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Background: Wine-induced asthmatic symptoms may be caused by sulphite additives. Prostaglandin (PG)D2 and cysteinyl leukotrienes (cysLT) are important mediators of asthmatic responses. To determine whether the sulphite additives in wine alter the production of PGD2 and cysLT, asthmatic patients with compelling histories of wine sensitivity were challenged with high- and low-sulphite wines; the urinary metabolites of PGD2 and cysLT were measured before and after challenge. Methods: Eight self-reporting wine-sensitive asthmatic patients completed double-blind challenges with high- and low-sulphite wines on separate days. Urine samples were collected before and after consumption of 150 ml of wine. Urinary concentrations of 9α,11β-PGF2 and leukotriene (LT)E4 were measured by enzyme immunoassay. Results: Urinary 9α,11β-PGF2 concentrations increased in all subjects following challenge with high-sulphite wine, and the median concentration increased 1.6-fold (p < 0.01). Urinary 9α,11β-PGF2 also increased 1.5-fold after low-sulphite wine challenge, although this did not reach statistical significance (p = 0.08). The median difference in 9α,11β-PGF2 concentration after high-sulphite wine challenge was not significantly different compared with that after low-sulphite wine challenge. Median urinary LTE4 concentrations did not change significantly after either wine challenge. Conclusions: Increased urinary 9α,11β-PGF2 concentrations following wine challenge suggest mast cell activation as a possible mechanism for wine-induced asthma, although this did not appear to be related to the sulphite additives in wine. Urinary 9α,11β-PGF2 may warrant further assessment as a potential biomarker of reactivity to wine in asthmatic subjects.

Eosinophils contribute to asthmatic airway inflammation by releasing cysteinyl leukotrienes (cysLT) and other inflammatory mediators, and bradykinin (BK) induces bronchoconstriction in asthmatic patients. The aims of this study were to investigate kinin receptor expression on eosinophils of asthmatic and healthy subjects and to assess the effects of kinin stimulation on eosinophils, which were isolated from peripheral blood of asthmatic (n=27) and healthy subjects (n=14). Kinin B1 and B2 receptors (B1R and B2R, respectively) and mRNA expression were investigated by quantitative confocal microscopy, flow cytometry, and RT-PCR. Intracellular Ca2+ was assessed by live-cell fluorescence confocal microscopy. Production of cysLT and eosinophil migration in response to BK and Lys-des[Arg9]-BK were assessed. Eosinophils expressed kinin B1R and B2R mRNA and proteins. Quantitative immunofluorescence analysis indicated that expression of B1R and B2R proteins was significantly greater in eosinophils of asthmatic patients compared with those of nonasthmatic subjects. However, kinin B1R and B2R mRNA expression did not differ significantly between these groups. Expression of kinin B1R and mRNA was decreased in patients using high doses of inhaled corticosteroids and in eosinophils treated with a corticosteroid in vitro. Kinin B1 and B2 agonists up-regulated expression of their respective receptors but did not increase intracellular Ca2+ or the production of cysLT or enhance eosinophil migration significantly. Up-regulation of kinin receptor expression in eosinophils of asthmatic patients may be a consequence of inflammation, whereby enhanced release of kinin peptides has a positive-feedback effect on kinin receptor expression. Importantly, anti-inflammatory corticosteroids down-regulated the expression of the kinin B1R.

Background: Secretory phospholipases A2 (sPLA2) have functions relevant to asthmatic inflammation, including eicosanoid synthesis and effects on dendritic cells and T cells. The aim of this study was to measure sPLA2 activity in patients with stable and acute asthma and to assess potential associations with body mass index (BMI), and plasma cholesterol and vitamin C concentrations. Conclusions: Plasma sPLA2 may provide a biological link between asthma, inflammation, increased BMI, lipid metabolism and antioxidants. Interactions among these factors may be pertinent to the pathophysiology and increasing prevalence of both asthma and obesity. Methods: Plasma sPLA2 activity and concentrations of cholesterol and vitamin C were measured in 23 control subjects and 61 subjects with stable asthma (42 mild to moderate, 19 severe). In addition, sPLA2 activity was measured in 36 patients experiencing acute asthma and in 22 of these patients after recovery from the acute attack. Results: sPLA2 activity was not significantly greater in severe (499.9 U; 95% confidence interval (CI) 439.4 to 560.4) compared with mild to moderate asthmatic subjects (464.8; 95% CI 425.3 to 504.3) or control subjects (445.7; 95% CI 392.1 to 499.4), although it was higher in patients with acute asthma (581.6; 95% CI 541.2 to 622.0; p,0.001). Male gender, high plasma cholesterol, increased BMI and atopy were associated with increased sPLA2 activity, while plasma vitamin C was inversely correlated with sPLA2 activity in patients with stable asthma and in control subjects. There were significant interactions between gender and plasma cholesterol and between gender and vitamin C in relation to sPLA2 activity.

