344 Clopidogrel Reduces Posttransplant Obliterative Airway Disease (OAD)

Abstracts 342 Azithromycin Inhibits Mesenchymal Transition of Allograft Epithelium B. Banerjee,2 M.M. Musk,3 S.T. Yerkovich,1 P.M. Hopkins,1 S.M. Stick,2,4,5 A. Kicic,2,4,5 D.C. Chambers.1 1Queensland Centre for Pulmonary Transplantation & Vascular Disease, The Prince Charles Hospital, Brisbane, QLD, Australia; 2University of Western Australia, Perth, WA, Australia; 3Western Australia Lung Transplant Unit, Royal Perth Hospital, Perth, WA, Australia; 4Telethon Institute of Child Health Research, Perth, WA, Australia; 5Respiratory Medicine, Princess Margaret Hospital, Perth, WA, Australia. Purpose: Epithelial mesenchymal transition (EMT) following bronchiolar injury may represent the final common pathway to fibrotic obliteration in obliterative bronchiolitis (OB) and is therefore an attractive therapeutic target. We hypothesised that compounds with apparent therapeutic efficacy in OB may act by inhibiting EMT. Methods and Materials: Primary cultures of small (SAEC) and large airway epithelium (LAEC) were established from post-transplant brushings (n⫽ 18 patients, all stable BOS 0) and EMT induced by incubating with TGF␤1 (50ng/ml). EMT inhibition was assessed by incubating with diluent alone or with azithromycin (1mg/L, 10mg/L, 50mg/L), mycophenolate (0.05mg/L, 0.5mg/L, 5.0mg/L) or RAD001 (0.01nM, 0.1nM, 1.0nM) (n⫽6 for each). EMT was assessed by expression of epithelial (ZO-1, Ck-19) and mesenchymal proteins (Eda-Fn, Vim) measured via western blot as well as by MMP 2 & 9 activity via zymography. Results: Azithromycin potently inhibited TGF␤1-induced EMT of both SAEC and LAEC (reduced expression of mesenchymal markers and preserved expression of epithelial markers, p⬍ 0.05 at all doses at 48h and 96h time points), with mycophenolate demonstrating some activity (p⬍ 0.05 at 0.5 mg/L and 5.0 mg/L dose at 96h time point) and RAD001, no activity (Fig 1).

Conclusions: The in vitro model we have developed is suitable for assessing the activity and potency of candidate therapeutics. Inhibition of TGF␤1-induced bronchiolar EMT by azithromycin may explain the apparent clinical efficacy of this agent in OB. 343 Decreased Efferocytosis (Phagocytosis of Apoptotic Cells) and Mannose Binding Lectin in the Airways of Patients with Bronchiolitis Obliterans Syndrome S. Hodge,1,2 M. Dean,3 G. Hodge,1,2 M. Holmes,1,2,4 P.N. Reynolds.1,2 1 Lung Research, Hanson Institute, Adelaide, SA, Australia; 2Thoracic Medicine, Royal Adelaide Hospital, Adelaide, SA, Australia; 3Research and Business Development, Australian Red Coss Blood Service, Brisbane, QLD, Australia; 4South Australian Lung Transplant Services, Adelaide, SA, Australia. Purpose: Mannose binding lectin (MBL) is a key mediator of both innate immunity and efferocytosis (phagocytosis of apoptotic cells) in the airway. Defective efferocytosis results in a net increase in apoptotic material that can undergo secondary necrosis, leading to tissue damage and chronic inflammation. We have shown reduced MBL and efferocytosis in other chronic inflammatory lung diseases; we therefore hypothesized that reduced MBL and

