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Background Cyclooxygenase (COX)-2 is an inducible enzyme responsible for catalysing the formation of prostaglandins (PGs) in settings of inflammation. Single nucleotide polymorphisms (SNPs) of the COX-2 gene may influence gene transcription and PG production in the asthmatic airway. Objective To evaluate the frequencies of COX-2 SNPs in an Australian Caucasian population, and determine potential associations between common COX-2 promoter SNPs and asthma, asthma severity and aspirin-intolerant asthma (AIA). Methods The frequencies of 25 COX-2 SNPs were determined in a random population (n = 176). The SNPs with a minor allele frequency of > 10% were then studied in asthmatic (n = 663), non-asthmatic controls (n = 513) and AIA subjects (n = 58). Genotype, allele and haplotype associations were assessed. Functional assessment of SNPs was performed by transfection into HeLa cells measured using the luciferase dual-reporter assay system. Results Eighteen COX-2 SNPs were not detected, five were rare and two promoter SNPs, - 1195G > A (rs689465), and - 1290A > G (rs689466), were further studied. The A allele of the - 1195 SNP was present at a significantly higher frequency among all asthmatic subjects (P= 0.012). Over 60% of the asthmatic individuals were - 1195A homozygotes compared with 54.6% of the control subjects (odds ratio, 1.35; 95% CI, 1.06-1.72, P= 0.03). After classifying for severity, the mild asthmatics represented 64.6% of - 1195AA individuals, the highest of all the asthma groups compared with 54.6% of the control subjects (odds ratio, 1.5; 95% CI, 1.12-2.02, P = 0.02). The - 1290A/- 1195G/ - 765G haplotype was associated with a reduced incidence of asthma (odds ratio, 0.76; 95% CI, 0.61-0.95, P= 0.017). Conclusion The - 1195G > A polymorphism appears to be associated with asthma, and in particular with mild asthma.

Summary Background Previous studies suggest that challenge of most wine-sensitive asthmatic patients may not result in a reduction in forced expiratory volume in 1 s (FEV1). Objective The aim of this study was to assess whether changes in bronchial hyperresponsiveness (BHR) occur following wine challenge of asthmatic patients who report sensitivity to wine, and whether such changes could help clarify the role of sulphite additives in wine-induced asthmatic responses. Methods Eight self-reporting wine-sensitive asthmatic patients completed double-blind challenges with high- and low-sulphite wines on separate days. FEV1 and histamine PC20 were measured before and after consumption of 150 mL of wine. Results None of the eight subjects demonstrated a clinically significant (≥15%) reduction in FEV1 following challenge with either high- or low-sulphite wine. In contrast, one patient demonstrated clinically significant increase in BHR following challenge with both high- and low-sulphite wines, and a smaller increase in BHR following placebo challenge. A second patient showed a significant increase, while another showed a significant decrease in BHR following challenge with low-sulphite wine. A fourth patient showed borderline increases in BHR following challenge with both high- and low-sulphite wines. Conclusions Although changes in BHR, in the absence of reductions in FEV1, were observed in some asthmatic patients following wine challenge, these changes were not consistent with a single aetiology. Consequently, this study did not support a major role for the sulphite additives in wine-induced asthmatic responses in the patients studied. The aetiology of wine-induced asthma is likely to be complex and appears to vary among individuals who are sensitive to these drinks.

