Thematic Poster Session

S UNDAY, S EPTEMBER 3 RD 2006

Hall B2-14 - 12:50-14:40

113. Cellular mechanisms in the pathogenesis of asthma and rhinitis

P1342 Toll-like receptor 3-mediated synthesis of eotaxin-1 from human bronchial smooth muscle cells stimulated with double-stranded RNA Kyoko Niimi, Koichiro Asano, Yoshiki Shiraishi, Takahisa Takihara, Junko Kagyo, Misa Wakaki, Takeshi Nakajima, Koichi Fukunaga, Tsuyoshi Oguma, Koichi Sayama, Akitoshi Ishizaka. Department of Medicine, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan Respiratory infections with RNA viruses are a major cause of asthma exacerbation, accompanied eosinophilic inammation of the airways. We studied the effects of double-stranded RNA (dsRNA) synthesized during RNA virus replication, and of its receptor, TLR3, on the synthesis of eosinophilic chemokines in bronchial smooth muscle cells (BSMC). Synthetic dsRNA [poly(I:C)] induced the synthesis of eosinophilic chemokines, eotaxin-1 and RANTES, from primary cultures of human BSMC, and IL-4 increased synergistically the synthesis of eotaxin-1. A robust eosinophil chemotactic activity was released from BSMC stimulated with poly(I:C) and IL-4, which was mostly inhibited by pre-incubation with an antieotaxin-1 antibody. Although the immunoreactivity of TLR3 was detectable on the cellular surface of BSMC by ow cytometric analysis, pretreatment with an anti-TLR-3 neutralizing antibody failed to block the poly(I:C)-induced synthesis of eotaxin-1. We have determined by confocal laser-scanning microscopy that the immunoreactivity of TLR3 was aggregated intracellularly in poly(I:C)-stimulated BSMC, co-localizing with uorescein-labeled poly(I:C). The poly(I:C)-induced synthesis of eotaxin-1 was prominently inhibited by TLR3-specic siRNA or balomycin A1, an endosomal acidication inhibitor, further supporting the essential role played by intracellular TLR3 in the synthesis of poly(I:C)-induced eotaxin-1 in BSMC. In conclusion, these observations suggest that, by activating intracellular TLR3 in BSMC, respiratory RNA virus infections stimulate the production of eotaxin-1 and enhance eosinophilic inammation of the airways in the Th2-dominant microenvironment. P1343 Attenuated IL-10 release in blood from allergic subjects following allergen stimulation Kristin Blidberg, Lena Palmberg, Kjell Larsson. Lung and Allergy Research, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Background Allergic disease is characterised by a Th2 dominated cytokine response. Interleukin (IL)-10 is involved in the regulation of cytokine production and has anti-inammatory effects. IL-10 is produced by various cell types including T regulatory cells. In the present study IL-10 response to rat allergen stimulation was investigated in vitro in subjects allergic to rats and in a group of healthy controls. Method Eight persons allergic to rats with allergic symptoms, positive skin prick test and > 0.35 kUA/L sIgE (to rat) and 8 healthy volunteers participated in the study. Whole blood was collected in heparinized tubes and directly stimulated with lipopolysaccharide (LPS) (1 g/mL) or rat allergen (1, 5 or 50 g/mL). Production of IL-10 was measured using ELISA technique after 4, 8 and 24 hours of stimulation. Results Rat allergen induced increased IL-10 production over time at 5 and 50 g/mL (p< 0.05) but not at 1 g/mL. Furthermore, IL-10 levels increased with increasing allergen dose (p<0.05). In blood from allergic subjects the allergen induced IL-10 release was signicantly attenuated compared to non-allergic subjects at 1 and 5 g/mL at all but one (4h, 5 g/mL) time points. At the highest allergen dose (50 g/mL) the difference between the groups did not reach signicance (p=0.059). LPS stimulation increased the IL-10 levels over time in both groups (p<0.001) but there was no difference between the groups at any time point. Conclusion: An attenuated IL-10 response in allergic subjects may partly explain the Th2 dominated cytokine response observed in allergic disease. P1344 T-cell susceptibility to glucocorticoid (GC)-induced apoptosis: the role in asthma pathogenesis and therapy Sergei V. Boichuk 1 , Ilshat G. Mustan 2 , Pavel D. Dunaev 1 , Rustem S. Fassahov 3 . 1 Pathophisiology, Kazan State Medical University, Kazan, Russia; 2 Immunology, Kazan AIDS Center, Kazan, Russia; 3 Allergology, Kazan Postgraduate Academy, Kazan, Russia Background. The aim of the study was to compare sensitivity of T-cells puried from donors and asthmatics to GC-induced apoptosis. Methods. T cell phenotype and apoptosis were assessed by ow cytometry. Several apoptosis parameters were assessed: decrease of mitochondrial potential, membrane phosphatidylserine exposure, DNA fragmentation and apoptosis related

