Eosinophil-derived Neurotoxin Concentrations Research Articles

Montelukast treatment response according to eosinophil-derived neurotoxin level in children with allergic rhinitis

Background Eosinophil-derived neurotoxin (EDN) is an important biomarker of eosinophilic inflammation. Methods This study evaluated Montelukast treatment response according to EDN concentration in children with perennial allergic rhinitis (PAR). Fifty-two children with PAR were recruited and took a combination of Montelukast (5mg) and Levocetirizine (5mg) “Mont/Levo Group” or only Montelukast (5mg) “Mont Group” for 4 weeks. All caregivers were instructed to record rhinitis symptoms for 4 weeks. EDN was measured before and after treatment. Results Daytime nasal symptom scores (DNSS) significantly decreased in both the Mont/Levo (p = 0.0001; n = 20) and Mont Group (p < 0.0001; n = 20), but there were no significant differences between the two groups. EDN concentration also significantly decreased after treatment in both groups (p < 0.0001 and p < 0.001, respectively). For secondary analysis, children with a high initial EDN concentration (EDN ≥ 53 ng/mL) were placed in the “High EDN Group”, while those with a lower initial EDN concentration (EDN < 53 ng/mL) were put in the “Low EDN Group”. Both groups experienced significant reductions in DNSS after either treatment regimen (p < 0.0001 and p = 0.0027, respectively) but the High EDN Group had greater reductions. EDN concentrations in the High EDN Group decreased significantly from either treatment (p < 0.0001). Conclusion We found that children with AR and a high serum EDN concentration may respond well to Montelukast treatment. A therapeutic strategy using EDN concentrations in patients with AR to evaluate therapeutic response may help improve quality of care.

Eosinophil Granule Proteins Involvement in Acute Appendicitis-An Allergic Disease?

Several pieces of evidence point to an allergic component as a trigger of acute appendicitis. As the Th2 immune response is characterized by eosinophil mobilization to the target organ and release of their cationic granule proteins, it is reasonable to investigate if the degranulation of eosinophils could be associated with the local injury. The primary aim of this study is to evaluate the participation of eosinophils granules proteins in acute appendicitis, both at local and systemic levels and the secondary aim is to evaluate the diagnostic accuracy of eosinophils granules proteins for the detection of acute appendicitis, as well as for distinguishing between complicated and uncomplicated acute appendicitis. Eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP) and eosinophil peroxidase (EP) are the most well-known eosinophil granule proteins. From August 2021 to April 2022, we present a prospective single-center study to evaluate the EDN, ECP, and EP concentrations simultaneously in appendicular lavage fluid (ALF) and the serum of 22 patients with acute phlegmonous appendicitis (APA), 24 with acute gangrenous appendicitis (AGA), and 14 normal controls. Concerning EDN, no differences were found between groups. ECP concentrations in ALF and serum were significantly higher in the histologically confirmed acute appendicitis compared to the control groups (p < 0.0001 and p < 0.0001, respectively). In ALF, no differences were found between ECP levels in APA: 38.85 ng/mL (IQR 26.50-51.77) and AGA 51.55 ng/mL (IQR 39.55-70.09) groups (p = 0.176). In the serum, no difference was found between ECP levels at APA: 39 ng/mL (IQR 21.30-56.90) and AGA: 51.30 ng/mL (IQR 20.25-62.59) (p = 0.100). For EP, the concentrations in ALF (p < 0.001) and serum (p < 0.001) were both higher in acute appendicitis compared to the control. In ALF, no difference was found between APA: 240.28 ng/mL (IQR 191.2-341.3) and AGA: 302.5 (IQR 227.7-535.85) (p = 0.236). In the serum, no differences were found between APA: 158.4 ng/mL (IQR 111.09-222.1) and AGA: 235.27 (IQR 192.33-262.51) (p = 0.179). Globally, the ALF concentrations were higher than serum concentrations, reflecting an intense inflammatory local reaction in AA. The optimal ECP cut-off for discriminating between acute appendicitis and the controls was >11.41 ng/mL, with a sensitivity of 93.5%, but with a specificity for identifying appendicitis of 21.4%, good discriminative power (AUC = 0.880). For EP, the optimal cut-off was >93.20 ng/mL, with a sensitivity of 87%, but with a specificity of 14.3% (AUC = 0.901), excellent discriminative power. For the diagnosis of perforated AA, the discriminative power of ECP and EP serum concentrations are weak (AUC = 0.562 and AUC = 0.664, respectively). Concerning the presence of peritonitis, the discriminative power of ECP and EP serum concentrations is acceptable, respectively: AUC = 0.724 and AUC = 0.735. Serum levels of EDN (p = 0.119), ECP (p = 0.586) and EP (p = 0.08) in complicated appendicitis were similar to uncomplicated appendicitis. Serum concentrations of ECP and EP can be added to decision-making AA diagnosis. A Th2-type immune response is present in AA. These data bring forward the role of an allergic reaction in the pathogenesis of acute appendicitis.

