Major basic protein, but not eosinophil cationic protein or eosinophil protein X, is related to atopy in cystic fibrosis.

Increased eosinophil granule proteins have been described in serum and sputum samples of patients with cystic fibrosis (CF). It has been assumed that eosinophil degranulation is enhanced in atopic subjects - as in asthmatics. Since in CF no differences in eosinophil cationic protein (ECP), eosinophil protein X (EPX), and eosinophil peroxidase between atopic and nonatopic subjects have been detected, we investigated whether major basic protein (MBP) is increased in serum and sputum samples derived from atopic (n = 14) compared with nonatopic CF subjects (n = 26). In CF patients, high mean serum (sputum) levels of ECP 29.7 microg/l (2.7 mg/l), EPX 53.7 microg/l (7.9 mg/l), and MBP 984.6 microg/l but low sputum MBP levels (57.4 microg/l) were measured. In addition, in serum and in sputum samples, a significant correlation between MBP and ECP (P<0.03 and P<0.0001, respectively) or EPX (P<0.05 and P<0.0004, respectively) was detected. By subdivision of the patients into allergic and nonallergic subjects, significant differences were found for serum MBP values only(mean 1382.2 microg/l vs. 770.5 microg/l; P<0.0001), but not for ECP or EPX serum levels or for eosinophil proteins in sputum. Although no differences between atopic and nonatopic CF patients in ECP and EPX were found, serum MBP levels were higher in patients sensitized to inhalant allergens than in nonsensitized subjects. These results indicate differential release of eosinophil granule proteins in peripheral blood from eosinophils, and they also indicate that MBP in serum likely is to be a better discriminator of atopy in CF.     

Human eosinophil cationic proteins (ECP and EPX) and their suppressive effects on lymphocyte proliferation

ECP (eosinophil cationic protein) and EPX (eosinophil protein X) are two highly basic proteins contained in the granules of human eosinophils. In this study, the effect of ECP and EPX upon lymphocyte proliferation in vitro has been investigated. Peripheral blood mononuclear cells from normal donors were cultured in medium containing ECP or EPX at concentrations from 10(-10) to 10(-7) M. 3H-Thymidine incorporation in PHA-blasts or MLR-blasts was dose-dependently inhibited by both ECP and EPX. The effect was irreversible and was not due to cytotoxic damage. The suppressive effect of EPX may involve suppressor cells. The effect of ECP and EPX on lymphocyte proliferation at relevant in vivo concentrations suggests a regulatory role for the eosinophil in immunological reactions.     

Serum eosinophil cationic protein, urinary eosinophil protein X and urinary N-methylhistamine during acute asthma in children]

Serum eosinophil cationic protein (ECP), urinary eosinophil protein X (EPX) and urinary N-methylhistamine were investigated in 24 asthmatic children during acute asthma to determine the sequential variation in the parameters. Serum ECP levels and urinary EPX levels were high during acute asthma, and decreased gradually with improvement of acute asthma, however, these levels of eosinophil drived proteins were significantly higher than those in non-atopic children. There was no significant variation of urinary NMH levels. Significant inverse correlation was found between lung function and urinary EPX level. The present study suggests that serum ECP and urinary EPX may be useful for monitoring airway inflammation during acute asthma, and activation of eosinophils may persist after improvement of acute asthma.     