Kinins have been implicated in the pathophysiology of asthma and activation of kinin receptors stimulates neutrophil chemotaxis. However, the expression of kinin receptors on neutrophils of asthmatic subjects has not been assessed. The aim of this study was to compare the expression of kinin B(1) and B(2) receptor mRNA and proteins in neutrophils of asthmatic and non-asthmatic subjects, and to assess whether inhaled corticosteroid treatment may influence expression of the kinin receptors. Neutrophils were isolated from peripheral blood of asthmatic (n=27) and non-asthmatic subjects (n=14). The presence of kinin B(1) and B(2) receptor protein on neutrophils was confirmed by immunolabeling with specific antibodies followed by immunoperoxidase, immunofluorescence and FACS detection. Kinin B(1) and B(2) receptor mRNA expression was assessed by RT-PCR. Quantitative image analysis of fluorescence immunolabeled neutrophils showed no differences in kinin B(1) or B(2) receptor protein expression between asthmatic and non-asthmatic subjects. Similarly, quantitative real time RT-PCR analysis demonstrated no differences in expression of mRNA for the kinin B(1) or B(2) receptors between asthmatic and non-asthmatic subjects. However, B(1) receptor mRNA expression was significantly lower in asthmatic subjects using > or =2000 microg of inhaled corticosteroid per day (p

The lungs of asthmatic patients are exposed to oxidative stress due to the generation of reactive oxygen and nitrogen species as a consequence of chronic airway inflammation. Increased concentrations of NO*, H2O2 and 8-isoprostane have been measured in exhaled breath and induced sputum of asthmatic patients. O2*-, NO*, and halides interact to form highly reactive species such as peroxynitrite and HOBr, which in turn cause nitration and bromination of protein tyrosine residues. Oxidative stress may also reduce glutathione levels and cause inactivation of antioxidant enzymes such as superoxide dismutase, with a consequent increase in apoptosis, shedding of airway epithelial cells and airway remodelling. The oxidant/antioxidant equilibrium in asthmatic patients may be further perturbed by low dietary intakes of the antioxidant vitamins C and E, selenium and flavonoids, with a consequent lowering of the concentrations of these and other non-dietary antioxidants such as bilirubin and albumin in plasma and airway epithelial lining fluid. Although supplementation with vitamins C and E appears to offer protection against the adverse effects of ozone, recent randomised, placebo-controlled trials of vitamin C or E supplements for patients with mild asthma have not shown significant benefits over standard therapy. However, genetic variation in glutathione S-transferase may influence the susceptibility of asthmatic individuals to oxidative stress and the extent to which they are likely to benefit from antioxidant supplementation. Long-term prospective trials are required to determine whether modification of dietary intake will benefit asthma patients and reduce the socio-economic burden of asthma in the community.

Low antioxidant levels and oxidative stress due to airway inflammation may be important determinants of asthma severity. The objective of the present study was to determine whether lower antioxidant intake and plasma antioxidant concentrations are associated with more severe asthma. Dietary antioxidant intakes and asthma severity were assessed using questionnaires, and plasma concentrations of ascorbic acid, vitamin E, carotenoids, bilirubin, albumin, uric acid and total antioxidant status were measured in 53 mild-to-moderate and 28 severe asthmatic patients and in 43 nonasthmatic subjects. Vitamin C and carotene intakes were lower in males than females and were particularly low in males with severe asthma. Plasma ascorbic acid was lower in severe (31.9+/-3.6 microM) compared with mild-to-moderate asthmatic (52.3+/-2.6) or control subjects (52.7+/-2.9). Low plasma ascorbic acid (odds ratio (OR) 0.93; 95% confidence interval (CI) 0.9-0.97), bilirubin (OR 0.69; 95% CI 0.51-0.93) and increased plasma cholesterol (OR 1.98; 95% CI 1.05-3.73) were independently associated with severe asthma. Albumin was positively and cholesterol negatively correlated with lung function. Low plasma concentrations of specific antioxidants are associated with more severe asthma. Increased antioxidant intake may help reduce the burden of severe asthma, particularly in males.