Multiplex Specific IgE Profiling in Neonatal Stool of Preterms Predicts IgE-Mediated Disease

Background: The natural history of immunoglobulin (Ig) E-mediated diseases in preterm infants is still elusive. We aimed at developing a non-invasive tool for detecting specific IgE (sIgE) and eosinophil-derived neurotoxin (EDN) in neonatal fecal samples and evaluating its predictive value for the development of IgE-mediated diseases during the first year of life. Methods: We developed a stool extraction protocol, followed by freeze-drying and solubilization. The sIgEs were investigated in neonatal fecal samples from 21 preterm infants with a 300-allergen multiplex and confirmed by a capillary Western blot with a nano-immunoassay. EDN concentration was used to investigate the local eosinophilic component. Results: The multiplexed allergen assay detected sIgE in all of the samples. A Western blot was used to confirm the results. The frequency and levels of sIgE in the neonatal fecal samples differed between the infants who developed IgE-mediated diseases and the controls. Allergen specificity was associated with the development of cow’s milk allergy (CMA) and asthma. The development of CMA was predicted by the sIgE response to milk proteins (sensitivity was 88%; specificity was 78%). The EDN levels predicted the development of IgE-mediated diseases (sensitivity was 100%; specificity was 75%). Conclusion: The non-invasive investigation of neonatal fecal sIgE is a promising tool for predicting the subsequent development of IgE-mediated diseases. Clinical Implications: The non-invasive sIgE and EDN profiling of neonatal fecal samples from preterm infants can predict the development of IgE-mediated diseases.

INCREASED EOSINOPHIL GRANULE PROTEIN PRODUCTION IN CHRONIC SPONTANEOUS URTICARIA

<h3>Introduction</h3> Chronic spontaneous urticaria (CSU) lesional and non-lesional skin reveals increased numbers of eosinophils, but the exact role of eosinophils in CSU pathogenesis remains unknown. A pathogenic role for eosinophils in CSU was supported by the results of a recent clinical trial demonstrating the efficacy of benralizumab in CSU. Previous studies have identified increased levels of major basic protein (MBP) and eosinophilic cationic protein (ECP) in the sera and skin of CSU subjects; however, eosinophil peroxidase (EPO), the granule protein uniquely expressed by eosinophils, and eosinophil-derived neurotoxin (EDN) were not examined. Therefore, we sought to determine whether eosinophil granule proteins (EGPs) are increased in CSU. <h3>Methods</h3> Sera from CSU subjects (n=11) and healthy controls (n=1) were collected and sent to the NIH Human Eosinophil Section for analysis with their novel multiplex immunoassay which simultaneously measures concentrations of MBP, ECP, EDN, and EPO using Luminex xMAP technology. CSU subjects completed disease activity surveys and complete blood count differentials were analyzed. <h3>Results</h3> Mean concentrations of MBP (1329.23 vs 399.55ng/ml), ECP (285.87 vs 45.31ng/ml), EDN (349.43 vs 38.53ng/ml), and EPO (54.96ng/ml vs undetectable) were markedly increased in CSU subjects compared to the healthy control. We found no correlation between eosinophil counts and granule protein levels, or with Urticaria Activity Scores. <h3>Conclusions</h3> Our results suggest that unique pathways may be activated in a subset of patients with CSU which led to the production of large quantities of EGPs. Additional research is needed to understand the pathways responsible for producing these granule proteins in CSU and their clinical impact.

S394 Esophageal Cytology Collection Device for Assessment of Esophageal Eosinophilia in Eosinophilic Esophagitis: Real World Experience

