Kavita Reginald

Immunoglobulin E antibody reactivity to bacterial antigens in atopic dermatitis patients

Cite this as: K. Reginald, K. Westritschnig, T. Werfel, A. Heratizadeh, N. Novak, M. Focke‐Tejkl, A. M. Hirschl, D. Y. M. Leung, O. Elisyutina, E. Fedenko and R. Valenta, Clinical & Experimental Allergy, 2011 (41) 357–369.

Staphylococcus aureus fibronectin-binding protein specifically binds IgE from patients with atopic dermatitis and requires antigen presentation for cellular immune responses.

BACKGROUND Staphylococcus aureus superinfections occur in more than 90% of patients with atopic dermatitis (AD) and aggravate skin inflammation. S aureus toxins lead to tissue damage and augment T-cell-mediated skin inflammation by a superantigen effect. OBJECTIVE To characterize IgE-reactive proteins from S aureus. METHODS A genomic S aureus library was screened with IgE from patients with AD for DNA clones coding for IgE-reactive antigens. One was identified as fibronectin-binding protein (FBP). Recombinant FBP was expressed in Escherichia coli, purified, and tested for specific IgE reactivity in patients with AD. Its allergenic activity was studied in basophil activation experiments and T-cell cultures. The in vivo allergenic activity was investigated by sensitizing mice. RESULTS Using IgE from patients with AD for screening of a genomic S aureus library, an IgE-reactive DNA clone was isolated that coded for FBP. Recombinant FBP was expressed in E coli and purified. It reacted specifically with IgE from patients with AD and exhibited allergenic activity in basophil degranulation assays. FBP showed specific T-cell reactivity requiring antigen presentation and induced the secretion of proinflammatory cytokines from PBMCs. Mice sensitized with FBP mounted FBP-specific IgE responses, showed FBP-specific basophil degranulation as well as FBP-specific T-cell proliferation, and mixed T(h)2/T(h)1 cytokine secretion. CONCLUSION Evidence is provided that specific humoral and cellular immune responses to S aureus antigens dependent on antigen presentation represent a novel mechanism for S aureus-induced skin inflammation in AD. Furthermore, FBP may be used for the development of novel diagnostic and therapeutic strategies for S aureus infections.

Trimolecular Complex Formation of IgE, FcεRI, and a Recombinant Nonanaphylactic Single-Chain Antibody Fragment with High Affinity for IgE

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 β-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 KD of 1.52 × 10−10 M, which is almost as high as the affinity of IgE for FcεRI, and that the ScFv is able to recognize FcεRI-bound IgE and to prevent IgE binding to FcεRI. 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 FcεRI- or FcεRII-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, FcεRI, 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.

Different modes of IgE binding to CD23 revealed with major birch allergen, Bet v 1-specific monoclonal IgE.

We investigated the binding of IgE and different types of allergen−IgE complexes to CD23‐expressing human B cells. We performed the experiments using chimeric Bip 1 (CB1), a chimeric humanized IgE specific for the major birch allergen, Bet v 1, together with monomeric and oligomeric forms of recombinant Bet v 1 (rBet v 1), and Bet v 1‐specific IgG antibodies. In this model IgE binding to CD23 was independent of variations in antibody affinities towards monomeric and oligomeric Bet v 1 as demonstrated by plasmon surface resonance. CB1 alone or in the form of small immune complexes consisting of one molecule of CB1 plus allergen, showed comparable binding to CD23 on B cells. Using anti‐IgE antibody probes discriminating CD23‐bound from CD23‐unbound IgE, it is demonstrated that in large immune complexes obtained with oligomeric Bet v 1 or by super‐crosslinking of small immune complexes with Bet v 1‐specific IgG, anti‐IgE staining of B cells increased. This increase of staining was due to the presence of IgE antibodies in the immune complexes that were not directly engaged in CD23 binding, and thus available for IgE detection. Our study thus reveals that CD23 can bind in a comparable manner to free IgE and IgE−allergen complexes of different size and composition, which may also include allergen‐specific IgG. The interplay of free IgE with IgE–allergen immune complexes of different sizes and composition with CD23 binding represents a mechanism for the modulation of CD23‐mediated immune responses such as IgE‐facilitated allergen presentation in allergic diseases.

IgE antibody reactivity to bacterial antigens in atopic dermatitis patients

Cite this as: K. Reginald, K. Westritschnig, T. Werfel, A. Heratizadeh, N. Novak, M. Focke‐Tejkl, A. M. Hirschl, D. Y. M. Leung, O. Elisyutina, E. Fedenko and R. Valenta, Clinical & Experimental Allergy, 2011 (41) 357–369.

Immunoglobulin E antibody reactivity to bacterial antigens in atopic dermatitis patients: Bacterial antigens in atopic dermatitis

Cite this as: K. Reginald, K. Westritschnig, T. Werfel, A. Heratizadeh, N. Novak, M. Focke‐Tejkl, A. M. Hirschl, D. Y. M. Leung, O. Elisyutina, E. Fedenko and R. Valenta, Clinical & Experimental Allergy, 2011 (41) 357–369.