A new variant of the basophil activation test for allergen-induced basophil CD63 upregulation. The effect of cetirizine.

OBJECTIVE: The aim of our study was to determine the diagnostic usefulness of a newly developed basophil activation test (BAT) in patients allergic to Dermatophagoides pteronyssinus and pollens. We also analyzed the influence of cetirizine on CD63 upregulation. This popular antihistamine strongly inhibits skin tests, but its impact on BAT sensitivity remains unknown and deserves at least preliminary determination. METHODS: The study sample comprised 22 patients allergic to house dust mite and pollens and 19 healthy controls. All participants underwent skin prick testing and the newly developed flow-cytometric basophil activation test. The protocol for allergen-induced basophil CD63 upregulation consisted of whole blood samples that were processed and stained with anti-CCR3/CD63 antibodies added to the buffer at the beginning of stimulation. Skin prick tests and BAT were performed twice--before and 2 hours after ingestion of 10 mg of cetirizine. RESULTS: The new BAT is characterized by its short processing time, easy basophil gating, and strong CD63 upregulation with very high sensitivity and excellent specificity. Our results suggest that allergen-induced CD63 upregulation by higher doses of allergens is not inhibited 2 hours after administration of cetirizine (unlike skin prick tests). CONCLUSION: The BAT is a very useful and precise method for the diagnosis of allergy to aeroallergens. It is not influenced by cetirizine.

[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.

Membrane proteins at the interface of erythrocytes fused by treatment with polyethylene glycol.

Fusion of human red cells through the action of polyethylene glycol gives rise to pairs or higher clusters with a common membrane envelope, in which a barrier at the position of the original interface can be seen in phase contrast. At early times this septum contains lipids, as judged by labelling with a fluorescent lipophile, and transmembrane protein; this was shown by the presence of the preponderant component, band 3, detected by a fluorescent label, covalently attached before fusion at an extracellular site, or by immunofluorescence with anti-band 3 antibody. Ankyrin, which is bound to band 3, is also observed in the septum. The lipid thereafter disappears from the interface, carrying most of the band 3 with it. A continuous membrane skeletal network, defined by the presence of spectrin (detected by immunofluorescent staining in epifluorescence and confocal microscopy) appears to persist for long periods, but in many cells interruptions develop in the septum. In other fused pairs, particularly at longer times, the interface is seen to have vanished completely. Protease inhibitors have no discernible effect on any of these observations. The results suggest a model for the events that follow fusion. Covalent cross-linking of membrane proteins beyond a critical level causes inhibition of fusion, suggesting that proteins, probably the membrane skeletal network, regulate the fusion process. The efficiency of fusion is strikingly dependent on the composition of the isotonic medium, being relatively high at an orthophosphate concentration of 5 mM and minimal at 20 mM.

Comparative study between skin prick tests and TOP-CAST allergen leukocyte stimulation in diagnosis of allergic status.

A recently developed CAST-ELISA method was applied to determine allergen-induced leukocyte stimulation. This method is based on the measurement of sulfidoleukotriene secretion by peripheral blood leukocytes previously stimulated with specific allergen in the presence of interleukin 3. 24 patients allergic to different aeroallergens and some food allergens were included in this study. Subjects received no medication 2 weeks before the testing. Leukocytes were isolated by dextran sedimentation. After incubation with allergen at two concentrations, the cells were centrifuged and the concentration of sulfidoleukotrienes in supernatant was determined. The results were expressed in pg/ml after subtraction of values of spontaneous sulfidoleukotriene production in portions incubated without allergen. We observed a wide range of sulfidoleukotriene secretion upon allergen stimulation. Concentrations of leukotrienes ranged from 0 to 5780 pg/ml at lower allergen concentration and from 210 to 5680 at higher allergen concentration. On the basis of observed results, we conclude that the better allergen concentration is the higher one because there was no appearance of negative cell stimulation. In the lower allergen concentration we observed negative results in two cases. Sixteen healthy control subjects were also included in this study. Eleven subjects had negative skin prick tests (SPT) as well as CAST results. In 5 healthy subjects with positive SPT, we also observed positive CAST results in 4 persons. In 1 healthy person with positive SPT results, CAST results were negative. We conclude that TOP-CAST allergen is a valuable mixture of different aero and food allergens for determining the allergic status in patients with suspicious allergic status, although it cannot differentiate between health and disease states.

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.

CAST-ELISA test in diagnosis of mite allergy.

