Targeted eicosanoid lipidomics of exhaled breath condensate provide a distinct pattern in the aspirin-intolerant asthma phenotype.

BACKGROUND: Eicosanoids, important signaling and inflammatory molecules, are present in exhaled breath condensate (EBC) in very low concentrations, requiring highly sensitive analytic methods for their quantification. OBJECTIVE: We sought to assess a vast platform of eicosanoids in different asthma phenotypes, including aspirin-intolerant asthma, by means of a recently developed analytic approach based on mass spectrometry. METHODS: EBC from 115 adult asthmatic subjects (62 with aspirin intolerance) and 38 healthy control subjects were assessed quantitatively for 19 eicosanoids by using complementary HPLC, gas chromatography-mass spectrometry, or both. Palmitic acid concentrations were used as a marker for dilution of condensate samples. RESULTS: Asthma was characterized by an increase in arachidonate lipoxygenase products and cysteinyl leukotrienes. The COX pathway was also significantly upregulated in asthmatic subjects. Subjects with aspirin-intolerant asthma were distinguished by a sharp increase in the level of prostaglandin D(2) and E(2) metabolites; their 5- and 15-hydroxyeicosateraenoic acid levels were also higher than in aspirin-tolerant subjects. A classical discriminant analysis permitted us to classify correctly 99% of asthmatic subjects within the study population; the specificity of the analysis was 97%. The eicosanoid profiling allowed for 92% correct classification of aspirin-intolerant subjects. CONCLUSIONS: The highly sensitive eicosanoid profiling in EBC makes it possible to detect alterations in asthma, especially in its distinct phenotype characterized by hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs. This permits us to discriminate asthmatic subjects from healthy subjects, as well as to distinguish the 2 asthma phenotypes based on the presence or absence of aspirin hypersensitivity.

The course of asthma in Churg-Strauss syndrome.

OBJECTIVES: Asthma is one of the key features of Churg-Strauss syndrome (CSS); however its course in the disease is unclear. The aim of this study was to analyze the asthma course in CSS patients. PATIENTS AND METHODS: We conducted a retrospective study of 22 CSS patients. Medical documentation was studied and patients were questioned about asthma symptoms during follow-up visits, which took place at four points of the disease. These points, at which lung function tests were performed, were CSS diagnosis; introduction of treatment at hospital discharge; and assessment of the first and last clinical and laboratory CSS remissions. Asthma control and severity were assessed in compliance with current guidelines. RESULTS: In the asthmatic group examined, 21 patients had an adult-onset asthma, with a mean age of onset 35.5 ± 11.8 years, preceded by sinusitis (100%) and nasal polyposis (36%). Asthma at its onset was severe in 68.5%, moderate in 27%, and very poorly controlled in 21 patients. Atopy was present in 64% of patients. Onset of vasculitic symptoms and CSS diagnosis was accompanied by high blood eosinophilia (maximal 7.2 x 10(3)/l [1.2 - 32], asthma exacerbation with airway obstruction in 16 patients (mean values of FEV1 68.8 ± 17.5% and FVC 84.8 ± 19.6%), and lung involvement. After introducing the treatment and achieving stable remission, asthma severity/control and lung function tests (forced expiratory volume in 1 s 92.7 ± 13.3%, and forced vital capacity 101 ± 15.5%; p < .001) improved. CONCLUSIONS: Asthma in CSS, although severe as its onset improves after achieving CSS remission.

Targeted eicosanoids lipidomics of exhaled breath condensate in healthy subjects.

