published_or_final_version / Medicine / Master / Doctor of Medicine

Measuring the level of exhaled NO (eNO) in the breath is a new method to monitor airway inflammation in asthma and may have a role in the management of asthma. The hypotheses were that eNO will reflect the degree of inflammation in chronic asthma, and will indicate how anti- inflammatory therapy should be altered to improve asthma control. Three studies were performed to test the hypotheses. A cross sectional study was performed to define the normal range of eNO and to compare this range with those who have asthma or atopy. The second study was observational, to compare the level of eNO during and after an exacerbation of asthma. The third study was an interventional study to evaluate eNO in management of paediatric asthma. In this latter study the level of eNO was measured to monitor airway inflammation in asthmatic children with the intention of adjusting antiinflammatory drugs (inhaled glucocorticosteroids) according to the level of eNO. These studies have shown that the mean level of eNO was significantly higher in asthmatic compared with normal subjects, but not significantly different when compared with atopic non-asthmatic subjects. eNO was correlated with the number of positive skin prick tests in atopic subjects whether asthmatic or nonasthmatic. The eNO level was increased during acute exacerbations of asthma and decreased after two weeks with therapy of GCS. In a pilot study eNO appeared to be superior to FEV1 in adjusting the dose of iGCS to control asthmatic children, but this needs to be confirmed with a larger sample size. Another non-invasive method to detect inflammatory markers is the technique of exhaled breath condensate (EBC). Although NO is degraded to NOx, it was found that eNO had no significant correlation with EBC NOx but had a significant correlation with pH. Hypertonic saline challenge, an artificial model of an asthmatic exacerbation was associated with an increase in EBC volume and the release of histamine, implicating mast cell activation. These novel findings suggest that non-invasive markers can be used both for clinical and mechanistic proposes.

Abstract not available

[Tuncated abstract] There has been longstanding concern over whether endobronchial biopsies adequately represent inflammation throughout the bronchial tree in diseases such as asthma, despite the endobronchial biopsy technique having been used frequently to assess airway inflammation in research settings. There has also been ongoing debate about whether endobronchial biopsies should be assessed by new, unbiased, three-dimensional (3D) stereological techniques instead of traditional, two-dimensional (2D) non-stereological techniques. Therefore, the aims of this study were: (i) to investigate whether endobronchial biopsies represent the density of mast cells in the large and small airways, in alveolar walls and in the lung as a whole (ii) to use both stereological and non-stereological methods to address this question, and where possible, to compare the results of these two approaches. '...' Mast cell density in biopsies was not related to mast cell density immediately adjacent to the biopsy site or to mast cell density in the total airway wall in the large airways, the inner airway wall in the small airways, the walls of the alveoli or the lung as a whole. In general, measurements of mean mast cell density on biopsies to a depth of 100µm below the basement membrane were poorly related to mean mast cell density in other compartments of the lung. Mean 3D and 2D mast cell densities were strongly correlated (r 0.9, p < 0.005) and where both methods were used, results were similar. The mean height and area profile of a mast cell were approximately 12µm and 68µm2 respectively. In disk-shaped IUR lung samples, percent shrinkage in height due to paraffin processing was systematically greater than percent radial shrinkage by an average of approximately 4 times. Cavalieri lung volumes were systematically smaller than displacement volumes by an average of 14%. Any given endobronchial biopsy is unlikely to represent mast cell density around the airway wall generally in the vicinity of the biopsy site. However, the average of at least 4 biopsies from different sites in the proximal airways can be used to both represent mean mast cell density in the inner airway wall of the large airways, and act as the basis for inter-subject comparisons of mean mast cell density in the total airway wall of the small airways. On biopsies, mast cell counts should be measured over the entire inner airway wall not just to a depth of 100µm or less below the basement membrane. 3D mast cell densities obtained by stereological methods are closely related to 2D mast cell densities obtained by non-stereological methods and are likely to result in similar conclusions. Lung volumes are smaller when measured by the Cavalieri method than when measured by fluid displacement. Shrinkage of isotropic uniform random samples of human lung tissue due to paraffin processing is anisotropic. The mean volume of a mast cell in the human lung is likely to be much smaller than that reported previously for monkey lungs.

