Production of leukotriene C4 by peripheral blood leukocytes stimulated with anti-fcepsilonRI antibody, PMA, and fMLP does not correlate with irreversible airway obstruction in asthmatics.

BACKGROUND: Airway remodeling has recently emerged as a major problem in an increasing percentage of patients with asthma. Reasons for great diversity in the progression of irreversible bronchoconstriction among asthmatics remain unclear. OBJECTIVE: The aim of this study was to assess whether the potential ability of leukocytes to produce cysteinyl leukotrienes in response to various stimuli is correlated with magnitude of irreversible airway obstruction in asthmatics. MATERIALS AND METHODS: The study sample comprised 76 asthmatics (34 males, mean +/- SD age 52 +/- 13 years), and 35 healthy controls (18 males, 38.2 +/- 15 years). Each subject underwent 2 pulmonary function tests: before and after bronchodilator administration. In addition, approximate annual decline in forced expiratory volume in 1 second (FEV1) (% of predicted) was calculated. Leukotriene C4 (LTC4) production was assessed combining a cellular antigen stimulation test and enzyme-linked immunosorbent assay using Bulhmann Laboratories AG kits. Leukocytes isolated from peripheral blood were stimulated with anti-FcepsilonRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). In separate tubes each subject's leukocytes were tested for spontaneous LTC4 production. Finally, stimulated LTC4 production was expressed in pg/mL after subtraction of values of spontaneous production. RESULTS: In asthmatics, baseline FVC% and FEV% values ranged from 24.4% to 122.4% (mean, 75.5%) and from 23.4% to 126.6% (mean, 74.4%), respectively. There were no significant differences between asthmatics and controls in LTC4 production stimulated by anti-FcepsilonRI antibody (P = .79), fMLP (P = .33) or PMA (P = .86). We found no correlation between stimulated LTC4 production and spirometric parameters at baseline or after bronchodilator administration or annual decline in FEV1%. CONCLUSION: Our results do not confirm the hypothesis that airway remodeling in asthma might be related to enhanced ability of leukocytes to produce cysteinyl leukotrienes in response to various stimuli.

Relevance of the selected cytokine release (TNF-alpha, IL-6, IFN-gamma, and IFN-alpha) to the exacerbation of bronchial asthma from airway mycotic infections. Predominant role of TFN-alpha?

Airway fungal infections are often associated in asthmatics with the exacerbation of asthma symptoms. However, the pathomechanism of this phenomenon has not been fully understood. The aim of our study was to assess whether antimycotic treatment can influence the capacity of bronchoalveolar (BAL) leukocytes to release proinflammatory cytokines, which could contribute to increase in asthma severity. Ten patients with bronchial asthma complicated by airway fungal infections (Candida albicans and/or Aspergillus fumigatus) were included in the study. Seven asthmatics were treated with systemic and inhaled corticosteroids, whereas the remaining three with inhaled ones only. All subjects underwent several courses of therapy with antibiotics due to respiratory infections. BAL leukocytes obtained from the patients were cultured in the absence or presence of lipopolysaccharide E.coli (LPS) or Newcastle disease virus (NDV). The BAL procedure and measurement of the levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (II-6), interferon-gamma (IFN-gamma), and interferon-alpha (IFN-alpha) by specific bioassays were performed twice: before antimycotic treatment and after 3 weeks of therapy with 8 mg of nebulized fluoconazole and 400 mg of oral ketoconazole per day. The elimination of fungi from respiratory tract resulted in an apparent clinical improvement. This coincided with diminished production of TNF-alpha in response to LPS and the production of IFN-alpha in response to NDV, which were initially high and subsided significantly after antimycotic therapy (p = 0.035, and 0.011, respectively). Such changes were not observed in the case of IFN-gamma and IL-6. This may suggest that TNF-alpha as well as IFN-alpha are secreted by fungi-prestimulated leukocytes from the lower respiratory tract and may be involved in the processes of exacerbation of asthma complicated by fungal infections. Further analyses of relationships between changes in cytokine levels and clinical parameters indicated that IFN-alpha seems to be of particular interest in fungal stimulation of asthma.

Skin prick test response to enzyme enolase of the baker's yeast (Saccharomyces cerevisiae) in diagnosis of respiratory allergy.