BACKGROUND: Exhaled breath condensate collection is a non-invasive method of sampling the respiratory tract that can be repeated several times in a wide range of clinical settings. Quantitation of non-volatile compounds in the condensate requires highly sensitive analytical methods, e.g. mass spectrometry. OBJECTIVE: To validate cross-platform measurements of eicosanoids using high performance liquid chromatography or gas chromatography coupled with mass spectrometry in exhaled breath condensate sampled from 58 healthy individuals. METHODS: Twenty different eicosanoid compounds, representing major arachidonic acid lipoxygenation and cyclooxygenation pathways were measured using a stable isotope dilution method. We applied a free palmitic acid concentration as a surrogate marker for the condensate dilution factor. RESULTS: Eicosanoids concentrations in the condensates were consistent with their content in other biological fluids. Prostaglandin E(2) was the most abundant mediator, represented by its stable metabolite tetranor-PGEM. Prostaglandin D(2) products were at low concentration, while hydroxyacids derived from lipoxygenation were abundant. 5-HETE was elevated in current tobacco smokers. Leukotriene B(4) has the highest concentration of all 5-LO products. 15-LO analogues of cysteinyl leukotrienes-eoxins were detectable and metabolized to eoxin E(4). Two main vascular prostanoids: prostacyclin and thromboxane B(2) were present as metabolites. A marker for non-enzymatic lipid peroxidation, 8-iso-PGF(2alpha) isoprostane was increased in smokers. CONCLUSION: Presented targeted lipidomics analysis of exhaled breath condensate in healthy subjects justifies its application to investigation of inflammatory lung diseases. Measurements of non-volatile mediators of inflammation in the condensates might characterize disease-specific pathological mechanisms and responses to treatment.

Genetic variability of the high-affinity IgE receptor alpha-subunit (FcepsilonRIalpha).

Our knowledge on the variability of FCER1A gene encoding for alpha-subunit of the high-affinity immunoglobulin E receptor (FcepsilonRI) that plays a central role in the pathogenesis of allergy and related disorders, has been recently much extended. Last findings from FCER1A mutational screening and genetic association studies, followed by functional analyses of the polymorphisms, are briefly summarized in this mini-review. The association between FCER1A gene variants and total serum IgE levels seems especially interesting and, supported by functional analyses of polymorphisms, may provide a rationale for pharmacogenetic studies on anti-IgE therapy that indirectly suppresses FcepsilonRI expression.

Common polymorphisms of cyclooxygenase-2 and prostaglandin E2 receptor and increased risk for acute coronary syndrome in coronary artery disease.

The arachidonic acid metabolites participate in development of coronary artery disease (CAD) and the plaque's instability. We assessed two common genetic polymorphisms: of cyclooxygenase-2 (COX-2) (COX2.8473, rs5275) and prostaglandin EP2 receptor gene (uS5, rs708494) in patients with CAD. Out of 1,368 patients screened by coronary arteriography, two groups fulfilled the entry criteria and were studied: stable coronary disease (sCAD, n = 125) and acute coronary syndromes (ACS, n = 63). They did not differ in the main characteristics. All patients were on aspirin at least seven days prior to the study. In 70 control subjects, the same genotypes were ascertained, expression of cyclooxygenases in peripheral blood monocytes was assessed by flow cytometry, and in-vitro biosynthesis of PGE(2) was measured by mass spectrometry. COX-2 CC homozygotes (variant allele), were more common, while EP2 GG homozygotes (wild-type) were less common in ACS (p = 0.03 and p = 0.017) than in the sCAD group. A combined genotype characterized by the presence of the wild-type COX2.8743T allele and the wild type homozygous EP2uS5 genotype (TT or CT | GG) decreased risk ratio of ACS in CAD patients (relative risk 0.41; 95% confidence interval 0.21-0.81). COX-2 polymorphism in control subjects did not affect the enzyme expression or PGE(2) production by peripheral blood monocytes, but production of PGE(2) increased by 40.1% in the subjects homozygous for EP2 receptor allele uS5A following lipopolysaccharide stimulation. In conclusion, the combined COX-2 (COX2.8473) and the EP2 receptor (uS5) genotypes seem to influence CAD stability, but in peripheral blood monocytes only EP2 receptor modulates PGE(2) production.

Eicosanoids in exhaled breath condensates in the assessment of childhood asthma.

