Eosinophil-derived neurotoxin levels in early childhood and association with preschool asthma - A prospective observational study.

INTRODUCTION: Eosinophil-derived neurotoxin (EDN) is related to childhood asthma, while normal values are lacking. We aimed to document serum EDN levels at 1 and 3 years in general and in non-atopic children, and explore if EDN levels differed by sex or were associated with preschool asthma at 3 years. METHODS: From the PreventADALL birth cohort, we included 1233 children with EDN analysed using ImmunoCAP at 1 and/or 3 years. Non-atopic children had no history of wheeze, asthma, allergic sensitization or atopic dermatitis. Preschool asthma was defined as having ≥3 episodes of bronchial obstruction between 2 and 3 years, plus doctor diagnosed asthma and/or asthma medication use by 3 years. The upper limit of normal (ULN) of EDN was defined as the 95th percentile. With Youden Index we calculated EDN cut-off levels for risk of preschool asthma. RESULTS: The overall median (ULN) EDN levels were 27.4 (121) µg/L at 1 year (n = 787), and 20.1 (87.8) µg/L at 3 years (n = 857). Non-atopic children had EDN levels of 24.0 (107) µg/L at 1 year (n = 147), and 17.3 (84.6) µg/L at 3 years (n = 173). EDN levels were higher in boys compared to girls; 32.0 (133) versus 24.5 (97.0) µg/L at 1 year, and 20.9 (96.3) versus 19.0 (72.4) µg/L at 3 years. Preschool asthma was observed in 109/892 (12.2%) children. Higher EDN levels at 1 (>26.7 µg/L) and 3 (≥20.5 µg/L) years were associated with preschool asthma; adjusted OR (95% CI) 2.20 (1.09, 4.41) and 4.68 (2.29, 9.55), respectively. CONCLUSION AND CLINICAL RELEVANCE: We report EDN values in early childhood, demonstrating higher levels at 1 compared to 3 years and in boys compared to girls at both ages. Higher EDN levels at both ages were associated with preschool asthma. However, EDN cut-off levels for preschool asthma were overall lower than the ULN of non-atopic children, limiting translation into clinical practice.

Serum Eosinophilic Cationic Protein Is a Reliable Biomarker for Childhood Asthma.

BACKGROUND: Eosinophilic cationic protein (ECP) is associated with airway inflammation and asthma. However, the clinical value of measuring ECP in childhood asthma is not fully known. We aimed to study the diagnostic performance of serum ECP and other common asthma biomarkers, individually and in combinations. METHODS: In a cross-sectional study, 5-16-year-old children with current asthma (CA) (n = 37), transient asthma (TA) (n = 43), (previous history of wheezing/asthma), and healthy children (HC) (n = 86) were investigated for ECP, blood eosinophil count (B-Eos), fractional exhaled nitric oxide (FeNO), and lung function, i.e., spirometry (forced expiratory volume during the first second [FEV1]/forced vital capacity [FVC] ratio). RESULTS: Both ECP and B-Eos were higher in CA compared to TA (p < 0.01) and HC (p < 0.0001). ECP and B-Eos were also higher in TA compared to HC (p < 0.05 and p < 0.001, respectively). FeNO was higher in CA (p < 0.0001) and TA (p < 0.01) compared to HC but similar between the asthma groups. The FEV1/FVC ratio was lower in CA compared to TA and HC (both p < 0.01) but similar between TA and HC. The best diagnostic performance regarding CA was found for ECP and B-Eos with receiver operating characteristics area under curve (AUC) of 0.801 and 0.810, respectively. The optimal cutoff for ECP (29 µg/L) yielded a sensitivity and specificity of 70.3% and 81.4%. The corresponding AUCs for FeNO and FEV1/FVC were 0.732 and 0.670, respectively. ECP and B-Eos showed the highest AUCs (0.669 and 0.673) for differentiation between CA and TA. Combining ECP with FeNO and FEV1/FVC increased the odds ratio (OR) for having CA from OR 3.97-10.3 for the single biomarkers to OR 20.2 (95% confidence interval: 5.76-68.6). CONCLUSION: Our results show that serum ECP is a reliable biomarker in the diagnosis of childhood asthma, with additional value in combination with FeNO and FEV1/FVC, and that ECP can be an alternative to B-Eos.

Human Allergen-Specific IgG Subclass Antibodies Measured Using ImmunoCAP Technology.

