Showing papers by "Donna E. Davies published in 2017"

Phenotypic and genetic aspects of epithelial barrier function in asthmatic patients.

Abstract: The bronchial epithelium is continuously exposed to a multitude of noxious challenges in inhaled air. Cellular contact with most damaging agents is reduced by the action of the mucociliary apparatus and by formation of a physical barrier that controls passage of ions and macromolecules. In conjunction with these defensive barrier functions, immunomodulatory cross-talk between the bronchial epithelium and tissue-resident immune cells controls the tissue microenvironment and barrier homeostasis. This is achieved by expression of an array of sensors that detect a wide variety of viral, bacterial, and nonmicrobial (toxins and irritants) agents, resulting in production of many different soluble and cell-surface molecules that signal to cells of the immune system. The ability of the bronchial epithelium to control the balance of inhibitory and activating signals is essential for orchestrating appropriate inflammatory and immune responses and for temporally modulating these responses to limit tissue injury and control the resolution of inflammation during tissue repair. In asthmatic patients abnormalities in many aspects of epithelial barrier function have been identified. We postulate that such abnormalities play a causal role in immune dysregulation in the airways by translating gene-environment interactions that underpin disease pathogenesis and exacerbation.

Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma

Abstract: Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy.

The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis

Abstract: Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects.

Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double-stranded RNA.

Abstract: Introduction: The epithelial and endothelial barriers of the airway mucosa are critical for regulation of tissue homeostasis and protection against pathogens or other tissue damaging agents. In response to a viral infection, epithelial cells must signal to the endothelium to initiate immune cell recruitment. This is a highly temporal regulated process; however, the mechanisms of this cross-talk are not fully understood. Methods: In a close-contact co-culture model of human airway epithelial and endothelial cells cellular crosstalk was analysed using transepithelial electrical resistance (TER) measurements, immunofluorescence, electron microscopy and ELISA. Viral infections were simulated by exposing airway epithelial cells apically to double-stranded RNA (Poly(I:C)). Using a microfluidic culture system the temporal release of mediators was analysed in the co-culture model. Results: Within 4h of challenge, double-stranded RNA induced the release of TNF-a by epithelial cells. This activated endothelial cells by triggering the release of the chemoattractant CX3CL1 (fractalkine) by 8h post-challenge and expression of adhesion molecules E-selectin and ICAM-1. These responses were significantly reduced by neutralising TNF-a. Conclusion: By facilitating kinetic profiling, the microfluidic co-culture system has enabled identification of a key signalling mechanism between the epithelial and endothelial barriers. Better understanding of cell-cell cross-talk and its regulatory mechanisms has the potential to identify new therapeutic strategies to control airway inflammation.

Functional characterization of a novel 3D model of the epithelial-mesenchymal trophic unit

Abstract: Background/Aim: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. Materials and Methods: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). Results: The outgrowths formed as a multicellular sheet with motile cilia becoming evident as the Matrigel was remodeled to provide an air interface; cultures were viable for more than one year. Immunofluorescence and electron microscopy (EM) identified an upper layer of mucociliary epithelium and a lower layer of ...

Mast cells are permissive for rhinovirus replication: potential implications for asthma exacerbations

Abstract: BACKGROUND: Human rhinoviruses (HRVs) are a major trigger of asthma exacerbations, with the bronchial epithelium being the major site of HRV infection and replication. Mast cells (MCs) play a key role in asthma where their numbers are increased in the bronchial epithelium with increasing disease severity. OBJECTIVE: In view of the emerging role of MCs in innate immunity and increased localisation to the asthmatic bronchial epithelium, we investigated whether HRV infection of MCs generated innate immune responses which were protective against infection. METHODS: The LAD2 MC line or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV at increasing multiplicities of infection (MOI) without or with IFN-? or IFN-?. After 24 h, innate immune responses were assessed by RT-qPCR and IFN protein release by ELISA. Viral replication was determined by RT-qPCR and virion release by TCID50 assay. RESULTS: HRV infection of LAD2 MCs induced expression of IFN-?, IFN-? and IFN-stimulated genes. However, LAD2 MCs were permissive for HRV replication and release of infectious HRV particles. Similar findings were observed with CBMCs. Neutralisation of the type I IFN receptor had minimal effects on viral shedding suggesting that endogenous type I IFN signalling offered limited protection against HRV. However, augmentation of these responses by exogenous IFN-?, but not IFN-?, protected MCs against HRV infection. CONCLUSION AND CLINICAL RELEVANCE: MCs are permissive for the replication and release of HRV which is prevented by exogenous IFN-? treatment. Taken together these findings suggest a novel mechanism whereby MCs may contribute to HRV-induced asthma exacerbations

In our opinion.

