Network analysis of quantitative proteomics on asthmatic bronchi: effects of inhaled glucocorticoid treatment.

BACKGROUND: Proteomic studies of respiratory disorders have the potential to identify protein biomarkers for diagnosis and disease monitoring. Utilisation of sensitive quantitative proteomic methods creates opportunities to determine individual patient proteomes. The aim of the current study was to determine if quantitative proteomics of bronchial biopsies from asthmatics can distinguish relevant biological functions and whether inhaled glucocorticoid treatment affects these functions. METHODS: Endobronchial biopsies were taken from untreated asthmatic patients (n = 12) and healthy controls (n = 3). Asthmatic patients were randomised to double blind treatment with either placebo or budesonide (800 µg daily for 3 months) and new biopsies were obtained. Proteins extracted from the biopsies were digested and analysed using isobaric tags for relative and absolute quantitation combined with a nanoLC-LTQ Orbitrap mass spectrometer. Spectra obtained were used to identify and quantify proteins. Pathways analysis was performed using Ingenuity Pathway Analysis to identify significant biological pathways in asthma and determine how the expression of these pathways was changed by treatment. RESULTS: More than 1800 proteins were identified and quantified in the bronchial biopsies of subjects. The pathway analysis revealed acute phase response signalling, cell-to-cell signalling and tissue development associations with proteins expressed in asthmatics compared to controls. The functions and pathways associated with placebo and budesonide treatment showed distinct differences, including the decreased association with acute phase proteins as a result of budesonide treatment compared to placebo. CONCLUSIONS: Proteomic analysis of bronchial biopsy material can be used to identify and quantify proteins using highly sensitive technologies, without the need for pooling of samples from several patients. Distinct pathophysiological features of asthma can be identified using this approach and the expression of these features is changed by inhaled glucocorticoid treatment. Quantitative proteomics may be applied to identify mechanisms of disease that may assist in the accurate and timely diagnosis of asthma. TRIAL REGISTRATION: ClinicalTrials.gov registration NCT01378039.

The effect of induced sputum and bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease on neutrophil migration in vitro.

OBJECTIVE: The aim of study was to investigate a chemotactic effect of induced sputum and bronchoalveolar lavage fluid on blood neutrophils in patients with chronic obstructive pulmonary disease (COPD) and healthy individuals. MATERIAL AND METHODS: Forty-three smokers with COPD, 19 ex-smokers with COPD, 13 healthy smokers, and 17 healthy nonsmokers were recruited to the study. Neutrophils were isolated from peripheral blood of study individuals. For the same experimental conditions, pooled induced sputum and bronchoalveolar lavage fluid of 20 COPD patients were used. Neutrophil chemotaxis in vitro was performed in cell-transmigration chamber. Substances tested for chemoattraction (interleukin-8, induced sputum, bronchoalveolar lavage fluid directly or in addition to interleukin-8) were added to lower wells. Upper wells were filled with 2.5 x 10(6)/mL of neutrophil culture and incubated for 2 hours. Migration was analyzed by flow cytometry. RESULTS: Interleukin-8 (10-100 ng/mL) induced a dose-dependant neutrophil migration in all the groups. Only 100 ng/L of interleukin-8 induced more intensive chemotaxis of neutrophils from COPD smokers as compared to ex-smokers (P<0.05). Such difference between healthy individuals was obtained using 30 ng/mL of interleukin-8 (P<0.05). Induced sputum/interleukin-8 (10-100 ng/mL), as well as induced sputum directly, induced neutrophil migration (P<0.05). Chemotaxis of neutrophils isolated from COPD patients and healthy nonsmokers did not depend on additional interleukin-8 concentration. Bronchoalveolar lavage fluid/interleukin-8 (30-100 ng/mL) induced more intensive migration of neutrophils from COPD patients than bronchoalveolar lavage fluid (P<0.05) alone. CONCLUSIONS: Migration of neutrophils isolated from patients with COPD was more intensive compared to healthy individuals. Induced sputum and bronchoalveolar lavage fluid directly and with addition of interleukin-8 stimulated chemotaxis, and it was higher in neutrophils from COPD patients. Migration of neutrophils did not depend on smoking status.