Regulation of the release of eosinophil cationic protein by eosinophil adhesion

BACKGROUND: Varying release of eosinophil granule proteins depending on the stimulus and environmental factors has previously been reported. OBJECTIVE: To investigate the degranulation from adherent eosinophils by using mixed granulocytes. METHODS: Granulocytes isolated by Percoll gradient centrifugation were incubated on plates coated with plasma and tissue fibronectin, fibrinogen or human serum albumin (HSA) and stimulated with Mn2+, phorbol-myristate-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (f-MLP) and combinations thereof, respectively. The release of eosinophil cationic protein (ECP) was measured by radioimmunoassay. RESULTS: Unstimulated eosinophils incubated in wells coated with plasma and tissue fibronectin, fibrinogen or HSA did not release any ECP. Furthermore, Mn2+ (5 mmol/L) did not induce release of ECP despite the fact that adhesion of eosinophils to these four proteins was induced. PMA stimulated a dose-dependent release of ECP. Contemporaneous stimulation of eosinophils with PMA and Mn2+ induced a dramatically increased release of ECP regardless of which protein the eosinophils were adhering to. A small but significant release of ECP was found when eosinophils incubated on plates coated with fibrinogen and HSA were stimulated by f-MLP. Contemporaneous stimulation of eosinophils with f-MLP and Mn2+ did not induce any synergistic effect on the release of ECP. On the contrary, Mn2+ inhibited the release of ECP induced by f-MLP from eosinophils. Serum-opsonized Sephadex particles stimulated a potent increase of the release of ECP up to 12%-14% in the presence of plasma fibronectin and, in particular, fibrinogen. The kinetics of eosinophil adhesion and degranulation showed that the cellular adhesion preceded the degranulation response and that the degranulation patterns depend on the stimuli and environment. CONCLUSION: The present study indicated that cellular adhesion plays an important role in the regulation of eosinophil degranulation, but that adhesion and degranulation can be induced separately.     

Immunoelectron microscopic evidence for release of eosinophil granule matrix protein onto microfilariae of Onchocerca volvulus in the skin after exposure to amocarzine

The involvement of eosinophils in the host reaction to microfilariae (mf) of Onchocerca volvulus was studied by immunohistochemistry and immunoelectron microscopy. Skin biopsies were obtained from patients after transepidermal administration of the microfilaricide amocarzine. At 20–28 h after the application of amocarzine, mf were degenerated or dead and a marked eosinophil-parasite adherence (EPA) reaction was seen, with intense staining for intra- and extracellular eosinophil granule proteins such as eosinophil cationic protein (ECP) surrounding the mf. Immunoelectron microscopically the eosinophil granule matrix in intact and necrotic eosinophils was specifically labeled, whereas granules whose matrix had dissolved showed no specific gold particle binding. As specific labeling was seen on lowly electron-dense material adjacent to matrix-depleted granules, the material was regarded as released eosinophil granule matrix material. Intact and necrotic eosinophils, matrix-containing as well as matrix-depleted eosinophil granules, and released eosinophil granule matrix material were observed on the surface of damaged mf and between collagen fibers. The coincidence of mf degeneration, EPA reaction, and release of eosinophil granule matrix material on damaged mf and collagen fibers indicated a role of eosinophils and eosinophil granule matrix protein in the host reaction to mf after amocarzine application. Received: 3 March 1998 / Accepted: 13 March 1998     

C3b-induced eosinophil degranulation involves PI3-kinases and is inhibited by protein kinase C activity

Carlson M, Venge P, Lampinen M. C3b‐induced eosinophil degranulation involves PI3‐kinases and is inhibited by protein kinase C activity. APMIS 2010; 119: 119–26. Selective release of individual eosinophil granule proteins has been demonstrated in eosinophilic conditions and in vitro using different stimuli. The aim of this study was to investigate if selective release of eosinophil cationic protein (ECP), eosinophil protein X/eosinophil derived‐neurotoxin (EPX/EDN) and eosinophil peroxidase (EPO) could be due to the involvement of different signal transduction pathways. Peripheral blood granulocytes from healthy donors were incubated with Wortmannin, LY294002, Genistein, Staurosporine, GÖ6976 or PD98059 prior to the induction of degranulation by C3b. The released amounts of ECP, EPO and EPX/EDN were determined by immunoassays, and related to the total cell content of respective protein. Wortmannin caused a significant, dose‐dependent inhibition of all three granule proteins. LY294002 (10^?6 M) also inhibited the release of all proteins. Genistein (10^?6 M) inhibited the release of ECP, whereas the release of EPO was increased. However, there was a tendency towards similar concentration‐dependent patterns of release of all three proteins. Staurosporine (10^?7 M), GÖ6976 (10^?6 M) and PD98059 (10^?5 M) caused an increased release of the three proteins. PI3‐kinases play an important role in the C3b‐induced release of ECP, EPO and EPX/EDN, whereas protein kinase C seems to have inhibitory effects on C3b‐induced degranulation.     

