Nadja Balic

Characterization of Der p 21, a new important allergen derived from the gut of house dust mites.

Background:  The house dust mite (HDM) Dermatophagoides pteronyssinus is a major allergen source eliciting allergic asthma. The aim of the study was to identify new important HDM allergens associated with allergic asthma.

Different allergenic activity of grass pollen allergens revealed by skin testing

Background  Grass pollen is one of the most important allergen sources. The aim of this study was to compare the in vivo allergenic activity of two recently characterized major grass pollen allergens, Phl p 4 and Phl p 13, with three established major grass pollen allergens, Phl p 1, Phl p 2 and Phl p 5 as a basis for the formulation of a grass pollen allergy vaccine based on purified allergens.

Molecular Characterization of Polygalacturonases as Grass Pollen-Specific Marker Allergens: Expulsion from Pollen via Submicronic Respirable Particles

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of ∼42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients’ IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

Microarray and allergenic activity assessment of milk allergens

Background Cows milk is one of the most common causes of food allergy affecting approximately 2.5% of infants in the first years of their life. However, only limited information regarding the allergenic activity of individual cows milk allergens is available.

Carrier‐bound Alt a 1 peptides without allergenic activity for vaccination against Alternaria alternata allergy

The mould Alternaria alternata is a major elicitor of allergic asthma. Diagnosis and specific immunotherapy (SIT) of Alternaria allergy are often limited by the insufficient quality of natural mould extracts.

Patients suffering from non-IgE-mediated cow’s milk protein intolerance cannot be diagnosed based on IgG subclass or IgA responses to milk allergens

To cite this article: Hochwallner H, Schulmeister U, Swoboda I, Twaroch TE, Vogelsang H, Kazemi‐Shirazi L, Kundi M, Balic N, Quirce S, Rumpold H, Fröschl R, Horak F, Tichatschek B, Stéfanescu CL, Szépfalusi Z, Papadopoulos NG, Mari A, Ebner C, Pauli G, Valenta R, Spitzauer S. Patients suffering from non‐IgE‐mediated cow’s milk protein intolerance cannot be diagnosed based on IgG subclass or IgA responses to milk allergens. Allergy 2011; 66: 1201–1207.

Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1

Background A considerable proportion of animal‐allergic patients are sensitized to both cat and dog allergens but knowledge about cross‐reactive allergens in cat and dog dander is limited.

Allergen cleavage by effector cell-derived proteases regulates allergic inflammation

The key event of allergic inflammation, allergen‐induced crosslinking of mast cell‐bound IgE antibodies, is accompanied by release of inflammatory mediators, cytokines, and proteases, in particular β‐tryptase. We provide evidence that protease‐mediated cleavage of allergens represents a mechanism that regulates allergen‐induced mast cell activation. When used in molar ratios as they occur in vivo, purified β ‐tryptase cleaved major grass and birch pollen allergens, resulting in defined peptide fragments as mapped by mass spectrometry. Tryptase‐cleaved allergens showed reduced IgE reactivity and allergenic activity. The biological relevance is demonstrated by the fact that lysates from activated human mast cells containing tryptase levels as they occur in vivo cleaved allergens. Additionally, protamine, an inhibitor of heparin‐dependent effector cell proteases, augmented allergen‐induced release of mediators from effector cells. Protease‐mediated allergen cleavage may represent an important mechanism for terminating allergen‐induced effector cell activation.—Rauter, I., Krauth, M.‐T., Flicker, S., Gieras, A., Westritschnig, K., Vrtala, S., Balic, N., Spitzauer, S., Huss‐Marp, J., Brockow, K., Darsow, U., Ring, J., Behrendt, H., Semper, H., Valent, P., Valenta, R. Allergen cleavage by effector cell‐derived proteases regulates allergic inflammation. FASEB J. 20, E61–E69 (2006)

Visualization of clustered IgE epitopes on α-lactalbumin

BACKGROUND alpha-Lactalbumin (alpha-La) is a major cows milk (CM) allergen responsible for allergic reactions in infants. OBJECTIVE We performed molecular, structural, and immunologic characterization of alpha-La. METHODS Recombinant alpha-lactalbumin (ralpha-La) was expressed in Escherichia coli, purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides. RESULTS According to circular dichroism analysis, ralpha-La represented a folded protein with a high thermal stability and refolding capacity. ralpha-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. ralpha-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of alpha-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch. CONCLUSIONS ralpha-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy.

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.