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95 Delayed Therapy with Clopidogrel and Everolimus Substantially Inhibits the Development of Transplant Arteriosclerosis
Abstracts Results: In each group, one beagle has died due to severe pulmonary edema after 2 h of reperfusion. Lung oxygenation significantly improved with normothermic EVLP (p⬍0.01), and lung oxygenation at the end of EVLP reflected lung oxygenation at the end of reperfusion (R⫽0.99, p⬍0.01). In details, PaO2 at the beginning, the end of EVLP, and 4 h of reperfusion were 437⫾68 mmHg, 558⫾35 mmHg, and 576⫾63 mmHg, respectively. Other lung functions (dynamic pulmonary compliance, peak airway pressure, pulmonary vascular resistance) were stable during EVLP. The posttransplant oxygenation in the EVLP group was significantly better than that in the SCS group (p⬍0.05). The dynamic pulmonary compliance at 4 h of reperfusion in the EVLP group was significantly better than that in the SCS group (EVLP 13.02⫾0.99 ml/mmHg, SCS 11.26⫾0.99 ml/ mmHg; p⬍0.05). The wet to dry lung weight ratio in the EVLP group was significantly lower than that in the SCS group (EVLP 6.30⫾0.54, SCS 10.92⫾0.86; p⬍0.01). Conclusions: Normothermic ex vivo lung perfusion could resuscitate NHBD lungs injured by 4 h of warm ischemia. 92 Donor Accidental Aspiration Injury – Can We Correct It by Ex-VivoLung-Perfusion (EVLP)? S. Wipper, A. Lüke, J. Sandte, J. Schirmer, H. Reichenspurner, F.M. Wagner. Department of Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany. Purpose: After establishing a porcine pulmonary aspiration injury model we evaluated reconditioning/treatment options during ex vivo lung perfusion. Methods and Materials: After inducing aspiration injury lungs were perfused for 6 hrs in our ex-vivo lung perfusion circuit (leucocyte filter, heparin coated system, perfusate Steen solution ⫹ erythrocytes, Hb5mg/dl) according to standardized protocol. Three groups (n⫽7 each) were studied: Gr.1 reperfusion only, Gr.2 reperf.⫹extensive medical treatment incl. NO ventilation, Gr.3 like Gr.2⫹ATG. Respiratory and hemodynamic parameters were monitored pre-harvest and hourly during reperfusion. Pre- and postreperfusion wet-dry ratio was calculated, histology performed. Judgement of transplantability based on standard donor criteria plus evaluation of pulmonary deflation index (PDI) ⱕ2 (index grading 1-5; 1⫽normal to 5⫽macroscopic frothy edema). Results: In Gr.1 one lung failed after 4hrs of reperfusion, while in Gr.2 and 3 all lungs were perfused for 6hrs. Compliance was equal in Gr.2 and 3 (59⫾8 vs. 59⫾13 ml/mbar), but significantly better than in Gr.1 (32⫾16 ml/mbar) at study end-point; PVR was lowest using ATG (1590⫾412 Gr.1 vs. 585⫾150 Gr.2 vs. 357⫾225 Gr.3 dynes). Pulmonary Oxygenation Capacity decreased in Gr.1 and remained stable in Gr.2/3 (172⫾115 Gr.1 vs. 383⫾45 Gr.2 vs. 322⫾57 mmHg Gr.3). Macroscopic evaluation revealed significantly better results in Gr.3 (PDI: 3.8⫾0.8 Gr.1 vs. 2.3⫾0.6 Gr.2 vs. 1.6⫾0.5 Gr.3). Histology and wet-dry ratio confirmed results. In Gr.1 all lungs failed transplantability criteria, in Gr.2 47% were transplantable, in Gr.3 using ATG even 86%. Conclusions: Using appropriate medical treatment including ATG administration EVLP allows reversal even of pulmonary aspiration injury. 93 Histological and Functional Evaluation of Lungs Reconditioned by Ex Vivo Lung Perfusion I.L. Medeiros, P.M. Pego-Fernandes, A.W. Mariani, F.G. Fernandes, F.V. Unterpertinger, M. Canzianba, F.B. Jatene. Heart Institute (InCor), São Paulo, Brazil. Purpose: Only 15% of donor lungs are considered suitable for transplantation (LTx). Ex vivo lung perfusion (EVLP) has been developed to repair damaged lungs. Several studies have shown that EVLP can ameliorate the injury process caused by prolonged ischemia. We report our experience with EVLP in nonacceptable lungs and evaluate its ability to recondition them. Methods and Materials: We studied lungs from 16 donors, rejected for LTx based on current clinical criteria. After harvesting, the lungs were stored at 4°C for 10 hours, and subjected to EVLP at 37°C with Steen Solution® for 1 hour. For functional evaluation, the following variables
