Raimundas Sakalauskas

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

BackgroundProteomic 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.MethodsEndobronchial 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.ResultsMore 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.ConclusionsProteomic 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 registrationClinicalTrials.gov registration NCT01378039

Effect of smoking cessation on cough reflex sensitivity.

Recent studies have shown that cigarette smokers have diminished cough reflex sensitivity compared with nonsmokers. The current authors proposed a mechanism of chronic cigarette smoke-induced desensitisation of airway cough receptors. To investigate this hypothesis, cough sensitivity to inhaled capsaicin (C5) in chronic smokers was measured both while they were actively smoking and 2, 6, 12 and 24 weeks after smoking cessation. In total, 29 subjects underwent baseline capsaicin challenge while smoking and 2 weeks after smoking cessation. Mean±sem log C5 fell from 1.86±0.12 to 1.60±0.12, demonstrating significant enhancement of cough reflex sensitivity. Of the total, 20, 18 and 14 subjects successfully abstained from smoking for 6, 12 and 24 weeks, respectively. Mean log C5 values after 12 and 24 weeks of smoking cessation were significantly diminished from baseline. In a control group of smokers, mean log C5 did not decrease from baseline after 6, 12 and 24 weeks. Overall, the log C5 profile of the smoking cessation group showed a clear, linearly decreasing trend over time compared with the control group. Even after many years of smoking, cough sensitivity is enhanced as early as 2 weeks after smoking cessation. Given the importance of an intact cough reflex, these changes may provide clinical benefit.

Peripheral blood Th9 cells and eosinophil apoptosis in asthma patients.

BACKGROUND AND OBJECTIVE Th9 cells producing interleukin (IL) 9 are novel subset of CD4+ T helper cells, which might contribute to airway inflammation in asthma. Moreover, the effect of IL-9 on eosinophils is still not fully understood. Study aim was to evaluate peripheral blood Th9 cells and eosinophil apoptosis in allergic asthma patients. MATERIALS AND METHODS Eighteen patients with allergic asthma and fourteen patients with allergic rhinitis were examined. Control group included sixteen healthy subjects. Allergic asthma and rhinitis patients did not use corticosteroids and antihistamines at least for 1 week. Peripheral blood eosinophils and CD4(+) cells were isolated by high density gradient centrifugation and magnetic separation. Th9 cells and apoptotic eosinophils were estimated by flow cytometer. Serum IL-9 and IL-5 concentration were determined by ELISA. RESULTS Peripheral blood Th9 cells percentage was increased in allergic asthma group compared with allergic rhinitis and control group (0.74%±0.32% vs. 0.19%±0.10% and 0.15%±0.08%, respectively, P<0.05). The same tendency was observed for IL-9 (P<0.01). Percentage of peripheral blood apoptotic eosinophils was decreased in allergic asthma and allergic rhinitis groups compared with control group (P<0.05). IL-9 concentration correlated with percentage of Th9 cells (r=0.64, P<0.05) and negatively with percentage of apoptotic eosinophils in allergic asthma group (r=-0.58, P<0.05). Negative correlation was found between apoptotic eosinophils count and IL-5 concentration in allergic asthma group (r=-0.76, P<0.05). CONCLUSIONS Patients with allergic asthma demonstrate increased peripheral blood Th9 cells count and serum IL-9, while eosinophil apoptosis is inversely related to IL-9 concentration.

The Role of β2-Adrenergic Receptors in Inflammation and Allergy

: Essential role of beta(2)-adrenoreceptor (beta(2)AR) in airway relaxation is well established. Nevertheless, beta(2)AR seems playing an actual role in allergy and inflammation. Interaction between beta(2)AR and proinflamatory cytokines in airway smooth muscle has been revealed. Being located on proinflamatory cells, beta(2)ARs may influence function of these cells in vivo. It was clear established, that stimulation of beta(2)AR inhibits release of proinflamatory mediators from mast cells, influences T-cell growth and function, eosinophil survival and function, including GM-CSF- or PAF-induced degranulation. Stimulation of beta(2)ARs, located on alveolar macrophages and epithelial cells, has ambiguity influence on their regulation and function, including phagocytosis and mediator secretion, in vivo. Vascular responses, resulting in inhibition of plasma exudation were confirmed, but modulation of sensory nerves via beta(2)AR is not certain yet. beta(2)AR-agonists are effective in treatment of immediate allergic reactions, but desensitisation of beta(2)ARs on inflammatory cells may result in paradoxical effects, especially in asthma. In summary, it is clear that beta(2)ARs may play an anti-inflammatory role in vitro. Unfortunately, in vitro data have shown limited applicability in vivo; therefore further research in this field is required.

