title: Reduction of cross-reactive carbohydrate determinants in plant foodstuff: elucidation of clinical relevance and implications for allergy diagnosis.
abstract: BACKGROUND
  A 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).
  
  
  RESULTS
  Tomato 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
  CCD-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.
authors.affiliation: Institute of Plant Biology and Biotechnology, University of Münster, Münster, Germany.
authors.forename: Heidi
authors.initials: H
authors.lastname: Kaulfürst-Soboll
authors.forename: Melanie
authors.initials: M
authors.lastname: Mertens
authors.forename: Randolf
authors.initials: R
authors.lastname: Brehler
authors.forename: Antje
authors.initials: A
authors.lastname: von Schaewen
mesh_terms: D000485:Allergens / Q000276:immunology; D001491:Basophils / Q000276:immunology; D002241:Carbohydrates / Q000276:immunology*; D003429:Cross Reactions / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D005512:Food Hypersensitivity / Q000097:blood / Q000175:diagnosis* / Q000276:immunology*; D005638:Fruit / Q000737:chemistry; D020868:Gene Silencing; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D018551:Solanum lycopersicum / Q000276:immunology*; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D010936:Plant Extracts; D010940:Plant Proteins / Q000276:immunology; D015203:Reproducibility of Results; D011198:Solanum tuberosum / Q000276:immunology*
chemical_list: D000485:Allergens; D002241:Carbohydrates; D000939:Epitopes; D007074:Immunoglobulin G; D010936:Plant Extracts; D010940:Plant Proteins; D007073:Immunoglobulin E; D017351:N-Acetylglucosaminyltransferases