Smoking affects eotaxin levels in asthma patients.

BACKGROUND: Chronic airway inflammation is most important pathological finding in asthma. Cigarette smoking may modify type of inflammation as well as may influence disease severity and response to the treatment. OBJECTIVE: Thus the aim of this study was to investigate whether cigarette smoking may have an influence on the levels of eotaxin-1, eotaxin-2, eotaxin-3 and IL-5 in patients with stable mild/moderate asthma. METHODS: 45 steroid naive asthmatics (mean age: 55.2 +/- 2.2 yrs) and 23 "healthy" smokers and non-smokers control subjects (mean age: 54.4 +/- 9.7 yrs) were investigated. Asthmatics were divided into two subgroups according to their smoking histories: asthmatic smokers (n = 19) who currently smoke and have a history of > 10 pack-years and asthmatic never-smokers (n = 26). BAL and induced sputum were performed. Cytospins of induced sputum and BAL were stained with May-Grunwald-Giemsa for differential cell counts. Eotaxin-1, eotaxin-2, eotaxin-3 and IL-5 concentrations in serum, sputum and BAL supernatant was measured using a commercial ELISA kit. RESULTS: In sputum supernatant from asthma smokers was significantly higher concentration of eotaxin-1 than in non-smokers asthmatics (203.4 +/- 10.0 vs. 140.2 +/- 9.5 respectively, p < 0.05). In non-smokers asthma patients levels of BAL eotaxin-1 strongly related to percent and absolute numbers of BAL eosinophils and neutrophils (Rs = 0.737 and Rs = 0.514 respectively, p < 0.05). The number and percent of sputum neutrophils and eosinophils, obtained from smokers asthmatics, significantly correlated with eotaxin-2 concentration in sputum supernatant (Rs = 0.58 and Rs = 0.75 respectively, p < 0.05). IL-5 levels in the serum and sputum from asthmatic never-smokers were significantly higher than they were from asthmatic smokers and "healthy" smokers. Asthmatic never-smokers showed a significantly higher amount of IL-5 in serum and sputum than the asthmatic smokers showed. CONCLUSIONS: This study showed the elevated levels of sputum eotaxin-1 as well as serum, sputum and BAL eotaxin-2 in asthmatic smokers without a significant increase of eosinophils compared to asthmatic never-smokers. The eotaxin concentrations were related not only with number of eosinophils but also with the number of neutrophils in all the studied tissue compartments. The data herein permits a suggestion that smoking may influence change in asthmatic airway inflammation by stimulating the production of eotaxins.

Patterns of airway inflammation and MMP-12 expression in smokers and ex-smokers with COPD.

BACKGROUND: Smoking activates and recruits inflammatory cells and proteases to the airways. Matrix metalloproteinase (MMP)-12 may be a key mediator in smoke induced emphysema. However, the influence of smoking and its cessation on airway inflammation and MMP-12 expression during COPD is still unknown. We aimed to analyse airway inflammatory cell patterns in induced sputum (IS) and bronchoalveolar lavage (BAL) from COPD patients who are active smokers and who have ceased smoking >2 years ago. METHODS: 39 COPD outpatients - smokers (n = 22) and ex-smokers (n = 17) were studied. 8 'healthy' smokers and 11 healthy never-smokers were tested as the control groups. IS and BAL samples were obtained for differential and MMP-12+-macrophages count analysis. RESULTS: The number of IS neutrophils was higher in both COPD groups compared to both controls. The amount of BAL neutrophils was higher in COPD smokers compared to healthy never-smokers. The number of BAL MMP-12+-macrophages was higher in COPD smokers (1.6 +/- 0.3 x 106/ml) compared to COPD ex-smokers, 'healthy' smokers and healthy never-smokers (0.9 +/- 0.4, 0.4 +/- 0.2, 0.2 +/- 0.1 x 106/ml respectively, p < 0.05). CONCLUSION: The lower amount of BAL neutrophils in COPD ex-smokers, compared to COPD smokers, suggests positive alterations in alveolar compartment after smoking cessation. Smoking and disease itself may stimulate MMP-12 expression in airway compartments (IS and BAL) from COPD patients.