The recently developed CAST-ELISA method was applied to determine allergen-induced leukocyte stimulation. This method is based on the measurement of sulfidoleukotriene secretion by peripheral blood leukocytes stimulated with specific allergen in the presence of interleukin 3.23 patients allergic to Dermatophagoides pteronyssinus were included in this study. All subjects received no medication 2 weeks before the testing. Leukocytes were isolated by dextran sedimentation. After incubation with allergen the cells were centrifuged and the concentration of sulfidoleukotrienes in supernatant was determined. The results were expressed in pg/ml after subtracting the values of spontaneous sulfidoleukotriene production in portions incubated without allergen. We observed a wide range of sulfidoleukotriene secretion upon allergen stimulation. Concentrations of leukotrienes ranged from 0 to 3440 pg/ml. The mean level in the whole group was 1521.3 pg/ml at the optimal allergen concentration. Positive results were observed in 21 subjects. In 2 subjects we could not demonstrate leukotriene secretion by stimulation with allergen. 15 healthy control subjects were also included in this study. 12 subjects had negative skin prick tests (SPT) as well as CAST results. In 2 healthy subjects with positive SPT, we also observed positive CAST results. However, leukotriene secretion was lower than 300 pg/ml in these subjects. We conclude that CAST-ELISA is a valuable method for determining the allergic status in patients with mite allergy.

[Spontaneous histamine release by basophils and bronchial hyperreactivity in patients with hay fever]. / Spontaniczne uwalnianie histaminy z bazofilów oraz nadreaktywnosc oskrzeli u chorych na pylkowice.

Basophil spontaneous histamine release test was performed in seventeen patients suffered from seasonal allergic rhinitis before, 2 weeks after immunotherapy and during pollen season. During pollen season at the same day non-specific bronchial hyperreactivity to histamine was estimated. Values of spontaneous basophil histamine release before immunotherapy were not different from control group but significantly increased after immunotherapy and during pollen season. There was no correlation between degree of nonspecific bronchial hyperreactivity and values of spontaneous histamine release from basophils.

Specific pollen allergen activates eosinophils of the patient with chronic allergic contact urticaria.

The aim of the study was to evaluate the activation of eosinophils in an unique case of a young man with atopy manifested as chronic pollen contact urticaria. In order to reveal the role of eosinophils in that case, the study was performed by means of monoclonal antibodies EG2 and chemiluminescence. In addition, comparative electron microscopic study of peripheral blood and skin infiltrating eosinophils were performed for which the name ultrastructural morphometric analysis of intracytoplasmic eosinophil granules has been proposed. The results indicated, that 40% of peripheral blood eosinophils were activated spontaneously and they were more active than those in skin infiltrates. Specific pollen allergen caused activation of 100% of peripheral blood eosinophils. The study suggests presence of a systemic pattern of eosinophil activation in atopy.

The behavior of T and B lymphocyte subpopulations in infectious and atopic bronchial asthma.

In 40 patients with infectious bronchial asthma and in 40 patients with atopic form of the disease, the behavior of T and B lymphocyte populations was studied by the E and EAC rosette tests. The control group consisted of 20 students unaffected by allergic diseases. A significant decrease in the percentage of E rosette forming T lymphocytes was found in the group of patients with atopic bronchial asthma, as compared with the control and infectious bronchial asthma groups (p less than 0.001). The proportions of EAC forming B lymphocytes showed no significant differences in all groups under examination.

Effect of histamine on the spontaneous E rosette formation after blockade of histamine H2 receptors with cimetidine.

Thirteen patients with clinical diagnosis of hay fever, were examined. T lymphocytes subpopulation was determined by the E rosette test. The present study is concerned with the effect of histamine on the ability of T lymphocytes for E rosette formation after blockade of the histamine H2 receptor with its inhibitor cimetidine. It has been found that histamine effect is expressed by the histamine H2 receptor, as its blockade with cimetidine leads to the failure of histamine to inhibit rosette formation by T lymphocytes.

T and B lymphocytes in patients with hay fever.

In 20 patients with hay fever and 19 control subjects the rosette formation test was carried out to determine T and B lymphocyte subpopulations. T and B lymphocyte subpopulations are expressed by the percentage of E, EA and EAC rosettes, respectively. Statistical analysis revealed a significant reduction in the number of E rosettes (p < 0.001), and a slight, but statistically significant decrease in the number of EAC rosettes (p < 0.05) in hay fever patients.