We conclude that divergent allergenicity of apple strains mainly depends on different expression levels of the major allergen. Introduction of a proline residue in position 111 of Mal d 1 and in position 112 of Bet v 1 led to a drastic reduction of allergenicity of both the pollen and the food allergen, obviously also removing the cross-reactive epitope. Mutants with reduced IgE-reactivity but maintained T-cell reactivity may represent new candidates for a safer specific immunotherapy with reduced side-effects.

A stable 'humanized' basophil cell line was established that will be a useful tool for the standardization and batch control of allergen extracts. Because of its high sensitivity, it can also be used to detect minute quantities of potentially allergenic proteins, e.g. in processed foods. In addition, the test may support the development of novel allergy vaccines, such as recombinant hypoallergenic molecules.

Background: This study was performed to get further insights into antibody responses to cross–reactive carbohydrate determinants (CCD), including initial experiments to prove the biological activity of anti–CCD IgE. Earlier studies have shown that IgE specific for CCD occurs in about 25% of celery–allergic patients. The clinical significance of these antibody specificities is doubtful. Methods: Patient sera were selected on the basis of a positive case history of celery allergy and multiple binding to high molecular weight celery allergens on immunoblots. Specific IgE to native and heated celery tuber was determined by the enzyme allergosorbent test (EAST). N–glycans were purified after extensive digestion of specific glycoproteins, such as pineapple stem bromelain, bovine fibrin, and human IgG, and used as antigens in an IgE ELISA as well as in EAST and immunoblotting inhibition experiments. Dose–related histamine release was performed with BSA neoglycoproteins containing 3–4 units of the purified glycopeptides. Results: Seven celery–allergic patients were identified who clearly presented IgE against the N–glycan purified from bromelain which is a common structure within the plant kingdom. Chemical defucosylation showed that α1,3–fucose is a key structure for IgE binding. In patients with anti–CCD IgE, the maximal inhibition of celery EAST by the bromelain glycan ranged from 22 to 100%. Inhibition of celery immunoblots by preincubation of patient serum with this glycan led to a quenching of multiple bands at masses >40 kD. After linking the bromelain glycopeptide to BSA, a strong dose–related histamine release was obtained in a celery–allergic patient, occurring at lower concentrations than with the recombinant major protein allergen from celery, Api g 1. Conclusions: Our results demonstrate that IgE specific for CCD is common in celery–allergic patients, and can represent the major proportion of IgE against this food. α1,3–fucose was confirmed to be an essential part of the IgE epitope. Immunoblotting inhibition indicated the presence of this carbohydrate determinant on multiple glycoproteins in celery extract. Although histamine release was only performed in 1 patient, our data show that proteins carrying multiple glycan units can be biologically active in patients sensitized to CCD.

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