Introduction: Eosinophilic esophagitis (EoE) is a chronic condition characterized by symptoms of esophageal dysfunction and tissue eosinophilia. One barrier to clinical care in EoE is the need for repeat upper endoscopy (EGD) with biopsies to assess for tissue esophageal eosinophilia. A non-endoscopic esophageal cytology collection device (CytospongeTM) may be a viable alternative to assess for esophageal eosinophilia, but few studies have examined its use in EoE. Methods: We first performed a pilot study in which individuals with confirmed EoE underwent CytospongeTM procedure prior to a clinically-indicated EGD. The cytology results were compared to histology obtained via EGD (gold standard). Cytology and histology from mucosal biopsies were classified as active disease (≥1 eos/hpf cytology; ≥15 eos/hpf, respectively) or remission (0 eos/hpf; < 15 eos/hpf, respectively). The eosinophil-associated protein, eosinophil-derived neurotoxin (EDN) was assayed in the supernatant using ELISAs. We subsequently used the CytospongeTM to assess for mucosal eosinophils, in place of standard EGD with biopsy, in a subset of patients for clinical care. Results: For the pilot study, 7 patients (ages 20-53; 57% males) underwent the CytospongeTM procedure 1 hour prior to EGD. When using a cut-off of 1 eos/hpf on cytology, there was 100% concordance between the CytospongeTM cytology and esophageal biopsies for identifying active EoE and remission (3 active, 4 remission). There was also a trend towards higher concentrations of EDN in the CytospongeTM supernatant of active EoE (median 34.11 ng/mL) versus remission (median 9.5 ng/mL, p=0.057). Between September 2020 and May 2022, 15 CytospongeTM procedures were performed for clinical care (9 patients; ages 30-61; 56% male). Eight patients were undergoing food elimination diets and one was undergoing surveillance after sustained deep remission. No eosinophilia was seen in 10/15 cytology specimens and 5/15 had eosinophilia (range 1-15 eos)(Table). One patient underwent EGD shortly after CytospongeTM (1 eos on cytology), which demonstrated 10-30 eos/hpf on mucosal biopsy. The CytospongeTM procedure was well-tolerated with no significant adverse events. (Figure) Conclusion: The CytospongeTM is a novel mucosal assessment tool that is easily performed and correlates well with gold standard esophageal biopsies in EoE. It should be considered for use in eosinophilic esophagitis to avoid numerous EGD procedures.Figure 1.: a) Esophageal cytology collection device (CytospongeTM), b) Histology from standard esophageal biopsy, c) Cytology from CytospongeTM (same patient as b). Arrows point to eosinophils. Note: images previously published in McGowan EC, Aceves SS, CME Review: Noninvasive tests for eosinophilic esophagitis: Ready for use?. Ann Allergy Asthma Immunol 129 (2022); 27-34. Table 1. - Esophageal Cytology Collection Device (CytospongeTM) Results in Clinical Use Patient Age/Sex Current Treatment CytospongeTM Results Interpretation 1 61M Food Elimination Diet - reintroduced soy 0 eos/hpf Soy is not a trigger 2 50F Food Elimination Diet - reintroduced dairy 2 eos/hpf Dairy is a trigger 3a 30F Food elimination diet - reintroduced soy 0 eos/hpf Soy is not a trigger 3b Food elimination diet - reintroduced egg 0 eos/hpf Egg is not a trigger 3c Food elimination diet - reintroduced wheat 0 eos/hpf Wheat is not a trigger 4 44F Food elimination diet - four food elimination (4FED) 3 eos/hpf Not in remission on 4FED 5a 40F Food elimination diet - reintroduced wheat 0 eos/hpf Wheat is not a trigger 5b Food elimination diet - reintroduced dairy 1 eos/hpf Dairy is a trigger 6a 32M Food elimination diet - two food elimination (2FED) 0 eos/hpf Remission on 2FED Food elimination diet - reintroduced wheat 0 eos/hpf Wheat is not a trigger 7a 43M Food elimination diet - reintroduced fish 0 eos/hpf Fish is not a trigger Food elimination diet - reintroduced soy 0 eos/hpf Soy is not a trigger 8a 39M Food elimination diet - two food elimination (2FED) 6 eos/hpf Not in remission on 2FED Food elimination diet - four food elimination (4FED) 15 eos/hpf Not in remission on 4FED 9 36M Swallowed steroids twice daily and PPI 0 eos/hpf Maintenance of remission

Urine eosinophil-derived neurotoxin: A potential marker of activity in select eosinophilic disorders.

Biomarkers of eosinophilic disease activity, especially in the context of novel therapies that reduce blood eosinophil counts, are an unmet need. Absolute eosinophil count (AEC) does not accurately reflect tissue eosinophilia or eosinophil activation. Therefore, the aims of this study were to compare the reliability of plasma and urine eosinophil major basic protein 1, eosinophil cationic protein, eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase measurement and to evaluate the usefulness of eosinophil granule protein (EGP) measurement for the assessment of disease activity in patients with eosinophil-associated diseases treated with mepolizumab, benralizumab, or dexpramipexole. Eosinophil granule protein concentrations were measured in serum, plasma, and urine from healthy volunteers and patients with hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), and eosinophilic asthma using a multiplex assay. Urine EGP concentrations remained stable, whereas serum and plasma EGP concentrations increased significantly with delayed processing. Plasma (p) EDN, but not urine (u) EDN, concentration correlated with AEC and negatively correlated with prednisone dose. Both pEDN and uEDN decreased significantly following treatment of HES patients with benralizumab and EGPA patients with mepolizumab. uEDN appeared to increase with clinical relapse in both patient groups. Measurement of EGP in urine is noninvasive and unaffected by cellular lysis. Although plasma and urine EDN concentrations showed a similar pattern following benralizumab and mepolizumab treatment, the lack of correlation between AEC or prednisone dose and uEDN concentrations suggests that measurement of uEDN may provide a potential biomarker of disease activity in patients with HES and EGPA.