Twenty-four-hour time course of eosinophil granule proteins ECP and EPX during bronchial allergen challenges in serum of asthmatic children

To study in vivo monitoring variables for bronchial allergen challenges, we investigated the time course of the eosinophil granule proteins, eosinophil cationic protein (ECP) and eosinophil protein × (EPX) after allergen provocation in serum. Thirty-two asthmatic children sensitive to house-dust mites and six healthy young adult controls were challenged by bronchial allergen provocations with Dermatophagoides pteronyssinus and D. farinae. Blood samples were taken at regular intervals up to 24 h. Base-line concentrations of ECP ( P <0.004), EPX ( P <0.002), and eosinophils ( P <0.001) were found to be increased in asthmatic children, as compared with healthy controls. ECP and EPX concentrations showed a uniform pattern with two characteristic features: 1) a rapid increase for both mediators up to 30 min after provocation over base-line values ( P <0.0001 and P <0.001), followed by a rapid decrease nearly to base-line values in the next 30 min; and 2) a steady increase for ECP and EPX up to 10 h ( P <0.02 and P <0.01), and even higher levels at 24 h, after challenge ( P <0.002 and P <0.003). We conclude that although eosinophils are activated in asthmatic children after bronchial allergen challenge, ECP and EPX concentrations are not suitable monitoring variables. Base-line eosinophils seem to predict the occurrence of a late-phase asthmatic reaction after allergen provocation.     

Extracellular deposit of the cationic proteins ECP and EPX in tissue infiltrations of eosinophils related to tissue damage

In a series of eosinophil inflammatory states affecting various organs (heart, gut, bladder and skin) we performed an immunohistochemical study of the eosinophil cationic proteins ECP and EPX. A strong correlation was noted between the liberation of ECP and EPX and tissue necrosis in all organs. In most cases ECP and EPX were found on the same location. However, one case indicated a possible differential release. Extracellular ECP and EPX were revealed concurrently with the two polyclonal antibodies and the monoclonal EG2 antibody. The latter binds to both ECP and EPX, but only during secretion. Since EG2 does not differentiate between ECP and EPX, but only during secretion. Since EG2 does not differentiate between EXP and EPX, it is for the first time demonstrated that both cationic proteins are correlated to tissue damage. The chymotrypsin-like cationic protein (CCP), related to neutrophils, showed a low correlation with the eosinophil cationic proteins in cases of tissue damage. The hypothesis is put forward that the release of eosinophil granule proteins and especially ECP results in a non-specific tissue damage.     

Eosinophilic gastroenteritis: ultrastructural evidence for a selective release of eosinophil major basic protein.

Ultrastructural study of mucosal eosinophils in a case of eosinophilic gastroenteritis involving stomach, duodenum and ileum showed an altered structure in ulcerated duodenal areas. The electron core density of eosinophil granules was inverted or disappeared and tubulovesicular structures occurred. Using immunogold staining with specific antibodies, major basic protein was detected diffusely in the matrix of eosinophil granules and out of the granules in tight association with extragranular membrane formations. In contrast, eosinophil cationic protein and eosinophil peroxidase were normally distributed in the granule matrix. When compared with the eosinophils in macroscopically normal duodenal mucosa in the same patient, these changes support a role for major basic protein in tissue damage in eosinophilic gastroenteritis. The diffusion of one granule protein from the granules to the exterior of the cells favours the view of a selective release of eosinophil mediators.     

The production of the eosinophil proteins ECP and EPX/EDN are regulated in a reciprocal manner

Previous studies showed that the biological activity and the eosinophil content of eosinophil cationic protein (ECP, RNase 3) are determined by single‐nucleotide polymorphisms (SNPs) in the ECP (RNase3) gene . In this study, we report the prevalence of a common SNP in the eosinophil protein x/eosinophil‐derived neurotoxin (EPX/EDN, RNase2) and the association with the cellular contents of EPX/EDN and ECP. The genes were sequenced and the EPX/EDN405(G>C) rs2013109 SNPs were also determined by TaqMan 5′nuclease allelic discrimination assay. ECP and EPX/EDN in purified eosinophils or in whole blood extracts were analysed by sensitive immunoassays. The study included 379 non‐allergic and allergic subjects. The genotype prevalence of the EPX/EDN405(G>C) polymorphism was GG 59%, GC 36% and CC 6%. The cellular contents of ECP and EPX/EDN were related in a reciprocal fashion with the sums of the protein contents being constant. The contents were associated with the ECP562(G>C) rs2233860 and EPX/EDN405(G>C) gene polymorphisms. The cellular content of eosinophil peroxidase (EPO) was not associated with the ECP and EPX/EDN genotypes. The prevalence of the EPX/EDN405(G>C) genotypes and the contents of the proteins were similar in non‐allergic and allergic subjects. The production and storage of the two ancestral proteins, ECP and EPX/EDN likely share common regulatory mechanisms, which result in opposing productions of the two proteins.     

