Małgorzata Proboszcz

Proposal of a new method for subtyping of Mycobacterium kansasii based upon PCR restriction enzyme analysis of the tuf gene

Within this study, a new, rapid method for subtyping of Mycobacterium kansasii was developed based on the sequence analysis of the tuf gene coding for the Tu (thermo-unstable) elongation factor (EF-Tu). The method involves PCR amplification of ca. 740-bp tuf gene fragment, followed by digestion with the MvaI restriction endonuclease.

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.

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.

Clinical, radiological and molecular features of Mycobacterium kansasii pulmonary disease

BACKGROUND Studies concerning sociodemographic, clinical, and laboratory features of Mycobacterium kansasii pulmonary disease are few and based on small patient cohorts. The objective of the study was to evaluate characteristics of patients from whom M. kansasii respiratory isolates were recovered and to provide a detailed description of M. kansasii disease. BASIC PROCEDURES Retrospective review of electronic medical records of all patients for whom at least one positive M. kansasii culture was obtained at the Department of Internal Medicine, Pulmonology and Allergology of the Warsaw Medical University between the year 2000 and 2015. Patients were categorized as having mycobacterial disease or as isolation cases based on the American Thoracic Society and Infectious Diseases Society of America (ATS/IDSA) diagnostic criteria. MAIN FINDINGS The study comprised of 105 patients (63 females, 42 males, mean age 64.6 ± 17.8 years). Of these, 86 (81.9%) were diagnosed as having M. kansasii disease. The proportion of positive smear microscopy was significantly higher in patients with M. kansasii disease compared to M. kansasii isolation (P < 0.001). There were no statistically significant differences between M. kansasii disease and isolation cases in terms of clinical symptoms or comorbidities. Patients with M. kansasii disease presented most commonly (43/86, 50%) fibro-cavitary disease upon radiology. Lesion distribution usually showed bilateral upper lobe involvement. Among the 191 isolates genotyped, all were identified as M. kansasii type I. PRINCIPAL CONCLUSIONS The findings from this study support the relaxation of the diagnostic criteria for the definition of M. kansasii disease, set forth by ATS/IDSA. Molecular typing did not differentiate isolates from patients with true disease from those with isolation only; the role of bacterial virulence factors thus remains elusive.

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.

A comparative study of sTREM-1, IL-6 and IL-13 concentration in bronchoalveolar lavage fluid in asthma and COPD: A preliminary study

BACKGROUND Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is a cell-surface receptor presented on neutrophils, macrophages and monocytes. Elevated sTREM-1 levels are a marker of acute microbial infections, and have also been reported in chronic lung diseases. IL-6 and IL-13 are effective markers in distinguishing these diseases. IL-6 has been shown to play an important role in chronic obstructive pulmonary disease (COPD), while IL-13 is described as one of the key mediators of allergic asthma. OBJECTIVES The aim of the study was to evaluate the level of sTREM-1 in bronchoalveolar lavage fluid (BALF) from stable mild-to-moderate asthma and COPD patients and to compare the utility of BALF sTREM-1 measurements in asthma/COPD differentiation with those of IL-6 and IL-13. MATERIAL AND METHODS The concentration of sTREM-1, IL-6 and IL-13 was evaluated by ELISA in the BALF of stable mild-to-moderate asthma and COPD patients. RESULTS There were no significant differences in BALF sTREM-1 levels between asthma and COPD patients (52.5 pg/mL for asthma vs 73.4 pg/mL for COPD, p = 0.492), in contrast to the differences in the IL6/IL13 ratio (0.68 in COPD, 0.22 in asthma, p = 0.043). CONCLUSIONS The study showed that BALF sTREM-1 levels do not discriminate between mild-to-moderate asthma and COPD. In contrast, the IL-6/IL-13 ratio measured in BALF effectively differentiated these two diseases in their stable state. These results suggest that the most important marker of inflammation in mild-to-moderate obstructive lung disease is not microbiology-dependent.