227s
Abstract printing supported by Nonin Medical, Inc. Visit Nonin Medical, Inc. at stand C09

Thematic Poster Session

S UNDAY, S EPTEMBER 3 RD 2006
markers (Bcl-2, Bax). To examine the mechanisms of GC-induced apoptosis T-cells from 32 allergic asthmatics and 18 control subjects were treated with dexamethason (DEX). Results. DEX induced apoptosis of T lymphocytes from asthmatics and control subjects in a dose-dependent manner in vitro. CD8+ T cells were more sensitive to DEX-induced apoptosis than CD4+ T cells. The apoptotic susceptibility of T cells to DEX was signicantly higher in asthmatics when compared to healthy subjects (p<0.01). DEX effect was associated with an increase of Bax/Bcl-2 ratio and decrease of Bcl-2 expression. T-cells in asthmatics expressed higher levels of activation markers (CD25, VLA-1, HLA-DR) when compared to healthy subjects. The percentages of apoptosis in DEX-treated T cells were found to be signicantly higher in activated T-cells(p<0.01). To modulate T-cell activation responsible for the susceptibility to GC-induced apoptosis we puried resting T-cells. The percentages of apoptosis in DEX-treated resting T cells were found to be signicantly lower when compared to anti-CD3-activated T-cells. Conclusion: Activated T cells are more susceptible than resting cells to GCinduced apoptosis. Asthmatic T lymphocytes might be preactivated in vivo and, hence, acquire increased susceptibility to apoptosis induction by DEX in vitro.

Hall B2-14 - 12:50-14:40

P1347 Histamine-stimulated monocytes release microparticles that upregulate IL-8 synthesis by human alveolar ephitelial cells Chiara Cerri, Tommaso Neri, Rosalia Tedone, Ilaria Conti, Pierluigi Paggiaro, Alessandro Celi. Dipartimento Cardiotoracico, University of Pisa, Pisa, Italy Background: Microparticles (MP) are cell derived fragments that play critical roles in numerous physiological processes, including blood coagulation and inammation. Aim: We investigated a potential novel proinammatory role of histamine mediated by MP induction in monocytes. Methods: Monocytes were isolated from normal donors following Ficoll/Hypaque gradient centrifugation and adhesion to plastic. MP release was stimulated with histamine (30M) for 15. The MP containing supernatant was harvested and incubated with cells of the alveolar line A549. Following a 20-hour incubation, the conditioned medium was analyzed for IL-8 release by ELISA and cells were analyzed for IL-8 mRNA by rt-PCR. Results: The supernatant of histamine-stimulated monocytes upregulates IL-8 expression; the effect is greatly diminished by ltration (0.2m cut-off) suggesting that particulate matter, rather then soluble molecules, is responsible for the effect. rt-PCR showed that mRNA expression parallels protein secretion. Fig. 1 shows the results from one experiment representative of 3.

P1345 The abnormalities in spontaneous and Fas-induced apoptosis in atopic bronchial asthma (ABA): the key to chronic lung inammation Sergei V. Boichuk 1 , Ilshat G. Mustan 2 , Rustem S. Fassahov 3 , Pavel D. Dunaev 1 . 1 Pathophysiology, Kazan State Medical University, Kazan, Russia; 2 Immunology, Republic AIDS Center, Kazan, Russia; 3 Allergology, Kazan State Postgraduate Academy, Kazan, Russia Background: The aim of the study was investigate the mechanisms of spontaneous and Fas-induced apoptosis of peripheral blood lymphocytes (PBLs) in asthmatics and donors. Methods: Apoptosis and its related markers (decrease of mitochondrial potential (MP); BcL-2 protein and Fas expression, out membrane phosphatidylserine exposure (PS), DNA fragmentation) were assessed by FACs. Results: PBLs in asthmatics were resistant to spontaneous apoptosis. Signicant decrease of Fas-expression was observed on CD4+ - and CD8+ -PBLs (not CD16+ and CD20+ ) in ABA. Activation of PBLs by anti-CD3+CD28 mAbs (assessed by CD69, CD25, HLA-DR) increased Fas-expression on T-cell subsets in asthmatics and donors. Activated PBLs demonstrated increased susceptibility to Fas-mediated apoptosis. Nevertheless, PBLs in asthmatics (even activated) were resistant to Fasinduced apoptosis when compared to healthy donors (p<0.01). The reduction of MP was earliest feature of spontaneous and Fas-mediated apoptosis. PS exposure reversibly depended from the MP level and correlated with BcL-2 expression. Conclusion: The resistance of PBLs to spontaneous apoptosis may be the main prerequisite for the chronic inammation in ABA. The resistance of TCR-activated PBLs to Fas-induced apoptosis in asthmatics can play the key role in defective elimination of T-cells, responding to antigens in ABA. Involvement of mitochondria in spontaneous and Fas-induced apoptosis indicates that this organelle may serve as an universal integrator of different apoptotic stimuli and promote the following recognition and engulfment of apoptotic cells by inducing the appearance of PS resides on their cell membrane.

