Urinary eosinophil-derived neurotoxin is associated with reduced lung function in pediatric asthma.

INTRODUCTION: Eosinophil-derived neurotoxin (EDN) is a biomarker for eosinophilic activation. Urinary (u) EDN may allow non-invasive monitoring of asthma, but clinical recommendations are lacking. We assessed the potential of uEDN as a marker of disease activity in pediatric asthma. METHODS: We assessed urine samples of 371 children from the German ALLIANCE study cohort, from which we had: 169 preschool wheezers (<6 years), 80 asthmatics (≥6 years), and 122 healthy controls using the ImmunoCAP™ EDN Assay. Creatinine (Cr)-adjusted uEDN values were analyzed using correlations, association tests, (non) parametric statistics, multiple linear, and multivariable regression. RESULTS: uEDN/uCr values were higher in atopic versus non-atopic preschool-aged subjects (p = .035) and associated with the sum of allergen-specific IgE in younger (r = 0.24, p = .003), and older subjects (r = 0.23, p = .043). uEDN/uCr was marginally a good determinant for atopy (p = .078, for subjects aged <6 years, and p = .058 for subjects ≥6 years). Children with the T2-high phenotype had higher uEDN/uCr (p < .001) versus T2-low-irrespective of using uEDN/uCr or blood eosinophils in combination to allergen sIgE for disease phenotyping. uEDN/uCr significantly correlated with reduced lung function among asthmatics (FEV1 z-scores: r = -0.30, p = .007, and FEV1/FVC z-scores: r = -0.24, p = .038). Using multivariable modeling, uEDN/uCr was an independent determinant of FEV1 (p = .038), and to a lesser extent, FEV1/FVC (p = .080). CONCLUSIONS: uEDN/uCr may serve as a non-invasive biomarker for clinical features such as lung function in pediatric asthma. We highlight the utility of uEDN/uCr as a biomarker that can be easily assessed using widely available robust diagnostic immunoassays.

Fixed airflow obstruction relates to eosinophil activation in asthmatics.

BACKGROUND: Some asthmatics develop irreversible chronic airflow obstruction, for example, fixed airflow obstruction (fixed-AO). This is probably a consequence of airway remodelling, but neither its relation to inflammation nor which asthma biomarkers can be clinically useful are elucidated. We hypothesized that the presence of type 2 inflammation relates to fixed-AO. OBJECTIVES: To evaluate the presence of four markers for type 2 inflammation in fixed airflow obstruction among asthmatics. METHODS: This was a cross-sectional study of 403 participants with asthma, aged 17-75 years, from three Swedish centres. Fixed airflow obstruction was defined as forced expiratory volume during the first second (FEV1 ) over forced vital capacity (FVC) being below the lower limit of normal (LLN). The following type 2 inflammation markers were assessed: exhaled nitric oxide (FeNO), serum periostin, serum eosinophil cationic protein (S-ECP), and urinary eosinophil-derived neurotoxin (U-EDN). RESULTS: Elevated U-EDN (values in the highest tertile, ≥65.95 mg/mol creatinine) was more common in subjects with fixed-AO vs. subjects without fixed-AO: 55% vs. 29%, P < 0.001. Elevated U-EDN related to increased likelihood of having fixed-AO in both all subjects and never-smoking subjects, with adjusted (adjusted for sex, age group, use of inhaled corticosteroids last week, atopy, early-onset asthma, smoking history, and packyears) odds ratios (aOR) of 2.38 (1.28-4.41) and 2.51 (1.04-6.07), respectively. In a separate analysis, having both elevated S-ECP (>20 µg/L) and U-EDN was related to having the highest likelihood of fixed-AO (aOR (95% CI) 6.06 (2.32-15.75)). Elevated serum periostin or FeNO did not relate to fixed-AO. CONCLUSIONS AND CLINICAL RELEVANCE: These findings support that type 2 inflammation, and in particular eosinophil inflammation, is found in asthma with fixed-AO. This could indicate a benefit from eosinophil-directed therapies. Further longitudinal studies are warranted to investigate causality and relation to lung function decline.

Urinary eosinophil protein X in childhood asthma: relation with changes in disease control and eosinophilic airway inflammation.