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-Grünwald-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.

Screening for alpha1-antitrypsin deficiency in Lithuanian patients with COPD

BACKGROUND Alpha1-antitrypsin (AAT) deficiency is an under-diagnosed condition in patients with chronic obstructive pulmonary disease (COPD). The objective of the present screening was to estimate the AAT gene frequency and prevalence and to identify AAT deficiency cases in a large cohort of Lithuanian patients with COPD. METHODS A nationwide program of AAT deficiency was conducted in 1167 COPD patients, defined according to the GOLD criteria. Patients were collected from outpatient clinics in five different Lithuanian regions (Kaunas, Vilnius, Siauliai, Klaipeda and Alytus). AAT serum concentrations were measured by nephelometry; PI-phenotypes characterized by isoelectric-focusing. RESULTS Mean age and FEV(1) were 62.0 (10.3) and 54.7% (10.9), respectively. Ninety-one AAT deficiency genotypes (40 MZ, 39 MS, 1 SS, 3 SZ and 8 ZZ) were identified. Calculated PI(*)S and PI(*)Z frequencies, expressed in per 1000, were 18.8 (95% CI: 13.9-25) and 25.3 (95% CI: 19.4-32.7), respectively. The calculated AAT gene prevalence (Hardy-Weinberg principle) was: 1/1.09 for MM, 1/28 for MS, 1/2814 for SS, 1/20 for MZ, 1/1049 for SZ and 1/1565 for ZZ. Calculated Odds ratio (OR) for PI(*)Z in COPD vs. Lithuanian healthy people was of 1.87 (P=0.004). CONCLUSION The OR for each genotypic class demonstrated a significant increase of MZ, SZ and ZZ genotypes in COPD patients. The results of the present study, with a significant number of ZZ individuals detected, support the general concept of targeted screening for AAT deficiency in countries like Lithuania, with a large population of COPD patients and low awareness among care-givers about this genetic condition.

Peculiarities of clinical profile of snoring and mild to moderate obstructive sleep apnea–hypopnea syndrome patients

PurposeThe purpose of this study is to perform comprehensive evaluation of the snoring and mild to moderate obstructive sleep apnea–hypopnea syndrome (OSAHS) patients for their anatomical, functional, and psychoemotional clinical properties.MethodsSeventy-four snoring patients, aged 24 to 64 (mean 41.83 ± 11.01) years underwent full-night polysomnography, nasopharyngoscopy, and Mueller maneuver. Clinical tests battery consisting of visual analogue scales (VAS) scales, Lithuanian version of Sleep Apnea Quality of Life Index (SAQLI-LT), Spielberg’s State-Trait Anxiety Inventory (STAI), Beck Depression Inventory—Second Edition (BDI-II), and Epworth Sleepiness Scale (ESS) were applied to assess their distinctive clinical properties.ResultsThe total group of snoring and mild to moderate OSAHS patients presented with considerably enlarged VAS snoring and daytime sleepiness scores (mean 66.32 ± 19.07 and 35.03 ± 27.83 points), mild BDI-II scores (mean 10.96 ± 9.42 points), and moderate trait anxiety scores (mean 41.51 ± 8.62 points). All the scores of daytime complaints measured with the VAS correlated statistically significantly with the mean scores of the ESS, SAQLI-LT, trait anxiety, and BDI-II. Both groups, of snoring and mild to moderate OSAHS patients, indicated similar intensity of the major complaints according to the VAS, same as similar BDI-II, STAI, and SAQLI-LT scores. A higher Friedman’s score of palatal tonsils was found in the group of snoring patients, comparing to that of the group of mild to moderate OSAHS patients (p < 0.05).ConclusionsSnoring and mild to moderate OSAHS patients have resemblances in their distinctive anatomical and clinical properties. This group of the patients revealed mild depression and moderate trait anxiety scores when measured with the BDI-II and STAI, which correlated significantly with the severity of the patients’ daytime complaints measured with the VAS.