Conclusions: Histamine induces monocytes to release MP capable of modulating airway inammation.

P1348 Apoptosis of lymphocytes in rhinal secretion of allergic rhinitis patients Olga P. Uhanova. Health Service Ministry, Stavropol Regional Clinical Center of Specialized Medical Care, Stavropol, Russia The aim of this research is the study of apoptosis mechanisms of lymphocytes in nasal cavity during the allergic rhinitis exacerbation and the topical glucocorticoid therapy. 50 people (15-60yrs) were examined. The control group consisted of 17 healthy people. 33 people suffered from seasonal allergic rhinitis caused by weed blooming in Stavropol region in August-September. They were classied into 2 groups according to the severity of clinical course. During 20 days they had been given the uticasone propionate (FP) therapy in the form of nasal spray Flixonase (GSK) 100 mgm a day. All of them received clinical, allergic, immunologic cytologic diagnostics. The expression of the apoptotic markers was determined by the ow cytouometry method. After FP therapy the reduction of the IgE level is to be observed, and that correlates with patients amelioration and reduction of clinical symptoms. The unilateral tendency to higher expression of AnnexinV, CD95, CD95-L, Bcl-2 in comparison with the control group were observed in rhinal secretion lymphocytes. The effect of FP on the expression of AnnexinV, CD95, CD95-L, Bcl-2 turned out to be opposite to the effect of allergen. One can observe the reduction of the expression of Bcl-2 under AR with mean degree 57.414.8% and grave condition from 90.14.3% to 15.95.1% and 14.14.5% (p<0.0001), that is considerably lower than the gures in control group. The same dynamics can be observed in the expression of CD95 and CD95-L.The results show the rhinal secretion lymphocytes stability in the AR patients to the process of apoptosis. At the same time the lymphocytes of the AR patients regardless of the severity level are susceptible to the apoptotic effect of FP.

P1346 Selective up-regulation of metalloproteinase-7 (matrilysin) in human blood and bronchoalveolar T-lymphocytes by the soluble nonpeptidic mycobacterial phosphoantigen (isopentenyl pyrophosphate) Grefachew Workalemahu, Foerster Martin, Kroegel Claus. Medical Clinic I, Pneumology & Allergology, Friedrich-Schiller-University, Jena, Germany Introduction. Human T-lymphocytes are believed to regulate local immune defense as well as enhance resistance towards invading microbes, although their precise function remains unknown. Herein we addressed the question whether T-lymphocytes mediate these processes via synthesis of MMP-7, a protease closely associated with both epithelial repair and mucosal defense. Methods. Blood and bronchoalveolar T-lymphocytes were cultured in the absence and presence of isopentenyl pyrophosphate (IPP) or TGF-1 /IL-15 for 24 h, and assessed for the expression and synthesis of MMP-1, MMP-7, and MMP-9. Results. Resting human T-lymphocytes constitutively expressed MMP-9 mRNA, marginal or no MMP-7 and MMP-1 mRNA. In the presence of IPP (3 g/ml), expression of MMP-7 mRNA signicantly increased whereas TGF1 /IL-15 had no effect. Further, quiescent T-lymphocytes obtained from bronchoalveolar lavage (BAL) uid showed either a weak or no MMP-7 mRNA signal which was raised signicantly following stimulation with IPP. In Western blot analysis, a 28-kDa pro-matrilysin could be detected both in cell lysates (2 days) and supernatants (5 days) with a 4- to 7-fold increased signal following IPP-stimulation of the T-lymphocytes. Conclusion. The data demonstrate that human T-lymphocytes are capable of differentially expressing and synthesizing MMP-7 in response to IPP and TGF1 /IL-15. This provides a new perspective for the understanding of T-lymphocyte function.

