Allergens and Antigens - Their Characterization and Biological Effects in
Mice after Inhalation Exposure
Principal Investigator: Anna-Liisa Pasanen, PhD, docent, University of Kuopio, Department of Environmental Sciences, -mail: firstname.lastname@example.org
The non-allergic symptoms that are reported by people occupying moldy buildings are assumed to be caused by microbial metabolites and components, such as fungal b(1‹3)-D-glucans, even though the biological effects of these agents and their relationship with fungal antigens have not yet been clarified sufficiently. It is also possible that the reactions that are reported to be caused by fungal antigens might be mediated by mechanisms other than those of immediate type of allergy. In this context, it is interesting that the molecular structure of only few fungal allergens is known so far, and that the cross-reactivities between different fungal species have been weakly characterized. On the other hand, skin and serological tests are generally performed in the clinical work with unstandardized fungal extracts without the clear comprehension, how the results should be interpreted. The specific knowledge on the composition of fungal allergens would also be useful for the development of specific detection methods so that the most harmful fungi, e.g. Stachybotrys chartarum, could be easily identified in the environment.
This research project is divided into two parts. In Part 1, Characterization of allergenic components of some mold species and the development of a specific detection method for Stachybotrys chartarum, the antigenic compositions of four to six fungal species (S. chartarum, A. versicolor, P. brevi-compactum, C. cladosporioides, and two yeasts) the exposure to which is common in moldy buildings or agriculture, is characterized by SDS-PAGE and immunoblotting using immune and human sera. The cross-reactivities of the fungi between each other and with other common fungal species are investigated by the same method, and the specific components are determined. Polyclonal and/or monoclonal antibodies are created against these components. ELISA methods for measuring mold-specific antibodies and ELISA inhibition methods for measuring the antigenic components of these fungi are also developed. The important objective of the study is to produce the specific components of S. chartarum and possibly of some other fungus as recombinant proteins. For identifying these proteins, the cDNA library is created and the library is screened by specific antibodies. The gene coding for the specific component is transformed in the Pichia pastoris yeast for production. This approach allows the definition of the nucleotide and amino acid sequences as well as the production of the protein in great amounts and, thus, enables the development of a rapid detection method for the fungi, particularly for S. chartarum.
In Part 2, Exposure of mice by inhalation to fungi, irritating effects in the respiratory tract and immune responses, mice sensitized to the fungi mentioned above, are repeatedly exposed to various amounts of fungal antigens, glucans and volatile metabolites by inhalation, and the respiratory functions of mice are monitored continuously during the experiment. The irritating potencies of the agents are determined. After the exposure, the levels of specific antibodies (IgG, IgE, IgA) in serum and inflammatory mediators (IL-1, IL-6, TNF-a) in bronchoalveolar lavage fluid are measured. Histological analyses are performed from the tissue samples of the upper and lower airways. According to the results, the biological effects of fungal agents can be elucidated.