The aim of this study was to assess cross-sectional and longitudinal correlations between uEPX and other markers of asthma control and eosinophilic airway inflammation. Methods. We measured uEPX at baseline, after 1 year and after 2 years in 205 atopic asthmatic children using inhaled fluticasone. At the same time points, we assessed symptom scores (2 weeks diary card), lung function (forced expiratory volume in one second (FEV(1))), airway hyperresponsiveness (AHR), and percentage eosinophils in induced sputum (% eos). Results. We found negative correlations between uEPX and FEV(1) at baseline (r = -0.18, P = 0.01), after 1 year (r = -0.25, P < 0.01) and after 2 years (r = -0.21, P = 0.02). Within-patient changes of uEPX showed a negative association with FEV(1) changes (at 1 year: r = -0.24, P = 0.01; at 2 years: r = -0.21, P = 0.03). Within-patient changes from baseline of uEPX correlated with changes in % eos. No relations were found between uEPX and symptoms. Conclusion. In this population of children with atopic asthma, uEPX correlated with FEV(1) and % eos, and within-subjects changes in uEPX correlated with changes in FEV(1) and % eos. As the associations were weak and the scatter of uEPX wide, it seems unlikely that uEPX will be useful as a biomarker for monitoring asthma control in the individual child.

Elevated eosinophil protein X in urine from healthy neonates precedes development of atopy in the first 6 years of life.

RATIONALE: Biomarkers predicting development of atopic disease are needed for targeted preventive measures and to study if disease pathology may be active before onset of symptoms. OBJECTIVES: To investigate whether eosinophil protein X, leukotriene-C4/D4/E4, and 11ß-prostaglandin (PG) F2α (PGD2 metabolite) assessed in urine from healthy at-risk neonates precede development of atopic disease during the first 6 years of life. METHODS: We measured eosinophil protein X (n = 369), leukotriene-C4/D4/E4 (n = 367), and 11ß-PGF2α (n = 366) in urine from 1-month-old children participating in the Copenhagen Prospective Studies on Asthma in Childhood birth cohort. Clinical data on development of allergic sensitization, allergic rhinitis, nasal eosinophilia, blood eosinophilia, eczema, troublesome lung symptoms (significant cough or wheeze or dyspnea), and asthma were collected prospectively until age 6 years. Associations between urinary biomarkers and development of atopic traits were investigated using general estimating equations, logistic regression, and Cox regression. MEASUREMENTS AND MAIN RESULTS: Eosinophil protein X in the urine of the asymptomatic 1-month-old neonates was significantly associated with development of allergic sensitization (odds ratio, 1.49; 95% confidence interval [CI], 1.08­1.89), nasal eosinophilia (odds ratio, 3.2; 95% CI, 1.2­8.8), and eczema (hazard ratio, 1.4; 95% CI, 1.0­2.0), but not with allergic rhinitis, asthma, or blood eosinophilia. Neither leukotriene-C4/D4/E4 nor 11ß-PGF2α was associated with any of the atopic phenotypes. CONCLUSIONS: Eosinophil protein X in urine from asymptomatic neonates is a biomarker significantly associated with later development of allergic sensitization, nasal eosinophilia, and eczema during the first 6 years of life. These findings suggest activation of eosinophil granulocytes early in life before development of atopy-related symptoms.

Increased bronchial NO output in severe atopic eczema in children and adolescents.