The value of measurements of eicosanoids in exhaled breath condensate (EBC) for the evaluation of childhood asthma is still inconclusive most likely because of the limited value of the methods used. In this case-control study in 48 asthmatic and 20 healthy children, we aimed to characterize the baseline profile of the inflammatory mediators cysteinyl leukotrienes (cysLTs), 9(alpha)11(beta)PGF(2), PGE(2), PGF(2alpha), 8-isoprostane (8-iso-PGF(2alpha)) within EBC in asthmatic compared with healthy children using new methods. In addition, we investigated their relation to other inflammatory markers. The assessment included collection of EBC, measurement of fractional exhaled nitric oxide (FE(NO)) and evaluation of urinary excretion of leukotriene E(4.) cysLTs were measured directly in EBC by radioimmunoassay and prostanoids were measured using gas chromatography negative-ion chemical ionization mass spectrometry. Only cysLT levels were significantly higher in asthmatic compared with healthy children (p = 0.002). No significant differences in cysLTs were found between steroid naïve and patients receiving inhaled corticosteroids. In contrast, FE(NO) was significantly higher in steroid naïve compared with steroid-treated asthmatic and healthy children (p = 0.04 and 0.024, respectively). The diagnostic accuracy of cysLTs in EBC for asthma was 73.6% for the whole group and 78.2% for steroid-naïve asthmatic children. The accuracy to classify asthmatic for FE(NO) was poor (62.9%) for the whole group, but improved to 79.9% when only steroid-naïve asthmatic children were taken into consideration. cysLTs in EBC is an inflammatory marker which distinguishes asthmatics, as a whole group, from healthy children.

Two different transcription factors discriminate the -315C>T polymorphism of the Fc epsilon RI alpha gene: binding of Sp1 to -315C and of a high mobility group-related molecule to -315T.

The alpha-chain is a specific component of FcepsilonRI, which is essential for the cell surface expression of FcepsilonRI and the binding of IgE. Recently, two single nucleotide polymorphisms (SNPs) in the alpha-chain promoter, -315C>T and -66T>C, have been shown by statistic studies to associate with allergic diseases. The effect of -66 SNP on GATA-1-mediated promoter activity has been already indicated. In the present study, to investigate roles of the -315 SNP on the alpha-chain promoter functions, the transcription activity was evaluated by reporter assay. The alpha-chain promoter carrying -315T (minor allele) possessed significantly higher transcriptional activity than that of -315C (major allele). EMSA indicated that the transcription factor Sp1, but not Myc-associated zinc finger protein (MAZ), was bound to the -315C allele probe and that a transcription factor belonging to a high mobility group-family bound to the -315T allele probe. The chromatin immunoprecipitation assay suggested that high mobility group 1, 2, and Sp1 bound around -315 of FcepsilonRIalpha genomic DNA in vivo in the human basophil cell line KU812 with -315C/T and in human peripheral blood basophils with -315C/C, respectively. When cell surface expression level of FcepsilonRI on basophils was analyzed by flow cytometry, basophils from individuals carrying -315T allele expressed significantly higher amount of FcepsilonRI compared with those of -315C/C. The findings demonstrate that a -315 SNP significantly affects human FcepsilonRI alpha-chain promoter activity and expression level of FcepsilonRI on basophils by binding different transcription factors to the SNP site.

The presence of rhinovirus in lower airways of patients with bronchial asthma.

RATIONALE: The common cold virus, human rhinovirus (HRV), is the most frequent cause of asthma exacerbations. However, a possible contribution of HRV to the pathogenesis of chronic, persistent asthma has not been defined. OBJECTIVES: To determine if patients with stable asthma, who are free of clinical signs of a respiratory infection for at least 3 weeks, harbor HRV in their bronchi more frequently than nonasthmatic control subjects, and whether clinical features of asthma are associated with the presence of HRV. METHODS: Immunohistochemistry and the indirect in situ reverse transcription-polymerase chain reaction method were used to detect the presence of HRV in bronchial mucosal biopsies in patients with asthma and nonasthmatic control subjects. MEASUREMENTS AND MAIN RESULTS: HRV was found by immunohistochemistry in 9 of 14 bronchial biopsies from subjects with asthma (64.3%) and 2 of 6 nonasthmatic control subjects (33.3%) (P = 0.38). With the more sensitive indirect in situ reverse transcription-polymerase chain reaction method, HRV was found in the mucosal biopsies of 73% of patients with asthma and 22% of nonasthmatic control subjects (P < 0.001). Subjects positive for HRV had lower pulmonary function, higher numbers of blood eosinophils and leukocytes, and eosinophilic infiltration in bronchial mucosa. CONCLUSIONS: HRV was detected in the lower airway tissue of patients with asthma significantly more often than in nonasthmatic subjects, and its presence was associated with clinical features of more severe disease.

