Molecular Mechanism of Asthma

We aim to investigate these separate cell types in the blood of subjects with asthma and identify which genes are more highly expressed when compared to cells obtained from patients without asthma. We will investigate corticosteroid sensitivity on gene expression in asthma (corticoid naive to corticosteroid resistant asthma). We will also investigate the lung cells (macrophages) from these subjects to identify whether the same or different genes are expressed in these cells. We will isolate different leukocyte populations from the blood and extract ribonucleic acid (RNA) from these samples. The type and quantity of RNA in these samples is a reflection of the specific genes expressed in these cells. Gene expression is also evaluated by ELISA.This RNA will be sent to Gene Logic in the USA and this company will test these samples to identify which genes have been expressed using Gene Arrays. This technique will examine the expression of all 33,000 human genes in each sample to examine activation of interacting pathways rather than individual genes at a time. Samples will be analysed in the USA and preliminary data shows that there are no problems involving sample transportation or degradation. Similar experiments will be performed using cells (macrophages) obtained following bronchoalveolar lavage of these subjects. We would aim to examine the responses of cells from two groups of subjects, namely (i) non-asthmatics controls and (ii) asthmatic subjects. The isolated cells will either be immediately solubilized in solutions to purify RNA or we will then use these isolated cells in vitro and following stimulation investigate whether different genes are expressed or at a differential rate in the disease state. We will examine both cells derived from peripheral blood and cells (macrophages) obtained from bronchoalveolar lavage with the aim to determine whether differences attributable to disease can be identified in both circulating cells and those at the site of disease. This is a preliminary study to determine the profile of inflammatory mediator expression from leukocytes and as such power calculations to determine the number of subjects is not appropriate. The objective of the study is to identify which genes are specifically expressed in important cells in patients with asthma with a view to identify novel targets for drug therapy.