Patrycja Nejman-Gryz

Comparative study of periostin expression in different respiratory samples in patients with asthma and chronic obstructive pulmonary disease.

INTRODUCTION Periostin is considered to be a marker of eosinophilic inflammation in patients with asthma. However, there are no literature data on periostin in patients with chronic obstructive pulmonary disease (COPD). OBJECTIVES The aim of the study was to evaluate periostin expression and to compare its concentrations in various materials in patients with mild-to-moderate asthma and COPD, as well as to evaluate the potential association between periostin and clinical features of both diseases. PATIENTS AND METHODS Using an enzyme-linked immunosorbent assay, we measured periostin concentrations in serum, induced sputum (IS), exhaled breath condensate (EBC), and bronchoalveolar lavage fluid (BALF) as well as periostin expression in bronchial biopsy samples in 24 patients with asthma, 36 patients with COPD, and 12 controls. Correlations between periostin levels in different materials were also analyzed and periostin concentrations were compared between patients with asthma and those with COPD. RESULTS Periostin levels were detectable in serum, IS, EBC, and BALF from patients with asthma, COPD, and controls. EBC periostin levels correlated with tissue periostin expression and were significantly higher in asthma than in COPD (P = 0.04). Periostin expression in bronchial mucosa was higher in asthma than in COPD (P <0.001), as well as in asthma and COPD patients compared with controls (P <0.001). No significant correlations between tissue periostin expression and BALF, IS, or serum periostin levels were found. There were no differences in serum, IS, BALF, or EBC periostin concentrations between patients with different phenotypes of both diseases. CONCLUSIONS Periostin may be detected not only in serum, IS, and airway tissue samples, but also in EBC and BALF. EBC periostin levels and tissue periostin expression are higher in patients with asthma than in those with COPD. EBC periostin levels may serve as a potential surrogate marker for tissue periostin expression.

Expression of eotaxins in the material from nasal brushing in asthma, allergic rhinitis and COPD patients

BACKGROUND Asthma and COPD are non-infectious inflammatory diseases of the respiratory tract. Allergic rhinitis can be assumed as an intermediate condition between healthy and asthmatic state. Eotaxins are important indicators of allergic reaction. They are strong chemoattractants mainly for eosinophils but also for other cells. OBJECTIVE We measured the level of eotaxin expression and inflammatory cell count in the material from nasal brushing in healthy controls and in patients with allergic rhinitis, asthma, and COPD. We studied the correlation between the eotaxin gene expression level in the material from nasal brushing and respiratory tests in asthma and COPD patients. METHODS Expression of eotaxins was measured using quantitative RT-PCR. Number of eotaxin transcript copies was evaluated using real time PCR standard curve method. RESULTS Of all eotaxins CCL24 had the highest expression in the material from nasal brushing, and its level was increased in allergic asthma. CCL11 was significantly increased in the material from nasal brushing of COPD patients. Increased levels of all three eotaxins were observed in the material from nasal brushing of patients with allergic rhinitis in season. The levels of CCL26 expression and FEV1/FVC factor were correlated negatively in the asthma group and positively in the COPD group. CONCLUSIONS Eotaxins are crucial factors of allergic, asthmatic and also COPD inflammatory reactions. Our results suggest a dual role of CCL26 - it can act as a negative regulator for neutrophils in COPD, while in asthma it may act as a chemoatractant of eosinophils and other cells into the lung.

Eosinophilic and Neutrophilic Airway Inflammation in the Phenotyping of Mild-to-Moderate Asthma and Chronic Obstructive Pulmonary Disease

ABSTRACT Asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous diseases with different inflammatory phenotypes. Various inflammatory mediators play a role in these diseases. The aim of this study was to analyze the neutrophilic and eosinophilic airway and systemic inflammation as the phenotypic characterization of patients with asthma and COPD. Twenty-four patients with asthma and 33 patients with COPD were enrolled in the study. All the patients were in mild-to-moderate stage of disease, and none of them were treated with inhaled corticosteroids. Concentrations of IL-6, neutrophil elastase (NE), matrix metalloproteinase 9 (MMP-9), eosinophil cationic protein (ECP), and IL-33 and IL-17 in serum and induced sputum (IS) were measured by enzyme-linked immunosorbent assay (ELISA). The cellular composition of blood and IS was evaluated. Hierarchical clustering of patients was performed for the combination of selected clinical features and mediators. Asthma and COPD can be differentiated based on eosinophilic/neutrophilic systemic or airway inflammation with unsatisfactory efficiency. Hierarchical clustering of patients based on blood eosinophil percentage and clinical data revealed two asthma clusters differing in the number of positive skin prick tests and one COPD cluster with two subclusters characterized by low and high blood eosinophil concentrations. Clustering of patients according to IS measurements and clinical data showed two main clusters: pure asthma characterized by high eosinophil/atopy status and mixed asthma and COPD cluster with low eosinophil/atopy status. The neutrophilic phenotype of COPD was associated with more severe airway obstruction and hyperinflation.