Immunofluorescence analysis of cytokine and granule protein expression during eosinophil maturation from cord blood–derived CD34 progenitors

Background: In allergic inflammation and asthma, eosinophils are major effector cells. They have been shown to synthesize at least 23 cytokines, some of which are stored intracellularly in their unique crystalloid granules together with cationic granule protein. Little is known about the synthesis and storage of cytokines relative to cationic granule proteins in maturing eosinophils during eosinophilopoiesis. Objective: Our purpose was to analyze the expression of eosinophil-derived mediators, major basic protein (MBP), eosinophil cationic protein (ECP), IL-6, and RANTES, during early stages of eosinophil maturation in CD34⁺ cell-derived colonies. Methods: Purified human cord blood CD34⁺ cells were grown in methylcellulose cultures in the presence of recombinant human IL-3 and IL-5. By confocal laser scanning microscopy, the coexpression of eosinophil granular proteins MBP and ECP was determined concurrently with IL-6 and RANTES during eosinophil maturation on days 16, 19, 23, and 28 of culture. Results: Immunoreactivity against MBP, ECP, IL-6, and RANTES was not detectable in freshly purified CD34⁺ cells. Maturing eosinophils (>95%) exhibited positive immunostaining for all these proteins between days 16 and 28 of culture. At early stages of culture, discrete immunostaining was observed around the periphery but not in the center of granular structures. By day 28 cultured eosinophil-like cells showed evidence of the acquisition of crystalloid granule-like structures, analogous to those observed in mature peripheral blood eosinophils. Conclusions: Eosinophils express and store cytokines simultaneously with cationic granule proteins during the process of maturation. We propose that the storage of cytokines during the development of eosinophils is an early event and it may be integral to inflammatory responses involving these cells. The results of this study suggest a potential immunoregulatory function for maturing eosinophils. (J Allergy Clin Immunol 2000;105:1178-84.)     

The human eosinophil in inflammation

Active research during recent years has clearly shown that the eosinophil is a potent inflammatory cell taking active part in almost all kinds of inflammatory processes. The activity of the human eosinophil is mediated by the secretion of four well characterized cytotoxic proteins, ECP, EPO, EPX/EDN and MBP in addition to lipid mediators such as LTC₄ and PAF and toxic oygen metabolites. The cytotoxic potential of the eosinophil has been demonstrated in a number of diseases with a close association of eosinophil accumulation with secretion of granule proteins and tissue injury. Also the measurements of the proteins in various body fluids have provided evidence for the active participation of eosinophils in a number of diseases such as asthma, ulcerative colitis, rheumatoid arthritis, psoriasis to mention a few. The identification of the principles that attract eosinophils to the sites of inflammation must be a major goal in our attempts to control the activity of this potents cell.     

Piecemeal degranulation of peripheral blood eosinophils: a study of allergic subjects during and out of the pollen season

The variability of serum and plasma levels of eosinophil granule proteins in different clinical conditions, interpreted as the result of different patterns of cytokine priming, suggests a selective mobilization of granule proteins. Inasmuch as piecemeal degranulation (PM) is the mechanism proposed for the differential release of eosinophil granule proteins, we decided to investigate whether blood eosinophils from allergic subjects show characteristics of PM during natural allergen challenge. Eosinophils from three birch-sensitive subjects were studied before and during the pollen season. Electron microscopy analysis showed that during the season, eosinophils presented morphologic features of PM. By immunogold labeling, eosinophil cationic protein (ECP) was detected not only in normal specific granules but also in the cytoplasm, in the vicinity of partially lucent specific granules. These results were confirmed by subcellular fractionation, where the amount of ECP associated with compartments containing small vesicles increased 2-fold during the pollen season. A study of the distribution of ECP, eosinophil peroxidase, and hexosaminidase in eosinophils of different densities showed that the profile of each of these proteins differed depending on cell density. All of these proteins decreased in the specific granule of hypodense cells and increased in other cell compartments. We conclude that allergen exposure causes PM of the peripheral blood eosinophils of allergic subjects, and that the density of these cells reflects the degree of degranulation. Our results provide novel information for the understanding of the selective mobilization of granule proteins into the circulation.     