S39 were assessed: arterial oxygen partial pressure (PaO2), pulmonary vascular resistance (PVR) and lung compliance (LC). For histological assessment, lung biopsy was done before harvest and after EVLP. Tissue samples were examined under light microscopy: a lung injury score (LIS) was defined with parameters as interstitial edema and inflammatory cell infiltration. To detect and quantify apoptosis, fluorescent in situ end labeling of DNA fragments (TUNEL) was used. Results: Thirteen lung donors were refused for having impaired lung function. The mean PaO2 obtained in the organ donor at the referring hospital was 193.7mmHg and rose to 489mmHg after EVLP (p⬍.001). During EVLP the mean PVR was 652.5dynes/sec/cm5 and mean LC was 48mL/cmH2O. Lung histology was preserved after 10 hours of cold ischemia and 1 hour of EVLP. There was no significant difference between the mean LIS before harvest and after EVLP (3.81 vs. 4.37, p⫽.456). There was a trend toward a reduction in the median number of apoptotic cells after EVLP (200/mm2 vs. 100/mm2, p⫽.063). Conclusions: EVLP improved lung function of organs considered unsuitable for LTx. There was not deterioration in lung tissue structure after EVLP. It also appears to lessen lung injury, as there was a reduction in the number of apoptotic cells. Further studies, including measurement of inflammatory mediators, are needed to clarify the possible benefit of EVLP in minimizing ischemia-reperfusion injury. 94 Gamma Delta T Cells Contribute to Acute and Chronic Allograft Rejection Via IL-17 Induced Inflammation in a Murine Heart Transplantation Model N. Kimura,1 S. Nakae,2 S. Itoh,1 D.R. Merk,1 Y. Gong,1 X. Wang,1 B. Patlolla,1 E. Neophytou,1 P.A. Chang,1 R.C. Robbins,1 M.P. Fischbein.1 1Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA; 2Frontier Research Initiative, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. Purpose: The role of gamma delta (GD) T cells in allograft rejection remains poorly understood. We have reported that IL-17 contributes to allograft rejection in a murine heart transplant model. Because intragraft GD T cells are the predominant source of IL-17, we hypothesized GD T cells may play a key role in allograft rejection. Methods and Materials: Acute rejection: FVB donor hearts heterotopically transplanted into (a) GD T cell-deficient C57BL/6 (TCR G⫺/⫺) or (b) C57BL/6 (WT) recipients. Graft survival measured. Graft infiltrating cells detected with FACS analysis. Neutrophil activity accessed via myeloperoxidase (MPO) assay. Cytokine mRNA expression detected with Taqman PCR. Chronic rejection: C-H-2bm12KhEg (H-2bm12) donor hearts transplanted into (a) TCR G⫺/⫺ or (b) WT recipients. Graft survival assessed with graft beating scores (0 to 4). Grafts harvested day 52 and graft coronary artery disease (GCAD) evaluated with morphometric analysis (luminal occlusion, intima-to-media ratio, and percentage of diseased vessels). Graft infiltrating cells assessed by FACS. Results: Acute model: Graft survival was prolonged in TCR G⫺/⫺ recipients (n ⫽ 18) compared to WT recipients (n ⫽ 14) (14.4⫾8.2 days vs. 8.6⫾1.2 days, p ⬍ 0.01). Graft infiltrating CD45⫹, CD4⫹, CD8⫹ cells, neutrophils, and MPO activity were decreased in TCR G⫺/⫺ recipients (p ⬍ 0.05). FACS analysis revealed GD T cells were key IL-17 producers (WT); IL-17 producing cells were decreased in TCR G⫺/⫺ recipients (p ⬍ 0.05). Supporting these findings, IL-17 and IL-23 mRNA expression was decreased in TCR G⫺/⫺ recipients (p ⬍ 0.05). Chronic model: Graft survival was improved in TCR G⫺/⫺ recipients, and GCAD development was reduced in TCR G⫺/⫺ recipients. Graft infiltrating CD45⫹ cells were attenuated in TCR G⫺/⫺ recipients (p ⬍ 0.05). Conclusions: GD T cells are (1) crucial for the development of acute and chronic allograft rejection during heart transplants, (2) key producers of IL-17, and (3) important in inflammatory cell recruitment. 95 Delayed Therapy with Clopidogrel and Everolimus Substantially Inhibits the Development of Transplant Arteriosclerosis S. Eckl, R. Preidl, S. Abele-Ohl, C. Heim, M. Ramsperger-Gleixner, N. Koch, M. Weyand, S.M. Ensminger. Cardiac Surgery, University Hospital, Erlangen- Nürnberg, Germany.