P1349 The inuence of hydrogen peroxide on reactive oxygen species generation in monocytes of healthy children and with pollinosis G. Semenkova, A. Krjukov, S. Cherenkevich, M. Petuh, V. Zhernosek. Biophysics, Belarus State University, Minsk, Belarus The mechanisms of reactive oxygen species (ROS) generation in monocytes of children with pollinosis before and after specic immunotherapy (SIT) were studied. 15 pollinosis patients and 20 healthy people 9-17 years old were included in this study. Cells were stimulated by the adhesion to glass and fMLP in the pres-

228s
Abstract printing supported by Nonin Medical, Inc. Visit Nonin Medical, Inc. at stand C09

Thematic Poster Session

S UNDAY, S EPTEMBER 3 RD 2006
ence and absence of H2 O2 . ROS production was measured by luminol dependent chemiluminescence (LDCL) method. It was previously shown that capability of monocytes to produce ROS was more in patients before SIT in comparison with control group. After SIT the yield of ROS generated by monocytes decreased, although monocyte response to priming agents and stimulation were still abnormal. After addition of H2 O2 (10 mol/l) the intensity of fMLP-induced LDCL in control group increased, but ROS generation in monocytes isolated from pollinosis patients decreased. Hydrogen peroxide (50 mol/l) induced the rise of free [Ca2+ ] in monocytes of healthy people on 32 nmol/l and on 8 nmol/l in patients. It was shown with the help of inhibitory analysis, that the increase of fMLP-induced ROS generation in monocytes of healthy children in the presence of hydrogen peroxide depended on the activity of phospholipase A2 , cyclooxygenases, MEK1/2. The addition of SB203580, an inhibitor of p38, to cells suspension of patients led to increase of ROS generation in monocytes at the stimulation of cells by fMLP in the presence of H2 O2 . Mechanisms of fMLP-induced ROS generation in monocytes of children with pollinosis in the presence of hydrogen peroxide are deranged that can be associated with the change of p38 activity.

Hall B2-14 - 12:50-14:40

P1352 In vitro analysis of mast cell and basophil responsiveness to modied and unmodied grass pollen extracts Laurie C. Lau 1 , Mark Gibson 1 , Pavels Gordins 1 , Dinesh Bagmane 1 , Simon Hewings 2 , David Borgars 2 , Andrew F. Walls 1 . 1 Infection, Inammation and Repair Division, University of Southampton, Southampton, United Kingdom; 2 Allergy Therapeutics, Worthing, United Kingdom In evaluating hypersensitivity to specic allergens or in assessing the allergenic potency of allergen extracts, there is a need for reliable in vitro tests of mast cell and basophil responsiveness. We have examined the ability of grass pollen extracts to stimulate secretion of histamine and basogranulin from blood basophils from allergic subjects and to alter expression of the basophil activation marker CD203c. In parallel studies we have investigated the potential of the extracts to stimulate histamine release from cells of the LAD2 mast cell line passively sensitised with serum from these subjects, and to provoke skin prick test responses. Allergen-induced mediator release from basophils and mast cells and up-regulation of CD203c expression was observed, with distinctive bell-shaped concentration response relationships. There were considerable differences between subjects in the concentrations that elicited mast cell and basophil activation. Basophil histamine and basogranulin responses were closely correlated, but there was no simple association with the expression of CD203c. The degree of histamine release from mast cells was not associated with any of the measures of basophil activation, but was closely correlated with the size of skin test responses. Glutaraldehyde modication of the grass pollen extract reduced the allergenicity as assessed by all methods, though the extent of the reduction varied between subjects, and in the basophil or mast cell models employed. The differences in ndings with the various methods call for the adoption of a broad approach in evaluating the potential for allergic responses in allergic subjects.

P1350 Role of dexamethasone and MAPkinases ERK and p38MAPK on T-bet expression and IFN release in peripheral blood lymphocytes from atopic asthmatics Maria Raidl 1 , Michael Lux 1 , Katja Mueller 1 , Hildegard Buening 2 , Sibille Humme 2 , Andrea Koch 1 . 1 University of Cologne, Medical Clinic III, Department of Pneumology, Cologne, NRW, Germany; 2 University of Cologne, Medical Clinic I, Center for Molecular Medicine Cologne, Cologne, NRW, Germany The transcription factor T-box-expressed-in-T-cells (T-bet) is required for TH1 lymphocyte differentiation, regulates the IL-12-induced expression of the TH1-specic cytokine IFN and may be dysregulated in asthmatics. The regulation of T-bet and of IFN by extracellular signal-regulated kinase (ERK)-1/-2, by p38mitogen-activated protein-kinase (MAPK) and by dexamethasone in IL-12-stimulated T-lymphocytes was assessed in 10 atopic asthmatics and 10 nonatopic normals using ELISA, western blotting and real-time RT-PCR methods. IFN production was dependent on phosphorylation of ERK-1/-2 and p38MAPK, as examined by PD089059, an inhibitor of the upstream activator of MAPKkinase (MKK-1), and SB203580, an inhibitor of p38MAPK. The inhibitory effect of PD098059 on IFN release was decreased in T-cells from asthmatics compared with normals. The IL-12-induced T-bet expression and the inhibitory effect of SB203580 were increased in T-cells from asthmatics compared with normals. Dexamethasone blocked the IL-12-induced T-bet expression in T-cells from asthmatics completely and decreased IL-12-induced IFN release by 50%, and this occurred to the same extent in T-cells from asthmatics and normals. In conclusion asthma is associated with enhanced IL-12-induced T-bet production, p38MAPK activation and a decreased ERK-1/-2 phosphorylation suggesting that these MAPkinase-pathways may play a more basic role in the pathogenesis of asthma. The therapeutic benet of dexamethasone on T-bet and IFN seems to be critical.