Serum Levels of Vascular Endothelial Growth Factor, Platelet Activating Factor and Eosinophil-Derived Neurotoxin in Chronic Spontaneous Urticaria-A Pilot Study in Adult Patients.

Chronic spontaneous urticaria (CSU) is a skin disease characterized by the presence of wheals, angioedema, or both for at least 6 weeks. Although, CSU is often regarded as autoimmune in nature, its etiology is not fully explained and interactions between various small molecules are still taken under account. The aim of this research was to investigate the mean serum concentration of vascular endothelial growth factor (VEGF), platelet activating factor (PAF), and eosinophil-derived neurotoxin (EDN) in relation to the disease activity and pruritus intensity in adult patients with CSU. Fifteen patients with CSU and 15 healthy subjects participated in this pilot study. Blood samples were taken to examine the mean serum levels of VEGF, PAF, and EDN by the enzyme-linked immunosorbent assay test (ELISA). The Urticaria Activity Score (UAS7) and The Visual Analogue Scale (VAS) were used to assess the disease activity and the pruritus intensity, respectively. Obtained results revealed that VEGF, PAF, and EDN concentrations were higher in patients with CSU compared with those of the control group, but only for VEGF it was statistically significant (p = 0.008). However, levels of all investigated cytokines were not significantly correlated neither with the disease activity nor with the pruritus intensity. Our results showed higher serum levels of VEGF, PAF, and EDN among CSU patients which may highlight a functional role of these cytokines in the disease’s pathogenesis. In contrast, VEGF, PAF, or EDN might not be useful to reflect the severity of symptoms.

Eosinophil-Derived Neurotoxin Predicts Response to Proton-Pump Inhibitor Treatment in Pediatric Eosinophilic Esophagitis.

There are no tests or patient factors to help predict the best treatment approach for a patient with eosinophilic esophagitis (EoE). The prevalence of proton-pump inhibitor (PPI) responsive EoE in children ranges from 30% to 71% with multiple studies showing similar characteristics in responders and nonresponders. Eosinophil-derived neurotoxin (EDN), an eosinophilic granule protein, measured in esophageal brushing has been shown to be a viable measure of disease activity in EoE. Our aim is to determine if EDN can help predict response to PPI in pediatric patients with EoE. We conducted a prospective cross-sectional study to compare EDN between PPI-responsive and PPI-nonresponsive EoE subjects from 2018 through 2020. Enrolled patients with active EoE were treated with high-dose PPI and underwent repeat endoscopy to determine PPI-responsiveness. EDN was measured at baseline endoscopy, before any treatment, and at follow up endoscopy, after PPI therapy. Subjects were divided into PPI-responsive and nonresponsive groups. EDN, endoscopic reference score (EREFS), and peak eosinophilic count (PEC) were compared. Fifteen out of the 36 enrolled subjects with EoE (age range 2-18 years, 73.3% male) were PPI-responsive and 21 (age range 2-19 years, 95.2% male) were PPI-nonresponsive. EDN concentration was significantly higher in the PPI-nonresponsive group than in the PPI-responsive group (219.1 ± 229 mcg/mL vs 75.7 ± 60 mcg/mL, respectively, P = 0.036). There was no difference between the two groups in EREFS (P = 0.55) or PEC (P = 0.15). EDN measured in esophageal epithelial samples obtained by brushing during endoscopy may predict PPI-responsiveness in children and young adults with EoE.

Japanese cedar pollen upregulates the effector functions of eosinophils.

BackgroundSymptoms of rhinitis and asthma can be exacerbated during Japanese cedar pollen (JCP)-scattering season, even in subjects who are not sensitized to JCP, suggesting that innate immune responses may contribute to this process. We previously reported that house dust mite directly activates the effector functions of eosinophils. Similar mechanisms may play roles in the JCP-related aggravation of allergic diseases.ObjectiveTo investigate whether JCP or Cry j 1, a major allergen of JCP, can modify the effector functions of eosinophils.MethodsEosinophils isolated from the peripheral blood of healthy donors were stimulated with either JCP or Cry j 1, and their adhesion to human intercellular adhesion molecule-1 was measured using eosinophil peroxidase assays. The generation of eosinophil superoxide anion (O2 −) was measured based on the superoxide dismutase-inhibitable reduction of cytochrome C. Concentrations of eosinophil-derived neurotoxin in the cell media were measured by enzyme-linked immunosorbent assay as a marker of degranulation.ResultsBoth JCP and Cry j 1 directly induced eosinophil adhesiveness, generation of O2 −, and release of eosinophil-derived neurotoxin. Both anti-αM and anti-β2 integrin antibodies blocked all of these eosinophil functions induced by JCP and Cry j 1. Similarly, PAR-2 antagonists also partially suppressed all of these effector functions induced by JCP and Cry j 1.ConclusionJCP and Cry j 1 directly activate the functions of eosinophils, and both αMβ2 integrin and partly PAR-2 are contributed to this activation. Therefore, JCP-induced eosinophil activation may play a role in the aggravation of allergic airway diseases in nonsensitized patients as well as in JCP-sensitized patients.