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The Journal of Heart and Lung Transplantation, Vol 30, No 4S, April 2011
Purpose: Our group has shown that combination of clopidogrel and everolimus dramatically reduced the development of transplant arteriosclerosis (TxA). The aim of this study was to investigate whether a delayed onset of treatment- similar to the clinical setting - could inhibit progression of TxA. Methods and Materials: Fully allogeneic C57BL/6 (H2(b)) donor aortas were transplanted into CBA (H2(k)) recipients. Recipients were treated with Clopidogrel (1 mg/kg/d) and Everolimus (0.05 mg/kg/d), alone or in combination, starting on postoperative days 1, 7 or 14. Grafts were analyzed by histology and morphometry on day 30 after transplantation. Results: In mice treated with clopidogrel alone, TxA was significantly reduced as compared to untreated controls when therapy was started on day 1 (intima proliferation 56%⫾11% vs. 81%⫾7% [control]/n⫽5). This effect was also seen when clopidogrel therapy was started on postoperative day 14 (intima proliferation 50%⫾4% vs. 81%⫾7% [control]/n⫽5). Combination of clopidogrel and everolimus dramatically reduced the formation of TxA when medication was started on day 1 (intima proliferation: 11% ⫾ 8% vs. 81% ⫾ 9% [control], n ⫽ 5) and showed remarkable reduction in both delayed onset groups (on day 7: intima proliferation: 36% ⫾ 10% vs. 81% ⫾ 9% [control], n ⫽ 5 and on day 14: intima proliferation: 44% ⫾ 3% vs. 81% ⫾ 9% [control], n ⫽ 5). Conclusions: These results demonstrate that even delayed onset of treatment with clopidogrel and everolimus – representative for a clinical setting - results in a strong reduction of TxA in this murine aortic allograft model. 96 An Artificial Nanoemulsion Carrying Paclitaxel Decreases the Cardiac Allograft Vasculopathy. A Study in a Rabbit Graft Model N.A.G. Stolf, E.R. Tavares, C. Contreras, R.C. Maranhão. Heart Institute University of Sao Paulo Medical School, São Paulo, Brazil. Purpose: Cardiac Allograft Vasculopathy (CAV) is the leading cause of death after heart transplantation (HT) after the fifth year. It was previously shown that paclitaxel associated with lipidic nanoemulsions resembling LDL was able to recede atheromatous lesions in rabbits with atherosclerosis induced by high cholesterol diet. Determine whether treatment with LDE-paclitaxel reduces the incidence and degree of CAV and analyze biodistribution of LDE in rabbits undergoing HT. Methods and Materials: Cervical heterotopic HT was performed in 21 rabbits fed with regular chow added with 0.5% cholesterol and cyclosporine A for 6 weeks, divided in two groups: A) 10 rabbits treated with saline intravenously (control) and B) 11 rabbits treated with LDE-paclitaxel intravenously. LDE labeled with radioactive cholesteryl ether was counted in hearts and other tissues after I.V. injection. Cross-sectional areas of coronary arteries of both native and grafts was estimated by measuring the internal elastic lamina and the lumen area. Percentage of stenosis was calculated from the difference between the area of the vessel lumen and the area of internal elastic lamina. Results: Uptake of labeled LDE in transplanted hearts was fourfold higher than in native hearts (pⱕ0.0001). Grafts from animals treated with LDEpaclitaxel have had an improvement in the status of the coronary arteries, showed by threefold increase of the vascular lumen area (pⱕ0.031) and 45% reduction of stenosis (pⱕ0.0008). Conclusions: LDE is able to concentrate in the graft, which enables the targeting of paclitaxel. Treatment with LDE-paclitaxel markedly reduced CAV, which may open a new perspective to achieve longer survival after HT. 97 Autoimmunity to Cardiac Myosin Is Associated with Acute and Chronic Rejection of Cardiac Allografts in Non-Human Primates A.M. Azimzadeh,1 A. Newton,1 X. Cheng,1 K. Tahir,1 A. Ward,1 T. Zhang,1 S. Kelishadi,1 D. Fairweather,2 M. Cunningham,3 R.N. Pierson III.1 1Surgery, University of Maryland School of Medicine, Baltimore, MD; 2Environmental Health Sciences, Johns Hopkins University, Baltimore, MD; 3Microbiology & Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK. Purpose: Immune responses to autoantigens have been described in animal and human allograft recipients. We previously reported immunity to vi-