P1353 Novel cytosolic phospholipase A2 (cPLA2 ) promoter single nucleotide polymorphisms Milena Sokolowska 1 , Anna M. Lewandowicz 1 , Marek L. Kowalski 2 , Rafal Pawliczak 1 . 1 Department of Immunopathology, Medical University of Lodz, Lodz, Poland; 2 Department of Immunology, Rheumatology and Allergy, Medical University of Lodz, Lodz, Poland Background. cPLA2 is a critical cytoplasmic enzyme involved in the liberation of arachidonic acid from cellular membranes, which is followed by formation of prostaglandins, leukotriens and hydroxyeicosatetraenoic acids. Its promoter lacks a TATA-box, but it contains interferon- response elements, interferon- activated sequence and glucocorticoid response elements. Earlier analysis (Uozumi, N. et al. Nature 1997; 390:618-622) revealed that cPLA2 (-/-) mice developed signicantly reduced allergen-induced bronchoconstriction and have reduced bronchial reactivity to methacholine. Subsequently, changes in the cPLA2 promoter structure might result in the differences of cPLA2 transcription activity and could inuence inammatory response in bronchial asthma. Aim. Analysis of cytosolic phospholipase A2 (cPLA2 ) promoter polymorphisms and their potential role in the cPLA2 gene transcription. Methods. The study group consists of 52 patients with bronchial asthma. Genomic DNA was extracted from the peripheral blood leukocytes. PCR reactions with specic primers were performed. Primers were designed based on the published promoter sequence (No. U08374). To verify amplied fragments the Sangers DNA Sequencing with Chain-Terminating Inhibitors method was performed. Results. Three novel single nucleotide polymorphisms: -211G/A, -229 ins.T and -261del.T upstream from the transcriptional start site were found in studied cPLA2 promoter region in comparison to published sequence. Conclusions. These polymorphisms might have an impact on cPLA2 transcription, probably by interaction with transcription factors regulating the promoters activity. Founded by Medical University of Lodz grant 502-11-248

P1351 Opsonized zymosan stimulates CXC chemokine secretion from bronchial epithelial cells Adam Lightfoot, Anwar M. Hasan, Gordon Dent. Institute of Science & Technology in Medicine, Keele University, Keele, Staffordshire, United Kingdom Zymosan, a cell wall component of Saccharomyces cerevisiae, activates complement via the alternative pathway, leading to opsonization of the zymosan particles. Bound complement fragment C3b mediates the uptake of zymosan by several cell types, including phagocytes and venous endothelial cells, and their consequent activation [FEMS Immunol Med Microbiol 2003; 36:55-61]. Airway epithelial cells are exposed to opsonized microbes invading the respiratory tract. We therefore investigated whether bronchial epithelial cells (BEC) are activated by opsonized zymosan (OZ). Zymosan was opsonized by 30-min incubation at 37C with 20% pooled human serum. Growth-arrested H292 BEC were incubated with unopsonized zymosan (Z) or OZ for 24 h and secretion of chemokines was measured by ELISA and neutrophil chemotaxis assay. As an additional control, BEC were incubated with zymosan opsonized with serum that had been heat-inactivated at 56C for 15 min to remove complement (HZ). No signicant quantities of IL8 (3.0 2.3 fmol/well, n=4) or neutrophil chemotactic activity (NCA: chemotactic index [CI] = 1.2 0.16, n=3) were released from Z-treated BEC, while there was basal release of Gro (77 fmol/well) and ENA78 (144 fmol/well). OZ-treated BEC released substantial quantities of IL8 (95 49 fmol/well) and Gro (3,428 fmol/well), and slightly increased ENA78 (271 fmol/well) and NCA (CI = 1.5 0.12). Surprisingly, NCA release from HZ-treated cells was greater than that evoked by OZ (CI = 1.9 0.68). We conclude that opsonization of zymosan confers BEC-activating properties, as suggested previously [J Immunol 1991; 147:972-979]. It is unclear whether this process involves complement.