[Truncated abstract] Asthma genetic studies have identified many genes that contribute to the pathogenesis of asthma and related variables. Members of the secretoglobin family appear to play an important role in controlling airway inflammation but they have received relatively little attention in asthma genetic research. In this thesis, I have investigated the genes of two members of the secretoglobin family (16 kDa Clara cell secretory protein (CC16) and secretoglobin 3A2 (SCGB3A2)) that are expressed at high levels in the airways and are important anti-inflammatory agents. The overall aim of these studies was to investigate the genetic variability of the CC16 and SCGB3A2 genes and their influence on airway inflammatory disease. The main hypothesis was that genetic variability in the genes for CC16 and SCGB3A2 exert an influence on airway inflammatory disease. Three populations were investigated: (1) a paediatric case control population (n=99), (2) an unselected birth cohort followed longitudinally at ages 1 month (n=244), six (n=123) and 11 years (n=195) and (3) an unselected Aboriginal Australian population (n=251). The case-control population was screened for novel DNA sequence variants in the CC16 promoter and the SCGB3A2 5’UTR and exons. No novel sequence variants were identified in the CC16 promoter and two were identified in the SCGB3A2 5’UTR (G- 811A and G-205A). A single nucleotide polymorphism previously identified in the CC16 gene (A38G) and the two polymorphisms identified in the SCGB3A2 gene were genotyped in both unselected populations. Genotype/phenotype associations were identified with adjustment for potential confounders such as age, gender, height and maternal tobacco smoking, where appropriate. This was due to the contribution of these factors to the aetiology of asthma, atopy and related phenotypes. All three polymorphism frequencies were significantly different between these two ethnically diverse populations

The allergic disease of asthma is characterized by an infiltration of inflammatory cells to the lung, a process co-ordinated by T-helper (TH) cells. The TH2 cytokine Interleukin (IL)-4 promotes infiltration of eosinophils to sites of inflammation. Eosinophil-selective chemoattractant cytokines (eg. eotaxins) are synthesized by lung epithelial cells. Eotaxin-3 is expressed at high levels in the asthmatic lung, predominantly after IL-4 stimulation. Eotaxin-3 is therefore a marker of inappropriate airway inflammation. Polyphenolic (PP) compounds found in high concentrations in berries may have beneficial effects in inflammatory conditions. Plant and Food Research produced high-PP extracts of blackcurrant (BC) cultivars that were tested for inflammation modulating effects. Since high doses of PPs have been shown to cause cell death, we tested two BC cultivars at a range of concentrations in a cell viability (WST-1) assay. While no toxic effects were attributable to the BC extracts (1-50µg/ml), a dose-related trend in cell death was observed and therefore 10µg/ml was chosen for further experiments Ten BC cultivars were compared for efficacy by measuring eotaxin-3 production in IL-4 stimulated human lung epithelial (A549) cells in vitro. Cells were incubated with BC extracts (10µg/ml) and IL-4 (10ng/ml) for 24 hours. The supernatants were then quantified for eotaxin-3 levels by an enzyme-linked immunosorbent assay (ELISA). All ten BC extracts reduced eotaxin-3 levels after stimulation with IL-4, and six BC extracts were effective by statistically significant levels (P<0.05), (BC cultivars -01, -02, -03, -05, -09 & -10). Of those, BC extracts of four cultivars demonstrated a reduction of more than 65% from the IL-4 stimulated control. In addition, a positive trend in inflammation modulation vs. one anthocyanin (ACN) in the BC extracts was shown. This study has demonstrated the beneficial inflammation modulatory effects of polyphenolic BC extracts, which could be related to cyanidin 3-O-rutinoside content. These results may have therapeutic potential for asthma.