mentin with development of cardiac allograft vasculopathy (CAV) in non-human primates. Here we report immunity to cardiac myosin, a heartspecific autoantigen, after transplantation in this model. Methods and Materials: Cynomolgus monkeys received a heterotopic heart allograft and were separated into 3 groups based on graft outcome: 1) Acute rejection (AR, median survival time [MST] ⬍20 days), after antiCD28 costimulation blockade, subtherapeutic Cyclosporine A (CsA), or anti-CD20 monotherapy; 2) chronic rejection (MST ⬍90 days), seen with therapeutic CsA (trough 400-600 ng/ml); and 3) graft acceptance (MST ⬎90 days), observed with CsA⫹anti-CD20. Antibodies (IgM and IgG) against human and monkey CM were assayed by ELISA until the time of graft explant. Positive samples were assayed for ␣CM IgG by western-blot. Results: AR was associated with increased ␣CM IgM (9/15) and/or IgG (7/15) levels, detected within 30 days post transplantation. CsA was associated with moderate to severe CAV (CAV score ⬎2); CM Abs were detected in 83% (5/6). When AR and CAV were prevented with CsA⫹Bcell depletion (CAV score ⬍0.4), CM Ab were not detected. Detection of immunity to CM correlated closely with detection of both serum alloantibody and C4d deposition in the graft. By western-blot, reactivity to the CM heavy chain was detected. Conclusions: Detection of humoral immunity to cardiac myosin is closely associated with both acute and chronic rejection of cardiac allografts, but is not detected when CAV is prevented. We conclude that increasing titer of antibody against CM is a biologic marker for chronic cardiac allograft injury. Whether loss of immunoregulation (tolerance) to the autologous CM protein is an epiphenomenon or pathogenic remains to be determined. If immunity against CM contributes significantly to allograft injury, inducing robust tolerance to CM may help attenuate heart allograft injury. 98 Role of Antibodies to HLA, Collagen-V and K-␣1-Tubulin in Antibody Mediated Rejection and Cardiac Allograft Vasculopathy V. Tiriveedhi,1 D.S. Nath,2 D. Phelan,3 N. Moazami,4 G. Ewald,5 T. Mohanakumar.1,6 1Department of Surgery, Washington University School of Medicine, St. Louis, MO; 2Department of Surgery, Children’s National Medical Center, Washington, DC; 3HLA Laboratory, BarnesJewish Hospital, St. Louis, MO; 4Surgery, Minneapolis Cardiothoracic Surgery Consultants, Minneapolis, MN; 5Cardiovascular Division, Washington University School of Medicine, St. Louis, MO; 6Depatment of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO. Purpose: Donor specific antibodies (DSA) to mismatched HLA and cardiac self antigens have been implicated in the pathogenesis of acute and chronic allograft rejection following human adult heart transplantation (HTx). We determined the role of DSA and antibodies (Abs) to self antigens, collagen-V (Col-V) and K-␣1-Tubulin (KAT) in the pathogenesis of acute antibody mediated rejection (AMR) and cardiac allograft vasculopathy (CAV) following HTx. Methods and Materials: 137 HTx recipients - 60 early period (EP, ⬍12 months) and 77 late period (LP, ⬎12 months) patients were enrolled. Circulating DSA was determined using LUMINEX. Abs against Col-I, II, IV, V and KAT were measured using ELISA. Frequency of CD4⫹ T helper cells (CD4⫹Th) secreting IFN-␥, IL-5, IL-10 or IL-17 specific to Col-V or KAT were determined using ELISPOT. Results: A significant association between AMR and DSA (AMR(⫹): 78%, AMR(-):18%, p⫽0.03) and Abs to Col-V (AMR(⫹): 383⫾72g/ mL, AMR(-):172⫾49g/mL, p⫽0.033) and KAT (AMR(⫹): 252⫾49g/ mL, AMR(-): 61⫾21g/mL, p⫽0.014) was demonstrated. AMR(⫹) patients demonstrated increased frequencies of CD4⫹Th cells secreting IFN-␥ and IL-5 with reduction in IL-10 specific for Col-V and KAT. CAV(⫹) patients developed DSA (CAV(⫹):79%, CAV(-):25%, p⫽0.03) and Abs to Col-V (CAV(⫹):835⫾142g/mL, CAV(-): 242⫾68g/mL, p⫽0.025) and KAT (CAV(⫹): 768⫾206g/mL, CAV(-): 196⫾72g/mL p⫽0.001) with increased frequencies of CD4⫹Th cells IL-17 with reduction in IL-10 specific for Col-V and KAT. Conclusions: We conclude that development of Abs to HLA and self antigens were associated with marked increases in CD4 T cells secreting IFN-␥ and IL-5 specific for self antigens in AMR and IL-17 in CAV with reduction in T cells secreting IL-10 in both AMR and CAV.