BACKGROUND: Knowledge of human IgG subclass antibody responses to various allergens has been hampered by a lack of reliable standardized assays. The aim here was to develop quantitative immunoassays for human IgG1, IgG2, and IgG3 antibodies using ImmunoCAP® technology and to evaluate their application. METHODS: Enzyme conjugates with isotype-specific monoclonal antibodies and calibrators composed of purified myeloma paraproteins were developed for each assay and used together with other standardized assay reagents for the Phadia® 100 instrument. The calibrators were adjusted to the international reference preparation IRP 67/86. The assays were characterized and used together with other standard ImmunoCAP assays to measure antibodies to various allergens in preliminary studies. RESULTS: The new assays had limits of quantitation of 1.0 (IgG1), 4.6 (IgG2), and 0.04 mgA/L (IgG3), and coefficients of variation of <20%. Only some minor cross-reactivity with IgG2 was observed for the specific IgG1 assay. The specific IgG2 assay showed a bias for the allotype G2m(23) and compensation factors were used to adjust the measured concentrations accordingly. Preliminary studies indicated a strong and stable IgG4 antibody response to ß-lactoglobulin in healthy individuals, a high IgG1 and even higher IgG2 antibody response to house dust mite in sensitized and nonsensitized subjects, and a mixed IgG subclass response to venom allergens in allergic patients with increasing IgG4 antibody levels during venom immunotherapy. CONCLUSIONS: The new research assays are valuable tools for immunological studies, enabling the characterization of antibody profiles using a standardized approach, and facilitating data interpretation and the comparison of results across studies.

Monitoring Ara h 1, 2 and 3-sIgE and sIgG4 antibodies in peanut allergic children receiving oral rush immunotherapy.

BACKGROUND: The aim was to study the clinical efficacy and safety of rush oral immunotherapy (OIT) for severe peanut-allergic children and to measure the antibody responses. METHODS: Eighteen Japanese children were enrolled after a positive double-blind, placebo-controlled food challenge (DBPCFC). The patients ingested peanuts up to 3-5 times a day every 30 min, increasing the dose by 20% every time. The goal dose was 3.5-7 g. IgE, IgG, and IgG4 antibody levels to peanut, and peanut allergen components were measured during up to 3 yr of maintenance treatment. RESULTS: Two children dropped out due to side effects. Sixteen patients (14 boys and two girls, median: 9 yr range: 5-14 yr) achieved the goal dose after a median of 11 days (range: 4-19 days). Their median threshold dose at DBPCFC was 0.20 g (range: 0.015-1.0 g). All were sensitized to Ara h 2. Fourteen of them had a history of previous anaphylaxis. In total, 173 adverse events were observed during the treatment (27% of the total ingestions) of which 74 needed medications. The median IgE, IgG, and IgG4 antibody levels to peanut increased during rush OIT. The IgG4 levels were high during the whole maintenance phase. IgE and IgG4 antibodies to Ara h 2 dominated the serological response during the treatment. CONCLUSIONS: The present rush OIT protocol for children with severe peanut allergy was effective and relatively safe. A sustained Ara h 2-specific IgG4 antibody response characterized the treatment.

Fecal and Serum Granulocyte Protein Levels in Inflammatory Bowel Disease and Irritable Bowel Syndrome and Their Relation to Disease Activity.

INTRODUCTION: Neutrophilic calprotectin (CP) and myeloperoxidase (MPO), neutrophil gelatinase-associated lipocalin (NGAL), and eosinophil-derived neurotoxin (EDN) are suggested proxy markers for gut inflammation. However, there are insufficient supporting data for MPO, NGAL, and EDN. METHODS: In a cross-sectional investigation including adult patients, we studied the ability of CP, MPO, NGAL, and EDN, measured in fecal and serum samples, to differentiate between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), and to predict disease activity. RESULTS: Fifty-nine patients had ulcerative colitis (UC), 38 had Crohn's disease, and 100 patients had IBS. The protein concentrations were higher in patients with IBD in the fecal samples ( P < 0.001) and the serum samples ( P < 0.01), and they correlated weakly (r s ≤0.38) between the sample sources. Fecal EDN was higher in patients with Crohn's disease compared with UC (1.79 vs 0.50 mg/kg, P = 0.016). The neutrophilic proteins were superior to EDN in the fecal samples for differentiating between patients with IBD and IBS. Fecal MPO (cutoff: 0.86 mg/kg) had the highest sensitivity (74.7%) and specificity (84.6%). Combining fecal CP and MPO increased the sensitivity to 82.3% (specificity: 73.6%). NGAL (cutoff: 196.9 µg/L) showed the best discriminating performance in serum (sensitivity: 62.9%; specificity: 68.0%). Serum NGAL (cutoff: 272.4 µg/L) predicted active disease in UC (Partial Mayo Score ≥2) with a sensitivity and specificity of 57.1% and 83.3%, respectively. DISCUSSION: Fecal MPO and serum NGAL are promising novel biomarkers, in addition to fecal CP, for differentiating between IBD and IBS. Serum NGAL may also predict disease activity in patients with UC.

Respiratory symptoms, exacerbations and sleep disturbances are more common among participants with asthma and chronic airflow limitation: an epidemiological study in Estonia, Iceland and Sweden.