Using Eosinophil Biomarkers From Rectal Epithelial Samples to Diagnose Food Protein-induced Proctocolitis: A Pilot Study.

The gold standard diagnostic procedure for food protein-induced proctocolitis (FPIP) requires flexible sigmoidoscopy (FS). To date there is no validated, noninvasive test to confirm FPIP diagnosis. Eosinophil-derived neurotoxin (EDN), a product of eosinophil (EOS) degranulation, has been shown to correlate with eosinophil infiltration in other tissues. Our objective was to compare EDN concentrations in rectal epithelial samples from infants with FPIP with those from a control population. Children who underwent routine FS at Arnold Palmer Hospital for Children were enrolled in an IRB-approved, prospective, open-label pilot study between July 2017 and May 2019. We obtained rectal epithelial samples via: rectal swab, cytology brushing through FS, and rectal biopsy through FS. We then measured EDN levels in the samples and compared levels found in infants with FPIP against levels found in the control group. FPIP was defined as more than 60 EOS per 10 high-power fields (HPF) in rectal epithelial tissue obtained via rectosigmoid biopsy. Twenty-four patients were enrolled. The control group (n = 13) included patients with normal histopathology (84% boys, mean age 19 months, SD 6 months) and the FPIP group (n = 11) included patients with FPIP confirmed via biopsy (45% boys, mean age 6.9 months, SD 9 months). EDN concentration was significantly higher in the FPIP group than in the control group, for 2 sampling methods: rectal biopsy (183.6 ± 114.6 vs 76.6 ± 71.0 μg/mL; P = 0.010) and rectal swab (66.2 ± 64.8 vs 20.4 ± 22.2 μg/mL; P = 0.025). EDN concentrations measured from rectal swab and rectal biopsy samples is elevated and may be a useful tool to screen for FPIP in children.

Serum Eosinophil-Derived Neurotoxin Better Reflect Asthma Control Status Than Blood Eosinophil Counts

Although several biomarkers have been proposed for eosinophilic asthma, biomarkers for reflecting asthma control status remain controversial. Eosinophil-derived neurotoxin (EDN), a degranulated eosinophil protein, is an emerging biomarker in asthmatic patients. This study analyzed serum EDN concentrations in asthmatics and compared its performance with that of blood eosinophil count as an indicator of asthma control status. We enrolled 75 uncontrolled asthmatics, 56 controlled asthmatics, and 43 healthy controls from Asan Medical Center. Serum EDN levels (ng/mL) were measured using an enzyme-linked immunosorbent assay kit. The predictability of EDN for asthma control status was analyzed by univariate and multivariable logistic regression analyses. A receiver operating characteristic (ROC) curve analysis was conducted to compare the performances of a serum EDN level and blood eosinophil count as indicators of uncontrolled asthma status. The mean serum EDN level in the uncontrolled asthma group was higher than that in the controlled asthma and healthy groups (103.2 ± 60.2 vs 60.8 ± 49.7 vs 49.6 ± 28.3 ng/mL, P < .001). Serum EDN level was the significant parameter related to asthma control status in univariate and multivariable analysis (both P < .001). Serum EDN levels correlated with blood eosinophil counts (r= 0.510, P < .001). However, in the ROC analysis, serum EDN level showed a significantly better performance for predicting uncontrolled asthma status (area under the curve, 0.726 vs 0.628, P= .024). Serum EDN levels significantly differed between patients with controlled and uncontrolled status in adult asthmatics. To our knowledge, this is the first study to identify EDN as a better indicator of asthma control status than blood eosinophil count.

Relationship between airway inflammation and airflow limitation in elderly asthmatics.