Blood coagulation at the site of microvascular injury in healthy and coumadin-treated subjects heterogenous for factor V Leiden mutation.

There is little knowledge regarding the in-vivo course of coagulation reactions in subjects with factor (F)V Leiden (G1691A, R506Q). The aim of the current study was to evaluate the effect of FV Leiden on coagulant reactions triggered by vascular injury. At the site of microvascular injury, prothrombin activation and FVa formation and inactivation have been evaluated in 16 apparently healthy subjects and 16 patients on long-term anticoagulation with acenocoumarol, with eight heterogenous carriers of FV Leiden in the healthy and in the anticoagulated group. Thrombin formation, measured by levels of thrombin-antithrombin complexes (TAT) and thrombin B-chain, proceeded at similar rates in the bleeding-time blood in carriers and non-carriers of FV Leiden. The onset and rate of FV activation did not differ significantly among the four groups. When healthy carriers of FV Leiden were compared to healthy non-carriers, the onset of FVa inactivation by activated protein C (APC) was delayed by 60 to 90 seconds (p=0.01). Anticoagulated individuals also showed the same pattern of FV Leiden associated differences in the time of occurrence of the 30 kD FVa heavy-chain fragment (p=0.021). Our results indicate that when blood clotting is triggered by microvascular injury, the heterozygous expression of FV Leiden has no effect on thrombin generation in healthy or coumadin-treated subjects. Inactivation of FVa by the APC mechanism is attenuated significantly in carriers of FV Leiden but the magnitude of this effect is smaller than that observed in most purified systems.

Genetic variability of the high-affinity IgE receptor alpha subunit (Fc epsilon RI alpha) is related to total serum IgE levels in allergic subjects.

Known susceptibility genes to atopy and asthma have been identified by linkage or associations with clinical phenotypes, including total serum IgE levels. IgE-mediated sensitivity reactions require a high-affinity IgE receptor (FcepsilonRI), which immobilizes the immunoglobulin on the surface of the effector cells, mostly mast cells and basophils. In this mini-review, recent findings are presented on genetic variation of this receptor, as related to atopy. Transcription of FCER1A gene encoding the receptor alpha subunit can be initiated from two separate promoters, the proximal one and the distal one, which results in a transcript containing two novel untranslated exons (1A, 2A). Our knowledge on the role of this mechanism in allergic diseases is still at an infancy stage. Within regulatory elements of FCER1A some common single nucleotide polymorphisms have functional associations, which were recently reported and replicated in different ethnical groups. Interestingly, these associations do not confer susceptibility to allergic diseases, but rather modulate serum concentrations of IgE. Similarly to the previously investigated beta subunit of the receptor, FCER1A is a good candidate for a quantitative trait locus (QTL) in allergic diseases, and appears to participate in the systemic regulation of IgE levels.

The prevalence of alpha(1)-antitrypsin deficiency in a representative population sample from Poland.

AIM: Severe alpha(1)-antitrypsin (AAT) deficiency is one of the most common genetic disorders in Caucasians. The aim of the present study was to assess an unbiased frequencies of PI*S and PI*Z alleles using genotyping of a representative sample from the general population of Poland. METHODS: A random sample of age- and gender-stratified residents, aged 20 years or older, was drawn from the municipal directory of Kraków, Poland. The two most common deficiency alleles: PI*S and PI*Z were genotyped with qualitative real-time PCR using degenerative dual-labeled allele-specific fluorescent probes. RESULTS: In the total population of 859 adult subjects (mean age: 49.5 years; range: 20-90), 28 heterozygotes MS, 18 heterozygotes MZ and one homozygote S were diagnosed. The frequency of PI*S allele was 17.5 (95% CI: 11.6-23.9) per 1000; and that of PI*Z was 10.5 (95% CI: 5.8-15.7) per 1000. Therefore, the estimated prevalence of inherited severe AAT deficiency (homozygotes Z) in Poland is 1/9110 (95% CI: 1/4057-1/29,727). CONCLUSIONS: In the whole population of Poland comprising 38 millions, one may expect of about 4189 (95% CI: 1284-9406) subjects with severe AAT deficiency. These numbers are high enough to consider genetic testing being introduced into a common clinical practice.