Abstract: The misleading statements made in the British Dental Journal in the December 2016 issue relating to dental age assessment are assessed for inaccuracies and negligent omission of the issue of Child Protection. It is emphasised that there is a need for the approach of objective knowledge viz. not influenced by personal feelings or opinions in considering and representing facts. The article by the Chair of the Education, Ethics, and Team Working Group implies that unsatisfactory consent procedures are followed. The DARLInG (Dental Age Research London Information Group) have followed a carefully prescribed procedure that fulfils all the requirements of the advice given by the Consent Committee at King's College Hospital. In addition, the active support in the form of independent support workers and lawyers assisted by interpreters is described. The issue of the lawful use of ionising radiation is described with correct information about where this information can be obtained. The seriously misleading statements made by the Chair of the Education, Ethics and Education Working Group are identified. An unacceptable oversight is the failure of the BDA representatives to draw attention to the need for child protection. The potential benefit of dental age estimation in terms of appropriately providing support for asylum seekers is described.The failure of the BDA Ethics group to be up to date with recent research which shows a high level of certainty in assigning age disputed subjects to above (or below) the 18-year threshold is discussed and the importance of this in reliably determining, in an objective way, the age status of asylum seekers. The incorrect and salacious use of the term 'X-rated' is highlighted and a formal request for its withdrawal has been made.

S89 Soluble adam33 augments the pulmonary immune response promoting allergic airway sensitivity

Abstract: Rational A disintegrin and metalloproteinase 33 (ADAM33) was discovered in 2002 as an asthma susceptibility gene. Genetic associations have been made between ADAM33 polymorphisms and asthma disease severity, bronchial hyperresponsivenss (BHR) and rate of lung function decline in both adults and children. A soluble, catalytically active form of the protein (sADAM33) has been identified in the bronchoalveolar lavage fluid of patients, levels of which correlate with disease severity (Lee JY et al, AJRCCM 2006). To study the role sADAM33, a lung specific, doxycycline (Dox) inducible, transgenic mouse, expressing the human pro and metalloproteinase domains of the full-length protein was generated (Ccsp-rtTA/Otet-ADAM33-Pro-MP). Induction of sADAM33, followed by house dust mite (HDM) sensitisation and challenge, resulted in increased BHR and airway inflammation (Davies ER et al, JCI-Insight 2016). The mechanisms by which ADAM33 promotes this susceptibility are unclear. The aim of this work is to identify pathways that are augmented by the induction of sADAM33, which promote increased sensitivity to allergen. Methods RNA samples from whole lung of adult mice, where sADAM33 had been induced for 4 or 8 weeks, were analysed by next generation RNA sequencing. Identified genes were confirmed across experimental time points (72 hour, 7 day, 4 and 8 weeks on Dox) in wider sample cohorts of Ccsp-rtTA/Otet-ADAM33-Pro-MP and control mice through RTqPCR. Results The predominant signal from the RNAseq output was for modulation of immune response genes at 4 weeks of sADAM33 expression (GO:0006955 Immune response: 31 genes, 58.49% coverage, FDR p value=7.09 E-22). Genes associated with an immune activation signature (Ccl5, Irgm1, Gm12250, Gzmb, Ncr1) were validated at least one time point. By 8 weeks of sADAM33 expression genes associated with negative regulation of the response were also validated (Ido1, Cd274). Conclusion Induction of sADAM33 in murine lungs, without allergic sensitisation, augmented underlying immune processes in this transgenic mouse model, which may contribute to increased susceptibility to allergic airway inflammation and BHR when challenged with allergen. Further work is required to delineate how sADAM33 affects immune cell populations and their behaviour in the lung.

Evaluation of novel LOXL2-selective inhibitors in models of pulmonary fibrosis

Abstract: Background: Increased tissue stiffness is a consequence and a driver of fibrosis. Tissue stiffness is modulated by the lysyl oxidase (LOX) family of enzymes, which cross-link collagen and elastin fibres. Increased expression of LOX-like-2 (LOXL2) is associated with the development of fibrosis and therefore a therapeutic target. We have profiled novel orally bioavailable LOXL2-selective small molecule inhibitors in in vitro and in vivo models of lung fibrosis. Methods: In the in vitro model, lung fibroblasts from IPF patients were cultured under optimised conditions for mature collagen matrix deposition. Following TGF-β1 treatment, multicellular foci formed which were histochemically similar in organization to fibroblastic foci in vivo (Jones et al., AJRCCM 191;2015:A4912). Transient overexpression of active TGF-β1 by adenovector gene transfer in rat lungs results in severe progressive fibrosis (Sime et al JCI 1997;100:768–776). The effects of treatment with a LOXL2-selective inhibitor (days 2-28) on histology and lung function were assessed at day 28. Results: Treatment with LOXL2-selective inhibitors dose-dependently reduced cross-link formation, altered the organisation of collagen matrix and reduced matrix stiffness (assessed by parallel plate compression) in the in-vitro model. In vivo, treatment at 15 or 30 mg/kg significantly lowered lung elastance compared to vehicle control from 2.2 (0.39) to 1.7 (0.31) or 1.6 (0.31) respectively (mean (SD) cmH2O/ml; p Conclusions The results suggest that inhibition of LOXL2 using these novel inhibitors has the potential to improve lung function in patients with lung fibrosis by reducing tissue stiffness.