The expression of the eotaxins IL-6 and CXCL8 in human epithelial cells from various levels of the respiratory tract

Airway epithelium acts as multifunctional site of response in the respiratory tract. Epithelial activity plays an important part in the pathophysiology of obstructive lung disease. In this study, we compare normal human epithelial cells from various levels of the respiratory tract in terms of their reactivity to pro-allergic and pro-inflammatory stimulation. Normal human nasal, bronchial and small airway epithelial cells were stimulated with IL-4 and IL-13. The expressions of the eotaxins IL-6 and CXCL8 were evaluated at the mRNA and protein levels. The effects of pre-treatment with IFN-γ on the cell reactivity were measured, and the responses to TNF-α, LPS and IFN-γ were evaluated. All of the studied primary cells expressed CCL26, IL-6 and IL-8 after IL-4 or IL-13 stimulation. IFN-γ pre-treatment resulted in decreased CCL26 and increased IL-6 expression in the nasal and small airway cells, but this effect was not observed in the bronchial cells. IL-6 and CXCL8 were produced in varying degrees by all of the epithelial primary cells in cultures stimulated with TNF-α, LPS or IFN-γ. We showed that epithelial cells from the various levels of the respiratory tract act in a united way, responding in a similar manner to stimulation with IL-4 and IL-13, showing similar reactivity to TNF-α and LPS, and giving an almost unified response to IFN-γ pre-stimulation.

The effect of 1,25-dihydroxyvitamin D3 on TSLP, IL-33 and IL-25 expression in respiratory epithelium.

BackgroundAirway epithelium is an active and important component of the immunological response in the pathophysiology of obstructive lung diseases. Recent studies suggest an important role for vitamin D3 in asthma severity and treatment response.ObjectiveOur study evaluated the influence of an active form of vitamin D3 on the expression of selected mediators of allergic inflammation in the respiratory epithelium.Material and MethodsPrimary nasal and bronchial epithelial cells were exposed to1,25D3 for 1 hour and were then stimulated or not with IL-4, TNF-α, LPS, and poly I:C. After 24 hours TSLP, IL-33, and IL-25 protein levels were measured in culture supernatants usingELISAandmRNAlevels in cells by real time PCR.Results1,25D3 increased TSLP concentration in unstimulated nasal epithelial cells, but did not influence IL-33 and IL-25 expression. In IL-4-stimulated epithelial cell cultures 1,25D3 mostly inhibited TSLP and IL-33 expression. In LPS-treated cultures 1,25D3 decreased IL-33 expression. Simultaneously 1,25D3 augmented IL-25 production in the same model of stimulation.ConclusionOur study revealed the dual nature of vitamin D3 manifested in both pro- and anti-inflammatory properties observed in airway epithelial cells.

Expression of Inflammatory Mediators in Induced Sputum: Comparative Study in Asthma and COPD

Asthma and COPD are the most common obstructive lung diseases characterized by inflammation in the lower airways which contribute to airflow limitation. Different inflammatory mediators are thought to play a key role in these diseases. This study was conducted in 13 patients with asthma, 12 patients with COPD, and 13 control subjects. The expression of mRNA of IL-6, IL-13, CXCL8, TSLP, IL-33, IL-25, IL-17, ECP, mast cell tryptase, CCL24, and CCL26 was assessed in induced sputum cells by real time PCR. We found that CXCL8 was strongly related to the neutrophil percentage but differed significantly in COPD and asthma patients. The expression of IL-17 was lower in patients with atopic asthma compared to non-atopic asthma. The percentage of macrophages correlated negatively with the expression of mast cell tryptase and ECP in COPD, and with CXCL8 in asthma. The expression of ECP correlated negatively with the severity of COPD symptoms measured by CAT. We conclude that asthma and COPD demonstrate a significant overlap in the airway cytokine profile. Thus, differentiation between the two diseases is difficult as based on a single cytokine, which suggests the coexistence of phenotypes sharing a common cytokine network in these obstructive lung diseases.

Comparison of Thymic Stromal Lymphopoietin Concentration in Various Human Biospecimens from Asthma and COPD Patients Measured with Two Different ELISA Kits

Thymic stromal lymphopoietin (TSLP) seems a promising asthma biomarker. In earlier studies, mainly the serum concentration of TSLP was investigated. The aim of the present study was to compare the TSLP concentration measured by two different ELISA kits in the serum, induced sputum, and exhaled breath condensate in asthma, COPD, and control subjects. The study included 24 asthmatics, 36 patients with COPD, and 12 controls. TSLP concentration was measured with the use of R&D and EIAab commercial ELISA kits. The results obtained with the EIAab kit were 3 to even 45-fold higher than those measured with the R&D kit. Significant differences between the investigated groups were found only for the TSLP concentration in induced sputum. When the R&D kit was used, the highest TSLP levels in induced sputum were found in asthmatics, while the EIAab kit showed the highest TSLP levels in controls. The distribution of results in the Bland-Altman plot was typical for a proportional constant error. TSP concentration in induced sputum might be a more reliable asthma biomarker than serum TSLP. We conclude that TSLP level is highly dependent on the ELISA kit used for the measurement. Thus, judgement on TSLP results obtained with different assays might be confusing and lead to wrong conclusions.

