M. Joskova

Experimental Model of Allergic Asthma

The aim of the study was to prepare and evaluate the experimental model of allergic asthma. Changes in chough reflex, bronchoconstriction and the degree of inflammation were studied in ovalbumin (OVA) sensitized guinea pigs after 0, 7, 14, 21 days of exposure. The cough reflex was induced by citric acid inhalation in conscious animals in a double chamber body plethysmograph. Tracheal smooth muscle reactivity was assessed by examining the in vitro response to histamine (H) (10(-8)-10(-3) mol/l) and in vivo to H nebulization (10(-6) mol/l). BALF levels of IL-4, IL-5 and the eosinophil count were used as parameters of airway inflammation. After 7 days of OVA sensitization, there was an increase in tracheal smooth muscle contractility in vitro to cumulative concentration of H and an increase in cough parameters. After 14 days of OVA sensitization, there was a further increase in tracheal smooth muscle contractility to H, an increase in airway resistance, and a small increase in cough parameters. After 21 day of OVA sensitization, cough parameters were significantly reduced, airway resistance after H inhalation was increased, and there were significant increases in IL-4, IL-5, and eosinophils in BALF. In conclusion, progress in asthmatic inflammation during 21-day OVA sensitization caused a gradual increase in inflammatory mediators, a decline in cough reflex, and enhanced bronchoconstriction. This experimental model of allergic asthma can be used for pharmacological modulations of defense reflexes and inflammation.

Effects of selective inhibition of PDE4 and PDE7 on airway reactivity and cough in healthy and ovalbumin-sensitized guinea pigs.

Phosphodiesterases (PDEs) are enzymes responsible for degradation of cAMP and cGMP in cells. Thus, PDE inhibitors may have significant clinical benefit in respiratory diseases associated with inflammation. The aim of the present study was to evaluate the effects of selective PDE4 (rolipram, ROL) and PDE7 inhibitors (BRL50481, BRL) on citric acid-induced cough, in vivo and in vitro airway smooth muscle reactivity in both healthy and ovalbumin sensitized guinea pigs. The drugs tested were administered intraperitoneally to male guinea pigs once daily for 7 days - ROL 1 mg/kg, BRL 1 mg/kg, and ROL+BRL 0.5 mg/kg. Double chamber whole body plethysmography was used for the evaluation of citric acid (0.6 M)-induced cough and specific airway resistance. An organ bath method was used for the measurement of tracheal and lung tissue strip contractions evoked by cumulative doses (10(-8)-10(-3) mol/L) of acetylcholine (ACH) and histamine (HIS). In healthy guinea pigs, the only significant relaxation was observed after ROL in ACH-induced contractions in vitro and the effect on cough was negligible. In ovalbumin-sensitized animals, more pronounced in vitro relaxing effects of BRL in HIS-induced contractions and of combination (ROL+BRL) in ACH-induced contractions were observed, with similar results in vivo, and no significant change in the number of cough efforts was observed in any of the groups tested. The results suggest that PDE4 and PDE7 inhibitors have stronger anti-inflammatory effects compared with direct effects on smooth muscle and cough, with a potential benefit of their concomitant administration.

Polyphenols and Their Components in Experimental Allergic Asthma

The aim of the study was to investigate the potential anti-inflammatory effects in -experimental allergic asthma of natural polyphenolic compounds or their single major components. The experiment was performed after 21-days sensitization of guinea pigs with ovalbumin suspension. Changes in airway reactivity after the long-term treatment with the polyphenolic compounds Provinol and Flavin-7 and their single major components quercetin and resveratrol during were assessed using a whole body plethysmography. Reactivity of tracheal smooth muscle was studied in vitro in response to cumulative doses of the bronchoconstrictive mediators histamine and acetylcholine. Furthermore, concentrations of the inflammatory cytokines IL-4 and IL-5 were measured in bronchoalveolar lavage fluid. The results demonstrate significant anti-inflammatory effects of Provinol and Flavin-7 exerted in the airways. In contrast, chronic treatment with quercetin and resveratrol, single components of the two polyphenols, did not show such activity. We conclude that polyphenolic compounds are more effective in the anti-inflammatory effects in the airways than their separate components.

Echinacea complex--chemical view and anti-asthmatic profile.