BackgroundThe prevalence of asthma in elderly population has been increasing. Previous studies have demonstrated clinical characteristics of elderly asthmatics (EA). However, little is known regarding the influence of immunological change on the physiological status of EA.ObjectiveWe investigated the relationship between inflammatory mediators and the pulmonary function (PF) of EA.MethodsEligible adult asthmatics recruited from the Allergy Center of Saitama Medical University Hospital were classified into a non-EA group (<40 years old, n = 15) and an EA group (≥60 years old, n = 43). Sputum induction and PF tests were performed. Concentrations of an eosinophil-derived neurotoxin (EDN) and neutrophil elastase (NE) in sputum supernatants were measured by enzyme-linked immunosorbent assay and a fluorometric assay using a commercial assay kit, respectively. Cell counts and EDN and NE concentrations in sputum were compared between the 2 groups. The association among those parameters and PF were analyzed in each group.ResultsThe EA group had a significantly higher severe asthmatics proportion (p = 0.01), a lower current smokers proportion (p = 0.002), lower sensitization rate to aeroallergens (p = 0.012), several PFs deterioration (p < 0.0001) and lower total IgE levels (p = 0.001) than the non-EA group. Sputum neutrophil counts and NE concentrations were significantly higher in the EA group than those in the non-EA group (median neutrophil: 4.11 vs. 2.74 ×105/mL, p = 0.03; NE: 2.0 vs.1.6 µg/mL, p < 0.05, respectively), whereas sputum eosinophil counts and EDN concentrations were not. Sputum EDN concentrations were significantly positively correlated with sputum neutrophil counts (r = 0.39, p = 0.031) and NE concentrations (r = 0.57, p < 0.0001) only in the EA group. Eosinophil-related parameters were negatively correlated with several PFs in the 2 groups. Neutrophil-related parameters were negatively correlated with PFs only in the non-EA group.ConclusionThis study determines that in EA, persistent active eosinophilic airway inflammation is accompanied by advanced neutrophilic inflammation, which may contribute to deteriorated PF. This distinct airway inflammation may increase the severity of asthma in EA.

Prognostic significance of faecal eosinophil granule proteins in inflammatory bowel disease

Background: Non-invasive markers for predicting relapse would be a useful tool for the management of patients with inflammatory bowel disease. Eosinophil granulocytes and their granule proteins eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) have previously been shown to reflect disease activity in Crohn’s disease and ulcerative colitis.Aim: To examine the capacity of faecal ECP and EDN to predict relapse in ulcerative colitis and Crohn's disease, and to compare these proteins with faecal calprotectin.Methods: Patients with Crohn's disease (n = 49) and ulcerative colitis (n = 55) were followed prospectively until relapse or end of the two-year study period. Faecal samples were obtained every third month. The predictive value of ECP and EDN was assessed in Cox regression models.Results: In ulcerative colitis, a doubled EDN or ECP concentration was associated with a 31% and 27% increased risk of relapse, respectively. EDN levels were increased both at relapse and three months prior. By contrast, in Crohn's disease, the concentration of EDN was higher among patients in remission than in those who relapsed. Correlations between faecal calprotectin, ECP and EDN were observed in both diseases.Conclusions: We demonstrate that the risk of relapse in ulcerative colitis can be predicted by consecutively measuring faecal EDN every third month, and suggest EDN as a complementary faecal marker to calprotectin to predict future relapse in ulcerative colitis. Our finding of higher EDN in Crohn’s disease-patients staying in remission than in those who relapsed indicates different functions of the protein in ulcerative colitis and Crohn’s disease.

Analysis of the predicting factors of recurrent wheezing in infants

BackgroundClinically, asthma in children under 5 years old is under estimated because lack of diagnostic criteria. The current study was, therefore, designed to identify the predicting factors for recurrent wheezing in infants.MethodsOne hundred forty-five infants under 3-year old hospitalized with respiratory diseases were enrolled into this study. Patients were followed up for one-year period after being discharged from the hospital and were, then, divided into recurrent wheezing group and non-recurrent wheezing group based on whether there was recurrent wheezing or not. Wheezing or recurrent wheezing was specifically monitored in addition to blood tests for allergic and respiratory diseases.ResultsThe prevalence of eczema and respiratory syncytial virus (RSV) infection were significantly higher in recurrent wheezing group than in control group (74.2% vs 45.8%; 32.3% vs. 13.3%, respectively, both P < 0.05); the percentage of blood eosinophil and serum eosinophil-derived neurotoxin (EDN) concentration at admission were also higher in recurrent wheezing group than in control group (3.10 ± 2.54% vs. 1.31 ± 1.15%; 68.67 ± 55.05 ng/mL vs. 27. 36 ± 19.51 ng/mL; respectively, both P < 0.001). Multivariate logistic regression analysis on eosinophil count and serum EDN concentration in predicting recurrent wheezing revealed that the eosinophil count showed the lowest sensitivity (51.6%) and highest specificity (90.4%), with the area under the ROC curve (AUC) of 0.752 ± 0.041; and that, in contrast, the serum EDN showed the highest sensitivity (88.7%) and lowest specificity (56.6%), with AUC of 0.795 ± 0.037.ConclusionCombination of eosinophil count and serum EDN measurement may be better to predict the risk of recurrent wheezing in early life of childhood.

Expressions of Eotaxin-3, Interleukin-5, and Eosinophil-Derived Neurotoxin in Chronic Subdural Hematoma Fluids.

