Abstract
BackgroundA longstanding debate in allergy is whether or not specific immunoglobulin-E antibodies (sIgE), recognizing cross-reactive carbohydrate determinants (CCD), are able to elicit clinical symptoms. In pollen and food allergy, ≥20% of patients display in-vitro CCD reactivity based on presence of α1,3-fucose and/or β1,2-xylose residues on N-glycans of plant (xylose/fucose) and insect (fucose) glycoproteins. Because the allergenicity of tomato glycoallergen Lyc e 2 was ascribed to N-glycan chains alone, this study aimed at evaluating clinical relevance of CCD-reduced foodstuff in patients with carbohydrate-specific IgE (CCD-sIgE).Methodology/Principal FindingsTomato and/or potato plants with stable reduction of Lyc e 2 (tomato) or CCD formation in general were obtained via RNA interference, and gene-silencing was confirmed by immunoblot analyses. Two different CCD-positive patient groups were compared: one with tomato and/or potato food allergy and another with hymenoptera-venom allergy (the latter to distinguish between CCD- and peptide-specific reactions in the food-allergic group). Non-allergic and CCD-negative food-allergic patients served as controls for immunoblot, basophil activation, and ImmunoCAP analyses. Basophil activation tests (BAT) revealed that Lyc e 2 is no key player among other tomato (glyco)allergens. CCD-positive patients showed decreased (re)activity with CCD-reduced foodstuff, most obvious in the hymenoptera venom-allergic but less in the food-allergic group, suggesting that in-vivo reactivity is primarily based on peptide- and not CCD-sIgE. Peptide epitopes remained unaffected in CCD-reduced plants, because CCD-negative patient sera showed reactivity similar to wild-type. In-house-made ImmunoCAPs, applied to investigate feasibility in routine diagnosis, confirmed BAT results at the sIgE level.Conclusions/SignificanceCCD-positive hymenoptera venom-allergic patients (control group) showed basophil activation despite no allergic symptoms towards tomato and potato. Therefore, this proof-of-principle study demonstrates feasibility of CCD-reduced foodstuff to minimize ‘false-positive results’ in routine serum tests. Despite confirming low clinical relevance of CCD antibodies, we identified one patient with ambiguous in-vitro results, indicating need for further component-resolved diagnosis.
Highlights
Specific immunoglobulin-E antibodies directed against plant-derived carbohydrate epitopes are ubiquitous among patients with confirmed pollen or food allergy [2]
Our RNAi-based approaches represent more or less the physiological situation after contact with or ingestion of plant foods, in contrast to previous studies that reported histamine release assays conducted with only a single native, glycosylated allergen versus the recombinant, non-glycosylated allergen form [7,14,15,16]. In this context it is noteworthy that tomato glycoallergen Lyc e 2, whose effector-cell triggering depends on cross-reactive carbohydrate determinants (CCD) epitopes alone [7,16], was no key player for reducing the allergenic potential of tomato fruits
An explanation for this finding is given by the recent study of Mertens and coworkers, revealing that CCD may induce basophil activation without clinical relevance in hymenoptera venom allergy [34]
Summary
Specific immunoglobulin-E antibodies (sIgE) directed against plant-derived carbohydrate epitopes (cross-reactive carbohydrate determinants, CCD [1]) are ubiquitous among patients with confirmed pollen or food allergy (reviewed by Altmann) [2]. The main motifs of these carbohydrate epitopes are asparagine (N)-linked glycan chains carrying core a1,3-fucose and b1,2-xylose residues [7]. They form essential parts of two independent complex N-glycan epitopes found on glycoproteins of plants and lower animals, and occur in pollen, natural rubber latex, vegetables and fruits, hymenoptera venoms (only a1,3-fucose), and in some pathogenic worms but not in mammals (see Altmann) [2]. Because the allergenicity of tomato glycoallergen Lyc e 2 was ascribed to N-glycan chains alone, this study aimed at evaluating clinical relevance of CCD-reduced foodstuff in patients with carbohydrate-specific IgE (CCD-sIgE)
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