Natural Polyphenols May Normalize Hypochlorous Acid-Evoked Hemostatic Abnormalities in Human Blood.

During pathogen invasion, activated neutrophils secrete myeloperoxidase (MPO), which generates high local concentrations of hypochlorous acid (HOCl), a strong antimicrobial agent. Prolonged or uncontrolled HOCl production may, however, affect hemostasis, manifesting in inhibition of platelet aggregation and thrombus formation and in elevated fibrin density and attenuated fibrinolysis. In this report, we investigated whether three plant-derived polyphenols with well-known antioxidant properties, i.e., quercetin (Que), epigallocatechin gallate (EGCG), and resveratrol (Resv), at concentrations not affecting platelet responses per se, may normalize particular aspects of hemostasis disturbed by HOCl. Specifically, Que (5-25 µM) and EGCG (10-25 µM) abolished HOCl-evoked inhibition of platelet aggregation (assessed by an optical method), while the simultaneous incubation of platelet-rich plasma with Resv (10-25 µM) enhanced the inhibitory effect of HOCl. A similar effect was observed in the case of thrombus formation under flow conditions, evaluated in whole blood by confocal microscope. When plasma samples were incubated with HOCl, a notably higher density of fibrin (recorded by confocal microscope) was detected, an effect that was efficiently normalized by Que (5-25 µM), EGCG (10-25 µM), and Resv (5-25 µM) and which corresponded with the normalization of the HOCl-evoked prolongation of fibrinolysis, measured in plasma by a turbidimetric method. In conclusion, this report indicates that supplementation with Que and EGCG may be helpful in the normalization of hemostatic abnormalities during inflammatory states associated with elevated HOCl production, while the presence of Resv enhances the inhibitory action of HOCl towards platelets.

The myeloperoxidase product, hypochlorous acid, reduces thrombus formation under flow and attenuates clot retraction and fibrinolysis in human blood.

Hypochlorite (HOCl), a strong oxidant and antimicrobial agent, has been proposed to be associated with hemostatic abnormalities during inflammatory response. However, its complex impact on hemostasis is not completely understood. In this report we studied the effect of clinically relevant (micromolar) HOCl concentrations on thrombus formation under flow, kinetics of platelet-fibrin clot formation, its architecture, retraction, and lysis. We found that HOCl (up to 500 µM) did not affect kinetics of coagulation measured in whole blood. HOCl (500-1000 µM) markedly diminished thrombus formation under flow. Clot retraction rate was reduced by HOCl dose-dependently (50-500 µM). HOCl (125-500 µM) inhibited fibrinolysis in whole blood and in platelet-depleted plasma, dose-dependently. Activity of plasmin was reduced by HOCl at concentrations started from 500 µM. HOCl (up to 500 µM) did not reduce plasminogen binding to fibrin under flow. HOCl (125-500 µM) modulated architecture of fibrin- and platelet-fibrin clots towards structures made of thin and densely packed fibers. Exposure of pure fibrinogen to HOCl (10-1000 µM) resulted in formation of dityrosine and was associated with altered fibrin structure derived from such modified fibrinogen. HOCl-altered fibrin net structure was not related with modulation of platelet procoagulant response, thrombin generation, and factor XIII activity. We conclude that, in human blood, clinically relevant HOCl concentrations may inhibit thrombus formation under flow, clot retraction and fibrinolysis. Fibrinolysis and clot retraction seem to be the most sensitive to HOCl-evoked inhibition. HOCl-modified fibrinogen and altered clot structure associated with it are likely to be primary sources of attenuated fibrinolysis.

Markers of anaphylaxis - a systematic review.

Anaphylaxis is defined as severe, life-threatening, systemic or general, immediate reaction of hypersensitivity, with repeatable symptoms caused by the dose of stimulus which is well tolerated by healthy persons. The proper diagnosis, immediate treatment and differential diagnosis are crucial for saving patient's life. However, anaphylaxis is relatively frequently misdiagnosed or confused with other clinical entities. Thus, there is a continuous need for identifying detectable markers improving the proper diagnosis of anaphylaxis. Here we presented currently known markers of anaphylaxis and discussed in more detail the most clinically valuable ones: tryptase, platelet activacting factor (PAF), PAF-acethylhydrolase, histamine and its metabolites.

Reduced clot retraction rate and altered platelet energy production in patients with asthma.

