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Notes: Blood eosinophils, and serum levels of the eosinophil proteins, eosinophil cationic protein (ECP) and eosinophil protein X (EPX) were measured in childhood asthma. Seventeen patients mean age 11–9 years who were symptomatic with asthma, were enrolled in a study examining the eosinophil counts and eosinophil proteins at the onset of study and after treatment in relation to changes in their baseline forced expiratory volume at 1 second (FEV1) and % predicted FEV1. The patients with symptomatic asthma were compared with 17 patients mean age 12.0 years with asymptomatic asthma maintained on daily inhaled steroid and 13 patients, mean age 12.0 years, without asthma but with urticaria who served as non-asthma controls. Patients with symptomatic asthma did not have significantly higher initial eosinophil counts compared with those with asymptomatic asthma (0.43 × 109/1 vs 0.26 × 109/1, P= 0.09) but had higher serum ECP levels (28.9 μ/1 vs 18.5 μg/1). Both asthma patient groups had significantly higher serum ECP levels (P〈0.01) than the controls (9.8 μg/1). After therapy consisting of increased dose of inhaled steroids and/or oral steroids, patients in the symptomatic asthma group demonstrated a significant rise in FEV1 (1.67 1/sec at Visit 1 vs 2.08 1/ sec at Visit 2, 1〈0.01). A similar rise was seen for % predicted FEV1. Patients in the asymptomatic asthma group showed no significant change in FEV1 between visits (2.23 1/sec vs 2.37 1/sec), which was verified with the % predicted FEV1, Patients in the symptomatic asthma group showed a significant decrease in ECP level following treatment (28.9 μ/1 to 9.6 μ/1. P〈0.001) while the values in the asymptomatic group did not change (18.6 μ/1 to 15.2 μ/1 not significant). There was a significant correlation between the initial ECP level in the symptomatic asthma group and the change in the FEV| with treatment. Serum EPX levels showed similar trends but there was no significant correlation between the initial EPX levels and the changes in FEV1. Neither did blood eosinophil counts show such a correlation. This data suggests that the changes in serum ECP levels correlate with the changes in lung function subsequently to anti-inflammation therapy.

Notes: A radio immunoassay was developed allowing measurement of the cytotoxic cationic ECP. The assay, which has a total incubation time of 3.5 hr, is a double antibody assay with radiolabelled ECP. covering the concentration range of 2–200 μ/l. Performance data show a detection limit of 〈 2 μg/1 and a cross-reactivity with eosinophil protein X (EPX/EDN) of 〈0.06%. The coefficient of variation (%) within the measuring range was, within assay 4.8–10.4, and total 6.6–12.0. The assay is useful for measurement in various body fluids including serum, nasal secretions and bronchoalveolar lavage fluid, and dilution of samples prior to analysts was generally not required. Sera from 100 apparently healthy individuals revealed a geometric mean of 6.0 μg ECP/1 and a range (95%) of 2.3–15.9 μg/1. The elimination rate of ECP, t12. in vivo was estimated to be 65 min when ECP was measured in serum. Comparisons between this assay and a method previously described showed that the new method is superior with regard to precision and assay procedure.

Notes: Background and Objectives Eosinophils are associated with bronchial asthma, but the role of the eosinophil is not fully understood. This study was initiated in order to study the influence of endogenous cortisol on eosinophil recruitment and activation in allergic inflammation in the lower airways in the pig.Methods Polyclonal and monoclonal antibodies against porcine eosinophil peroxidase (EPO) were raised. Detection of eosinophils in blood smears and lung biopsy specimens was achieved using the polyclonal antibody. For determination of porcine EPO in bronchoalveoiar lavage (BAL) fluid, a sandwich enzyme-linked immuno-sorbent assay (ELISA) with a detection limit of 0.15μig/L was developed. No cross-reactivity with porcine myeloperoxidase was found. Pigs that had been actively sensitized with repeated subcutaneous injections of Ascaris suum antigen were acutely challenged with antigen in the lower airways under pentobarbitone anaesthesia.Results Control animals with plasma cortisol levels of approximately 400 nM did not exhibit infiltration of eosinophils into lung parenchyma or EPO-release in the bronchial lumen within 8 h after challenge. However, in pigs treated with a cortisol-synthesis inhibitor (metyrapone), resulting in plasma cortisol levels of approximately 40 nM, there was a marked eosinophil infiltration into lung tissue at 8 h. Furthermore, EPO levels in BAL fluid were increased in some, although not all, low-cortisol animals. There was no infiltration of eosinophils into skin tissue in these animals.Conclusions It is concluded that, after allergen challenge in the lower airways of metyrapone-treated pigs, newly recruited eosinophils infiltrate lung tissue specifically. Furthermore, a cortisol-sensitive release of the eosinophil-derived cationic protein EPO, into the bronchial lumen was established. This is, to our knowledge, the first description of direct measurements of the release of an eosinophil granule protein in a large animal model of allergy.