The additive antiplatelet action of clopidogrel in patients with coronary artery disease treated with aspirin.

We searched for additional anti-platelet effects of clopidogrel in coronary artery disease (CAD) patients treated with aspirin. Response to clopidogrel was also stratified according to aspirin resistance. Out of 76 screened aspirin-treated CAD male patients, five were aspirin-resistant based on arachidonic acid (AA) and ADP aggregometry. These five patients and 15 aspirin-sensitive patients entered the proper study. Platelet function was assessed at baseline and after one week of additional clopidogrel treatment using aggregometry, flow cytometry (ADP, TRAP-6) and platelet reactivity index (PRI) based on VASP (vasodilatorstimulated phosphoprotein) expression. We evaluated the same markers in 15 healthy men after aspirin treatment. In healthy subjects aspirin did not affect resting or ADP-induced activated GPIIb/IIIa and P-selectin expression. The P-selectin expression on ADP-activated platelets was increased (p < 0.01) in aspirin treated ASA-resistant CAD patients as compared to ASA-sensitive group or aspirin-treated healthy subjects. Clopidogrel significantly decreased ADP and AA-induced platelet aggregation and overcame aspirin resistance in four of five patients. Expression of ADP-induced activation markers was significantly lowered after clopidogrel in all patients. Out of 20 patients, five did not respond to clopidogrel (<10% inhibition of ADP aggregation), and this group showed no change in expression of ADP-induced activation markers after clopidogrel. Clopidogrel treatment significantly reduced PRI only in the clopidogrel-sensitive group. In conclusion, the addition of clopidogrel to aspirin provides greater inhibition of platelets and can overcome aspirin resistance. Flow cytometric analysis of platelets is useful for monitoring of clopidogrel therapy.

Psychopathology and personality factors modify the perception of dyspnea in asthmatics.

We studied the relationship between the intensity of dyspnea and psychopathological and personality dimensions in 74 women and 38 men (mean age 49.7 years) with asthma. The women had higher values for the following variables: depression, anxiety-trait, and neuroticism, and they proved to have a greater external control location than the men. After checking the spirometry results, body mass index (BMI) and gender, it was found that dyspnea correlated with anxiety-trait and anxiety-state, neuroticism, and depression. In the men, anxiety-trait modified the relationship between dyspnea and the duration of the disease. This effect was not found in the women. A high level of anxiety-trait seems to be responsible for the escalation of dyspnea during the progression of the disease in men, whereas low anxiety may protect them against the increase of dyspnea. FEV(1), BMI, and anxiety-trait were predictors of dyspnea.

Zileuton added to low-dose inhaled beclomethasone for the treatment of moderate to severe persistent asthma.

OBJECTIVE: To assess the therapeutic effects of oral zileuton tablets combined with low-dose beclomethasone compared to doubling the dose of beclomethasone, in improving lung function and reducing asthma symptoms. METHODS: Randomized, active-control, double-blind, parallel, multi-center study of zileuton (400 or 600 mg QID)+200 microg beclomethasone dipropionate (BDP) BID versus placebo+BDP 400 microg BID in asthmatics with baseline FEV(1) percent predicted values between 40% and 80% following a single-blind ICS (BDP 200 microg BID) 2-week run-in. During the 3-month double-blind treatment period, assessments included safety, daytime and nighttime symptoms, acute asthma exacerbations, beta(2)-agonist use, AM and PM peak expiratory flow (PEF) and FEV(1). RESULTS: The addition of a 5-lipoxygenase (5-LO) inhibitor added to a low-dose of BDP showed no significant difference in FEV(1) compared to doubling the dose of BDP. FEV(1) improved in all 3 treatment groups, with mean increases of 10% with zileuton 600 mg QID+BDP 200 microg BID, 12% with zileuton 400mg QID+BDP 200 microg BID, and 11% with BDP 400 microg BID by study end. Within each treatment group, there were significant improvements in asthma symptoms and AM and PM PEF compared to baseline. No significant differences were observed between groups with regards to salbutamol use, acute asthma exacerbations, the requirement for oral/parenteral corticosteroids and adverse clinical events. CONCLUSIONS: The addition of a 5-LO inhibitor added to low-dose beclomethasone may be an alternative to higher-doses of ICS in patients unable to achieve sufficient asthma control on low-dose ICS therapy.