ETHNOPHARMACOLOGICAL RELEVANCE Echinacea purpurea (L.) Moench is one of the mostly used herbs in the traditional medicine for the treatment of respiratory diseases. Modern interest in Echinacea is directed to its immunomodulatory activity. Recent studies have shown that secretion of asthma-related cytokines in the bronchial epithelial cells can be reversed by Echinacea preparations. AIM OF THE STUDY To examine the pharmacodynamics profile of Echinacea active principles, a complex has been isolated from its flowers by alkaline extraction and has been tested using an animal model of allergic asthma. MATERIAL AND METHODS The structural features of Echinacea purpurea complex was determined using chemical and spectroscopic methods. Allergic inflammation of the airways was induced by repetitive exposure of guinea pigs to ovalbumin. Echinacea complex was then administered 14 days in 50mg/kg b.w. daily dose perorally. Bronchodilatory effect was verified as decrease in the specific airway resistance (sRaw) in vivo and by reduced contraction amplitude (mN) of tracheal and pulmonary smooth muscle to cumulative concentrations of acetylcholine and histamine in vitro. The impact on mucociliary clearance evaluated measurement of ciliary beat frequency (CBF) in vitro using LabVIEW™ Software. Anti-inflammatory effect of Echinacea complex was verified by changes in exhaled NO levels and by Bio-Plex® assay of Th2 cytokine concentrations (IL-4, IL-5, IL-13 and TNF-alpha) in serum and bronchoalveolar lavage fluid (BALF). RESULTS Chemical and spectroscopic studies confirmed the presence of carbohydrates, phenolic compounds and proteins, as well as the dominance of rhamnogalacturonan and arabinogalactan moieties in Echinacea complex. The significant decrease in sRaw values and suppressed histamine and acetylcholine-induced contractile amplitude of isolated airways smooth muscle that were similar to effects of control drug salbutamol confirmed Echinacea complex bronchodilatory activity. The anti-inflammatory effect was comparable with that of control agent budesonide and was verified as significantly reduced exhaled NO levels and concentration of Th2 cytokines in serum and BALF. The values of CBF were changed only insignificantly on long-term administration of Echinacea complex suggested its minimal negative impact on mucociliary clearance. CONCLUSION Pharmacodynamic studies have confirmed significant bronchodilatory and anti-inflammatory effects of Echinacea complex that was similar to effects of classic synthetic drugs. Thus, results provide a scientific basis for the application of this herb in traditional medicine as a supplementary treatment of allergic disorders of the airways, such as asthma.

Effects of Flavin7 on allergen induced hyperreactivity of airways

Some studies have suggested that the polyphenolic compounds might reduce the occurrence of asthma symptoms. The aim of our experiments was to evaluate the effects of 21 days of the flavonoid Flavin7 administration on experimentally induced airway inflammation in ovalbumin-sensitized guinea pigs. We assessed tracheal smooth muscle reactivity by an in vitro muscle-strip method; changes in airway resistance by an in vivo plethysmographic method; histological picture of tracheal tissue; and the levels of interleukin 4 (IL-4), and interleukin 5 (IL-5) in bronchoalveolar lavage fluid (BALF). Histological investigation of tracheal tissue and the concentrations of the inflammatory cytokines IL-4 and IL-5 in BALF were used as indices of airway inflammation. Administration of Flavin7 caused a significant decrease of specific airway resistance after histamine nebulization and a decline in tracheal smooth muscle contraction amplitude in response to bronchoconstricting mediators. Flavin7 minimized the degree of inflammation estimated on the basis of eosinophil calculation and IL-4 and IL-5 concentrations. In conclusion, administration of Flavin7 showed bronchodilating and anti-inflammatory effects on allergen-induced airway inflammation.

Bronchodilatory, antitussive and anti-inflammatory effect of morin in the setting of experimentally induced allergic asthma.

Using an experimental model of allergic asthma, we evaluated the anti‐asthmatic potential of polyphenol flavonol derivate morin after either acute or long‐term treatment of male OVA‐sensitised guinea pigs.

Pulmonary surfactant in the airway physiology: a direct relaxing effect on the smooth muscle.

Beside alveoli, surface active material plays an important role in the airway physiology. In the upper airways it primarily serves in local defense. Lower airway surfactant stabilizes peripheral airways, provides the transport and defense, has barrier and anti-edematous functions, and possesses direct relaxant effect on the smooth muscle. We tested in vitro the effect of two surfactant preparations Curosurf® and Alveofact® on the precontracted smooth muscle of intra- and extra-pulmonary airways. Relaxation was more pronounced for lung tissue strip containing bronchial smooth muscle as the primary site of surfactant effect. The study does not confirm the participation of ATP-dependent potassium channels and cAMP-regulated epithelial chloride channels known as CFTR chloride channels, or nitric oxide involvement in contractile response of smooth muscle to surfactant.By controlling wall thickness and airway diameter, pulmonary surfactant is an important component of airway physiology. Thus, surfactant dysfunction may be included in pathophysiology of asthma, COPD, or other diseases with bronchial obstruction.

