A number of structurally different allergens trigger the release of mediators from basophils by cross-linking of IgE receptors. In this study, we analyzed the effects of cyclosporine A (CSA) and FK-506 on allergen-induced histamine release in human blood basophils obtained from birch- or grass-pollen-allergic donors (n = 12). Preincubation of basophils with CSA (0.003–3 μg/ml) or FK-506 (0.003–3 μg/ml) led to inhibition of histamine release induced by purified recombinant tree pollen allergens (r Bet v 1, r Bet v 2) and timothy grass pollen allergens (r Ph1 p 1, r Ph1 p 2, r Ph1 p 5). The effects of CSA and FK-506 were dose dependent, with IC50 values ranging between 0.03 and 0.3 μg/ml for both CSA and FK-506. Cyclosporine H, an inactive CSA analog, did not show any effect on allergen-induced histamine secretion. IgE dependency of the reaction was demonstrated in passive transfer experiments using highly enriched human basophils (> 95% pure) and specific IgE from a patient allergic to Bet v 2. In summary, our data show that CSA and FK-506 inhibit recombinant-allergen-induced histamine release from peripheral blood basophils in allergic donors.
cDNAs coding for the major allergen of alder (Alnus glutinosa) pollen Aln g 1, for nine isoforms of Bet v 1, the major birch (Betula verrucosa) pollen allergen, and for four isoforms of Cor a 1, the major allergen of hazel (Corylus avellana) pollen, were inserted into the plasmid pMW175 or pMW 172 and expressed in Escherichia coli as recombinant non-fusion proteins. These constructs produced between 20 and 160 mg protein/l. The recombinant tree pollen isoallergens were tested in immunoblots for their antibody binding properties. For this purpose, we used two monoclonal antibodies (BIP 1 and BIP 4) raised against natural Bet v 1, a polyclonal rabbit anti-recombinant Bet v 1a, as well as serum IgE from allergic patients. Our results show that this expression system is suitable for the production of milligram amounts of tree pollen isoallergens which can be used for the characterization of allergenic epitopes recognized by T and B cells.
for each drug for any change in indications and dosage and for added warnings and precautions.This is particularly important when the recommended agent is a new and/or infrequently employed drug.
In the past 10 years, a considerable number of cDNAs coding for allergens have been isolated and expressed. Intensive investigations showed that recombinant allergens and their respective natural counterparts possess comparable properties with respect to structure, function and interaction with the immune system. Recent studies documented that in vitro as well as in vivo diagnosis of IgE–mediated allergic diseases can be successfully improved by the application of recombinant allergens. In addition, new strategies for a safer specific immunotherapy (SIT) have been developed based on the knowledge of the primary structures of allergens. Naturally occurring isoforms of allergens as well as recombinant allergens with modified amino acid sequences show very low IgE binding capacity but strong T cell–stimulatory activity and represent possible candidates. In case of Bet v 1, the major birch pollen allergen, isoforms d, g and l and a Bet v 1a mutant, produced by site–directed mutagenesis resulting in 6 amino acid exchanges, fulfilled the above mentioned criteria. In a third approach, two adjacent peptides covering the entire Bet v 1a sequence were produced in an <i>Escherichia coli</i> expression system. These peptides contained most of the relevant T cell epitopes, but lost their IgE binding capacity and, thus, their ability to activate mast cells and basophils of sensitized patients. Our results suggest that allergen variants (isoforms, mutants, T cell epitope–containing peptides) may be used as ‘hypoallergenic agents’ in SIT.
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