[Genetic features during chronic obstructive pulmonary disease]. / Genetiniu pokyciu ypatybes sergant letine obstrukcine plauciu liga.

Chronic obstructive pulmonary disease is one of the leading causes of mortality, which is caused by the interaction of genetic susceptibility with environmental factors (especially smoking); however, genetic features of chronic obstructive pulmonary disease still remain unknown. The only proven genetic risk factor is severe deficiency of plasma protease inhibitor, alpha-1 antitrypsin. A large number of candidate genes for chronic obstructive pulmonary disease have already been identified in many experimental and clinical studies; however, polygenic etiology of chronic obstructive pulmonary disease is the main reason for conflicting molecular and genetic findings. This review describes the correlation between single nucleotide polymorphism of alpha-1 antitrypsin, alpha-1 antichymotrypsin, microsomal epoxide hydrolase-1, matrix metalloproteinases-1,-9,-12, glutathione S-transferases, heme oxygenase-1, tumor necrosis factor-alpha, transforming growth factor-beta, interleukins-1,-4,-13, beta2-adrenergic receptor, G-globulin genes and chronic obstructive pulmonary disease. Regions on chromosomes 1, 2, 12, and 17 are indicated as candidate chromosomal regions influencing the decrease in spirometric parameters. Genome-wide scan shows direct evidence for linkage of decreased FEV1/FVC ratio to one or more genes on chromosome 2q influencing the development of airflow obstruction. Genetic markers on chromosome 12p suggest evidence for linkage of FEV1 to this region, and observations on chromosome 1p show the relationship between FVC and genes of this region. All these findings also need to be proven, and linkage analysis among combinations of gene candidates for chronic obstructive pulmonary disease has to be done.

[Effect of pyruvate on the dependence of electromechanical activity on stimulation frequency in human myocardium]. / Piruvato itaka zmogaus miokardo elektromechaninio aktyvumo priklausomumui nuo dirginimo daznio.

The aim of the study was to determine the effect of pyruvate, a substrate of energetic metabolism, on the dependence of electromechanical activity on stimulation frequency in human myocardium. The experiments were performed on human ventricle myocardium from patients undergoing cardiac corrective open heart surgery at Clinic of Cardiac Surgery in Kaunas University of Medicine by using standard method of registration of myocardium electromechanical activity. The stimulation frequency of myocardial strips were 0.2, 0.5, 1.0, 1.5, 2.0, 2.5 and 3 Hz, duration of pulses -2-5 ms. In control, i.e. at perfusion of mycoardial strips by Tyrode solution and stimulation frequency 1 Hz, an average of contraction force (P) was 0.94+/-0.18 mN (n=12), half time of relaxation (t(a)) - 178.88+/-9.31 ms (n=12), and action potential duration measured at 50% repolarization level (VP(50)) - 294.75+/-27.95 ms (n=9). Pyruvate (10 mM) increased P to 159.9+/-13.4% (p<0.00), t(a)- 106.9+/-2.2% (n=12) (p<0.001), and VP(50)- 111.1+/-7.4% (n=5) (p<0.05), as compared to control. In the absence of pyruvate values of P, t(a) and VP(50) of myocardial strips were higher at 0.2 Hz and 0.5 Hz and lower at 3 Hz compared with those at 1 Hz of stimulation frequency. Although the dependence of these parameters on stimulation frequency in the presence of pyruvate (10 mM) was not altered but their values were persistently increased at all stimulation frequencies. The present data indicate that pyruvate is an effective inotropic agent, which can improve contractility function in wide-ranging diapason of stimulation frequency in failing human heart.

Biogenic amines activate blood leukocytes via trace amine-associated receptors TAAR1 and TAAR2.

