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Growth temperature strongly influence the allergenicity of Aspergillus fumigatus spores
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Growth temperature strongly influence the allergenicity of Aspergillus fumigatus spores

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  • 1. Growth temperature strongly influence the allergenicity of Aspergillus fumigatus sporesSwee Yang Low1, Karen Dannemiller1, MaoshengYao2, Naomichi Yamamoto1, and Jordan Peccia11YaleUniversity, New Haven, CT, USA2Peking University, Beijing, China NIEHS RES015312A
  • 2. Microbial Material in the Air•  Not a well researched subject- as opposed to microbiological content in soils and water l very dilute concentrations l poor cross-training between aerosol scientists and biologists l historical bias against the airborne route of infection
  • 3. Measurement of allergens ●Allergenic disease is a major cause of illness and disability in the US. Up to 40% of the members of the general public have developed IgE antibodies against environmental antigens (Pope et al., Indoor Allergens 1993).●Type 1 allergenic disease isincreasing (Ring, J., et al., Current Opinion in Immunology, 2001. 13: p. 701). Peccia, Milton, Reponen, and Hill. Environmental Science and Technology, 2008● >700 different biological allergens. (I.U.I.S., The official List of Allergens.2011).
  • 4. Allergenic fungal diversity through 454 pyrosequencing Stemphylium $!!"# Stachybotrys Rhodotorula Aspergillus total fungal ,!"# Penicillin Alternaria sequences +!"# Pleosporaallergen fraction *!"# Cryptococcus ambiguous )!"# Epicoccum allergenic classifications fungi (!"# non-allergenic !"# fungi &!"# Cladosporium Epicoccum %!"# $!"# !"#
  • 5. Complexity of biological allergens IgE MAST CELLS/ BASOPHILS MAST cells and BASOPHILS with attached IgE releases histamine or other inflammatory agents upon binding with allergenic epitope Asp f 1
  • 6. Allergenic spores have multiple IgE binding sites Aspergillus fumigatus has 23 allergenic proteins. (proteins have diversity of functions e.g. ribotoxin, heat shock 3 protein) IgE can bind to multiple sites within a 1 specific proteinTTGGSSSTPHGKDDHYLLEFFWTCINGIVAHQR
  • 7. Hypothesis and ObjectivesHYPOTHESIS: Allergen potency is a function of the environment inwhich a spore is producedObjective 1: Determine if the allergen potency (IgE binding capacity)of A. fumigatus spores is a function of temperature.Objective 2: Track the expression of allergen encoding genes infungal spores produced at variable temperatures.
  • 8. Human IgE binding assay for allergenictyApproach•  Allow Aspergillus fumigatus conidia to form at temperatures rangi32°C and determine the relative allergenicity per spore. Fi Bind A. fumigatus pe spores with IgE from the sensitized patients bin fro ai Unbound IgE is mixed fum with immobilized A. ex fumigatus extract high allergenicity low allergenicity
  • 9. Characteristic dose-response curve,IgE bind to A. fumigatus proteins 450 400 17 oC 350 Relative Allergenicity 300 250 200 150 100 50 0 -50 1.0E+05 105 1.0E+06 106 1.0E+07 107 Spore Concentration (spores/ml)
  • 10. Effects of temperature on allergen potency (P < 0.01, ANOVA)
  • 11. Effects of temperature on culturability of A. fumigatus (P < 0.005, t test)
  • 12. spores with IgE from sensitized patients binding of human IgE Gene expression microarrays of A. potency Effects of temperature on allergen fumigatus from sensitize patients to a immobilized Aspergillus sporulating at immobilizedmixed 32 C Unbound IgE is and with 17oC A. o fumigatus allergen extract. fumigatus extract high allergenicity low allergenicity•  Quantify the transcription levels of allergen encoding genes in spores grown at 17°C to 32°C todetermine if the variation in allergenicity has a transcriptional basis. 17°C Figure 2. Gene expression microarrays were use toSporulate at compare global and allergen Extract Label cDNAdifferent mRNA with Cy5 & Cy3 encoding gene transcription intemperatures A. fumigatus that sporulated at 17°C and 32°C. 32°C
  • 13. Global expression pattern – proteinproduction upregulated at lower temperatures
  • 14. Expression of allergen encoding genes
  • 15. Investigations in progress:●How do indoor and atmospheric pollutants impact theallergenicity of different fungal allergens? (1) O3 NOx NO2 (2) Low oxidant levels increase the production of peroxisomal membrane protein (Asp f3). Homologs of the genes encoding for the PMP are found in 23 or 25 allergenic fungi in with genomes have been sequenced.
  • 16. Conclusions● Environmental conditions during sporulation influencethe allergenicity “IgE binding capacity” and allergenproduction in A. fumigatus spores. Human IgE Total and Microarray and binding allergenic qPCR gene protein expression● Culturabilityand qPCR can underestimate (5 to 50times) the allergenicity of A. fumigatus spores that wereproduced at lower temperature.