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.

Phospholipase A2 (PLA2) activity is usually assayed with expensive radioactive or chromogenic substrates unsuitable for performing large numbers of assays. We have designed a simple microplate assay for human serum PLA2 using the chromogenic substrate 4-nitro-3-octanoyloxy-benzoic acid. Using this substrate, serum PLA2 activity was similar to that measured with the previously characterized chromogenic phospholipid substrate 1,2-bis-heptanoylthio-glycerophosphocholine. However, the assay described here appears to be more sensitive. The mean PLA2 activity in serum from healthy volunteers (n = 30) measured by this assay was 10.4 +/- 1.6 micromol x h(-1) x ml(-1). The assay is reproducible and is suitable for the analysis of large numbers of samples in a clinical setting. We have also demonstrated that 94% of the PLA2 activity in normal human serum is associated with high-density lipoproteins and that serum PLA2 activity is positively correlated with the lipoprotein parameters total triglyceride (P < 0.0001), total cholesterol (P < 0.0001), and atherogenic index (P = 0.008). The serum PLA2 activity was calcium dependent and was inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin (EC(50) = 0.4 mM). The PLA2 activity characterized here is unlikely to be due to plasma platelet-activating factor acetylhydrolase or low molecular weight His-Asp sPLA2, and may represent a new sPLA2 type.

Despite the central role of γ-glutamylcysteine synthetase (γGCS) in lung antioxidant defenses, the limited studies of the activity of this enzyme in respiratory cells have produced variable results. This study has examined the factors, which may influence the measurement of γGCS activity in cultured human lung epithelial cells (A549). Although a source of potential error, γGCS activity in A549 cell extracts did not vary significantly when appropriately assayed by three different methods or after removal of the endogenous inhibitor, glutathione (GSH). However, γGCS activity did increase significantly during the early stages of cell proliferation (3.50 ± 0.31 vs. 2.35 ± 0.16 nmol/min/106 cells for baseline, p

Background: Apoptosis represents a mechanism by which the accumulation and inflammatory potential of eosinophils in asthma might be limited. Mediators derived from the airway epithelium may influence the rate of eosinophil apoptosis. Objective: We have investigated the effects on eosinophil apoptosis of 3 mediators that are likely to be produced by the airway epithelium, namely PGE 2 , TNF-α, and nitric oxide. Methods: Peripheral blood eosinophils from healthy adult volunteers were purified by density gradient centrifugation and negative immunomagnetic selection. Eosinophils were cultured for 16 or 40 hours with PGE 2 (10 nmol/L), dibutyryl cyclic adenosine monophosphate (AMP; 100 μmol/L), TNF-α (500 U/mL), the nitric oxide donors, S-nitroso-N-acetylpenicillamine (100 μmol/L), and 2,2 -(hydroxynitrosohydrazono)bis-ethanamine (1 mmol/L), or dibutyryl cyclic guanosine monophosphate (100 μmol/L). Control cultures consisted of untreated, IL5–treated (100 U/mL), and anti–Fas-treated (400 ng/mL) cells. Eosinophil apoptosis was assessed by flow cytometric analysis of annexin V-FITC binding to externalized phosphatidylserine, by electrophoresis of phosphorus 32 end-labeled DNA fragments, and by flow cytometric assessment of hypodiploid DNA with propidium iodide. Results: PGE 2 and cyclic AMP inhibited spontaneous eosinophil apoptosis at both 16 and 40 hours as did the PGEP 2 receptor agonist, 11-deoxy PGE 1 , at 40 hours, but these effects were not inhibited by a protein kinase A antagonist. TNF-α delayed apoptosis in eosinophil cultures at 16 hours, whereas S-nitroso- N-acetylpenicillamine, 2,2 -(hydroxynitrosohydrazono)bis-ethanamine, and cyclic guanosine monophosphate had little effect. Anti-Fas had little effect on spontaneous eosinophil apoptosis but significantly reduced the inhibitory effects of PGE 2 , cyclic AMP, and TNF-α. Assessments of apoptosis by DNA fragmentation gave similar but quantitatively less sensitive results. Conclusion: Inhibition of spontaneous eosinophil apoptosis by PGE 2 appears to be mediated by EP 2 receptors but is not protein kinase A dependent. By enhancing eosinophil survival, PGE 2 may increase the proinflammatory potential of these cells in chronic asthma. (J Allergy Clin Immunol 1999;104:153-62.)