S39 were assessed: arterial oxygen partial pressure (PaO2), pulmonary vascular resistance (PVR) and lung compliance (LC). For histological assessment, lung biopsy was done before harvest and after EVLP. Tissue samples were examined under light microscopy: a lung injury score (LIS) was defined with parameters as interstitial edema and inflammatory cell infiltration. To detect and quantify apoptosis, fluorescent in situ end labeling of DNA fragments (TUNEL) was used. Results: Thirteen lung donors were refused for having impaired lung function. The mean PaO2 obtained in the organ donor at the referring hospital was 193.7mmHg and rose to 489mmHg after EVLP (p⬍.001). During EVLP the mean PVR was 652.5dynes/sec/cm5 and mean LC was 48mL/cmH2O. Lung histology was preserved after 10 hours of cold ischemia and 1 hour of EVLP. There was no significant difference between the mean LIS before harvest and after EVLP (3.81 vs. 4.37, p⫽.456). There was a trend toward a reduction in the median number of apoptotic cells after EVLP (200/mm2 vs. 100/mm2, p⫽.063). Conclusions: EVLP improved lung function of organs considered unsuitable for LTx. There was not deterioration in lung tissue structure after EVLP. It also appears to lessen lung injury, as there was a reduction in the number of apoptotic cells. Further studies, including measurement of inflammatory mediators, are needed to clarify the possible benefit of EVLP in minimizing ischemia-reperfusion injury. 94 Gamma Delta T Cells Contribute to Acute and Chronic Allograft Rejection Via IL-17 Induced Inflammation in a Murine Heart Transplantation Model N. Kimura,1 S. Nakae,2 S. Itoh,1 D.R. Merk,1 Y. Gong,1 X. Wang,1 B. Patlolla,1 E. Neophytou,1 P.A. Chang,1 R.C. Robbins,1 M.P. Fischbein.1 1Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA; 2Frontier Research Initiative, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. Purpose: The role of gamma delta (GD) T cells in allograft rejection remains poorly understood. We have reported that IL-17 contributes to allograft rejection in a murine heart transplant model. Because intragraft GD T cells are the predominant source of IL-17, we hypothesized GD T cells may play a key role in allograft rejection. Methods and Materials: Acute rejection: FVB donor hearts heterotopically transplanted into (a) GD T cell-deficient C57BL/6 (TCR G⫺/⫺) or (b) C57BL/6 (WT) recipients. Graft survival measured. Graft infiltrating cells detected with FACS analysis. Neutrophil activity accessed via myeloperoxidase (MPO) assay. Cytokine mRNA expression detected with Taqman PCR. Chronic rejection: C-H-2bm12KhEg (H-2bm12) donor hearts transplanted into (a) TCR G⫺/⫺ or (b) WT recipients. Graft survival assessed with graft beating scores (0 to 4). Grafts harvested day 52 and graft coronary artery disease (GCAD) evaluated with morphometric analysis (luminal occlusion, intima-to-media ratio, and percentage of diseased vessels). Graft infiltrating cells assessed by FACS. Results: Acute model: Graft survival was prolonged in TCR G⫺/⫺ recipients (n ⫽ 18) compared to WT recipients (n ⫽ 14) (14.4⫾8.2 days vs. 8.6⫾1.2 days, p ⬍ 0.01). Graft infiltrating CD45⫹, CD4⫹, CD8⫹ cells, neutrophils, and MPO activity were decreased in TCR G⫺/⫺ recipients (p ⬍ 0.05). FACS analysis revealed GD T cells were key IL-17 producers (WT); IL-17 producing cells were decreased in TCR G⫺/⫺ recipients (p ⬍ 0.05). Supporting these findings, IL-17 and IL-23 mRNA expression was decreased in TCR G⫺/⫺ recipients (p ⬍ 0.05). Chronic model: Graft survival was improved in TCR G⫺/⫺ recipients, and GCAD development was reduced in TCR G⫺/⫺ recipients. Graft infiltrating CD45⫹ cells were attenuated in TCR G⫺/⫺ recipients (p ⬍ 0.05). Conclusions: GD T cells are (1) crucial for the development of acute and chronic allograft rejection during heart transplants, (2) key producers of IL-17, and (3) important in inflammatory cell recruitment. 95 Delayed Therapy with Clopidogrel and Everolimus Substantially Inhibits the Development of Transplant Arteriosclerosis S. Eckl, R. Preidl, S. Abele-Ohl, C. Heim, M. Ramsperger-Gleixner, N. Koch, M. Weyand, S.M. Ensminger. Cardiac Surgery, University Hospital, Erlangen- Nürnberg, Germany.
