Characterisation of gene expression in the airway epithelium of children with asthma

Catherine Lane

    Research output: ThesisDoctoral Thesis

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    Abstract

    Most information regarding common childhood diseases has come from studies in adults. This is primarily due to the difficulty in obtaining target organ tissue from children. Studies in adults have established a clear role for the airway epithelium in a number of respiratory diseases, but at present there are few data regarding the paediatric epithelium. The current study has addressed this deficit by developing a method to sample the airway epithelium in an unselected population of children. The use of non-bronchoscopic brushing to safely sample useful quantities of epithelial cells for use in multiple investigative techniques was evaluated. Whilst children were anaesthetised and intubated for elective surgery, a small cytology brush was inserted directly into the endotracheal tube and brushed against the airway to sample epithelial cells. Children undergoing brushing experienced no complications. Cells sampled from the airway were identified as 95% epithelial, and were successfully used for immunocytochemistry and extraction of RNA and protein. This thesis has, for the first time, outlined a method which allows the study of multiple aspects of airway epithelial cell biology, using a single sample of epithelial cells, taken quickly and safely from children. This method was then applied to the investigation of the epithelium in asthma. Of the children recruited for this study, 23% had atopic asthma, 25% were healthy atopic, and 52% were not atopic or asthmatic. The airway epithelium is clearly abnormal in adult asthma; however, it was unclear whether differences in gene or protein expression would be detectable in the epithelium of asthmatic children. This study aimed to determine whether non-bronchoscopic brushing could be used in children to investigate epithelial abnormalities in asthma. Three areas of fundamental importance to asthma were investigated: inflammation, damage and repair.
    Firstly, inflammation was investigated by examining eosinophils in BAL fluid, exhaled nitric oxide (FeNO), and epithelial expression of the nitric oxide synthase (NOS) enzymes. Although FeNO was increased in asthmatic children, this was likely due to atopy, rather than asthmatic inflammation, as there was no difference between FeNO in healthy atopics and atopic asthmatics. Neither the number of eosinophils in BAL fluid, nor the expression of NOS in the epithelium provided any evidence of asthmatic inflammation in these children. Secondly, epithelial damage was investigated by examining epithelial cell shedding, and expression of the cellular adhesion molecule CD44. Neither of these markers was increased in the asthmatic children, suggesting that the airway epithelium was not damaged. Finally, epithelial repair was examined. EGF and EGF-R are central to the epithelial repair response, and are over-expressed in severe and adult asthma, yet expression of the genes encoding both of these molecules was significantly decreased in the epithelium of children with mild asthma. Despite this, epithelial proliferation, as indicated by immunoreactivity to PCNA, was increased in the asthmatic children.
    These findings demonstrate that bronchial brushing can be used in conjunction with techniques such as real-time PCR and immunohistochemistry, to investigate airway diseases. This study provides evidence for abnormalities in epithelial function in children with asthma, and suggests that mild childhood asthma may have a different aetiology to either severe childhood asthma, or adult asthma. To provide proof of concept, the scope of this research was initially restricted to individual genes and pathways known to be important to asthma. However, in order to extend our knowledge of airway diseases, and identify novel pathways and genes of importance, the possibility of using microarray technology to investigate broader epithelial function was examined.
    Gene expression was analysed in 9 asthmatic and 7 healthy children using Affymetrix U133A microarrays. Preliminary data from the microarrays showed that gene expression in epithelial cells from asthmatic subjects was significantly different to that in healthy subjects. Six databases of gene sets were analysed. Significantly enriched gene sets were found in all databases. In total, 50 gene sets were enriched in the asthmatics, and 29 gene sets were enriched in the healthy non-atopics. Based on gene expression, children with mild asthma have significant differences in their airway epithelium as compared to their healthy counterparts. This study has demonstrated that bronchial brushing can be used in conjunction with techniques such as PCR, immunohistochemistry and microarray to examine the airway epithelium in respiratory diseases. Even though the asthmatic children involved in this study had mild, asymptomatic airway disease, a number of novel, and important differences were identified. Further investigation is needed to directly compare this cohort with healthy and asthmatic adults, and also with severely asthmatic children. The microarray data have also presented further opportunity for research, and prompt detailed investigation of the pathways implicated in asthma, using cell culture, real-time PCR, and western blotting.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Publication statusUnpublished - 2007

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