P1354 Activation of airway epithelial cell inhibited by peroxisome proliferator-activated receptor Wei Han, Xin Zhou. Department of Respiratory Medicine, Shanghai Jiaotong University, Shanghai, China; Department of Respiratory Medicine, Shanghai Jiaotong University, Shanghai, China Background Recent studies suggest that the peroxisome proliferator-activated receptor(PPAR-) ligands can modulate inammatory and remodeling of asthma through different ways, but the expression and mechanism of PPAR in asthma was not claried yet. Methods To investigated the expression of PPAR and effect of PPAR agonist in allergic airway disease, the human primary airway epithelial cells stimulated with bronchoalveolar lavage uids (BALF) from asthma patients were cultured and administrated with rosiglitazone alone (in several dosages), or combination with budesonide respectively. Result: The nuclear expression of PPAR and glucocorticoid receptor (GR) were all increased slightly by BALF stimulation, but steeply rose after administrated with rosiglitazone and combined therapy. The MMP-9 gelatinolytic activity, eotaxin expression and concentrations of procollagen in supernatant were inhibited in a concentration dependent manner. There was no signicant difference among the

229s
Abstract printing supported by Nonin Medical, Inc. Visit Nonin Medical, Inc. at stand C09

Thematic Poster Session

S UNDAY, S EPTEMBER 3 RD 2006
high dose group and combination group, which implied that a certain synergistic effect between the two agonists. Conclusion Our data suggests that the mechanism that PPAR agonist inhibited allergic airway inammation and remodeling includes (1) inducing nuclear expression of PPARand activating PPARdirectly, (2) enhancing the nuclear location of GR and exerting synergism. The interaction between these two receptors may provide a potential target for pharmacological intervention of asthma.

Hall B2-14 - 12:50-14:40

P1355 Interferon--pretreated dendritic cells induced dual Th1 and Th2 responses and failed to prevent development of allergic airway inammation Chang Zheng Wang, Jun Bo Xia, Kun Sun. Institute of Respiratory Disease, Xinqiao Hospital, Chongqing, China Background: Dendritic cells (DCs) are crucial for the induction of Th2 immune response in asthma. However, many cytokines may modulate the functions of DCs. We investigated which priming DCs by interferon (IFN)- modulated T cell responses and allergic airway inammation. Methods: Marrow-derived DC were generated from DC precursors in mice marrow by incubation with GM-CSF. Presence in IFN- promoted DCs further mature in dose dependent manner with increase of the expression of CD80 and MHCIIon DCs. Then, pre-treated DCs in presence of IFN- were incubated with T cells from mice sensitized by ovalbumin(OVA) and ovalbumin antigen for 48hr. Furthermore, IFN--primed DCs were injected intratracheally into sensitized mice and challenged by OVA subsequently. Results: IFN- pre-treatment enhanced signicantly DCs to release IL-12. However, IFN--primed DCs induced Th1 as well as Th2 response. Moreover, adaptive transfer of IFN-/DCs could not remarkably reduce total cell counts and eosinophils in BALF and attenuate eosinophil inltration in histological analysis for lung sections compared with PBS/DC-transferred and OVA-sensitized mice. Conclusions: IFN--primed DCs induced dual allergen-specic Th1 and Th2liked responses, which suggests that IFN- is also a pro-inammatory cytokine. Moreover, IFN--primed DCs failed to prevent development of allergic airway in sensitized mice exposed to antigen.

ences in whole-cell Kv1.3. conductability (gK) in T cells from three groups of patient: healthy (N=42), atopic (pollinosis: during allergen exposition N=14 and out of the pollen season N=24), and asthma (chronic N=72 and aspirin induced asthma - ASA N=10). Results: The gK in atopy (1.871.17nS) was higher then in control group (p<0.0001) The gK did not differ between pollinotics during and out of season (gK 3.943.45 and 3.382.01 nS respectively). No signicant differences in the Kv1.3. channels gK between healthy donors and asthmatic patients (1.831.03 nS) were observed. No signicant differences in the Kv1.3. channels gK in patients with different types of asthma that is: chronic asthma and ASA were found. Conclusions: We found out that the activity/expression of Kv1.3. on T cells of atopic patients is elevated.