Eosinophils induce inflammation by releasing cytokines and cytotoxic granule proteins. Infiltration of eosinophilic granulocytes occurs in the outer membrane of chronic subdural hematomas (CSDHs). Eosinophils play an important role in the growth of CSDHs. However, the manner in which eosinophils accumulate within CSDH fluid remains undetermined. In the current study, we assessed the expression of eosinophil chemoattractants in CSDH fluids according to growth stage of CSDHs and examined the correlation between the two. CSDH fluids were obtained from 38 patients during trepanation surgery. Ecalectin, eotaxin-3, interleukin-5 (IL-5), and eosinophil-derived neurotoxin (EDN) concentrations were measured using enzyme-linked immunosorbent assay kits. For use as controls, serum samples were collected from 5 healthy adults, and cerebrospinal fluid (CSF) samples were collected from 5 adults with unruptured aneurysms. The percentage of eosinophils (%eosinophil) in CSDH fluids was calculated using Giemsa staining. Concentrations of ecalectin, eotaxin-3, IL-5, and EDN were nearly equivalent in serum and CSF samples; however, their concentrations were high in CSDH fluids. In particular, ecalectin and EDN levels in CSDH fluids were significantly higher than those in serum and CSF. Levels of eotaxin-3, IL-5, EDN, and %eosinophil were significantly higher in laminar type of CSDH, whereas that of ecalectin was not. The correlations between eotaxin-3 and IL-5, IL-5 and EDN, and EDN and %eosinophil were statistically significant (p < 0.01). Our data suggest that eotaxin-3 is a chemoattractant of eosinophils. IL-5 induces the activation of eosinophils subsequent to degranulation of EDN into CSDH fluids. These factors may serve as novel therapeutic targets for managing CSDH.

Eosinophilic biomarkers for detection of acute exacerbation of chronic obstructive pulmonary disease with or without pulmonary embolism.

Eosinophilia has been implicated in the pathophysiology of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). However, the role of eosinophil activation in the development of AECOPD remains unclear. In the present study, the reliability of plasma levels of eosinophil activation markers, including eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPX), were measured and used as diagnostic biomarkers of AECOPD with or without pulmonary embolism (PE). A total of 47 patients with AECOPD, 30 patients with AECOPD/PE and 35 healthy adults were enrolled in the present study. Plasma levels of ECP, EDN, EPX and MBP were measured using commercial ELISA kits. The mean concentrations of plasma ECP, EDN, EPX and MBP in the patients with AECOPD was significantly 2.87-, 3.06-, 1.60- and 1.92-fold higher, respectively, compared with the control group (P<0.05). Similar results were obtained in patients with AECOPD/PE, for whom plasma levels of ECP, EDN, EPX and MBP were significantly 2.06-, 2.21-, 1.42- and 2.42-fold higher, respectively, compared with the controls (P<0.05). No significant differences were observed in the levels of these proteins between patients with AECOPD or AECOPD/PE. Among the four potential markers, ECP was determined to be the optimal marker for distinguishing patients with AECOPD or AECOPD/PE from the controls. No significant correlation was observed between marker concentrations and gender, age or disease severity. The results of the present study may have clinical applications in the diagnosis of AECOPD using these novel biomarkers.

Eosinophil Cationic Protein (ECP), a predictive marker of bullous pemphigoid severity and outcome

Bullous Pemphigoid (BP) is an inflammatory rare autoimmune bullous dermatosis, which outcome cannot be predicted through clinical investigations. Eosinophils are the main immune infiltrated cells in BP. However, the release of Major Basic Protein (MBP), Eosinophil Derived Neurotoxin (EDN), and Eosinophil Cationic Protein (ECP) upon eosinophil activation has still not been evaluated with respect to BP development. MBP, EDN and ECP were measured by ELISA in serum (n = 61) and blister fluid (n = 20) of patients with BP at baseline, and in serum after 2 months of treatment (n = 41). Eosinophil activation in BP patients was illustrated at baseline by significantly higher MBP, EDN and ECP serum concentrations as compared with control subjects (n = 20), but without distinction according to disease severity or outcome. EDN and ECP values were even higher in the blister fluids (P < 0.01 and P < 0.05, respectively), whereas MBP values were lower (P < 0.001). ECP serum concentration decreased after 60 days of treatment in BP patients with ongoing remission but not in patients who later relapsed (P < 0.05). A reduction of at least 12.8 ng/mL in ECP concentrations provided a positive predictive value for remission of 81%, showing that ECP serum variation could be a useful biomarker stratifying BP patients at risk of relapse.

