Jaakko Rautiainen

Recombinant allergen fragments as candidate preparations for allergen immunotherapy

BACKGROUND Lately, renewed interest has arisen in the new forms of allergen immunotherapy because they may offer alternatives for drug treatment. OBJECTIVE The purpose of this study was to develop a well-characterized preparation of the main respiratory cow dander allergen, Bos d 2, with attenuated allergenic activity. METHODS The immunologic characteristics of Bos d 2 preparations were studied by indirect IgE ELISA, ELISA inhibition, Western blotting, histamine release, skin prick tests, and the proliferation tests of allergen-specific T-cell clones. RESULTS The complete recombinant Bos d 2 was observed to bind effectively, IgE of cow-allergic patients in indirect ELISA. In other experiments, the IgE-binding capacity of recombinant Bos d 2 proved to be lower compared with native Bos d 2. When the two overlapping recombinant fragments of Bos d 2 (corresponding amino acids 1-131 and 81-172, respectively) covering the whole molecule were compared with the complete recombinant Bos d 2 with several methods, only a low level of residual reactivity was observed. For example, recombinant fragments could not bind antibody at all in ELISA inhibition tests retaining, however, some reactivity in skin prick tests. In contrast, the fragments were able to stimulate vigorously Bos d 2-specific T-cell clones. CONCLUSION The approach we have taken may offer a simple and reproducible way to produce hypoallergenic preparations for immunotherapy, circumventing simultaneously some of the problems of other experimental methods such as individual T-cell epitope recognition in peptide-based immunotherapy.

Complementary DNA cloning of the predominant allergen of bovine dander: A new member in the lipocalin family

BACKGROUND A number of allergenic proteins in animal danders have been characterized at the molecular level, but little is known of their biologic functions. We have found that the prevalence of IgE antibodies among patients with cattle-associated asthma is highest against a dander protein referred to as BDA20. OBJECTIVE The study was performed to characterize the molecular structure of BDA20,* the predominant allergen in bovine dander. METHODS Clones encoding allergens were identified and isolated from a complementary DNA library by immunoblotting and DNA hybridization and sequenced. Recombinant proteins were produced in Escherichia coli. Immunoreactivity of the recombinant proteins and amino acid sequences of peptides obtained from native BDA20 after Lys-C cleavage were used to identify clones coding for BDA20. RESULTS In this article we report the cDNA and amino acid sequences of BDA20. Homology comparisons showed that BDA20 belongs to the family of lipocalins. CONCLUSIONS The results link a dander allergen to a group of functionally important proteins. Lipocalins are present in various body fluids and secretions of several animal species in which they function as carriers of small hydrophobic molecules, such as retinoids and pheromones. If allergenicity proves to be a property shared by lipocalins, our results will have considerable implications for allergen research.

Lipocalins as allergens

The term allergy refers to clinical conditions caused by an inappropriate immune response to innocuous proteins in genetically predisposed persons. Allergens of animal origin are responsible for a significant proportion of allergies. In recent years, it has become evident that practically all respiratory animal allergens characterized at the molecular level belong to the lipocalin family of proteins. The current list comprises the major allergens of horse, cow, dog, mouse, rat and cockroach as well as beta-lactoglobulin of cows milk. While the molecular structure of all these allergens is known, far less information is available regarding their immunological characteristics. Knowing the way the immune system recognizes these allergens and reacts to them might, however, be the key for discovering the common denominator of the allergenicity of lipocalins. The human body contains numerous endogenous lipocalins, and the immune system has to adapt to their presence. We have proposed that under these conditions the immune response against the lipocalin allergens which are structurally related to endogenous lipocalins might be the pathway to allergy in genetically predisposed persons. The same might well apply also to other allergens with homologous endogenous counterparts.

Allergy to lipocalins: a consequence of misguided T-cell recognition of self and nonself?

Abstract The molecular mimicry between lipocalin allergens and endogenous lipocalins at the T-cell level may explain why the immune response against lipocalins is Th2-dominated and results in allergy. This view is supported by recent studies of autoimmune and parasitic diseases and peptide analogues.

Tissue localization of bovine dander allergen Bos d 2

BACKGROUND Domestic mammals are important sources of indoor allergens. However, the origin at the tissue level and the biologic function of mammalian allergens are largely unknown. OBJECTIVE The aim of this study was to localize the source of the major bovine dander allergen, Bos d 2, in bovine tissues. METHODS Samples from several organs were tested for the presence of mRNA encoding Bos d 2 and Bos d 2 protein by using the reverse transcriptase polymerase chain reaction and immunohistochemical staining, respectively. RESULTS Skin proved to be the only tissue where mRNA encoding Bos d 2 was detected. This observation was confirmed by immunohistochemistry with a monoclonal anti-Bos d 2 antibody as the primary antibody. In the skin sections, Bos d 2 was found in the secretory cells of apocrine sweat glands and the basement membranes of the epithelium and hair follicles. Bos d 2 belongs to the family of lipocalins comprising a number of pheromone carrier proteins that are present, for example, in the secretions of the apocrine sweat glands. CONCLUSION Together with earlier data, our findings suggest that Bos d 2 is produced in sweat glands and transported to the skin surface as a carrier of the pheromone ligand. Because dander allergens of a number of mammalian species are lipocalins, the common biologic function of being pheromone carriers seems to be a common feature of an important group of aeroallergens.

Partial amino acid sequence of a cellulase-like component with IgE-binding properties from Stachybotrys chartarum

Background: The aim of this study was to characterize the amino acid sequence of a selected Stachybotrys chartarum component and to investigate human IgE reactivity against components of S. chartarum and nine other fungal species. Methods: Human IgE reactivity against S. chartarum and nine other fungal extracts was investigated by the immunoblotting method. For automated amino acid sequencing analyses, the S. chartarum extract was purified by ion exchange chromatography prior to in-gel alkylation and digestion with modified trypsin. Results: Human IgE reactivity was detected against eight components in the S. chartarum extract. Over 80% of the sera from the exposed subjects and less than 50% of the control sera recognized the 33-, 48- and 50-kD S. chartarum components. The human sera detected a 48- to 50-kD component from the extracts of eight fungal species. Nineteen peptide sequences were identified from the 48-kD component of S. chartarum. An analysis of the peptide sequences revealed homology with known fungal glycoside hydrolase enzymes (cellulases). Conclusions: The data showed human IgE reactivity against several S. chartarum components, including one at 48 kD. On the other hand, the human sera recognized 48- to 50-kD components from seven other fungal species, suggesting shared antigenic components (e.g. enolase) between the fungi. Thus, to our knowledge, this is the first antigen identified from S. chartarum.

Two new variants of the lipocalin allergen Bos d 2

Allergens from various sources have been shown to comprise several isoforms. In the present study, a series of chromatographic steps was carried out to separate the lipocalin allergen Bos d 2 isoforms present in cow dander. Subsequent HPLC-MS-MS analyses revealed two new Bos d 2 variants. In one of the proteins, tyrosine (Y83) was substituted by aspartic acid, and in the other protein valine (V102) was replaced by alanine. We propose the three Bos d 2 variants be named as Bos d 2.0101 (previously sequenced Bos d 2), Bos d 2.0102 and Bos d 2.0103. Our results suggest that molecular polymorphism is a common property among lipocalin allergens. Since allergen isoforms may show variation in their IgE binding and/or T-cell reactivity, all of the many allergen forms should be taken into account when planning preparations for immunotherapy.