Certain biogenic amines, such as 2-PEA, TYR, or T1AM, modulate blood pressure, cardiac function, brain monoaminergic systems, and olfaction-guided behavior by specifically interacting with members of a group of rhodopsin-like receptors, TAAR. A receptor that is absent from olfactory epithelia but had long been identified in the brain and a variety of peripheral tissues, TAAR1 has been found recently in blood B cells, suggesting a functional role of TAAR1 in these cells. With the present study, we have set out to clarify the expression and functional roles of TAAR in different isolated human blood leukocyte types. Here, we report the functional expression of TAAR1 and its closest relative TAAR2 in blood PMN and T and B cells. Both receptors are coexpressed in a subpopulation of PMN, where they are necessary for the chemosensory migration toward the TAAR1 ligands 2-PEA, TYR, and T1AM, with EC50 values of 0.43 ± 0.05 nM, 0.52 ± 0.05 nM, and 0.25 ± 0.04 nM, respectively. The same amines, with similar potencies, triggered cytokine or Ig secretion, in purified blood T or B cells, respectively. Notably, 2-PEA regulated mRNA expression of 28 T cell function-related genes, above all of the CCL5. In siRNA-guided experiments, TAAR1 and TAAR2 proved to be necessary for amine-induced blood leukocyte functions. In summary, our results demonstrate that biogenic amines potently regulate blood cell functions via TAAR1 and TAAR2 and open the perspective of their specific pharmacological modulation.

Distribution of γδ and other T-lymphocyte subsets in patients with chronic obstructive pulmonary disease and asthma.

The role of T lymphocytes in pathogenesis of chronic inflammatory airway diseases - asthma and chronic obstructive pulmonary disease (COPD) has been emphasized in recent years: the importance of αß T-cells (CD8+ and CD4+) has been widely described. A substantial fraction of γδ T-cells is a composite part of pulmonary T lymphocytes. Specific localisation of γδ T-cells in epithelium/mucosa-rich tissues implies their potential role in local inflammatory immune response, which occurs in chronic inflammatory airway diseases. An investigation was made of the T-lymphocyte subsets in induced sputum (IS), in bronchoalveolar lavage (BAL) and in peripheral blood from 20 patients with COPD (stages II-III; GOLD), 18 patients with asthma (persistent mild to moderate; GINA) and 14 healthy subjects. Relationship of γδ T-cells with lung function and smoking history was analysed. COPD patients had significantly higher numbers of CD8+T-cells in the airways of smokers compared to ex-smokers in the COPD group. A significant positive correlation was found between CD8+T-cells and pack-years of smoking. Differently, the COPD patients had significantly lower relative and absolute numbers of γδ T-cells in IS and in BAL compared to those from asthma or healthy subjects. The quantity of γδ T-cells negatively correlated with forced expiratory volume in 1 s and smoking (pack-years) only in COPD group. Our findings indicate a different local inflammatory response in COPD patients and in asthmatic groups. The reduced amount of γδ T-cells in IS and in BAL from COPD patients raises the hypothesis about their important role in pathogenesis of COPD.

Influence of smoking status on cough reflex sensitivity in subjects with COPD.

Cough reflex sensitivity has not been studied extensively in patients with chronic obstructive pulmonary disease (COPD). The aim of the study was to evaluate cough reflex sensitivity to capsaicin in current and former smokers with COPD and examine its association with potentially protussive mediators. Fifteen active smokers and 18 ex-smokers with moderate to severe COPD, 14 healthy active smokers, and 13 healthy never smokers were enrolled. Capsaicin aerosol was administered in order of ascending concentration until the concentrations inducing two or more coughs (C(2)) and five or more coughs (C(5)) were attained. The concentrations of leukotriene E(4) (LTE(4)), leukotriene B(4) (LTB(4)), and interleukin-8 (IL-8) in bronchoalveolar lavage (BAL) fluid were analyzed by ELISA. Cough reflex sensitivity in COPD smokers [mean log C(2) = 1.20 +/- 0.23 (SEM) microM; log C(5) = 1.85 +/- 0.21 microM] did not differ from that in COPD ex-smokers (log C(2) = 1.15 +/- 0.14 microM; log C(5) = 2.10 +/- 0.19 microM; p > 0.05). Mean C(2) and C(5) in both COPD groups were significantly lower than in healthy active smokers, but higher compared with the healthy never-smokers. BAL fluid concentrations of LTE(4) and LTB(4) were similar in all groups. IL-8 concentrations did not differ between COPD smokers, COPD ex-smokers, and healthy active smokers, but were significantly higher in all three groups compared with healthy never smokers. Cough reflex sensitivity to capsaicin does not differ between smokers and ex-smokers with COPD.