The production of platelet-activating factor (PAF) by inflammatory cells is regulated by lyso-PAF acetyltransferase, and the activity of this enzyme is increased in neutrophils of stable asthmatic patients. The aim of this investigation was to determine whether acetyltransferase activity is further upregulated in asthmatic patients experiencing acute symptoms. A radioenzymatic assay was used to measure the enzymatic affinity constant (Km) and maximal enzymatic activity (Vmax) for acetyltransferase from unstimulated and Ca2+ ionophore (A23187)-stimulated neutrophils from 16 patients with acute asthma, and the measurement was repeated at the time of discharge (n=9) and after recovery from the acute episode (n=13). During acute asthma, Km (median 93.8 (interquartile range 64.1-109.7) microM) was lower than that measured in nonasthmatic subjects in a previous study using identical methods (155.1 (122.2-179.9) microM; p=0.0001), and in 10 out of 13 acute patients Km for unstimulated neutrophils increased following recovery. In A23187-stimulated neutrophils, Km during acute asthma (84.3 (73.6-100.2) microM) and at discharge (83.9 (83.1-94.8) microM) were similar, but Km after recovery was increased (115.0 (95.6-119.5) microM; p=0.02). The change in Km following stimulation with A23187 was also significantly less during acute asthma than previously measured in nonasthmatic subjects (p=0.003). Although Vmax during acute asthma (12.9 (interquartile range 10.5-22.5) nmol x min(-1) x mg(-1) protein) did not differ significantly from that at discharge (14.4 (12.3-20.4) nmol x min(-1) x mg(-1)) or after recovery (17.3 (12.3-18.4) nmol x min(-1) x mg(-1)), both median Km and Vmax tended to be lowest during acute asthma and increase at discharge and after recovery. An increase in lyso-PAF acetyltransferase activity alone may not account for increased systemic PAF concentrations during acute asthma. However, the reduction in the enzymatic affinity constant and its smaller change following in vitro stimulation suggest that alterations in the affinity of acetyltransferase for acetylcoenzyme A (CoA) and in the regulation of enzyme activity may be occurring during acute 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

Investigation of the pathogenesis of inflammatory diseases has involved assessment of polymorphonuclear leukocyte (PMN) activity and a variety of techniques for separating PMNs from whole blood have been described. In this study the effects of Percoll gradient, Ficoll-Hypaque/Dextran sedimentation (FH/DS) and Mono-Poly Resolving Medium (M-PRM) on activation and function of PMNs were compared. All three separation techniques gave similar cell yield and purity. The mean (+/- SEM) percentage of cells demonstrating pseudopodia formation for Percoll, FH/DS and M-PRM were 39 +/- 9, 57 +/- 6 and 63 +/- 5, respectively, while superoxide release from resting cells was 1.9 +/- 0.9, 7.2 +/- 3.5 and 11 +/- 4.8 pmols per 10(6) cells min-1, respectively, indicating that activation of cells during separation may be less with Percoll compared to the other methods. The functional capacity of the cells to respond to a stimulus was similar for all methods as indicated by similar EC50 values for chemotaxis to zymosan-activated serum and similar superoxide production induced by tetradecanoyl phorbol acetate. All three separation techniques produce functionally active PMNs of high purity but the use of Percoll gradients may be preferable when a quick method of separation which causes minimum pre-activation of PMNs is required.

Tissue kallikrein (KLK1) and plasma kallikrein (KLKB1) may regulate the growth and proliferation of tumours of the lung and pleura, through the generation of kinin peptides that signal through the kinin B1(BDKRB1) and B2(BDKRB2) receptors. The development and progression of cancer results from genetic mutations, as well as epigenetic changes that include methylation of DNA at CpG islands. The aim of this study was to assess whether expression of the kallikrein-kinin genes in lung cancer and mesothelioma cells is regulated by DNA methylation. Quantitative reverse transcriptase-PCR and immunocytochemistry showed differences in the basal expression of the kallikrein-kinin genes and proteins in lung carcinoma and mesothelioma cells, compared with non-malignant lung epithelial and mesothelial cells, respectively. Following treatment with the demethylating agent, 5-azacytidine (5-AZA),KLKB1mRNA expression was consistently increased in both lung carcinoma and mesothelioma cells, whereasKLK1,BDKRB1andBDKRB2mRNA expression was decreased or unchanged. Increased expression ofKLKB1after 5-AZA treatment suggests it may function as a tumour suppressor gene in cancers of the lung and pleura. Studies on DNA methylation of the kallikrein-kinin genes will enhance understanding of their role in carcinogenesis and provide insights into the importance of kallikreins as tumour biomarkers.