BACKGROUND: The aim of the study is to prove that Saccharomyces cerevisiae enolase, the major allergen of the baker's yeast, induces allergic immediate response in patients with inhalant allergy sensitized to Candida albicans extract. MATERIAL AND METHODS: The study was performed in three groups of patients: I. 20 atopic patients with respiratory allergy sensitized to Candida albicans and inhalant allergens (mite, feather, pollens) II. 30 patients with respiratory allergy, positive skin tests to inhalant allergens but negative skin tests to Candida albicans and other fungi; III. 20 nonatopic, healthy individuals. Skin prick test of purified enolase from Saccharomyces cerevisiae (bakers yeast) at concentration 1 and 10 mg/ml was performed in all groups. The results were documented planimetrically. RESULTS: 95% of patients sensitized to Candida albicans extract showed positive skin reactions to Saccharomyces cerevisiae enolase, 10% of patients of group II and none of the patients of the control group had positive skin responses to enolase. The mean wheal size (mm2) in skin prick test to Candida albicans, Saccharomyces cerevisiae enolase at concentration 10 mg/ml was x = 15.17 +/- 11.08, 15.76 +/- 19.67 and at concentration 1 mg/ml 10.02 +/- 10.49, respectively. CONCLUSIONS: 1. Saccharomyces cerevisiae enolase induces an immediate allergic reaction in skin in subjects with respiratory allergy and positive skin prick test results to Candida albicans and other fungi. 2. Enolase can be an important allergenic component of the Candida albicans extract.

[Bronchial hyperreactivity to histamine and levels of serum eosinophil cationic protein in patients with non-atopic asthma]. / Nadreaktywnosc oskrzeli na histamine a stezenie eozynofilowego bialka kationowego w surowicy chorych na astme oskrzelowa nieatopowa.

Pathogenesis of non-atopic bronchial asthma remains still an open question. Until now there have been very few studies concerning relationship between eosinophil activation and nonspecific bronchial hyperreactivity to histamine in this form of asthma. Sixteen subjects with mild nonatopic bronchial asthma entered the study. Evaluations of PC 20 for histamine and serum eosinophil cationic protein (ECP) concentration have been performed in all patients. In spite of fact that all patients were considered as mild asthmatic we have observed wide range of PC 20 and serum ECP concentration. Moreover we were able to find statistically significant inverse correlation between PC 20 for histamine and serum eosinophil cationic protein concentration: r = -0.498, p < 0.05. We conclude that eosinophil activation plays an important role in pathophysiology of nonspecific bronchial hyperreactivity in nonatopic bronchial asthma.

[Validation of the Polish version of St. George's respiratory questionnaire in patients with bronchial asthma]. / Ocena polskiej wersji St. George's respiratory questionnaire u chorych na astme oskrzelowa.

St George's Respiratory Questionnaire (SGRQ) has been widely used in the assessment of health related quality of life (HRQOL) in patients with asthma and chronic obstructive pulmonary disease. Introduction of the new language version of the HRQOL questionnaire needs to be preceded by a highly structured process of validation. We aimed to validate the Polish version of SGRQ in the group of 83 patients with asthma. Following the comprehension study, we thus evaluated reliability, validity, reproducibility, responsiveness, and measurement equivalence of the Polish version of SGRQ. Disease severity and health status were also concurrently assessed. The reliability was good, with Cronbach's alpha coefficient exceeding 0.75 for global and all subscale scores. There have been highly significant correlations between spirometric parameters, intensity of symptoms, health status self-assessment, and the degree of depression, and quality of life scores. Reproducibility, stability and responsiveness were confirmed in the follow-up study. Minimal clinically important difference was found to be 5.3 points. Polish version of SGRQ was found to be psychometrically equivalent to four other versions of SGRQ, which underscores its validity in the population of Polish asthmatics.

Bacterial lipopolysaccharide-induced sulfidoleukotriene release from peripheral blood leukocytes in patients with asthma and chronic obstructive pulmonary disease.