Atopic children have an increased risk for asthma, which is preceded by bronchial inflammation. Exhaled nitric oxide (NO) measured at multiple exhalation flow rates can be used to assess alveolar NO concentration and bronchial NO flux, which reflect inflammation in lung periphery and central airways, respectively. Exhaled breath condensate is another non-invasive method to measure lung inflammation. The purpose of the present study was to find out if the severity of atopic eczema is associated with lung inflammation that can be observed with these non-invasive tests. We studied 81 patients (7-22 yr old) with atopic eczema and increased wheat-specific IgE (>or=0.4 kUA/l) and no diagnosis of asthma. Exhaled NO was measured at multiple exhalation flow rates, and bronchial NO flux and alveolar NO concentration were calculated. Cysteinyl-leukotriene concentrations were measured in exhaled breath condensate. The patients were divided into two groups according to the severity of atopic eczema. Patients with severe atopic eczema had enhanced bronchial NO output as compared with patients with mild eczema (2.1 +/- 0.5 vs. 0.9 +/- 0.1, p = 0.003). No statistically significant differences in alveolar NO concentrations were found between the groups. In the whole group of patients, the bronchial NO output correlated positively with serum eosinophil protein X (r(s) = 0.450, p < 0.001), serum eosinophil cationic protein (r(s) = 0.393, p < 0.001), serum total IgE (r(s) = 0.268, p = 0.016) and with urine eosinophil protein X (r(s) = 0.279, p = 0.012), but not with lung function. Alveolar NO concentration correlated positively with serum eosinophil protein X (r(s) = 0.444, p < 0.001) and with serum eosinophil cationic protein (r(s) = 0.362, p = 0.001). Measurable cysteinyl-leukotriene concentrations in exhaled breath condensate were found only in one-third of the patients, and there were no differences between the two groups. The results show that increased bronchial NO output is associated with eosinophilic inflammation and severe atopic eczema in patients without established asthma.

Bifidobacterium pseudocatenulatum is associated with atopic eczema: a nested case-control study investigating the fecal microbiota of infants.

BACKGROUND: Exposure to specific bacterial bowel commensals may increase/reduce the risk of atopic diseases. OBJECTIVE: To compare fecal bacterial communities of young infants with/without eczema. METHODS: Nested case-control study. Infants age 3 to 6 months with eczema (cases, n = 37) and without (controls, n = 24) were matched for sex, age, feeding (breast/bottle/mixed/solids), ethnicity. Information was collected on maternal/infant antibiotic exposure, feeding, gastrointestinal symptoms, family history of allergy. Eczema severity scoring was used (Severity Scoring of Atopic Dermatitis index). Samples were taken for determination of allergen-specific serum IgE (cases) and urinary/fecal eosinophilic protein X. Gastrointestinal permeability was measured. The compositions of fecal bacterial communities were analyzed (culture-independent, nucleic acid-based analyses). RESULTS: There was no difference in overall profiles of fecal bacterial communities between cases and controls. Family history of allergy increased likelihood of bifidobacteria detection (history, 86%; no history, 56%; P = .047); breast-fed infants were more likely to harbor Bifidobacterium bifidum (odds ratio, 5.19; 95% CI, 1.47-18.36; P = .01). Bifidobacterium pseudocatenulatum was detected more commonly in feces of non-breast-fed children (odds ratio, 5.6; 95% CI, 1.3-24.3; P = .02) and children with eczema (eczema, 26%; no eczema, 4%; P = .04). There were no significant associations between clinical measurements and detection of B pseudocatenulatum. CONCLUSION: Presence of B pseudocatenulatum in feces was associated with eczema and with exclusive formula-feeding; B bifidum was associated with breast-feeding.

Urinary eosinophil-derived neurotoxin concentrations in patients with atopic dermatitis: a useful clinical marker for disease activity.

BACKGROUND: It has been reported that measurements of eosinophil-derived neurotoxin (EDN) may be useful for identifying eosinophil activities in allergic diseases including atopic dermatitis. METHODS: EDN concentrations in the urine were measured by enzyme-linked immunosorbent assay, and the number of eosinophils in the peripheral blood was counted in 30 patients with atopic dermatitis. The severity of atopic dermatitis was graded on the criteria proposed by Rajka and Langeland. The disease activity was assessed by each patient on a visual analogue scale (VAS). RESULTS: Urinary concentrations of EDN in patients with atopic dermatitis showed a significant positive correlation with disease severity. Urine EDN concentrations also correlated with VAS scores for itching, skin condition, overall skin symptoms and total VAS score, but not with the VAS score for skin dryness. Urinary EDN concentrations did not correlate with the number of eosinophils in the peripheral blood. CONCLUSIONS: The urinary EDN concentration in patients with atopic dermatitis is a useful clinical marker for monitoring disease activity.

Urinary eosinophil protein X in children with atopic asthma.