It is increasingly recognized that the morphological changes of apoptosis vary between cell types. This heterogeneity reflects the wide range of cellular proteins and enzymes involved in apoptotic pathways. Fibroblast apoptosis is crucial to the regression of scars and the restitution of healthy tissue during wound repair and may be aberrant in diseases such as idiopathic pulmonary fibrosis (IPF). The biochemical and morphological changes characterizing fibroblast apoptosis are unknown and may provide insights into the specific enzymatic mediators activated in these cells. This study aimed to examine the morphological changes of fibroblast apoptosis in both primary normal lung fibroblasts (normal-Fb) and fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF-Fb) and to correlate these changes with conventional biochemical markers. Transmission electron microscopy (TEM) and video time-lapse microscopy demonstrated no difference in the duration of fibroblast apoptosis in response to FasL (6 +/- 0.3 h in normal-Fb and 6.4 +/- 0.2 h in IPF-Fb). However, IPF-Fb were more resistant to FasL-induced apoptosis compared with normal-Fb. Although the majority of morphological changes of normal-Fb and IPF-Fb were similar, the formation of filopodia and condensation of the cytoskeletal bundles in IPF-Fb, and more prominent vacuolation in normal-Fb, were the significant differences between these cell subtypes. Loss of the mitochondrial membrane potential occurred prior to caspase-3 activation, while phosphatidylserine expression, cytokeratin-18 cleavage, and DNA fragmentation commenced after caspase-3 activation. These observations not only suggest that specific enzymatic effectors may be preferentially activated during fibroblast apoptosis, but also provide potential insights into the pathogenesis of IPF.

BACKGROUND Airway inflammation in asthma is associated with cysteinyl leukotriene and prostaglandin D(2) 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)E(4) and 9 alpha, 11 beta-prostaglandin (PG)F(2) 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 LTE(4) and 9 alpha, 11 beta-PGF(2) concentrations were measured in urine samples from these patients and from 175 control subjects using enzyme immunoassays. RESULTS Urinary LTE(4) was correlated with 9 alpha, 11 beta-PGF(2) in both control subjects and asthmatic patients (P

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 gamma-glutamylcysteine synthetase (gamma-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. gamma-GCS activity was measured by HPLC assay and changes in gamma-GCS mRNA by Northern blotting. RESULTS Exposure to menadione (MQ, 10-200 microm, 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 microm, 30 min), increased c. two fold over baseline levels (P < 0.001). gamma-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, gamma-GCS mRNA expression increased 1.3-1.6-fold relative to GAPDH mRNA, 3-6 h after MQ treatment. The MQ-induced increase in gamma-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 gamma-GCS mRNA transcription and enzyme activity.

The release of PGE(2) and nitric oxide (NO) from the respiratory epithelium may act to dampen inflammation. In other tissues, oncostatin M (OSM), a potent inducer of epithelial antiproteases, has also been shown to interact with IL-1beta to stimulate PGE(2) release. However, whether OSM interacts with pro-inflammatory cytokines and proteases in the production of anti-inflammatory eicosanoids and NO from airway epithelium is unknown. The effect of OSM and the related cytokine leukaemia inhibitory factor (LIF) on PGE(2) and NO production by the respiratory epithelial cell line, A549 in response to pro-inflammatory cytokines as well as protease-rich house dust mite (HDM) fractions and a protease-deficient rye grass pollen extract was examined by immunohistochemistry, cell culture, ELISA and enzyme-immunoassay. Cells treated with a mixture of IL-1beta, IFNgamma and LPS for 48 h produced a 9 fold increase in PGE(2) and a 3 fold increase in NO levels (both P

Aims Aspirin has proven clinical efficacy in limiting the thrombotic complications of atherosclerotic vascular disease but its mechanism of action remains unclear. Recent evidence suggests the anti-platelet action of aspirin may be partly mediated by neutrophil derived nitric oxide (NO). The aim of the study was to determine the effects of aspirin on thrombin-induced platelet expression of the α-granule membrane protein, P-selectin, and the platelet surface glycoprotein required for aggregation, GPIIb-IIIa, and to assess whether this was enhanced by the presence of neutrophils. Methods Platelet P-selectin and GPIIb-IIIa receptor expression were assessed by flow cytometric analysis of washed platelets stimulated with thrombin (0.025 iu ml−1, sub aggregatory concentration) alone or after pre-incubation with aspirin (0.05, 0.1, 0.5, 1.0 mg ml−1 ) either in the presence or absence of neutrophils (100 platelets per neutrophil). NO release was determined by assay of nitrite in the supernatants from parallel samples. Results In preliminary aggregation studies, aspirin at all concentrations inhibited arachidonic acid but not thrombin-induced platelet aggregation. Similarly, aspirin at all concentrations failed to inhibit thrombin-induced platelet P-selectin or GPIIb-IIIa expression and this was not influenced by the presence of neutrophils. A reduction in P-selectin and GPIIb-IIIa receptor density on non-activated platelets co-incubated with unstimulated neutrophils was associated with NO release from neutrophils, but this was not enhanced by the addition of aspirin. Conclusions These results confirm that thrombin-induced platelet α-granule release, with consequent P-selectin expression, and platelet GPIIb-IIIa expression, are not affected by aspirin inhibition of cyclo-oxygenase and suggest that the anti-thrombotic efficacy of aspirin in vivo may partly depend on other mechanisms. This study did not demonstrate an effect of neutrophils or neutrophil derived NO on aspirin inhibition of platelet adhesion receptor expression.