S119 efferocytosis in the airways may be a determinant of bronchiolitis obliterans syndrome (BOS) following lung transplantation. Methods and Materials: We investigated MBL (ELISA), MBL-mediated complement deposition (UC4, ELISA) and efferocytosis of apoptotic bronchial epithelial cells (flow cytometry) in bronchoalveolar lavage (BAL) and peripheral blood from 75 lung transplant recipients (16 with stable graft function, 34 stable with proven infection, 25 with BOS) and 14 healthy controls. Results: In plasma, MBL levels were highly variable (0-17.538 mg/mL), but increased in infected subjects vs control (p⫽0.09) or stable groups (p⫽0.003). There was a similar increase in UC4 in infected patients and a significant correlation between MBL and UC4. There was no correlation between MBL and time post-transplant. In BAL, MBL levels were less variable (0-73.3 ng/mL) and significantly reduced in patients with BOS vs controls and stable groups. Efferocytosis was significantly reduced in the BOS group vs control and stable groups (Mean [SEM] Control: 20% [1.3%], Stable 20.5% [2.5%], Infected 17.3% [2.8%], BOS 11.3% [1.5%, p⫽.04]). Conclusions: Low levels of MBL in the airway may play a role in reduced efferocytosis, subsequent tissue damage and BOS following lung transplantation. 344 Clopidogrel Reduces Posttransplant Obliterative Airway Disease (OAD) R. Preidl, S. Eckl, M. Ramsperger-Gleixner, N. Koch, M. Weyand, S.M. Ensminger. Department of Cardiac Sugery, University Hospital, Erlangen- Nürnberg, Germany. Purpose: Survival after lung transplantation is mainly limited by chronic rejection with its main feature Obliterative Airway Disease (OAD). The aim of this study was to investigate if platelet inhibition with the P2Y12antagonist Clopidogrel has any influence on the development of OAD. Methods and Materials: Fully major histocompatibility complex-mismatched C57BL/6 (H2(b)) donor tracheas were orthotopically transplanted into CBA (H2(k)) recipients. Mice received either 1mg/kg/d or 20mg/kg/d Clopidogrel. Grafts were analysed by histology and morphometry on postoperative days 15, 30 or 60. In addition, alloantibody-production was analysed by FACS. Results: Untreated mice showed significant amounts of luminal obliteration on postoperative days 15, 30 and 60. Mice treated with 1mg/kg/d Clopidogrel for 30 days showed reduced tracheal obliteration as compared to untreated controls (42.2%⫾12.8% (1mg/kg Clopidogrel) vs. 45.8⫾9.9%(control), n⫽5). An increased dose of 20mg/kg/d Clopidogrel resulted in even stronger reduction of OAD (day 30: 34.4⫾8.4% (20mg/kg Clopidogrel) vs. 45.8⫾9.9% (control), n⫽5 and day 60: 37.7⫾14.0% (20mg/kg Clopidogrel) vs. 41.8⫾11.7% (1mg/kg Clopidogrel) vs. 46.1⫾13.1% (control), n⫽5). In addition, we could demonstrate that platelet inhibition with Clopidogrel resulted in reduced alloantibody production on postoperative days 30 and 60 (day 30: 17992⫾6864 MFI (1mg/kg Clopidogrel) vs. 25754⫾7201 MFI (control), n⫽6 and day 60: 26857⫾3901 MFI (20mg/kg Clopidogrel) vs. 31027⫾5391 MFI (control), n⫽5). Conclusions: Here we can show for the first time that platelet inhibition with clopidogrel can have a significant effect on the development of OAD, a major life-limiting complication after lung transplantation. 345 Ligation of Human Airway Epithelial Cells Antibodies Specific to K␣1-Tubulin Leads to Activation of HIF-1␣ Dependent Induction of Fibrinogenic Growth Factors: Role in the Pathogenesis of Chronic Rejection Following Human Lung Transplantation V. Tiriveedhi,1 T. Mohanakumar.1,2 1Department of Surgery, Washington University School of Medicine, St. Louis, MO; 2Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO. Purpose: Long term survival of the human lung transplant is hindered by chronic rejection manifested clinically as Bronchiolitis Obliterans Syndrome (BOS). We previously identified development of antibodies (Abs) to