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Notes: 1. Platelet-activating factor (PAF) may be involved in the pathogenesis of asthma, and therefore the effects of the anti-asthma drugs theophylline and enprofylline on human platelet aggregation and adenosine triphosphate (ATP) release induced by PAF and adenosine diphosphate (ADP) were studied.2. Enprofylline (50% inhibitory concentration [IC50] = 94.8±13.2 μmol/L) was more potent than theophylline (IC50= 934.1±40.1 μmol/L) as an inhibitor of PAF-induced aggregation, and the xanthines were twice as potent as inhibitors of PAF-induced aggregation when compared with ADP-induced aggregation. ATP release was 1.4 times more sensitive to inhibition by the xanthines than aggregation.3. Although high concentrations of xanthines inhibited platelet aggregation and ATP release induced by PAF, therapeutic concentrations are unlikely to inhibit PAF-induced effects.

Notes: Background Cyclooxygenase (COX)-2 is a key inducible enzyme that regulates the production of anti-inflammatory prostaglandin E2. A single-nucleotide polymorphism, −765G〉C, located within a stimulatory protein-1 binding site in the COX-2 promoter region, has been shown to have significantly lower promoter activity in vitro compared with the wild-type and was associated with decreased plasma levels of C-reactive protein after coronary artery bypass surgery. We hypothesized that this polymorphism, which may result in decreased COX-2 transcription, could be associated with more severe asthma, and/or aspirin-intolerant asthma (AIA).Objective To determine the association between the −765G〉C COX-2 polymorphism and asthma, disease severity and AIA in a large, well-phenotyped Australian population.Methods PCR and restriction fragment length polymorphism analysis was used to characterize the polymorphism in an Australian Caucasian population of patients with mild (n=322), moderate (n=254) or severe (n=88) asthma and in non-asthmatic control subjects (n=512), as well as in patients with AIA (n=58). Genotype and allele association analyses were performed using χ2 tests.Results The polymorphic −765C allele was present in approximately 30% of asthmatic patients and non-asthmatic controls. There was no association between the −765G〉C polymorphism and asthma (P=0.920), disease severity (P=0.840), atopy (P=0.655) or AIA (P=0.841) in this population.Conclusion Although the −765G〉C polymorphism may have lower promoter activity and result in decreased COX-2 expression, it is not associated with asthma, disease severity, AIA or atopy in this Australian population.

Notes: Background Asthmatic inflammation results in increased oxygen free radical generation and assessment of the activity of the selenitim (Se) dependent anti-oxidant enzyme, glutathione peroxidase (GSH-Px) in asthma may therefore be important. Objective To test the hypothesis that reduced GSH-Px activity and Se intake contribute to asthmatic infiammation, platelet and whole blood GSH-Px activities and serum and whole blood Se concentrations were measured and compared in atopic and non-atopic asthmatic patients and non-asthmatic control subjects.Methods GSH-Px activities of whole blood and isolated platelets were assessed in 41 asthmatic patients (33 atopic) and 41 age- and sex-matched non-asthmatic sttbjects (15 atopic) by spectrophotometric assay based oti the oxidation of NADPH. Se concentrations were determined by semi-automated fluorimetric assay. Results Mean (± sd) platelet GSH-Px activity was lower in asthmatic (89.5 ± 45.7 μmol NADPH oxidized min−1 g−1 of protein) than in non-asthmatic subjects (109,9 ± 41.9; P= 0.038) and in atopic (89.7 ± 45.1, n = 48) compared with non-atopie subiects (113.7 ± 40.9, n= 34: P= 0.016). Mean whole blood GSH-Px activity was also lower in atopic (12.2 ± 5.2 μmol NADPH oxidized min−1 g−1 of Hb) than in non-atopic subjects (14.5 ± 4.2; P= 0.038). In non-asthmatic subjects, the mean whole blood GSH-Px activity was lower in men (9.9 ± 3.5) than in women (14.5 ± 3.7; P = 0.0004) and was positively correlated with age (r= 0.51; P = 0.0006). Mean serum Se was lower in asthmatic (1.07 ± 0.12 μmol/L) than in non-asthmatic subjects (1.16 ± 0.31; P = 0.036), Using multiple linear regression, asthma was an independent predictor of decreased platelet GSH-Px after gender, age and serum Se were taken into account (P = 0.048) while atopy was a significant predictor of low whole blood GSH-Px independent of asthma, gender, age and whole blood Se (P = 0.033).Conclusions In addition to Se status, atopy, gender and uge all appear to influence GSH-Px activity, although the relative importance of these factors may difler in asthmatic and non-asthmatic populations. It seems likely that the reduced activity of this enzyme in platelets und hiood may reflect mechanisms associated with the pathogenesis and severity of asthma.

