real-time, label-free, high sensitivity sensing. The most popular approach to silicon nanowire fabrication uses electron-beam lithography to pattern silicon nanowires on SOI wafers. While this approach has the advantage of CMOS-compatibility, it has the disadvantage of high cost, because both the lithography and the SOI wafers are expensive. Recently, spacer nanowires patterned by a conventional anisotropic dry etch were used to form transistors, which tends to give a triangular shape. In this paper, we demonstrate a low cost, CMOS-compatible fabrication process of polycrystalline silicon nanowires for biosensor applications using a Bosch dry etch process. The nanowires produced in this way have a rectangular profile, which gives good control over the nanowire width and height.