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SUMMARYThis study was carried out at Chest and Pathology Departments, Faculty of Medicine, Zagazig University, during the period from March 2003 to March 2005. It included 60 subjects who were classified into two groups, (50) asthmatics who were randomly selected from the outpatients attendants and (10) control volunteer subjects.The aim of this work was evaluation of the role and the possible significance of local eosinophil apoptosis in bronchial asthma patients.? All subjects were submitted to the following :(1) Thorough medical history.(2) Full clinical examination, general and local chest examinations.(3) Plain chest X-ray postero-anterior and lateral views to exclude any associated radiological abnormalities.(4) Laboratory investigations :A- Hematological assessmentTotal leucocytic count (TLC) and differential leucocytic cell counts were assessed.(B) Arterial Blood Gases (ABGs) analysis(5) Spirometric pulmonary function testing :-This testing was done for all patients. It was carried out using computerized pulmonary function device (ZAN 1100, computerized pulmonary function apparatus) before and 20 minutes after inhalation of 200 ?g (2 puffs) salbutamol using MDI and spacer to measure : FEV1 % of predicted, FEV1/FVC% and FEF25-75% and reversibility of FEV1% ? 15%.(6) Skin prick testing :-Allergens used : Extracts of house dust, cotton dust, mixed pollens, mixed fungi, wool, feather, horse hair, cat hair, rabbit hair and dog hair.(7) Sputum induction, by hypertonic saline 2.7%(8) Measurement of BHR, by methacholine challenge test(9) Bronchoscopy and bronchial biopsy :Using fibroptic bronchoscopy (Olympus model BF type 20).• Two endobronchial mucosal biopsies were taken between carina and the right lower lobes using bronchoscopic forceps• Processing of bronchial mucosal samples.All bronchial mucosal samples were fixed in 10% formalin solution at room temperature. In the laboratory, the samples were embedded in paraffin to form paraffin block. Paraffin blocks were transected to form sections containing the biopsies. The sections were stained with :A- Chromtrope 2R stain Preparation :Melt 10 g phenol crystals by warming the flash under a hot water tap. Add 5 g Chromtrope 2R and mix with phenol. Dissolve the mixture in 1000 cm3 distilled water.B- Hematoxylin and Eosin, for the demonstration of the parenchymal and inflammatory cells.(10) Apoptotic cells can be counted versus total eosinophils according to morphological features of apoptosis1- Cell shrinkage2- Nuclear coalescence (shift from bilobbed to monolobbed nucleus).3- Nuclear condensation and extrusionThe results of this study showed the following:1. Ther was a highly significant difference between atopic and non atopic asthmatics regarding the age in years (p<0.001).2. The most prevalent respiratory symptoms among asthmatic patients were chest wheezes (82%), dyspnea (78%), cough (72%) and expectoration (30%) only.3. The ventilatory function parameters (FEV1 % of prediction, FVC% of prediction, FEV1/FVC% and FEF25-75%) showed lower values and higher significant difference between asthmatic and control groups (p <0.001). But there was a non significant difference between atopic and non atopic asthmatics as regards these ventilatory functions.4. As regards arterial blood gas parameters, only PaO2 was less than normal range in patients (79.16 mmHg) and showed a highly significant difference when compared with control subjects (p<0.001), also pH, PaCO2, and SaO2% (p< 0.01, 0.05, and 0.01) respectively, only HCO3 serum level showed nearly no difference between the two groups (p > 0.05).5. Eosinophil blood cell count was significantly higher in asthmatics than control group (p<0.001), the rest of measured blood picture (TLC, RBCs and Hb) showed a non significant difference between asthmatic and control groups (p > 0.05).6. There was statistically significant increase in eosinophilic tissue load in tissue biopsies in asthmatics than in controls (p<0.05), but there was a non significant difference as regards apoptotic cells and index in tissue biopsies between both groups (p>0.05).7. As regards eosinophil count, apoptotic cells and apoptotic index in sputum samples, there was a significant difference between both groups in all that parameters (p< 0.01 ,0.01, and 