The effects of varying concentrations of platelet-activating factor (PAF), arachidonic acid (AA) and collagen on the expression of the platelet activation markers CD63 and CD62P were assessed in 10 normal subjects using flow cytometry. CD63 expression was significantly greater than CD62P expression, with PAF (80 nM) inducing mean maximum CD63 expression of 32.9 ± 6.4% and mean maximum CD62P expression of 5.5 ± 1.8%. AA (1 mM) induced maximum CD63 expression of 37.7 ± 7% and maximum CD62P expression of 9.3 ± 1%. Collagen (2-80 pg/ml) induced minimal expression but 800 pg/ml induced mean CD63 expression of 33.1 ± 4.1% and mean CD62P expression of 6.1 ± 0.8%. Greater CD63 and CD62P expression were induced by phorbol myristate acetate (1.6 pM, 70.9 ± 11% and 69.4 ± 9.9%, respectively) and thrombin (0.1 U/ml, 70.7 ± 9.3% and 73.5 ± 5.4%, respectively). With PAF and collagen only one platelet population was detected whereas with 1 mM AA two populations were observed. These results indicate that expression of platelet adhesion receptors depends on the nature and concentration of agonist and that subpopulations of platelets may exist. Importantly, PAF concentrations inducing moderate CD63 and CD62P expression did not induce platelet aggregation, suggesting that platelets can be activated independently of aggregation.

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

Remote ischemic preconditioning (RIPC) has been shown to reduce ischemic-reperfusion injury and is induced by brief forearm ischemia. Kinins are known to be involved in RIPC and act via the G protein coupled B1 and B2 receptors. Interaction of the kinins with their respective receptors causes receptor internalization, thereby reducing the potential for further activation. This may be critical for the protective effect of RIPC and if so, we hypothesized, would significantly decrease the expression of kinin receptors on the surface of neutrophils.The study was performed on five healthy human volunteers. The left forearm was rendered ischemic for three 5-min periods, each separated by 5 min of reperfusion. Three venous blood samples were taken from the right arm, one before and two after RIPC. Neutrophil isolation, immunofluorescence labeling, and confocal microscopy were performed. Mean pixel intensity data were generated using a fixed circular area of interest (AOI, 40×40 μm). For every image, the AOI was placed over a cell and the mean pixel intensity was recorded. The mean intensity was expressed as pixel×10(2)/μm(2) and presented as mean±SEM. Immunofluorescence at the different time points was compared by one way analysis of variance with Bonferroni's post-hoc test. A P value0.05 was considered significant.The mean pixel intensity for kinin B1 receptors was decreased at 24 h after RIPC compared with both baseline and 15 min after RIPC (P0.001). Similarly, the intensity for B2 receptor labeling on neutrophils was significantly decreased 24 h after RIPC compared with the baseline value (P0.001).RIPC decreases expression of kinin receptors on circulating human neutrophils. Reduction in kinin surface receptors suggests internalization of receptors and is consistent with the concepts of kinin receptor activation and their role in RIPC.

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.

The kinins, bradykinin (BK) and Lys-des[Arg9]-BK, are important inflammatory mediators that act via two specific G protein-coupled kinins, B1 and B2 receptors (B2R). Kinins influence the activity of immune cells by stimulating the synthesis of cytokines, eicosanoids, and chemotactic factors. Whether human dendritic cells (DC) express kinin receptors and whether kinins influence DC function are unknown. Fluorescence immunocytochemistry and RT-PCR were used to demonstrate that immature human monocyte-derived DC (hMo-DC) constitutively expressed kinins B1R and B2R. Kinin receptor expression was induced on the 3rd and 4th days of culture during differentiation of hMo-DC from monocytes and was not dependent on the presence of IL-4 or GM-CSF. Although monocytes also expressed B2R mRNA, the protein was not detected. The kinin agonists BK and Lys-des[Arg9]-BK up-regulated the expression of their respective receptors. BK, acting via the B2R, increased intracellular Ca2+, as visualized by confocal microscopy using the fluorescent Ca2+ dye, Fluor-4 AM. Evaluation of migration in Trans-well chambers demonstrated significant enhancement by BK of migration of immature hMo-DC, which was B2R-dependent. However, kinins did not induce maturation of hMo-DC. The novel finding that kinin receptors are constitutively expressed in immature hMo-DC suggests that these receptors may be expressed in the absence of proinflammatory stimuli. BK, which increases the migration of immature hMo-DC in vitro, may play an important role in the migration of immature DC in noninflammatory conditions and may also be involved in the recruitment of immature DC to sites of inflammation.