Rapid and inexpensive detection of alpha1-antitrypsin deficiency-related alleles S and Z by a real-time polymerase chain reaction suitable for a large-scale population-based screening.

alpha(1)-Antitrypsin (AAT) deficiency is one of the most common genetic disorders in Caucasians, leading to early onset pulmonary emphysema and/or liver disorders. Accumulating data suggest that AAT deficiency is commonly under-recognized or misdiagnosed by physicians. The need for a rapid, timesaving, and relatively inexpensive but reliable detection method for the two most common deficiency alleles was developed using real-time polymerase chain reaction (PCR) genotyping. We designed and validated a 5'-nuclease assay for typing of the PI*S and PI*Z alleles using dual-labeled target-specific fluorescent probes. As a reference method, we used restriction fragment length polymorphism. The real-time PCR method was tested on a large, cross-sectional epidemiological trial. Overall, we genotyped about 1200 samples and found a very good concordance with AAT serum levels and restriction fragment length polymorphism results. In addition, external interlaboratory validation confirmed the accuracy of the real-time PCR method. In our experience, the real-time qualitative PCR using 5'-nuclease assay is suitable as a genetic test for AAT deficiency. This method offers an acceptable balance between reliability and expenses. It seems appropriate for both population-based screening and clinical diagnosis of the deficiency.

Antithrombotic effects of aspirin based on PLA1/A2 glycoprotein IIIa polymorphism in patients with coronary artery disease.

OBJECTIVE: The diallelic glycoprotein IIIa polymorphism P1A1/A2 was attributed to be an inherited risk factor for coronary events. Whether this polymorphism affects response to aspirin in patients with coronary artery disease is not known. METHODS: We assessed thrombin generation (prothrombin fragment F1+2) in consecutive blood samples collected from bleeding-time wounds in 28 men with coronary artery disease; P1A2 carriers, n=9; P1A1/A1, n=19. Thrombin generation and bleeding time were measured before and after 2 weeks of aspirin 300 mg/day. RESULTS: Aspirin-depressed thrombin generation in A1 homozygotes (p=0.04), but not in A2 carriers. Bleeding time after aspirin was also prolonged in A1 subjects only (p=0.02). CONCLUSION: Genotyping for glycoprotein IIIa polymorphism might be helpful in predicting antithrombotic action of aspirin in secondary prevention of coronary artery disease.

Molecular diagnostics of α1-antitrypsin deficiency.

Diagnosis of α1-antitrypsin (AAT) deficiency is based on serum protein level, phenotyping and genotyping. Each of these methods has serious limitations and not enough accuracy to be used alone. Combinations of two different techniques are recommended to increase the reliability of AAT deficiency testing. Changes of AAT serum levels can result from clinical conditions and the inflammatory response may increase protein level. Phenotyping discriminates AAT variants due to the altered isoelectric point of the protein. It can detect numerous mutations, but often fails in the presence of rare, unknown or null alleles. Genotyping assays can detect or exclude specific mutations. The results are reliable and definitive, but restricted to the most common variants. DNA techniques for AAT gene screening and detection of new variants are available, but these are of limited value as sequencing is required to confirm the results. This review describes common approaches used in AAT deficiency testing and discusses their strengths and weaknesses. A novel functional test for the rapid discrimination of patients with AAT deficiency would greatly enhance diagnostic algorithms.