Reiko Teshima

Proteomics-based allergen analysis in plants

UNLABELLEDnPlants may trigger hypersensitivity reactions when individuals with allergies consume foods derived from plant materials or inhale plant pollen. As each plant food or pollen contains multiple allergens, proteomics is a powerful tool to detect the allergens present. Allergen-targeted proteomics, termed allergenomics, has been used for comprehensive identification and/or quantification of plant allergens, because it is a simple and inexpensive tool for rapid detection of proteins that bind to IgE. There are increasing numbers of reports on the applications of allergenomics. In this review, we outline some of the applications of proteomics, including: (i) identification of novel allergens, (ii) allergic diagnoses, (iii) quantification of allergens, and (iv) natural diversity of allergens, and finally discuss (v) the use of allergenomics for safety assessment of genetically modified (GM) plants.nnnBIOLOGICAL SIGNIFICANCEnRecently, the number of allergic patients is increasing. Therefore, a comprehensive analysis of allergens (allergenomics) in plants is highly important for not only risk assessment of food plants but also diagnosis of allergic symptoms. In this manuscript, we reviewed the recent progress of allergenomics for identification, quantification and profiling of allergens. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Sensitization to acid‐hydrolyzed wheat protein by transdermal administration to BALB/c mice, and comparison with gluten

An increasing number of studies have shown that hydrolyzed wheat protein (HWP) can induce IgE‐mediated hypersensitivity by skin contact and/or food ingestion. However, there has been no study of the sensitizing potential of HWP. In this study, the possibility of transdermal pathway for sensitization to acid‐HWP (HWP1) was investigated using BALB/c mice, and compared with that of gluten.

Evaluation of Allergenicity of Acid-Hydrolyzed Wheat Protein Using an in vitro Elicitation Test

Background: We performed an in vitro elicitation test to determine the ability of different types of wheat-allergic patients’ IgE to induce humanized mast cell activation after the addition of various time-treated acid-hydrolyzed wheat proteins (HWPs). Methods: The reactivity of heat- and various time-treated acid-hydrolyzed glutens (acid-HGs) and commercial acid-HWP (HWP1), using serum IgE from wheat allergy accompanied by skin and rhinoconjunctival sensitization to HWP1 in the facial soap, pediatric subjects with food allergy to native wheat, adult wheat-dependent exercise-induced anaphylaxis subjects, and nonatopic healthy subjects, was elucidated by dot blot and a luciferase assay-based in vitro elicitation test (EXiLE test). Results: Serum from subjects sensitized with HWP1 reacted only to acid-HGs (acid-HGs treated for 0.5–3 or 6 h), but not native gluten, in the results of the dot blot. In contrast, sera from pediatric subjects sensitized with native wheat reacted to native gluten more strongly and showed only slight reactions to 0.5- to 1-hour-treated acid-HGs. The results of the in vitro elicitation test showed that acid hydrolyzation of the gluten attenuated antigen-induced luciferase expression in a time-dependent manner for sera from native-wheat-sensitized pediatric subjects. On the other hand, in the sera from HWP1-sensitized subjects, acid hydrolyzation of the gluten for 0.5 h dramatically increased luciferase expression. Conclusions: Even after prolonged hydrolyzation, acid-HGs still retained the ability to activate mast cells in the case of HWP1-sensitized subjects.

RNAi-mediated suppression of endogenous storage proteins leads to a change in localization of overexpressed cholera toxin B-subunit and the allergen protein RAG2 in rice seeds

Key messageRNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2.AbstractA purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2.

MucoRice-cholera Toxin B-subunit, a Rice-based Oral Cholera Vaccine, Down-regulates the Expression of α-Amylase/trypsin Inhibitor-like Protein Family as Major Rice Allergens