OBJECTIVE: Asthma enhances the risk of pulmonary embolism. The mechanism of this phenomenon is unclear. METHODS: We evaluated the kinetics of clot formation, clot retraction rate (CRR), clot volume at 40 min, the rate of lactate production (a marker of aerobic glycolysis in platelets in contracting clots), blood eosinophil count (EOS), nitric oxide in exhaled breath (FENO), and spirometry (FEV1) in 50 healthy controls and in 81 allergic asthmatics (41 subjects with steroid-naïve asthma and 40 with steroid-treated asthma). RESULTS: Thromboelastometry revealed that only steroid-treated asthmatics had slightly activated coagulation. Compared with healthy controls, whole asthmatics demonstrated (p < 0.05) reduced CRR, higher clot volume at 40 minutes, higher FENO, decreased FEV1, elevated EOS, and augmented lactate production in retracting clots. Reduced CRR was observed also in the absence of native plasma. In whole study population (asthmatics and healthy controls), CRR positively correlated with spirometry (rS = 0.668, p = <0.001) and negatively with FENO (rS = -0.543; p < 0.001), EOS (rS = -0.367, p < 0.002), and lactate production (rS = -0.791; p < 0.001). However, in steroid-treated asthmatics, the CRR did not correlate with FENO and EOS. In all study patients lactate production negatively correlated with FEV1 and positively with FENO. CONCLUSION: Collectively, this data is consistent with the hypothesis that, in asthmatics, reactive nitrogen species produced in the lungs may reduce platelet contractility (and CRR) through the diminution of platelet energy production. CRR inhibition would predispose asthmatics to pulmonary embolism.

Inflammatory markers and acid-base equilibrium in exhaled breath condensate of stable and unstable asthma patients.

BACKGROUND: Nitrosative and acid stress play an important role in the pathogenesis of asthma. The aim of this study was to evaluate whether, in asthmatics, a link exists between the concentrations of nitrite/nitrate, ammonia and pH values in exhaled breath condensate (EBC) and asthma severity, lung function, exhaled nitric oxide (F(ENO)), total IgE, eosinophil cationic protein (ECP) and blood eosinophilia. METHODS: The above-mentioned parameters were measured in 19 healthy volunteers and 91 allergic asthmatics divided into three groups, i.e. 22 subjects with steroid-naïve stable asthma, 35 with inhaled corticosteroid (ICS)-treated stable asthma and 34 with ICS-treated unstable asthma. RESULTS: Compared with healthy subjects, EBC from asthmatics had significantly lower pH values and ammonia concentrations and significantly higher levels of nitrite/nitrate. The extent of these changes was higher in patients with unstable than in patients with steroid-naïve and stable ICS-treated asthma. The EBC pH was positively correlated with ammonia and negatively correlated with nitrite/nitrate, F(ENO) or blood eosinophilia in all three groups of asthmatics. Significant positive correlations between EBC nitrite/nitrate and blood eosinophilia, ECP levels or F(ENO) were observed in all groups of asthmatics. Significant negative correlations between EBC ammonia and nitrite/nitrate, F(ENO), ECP concentrations or blood eosinophilia were demonstrated in the groups of ICS-naïve and ICS-treated stable asthmatics. CONCLUSIONS: In asthmatic patients there is a relationship between EBC pH, ammonia and nitrite/nitrate concentrations and other recognized markers of airway inflammation. EBC pH values, ammonia and nitrite/nitrate levels measured together may help to assess airway inflammatory status and asthma severity.

RANTES in exhaled breath condensate of patients with severe persistent allergic asthma during omalizumab therapy.

BACKGROUND: Omalizumab is a humanized monoclonal anti-IgE antibody developed for the treatment of IgE-mediated diseases, including asthma. The aim of the study was to determine the effect of omalizumab treatment on changes in RANTES in exhaled breath condensate and other inflammatory markers in patients with persistent severe asthma. METHODS: The study was conducted on a group of 19 patients with severe persistent allergic asthma treated with conventional therapy (according to GINA 2006) and with or without omalizumab (9 vs. 10 patients). Changes in inflammatory parameters [RANTES in exhaled breath condensate, exhaled nitric oxide, blood eosinophil count and serum eosinophil cationic protein (ECP)] were measured before and after 16 weeks of therapy. RESULTS: Omalizumab-treated patients showed a statistically significant decrease in the concentrations of RANTES in exhaled breath condensate, exhaled nitric oxide (F(ENO)), serum ECP, and blood eosinophil count compared with patients with conventional therapy after 16 weeks of treatment. In this group of patients, statistically significant correlations were revealed between the decrease in RANTES and a decrease in F(ENO) and between the decrease in F(ENO) and a decrease in ECP or blood eosinophil count after omalizumab therapy. CONCLUSIONS: Our results confirmed that during anti-immunoglobulin E therapy with omalizumab in patients with severe persistent allergic asthma, RANTES expression is decreased. This process in turn could lead to a limitation of airway inflammation and could be essential for the beneficial effect of anti-IgE therapy with omalizumab.