Bacterial endotoxins are seen to possess strong proinflammatory activities. These substances may intensify inflammation in the airways of patients with chronic obstructive pulmonary disease (COPD) and asthma by facilitating release of various mediators from different types of cells. Sulfidoleukotrienes (sLT) cause bronchoconstriction, increase vascular permeability and stimulate mucous secretion. The aim of our study was to evaluate sLT release from peripheral blood leukocytes stimulated by Klebsiella pneumoniae lipopolysaccharide (LPS) and obtained from COPD and asthma patients. Nineteen subjects with mild or moderate stable bronchial asthma, nine patients with COPD and 10 healthy controls entered the study. Cellular allergen stimulation test (CAST)-ELISA test was performed using Bühlmann Laboratories AG kits to determine sLT production. The differences between atopic (462.57 SD = 215.89 pg/ml) and nonatopic (474.25 SD = 158.02 pg/ml) asthmatics in comparison to healthy controls (191.55 SD = 53.2 pg/ml) were statistically significant (p < 0.005) upon LPS stimulation at the concentration of 10 micrograms/ml. At lower LPS concentration (1 microgram/ml) the difference was statistically significant only between nonatopic asthmatics and healthy subjects (p < 0.02). In the COPD group the sLT production in either LPS concentration was higher than in the controls but the difference was not significant. We suppose that leukocytes obtained from asthmatics and COPD patients are more susceptible to LPS than these cells from healthy individuals. An increased sLT production upon LPS stimulation during respiratory bacterial infection may intensify inflammation, bronchoconstriction and increase nonspecific bronchial hyperreactivity.

TNF-alpha, IL-6 and IFN-gamma secreted by bronchoalveolar leukocytes isolated from patients with bronchial asthma, complicated by fungal airways infections.

It is widely known that fungal airways infections may deteriorate the course of bronchial asthma. The mechanism of the phenomenon is still unclear. The aim of our study was to assess the effect of fungal infections on the secretion of selected cytokines by bronchoalveolar leukocytes. Five patients (group FA) with bronchial asthma and Candida albicans or Aspergillus fumigatus airways infections (confirmed by bronchoscopy and culture) were included in the study. All of them were on the chronic treatment with corticosteroids (10-20 mg of prednisone per day) and underwent several courses of therapy with antibiotics. The control groups comprised 5 previously untreated asthmatics without bronchial colonization with fungi (group A) as well as 5 healthy volunteers (group H). Leukocytes were isolated from bronchoalveolar lavage fluid (BALF) and cultured in the presence or absence of cytokine inducers such as phytohemagglutin L (PHA), lipo-polysaccharide (LPS) from E. coli. The activity of TNF-alpha, IL-6 and IFN-gamma were measured in the BAL cell culture supernatants by using specific bioassays. In comparison with healthy controls the spontaneous or induced secretion of cytokines were significantly augmented in patients from group A. In contrast the asthmatics who represented group FA demonstrated normal levels of spontaneous cytokine secretion. However, the tendency to increase LPS and PHA-induced production was observed in BAL leukocytes from the patients. The above results support the view that beneficial effect of corticosteroid treatment in bronchial asthma may act, at least in part, by inhibition of the high spontaneous secretion of proinflammatory cytokines. Nevertheless, fungal airways infections may lead to increase of LPS- or PHA-induced production of TNF-alpha, IL-6 or IFN-gamma (despite prednisone therapy) by prestimulation of the BAL cells with fungi.

CAST-ELISA test--a new diagnostic tool in pollen allergy.

We have applied a recently developed method called CAST-ELISA to evaluate the degree of leukocyte stimulation by specific allergen. This method is based on the measurement of sulfidoleukotriene levels in supernatants taken from previously stimulated peripheral blood leukocytes by specific allergen in the presence of interleukin 3.23 patients with pollinosis entered the study. All of them received no medication during 2 weeks before the test. Leukocytes were isolated by dextran sedimentation followed by single centrifugation. After removal of platelet rich plasma the cells were suspended in stimulation buffer and divided into portions incubated with or without specific allergen for 40 minutes at 37 degrees C. After the incubation, the cells were centrifuged and the evaluation of sulfidoleukotrienes in supernatant was performed. The results were expressed in pg/ml after subtraction of the value of spontaneous sulfidoleukotriene production in portions incubated without allergen. The concentration higher than 200 pg/ml of sulfidoleukotrienes above spontaneous production was regarded as a positive result. We have observed a large spectrum of the leukocyte response upon allergen stimulation. In the initial part of our study we established the optimal allergen concentration. The concentration of sulfidoleukotrienes in supernatants ranged from 10 to 5130 pg/ml. The mean sulfidoleukotriene concentration in the whole group was 1671.69. Positive results were observed in 20 persons. In 3 persons the results of allergen stimulation were negative. We conclude that CAST-ELISA is a reliable method to determine the allergic status in persons with pollinosis.

Is mast cell activation during asthmatic reaction reflected in the circulation?

