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IgE is a central molecule in allergic disease. We have isolated cDNAs coding for the heavy and light chains of a murine mAb specific to human IgE and expressed a recombinant single-chain variable fragment (ScFv) derived thereof in Escherichia coli. The purified recombinant ScFv has a molecular mass of 28 kDa as measured by mass spectrometry and shows a beta-sheet fold as determined by circular dichroism. In biosensor-based studies it was demonstrated that the ScFv rapidly and stably binds to human IgE with an affinity of K(D) of 1.52 x 10(-10) M, which is almost as high as the affinity of IgE for FcepsilonRI, and that the ScFv is able to recognize FcepsilonRI-bound IgE and to prevent IgE binding to FcepsilonRI. The ScFv reacts specifically with IgE but not with other isotypes, allows the measurement of allergen-specific IgE in serum samples, and specifically targets cells that contain FcepsilonRI- or FcepsilonRII-bound IgE or that secrete IgE. Using negative-stain electron microscopy we demonstrated the formation of bimolecular complexes consisting of two ScFv molecules and one IgE and trimolecular complexes consisting of IgE, FcepsilonRI, and ScFv in which only one ScFv is able to bind to IgE. Accordingly, we found that the ScFv does not cross-link basophil-bound IgE and hence does not induce histamine release or activation of basophils as demonstrated by FACS analysis of CD203c expression and by histamine release experiments. In vivo skin testing confirmed the lack of allergenic activity of the ScFv. The recombinant ScFv may represent a universal tool for the IgE-targeted treatment of allergies.

Background: Dasatinib is a multikinase inhibitor active against several tyrosine kinases including ABL, KIT, Lyn and Btk. Apart from its known antileukemic activity, the drug produces several side effects including edemas and pleural effusions, which are supposedly triggered by activated immune cells. Effusion formation can be treated effectively by glucocorticosteroids. We have recently shown that low concentrations of dasatinib (<0.1 μM) promote IgE-dependent secretion of histamine in basophils, especially in allergic individuals. In the current study, we asked whether glucocorticosteroids inhibit dasatinib-induced activation of basophils. Methods: Basophils were preincubated with dexamethasone, prednisolone and hydrocortisone for 24 h, and were then exposed to an anti-IgE antibody (normal basophils) or the allergens Bet v 1 and Phl p 5 (allergic patients) with or without low concentrations of dasatinib (0.025 μM). After incubation, basophils were examined for histamine release and expression of CD63 and CD203c. Results: All three glucocorticosteroids were found to counteract IgE-dependent and dasatinib-enhanced histamine release in basophils in nonallergic and allergic individuals. In addition, glucocorticosteroids were found to inhibit anti-IgE-induced upregulation of CD63 and CD203c in the presence or absence of dasatinib. The inhibitory effects of glucocorticosteroids were dose-dependent (effective range: 1-10 μM) and seen in all donors examined. Conclusions: Glucocorticosteroids rescue IgE receptor cross-linked basophils from additional costimulatory effects of low-dose dasatinib which may have clinical implications in dasatinib-treated patients. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy. [ABSTRACT FROM AUTHOR]