Mechanisms of Rhinovirus Induced Asthma Exacerbations

We, the investigators, hypothesise that there are distinct gene profiles in rhinovirus-induced acute exacerbations of asthma. We further hypothesise that these changes in gene expression involve both known mediators of the asthma phenotype as well as other molecules not previously associated with asthma. The primary objective of this study is to use gene array analysis to determine differentially expressed genes in bronchial epithelial cells and alveolar macrophages from normal and asthmatic subjects before and during rhinovirus infection in vivo. A secondary objective is to determine whether any altered expressions are related to symptom severity, virus load, lung function or airway inflammation in vivo. We plan to recruit 45 subjects: 15 healthy volunteers, 15 asthmatics naïve to inhaled corticosteroid therapy, and 15 asthmatics on inhaled corticosteroids who will undergo two bronchoscopies, one prior to infection with rhinovirus and the second 4 days post inoculation. Bronchial brushings, biopsies and bronchoalveolar lavage (BAL) will be performed. RNA will be extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) and purified by passage through RNeasy columns (Qiagen, Valencia, CA). Exon 1.0ST array chips (Affymetrix, Santa Clara, CA) will be used to analyse changes in gene expression. These are the most powerful genome expression tools available with 1.4 million probe sets and over 5.5 million features per array. Genes found to be significantly upregulated will be confirmed by quantitative RT-PCR. Using a novel method of collecting undiluted bronchial epithelial lining fluid (bronchosorption) large numbers of proteins will be measured with a MesoScale Discovery multiplexed array system (MesoScale Discovery, Gaithersburg, Md) allowing further confirmation of the gene array results as well as providing in vivo evidence of dysregulated protein production in asthmatics. Gene expression and protein levels will be correlated with viral load, symptom scores, lung function and airway inflammation in vivo. This study represents the first comprehensive evaluation of changes in bronchial epithelial gene expression during rhinovirus infection in vivo and therefore has the potential to provide significant insights into the host response in asthma and identify potential novel targets for further evaluation.

differentially expressed genes in bronchial epithelial cells and alveolar macrophages from normal and asthmatic subjects before and during rhinovirus infection

Inclusion Criteria for atopic asthmatics: Age 18-55 years Doctor diagnosis of Asthma PC20 < 8 µg/ml and worsening asthma symptoms with infection since last change in asthma therapy. Atopic on skin testing Treatment comprising short acting beta agonist (SABA) only (steroid naïve group) or SABA + inhaled corticosteroid (ICS) * (steroid treated group) subjects on inhaled corticosteroids must be on a daily dose of between 100mcg and 1000mcg fluticasone or equivalent. Exclusion Criteria: Smoking history over past 6 months or > 5 py history Current symptoms of allergic rhinitis Current or previous history of significant respiratory disease (other than asthma) Any clinically relevant abnormality on screening or detected significant systemic disease Pregnant or breastfeeding women Contact with infants or elderly at home or at work Exacerbation or virus within the previous 6 weeks Treatment with oral steroids now or in the previous 3 months Current use of nasal spray, anti-histamine, anti-leukotriene Antibodies to rhinovirus 16 in a titre >1:2