Eotaxin-1 in exhaled breath condensate of stable and unstable asthma patients.

BACKGROUND: Airway eosinophilia is considered a central event in the pathogenesis of asthma. Eotaxin plays a key role in selective eosinophil accumulation in the airways and, subsequently, their activation and degranulation. The study was undertaken to evaluate eotaxin-1 levels in the exhaled breath condensate (EBC) of asthmatics with different degrees of asthma severity and to establish the possible correlation of these measurements with other recognized parameters of airway inflammation. METHODS: EBC was collected from 46 patients with allergic asthma (14 with steroid-naïve asthma, 16 with ICS-treated, stable asthma, 16 with ICS-treated unstable asthma) and 12 healthy volunteers. Concentrations of eotaxin-1 were measured by ELISA. RESULTS: In the three groups of asthmatics, eotaxin-1 concentrations in EBC were significantly higher compared with healthy volunteers (steroid-naïve asthma: 9.70 pg/ml +/- 1.70, stable ICS-treated asthma: 10.45 +/- 2.00, unstable ICS-treated asthma: 17.97 +/- 3.60, healthy volunteers: 6.24 +/- 0.70). Eotaxin-1 levels were significantly higher in patients with unstable asthma than in the two groups with stable disease. We observed statistically significant correlations between the concentrations of eotaxin-1 in EBC and exhaled nitric oxide (F(ENO)) or serum eosinophil cationic protein (ECP) in the three studied groups of asthmatics. We also discovered a significantly positive correlation between eotaxin-1 in EBC and blood eosinophil count in the groups of patients with unstable asthma and steroid-naïve asthma. CONCLUSIONS: Measurements of eotaxin-1 in the EBC of asthma patients may provide another useful diagnostic tool for detecting and monitoring airway inflammation and disease severity.

Anti-IgE therapy with omalizumab decreases endothelin-1 in exhaled breath condensate of patients with severe persistent allergic asthma.

BACKGROUND: Omalizumab is a humanized monoclonal anti-IgE antibody, especially useful for the treatment of severe persistent allergic asthma, inadequately controlled despite regular therapy. OBJECTIVES: The aim of the study was to determine the effect of omalizumab treatment on changes in endothelin-1 (ET-1), which plays an important role in the development of airway inflammation and remodeling in exhaled breath condensate (EBC) in patients with severe asthma. METHODS: The study was conducted in a group of 19 patients with severe persistent allergic asthma treated with conventional therapy (according to the Global Initiative for Asthma, 2006) and with or without omalizumab (9 vs. 10 patients). Changes in ET-1 in EBC compared with other inflammatory parameters [exhaled nitric oxide - (FE(NO)), blood eosinophil count, and serum eosinophil cationic protein (ECP)] were measured after 16 and 52 weeks of therapy. RESULTS: Omalizumab-treated patients demonstrated a statistically significant decrease in the concentrations of ET-1 in EBC, FE(NO), serum ECP, and blood eosinophil count and an increase in spirometry parameters compared to patients with conventional therapy. In the group of omalizumab-treated patients, statistically significant correlations between the decrease in ET-1 in EBC and a decrease in FE(NO), ECP, and blood eosinophil count as well as the increase in forced expiratory volume in 1 s after omalizumab therapy were revealed. CONCLUSIONS: Our results confirmed that anti-IgE therapy with omalizumab in patients with severe persistent allergic asthma results in decreased expression of ET-1 in the airways. This could be very important in limiting airway inflammation and bronchial structural changes caused by such treatment in asthmatic patients.

Changes in high-sensitivity C-reactive protein in serum and exhaled breath condensate after intensive exercise in patients with allergic asthma.

BACKGROUND: Exercise-induced bronchoconstriction (EIB) in asthmatics depends on the presence of allergic inflammation. This study was performed to assess the possible association of EIB with low-grade systemic inflammation, whose presence was revealed in asthmatic patients. METHODS: The study was conducted in a group of 24 asthmatics (14 with EIB, 10 without EIB) and 8 healthy volunteers. Changes in serum and exhaled breath condensate (EBC) high-sensitivity C-reactive protein (hs-CRP) levels induced by intensive exercise were determined. Moreover, the possible correlation of these measurements with the results of other tests used in the diagnosis of asthma as well as laboratory tests commonly associated with asthma were investigated. RESULTS: In asthmatic patients with EIB, a statistically significant increase in hs-CRP levels both in serum and EBC after an exercise test was observed. Twenty-four hours after the exercise test in the group of asthmatics with EIB, a statistically significant increase in exhaled nitric oxide (F(ENO)), serum eosinophil cationic protein (ECP) concentrations and bronchial hyperreactivity to histamine was revealed. A statistically significant correlation between the maximum increase in hs-CRP levels both in serum and EBC after exercise and either baseline F(ENO) and an increase in serum ECP or F(ENO) 24 h after exercise in the group of asthmatics with EIB was revealed. CONCLUSIONS: We show that, as a result of intensive exercise leading to bronchoconstriction, an increase in serum and EBC hs-CRP occurs. Our observations could suggest that in asthmatic patients, as a consequence of exercise-induced bronchoconstriction, an intensification of low-grade systemic inflammation can be observed.

