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Growth temperature strongly influence the allergenicity of Aspergillus fumigatus spores
1. Growth temperature strongly influence the
allergenicity of Aspergillus fumigatus
spores
Swee Yang Low1, Karen Dannemiller1, Maosheng
Yao2, Naomichi Yamamoto1, and Jordan Peccia1
1YaleUniversity, New Haven, CT, USA
2Peking 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 is
increasing (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).
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 protein
TTGGSSSTPHGKDDHYLLEFFWTCINGIVAHQR
7. Hypothesis and Objectives
HYPOTHESIS: Allergen potency is a function of the environment in
which a spore is produced
Objective 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 in
fungal spores produced at variable temperatures.
8. Human IgE binding assay for allergenicty
Approach
• Allow Aspergillus fumigatus conidia to form at temperatures rangi
32°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
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 to
determine if the variation in allergenicity has a transcriptional basis.
17°C
Figure 2. Gene expression
microarrays were use to
Sporulate at compare global and allergen
Extract Label cDNA
different
mRNA with Cy5 & Cy3 encoding gene transcription in
temperatures
A. fumigatus that sporulated at
17°C and 32°C.
32°C
15. Investigations in progress:
●How do indoor and atmospheric pollutants impact the
allergenicity 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 influence
the allergenicity “IgE binding capacity” and allergen
production in A. fumigatus spores.
Human IgE Total and Microarray and
binding allergenic qPCR gene
protein expression
● Culturabilityand qPCR can underestimate (5 to 50
times) the allergenicity of A. fumigatus spores that were
produced at lower temperature.