UNLABELLED: The aim of this study was to investigate the relationship between urinary eosinophil protein X (uEPX) and asthma symptoms, lung function, and other markers of eosinophilic airway inflammation in asthmatic school children. METHODS: A cross-sectional study was performed in 180 steroid dependent atopic children with stable moderately severe asthma, who were stable on 200 or 500 microg of fluticasone per day. uEPX was measured in a single sample of urine and was normalized for creatinine concentration (uEPX/c). Symptom scores were kept on a diary card. FEV1 and PD20 methacholine were measured. Sputum induction was performed in 49 and FE(NO) levels measured in 24 children. RESULTS: We found an inverse correlation between uEPX/c and FEV1 (r = -.20, P = .01) and a borderline significant correlation between uEPX/c and PD20 methacholine (r = -.15, P = .06). Symptom score, %eosinophils and ECP in induced sputum and FE(NO) levels did not correlate with uEPX/c. CONCLUSION: uEPX/c levels did not correlate with established markers of asthma severity and eosinophilic airway inflammation in atopic asthmatic children.

Eosinophils play a major role in the severity of exercise-induced bronchoconstriction in children with asthma.

Exercise-induced bronchoconstriction (EIB) was associated with eosinophilic airway inflammation, bronchial hyperresponsiveness (BHR), atopy and airway obstruction. To understand the pathogenesis of EIB, we determine whether eosinophil is more related to the mechanism of EIB than atopy, BHR and airway obstruction. This study comprised 268 asthmatic children who underwent lung function test, methacholine challenge test, exercise challenge test, and blood tests for total IgE levels and total eosinophil counts (TEC). Urine eosinophil protein X (EPX) levels after exercise were measured by using ELISA method. EIB was observed in 195 of 268 asthmatics (72.8%). Asthmatics with EIB showed significantly increased TEC (P < 0.01) and decreased log PC(20) as compared with asthmatics without EIB. Maximal percent fall in FEV(1) after exercise was significantly correlated with TEC, log IgE, FEF(25-75%), log PC(20) (P < 0.001, respectively) and FEV(1) (P = 0.013). When the same study was carried out in nonatopic asthmatics, those with EIB showed significantly increased TEC (P = 0.01) compared with those without EIB; however, log PC(20), FEV(1), and FEF(25-75%) showed no significant differences between the two groups of nonatopic asthmatics. In addition, there was a significant correlation between the severity of EIB and TEC in nonatopic asthmatics. Urine EPX/Cr levels after exercise were correlated with the severity of EIB (r = 0.238, P = 0.014). Blood eosinophils and urine EPX/Cr after exercise correlate significantly with the maximal percent fall in FEV(1) after exercise, therefore EIB may reflect a state of eosinophilic inflammation in the airway of asthmatic children.

Measurements of eosinophil activation before and after food challenges in adults with food hypersensitivity.

BACKGROUND: Objective assessment of inflammatory reactions in the gastrointestinal tract could be useful in the diagnosis of food hypersensitivity. The aim of the present study was to investigate the involvement of eosinophils and mast cells in the inflammatory response of patients with food hypersensitivity before and after food challenges. METHODS: Eleven patients (4 with IgE-mediated allergy and 7 without) with food hypersensitivity and positive double-blind, placebo-controlled food challenge were subjected to food challenge in a single-blinded fashion. Four subjects with no known food hypersensitivity were recruited as controls. Placebo was given after a 1-week washout period followed by an active dose. Stool, urinary and serum samples were collected and symptoms were recorded in a diary. Fecal samples were analyzed for eosinophil protein X (F-EPX) and tryptase; urinary samples for EPX (U-EPX) and leukotriene E4 (U-LTE4) and serum samples were analyzed for eotaxin and food-specific IgE antibodies. RESULTS: Patients with IgE-mediated food allergy had increased levels of F-EPX compared to controls and tended to have lower serum levels of eotaxin compared to non-allergic patients and controls. U-LTE4 was significantly higher in allergic patients compared to non-allergic patients after challenge. Moreover, F-EPX correlated to U-LTE4 (p = 0.011). Reported symptoms, abdominal pain, distension, flatulence and nausea were similar in the allergic and non-allergic patients. CONCLUSION: The results strongly indicate that eosinophils are activated in the gastrointestinal tract of food-allergic patients but not in patients with non-allergic food hypersensitivity. Due to the inconsistent pattern of symptoms after placebo and active food challenge, it was not possible to relate the levels of inflammation markers to the recorded symptoms.