To develop a cold chain- and needle/syringe-free rice-based cholera vaccine (MucoRice-CTB) for human use, we previously advanced the MucoRice system by introducing antisense genes specific for endogenous rice storage proteins and produced a molecularly uniform, human-applicable, high-yield MucoRice-CTB devoid of plant-associated sugar. To maintain the cold chain-free property of this vaccine for clinical application, we wanted to use a polished rice powder preparation of MucoRice-CTB without further purification but wondered whether this might cause an unexpected increase in rice allergen protein expression levels in MucoRice-CTB and prompt safety concerns. Therefore, we used two-dimensional fluorescence difference gel electrophoresis and shotgun MS/MS proteomics to compare rice allergen protein expression levels in MucoRice-CTB and wild-type (WT) rice. Both proteomics analyses showed that the only notable change in the expression levels of rice allergen protein in MucoRice-CTB, compared with those in WT rice, was a decrease in the expression levels of α-amylase/trypsin inhibitor-like protein family such as the seed allergen protein RAG2. Real-time PCR analysis showed mRNA of RAG2 reduced in MucoRice-CTB seed. These results demonstrate that no known rice allergens appear to be up-reregulated by genetic modification of MucoRice-CTB, suggesting that MucoRice-CTB has potential as a safe oral cholera vaccine for clinical application.

Differential Analysis of Protein Expression in RNA-Binding-Protein Transgenic and Parental Rice Seeds Cultivated under Salt Stress

Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in protein expression under salt stress, NT and RBP rice were cultured with or without 200 mM sodium chloride. Only two protein spots differed between NT and RBP rice seeds cultured under normal conditions, one of which was identified as a putative abscisic acid-induced protein. In NT rice seeds, 91 spots significantly differed between normal and salt-stress conditions. Two allergenic proteins of NT rice seeds, RAG1 and RAG2, were induced by high salt. In contrast, RBP rice seeds yielded seven spots and no allergen spots with significant differences in protein expression between normal and salt-stress conditions. Therefore, expression of fewer proteins was altered in RBP rice seeds by high salt than those in NT rice seeds.

A New Reliable Method for Detecting Specific IgE Antibodies in the Patients with Immediate Type Wheat Allergy due to Hydrolyzed Wheat Protein: Correlation of Its Titer and Clinical Severity

BACKGROUNDnImmediate-type wheat allergy caused by a specific hydrolyzed wheat protein (HWP-IWA), Glupearl 19S (GP19S), typically develops food-dependent exercise-induced anaphylaxis (FDEIA), but is different from conventional FDEIA, or simple wheat allergy in many aspects. The skin prick test (SPT) is considered to be the most effective method for diagnosis of HWP-IWA. As SPT is a relatively qualitative method, we developed quantitative and high-throughput test method for HWP-IWA.nnnMETHODSnAn enzyme-linked immunosorbent assay (ELISA)-based GP19S-specific IgE assay was tested using sera from 14 HWP-IWA and five conventional wheat-dependent exercise-induced anaphylaxis (CO-WDEIA) patients, as well as five healthy subjects. Then a validation study at five different institutions was carried out using sera from 10 HWP-IWA and five CO-WDEIA patients, as well as five healthy subjects different from the previous studies.nnnRESULTSnThe mean unit values converted from measured absorbance of ELISA were 68.3, 1.3 and 1.1 respectively. Furthermore, the validation study revealed reproducible results across all five institutions, with the standard deviation (SD) being 0.3-0.4 for the healthy group, 0.2-0.6 for the CO-WDEIA group, and 3.8-9.6 for HWP-IWA group except for one case. One case of HWP-IWA was excluded from analysis due to the high SD of 53.3 units, indicating that samples with a unit value > 100.0 will affect inter-laboratory reproducibility.nnnCONCLUSIONSnOur findings suggest that the ELISA-based GP19S-specific IgE assay can be used to test HWP-IWA using venous blood samples, except for those with a unit value > 100.0.

Case of anaphylactic reaction to soy following percutaneous sensitization by soy‐based ingredients in cosmetic products

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Determining Food Allergens by Skin Sensitization in Mice

A food allergy is a chronic inflammatory disease against dietary antigens with high prevalence in industrialized countries. Because there is currently no cure for food allergies, avoiding the allergen is crucial for the prevention of an allergic reaction. Therefore, a further understanding of the pathogenesis and risk factors that augment the sensitization to food allergens is required. We have previously developed a food allergy mouse model using transdermal sensitization, which influences the susceptibility to food allergies. In this model, mice sensitized with partially hydrolyzed wheat protein (HWP) successfully resembled the major features of HWP‐sensitized and wheat allergy–induced patients. In this article, we describe transdermal sensitization of food allergens and induction of immediate‐type food allergies in mice. The methodology detailed here was mainly adapted from an original work by Adachi and colleagues with some modifications to the dressing methods to reduce stress.