Exhaled matrix metalloproteinase 9 in patients with stable COPD - an observational study

Introduction Data on the measurement of matrix metalloproteinase 9 (MMP‑9) in exhaled breath condensate (EBC) from patients with chronic obstructive pulmonary disease (COPD) are scarce and inconsistent. Objectives We aimed to assess the usefulness of enzyme‑linked immunosorbent assay (ELISA) and immunoenzymatic assay (IEA) for the measurement of MMP‑9 in EBC, the agreement between the results of both methods, and the relationships between total and active MMP‑9 in EBC and clinical and functional COPD characteristics. Patients and methods Total (ELISA and IEA) and active (IEA) MMP‑9 levels were assessed in EBC from 70 patients with stable COPD and 21 controls and correlated with pulmonary function and COPD symptom severity. Results MMP‑9 levels did not reach the sensitivity threshold of the ELISA kit in any of the COPD patients and in 11 controls. Total and active MMP‑9 (IEA) levels did not differ between COPD patients and controls. In COPD patients, total MMP‑9 levels correlated positively with forced expiratory volume in 1 second (FEV1) and FEV1 to forced vital capacity ratio and inversely with residual volume to total lung capacity ratio. A weak positive correlation between active MMP‑9 concentrations and COPD Assessment Test (CAT) score was found (r = 0.31, P = 0.01). Conclusions The utility of ELISA in MMP‑9 assessment in EBC is limited in COPD patients, while MMP‑9 measurement in EBC by IEA is feasible. The positive correlation between active MMP‑9 and CAT score in our patients and the inverse relationship between total MMP‑9 concentration and the degree of airway obstruction reflect the complex role of MMP‑9 in COPD.

Comparative Study of IL-33 and IL-6 Levels in Different Respiratory Samples in Mild-to-Moderate Asthma and COPD

ABSTRACT IL-6 and IL-33 are involved in the inflammatory process in obstructive lung diseases. In contrast to IL-6, few data on the expression of IL-33 in different biological samples from asthma and COPD patients are available. The aim was to evaluate the expressions of IL-33 and IL-6 in bronchial mucosa and to compare these expressions with the concentrations of both cytokines in various respiratory samples from patients with mild-to-moderate asthma and COPD. Serum, induced sputum and exhaled breath condensate IL-6 and IL-33 levels, as well as their expression in bronchial mucosa were evaluated in 22 asthma and 33 COPD patients. There were significant differences between bronchial mucosa IL-6, but not IL-33 expression in asthma and COPD. Serum and IS IL-6 concentrations were higher in COPD than in asthma (3.4 vs. 2.02 pg/mL, p = 0.002 and 16.5 vs. 12.7 pg/mL, p = 0.007, respectively); IL-33 levels reached similar values in asthma and COPD in all investigated samples. In both diseases, the lowest levels of IL-6 and IL-33 were found in EBC. EBC levels of both cytokines did not correlate with their expression in other materials. The IL-33 and IL-6 are detectable in serum, IS and EBC not only in asthma but also in COPD patients. In the COPD group, serum and IS IL-6 concentrations were statistically higher than in the asthma group. The tissue expression of IL-33 and IL-33 concentrations in the investigated biological samples were on a comparable level in both diseases. Our findings may suggest that IL-33 activation is a common pathway in asthma and COPD.

Sputum interleukin-25 correlates with asthma severity – a preliminary study

Introduction Interleukin 25 is an epithelial-derived cytokine associated with allergic Th2 inflammation. However, little is known about the role of IL-25 in different asthma phenotypes and its relationship with disease severity. Aim To evaluate and compare the mRNA and protein expression of IL-25 in patients with mild-to moderate/severe asthma and cough variant asthma (CVA). Material and methods Thirty-eight patients with stable asthma (11 patients with mild-to-moderate asthma, 14 patients with severe asthma and 13 patients with CVA) and 14 control subjects were enrolled. IL-25 protein concentration was measured in induced sputum (IS) supernatants by ELISA and IL-25 mRNA expression was evaluated in IS cells by real time PCR. Results No differences in IS IL-25 mRNA and IL-25 concentration between controls and the whole asthma group were found. In the detailed analysis, a lower IL-25 mRNA expression in sputum cells was observed in severe asthma compared to CVA and controls. IL-25 protein concentration in sputum supernatants was elevated in patients with severe asthma compared to controls, CVA and mild-to-moderate asthma. A sputum IL-25 level was increased in atopic vs. non-atopic asthma patients. The elevated IL-25 mRNA expression and protein concentration was associated with a lower eosinophil and higher neutrophil percentage in asthmatic airways. Conclusions Our results suggest that IL-25 is particularly associated with severe asthma. The relationship between IL-25 and neutrophilic airway inflammation suggests the pleiotropic role of IL-25 in the immune response in this disease.