Reactive Oxygen Species in Peripheral Blood and Sputum Neutrophils During Bacterial and Nonbacterial Acute Exacerbation of Chronic Obstructive Pulmonary Disease

Chronic airway inflammation can be mediated by an enhanced neutrophil oxidative burst. However, the role of bacteria in the pathogenesis of chronic obstructive pulmonary disease (COPD) exacerbations is highly controversial. The aim of this study was to evaluate the production of reactive oxygen species (ROS) in peripheral blood and sputum neutrophils during bacterial and nonbacterial acute exacerbations of COPD (AECOPD). A total of 40 patients with AECOPD, 10 healthy nonsmokers, and 10 “healthy” smokers were enrolled into the study. Peripheral blood and sputum samples were obtained during exacerbation and after recovery. Neutrophils were isolated by high-density gradient centrifugation and magnetic separation. ROS production by neutrophils was investigated after stimulation with phorbol-myristate-acetate and Staphylococcus aureus bacteria. ROS production by neutrophils was assessed as the mean fluorescent intensity using a flow cytometer. IL-8 levels in serum and induced sputum were determinant by ELISA. Spontaneous ROS production was significantly higher in neutrophils from the patients with bacterial AECOPD as compared with nonbacterial AECOPD and stable COPD (P <0.05). ROS production stimulated with PMA and with Staphylococcus aureus was significantly higher in neutrophils isolated from the patients with bacterial AECOPD as compared with nonbacterial and stable COPD (P <0.05). The serum and induced sputum IL-8 levels were significantly increased in the patients with bacterial AECOPD than nonbacterial AECOPD, stable COPS, and “healthy” smokers and nonsmokers (P <0.05) and higher in the induced sputum as the compared with serum in all studied groups (P <0.05). Enlarge CRP level was documented during AECOPD than in all other groups (P <0.05). A markedly increased ROS production in sputum neutrophils during bacterial AECOPD shows an inflammatory response reflecting enhanced local inflammation, which can be mediated by bacterial colonization.

The Burden of Allergic Asthma in Children: A Landscape Comparison Based on Data from Lithuanian, Latvian, and Taiwanese Populations

Asthma is one of the most common chronic respiratory diseases with an increasing prevalence and financial burden worldwide. This disease affects individuals in all countries and all ethnic groups; however, prevalence rates of asthma have been reported to vary significantly between different regions. To understand the origin of asthma and to manage it effectively, it is necessary to analyze the genetic and environmental factors that cause these geographic differences. Therefore, we aimed to review published data from the investigations of asthma patients in Eastern Europe, represented by Latvia and Lithuania, and of patients from Eastern Asia represented by Taiwan. We hope that some of the common factors can be identified and different variants can be compared among these three countries for development of a new strategy to prevent childhood asthma.

Distribution of CD4+ and CD8+ T cells in tumor islets and stroma from patients with non-small cell lung cancer in association with COPD and smoking

BACKGROUND AND OBJECTIVE The immune system plays an important role in non-small cell lung cancer (NSCLC) and chronic obstructive pulmonary disease (COPD). The aim of this study was to evaluate the infiltration patterns of CD4(+) and CD8(+) T cells in NSCLC and to analyze their relation to COPD, smoking status and other clinicopathologic variables. MATERIALS AND METHODS Lung tissue specimens from 50 patients who underwent surgery for NSCLC (stages I-III) and 10 control group subjects were analyzed immunohistochemically. RESULTS NSCLC patients had a greater number of CD4(+) and CD8(+) T cells infiltrating the lung tissue than the control group (P=0.001) with predominant infiltration in the tumor stroma. We found a significant association between the number of total and tumor stroma-infiltrating CD4(+) and CD8(+) T cells, and smoking status (P<0.05). There were more CD8(+) T cells in the tumor stroma and fewer in the tumor islets in NSCLC patients with COPD as compared to NSCLC patients without COPD (P<0.05). However, there was no such association between CD4(+) T cells and COPD status. A high level of CD8(+) T cell infiltration in the tumor stroma was independently associated with the coexistence of COPD in multivariate analysis (P<0.05). CONCLUSIONS According to our data, COPD but not smoking seems to be associated with higher infiltration of CD8(+) T cells in the tumor stroma of patients with NSCLC. It allows us to hypothesize that NSCLC patients with coexisting COPD may have a more favorable outcome due to anticancer properties of stromal CD8(+) T cells.