P1358 IL4 in patients of bronchial asthma induced by mould sensitization via early and late phase of atopy and delayed T-cell mediated hypersensitivity Vladimir V. Klimov, Lyudmila I. Volkova, Alina N. Shkatova. Clinical Immunology and Allergology, Siberian State Medical University, Tomsk, Russia Background. Mould sensitization in asthma involves different pathways of immune responses. Prevalence of early or late phase of atopic pathway and delayed T-cell mediated hypersensitivity is of interest to choose a proper method of immunointervention in asthma such as specic immunotherapy with mould allergen. Methods. Using culture uid in the course of lymphoblast transformation test as analyte, IL4 was determined by enzyme-linked immunosorbent assay in 39 patients of bronchial asthma. All patients were divided by skin allergic tests with mould allergens into persons with early atopic phase, early+late atopic phases and early+late atopic phases+delated type. There were no patients with delayed type only. Results.Value of IL4 as the Th2 keystone cytokine has been different from control (31,22,2 pg/ml, p<0,001) in all groups. Rate of IL4 has been higher in patients with early atopic phase (62,21,0 pg/ml) and early+late phases (63,60,8 pg/ml) compared to persons with early+late phases+delated type (50,82,1 pg/ml, p<0,001). Conclusions. The data display a high prevalence of atopic mechanism in asthma induced by mould sensitization if even the delayed T-cell mediated hypersensitivity is available.

P1356 The cell haemostasis changes under the glucocorticoid therapy inuence in patients with bronchial asthma Janna S. Savitskaya, Vasily I. Tromov, Natalia L. Shaporova, Tamara V. Shjukina. Hospital Therapy, Pavlovs State Medical University, Saint-Petersburg, Russia Because of broader using of glucocorticoid therapy in treatment of patients with bronchial asthma (BA) and frequent development of its complications, including system of haemostasis, problem of safety of the treatment remains actual. We studied the inuence of infusion glucocorticoid therapy (the average dose 60-120 mg per day) on factors of cellular haemostasis (the factors of intravascular activation of thrombocytes, aggregation of erythrocytes) in 83 patients with exacerbation of the bronchial asthma. The study of the glucocorticoid therapy inuence on thrombocytes aggregation revealed it positive inuence on thrombocytes activity, which was shown in reduction of the active forms of thrombocytes amount (r<0, 05), and increase of the inactive form of thrombocytes content. The analysis of the glucocorticoid therapy infusion inuence on erythrocytes aggregation revealed signicant growth of the percent of aggregations (before treatment 72,39 3,62, after treatment 80,63 3,34; r<0,05), increased amount of huge aggregates (before treatment 300,5 43,13, after treatment 456,25 40,75; r<0,05) and increased of branch shaped forms (branch shaped forms were revealed in 72% patients). Activation of the aggregation activity was conrmed by positive correlation between aggregation and doses of glucocorticoid hormones. Thus, we describe quite opposite inuence of glucocorticoid hormones on aggregation function of thrombocytes and erythrocytes. So, glucocorticoid therapy reduced the thrombocytes aggregation activity and enlarged it in erythrocytes.

P1359 Attenuation of human bronchial smooth muscle cell proliferation by rapamycin in Th2 cytokine dominant status Junko Kagyo 1 , Koichiro Asano 1 , Yusuke Suzuki 2 , Kyoko Niimi 1 , Misa Wakaki 1 , Takahisa Takihara 1 , Yoshiki Shiraishi 1 , Koichi Fukunaga 1 , Koichi Sayama 1 , Akitoshi Ishizaka 1 . 1 Division of Pulmonary Medicine, Department of Medicine, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, Japan; 2 Department of Medicine, Saitama Municipal Hospital, Saitama, Japan It is known that asthmatic patients lose their lung function year by year over their lifetime, which is caused by structural remodeling of their airways such as bronchial smooth muscle cell (BSMC) hyperplasia. It has been reported that BSMC taken from asthmatics proliferated twice as fast as those from non-asthmatics in vitro, and glucocorticoid failed to suppress asthmatic BSMC proliferation. Rapamycin is known to inhibit vascular smooth muscle cell proliferation and has already been clinically applied for that purpose. We have investigated if rapamycin suppresses the proliferation of human BSMC even in Th2 cytokine-dominant environment. BSMC was incubated with serum-free medium in the presence or absence of interleukin-13 (IL-13, 30 ng/ml) for 24 hrs. We pretreated BSMC with rapamycin (10-9 -10-6 M) for 1 hr and then stimulated with 5% serum. After 24hrs of incubation, DNA synthesis rate was evaluated by BrdU uptake assay. IL-13 alone showed variable effects on BSMC proliferation. In contrast, rapamycin constantly inhibited the serum-induced proliferation of BSMC regardless of the presence of IL-13 signicantly (3922% without IL-13 and 4321% with IL-13 at the rapamycin concentration of 10- 7 M, respectively, mean SD, n = 3, P<0.05). In conclusion, rapamycin can be useful to suppress airway remodeling in asthmatic patients.