0.05) respectively.8. When eosinophils ,apoptotic cells and index in bronchial biopsies and sputum were compared between controls and asthmatic subgroups (intermittent, mild persistent and moderate persistent) the previous items were significantly increased.9. There was a significant positive correlation between asthma severity and eosinophil load in bronchial biopsies (p<0.001), with no significant correlation as regard apoptotic cells and index compared with grades of severity.10. In sputum samples, asthma severity correlated positively with eosinophil count and apoptosis ,and negatively with apoptotic index.11. Evaluation of correlation between eosinophil cells and their apoptosis in bronchial biopsies and sputum samples in asthmatic subgroups showed a higher significant positive correlation in all subgroups in both specimens except in intermittent asthmatics as regards bronchial biopsies.12. There was a positive significant correlation between tissue load eosinophils and eosinophils count in sputum (p<0.001),also that result detected between apoptotic indices in bronchial biopsies and sputum (p<0.001), an inverse correlation between eosinophils count in bronchial biopsies and apoptotic index in sputum was found (p<0.001).13. There was a significant positive correlation between apoptotic cells and both eosinophil cell count and apoptotic index in bronchial biopsies (p < 0.001, 0.01) respectively.14. Eosinophils count in sputum showed a positive significant correlation with apoptotic cells (p<0.001), and negative significant correlation with apoptotic index (p<0.001) in sputum samples in asthmatic group.15. Bronchial hyperresponsiveness, which measured by methacholine showed a higher significant difference between asthmatic and control groups (p<0.001) as regard PC20 (mg/ml).16. Methacholine challenge test (PC20 mg/ml) showed a significant inverse association with eosinophils count either in biopsies or sputum (p<0.01).17. PC20 (mg/ml) showed a significant positive correlation with all measured ventilatory function parameters (FEV1% of peridected, FVC% of peridected, FEV1/FVC% and FEF25-75%), p values were (<0.001, 0.05, 0.01 and 0.001) respectively.18. A decreased eosinophil count in samples of (blood, bronchial biopsies and sputum) in corticosteroids users than non users in mild asthmatics, and increased apoptotic index in them in bronchial biopsies and sputum samples was evident but without significant difference.19. There was inverse correlation between eosinophils count in bronchial biopsies and ventilatory function parameters (FEV1% of peridected, FVC% of peridected, FEV1/FVC% and FEF25-75%), and significant positive correlation in bronchial biopsies between apoptotic index and ventilatory functions (p < 0.05).20. Also in sputum, a similar results were detected between ventilatory function parameters and eosinophil count and apoptotic index, with a higher significance in sputum than in bronchial biopsies.21. As regard the comparison between atopic and non atopic asthmatics in eosinophils count in blood, bronchial biopsies and sputum, and their apoptosis and apoptotic index in bronchial biopsies and sputum, it showed a non significant difference ,with higher number of eosinophils in all specimens .22. There was a significant positive correlation between eosinophils count in (blood, bronchial biopsies and sputum) and duration of the disease in asthmatic patients.23. A non significant correlation was detected between age of patients in years and eosinophils counts and their apoptosis either in bronchial biopsies or sputum.CONCLUSIONS1- Enhancement of eosinophil apoptosis may play an important role in the resolution of inflammation in bronchial asthma patients.2- The use of ICS may enhance the eosinophil apoptosis in bronchial asthma patients.3- Measuring sputum eosinophils may provide a useful additional information for disease monitoring.4- Local eosinophils count and simple ventilatory function indices could represent a clear idea about the methacholine challenge test.RECOMMENDATION• Multicentric and wide-scale prospective studies about eosinophil apoptosis must be done, searching for possible mechanisms and medications that enhance and maintain apoptosis esinophils in bronchial asthma patients.
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