Notes: Background Airway inflammation in asthma is associated with cysteinyl leukotriene and prostaglandin D2 production. Measurement of urinary metabolites of these eicosanoids may be useful for monitoring asthma patients. However, the influence of asthma phenotype and severity on basal urinary excretion of these metabolites is unknown.Objective To compare urinary leukotriene (LT)E4 and 9α, 11β-prostaglandin (PG)F2 concentrations in large groups of mild, moderate and severe asthmatic patients and healthy control subjects.Methods Asthma severity, treatment and aspirin sensitivity were assessed by questionnaire in 168 asthmatic patients. Basal LTE4 and 9α, 11β-PGF2 concentrations were measured in urine samples from these patients and from 175 control subjects using enzyme immunoassays.Results Urinary LTE4 was correlated with 9α, 11β-PGF2 in both control subjects and asthmatic patients (P〈0.002). Median LTE4 and 9α, 11β-PGF2 concentrations in patients with severe asthma were significantly reduced compared with mild asthmatic patients (P〈0.05 and 〈0.001, respectively). Urinary 9α, 11β-PGF2, but not LTE4 was lower in asthmatic patients using inhaled corticosteroids (P〈0.02). Multiple regression analysis indicated that urinary 9α, 11β-PGF2 concentration was negatively correlated with asthma severity (P=0.003) and also with % predicted FEV1 (forced expiratory volume in 1 s) (P=0.005).Conclusions Baseline urinary LTE4 and 9α, 11β-PGF2 concentrations are of limited value in discriminating between patients with different severities of asthma. Reduced urinary LTE4 and 9α, 11β-PGF2 in patients with severe asthma suggest that direct or indirect effects of high-dose corticosteroid therapy combined with other factors associated with severe asthma may influence eicosanoid production. However, the negative association of urinary 9α, 11β-PGF2 with lung function suggests an adverse effect of chronic PGD2 production on lung function in asthma, irrespective of severity.

Notes: Background Activation of platelets and expression of adhesion molecules (e.g. CD62P and CD63) which mediate interactions between platelets and other cells may be important in the pathogenesis of aspirin-sensitive asthma.Objective To determine the expression of CD62P and CD63 on platelets from aspirinsensitive asthmatic (ASA +), aspirin-tolerant asthmatic (ASA-) and normal subjects and to assess the modulatory effect of aspirin on platelet CD62P and CD63 expression following stimulation with either platelet-activating factor (PAF), arachidonic acid (AA) or collagen (COL).Methods Platelet-rich plasma was obtained from 10 ASA +, 10 ASA—and 10 normal control subjects, and expression of CD62P and CD63 was measured by flow cytometry. Platelets were stimulated with PAF (10, 80 nM), AA (0.1, 1 mM) or COL (80, 800 μg/mL) with or without aspirin (concentration range 0.4–4 mg/mL).Results In the absence of aspirin, CD62P expression induced by AA and COL was greater in ASA+ patients compared with control subjects (P〈0.001) while CD62P expression with PAF, AA and COL was reduced in ASA—when compared wilh ASA+ and control subjects (P 〈 0.001). CD63 expression with PAF and AA was reduced in both ASA+ and ASA- patients compared with control subjects (P〈0.001). Aspirin inhibited the expression of both CD62P and CD63 after agonist stimulatitin. Greater inhibition of CD62P expression was observed in ASA+ compared with ASA- patients (P〈0.001) and normal subjects (P〈0.05) while greater inhibition of CD63 expression was observed in normal subjects compared with both ASA+ and ASA- patients (P〈0.05). In ASA+ patients and normal subjects, stimulation with PAF and COL resulted in only one platelet population while in contrast with 1 mM AA two populations were observed.Conclusions Fnhanced AA- and collagen-induced platelet CD62P expression in ASA+ patients compared with normal subjects and greater inhibition by aspirin of CD62P expression in ASA+ may be relevant to the pathogenesis of this syndrome. Reduced expression of CD62P and CD63 in platelets of ASA- patients following stimulation with PAF and AA may also have implications for the role of platelets and these mediators in the pathogenesis of other forms of asthma.

Notes: Background The bronchial epithelium is exposed to reactive oxygen species (ROS) derived from cigarette smoke, air pollutants and activated leucocytes. Glutathione (GSH) prevents ROS-mediated loss of cell function, tissue injury and inflammation, and its synthesis is regulated by γ-glutamylcysteine synthetase (γ-GCS). However, the capacity of bronchial epithelial cells to adapt to oxidative stress and the mechanisms involved are not known.Objective To investigate the effects of oxidative stress on the regulation of GSH synthesis in human bronchial epithelial (NCI-H292) cells.Methods NCI-H292 cells were exposed to menadione and intracellular GSH concentrations were measured by spectrophotometry. γ-GCS activity was measured by HPLC assay and changes in γ-GCS mRNA by Northern blotting.Results Exposure to menadione (MQ, 10–200 µm, 30–120 min) decreased total cellular GSH content, measured immediately after exposure to MQ. However, GSH content measured 6–12 h after withdrawal of the oxidant stress (MQ, 50 µm, 30 min), increased c. two fold over baseline levels (P 〈 0.001). γ-GCS activity measured 6 h (21.7 ± 3.4 nmol/min/mg, SD, n = 5, P 〈 0.01) or 12 h (23.2 ± 4.6, P 〈 0.001) after MQ treatment was also significantly increased compared with untreated cells (12.8 ± 1.0). Similarly, γ-GCS mRNA expression increased 1.3–1.6-fold relative to GAPDH mRNA, 3–6 h after MQ treatment. The MQ-induced increase in γ-GCS mRNA expression was completely inhibited by actinomycin D.Conclusions Bronchial epithelial (NCI-H292) cells respond rapidly and sensitively to oxidant stress, and this adaptive response is mediated by increased γ-GCS mRNA transcription and enzyme activity.