Enhanced adhesion to laminin by apoptotic eosinophils

Apoptotic cells are regarded as inert bodies that turn off intracellular processes and functional capabilities. The objective was to study adhesion by eosinophils in relation to the apoptotic process. Eosinophils were cultured for up to 72 h. The living cells were separated from the apoptotic cells, and their adhesion to transfected cell lines expressing vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin and laminin was measured. To relate the functional studies with cell structure, the surface receptor expression of beta1- and beta2-integrins was investigated by flow cytometry. Apoptotic eosinophils evidenced an increased expression of the alpha-chain of the laminin receptor and CD49f and an increased ability to adhere to a laminin-coated surface. Adhesion to the endothelial cell adhesion receptors E-selectin, VCAM-1 and ICAM-1 was absent in apoptotic eosinophils and was paralleled by a low expression of CD11b, CD29, CD49d and CD66b. The specifically increased adhesion to laminin and expression of the laminin receptor alpha-chain is a unique feature of apoptotic eosinophils. When an eosinophil goes into apoptosis, it still possesses the ability to interact with its environment. Our results point to new ideas as to how the apoptotic eosinophil behaves in apoptosis.     

Eosinophil cationic protein(ECP) and eosinophil protein X (EXP) concentrations in serum and bronchial lavage fluid in asthma. effect of prednisolone treatment.

Background Eoswinophil granule proteins may contribute to hyperresponsiveness in asthma.
Objective To measure eosinophil cationic protein (ECP) and eosinophil protein X (EPX) in sereum and bronchial lavage fluid from 20 asthmatics and 16 control subjects. To asses the effect on these eosinophil proteins of corticosteroid treatment of asthma. To determine ehether serum ECP and EPX measured weekly in a longitudina study for 10 weeks reflected changes in lung function.
Methods Eosinophil granule proteins were measured by radiommunoassy of bronchial wash (BW), bronchoalveolar lavage (BAL) serum.
Results Eosinophils were elevated in BAL (P<0.01) , BW (P<0.01) and blood (P<0.01) from asthmatic compared with control subjects. Eosinophil cationic protein concentration was significantly elevated in BAL (P<0.05) and BW from asthmatics (P<0.01) and EPX was increased in BAL (P<0.05) and BW (P<0.01) . These changes were also reflected in elevated serum ECP (P<0.01) and EPX (P<0.01) concentrations is asthmatic subjects. There was no significant difference between sujects receiving prednisolone and the placebo group, but there was a fall in ECP in BW (P<0.05) and serum (P<0.01) and in EPX in BW (P<0.01) and serum (P<0.01) within the group receiving prednisolone. In the longitudinal study there was only significant difference between ECP values associated with highest and lowest peak expiratory flow rate (PEFR) (P<0.05).
Conclusion These data confirm a role for cosinophil activation in the airway in asthma pathogenesis, and add some support to the hypothesis that corticosteroids may inhibit cosinophil activation in asthma.     

The Gordon phenomenon induced by the eosinophil cationic protein and eosinophil protein X

Cerebellar Purkinje cell degeneration after intracerebral injection of eosinophil granulocytes or extracts thereof is known as the Gordon phenomenon. The reaction is said to be highly selective. An eosinophil-derived neurotoxin (EDN) has recently been reported to induce the Gordon phenomenon. However, we report here that two eosinophil-derived proteins, eosinophil cationic protein (ECP) and eosinophil protein X (EPX), may induce the Gordon phenomenon after intraventricular injection. The potency of ECP is far greater than that of EPX and the latter is possibly identical to EDN. The Fink-Heimer staining for degenerating nerve fibers and boutons, however, indicated that the selectivity of the Gordon phenomenon is not as specific as was previously thought, since this method revealed degeneration of all brain areas in proximity to the ventricular system.     