Mast cell activation and leukotriene secretion in wheezing infants. Relation to respiratory syncytial virus and outcome.

The persistence of wheezing after early wheezing episodes in infancy may be related to the virus involved and to the type of inflammation during the initial wheezing. The role of mast cell activation and leukotriene secretion in wheezing, and the relation to outcome, is not known. Our objective was to study markers of mast cell activation and leukotriene secretion from wheezing infants, and the relation to respiratory syncytial virus (RSV) infection and persistent wheezing. Urinary 9alpha,11beta-PGF(2), a marker of mast cell activation, and urinary leukotriene E4 were measured in 106 infants hospitalized for wheezing during their first year of life. Results were related to the presence of RSV infection and the persistence of wheezing at follow-up 20 months later. Levels of 9alpha,11beta-PGF(2) were higher in infants positive for RSV than in those with RSV negative wheezing, but both groups had higher levels than controls. Leukotriene E4 levels were higher in wheezing infants than in controls. Urinary 9alpha,11beta-PGF(2) levels were higher in infants with transient compared with persistent wheezing. We found a positive correlation between 9alpha,11beta-PGF(2) and leukotriene E4, strongest in infants with RSV negative disease and in infants with persistent wheezing. The results suggest that mast cells play an important role in infant wheezing, and may be a major source of leukotriene secretion in these infants. Mast cell activation and leukotriene secretion were not associated with persistent wheezing.

Proteomic-based discovery and characterization of glycosylated eosinophil-derived neurotoxin and COOH-terminal osteopontin fragments for ovarian cancer in urine.

PURPOSE: The objective was to identify and characterize low molecular weight proteins/peptides in urine and their posttranslational modifications that might be used as a screening tool for ovarian cancer. EXPERIMENTAL DESIGN: Urine samples collected preoperatively from postmenopausal women with ovarian cancer and benign conditions and from nonsurgical controls were analyzed by surface-enhanced laser desorption/ionization mass spectrometry and two-dimensional gel electrophoresis. Selected proteins from mass profiles were purified by chromatography and followed by liquid chromatography-tandem mass spectrometry sequence analysis. Specific antibodies were generated for further characterization, including immunoprecipitation and glycosylation. Quantitative and semiquantitative ELISAs were developed for preliminary validation in patients of 128 ovarian cancer, 52 benign conditions, 44 other cancers, and 188 healthy controls. RESULTS: A protein (m/z approximately 17,400) with higher peak intensities in cancer patients than in benign conditions and controls was identified and subsequently defined as eosinophil-derived neurotoxin (EDN). A glycosylated form of EDN was specifically elevated in ovarian cancer patients. A cluster of COOH-terminal osteopontin was identified from two-dimensional gels of urine from cancer patients. Modified forms EDN and osteopontin fragments were elevated in early-stage ovarian cancers and a combination of both resulted to 93% specificity and 72% sensitivity. CONCLUSIONS: Specific elevated posttranslationally modified urinary EDN and osteopontin COOH-terminal fragments in ovarian cancer might lead to potential noninvasive screening tests for early diagnosis. Urine with less complexity than serum and relatively high thermodynamic stability of peptides or metabolites is a promising study medium for discovery of the novel biomarkers which may present in many non-urinary tract neoplastic diseases.

U-EPX levels and wheezing in infants and young children with and without RSV bronchiolitis.

An association between severe infant bronchiolitis due to respiratory syncytial virus (RSV) and subsequent wheezing is well documented. High levels of urinary eosinophil protein X (U-EPX) have been related to active disease in asthmatic children. The aim of this study was to analyse whether RSV bronchiolitis leads to an increase in U-EPX levels and whether wheezing is more common in children with high U-EPX values. Seventeen infants requiring in-ward care for verified RSV lower respiratory tract infection were followed and compared with age-matched controls. A reference group without a history of RSV bronchiolitis was also included. At inclusion at mean age 3.3 months and at follow-up at mean age 32.9 months, U-EPX levels were comparable in the RSV group. However, at follow-up at mean age 6.4 months, the RSV group had significantly increased levels of U-EPX compared with inclusion (median 167.8; range 46.2-470.7 vs. 122.8; 43.7-266.0 microg/mmol creatinine; P=0.023) and also significantly increased compared with the 6-month-old controls (167.8 vs. 93.0; 19.0-204.0 microg/mmol creatinine; P=0.0095). RSV infected subjects that experienced wheezing had significantly higher U-EPX values both at inclusion and at age 32.9 months than those who did not. Also, in the reference group (mean age 18.4 months), the children who had wheezed during the preceding year had higher U-EPX levels than those who had not wheezed. In conclusion, RSV bronchiolitis severe enough to require in-ward care produces a significant, but transient increase in U-EPX. Furthermore, a high U-EPX at baseline appears to be associated with an increased risk of future wheezing.

