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• W2058045152 abstract "The fungus Alternaria, a common plant pathogen, occurs worldwide and releases allergen-bearing spores especially in late summer and early autumn.1Rodríguez-Rajo F.J. Iglesias I. Jato V. Variation assessment of airborne Alternaria and Cladosporium spores at different bioclimatical conditions.Mycol Res. 2005; 109: 497-507Crossref PubMed Scopus (102) Google Scholar Increased levels of Alternaria spores in the atmosphere have been linked to severe manifestations of respiratory allergy (ie, severe rhinitis and asthma), frequent occurrence of hospital delivery, and even asthma-related death.2Targonski P.V. Persky V.W. Ramekrishnan V. Effect of environmental molds on risk of death from asthma during the pollen season.J Allergy Clin Immunol. 1995; 95: 955-961Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar Alt a 1 is the most important Alternaria allergen. It reacts with serum IgE from more than 95% of Alternaria-sensitized patients, contains most of the Alternaria-specific IgE epitopes, and shows high allergenic activity. Several other Alternaria allergens have been characterized, but they are recognized by fewer patients and their allergenic activity appears to be much lower.3Simon-Nobbe B. Denk U. Pöll V. Rid R. Breitenbach M. The spectrum of fungal allergy.Int Arch Allergy Immunol. 2008; 145: 58-86Crossref PubMed Scopus (353) Google Scholar In this study, we investigated the ultrastructural localization of Alt a 1 and Alt a 8, another well-described Alternaria allergen which is less frequently recognized by Alternaria-sensitized patients than Alt a 1 (ie, 41%),3Simon-Nobbe B. Denk U. Pöll V. Rid R. Breitenbach M. The spectrum of fungal allergy.Int Arch Allergy Immunol. 2008; 145: 58-86Crossref PubMed Scopus (353) Google Scholar by immunogold electron microscopy. In order to obtain highly specific antibody probes recognizing the protein backbone of the allergens, peptides of Alt a 1 (aa 19-52) and Alt a 8 (aa 146-173) with predicted high surface accessibility were synthesized and used for immunization of rabbits as described.4Focke M. Mahler V. Ball T. Sperr W.R. Majlesi Y. Valent P. et al.Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination.FASEB J. 2001; 15: 2042-2044PubMed Google Scholar The Alternaria strain CBS 103.33 (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was cultivated on a Sabouraud glucose agar plate (Becton Dickinson, Heidelberg, Germany) for 7 days at room temperature. Pieces measuring 1 mm2 were excised from the dry mould and fixed overnight at 4°C in phosphate buffer (0.1 mol/L, pH 7.4) containing 4% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde. Samples were dehydrated through an ethanol series and afterwards infiltrated and polymerized in a LR White resin (Sigma, St Louis, Mo). Ultrathin sections were cut from embedded fungal tissue and mounted on gold grids. The sections were preincubated in 5% (w/v) BSA in 100 mM phosphate buffer (pH 7.4) and incubated with protein G purified (Pierce, Rockford, Ill) anti-Alt a 1 and anti-Alt a 8 antibodies. Sections were then exposed to goat anti-rabbit IgG antibodies coupled to 10-nm gold particles diluted in phosphate buffer (0.1 mol/L, pH 7.4). Interestingly, Alt a 1 was exclusively found in the cell wall, and there in the melanin layer of older spores (Fig 1, A) whereas no significant labeling was found within the cytoplasm, in the hyphae or in younger spores (data not shown). In contrast, Alt a 8 could be localized only in the cytoplasm of hyphae (Fig 1, B and C) but not in spores (data not shown). In the cytoplasm of hyphae it accumulated in electron-dense material within vacuole-like compartments. The distinct and exclusive localization of Alt a 1 in the cell wall observed by us is in contrast to data obtained in 2 earlier studies describing a diffuse labeling of the cytoplasm and cell wall.5Paris S. Debeaupuis J.P. Prévost M.C. Casotto M. Latgé J.P. The 31 kd major allergen, Alt a I1563, of Alternaria alternata.J Allergy Clin Immunol. 1991; 88: 902-908Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 6Ibarrola I. Suárez-Cervera M. Arilla M.C. Martínez A. Monteseirín J. Conde J. et al.Production profile of the major allergen Alt a 1 in Alternaria alternata cultures.Ann Allergy Asthma Immunol. 2004; 93: 589-593Abstract Full Text PDF PubMed Scopus (20) Google Scholar This diffuse localization observed in the earlier studies may have been caused by different culture conditions. In fact, we used air-grown mold mimicking natural growth conditions, whereas in the other studies molds were grown under artificial liquid culture conditions. In this context, it has been shown recently that different culture conditions can influence the formation of a hydrophobin layer that may affect immune recognition of fungi.7Aimanianda V. Bayry J. Bozza S. Kniemeyer O. Perruccio K. Elluru S.R. et al.Surface hydrophobin prevents immune recognition of airborne fungal spores.Nature. 2009; 460: 1117-1121Crossref PubMed Scopus (553) Google Scholar It is also possible that growth in liquid media may have caused elution and dislocation of Alt a 1 in liquid cultures, a phenomenon that has been found to cause diffusion of allergens also from pollen grains upon hydration.8Grote M. In situ localization of pollen allergens by immunogold electron microscopy: allergens at unexpected sites.Int Arch Allergy Immunol. 1999; 118: 1-6Crossref PubMed Scopus (38) Google Scholar The localization of Alt a 1 in the spore wall observed in this study is in agreement with data suggesting that Alt a 1 might be involved in spore germination.9Mitakakis T.Z. Barnes C. Tovey E.R. Spore germination increases allergen release from Alternaria.J Allergy Clin Immunol. 2001; 107: 388-390Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Spores are essential for the survival and dispersal of fungi and some of them—including those of Alternaria—are resistant against adverse climatic conditions. In fact, Alt a 1 also represents an extremely stable protein whose conformation and IgE epitopes even resist heat treatments of 95°C (Twaroch et al, unpublished data, 2011). The localization of the less frequently recognized Alt a 8 allergen in vacuole-like compartments of the hyphae is in accordance with its natural function as a mannitol dehydrogenase. The sugar alcohol mannitol is besides trehalose an important storage carbohydrate in fungi that constitutes approximately 11% of Alternaria alternata dry weight. It is tempting to speculate that the localization of Alt a 1 in the airborne spore fraction, and in particular in the cell wall of Alternaria spores, may contribute to symptoms in the upper airways and eventually to sensitization and boosting of IgE responses via the nasal mucosa and thus explain the high clinical relevance of this allergen." @default.
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• W2058045152 title "Predominant localization of the major Alternaria allergen Alt a 1 in the cell wall of airborne spores" @default.
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