Notes: Background Eosinophil peroxidase (EPO) is an eosinophilic basic protein, which leads to increased permeability and damage of bronchial epithelial cells in asthma.Objective As little is known about its local expression and release in humans the intracellular expression in lung and peripheral eosinophils and the concentrations of EPO in bronchoalveolar lavage (BAL) fluid and serum was investigated in patients with asthma.Methods Twelve mild atopic asthmatic and nine control subjects underwent segmental sham and allergen challenge. EPO concentrations in BAL fluid and serum were determined by immunoassay and flow cytometry was used to determine the intracellular expression of EPO in BAL-derived and peripheral eosinophils.Results In asthmatic patients a large increase in BAL eosinophils – total cells: median 9.5 × 106 (range: 0.5 to 455.0 × 106); relative: 38% (1 to 91%) – was detectable 24 h following allergen challenge, but peripheral blood eosinophil counts did not change. Concentrations of EPO in BAL fluid increased from 1 µg/L (1.0 to 6.8 µg/L) to 42 µg/L (5.6 to 379.6 µg/L; P 〈 0.01) after allergen but not after saline challenge (1.5 µg/L; 1.0 to 21.9 µg/L), whereas in control subjects all measurements were below the detection limit. Serum concentrations of EPO increased slightly from 18.3 µg/L (3.0 to 56.8 µg/L) to 27 µg/L (3.8 to 133.9 µg/L; P 〈 0.05) 24 h after allergen challenge in asthmatic patients. Furthermore, the intracellular expression of EPO (measured as mean fluorescence intensity) was decreased in BAL eosinophils compared with blood eosinophils (mean fluorescence intensity 29 (7 to 71) vs. 48 (20 to 85); P 〈 0.01) after allergen challenge.Conclusion The finding of increased EPO concentrations in the BAL fluid and decreased intracellular EPO expression in pulmonary eosinophils of asthmatic patients reflects the allergen-triggered release of EPO into the bronchial space.

Notes: Backgroumd Late airways obstruction and eosinophil infiltration after allergen challenge are often seen in human asthma and animal models of allergy. This inflammatory reaction, which may be a link between acute and chronic asthma, is blocked by glucocorticoid pretreatment. However, the role of eosinophils in late airways obstruction and the primary site of action of glucocorticoids, i.e. locally or systemically, have not been fully determined.Objectives This study was initiated to find out the role of eosinophils and neutrophils in allergen-induced late airways obstruction in the pig. The effect of pretreatment with budesonide (BUD) given locally or systemically on cellular responses seen within 8 h after allergen challenge was also studied.Methods Twenty-five minipigs were actively sensitized with Ascaris suum antigen and challenged under anaesthesia with antigen in the lower airways. Pigs were given BUD as an aerosol (l0μg/kg) or an intravenous infusion (5μg/kg) 1 h before allergen challenge. In one group, high doses of BUD (50μg/kg) were infused twice with a 3-h interval before allergen challenge. As a positive control, one group was given the BUD vehicle as an infusion and as a negative control, one group not treated with BUD was given the irrelevant antigen ovalbumin. Eosinophils and neutrophils in lung tissue specimens were detected and levels of eosinophil peroxidase (EPO) and myeloperoxidase (MPO) in bronchoalveolar lavage (BAL) fluid were measured using specific antibodies against porcine EPO and MPO.Results The number of eosinophils in lung tissue and BAL fluid and the level of EPO in BAL fluid were significantly increased 8 h after A. suum challenge in pigs not treated with BUD. With regard to possible recruitment and activation of neutrophils the only significant finding was an increase in the number of cells in BAL fluid. The eosinophil numbers and the level of EPO in BAL fluid were shown to be decreased by all BUD treatments in all the compartments studied compared to the positive control. However, the number of eosinophils in lung tissue and EPO levels in BAL fluid did not correlate with the magnitude of the late airways obstruction.Conclusion Although eosinophils are present in the bronchial wall and lumen and are apparently activated, a causative relationship between this granulocyte and the late bronchial obstruction could not be established in this model.

Notes: Background: Harlier in vitro studies have suggested that the eosinophil may release its granule proteins selectively depending on the stimulus to which the cell is exposed.Objective: The object of the present study was to study the question of selective release in vivo by means of serum measurements of the two eosinophil granule proteins eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) in acute infections.Methods: Fourty-six subjects with acute infections were studied before treatment, 20 with bacterial infections and 26 with viral infections. Serum ECP, EPO and MPO were measured by specific RIA.Results: In acute bacterial infections ECP, but not EPO. was significantly raised in serum (P 〈 0.0001) compared with non-infected healthy subjects. In acute bacterial infections ECP was significantly correlated to the levels of the neutrophil marker myeloperoxidase (MPO) (rs= 0.96, P 〈 0.0001) but not to EPO. In acute viral infections neither ECP nor EPO were on average raised. However, almost 20% the patients had elevated levels of both proteins. In the viral infections the serum-levels of ECP and EPO were correlated (rs= 0.63, P 〈 0.001), but no correlation was found with MPOConclusion: It is concluded that eosinophils are activated during acute bacterial infections and that this activation results in the preferential mobilisation of ECP. The simultaneous assay of the two eosinophil proteins, ECP and EPO. may give new insight into the role of the eosinophil in disease.