BACKGROUND: Chronic airflow limitation (CAL) is a hallmark of chronic obstructive pulmonary disease but is also present in some patients with asthma. We investigated respiratory symptoms, sleep and health status of participants with and without CAL with particular emphasis on concurrent asthma using data from adult populations in Iceland, Estonia and Sweden investigated within the Burden of Obstructive Lung Disease study. METHODS: All participants underwent spirometry with measurements of forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) before and after bronchodilation. CAL was defined as postbronchodilator FEV1/FVC below the lower limit of normal. IgE-sensitisation and serum concentrations of eosinophil-derived neurotoxin (S-EDN) were assessed in a subsample. The participants were divided into four groups: no self-reported doctor's diagnosed asthma or CAL, asthma without CAL, CAL without asthma and asthma and CAL: χ2 test and analysis of variance were used in bivariable analyses and logistic and linear regression when analysing the independent association between respiratory symptoms, exacerbations, sleep-related symptoms and health status towards CAL, adjusting for centre, age, sex, body mass index, smoking history and educational level. RESULTS: Among the 1918 participants, 190 (9.9%) had asthma without CAL, 127 (6.6%) had CAL without asthma and 50 (2.6%) had CAL with asthma. Having asthma with CAL was associated with symptoms such as wheeze (adjusted OR (aOR) 6.53 (95% CI 3.53 to 12.1), exacerbations (aOR 12.8 (95% CI 6.97 to 23.6), difficulties initiating sleep (aOR 2.82 (95% CI 1.45 to 5.48), nocturnal gastro-oesophageal reflux (aOR 3.98 (95% CI 1.79 to 8.82)) as well as lower physical health status. In these analyses, those with no asthma and no CAL were the reference group. The prevalence of IgE-sensitisation was highest in both asthma groups, which also had higher levels of S-EDN. CONCLUSION: Individuals with self-reported asthma with CAL suffer from a higher burden of respiratory and sleep-related symptoms, higher exacerbation rates and lower health status when compared with participants with asthma alone or CAL alone.

Evaluating the Performance of Two Automated Anti-drug Antibodies Assays for Infliximab and Adalimumab Without Acid Dissociation.

Monitoring anti-drug antibodies (ADAs) to infliximab and adalimumab is critical to treatment management in various autoimmune disorders. The growing need for proactive therapeutic monitoring further requires the detection of ADAs in the presence of measurable concentrations of infliximab or adalimumab. To provide robust analytical assays for clinical application, we evaluated two automated immunoassays developed using ImmunoCAP™ technology and based on the bridging format to measure serum ADAs to infliximab and adalimumab respectively. Without an acid-dissociation step, these research prototype assays can detect a positive control monoclonal ADA towards infliximab and adalimumab, ranging from < 25 ng/ml to 10,000 ng/mL. Both assays exhibit imprecision less than 20% at different ADA titer levels and can distinguish ADAs towards different drug targets. In method comparison using authentic patient samples, the quantitative results of the ADA assays are not directly comparable to two existing clinical immunoassays for ADAs (correlation coefficient rs = 0.673 for infliximab ADAs; rs = 0.510 for adalimumab ADAs), presumably due to the lack of commutable ADA standards and the polyclonal nature of ADAs. Nevertheless, there is qualitative agreement between the methods when evaluating putative positive and negative patient samples (overall agreement 0.83 for infliximab ADAs; 0.76 for adalimumab ADAs). Biotin and high levels of rheumatoid factors may interfere with the performance of the automated assays due to competitive binding with the biotinylated drug and non-specific formation of bridging complexes. The two ImmunoCAP assays can provide new analytical methods for proactive therapeutic monitoring of adalimumab and infliximab.

Reference values of serum total IgE in Uppsala - comparison over four decades.

Background: Total immunoglobulin E (IgE) analysis is a common tool in allergy diagnosis. Suggested reference values for IgE are divergent and sometimes based on outdated assay methods. We aimed to validate the published reference values (geometric mean [GM]: 13.2 kU/L, upper limit of normal [ULN], 114 kU/L) shown in an Uppsala cohort from 1974 using Phadebas IgE PRIST, and the suggested clinical threshold of 100 kU/L (Zetterström and Johansson 1981). Methods: Immunoglobulin E was measured in two Uppsala cohorts from 1997 (Blood bank) and 2011 to 2013 (the European community respiratory health survey part III [ECRHS III]) using ImmunoCAP™ Total IgE. For the reference value calculations, exclusion criteria were atopy (both cohorts), doctor's diagnosis of asthma and self-reported allergy (hay fever, rhinitis, rash) (only ECRHS III). Upper limit of normal was defined as mean + 2 standard deviations (SD) calculated using log-transformed values and back-transformation of the ULN prior to presentation. Common imputation methods for results below the assay range were evaluated. Results: The average GM was 14.2 kU/L (Blood bank, n = 63; imputation method range: 16.9-17.4 kU/L; ECRHS III, n = 113: 10.7-11.6 kU/L) and the overall mean ULN was 118 kU/L (Blood bank: 113-130 kU/L; ECRHS III: 104-128 kU/L). The clinical sensitivity and specificity of the 100 kU/L IgE threshold were 37.8 and 94.3% for atopy, 34.9 and 89.5% for doctor's diagnosis of asthma, and 24.5 and 97.3% for any self-reported allergy (ECRHS III). Conclusion: The calculated ULN values were similar between the cohorts. We conclude that the total IgE reference values shown for Uppsala subjects from 1974 are still valid and suitable also for the ImmunoCAP Total IgE assay. The 100 kU/L threshold for total IgE had a low sensitivity but high specificity for atopy, asthma, and allergy.