The allergic disease of asthma is characterized by an infiltration of inflammatory cells to the lung, a process co-ordinated by T-helper (TH) cells. The TH2 cytokine Interleukin (IL)-4 promotes infiltration of eosinophils to sites of inflammation. Eosinophil-selective chemoattractant cytokines (eg. eotaxins) are synthesized by lung epithelial cells. Eotaxin-3 is expressed at high levels in the asthmatic lung, predominantly after IL-4 stimulation. Eotaxin-3 is therefore a marker of inappropriate airway inflammation. Polyphenolic (PP) compounds found in high concentrations in berries may have beneficial effects in inflammatory conditions. Plant and Food Research produced high-PP extracts of blackcurrant (BC) cultivars that were tested for inflammation modulating effects. Since high doses of PPs have been shown to cause cell death, we tested two BC cultivars at a range of concentrations in a cell viability (WST-1) assay. While no toxic effects were attributable to the BC extracts (1-50µg/ml), a dose-related trend in cell death was observed and therefore 10µg/ml was chosen for further experiments Ten BC cultivars were compared for efficacy by measuring eotaxin-3 production in IL-4 stimulated human lung epithelial (A549) cells in vitro. Cells were incubated with BC extracts (10µg/ml) and IL-4 (10ng/ml) for 24 hours. The supernatants were then quantified for eotaxin-3 levels by an enzyme-linked immunosorbent assay (ELISA). All ten BC extracts reduced eotaxin-3 levels after stimulation with IL-4, and six BC extracts were effective by statistically significant levels (P<0.05), (BC cultivars -01, -02, -03, -05, -09 & -10). Of those, BC extracts of four cultivars demonstrated a reduction of more than 65% from the IL-4 stimulated control. In addition, a positive trend in inflammation modulation vs. one anthocyanin (ACN) in the BC extracts was shown. This study has demonstrated the beneficial inflammation modulatory effects of polyphenolic BC extracts, which could be related to cyanidin 3-O-rutinoside content. These results may have therapeutic potential for asthma.

The allergic disease of asthma is characterized by an infiltration of inflammatory cells to the lung, a process co-ordinated by T-helper (TH) cells. The TH2 cytokine Interleukin (IL)-4 promotes infiltration of eosinophils to sites of inflammation. Eosinophil-selective chemoattractant cytokines (eg. eotaxins) are synthesized by lung epithelial cells. Eotaxin-3 is expressed at high levels in the asthmatic lung, predominantly after IL-4 stimulation. Eotaxin-3 is therefore a marker of inappropriate airway inflammation. Polyphenolic (PP) compounds found in high concentrations in berries may have beneficial effects in inflammatory conditions. Plant and Food Research produced high-PP extracts of blackcurrant (BC) cultivars that were tested for inflammation modulating effects. Since high doses of PPs have been shown to cause cell death, we tested two BC cultivars at a range of concentrations in a cell viability (WST-1) assay. While no toxic effects were attributable to the BC extracts (1-50µg/ml), a dose-related trend in cell death was observed and therefore 10µg/ml was chosen for further experiments Ten BC cultivars were compared for efficacy by measuring eotaxin-3 production in IL-4 stimulated human lung epithelial (A549) cells in vitro. Cells were incubated with BC extracts (10µg/ml) and IL-4 (10ng/ml) for 24 hours. The supernatants were then quantified for eotaxin-3 levels by an enzyme-linked immunosorbent assay (ELISA). All ten BC extracts reduced eotaxin-3 levels after stimulation with IL-4, and six BC extracts were effective by statistically significant levels (P<0.05), (BC cultivars -01, -02, -03, -05, -09 & -10). Of those, BC extracts of four cultivars demonstrated a reduction of more than 65% from the IL-4 stimulated control. In addition, a positive trend in inflammation modulation vs. one anthocyanin (ACN) in the BC extracts was shown. This study has demonstrated the beneficial inflammation modulatory effects of polyphenolic BC extracts, which could be related to cyanidin 3-O-rutinoside content. These results may have therapeutic potential for asthma.