Summary 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.

Summary 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

1. The effect of increasing doses of orally administered aspirin (30-900 mg) on platelet aggregation and ATP release induced by arachidonic acid (AA), collagen and platelet activating factor (PAF) was assessed in 12 normal volunteers. 2. Aspirin ingestion was associated with a significant increase in EC50 for AA (P less than 0.0001) and collagen (P less than 0.0001) but not for PAF (P greater than 0.495) although the normal biphasic aggregation response for the latter was abolished. Maximum ATP release was reduced by aspirin for all three agonists. 3. The mean maximum degrees of inhibition of platelet aggregation induced by aspirin for AA, collagen and PAF were 100%, 48% and 21% of baseline, respectively. The corresponding mean maximum inhibition of ATP release was 100%, 63% and 57%. The minimum cumulative doses of aspirin producing these effects were 240, 240 and 90 mg for AA, collagen and PAF respectively. For collagen alone, there was a significant decrease in the degree of inhibition of aggregation between the last dose on day 1 (150 mg) and the baseline measurement on day 2. 4. Platelets from female subjects were more sensitive to collagen (P less than 0.05) and AA (P less than 0.01) stimulation compared with males. However, prior to aspirin ingestion, PAF produced a greater maximum response in platelets from females (P less than 0.02) while following aspirin ingestion PAF-induced activation was inhibited to a greater degree in females (P less than 0.02). 5. These results indicate that collagen- and PAF-induced platelet activation are only partially dependent on cyclo-oxygenase and for PAF this seems related only to the second phase of aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary Background Inflammation of the airways in asthma is associated with the production of cysteinyl leukotrienes (cysLT), prostaglandin (PG)E2, 8-isoprostane, nitric oxide and other mediators. However, the relationship between asthma severity or eosinophilic inflammation and the concentrations of mediators in sputum is unclear. Objective To assess sputum PGE2, cysLT, 8-isoprostane and nitrate concentrations, as well as urinary leukotriene (LT)E4 and 9α,11β-prostaglandin (PG)F2 concentrations, in patients with differing severities of asthma and eosinophilic or non-eosinophilic airway inflammation. Methods Inflammatory cells in sputum were assessed in 12 patients with mild, 14 with moderate and 12 with severe persistent asthma, as well as in 13 control subjects. Asthmatic patients were categorized into those with eosinophilic or non-eosinophilic airway inflammation. Sputum PGE2, cysLT and 8-isoprostane, and urinary LTE4 were extracted on immunoaffinity sorbents, and the concentrations of all mediators were measured using enzyme immunoassays. Sputum nitrate concentrations were measured on a chemiluminescence analyzer. Results Sputum PGE2 concentrations were higher in both moderate (1710 pg/mL) and severe asthmatic (1590 pg/mL) compared with control subjects (827 pg/mL) (P

The effect of eicosapentaenoic acid (EPA) on the inflammatory potential of neutrophils was investigated by supplementing the diets of 12 subjects with 2.16 g of EPA or 12 g of olive oil per day for 4 weeks in a double blind crossover study. Neutrophil function as assessed by luminol enhanced chemiluminescence responses to platelet-activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (FMLP) was significantly reduced after EPA but not after olive oil consumption in the subjects who consumed EPA first. In contrast, EPA had no significant effect on neutrophil chemiluminescence in the subjects who consumed olive oil first. Dietary supplementation with EPA inhibits neutrophil responses to inflammatory mediators such as PAF while other fatty acids appear to modify the effects of EPA.