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Transcript of Cross-Reactivity Between Recombinant Tropomyosin From Chortoglyphus and Natural Tropomyosin Of Other...
SUNDAY
J ALLERGY CLIN IMMUNOL
VOLUME 133, NUMBER 2
Abstracts AB101
350 Immunomodulatory Effects Of Rye Grass Pollen AllergenLol p 5 On The Prostaglandin E2 Pathway and Kallikrein-Kinin System Of Respiratory Epithelial Cells
Cecilia Tong, Alice Vrielink, Martha Ludwig, Geoffrey Stewart; Univer-
sity of Western Australia.
RATIONALE: Several important aeroallergens are known to modulate
respiratory epithelial (RE) function. The rye grass pollen Group 5 allergen
is a significant contributor to pollen allergy but its immunomodulatory
effects on RE function are unknown.
METHODS: Rye grass pollen allergen rLol p 5 and its N- and C-terminal
domains were expressed in E. coli. Physicochemical studies were conduct-
ed using SDS-PAGE, native-PAGE, HPLC, circular dichroism and Phyre2
modelling. Allergenicity was determined by ELISA and immunoblot, and
ribonuclease activity by enzyme assay. RE IL-8 release was examined us-
ing ELISA and A549, 16HBE14sigma- and Detroit562 cell lines. mRNA
level expression of enzymes/proteins associated with the prostaglandin
E2 (PGE2) pathway and kallikrein-kinin system (KKS) by RE cells were
determined using RT-PCR.
RESULTS: 3D-modelling showed rLol p 5 to be structurally similar to
Timothy grass pollenGroup 5 allergen; circular dichroism analysis showed
heat stable proteins with a predominance of a-helices and coils, consistent
with the 3D structures. HPLC and native PAGE indicated trimerization
(mature and N-terminal domain) and dimerization (C-terminal domain).
Each protein was enzymatically active, and reacted with IgE from seven
rye grass pollen sensitive patients. All three cell lines released IL-8, and
A549 cells showed PGE2 release. RT-PCR with A549 showed up-regula-
tion of COX-2, mPGES-1, mPGES-2 and cPGES in the PGE2 pathway,
and gC1qR, UPAR, UPA, HSP90a and PAI associated with the KKS.
CONCLUSIONS: The ribonuclease Lol p 5 and its component domains
were allergenic, existed as oligomers, induced IL-8 and PGE2 and up-regu-
lated proinflammatory PGE2 and KKS pathways by, as yet, unknown
mechanisms.
351 Development and Characterization Of a Murine Model OfRepeated Dry Exposure To Aerosolized Fungal Conidia
Dr. Ajay Nayak, PhD1, Dr. Amanda Buskirk, PhD1,2, Mr. W. Travis
Goldsmith3, Ms. Angela Lemons1, Dr. Justin Hettick, PhD1, Dr. Michael
Kashon, PhD4, Ms. Amy Cumpston3, Mr. Jared Cumpston3, Mr. Howard
Leonard3, Mr. Walter McKinney3, Dr. David Frazer, PhD3, Dr. Donald H.
Beezhold, PhD, FAAAAI1, Dr. Brett J. Green, PhD1; 1CDC/NIOSH/ACIB,
Morgantown, WV, 2West Virginia University, Morgantown, WV, 3CDC/
NIOSH/PPRB, Morgantown, WV, 4CDC/NIOSH/BEB, Morgantown, WV.
RATIONALE: Personal fungal exposures are associated with a variety of
adverse health outcomes, including invasive disease, allergic sensitization,
and asthma. Most murinemodels of fungal exposure have utilized aspiration
or inhalation of uncharacterized extracts or liquid conidial suspensions that
do not resemble natural human exposures. These studies were conducted to
characterize a novel dry aerosol repeated exposure model to Aspergillus fu-migatus with a goal towards defining the resulting immune response.
METHODS: In these studies, immunocompetent Balb/c mice were
repeatedly exposed to A. fumigatus wild-type (WT) or melanin-deficient
(Dalb1) conidia via aerosol exposure of dry conidia using an acoustical
generator. Flow cytometric and histopathologic analyses were conducted
to characterize the immune responses and the associated lung pathology
following repeated exposures.
RESULTS: Histological analysis demonstrated in vivo germination in mice
exposed to A. fumigatus conidia. WT exposure led to increased numbers of
adaptive immune system cells (B cells and T cells) and innate immune
effector cells (eosinophils, neutrophils, and macrophages). Importantly,
CD8+IL-17+ (Tc17) cells were also elevated in exposed mice, which ap-
peared to closely correlatewith the germination ofWT A. fumigatus conidia.
CONCLUSIONS: The data presented here are among the first to
characterize the immune responses to repeated dry fungal exposures in
immunocompetent animals. Dry aerosol exposures via the acoustical
generator may provide more accurate analyses of immune responses
following exposures to other environmentally prevalent fungi.
