77 Characterization of Immune Response In �-tubulin InducedMurine Autoimmune Hearing Loss

C. CAI, B. ZHOU, J. GLICKSTEIN, T. J. YOO; UNIVERSITY OF

TENNEESSEE, MEMPHIS, TN.

RATIONALE: Western blot analysis has shown that 59% of Meniere’s

disease patients produce antibodies to a 55 kD inner ear membranous and

neural protein identified to be �-tubulin.

METHODS: BALB/C mice were subcutaneously injected with �-tubulin

in dosage of 100, 200 and 300 �g with CFA per mouse, immunizations

were boosted in IFA with varying doses of tubulin twice at one-week

intervals. Control mice underwent subcutaneous injection of PBS and

CFA/IFA as well.

RESULTS: The antibodies activity to �-tubulin increased in dose depen-

dent compared with controls, all control subjects were relatively unre-

sponsive. Moreover, IFN-r level was markedly increased in both serum

and supernatant of lymphocytes, protein levels of, IL-4, IL-5 IL-10 and

IL-13 were significantly reduced following �-tubulin immunization. Flow

cytometric analysis of spleen cells from �-tubulin induced mice and con-

trol mice have been showed that 2.72% of total splenocytes in control

mice were CD25+CD4+ regulatory T cells (Treg cells), the population of

Treg cells was reduced in �-tubulin induced mice and followed dose

dependent (such as 1.68% in 300�g, 2.16% in 200 �g and 2.19% in 100

�g), the Treg cells in naïve mice is 2.6%. Moreover, IFN-r and IL-2 level

was markedly increased in supernatant of Treg cells culture, protein lev-

els of IL-4, IL-5, IL-10, and IL-13 were significantly reduced following

�-tubulin immunization.

CONCLUSIONS: These data indicate an immune reactivity against �-

tubulin, which might be responsible for the autoimmune inner ear hearing

loss.

Funding: NIH

78 Activin A Deficiency: Association with Autoimmune Lung Disease

M. J. Thomassen1, T. L. Bonfield2, B. P. Barna2, N. John2, M. S. Kavuru1;1Pulmonary and Critical Care, East Carolina University, Greenville, NC,2Dept of Pulmonary, Allergy & Critical Care Medicine, Cleveland Clinic

Foundation, Cleveland, OH.

RATIONALE: Because pulmonary alveolar proteinosis (PAP) is an

autoimmune lung disorder characterized by neutralizing autoantibodies to

granulocyte-macrophage colony stimulating factor (GM-CSF), we

searched for autoimmunity-related genes in bronchoalveolar lavage

(BAL) cells by global microarray.

METHODS: Total RNA was isolated from BAL cells of PAP patients and

healthy controls and analyzed by microarray and real-time PCR. Proteins

in BAL fluids and conditioned media from BAL cultures were examined

in ELISA assays. Proliferation and secretion of anti-GM-CSF antibodies

were evaluated in PAP peripheral blood B cells by Cylex and Luminex

microplate assays, respectively.

RESULTS: Activin A, a cytokine implicated in B cell regulation was

severely deficient in PAP BAL cells; microarray results indicated a

>1000-fold reduction in PAP compared to controls (p = 0.001). Real time

PCR confirmed activin A mRNA deficiency. BAL fluid protein level and

BAL cell protein synthesis were also markedly reduced. In PAP or control

BAL cells cultured for 24 hours with GM-CSF, activin A secretion

increased. Treatment of PAP B cells with activin A in vitro suppressed B

cell proliferation in a receptor-dependent manner and decreased secretion

of anti-GM-CSF autoantibody.

CONCLUSIONS: Deficiency of the B cell regulatory cytokine, activin A

may contribute to chronic autoantibody production in PAP.

Funding: NIH

79 [Withdrawn]

80 The Spontaneous and Induced Production of Fas-ligand byEosinophils from Subjects with Aspirin Hypersensitivity, AllergicRhinitis and Healthy Volunteers

I. Kuprys-Lipinska, M. Kupczyk, M. Bochenska-Marciniak, P. Gorski,

P. Kuna; Allergy and Pneumonology, Barlicki University Hospital, Lodz,

POLAND.

RATIONALE: One of the reasons for persistent tissue inflammation in

patients with aspirin hypersensitivity and allergic rhinitis is decrease of

cells apoptosis. The aim of this study is to investigate the spontaneous and

induced production of Fas-ligand - the factor participating in cells apop-

tosis- by the eosinophils derived from subjects with aspirin hypersensitiv-

ity, allergic rhinitis and healthy volunteers.

METHODS: Eosinophils were isolated form peripheral blood mononu-

clear cells by negative selection using MACS System. Then 24-hours

eosinophils cultures were performed. Both spontaneous and induced Fas-

ligand production was investigated. For eosinophils stimulation nasal

lavages from aspirin hypersensitivity subjects undergoing aspirin

intranasal challenge were used.

RESULTS: Spontaneous and stimulating production of Fas-ligand by

eosinophils from patients with allergic rhinitis was decreased comparing

to healthy subjects (0,032 ng/ml and 0,013 ng/ml as compared to 0,386

and 0,321 ng/ml respectively, p<0,05). There was no difference between

both spontaneous (0,105 ng/ml) and induced (0,386 ng/ml) Fas-ligand

production by eosinophils derived from aspirin hypersensitive patients

and healthy volunteers. In none of the groups no differences between

spontaneous and stimulated Fas-ligand production was observed.

CONCLUSIONS: As expected the production of apoptosis induced

factor by eosinophiles derived from allergic subjects was decreased.

Unchanged production of Fas-ligand by eosinophils from patients with

aspirin hypersensitivity may suggest both decrease and dysfunction of

Fas-receptor or dysfunction other beyond Fas apoptosis pathways.

Funding: Polish Scientific Research Committee 507-11-173

S20 Abstracts J ALLERGY CLIN IMMUNOL

FEBRUARY 2006SA

TU

RD

AY