Reductions in eosinophil biomarkers by benralizumab in patients with asthma

BackgroundEosinophilic inflammation is frequently associated with increased asthma severity. Benralizumab is a humanized, afucosylated, anti–interleukin-5Rα monoclonal antibody that selectively depletes eosinophils and basophils through enhanced antibody-dependent cell-mediated cytotoxicity. ObjectiveTo study effects of benralizumab on eosinophil counts and activity following administration to asthma patients. MethodsSera were collected from asthma patients enrolled in two clinical studies. Placebo or benralizumab was subcutaneously administered to patients in Phase I (100 or 200 mg, multiple doses; N = 14; NCT00659659) and Phase IIa (25, 100, or 200 mg every 4 weeks; N = 24; NCT00783289) studies. Sera were also collected from healthy volunteers (N = 20) for comparison. Blood eosinophils, IL-5, eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), eotaxin/chemokine (C–C motif) 11 (CCL11), eotaxin-2/CCL24, tumor necrosis factor (TNF), and interferon-γ (IFN-γ) were measured at baseline and post-treatment. ResultsIncreased EDN concentrations were observed in sera of patients from both studies relative to healthy volunteers (p < 0.05). At baseline, sera EDN concentrations correlated with blood eosinophil counts (rs = 0.5; p < 0.05). Benralizumab reduced blood eosinophil numbers and sera EDN and ECP relative to baseline (p < 0.05). No changes in TNF or IFN-γ were observed, while serum IL-5, eotaxin/CCL11, and eotaxin-2/CCL24 increased after benralizumab administration vs. placebo (p < 0.05). ConclusionsIn two independent studies, serum IL-5, EDN, and ECP were modulated following benralizumab. Eosinophil depletion after benralizumab also resulted in significant reductions in EDN and ECP concentrations, suggesting that cytotoxic granule proteins were not released after eosinophil reduction.

Eosinophil-derived neurotoxin, elastase, and cytokine profile in effusion from eosinophilic otitis media

BackgroundEosinophilic otitis media (EOM) is an intractable disease characterized by a remarkably viscous effusion and accumulation of numerous eosinophils in both the middle ear effusion and the mucosa. The key factors in EOM pathogenesis remain unclear. The purpose of this study is to identify the important factors involved in EOM pathogenesis. MethodsMiddle ear effusion samples were collected from 12 patients with EOM and 9 patients with secretory otitis media (SOM), as controls. Multiple cytokines in the effusion were measured using a Bio-Plex™ Human Cytokine 27-Plex panel. Eosinophil-derived neurotoxin (EDN) and elastase were measured by ELISA. The concentrations of EDN, elastase, and each cytokine were compared between the EOM and SOM groups. Furthermore, in the EOM group, each cytokine was examined for correlation with EDN and elastase. ResultsEDN and elastase concentrations were significantly higher in the EOM group than in the SOM group (p < 0.05). IL-5, IL-1β, MIP-1α, G-CSF, IL-1ra, IL-4, IFN-γ, MIP-1β, IL-10, TNF-α, VEGF, and IL-2 concentration was significantly higher in the EOM group than in the SOM group (p < 0.05). Significant positive correlations were found between EDN and IL-1ra, IL-2, IL-5, IL-9, IL-13, eotaxin, MIP-1α, PDGF-BB, and RANTES in the EOM group (p < 0.05). ConclusionsOur study showed that IL-5, IL-2, MIP-1α, and IL-1ra are the important factors involved in EOM pathogenesis. Furthermore, not only eosinophil, but also neutrophil are involved in middle ear inflammation of EOM.

Development of a suspension array assay in multiplex for the simultaneous measurement of serum levels of four eosinophil granule proteins

The concentrations of major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO) have been associated with eosinophilic disease severity. Whereas a variety of techniques have been used to measure individual eosinophil granule protein concentration, none of these methods efficiently measures MBP, ECP, EDN and EPO simultaneously. A multiplex suspension array system was developed to simultaneously measure the concentrations of MBP, ECP, EDN and EPO in serum. The assay showed excellent inter- and intra-assay reliability, and serum levels of MBP, ECP and EDN from eosinophilic subjects analyzed by ELISA and multiplex were highly correlated (r=0.8579; P<0.0001, r=0.6356; P=0.0006 and r=0.8600; P<0.0001, respectively, Spearman rank correlation). Moreover, the multiplex assay required 500-fold less serum than a single ELISA to achieve comparable sensitivity. Absolute eosinophil count and eosinophil surface expression of the activation marker, CD69, were significantly correlated with concentrations of MBP, EDN and EPO, but not ECP, in serum from eosinophilic subjects. Furthermore, subjects with eosinophilic gastrointestinal disorder and normal peripheral absolute eosinophil counts (<0.5×109/l) had significantly increased concentrations of MBP (P<0.0001), ECP (P<0.0001), EDN (P=0.0001) and EPO (P<0.0001) compared to normal donors. In summary, the eosinophil granule protein multiplex assay provides a rapid and reliable way to measure eosinophil granule protein levels and should prove useful in assessing patterns of degranulation in patients with eosinophilic disorders.