Notes: Background Immune-mediated food hypersensitivity affecting the gut is difficult to evaluate, and objective tools to diagnose local gastrointestinal (GI) inflammatory reactions are lacking.Objectives To determine whether allergic manifestations in adults with a history of food-related GI symptoms could be assessed in feces during symptomatic and non-symptomatic periods, using the surrogate markers, eosinophil cationic protein (ECP), eosinophil protein X (EPX) and myeloperoxidase (MPO).Methods Thirteen subjects with food hypersensitivity-related GI symptoms, confirmed by a positive double-blind placebo-controlled food challenge (DBPCFC), were subjected to an open kinetic food challenge design for 6 weeks. Symptoms were recorded and scored during the 3-week study period and stool samples were obtained every day. The surrogate markers ECP, EPX and MPO were measured in the supernatants from feces samples.Results A significant increase in abdominal pain, distension and flatulence was observed during challenge, with a gradual decrease during elimination diet. Both between days and subjects, EPX levels were more frequently increased compared to ECP and MPO. Individuals with a history of a short duration of symptoms had significantly higher mean levels of EPX and MPO than those with a longer duration of symptoms.Conclusions An overall increase in levels of eosinophil markers, in particular EPX, was observed in feces from patients with food-related GI symptoms. However, rather than being a tool to differentiate symptomatic from non-symptomatic periods, EPX might be used for detecting an ongoing clinical or subclinical chronic inflammation, that may have an impact on the patient's clinical course of GI symptoms.

Notes: Serum measurement of ECP (eosinophil cationic protein) is used as an indication of eosinophil activation in diseases such as asthma. The levels are dependent on sample handling, since a certain amount of ECP is released during storage. The mechanisms that induce this in vitro release are not known, but are supposed to be related to the coagulation process. The aim of this study was to investigate this further. ECP was measured in EDTA plasma and serum at 22 and 37°C from healthy individuals and patients with asthma and allergy. The serum levels of ECP increased with temperature. Recalcification of citrated plasma in the presence of granulocytes with increasing concentrations of Ca2+ showed a dissociation between the levels of ECP and the occurrence of coagulation. Further experiments indicated that plasma coagulation is not of any importance for the degranulation of eosinophils, nor did the addition of platelets or mononuclear cells affect the ECP levels. Incubations of granulocytes with fresh or frozen plasma and Ca2+suggested the existence of a freezing labile factor in plasma, necessary for the degranulation of healthy eosinophils, but not for allergic/asthmatic eosinophils. Further experiments with pure eosinophils indicated the existence of factors in serum and plasma which facilitate ECP secretion of an active, temperature-dependent nature. We conclude that the raised ECP levels in serum, as compared to EDTA plasma, are unrelated to the coagulation process, but are due to the continuous secretion ex vivo of ECP from active eosinophils. This process is time and temperature dependent and may be facilitated by eosinophil-activating components in the extracellular environment.

Notes: Background The purpose was to study activation markers of the eosinophil granulocytes in seasonal allergic rhinitis, and the impact of topical steroid therapy thereupon. Methods Sixty-three rhinitis patients with monoallergy to grass were examined before and at peak pollen season. Blood eosinophil count, eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO) in serum and nasal lavage fluid were measured. During the season, patients were randomized to treatment with intranasal fluticasone propionate 0.1 mg o.d. (n=26), 0.2 mg o.d. (n=25), or placebo (n = 12). Six healthy persons served as controls. Results During the season, all parameters, except nasal lavage ECP, increased in the placebo group (P〈0.001 – P〈0.05). Significant differences were seen between the steroid grotips and the placebo group for all parameters (P〈0.001–F〈0.05). Higher eosinophil count (P〈0.05), serum EPO (F〈0.02), and nasal lavage EPO (P〈0.05) were found in patients before season than in controls. The following winter, 44 patients returned for repeated measurement. Lower levels of nasal lavage EPO were observed for patients than levels at the beginning of the season (P〈0.0001). Conclusions Intranasal fluticasone propionate reduced inflammation of the nasal mucosa, demonstrated locally by nasal lavage ECP and EPO, and systemically by blood eosinophils, serum ECP, and serum EPO. EPO seemed more sensitive than ECP as indicator of allergic inflammation. EPO demonstrated some perennial eosinophil activity in hay fever patients, increasing locally during spring.