Oxidative stress and lipid-derived inflammatory mediators during acute exacerbations of cystic fibrosis REID DW, MISSO N, AGGARWAL S, THOMPSON PJ, WALTERS EH. Respirology 2007; 12: 63–69 Background and objective: In cystic fibrosis (CF) very few studies have assessed sputum 8-iso- PGF2α levels during pulmonary exacerbations as a direct measure of airway oxidative stress. The role of other lipid-derived inflammatory mediators, such as the cysteinyl leukotrienes (cys-LTs) and prostaglandin (PG)-E2, during exacerbations is also poorly defined and the effect of conventional antibiotic therapy on these components of the inflammatory process is unclear. Methods: Sputum 8-iso-PGF2α, total cys-LT and PGE2 levels were measured in 17 CF patients experiencing a pulmonary exacerbation and repeated analysis were performed in 15 of these patients after antibiotic treatment. Eight stable CF and nine healthy subjects provided control data. Results: Sputum 8-iso-PGF2α was significantly elevated in acute, but not stable CF patients versus healthy controls (P < 0.001). Similarly, sputum cys-LT and PGE2 levels were increased in acute compared with stable CF patients and healthy controls (P ≤ 0.001). Although substantially lower than in acute patients, sputum cys-LT levels in stable patients were also significantly higher than in normal controls (P = 0.01). There were strong associations between cys-LT levels and sputum total cell counts, and blood neutrophils in acute patients (r2 = 0.53, P = 0.001 and r2 = 0.33, P < 0.05, respectively). Overall in the CF patients, FEV1%predicted was strongly and negatively correlated with sputum 8-iso-PGF2α (r2 = 0.34, P = 0.006), cys-LT (r2 = 0.40, P = 0.002) and PGE2 (r2 = 0.52, P < 0.001) levels. Antibiotic treatment reduced sputum total cell count (P = 0.03), but did not affect 8-iso-PGF2α, cys-LT or PGE2 levels. Conclusions: CF exacerbations are characterized by increased oxidative stress and sputum concentrations of bioactive lipid mediators. Treatment does not modulate these aspects of inflammation and more targeted therapy needs further study.

The intracellular regulation and kinetics of prostaglandin (PG)E(2) synthesis in human airway epithelial (NCI-H292) cells was investigated. Interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) all induced PGE(2) synthesis (p0.001) and transient (5-15 min) phosphorylation of extracellular signal-regulated kinase (ERK). Phorbol myristate acetate (PMA) and calcium ionophore, A23187 further enhanced PGE(2) synthesis (p0.001) and caused phosphorylation of ERK that was sustained for up to 16 h. COX-2 protein expression and PGE(2) synthesis were increased following exposure to combinations of stimuli that increased intracellular Ca(2+), and activated protein kinase C as well as ERK. Inhibition of ERK almost completely abrogated PGE(2) synthesis in response to all stimuli. Sustained, maximum PGE(2) synthesis was observed when cells were stimulated such that ERK phosphorylation was concomitant with increased COX-2 protein expression. These results argue against redundancy in pathways for PGE(2) synthesis, and suggest that at various stages of inflammation different stimuli may influence ERK activation and COX-2 expression, so as to tightly regulate the kinetics and amount of PGE(2) produced by airway epithelial cells in response to lung inflammation.