An Extragranular Compartment of Blood Eosinophils Contains Eosinophil Protein X/Eosinophil-Derived Neurotoxin (EPX/EDN)

Serum and plasma profiles of eosinophil protein X (EPX/EDN) and those of other eosinophil proteins differ in various conditions, suggesting a different mobilisation from storage granules. This work studied the subcellular localisation of EPX/EDN in non-primed and in vivo primed blood eosinophils from healthy and allergic subjects, during and out of the pollen season. Primed eosinophils contain easily mobilisable secretory proteins. By fractionation on sucrose density gradients, EPX/EDN localised in the specific granules as well as in a cytoplasmic extra-granular compartment of low equilibrium density that partially overlapped with vesicular structures, cytosolic proteins and plasma membranes. This compartment was clearly separate from the low density peak of ECP that increases during the pollen season. There were no significant differences in the amounts of EPX/EDN present in the low density peak of healthy and allergic subjects. Immuno-gold labelling electron microscopy showed EPX/EDN in specific granules, cytoplasm and associated to plasma membranes. In conclusion, substantial amounts of EPX/EDN localise in an extra-granular, low equilibrium density compartment of human eosinophils.     

A sensitive high throughput ELISA for human eosinophil peroxidase: A specific assay to quantify eosinophil degranulation from patient-derived sources

Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings.     

Blood eosinophil number and activity in relation to lung function in patients with asthma and with eosinophilia.

Blood eosinophil count, serum concentrations of eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO), and peak expiratory flow (PEF) rate were studied in 23 patients with severe labile asthma characterized by eosinophilia at the start and end of a treatment period of 5 weeks. The mean blood eosinophil count was 808 x 10(6)/L at the start of the treatment period. Serum ECP and EPX were significantly raised compared with that of the references, whereas the mean serum MPO level was normal. The mean PEF was significantly and negatively correlated to both blood eosinophil count and serum ECP and EPX, but the predominant correlation was that between blood eosinophil count and PEF. At the end of the treatment period, PEF had increased and the blood eosinophil count and serum ECP and EPX were reduced when these values were compared with the values at the start of the treatment period. There was a significant and negative correlation of mean PEF to serum ECP but not to the blood eosinophil count. In individual subjects, the decreases in the blood eosinophil counts and serum EPX were significantly correlated to the individual increases of mean PEF. In conclusion, the present investigation indicates that in patients with asthma and pronounced eosinophilia, the lung function of the patients was principally related to the number of circulating eosinophils, whereas, when their eosinophilia was reduced to moderate levels, the patient's lung function was closer related to the activity of the eosinophils.     

Lack of relationship between eosinophil cationic protein and eosinophil protein X in nasal lavage and urine and the severity of childhood asthma in a 6-month follow-up study.

BACKGROUND: Recent studies suggest that eosinophil cationic protein (ECP) and eosinophil protein X (EPX) may be valuable markers of airway inflammation in various body fluids of asthmatic children. Most of these studies have relied on a single measure of inflammatory markers. OBJECTIVE: We measured ECP and EPX in nasal lavage fluids (NALF) and urine samples in children with asthma over a 6-month period to study the relationship between inflammatory markers and clinical severity. METHODS: Fourteen children with mild persisting asthma (mean age 11.7 years, SD 2.2) were recruited. All patients were on therapy including inhaled steroids. For a 6-month period asthma severity was monitored by at least monthly physical examination and pulmonary function tests. Daily morning and evening PEF, asthma symptoms and medication were recorded in diaries for the whole study period. Telephone interviews were performed between visits and additional visits were done in case of an increase in asthmatic symptoms or drop of PEF values under 80% of best value. An exacerbation was defined by a fall of FEV1 > 10% and an increase in asthma symptoms and additional need of beta2-agonist. NALF and urine samples were obtained at each visit and analysed for ECP (NALF only) and EPX. RESULTS: Mean observation time was 186.4 days (SD 19.8). Thirteen patients completed the study. During the study period 11 exacerbations were observed in six patients. No significant associations between PEF, PEF variability (amplitude % of mean), daily symptoms, additional beta2-agonist, FEV1 and MEF50 and nasal ECP, nasal EPX and urinary EPX were found. However, at exacerbations an average increase of nasal ECP (9.3 vs 50.3 microg/L) and EPX (nasal EPX 36.4 vs 141.7 microg/L, urinary EPX 46.4 vs 74.1 microg/mmol creatinine) was observed. CONCLUSION: Serial measurements of ECP and EPX in NALF and urine samples do not provide additional information for the practical management in monitoring childhood asthma.