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