[The role of interleukin 13 and interleukin 5 in asthma]. / Rola interleukin 13 i 5 w astmie.

Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play roles. Interleukins 5 (IL-5) and 13 (IL-13) are cytokines which play important roles in the pathophysiology of asthma. Selective accumulation and activation of eosinophils in the bronchial mucosa is considered a central event in the pathogenesis of asthma. IL-5 acts as a mediator of activation of eosinophils, influencing adhesion, membrane receptor expression, chemotaxis, and mediator synthesis. Airway eosinophilia has been related to bronchial hyperreactivity, asthma symptoms, and airway narrowing in subjects with asthma. IL-13 has a great influence on bronchial hyperreactivity, inflammation, and airway remodeling. Moreover, this cytokine drives many cellular responses relevant in asthma, including epithelial cell maturation and mucus production, synthesis of extracellular matrix proteins, and enhanced contractility of airway smooth muscle cells. In recent years, efforts have been underway to use substances acting as antagonists of these cytokines in the treatment of asthma. Many studies are being performed to assess the efficacy of anti-IL-5 and anti-IL-13 antibodies as well as substances inactivating receptors of these cytokines in asthma therapy. The results of these studies seem very interesting and induced the authors to discuss this issue.

[Anti-IgE therapy in persistent allergic asthma]. / Przeciwcialo monoklonalne anty-IgE w leczeniu przewleklej ciezkiej astmy.

Asthma is a chronic inflammatory disease of airways. Approximately 40% of asthma cases can be attributed to atopy. An increased immunoglobulin E (IgE) production is the strongest predisposing factor for the development of asthma. IgE is a key component of asthma pathophysiology and contributes to both the early- and late-phase inflammatory cascade in the airways. Omalizumab is a recombinant anti-IgE monoclonal antibody developed for the treatment of allergic diseases associated with high circulating IgE levels. By reducing serum IgE levels, as well as FceRI and FceRII receptor expression on inflammatory cells, omalizumab inhibits development of inflammatory cascade. Omalizumab is currently the only IgE-targeted therapy approved by EMEA (European Agency for the Evaluation of Medicinal Products) and FDA (Food and Drug Administration) for asthma treatment. It is efficacious in the treatment of moderate-to-severe and severe persistent allergic asthma poorly controlled with regular treatment. The drug reduces symptoms, exacerbations, emergency visits, hospitalizations, inhaled and systemic corticosteroid and rescue medication requirements and improves quality of life.

High-sensitivity C-reactive protein in the exhaled breath condensate and serum in stable and unstable asthma.

BACKGROUND: Asthma is a chronic airway inflammatory disease. Measurement of serum high- sensitivity C-reactive protein (hs-CRP) levels has suggested the involvement of low-grade systemic inflammation in several disorders, such as cardiovascular disease and diabetes mellitus. In recent years, there have been some reports concerning hs-CRP assessment as a useful tool for detecting systemic inflammation in asthma. The study was undertaken to evaluate hs-CRP levels in the exhaled breath condensate (EBC) of asthmatics with different degrees of asthma severity and their relationship to hs-CRP levels in serum, clinical characteristics, and the intensification of airway inflammation. METHODS: The study group was 62 patients with allergic asthma (20 with steroid-naïve mild asthma, 19 with ICS-treated, stable mild-to-moderate asthma, 23 with ICS-treated unstable, severe asthma) and 15 healthy volunteers. RESULTS: In the three groups of asthmatics hs-CRP concentrations in EBC and serum were significantly higher than in healthy volunteers. hs-CRP levels both in EBC and serum were significantly higher in patients with unstable asthma than in the two groups with stable disease. hs-CRP concentrations in EBC strongly correlated with those measured in serum. There was a significant correlation between hs-CRP levels both in EBC and serum and exhaled nitric oxide (F(ENO)) in the three groups of asthmatics or serum ECP in the group of patients with steroid-naïve mild asthma and unstable, severe asthma. CONCLUSION: The levels of hs-CRP in EBC are correlated with those measured in serum and may provide another useful diagnostic tool for detecting and monitoring low-grade inflammation in patients with asthma.