Expression, purification, characterization, and patient IgE reactivity of new macadamia nut iso-allergen.

Structural and functional information about food allergens is essential for understanding the allergenicity of food proteins. All allergens belong to a small number of protein families. Various allergens from different families have been successfully produced recombinantly in E. coli for their characterization and applications in allergy diagnosis and treatment. However, recombinant hexameric 11S seed storage protein has not been reported, although numerous 11S legumins are known to be food allergens, including the recently identified macadamia nut allergen Mac i 2. Here we report the production of a macadamia nut legumin by expressing it in E. coli with a substrate site of HRV 3C protease and cleaving the purified protein with HRV 3C protease. The protease divided the protein into two chains and left a native terminus for the C-terminal chain, resulting in a recombinant hexameric 11S allergen for the first time after the residues upstream to the cleavage site flipped out of the way of the trimer-trimer interaction. The 11S allergens are known to have multiple isoforms in many species. The present study removed an obstacle in obtaining homogeneous allergens needed for studying allergens and mitigating allergenicity. Immunoreactivity of the protein with serum IgE confirmed it to be a new isoform of Mac i 2.

Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4(+) T cell epitopes with other tree nuts.

BACKGROUND: Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T cell epitopes and T cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. OBJECTIVES: In this study, we characterized cashew-specific T cell responses in cashew-allergic subjects and examined cross-reactivity of these cashew-specific cells towards other tree nut allergens. METHODS: CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T cells were determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. RESULTS: CD4(+) T cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T cell epitopes were then identified. These epitopes elicited either TH 2 or TH 2/TH 17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. CONCLUSIONS: Phylogenetically diverse tree nut allergens can activate cashew-reactive T cells and elicit a TH 2-type response at an epitope-specific level. CLINICAL RELEVANCE: Lack of cross-reactivity between walnut and cashew suggests that cashew peptide immunotherapy approach may not be most effective for walnut.

Lipids are required for the development of Brazil nut allergy: the role of mouse and human iNKT cells.

BACKGROUND: Lipids are required for mice sensitization to Ber e 1, Brazil nut major allergen. Here, we characterized different lipid fractions extracted from Brazil nuts and the lipid-binding ability of Ber e 1. Further, we determined their in vivo ability to induce Ber-specific anaphylactic antibodies and the role of invariant natural killer T (iNKT) cells in this process. METHODS: Wild-type (WT) and iNKT cell-deficient mice were sensitized with Ber e 1 and specific lipid fractions, and anaphylactic antibodies were measured by enzyme-linked immunosorbent assay (ELISA) and passive cutaneous anaphylaxis (PCA). The lipid-binding characteristic of Ber e 1 (Ber) was established by using fluorescent probes and (15) N-labeled NMR. In vitro production of IL-4 was determined in Ber/lipid C-stimulated mouse iNKT cells and human T-cell lines containing NKTs primed with CD1d+C1R transfectants by flow cytometry and ELISA, respectively. RESULTS: Only one specific lipid fraction (lipid C), containing neutral and common phospholipids, induced Ber anaphylactic antibodies in mice. Ber e 1 has a lipid-binding site, and our results indicated an interaction between Ber e 1 and lipid C. iNKT-deficient mice produced lower levels of anaphylactic antibodies than WT mice. In vitro, Ber/lipid C-stimulated murine iNKT cells produced IL-4 but not IFN-gamma. Human T-cell lines derived from nut-allergic patients produced IL-4 to Ber/lipid C in a CD1d- and dose-dependent manner. CONCLUSION: Lipid fraction C from Brazil nut presents an essential adjuvant activity to Ber e 1 sensitization, and iNKT cells play a critical role in the development of Brazil nut-allergic response.

Basophil CD203c levels are increased at baseline and can be used to monitor omalizumab treatment in subjects with nut allergy.

RATIONALE: Basophils contribute to anaphylaxis and allergies. We examined the utility of assessing basophil-associated surface antigens (CD11b/CD63/CD123/CD203c/CD294) in characterizing and monitoring subjects with nut allergy. METHODS: We used flow cytometry to analyze basophils at baseline (without any activation) and after ex vivo stimulation of whole blood by addition of nut or other allergens for 2, 10, and 30 min. We also evaluated whether basophil expression of CD11b/CD63/CD123/CD203c/CD294 was altered in subjects treated with anti-IgE monoclonal antibody (omalizumab) to reduce plasma levels of IgE. RESULTS: We demonstrate that basophil CD203c levels are increased at baseline in subjects with nut allergy compared to healthy controls (13 subjects in each group, p < 0.0001). Furthermore, we confirm that significantly increased expression of CD203c occurs on subject basophils when stimulated with the allergen to which the subject is sensitive and can be detected rapidly (10 min of stimulation, n = 11, p < 0.0008). In 5 subjects with severe peanut allergy, basophil CD203c expression following stimulation with peanut allergen was significantly decreased (p < 0.05) after 4 and 8 weeks of omalizumab treatment but returned toward pretreatment levels after treatment cessation. CONCLUSIONS: Subjects with nut allergy show an increase of basophil CD203c levels at baseline and following rapid ex vivo stimulation with nut allergen. Both can be reduced by omalizumab therapy. These results highlight the potential of using basophil CD203c levels for baseline diagnosis and therapeutic monitoring in subjects with nut allergy.

cDNA Cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (prunus dulcis).

Tree nuts, including almond (prunus dulcis) are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP) was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control) possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.