鈴木 加余子

OBJECTIVE: Eosinophilic gastrointestinal disorders (EGIDs) are pathologically diagnosed by manually counting the eosinophils in biopsy tissue under microscopy. However, the skill of the individual examiner is considered to influence the accuracy of the resulting eosinophil count (EC). This study aimed to examine the effects of different examiners and histopathological staining types on the EC results of pathological tissues from patients with EGIDs. METHODS: Infiltrating eosinophils in lesion tissues from 10 eosinophilic esophagitis and 28 eosinophilic gastroenteritis cases were counted by three pathologists and one cytotechnologist. The intra- and inter-observer variabilities in ECs related to hematoxylin-eosin (HE), May-Grünwald Giemsa (MG), and direct fast scarlet (DFS) staining were investigated. The effects of examiner expertise and staining method on ECs were analyzed using a linear mixed effects model. The difference in color value (ΔE) for each staining method was obtained using the Commission International de l'Eclairage luminance-a-b model (L*a*b*). RESULTS: There was no significant intra-observer variability in eosinophil counting. Regarding inter-observer agreement, the examiner with the most EGIDs experience reported higher ECs than the other examiners for all three staining types (P<0.001). ECs were significantly higher with MG and DFS staining than with HE staining, regardless of the examiner (both P<0.001). Additionally, the ΔE values with DFS were higher than those with MG and HE. CONCLUSIONS: DFS staining offered the most selective visualization of eosinophils. ECs may vary depending on both the skill of the examiner and the staining method.

INTRODUCTION: Screening for ω-5 gliadin specific IgE antibody (sIgE) has high diagnostic utility in cases of suspected wheat-dependent exercise-induced anaphylaxis (WDEIA); however, negative cases may require confirmatory tests, such as the oral challenge test. Thus, newly identified allergens that can be used for the serological diagnosis of WDEIA are needed. This study aimed to identify additional sIgE biomarkers of WDEIA. METHODS: Forty-two patients with WDEIA (5 negative/37 positive for ω-5 gliadin sIgE) were enrolled. For comparison, 8 patients with immediate-type wheat allergy without WDEIA and 20 healthy controls without wheat allergy were also enrolled. Extracted wheat proteins were separated by 2D-PAGE. Proteins that reacted with serum IgE antibody in 2D Western blotting (2D-WB) were identified using mass spectrometry. Recombinant proteins were synthesized in Escherichia coli, and the antigenicity was tested using ELISA and the basophil activation test. RESULTS: In 2D-WB, nine proteins reacted with the serum IgE antibody from at least 60% of patients with WDEIA (n ≥ 25/42). ELISA revealed that alpha/beta gliadin MM1 exhibited the highest positive immunoreactivity in 23 of 26 patients who were positive for ω-5 gliadin sIgE (88%) and in 5 of 5 patients who were negative for ω-5 gliadin sIgE (100%). Alpha/beta gliadin MM1 exhibited significantly higher basophil activation in 14 patients with WDEIA when compared to 5 individuals without a wheat allergy. CONCLUSIONS: Alpha/beta gliadin MM1 sIgE exhibited the highest seropositivity, even among patients who were negative for ω-5 gliadin sIgE. The inclusion of alpha/beta gliadin MM1 in allergen-sIgE tests may improve the sensitivity for diagnosing WDEIA.

BACKGROUND: Immediate allergy caused by natto, a popular Japanese food prepared by fermenting soybeans with Bacillus subtilis var. natto, has been reported. Polygamma glutamic acid (PGA) in the sticky substance around natto beans has been reported to be a causative allergen of natto allergy. However, some of our patients with natto allergy were negative for PGA in the skin prick test (SPT). The sticky substance of natto beans contains a subtilisin family serine protease, nattokinase, along with PGA. In this study, we aimed to examine the antigenicity of nattokinase in natto allergy. METHODS: Eight patients, who developed symptoms after ingesting natto and positively reacted to natto (seven to the sticky substance in natto and one to the whole natto product) in their SPT, were enrolled in this study. To analyze IgE reactivity, we performed immunoblotting, ELISA, and SPT for natto (bean and sticky substance), and/or PGA, and/or nattokinase and/or cultured B. subtilis var. natto extract. RESULTS: In the SPT, four cases each were PGA-positive and PGA-negative. Immunoblotting of the sera from PGA-negative patients showed a protein band at 30 kDa, which was identified as nattokinase. Three PGA-negative cases, but not three PGA-positive cases, showed a positive reaction to nattokinase in the SPT and had a history of atopic dermatitis. The ELISA for nattokinase revealed a positive reaction of PGA-negative cases and negative reaction of PGA-positive cases in the SPT. CONCLUSIONS: We identified a subtilisin family serine protease, nattokinase, as a novel allergen in natto allergy patients unsensitized to PGA.