Neutrophil apoptosis is important for the resolution of airway inflammation in a number of lung diseases. Inflammatory mediators, endogenous reactive oxygen and nitrogen species, and intracellular and extracellular antioxidants may all influence neutrophil apoptosis. This study investigated the involvement of these factors during apoptosis of neutrophils cultured in vitro. Neutrophils undergoing spontaneous apoptosis in culture as assessed by annexin V binding generated significant amounts of nitrite. Incubation with agonistic anti-Fas monoclonal antibody or tumor necrosis factor-alpha (TNF-alpha) enhanced neutrophil apoptosis at 6 h, although it decreased nitrite accumulation. Although granulocyte-macrophage colony-stimulating factor significantly reduced neutrophil apoptosis, this was also associated with decreased nitrite accumulation. In contrast, inhibition of apoptosis at 16 h by dibutyryl cyclic adenosine monophosphate was associated with increased nitrite accumulation. Exogenous glutathione (GSH) or N-acetylcysteine significantly enhanced neutrophil apoptosis at 6 h and stimulated the production of H(2)O(2), which may mediate apoptosis through intracellular hydroxyl radical production. Intracellular GSH concentrations decreased in neutrophils undergoing apoptosis, and this was more marked in neutrophils treated with anti-Fas or TNF-alpha. These results suggest a causal association between reduced endogenous nitric oxide production, reduced intracellular GSH, and Fas- and TNF-alpha-mediated neutrophil apoptosis, whereas enhanced neutrophil survival mediated by dibutyryl cyclic adenosine monophosphate is associated with increased nitrite generation and maintenance of intracellular GSH. The interaction of endogenous reactive oxygen species with extracellular antioxidants such as GSH could also contribute to the complex processes regulating neutrophil apoptosis and hence the resolution of inflammation in the lung.

1. Close contact between platelets and neutrophils modulates their cellular interactions in thrombotic and inflammatory states, with stimulation of P-selectin expression on platelets by agonists such as thrombin and neutrophil-derived cathepsin G being critical in mediating platelet—neutrophil adhesion. This study compared the effects of thrombin and cathepsin G on platelet P-selectin expression and on P-selectin-mediated platelet—neutrophil adhesion. 2. Washed platelets and platelet—neutrophil mixed cell suspensions (platelet/neutrophil ratio, 10:1) were incubated with either the supernatant of activated neutrophils, purified cathepsin G or thrombin. Platelet P-selectin expression and platelet adhesion to neutrophils was quantified by flow fluorocytometric analysis. 3. The supernatant from activated neutrophils stimulated platelet P-selectin expression comparable to that produced by purified cathepsin G or thrombin. P-selectin expression induced by both activated neutrophil supernatant and purified cathepsin G was completely inhibited by α1-antichymotrypsin, a specific inhibitor of cathepsin G. Unlike thrombin, which induced maximum platelet P-selectin expression by 10 min, sustained to 120 min, cathepsin G induced an initial large increase in platelet P-selectin expression, followed by a progressive reduction over 30–60 min to baseline levels. 4. Co-incubation of neutrophils with thrombin-stimulated platelets resulted in a significant increase in P-selectin-mediated platelet—neutrophil adhesion, which was completely inhibited by preincubation of neutrophils with anti-sialyl Lewisx monoclonal antibody. Thrombin produced maximum platelet—neutrophil adhesion by 10 min which remained stable over 120 min. In contrast, cathepsin G-stimulated platelets did not adhere to neutrophils over 120 min of co-incubation. Addition of cathepsin G to thrombin-stimulated platelets caused a progressive reduction over 30–60 min to baseline levels of platelet—neutrophil adhesion. 5. Neutrophil-derived cathepsin G is a potent platelet activator, but unlike thrombin it causes a time-dependent loss of platelet P-selectin expression and inhibits P-selectin-mediated platelet—neutrophil adhesion. Therefore, cathepsin G may modulate thrombin-mediated platelet—neutrophil adhesive interactions in inflammation and thrombosis.

SUMMARY 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.

The relative rates of synthesis of 24-methylcholest-5-en-3β-ol and 24-ethylcholest-5-en-3β-ol inZea mays shoots were determined using [2-(14)C]mevalonic acid and [methyl-(14)C]methionine as substrates. The 24-ethylsterol had a higher specific activity and it apparently was synthesized at about 3-4 times the rate of the 24-methylsterol.(1)H NMR spectroscopy showed that the 24-ethylsterol was predominantly the 24α-epimer but the 24-methylsterol was a mixture of the 24α-epimer (30-40%) and the 24β-epimer (60-70%). The results are discussed in relation to the involvement of Δ(24(28))-, Δ(23), Δ(25)- and Δ(24(25))-sterol intermediates in 24-methyl- and 24-ethylsterol production.