Allergic asthmatic patients were challenged with specific allergen that resulted in early asthmatic reaction (EAR). Serum tryptase concentration (STC) and neutrophil chemotactic activity (NCA) were measured before and during EAR. A significant increase in neutrophil chemotactic activity was noticed in the 60th min, without an accompanying increase in serum tryptase concentration. The rise in neutrophil chemotactic activity in our study confirms previous observations in this field. However, because of the numerous cellular sources for neutrophil chemotactic activity, it does not seem to be a gold standard for measurement of mast cell activation. Undetectable levels of serum tryptase concentration during EAR in our study do not exclude the role of the mast cell in its pathogenesis. To our knowledge, only massive mast cell degranulation is reflected in the circulation as an increased tryptase concentration. In the case of upper respiratory allergic reactions, mast cell degranulation definitely takes place, but does not result in evident changes in serum tryptase concentration. We conclude that mast cell activation during allergen-induced EAR is poorly represented in the circulation.

[Concentration of eosinophil cationic protein in blood serum during early and late asthmatic reaction]. / Stezenie eozynofilowego bialka kationowego w surowicy podczas wczesnej i póznej reakcji astmatycznej.

Eosinophil cationic protein (ECP) was measured in blood serum of 15 atopic asthmatics during early (EAR) and late (LAR) asthmatic reaction triggered by specific allergen provocation. Nonspecific bronchial hyperreactivity was evaluated in histamine provocation test before and 48 hours after the allergen challenge. We observed dual asthmatic reaction (DAR) in 8 and an isolated EAR in 7 patients. The ECP serum level and nonspecific bronchial hyperreactivity were significantly higher (p < 0.05) in the DAR group when compared to EAR responders. An inverse correlation between PC20 for histamine and ECP level was shown before and after the allergen challenge in all examined subjects (R = -0.5472, p < 0.05).

[Peripheral blood eosinophilia in patients with bronchial asthma before and after bronchial provocation]. / Eozynofilia krwi obwodowej u chorych na astme oskrzelowa przed i po swoistej prowokacji oskrzeli.

The study was set up in order to check the usefulness of peripheral blood eosinophilia in asthma diagnosis and asthma therapy monitoring. Twenty mild asthmatics entered the study--ten atopic and ten nonatopic. Eosinophilia was estimated twice: on the day of admission and twenty four hours after bronchial provocation. Bronchial hyperreactivity was measured on both occasions. We showed, that there was no difference in eosinophilia between atopic and nonatopic subjects before provocation but the difference was significant 24 hrs after provocation. In both groups of asthmatics eosinophilia correlated with bronchial hyperreactivity before and after provocation. We concluded, that eosinophilia is an easy and valuable parameter in monitoring the degree of allergic inflammation in asthmatics.

Synthesis and pharmacological properties of derivatives of alpha-amino-beta-(p-chlorobenzoyl)-propionic acid and alpha-amino-gamma-(p-chlorophenyl)-tetrahydrofuran-2-one.

Several new alpha-aminoderivatives of gamma-(p-chlorophenyl)-tetrahydrofuran-2-one were synthesized. alpha-Aminoderivatives of beta-(p-chlorobenzoyl)-propionic acid 2-13 were used as the substrates. After the reduction with NaBH4 at 10-12 degrees C and cyclization the compounds were converted into the appropriate derivatives of tetrahydrofuran-2-one 16-26. In pharmacological tests compounds 9 and 26 abolished the aggressiveness in isolated mice while compound 8 showed antiinflammatory activity.

Studies on derivatives of 2-amino-4-p-chlorophenylthiazole-5-acetic acid. V. Syntheses of 2-N-aralkylidene and 2-N-aralkyl derivatives of 2-amino-4-p-chlorophenylthiazole-5-acetic acid.

A number of new N-aralkylidene and N-aralkyl derivatives of amides and hydrazides of 2-amino-4-p-chlorophenylthiazole-5-acetic acid (III-XXIX) have been synthesized. These compounds were obtained in two various ways depending on the character of substituents in the carboxyl group. The chemical structure of the newly obtained compounds was determined on the basis of the data of elementary and spectral analyses.

Synthesis of derivatives of 2-amino-4-p-chlorophenylthiazole-5-acetic acid.

In the course of studies on compounds with expected antiinflammatory and immunosuppressive action a number of new derivatives of 2-amino-4-p-chlorophenylthiazole-s-acetic acid have been synthetized. The chemical structure of these compounds has been confirmed by elemental analysis and IR spectra. The newly obtained compounds were investigated pharmacologically. It has been found that some of the derivatives possess antiinflammatory and immunosuppressive activity.