352 Cross-Reactivity Between Recombinant Tropomyosin FromChortoglyphus and Natural Tropomyosin Of Other Extracts
Dr. Jer�onimo Carn�es1, Dr. M. Angeles L�opez Matas1, Dr. Manuel
Boquete, MD2, Dr. Raquel Moya1, Dr. Victor Miguel Iraola1; 1Laborator-
ios LETI, Tres Cantos, Spain, 2Hospital Xeral de Calde, Lugo, Spain.
RATIONALE: Tropomyosin is a pan-allergen with high homology
among species, involved in cross-reactivity between mites, crustacean,
mollusks and insects. The objectives were to produce the recombinant
tropomyosin from Chortoglyphus arcuatus and to investigate the cross-
reactivity between different species.
METHODS: Recombinant C. arcuatus tropomyosin (r-tropomyosin) was
cloned, sequenced, expressed inEscherichia coli and purified byHPLC (ion ex-
change and affinity chromatography). Polyclonal anti-tropomyosin antibodies
wereproduced inmice. IgErecognition to r-tropomyosinwascheckedby immu-
noblotwith a pool of sera frompatients sensitized to storagemites fromGalicia.
Thenative tropomyosinwas identified in thecomplete extractofmitesby immu-
noblot inhibition after inhibiting the human pool of sera with r-tropomyosin.
Tropomyosin was also identified in shrimp, cockroach and Anisakis extracts
by immunoblot, incubating with anti-tropomyosin antibodies. Cross-reactivity
between r-tropomyosin and Der p 10 was studied by immunoblot inhibition.
RESULTS: A 40 kDa protein corresponding to tropomyosin (GeneBank,
JN596422), with a purity higher than 95% and a yield of 1.85 mg/l of bac-
terial culture, was obtained. r-tropomyosin was recognized by a pool of
sera from sensitized individuals. C. arcuatus tropomyosin was identified
in the whole extract. Tropomyosin was also identified in Anisakis, shrimp
and cockroach extracts. r-tropomyosin completely inhibited the recogni-
tion of Der p 10 by a monoclonal anti-Der p 10 antibody, therefore
cross-reactivity with Der p 10 was demonstrated.
CONCLUSIONS:
� Recombinant C. arcuatus tropomyosin was purified demonstrating its
capacity to recognize IgE by a specific pool of sera
� Cross-reactivity between mite tropomyosins was demonstrated.
� Anti-tropomyosin antibody recognized tropomyosin in other extracts
from invertebrates.
353 Identification Of The Cysteine Protease Amb a x As A NovelMajor Allergen From Short Ragweed Pollen (Ambrosiaartemisiifolia)
Dr. Philippe Moingeon, PhD1, Julien Bouley, PhD2, Maxime Le
Mignon, PhD2, V�eronique Baron-Bodo, PhD2, V�eronique Bordas, PhD2,
Laetitia Bussi�eres2, Marie-No€elle Couret3, Aur�elie Lautrette, PhD2, Thierry
Batard, PhD4, Rachel Groeme2, Henri Chabre, PhD2, Emmanuel Nony2;1Stallergenes SA, Antony, France, 2Stallergenes, France, 3Stallergenes,4Stallergenes, Antony, France.
RATIONALE: Allergy to pollen from short Ragweed (Ambrosia artemisii-folia) is a serious and ever expanding health problem in theUSand in Europe.
Herein, we investigated the presence of unrecognized allergens in Ragweed
pollen, with the goal to improve diagnostic and treatment modalities.
METHODS: A Ragweed pollen extract was analyzed by 2D gel
electrophoresis and IgE immunoblotting using 70 individual sera from
Ragweed pollen-allergic donors (from the US and Central Europe). IgE-
reactive protein spots were characterized by mass spectrometry.
RESULTS: Four distinct patternsof IgE sensitizationwere identified among
patients, in both American and European patients. High resolution 2D gel
electrophoresis allowed to identify a newallergen termedAmbax*,with IgE
reactivity confirmed in more than 60% of patients. Based on partial amino
acid sequencing, the genewas PCR cloned, demonstrating the high sequence
homology of Amb a x with known cysteine proteases, such as the house dust
miteDer p1 allergen.Ambaxwaspurified, fully characterizedbymass spec-
trometry and its three-dimensional structure established by homology
modeling. IgE reactivity was confirmed on purified natural and recombinant
forms of Amb a x. Our results suggest that the allergenic activity of Amb a x
was previously unrecognized and likely inappropriately ascribed toAmb a 1.
CONCLUSIONS: We identified Amb a x as a new major allergen
belonging to the cysteine protease family. Amb a x should be considered as
an essential component for diagnosis and specific immunotherapy of
Ragweed pollen allergy. * pending IUIS official nomenclature