Kestutis Malakauskas

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.

Th17 response to Dermatophagoides pteronyssinus is related to late-phase airway and systemic inflammation in allergic asthma

BACKGROUND Th17 cells may play a role in the development of late-phase allergen-induced airway and systemic inflammation in allergic asthma, although the mechanisms involved remain to be elucidated. METHODS A total of 36 subjects were enrolled into the study: 15 allergic asthma patients with early asthmatic reaction (n=7) or dual asthmatic reaction (n=8) developed to inhaled D. pteronyssinus, 13 patients with allergic rhinitis, and 8 healthy subjects. Peripheral blood and induced sputum were collected 24h before as well as 7h and 24h after a bronchial challenge with D. pteronyssinus. Th17 cells were analyzed by FACS; IL-17 levels were determined by ELISA. RESULTS At baseline, the percentage of peripheral blood Th17 cells and serum and sputum IL-17 levels were significantly higher in all groups of studied patients compared with those of healthy subjects. After the bronchial challenge, there was a significant increase in the percentage of peripheral blood Th17 cells and in serum and sputum IL-17 levels in rhinitis and asthma patients compared with their baseline values, particularly in allergic asthma patients with the dual asthmatic reaction. Positive correlations were found between the percentage of Th17 cells and IL-17 levels in serum (Rs=0.649; P=0.009) as well in sputum (Rs=0.583; P=0.022) in allergic asthma patients 24h after the bronchial challenge. CONCLUSIONS The Th17 response is associated with the development of late-phase airway and systemic inflammation after the inhalation of D. pteronyssinus in patients with allergic asthma.

Suppression of Eosinophil Integrins Prevents Remodeling of Airway Smooth Muscle in Asthma

Background: Airway smooth muscle (ASM) remodeling is an important component of the structural changes to airways seen in asthma. Eosinophils are the prominent inflammatory cells in asthma, and there is some evidence that they contribute to ASM remodeling via released mediators and direct contact through integrin–ligand interactions. Eosinophils express several types of outer membrane integrin, which are responsible for cell–cell and cell–extracellular matrix interactions. In our previous study we demonstrated that asthmatic eosinophils show increased adhesion to ASM cells and it may be important factor contributing to ASM remodeling in asthma. According to these findings, in the present study we investigated the effects of suppression of eosinophil integrin on eosinophil-induced ASM remodeling in asthma. Materials and Methods: Individual combined cell cultures of immortalized human ASM cells and eosinophils from peripheral blood of 22 asthmatic patients and 17 healthy controls were prepared. Eosinophil adhesion was evaluated using eosinophil peroxidase activity assay. Genes expression levels in ASM cells and eosinophils were measured using quantitative real-time PCR. ASM cell proliferation was measured using alamarBlue® solution. Eosinophil integrins were blocked by incubating with Arg-Gly-Asp-Ser peptide. Results: Eosinophils from the asthma group showed increased outer membrane α4β1 and αMβ2 integrin expression, increased adhesion to ASM cells, and overexpression of TGF-β1 compared with eosinophils from the healthy control group. Blockade of eosinophil RGD-binding integrins by Arg-Gly-Asp-Ser peptide significantly reduced adhesion of eosinophils to ASM cells in both groups. Integrin-blocking decreased the effects of eosinophils on TGF-β1, WNT-5a, and extracellular matrix protein gene expression in ASM cells and ASM cell proliferation in both groups. These effects were more pronounced in the asthma group compared with the control group. Conclusion: Suppression of eosinophil-ASM interaction via RGD-binding integrins attenuates eosinophil-induced ASM remodeling in asthma. Trial Registration: ClinicalTrials.gov Identifier: NCT02648074.

Dyspnea perception and reversibility of methacholine-induced unlimited airway narrowing in asthmatics.

The hypothesis was that asthmatics might experience impaired perception of dyspnea and salbutamol-induced reversibility during unlimited airway narrowing. A total of 38 asthmatics (18 to 59 years of age) were examined. All patients underwent the methacholine challenge test. According to the dose-response curve to methacholine, they were categorized as having either unlimited airway narrowing (UAN group) (n = 20) or response plateau (RP group) (n = 18). Reversibility of methacholine-induced bronchoconstriction was measured 20 minutes after the inhalation of 400 μg of salbutamol to compare postbronchodilator FEV1 with baseline FEV1. Dyspnea perception was evaluated using the Borg Scale to calculate a perception score at a 20% decrease in FEV1 (PS20) and the slope α of the regression line between the changes in Borg scores and the reduction in FEV1 as percentage of the baseline value. Subjects in the UAN group exhibited significantly lower PS20 compared with the RP group (1.45± 0.23 vs. 2.84 ± 0.35, p = 0.002); the mean of the slope values was higher in the RP group than it was in the UAN group (0.150 ± 0.015 vs. 0.095 ± 0.006, p = 0.003). Salbutamol-induced reversibility was significantly lower in the UAN group (81 ± 1.4 % of baseline FEV1) compared with patients from the RP group (91 ± 1.1% of baseline FEV1; p < 0.001). In conclusion, asthmatics during methacholine-induced unlimited airway narrowing exhibit diminished perception of dyspnea and lower bronchial reversibility to the baseline 20 minutes after inhalation of salbutamol. This suggests that more careful monitoring of the lung function for timely recognition of asthma deteriorations and adequate bronchodilatory therapy during severe acute attacks should be recommended for such patients.

Peripheral blood Th9 cells and eosinophil apoptosis in allergic asthma patients

Material and Methods 18 patients with allergic asthma (AA) and 14 patients with allergic rhinitis (AR) were examined. The control group included 16 healthy subjects (HS). All AA and AR patients did not take inhaled corticosteroids at least for one month and/or histamine antagonists at least for one 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 interleukin-9 (IL-9) and interleukin-5 (IL-5) level was determined by ELISA.