Potassium Ion Channels and Allergic Asthma

High-conductive calcium-sensitive potassium channels (BK+Ca) and ATP-sensitive potassium (K+ATP) channels play a significant role in the airway smooth muscle cell and goblet cell function, and cytokine production. The present study evaluated the therapeutic potential of BK+Ca and K+ATP openers, NS 1619 and pinacidil, respectively, in an experimental model of allergic inflammation. Airway allergic inflammation was induced with ovalbumine in guinea pigs during 21 days, which was followed by a 14-day treatment with BK+Ca and K+ATP openers. The outcome measures were airway smooth muscle cells reactivity in vivo and in vitro, cilia beating frequency and the level of exhaled NO (ENO), and the level of pro-inflammatory cytokines in the plasma and bronchoalveolar lavage fluid. The openers of both channels decreased airway smooth muscle cells reactivity, cilia beating frequency, and cytokine levels in the serum. Furthermore, NS1619 reduced ENO and inflammatory cells infiltration. The findings confirmed the presence of beneficial effects of BK+Ca and K+ATP openers on airway defence mechanisms. Although both openers dampened pro-inflammatory cytokines and mast cells infiltration, an evident anti-inflammatory effect was provided only by NS1619. Therefore, we conclude that particularly BK+Ca channels represent a promising new drug target in treatment of airways allergic inflammation.

Selective Inhibition of Phosphodiesterase 7 (PDE7) by BRL50481 in Healthy and Ovalbumin-Sensitized Guinea Pigs

Selective Inhibition of Phosphodiesterase 7 (PDE7) by BRL50481 in Healthy and Ovalbumin-Sensitized Guinea Pigs Phosphodiesterase (PDE) inhibitors may have significant clinical benefit in respiratory diseases associated with inflammation. The aim was to evaluate effects of selective PDE7 inhibitor (BRL50481) on citric acid induced cough, in vivo and in vitro airway smooth muscle reactivity in both healthy and ovalbumin sensitized guinea pigs, as well as its effectiveness in changes of blood cells count. Tested drugs were administered intraperitoneally to male guinea pigs once daily for 7 days either vehicle 10% DMSO (dimethyl sulfoxide) 3 ml/kg (as control) or BRL50481 1 mg/kg. Double chamber whole body plethysmograph was used for evaluation of citric acid (0.6 M) induced cough and specific airway resistance. Organ bath method was used for measurement of tracheal and lung tissue strips contractions evoked by cumulative doses (10-8 - 10-3 mol/L) of acetylcholine (ACH) and histamine (HIS). In healthy guinea pigs we did not observe significant effect of tested drug BRL50481 on in vitro contractions (similarly to in vivo conditions). The effect on cough was in healthy animals negligible. In ovalbumin-sensitized animals, more pronounced in vitro relaxing effect of BRL50481 in HIS induced contractions was observed with similar results in vivo and no significant change in number of cough efforts. Our results suggest that PDE7 inhibitors have stronger anti-inflammatory effect compared to its direct effect on smooth muscle and cough.

The effect of long-term administered CRAC channels blocker on the functions of respiratory epithelium in guinea pig allergic asthma model.

Previously, therapeutic potency of CRAC channels blocker was evidenced as a significant decrease in airway smooth muscle hyperreactivity, antitussive and anti-inflammatory effects. The major role of the respiratory epithelium in asthma pathogenesis was highlighted only recently and CRAC channels were proposed as the most significant route of Ca2+ entry into epithelial cells. The aim of the study was to analyse the impact of long-term administered CRAC channels blocker on airway epithelium, e.g. cytokine production and ciliary beat frequency (CBF) using an animal model of allergic asthma. Ovalbumin-induced allergic airway inflammation of guinea pigs was followed by long-term (14 days lasted) therapy by CRAC blocker (3-fluoropyridine-4-carboxylic acid, FPCA). The influence of long-term therapy on cytokines (IL-4, IL-5 and IL-13) in BALF and in plasma, immunohistochemical staining of pulmonary tissue (c-Fos positivity) and CBF in vitro were used for analysis. Decrease in cytokine levels and in c-Fos positivity confirmed an anti-inflammatory effect of long-term administered FPCA. Cytokine levels in BALF and distribution of c-Fos positivity suggested that FPCA was a more potent inhibitor of respiratory epithelium secretory functions than budesonide. FPCA and budesonide reduced CBF only insignificantly. All findings supported CRAC channels as promising target in the new strategy of antiasthmatic treatment.