Scoring atopic dermatitis in infants and young children: distinctive features of the SCORAD index.

BACKGROUND: Atopic dermatitis (AD) affects infants, children, and adults with a wide degree of severity; several scoring systems have been used in trials and clinical practice. Infants and young children have a typical distribution of the lesions, but a correlation among skin surface involvement, intensity and subjective symptoms has not been reported in age groups. AIMS OF THE STUDY: To evaluate the clinical features of AD in infants and young children, by using the SCORAD index. A simplified scoring method for clinical practice is also discussed. METHODS: The SCORAD index was assessed in 63 infants and young children with AD [mean age (+/-SD) 17.5 +/- 11.15 months]; the single parameters of the index were evaluated, and compared with each other. Serum eosinophil cationic protein (s-ECP) and urinary eosinophil protein X (u-EPX) levels were determined and correlated with the SCORAD index. RESULTS: The presence of erythema, edema/papulation, and oozing/crust was significantly high in these patients. A strong positive correlation resulted among the three SCORAD index parameters (extent-intensity: P <0.001; extent-subjective symptoms: P <0.001; intensity-subjective symptoms: P <0.001). S-ECP and u-EPX levels positively correlated to almost every single parameter of the SCORAD index as well as to its total. CONCLUSIONS: Distinctive intensity items were found in infants and young children with AD. A strong correlation resulted among the extent, intensity, and subjective symptoms; each of the three parameters was positively correlated with the total SCORAD. Immunologic parameters positively correlated to each of the SCORAD index items, which remains the gold standard for assessing disease severity in clinical trials.

Urinary eicosanoid and tyrosine derivative concentrations in patients with vasculitides.

BACKGROUND: Vasculitides are classified on the basis of the type of cell involved, namely, eosinophilic vasculitides such as Churg-Strauss syndrome (CSS) and noneosinophilic vasculitides. However, knowledge on inflammatory mediators and oxidative tissue damage associated with vasculitides is insufficient. OBJECTIVE: We measured the urinary concentrations of inflammatory mediators and tyrosine derivatives to assess biomarkers associated with the pathophysiology of vasculitides. METHODS: Urine was collected from 9 patients with CSS during acute exacerbation and during clinical remission, 24 patients with rheumatoid arthritis in stable condition, and 8 patients with vasculitis diseases (VDs) during acute exacerbation. Leukotriene E 4 (LTE 4 ), 9alpha,11beta prostaglandin F 2 , and eosinophil-derived neurotoxin (EDN) concentrations were determined by enzyme immunoassay. 3-Bromotyrosine (BrY) and 3-chlorotyrosine (ClY) concentrations were determined by gas chromatography-mass spectrometry. RESULTS: The urinary LTE 4 , EDN, BrY, and ClY concentrations were significantly higher in the patients with CSS during acute exacerbation than in healthy control subjects and, except for urinary ClY concentration, significantly decreased during clinical remission. The urinary EDN and BrY concentrations were significantly higher in patients with CSS during acute exacerbation than in patients with VD during acute exacerbation. Only urinary LTE 4 concentration was significantly different between the patients with rheumatoid arthritis in stable condition and the patients with VD during acute exacerbation. CONCLUSION: Oxidative tissue damage caused by eosinophil peroxidase is a pathophysiological characteristic of eosinophil-associated diseases such as CSS. Urinary LTE 4 concentration may reflect a pathophysiological event involved in eosinophilic and noneosinophilic vasculitides. Cysteinyl-leukotriene pathways are potential therapeutic targets for small-vessel vasculitides.