P1357 The conductability of potassium ion channels on lymphocytes T in atopic patients with pollinosis and in chronic asthma and aspirin induced asthma patients Maryla Krasnowska, Marita Nittner-Marszalska, Antonina Gawlik, Andrzej Teisseyre, Andrzej Fal. Department of Internal Medicine and Allergology, Medical University, Wroclaw, Poland Background: Ion channels constitute an important part of cellular signaling processes of the immune system. Activated T cells require high levels of intracellular calcium, which enters cells through calcium release-activated-calcium channels: the voltage-gated Kv1.3. and calcium dependent KCa3.1. Kv1.3 channels in activated T cells take over as the dominant functional K channels. Several groups have described high expression of Kv1.3 on T cells in autoimmune diseases and MS. In pathogenesis of atopy and asthma activated T cells play a fundamental role but so far this phenomenon has not been investigated. The aim of our study is an estimation of the conductability of Kv1.3.on T cells in atopy (pollinosis) and asthma. Material and method: Using the patch-clamp technique we studied the differ-

P1360 The effects of dendritic cell expressing FasL on CD4+ T cell in a mouse model of asthma Yan Wang, Changzheng Wang. Institute of Respiratory Diseases, Institute of Respiratory Diseases, Chongqing, China Background: Fas ligand (FasL) is a member of the tumor necrosis factor family, and it induces apoptosis of the cell by bounding to Fas. We investigated the effects of dendritic cell (DC) expressing FasL on CD4+ T cell in a mouse model of asthma. Methods: Mouse FasL (mFasL) gene was transfected into the normal DC and the expression of the mFasL gene was conrmed by immunocytochemistry and Western Blot. C57BL/6 mice were sensitized and challenged by house dust mite (HDM) extract. Two days after challenged, the mice were killed and CD4+ T cells was harvested. After cocultured with DC expressing mFasL (mFasL-DC) and HDM extract, CD4+ T cells were collected for the measurement of proliferation

230s
Abstract printing supported by Nonin Medical, Inc. Visit Nonin Medical, Inc. at stand C09

Thematic Poster Session

S UNDAY, S EPTEMBER 3 RD 2006
with MTT and apoptosis with Annexin V by FACS, the supernatants were used to assay the level of IL-4, IL-5 and IFN-gamma by ELISA. Results: The mFasL-DC expressed the mFasL mRNA and protein. The percentage of the apoptosis CD4+ T cells from asthmatic mice (82.416.7%) was higher signicantly than from control mice (29.58.1%). The mFasL-DC inhibited the proliferation of CD4+ T cell from asthmatic mice. The levels of IL-4, IL-5 decreased signicantly and the level of IFN-gamma increased slightly in the supernatants of CD4+ T cell from asthmatic mice after cocultured with mFasL-DC. Conclusion: The results suggested that mFasL-DC induced the apoptosis of CD4+ T cell from asthmatic mice and inhibited Th2 skewed immune response. Supported by National Natural Science Fundation of China (No. 30170403).

Hall B2-14 - 12:50-14:40

P1361 A differential role for ceramide kinase in antigen/Fc RI mediated mast cell activation and function Christopher A. Hewson, Rachel L. Grimley, Mark D. Fidock. Biomarkers & Translational Biology, Pzer Global R&D, Sandwich, Kent, United Kingdom Rationale: Mast cells are specialised secretory cells critically involved in allergic disease by release of multiple inammatory mediators. Activation initiates a complex kinase signalling-cascade. We have investigated a role for the novel Ceramide Kinase (CERK) in this cascade. Methods: The mast cell-line RBL-2H3 was stimulated via Fc RI or with the active product of CERK (ceramide-1-phosphate [C1P]) and multiple end-points of mast cell activation assayed by ELISA; histamine (pre-formed acute-phase mediator), prostaglandin D2 (PGD2 - rapidly metabolised acute-phase mediator) and IL-13 (de novo transcribed late-phase mediator). Specic siRNA was used to reduce CERK expression. Results: C1P induced a time and dose-dependent release of histamine and PGD2 independently (4-6h post-stimulation, p<0.001) and in synergy with FcRI mediated activation (1-2h post-stimulation, p<0.001), and inhibition of CERK abrogated this (p<0.001); with histamine more dependent on CERK activity. CERK-specic siRNA, reducing mRNA by 48%, reduced Fc RI-mediated histamine release by 40% (p<0.001). However, this level of silencing did not affect PGD2 release. Finally, using validated IC50 concentrations of specic inhibitors and C1P stimulation, we identied that, for histamine, CERK was downstream of the SYK, PI-3K and PLC kinases, but upstream of JNK (p<0.001); while for PGD2 CERK was positioned upstream of JNK, MEK and cyclooxygense (p<0.001). C1P did not stimulate IL-13, indicating a specic role in acute-phase activation. Conclusions: We have identied a novel and differential role for CERK in mastcell activation and have begun to elucidate its position in the mast cell-signalling cascade.

231s
Abstract printing supported by Nonin Medical, Inc. Visit Nonin Medical, Inc. at stand C09