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The Journal of Heart and Lung Transplantation, Vol 30, No 4S, April 2011
Purpose: Our group has shown that combination of clopidogrel and everolimus dramatically reduced the development of transplant arteriosclerosis (TxA). The aim of this study was to investigate whether a delayed onset of treatment- similar to the clinical setting - could inhibit progression of TxA. Methods and Materials: Fully allogeneic C57BL/6 (H2(b)) donor aortas were transplanted into CBA (H2(k)) recipients. Recipients were treated with Clopidogrel (1 mg/kg/d) and Everolimus (0.05 mg/kg/d), alone or in combination, starting on postoperative days 1, 7 or 14. Grafts were analyzed by histology and morphometry on day 30 after transplantation. Results: In mice treated with clopidogrel alone, TxA was significantly reduced as compared to untreated controls when therapy was started on day 1 (intima proliferation 56%⫾11% vs. 81%⫾7% [control]/n⫽5). This effect was also seen when clopidogrel therapy was started on postoperative day 14 (intima proliferation 50%⫾4% vs. 81%⫾7% [control]/n⫽5). Combination of clopidogrel and everolimus dramatically reduced the formation of TxA when medication was started on day 1 (intima proliferation: 11% ⫾ 8% vs. 81% ⫾ 9% [control], n ⫽ 5) and showed remarkable reduction in both delayed onset groups (on day 7: intima proliferation: 36% ⫾ 10% vs. 81% ⫾ 9% [control], n ⫽ 5 and on day 14: intima proliferation: 44% ⫾ 3% vs. 81% ⫾ 9% [control], n ⫽ 5). Conclusions: These results demonstrate that even delayed onset of treatment with clopidogrel and everolimus – representative for a clinical setting - results in a strong reduction of TxA in this murine aortic allograft model. 96 An Artificial Nanoemulsion Carrying Paclitaxel Decreases the Cardiac Allograft Vasculopathy. A Study in a Rabbit Graft Model N.A.G. Stolf, E.R. Tavares, C. Contreras, R.C. Maranhão. Heart Institute University of Sao Paulo Medical School, São Paulo, Brazil. Purpose: Cardiac Allograft Vasculopathy (CAV) is the leading cause of death after heart transplantation (HT) after the fifth year. It was previously shown that paclitaxel associated with lipidic nanoemulsions resembling LDL was able to recede atheromatous lesions in rabbits with atherosclerosis induced by high cholesterol diet. Determine whether treatment with LDE-paclitaxel reduces the incidence and degree of CAV and analyze biodistribution of LDE in rabbits undergoing HT. Methods and Materials: Cervical heterotopic HT was performed in 21 rabbits fed with regular chow added with 0.5% cholesterol and cyclosporine A for 6 weeks, divided in two groups: A) 10 rabbits treated with saline intravenously (control) and B) 11 rabbits treated with LDE-paclitaxel intravenously. LDE labeled with radioactive cholesteryl ether was counted in hearts and other tissues after I.V. injection. Cross-sectional areas of coronary arteries of both native and grafts was estimated by measuring the internal elastic lamina and the lumen area. Percentage of stenosis was calculated from the difference between the area of the vessel lumen and the area of internal elastic lamina. Results: Uptake of labeled LDE in transplanted hearts was fourfold higher than in native hearts (pⱕ0.0001). Grafts from animals treated with LDEpaclitaxel have had an improvement in the status of the coronary arteries, showed by threefold increase of the vascular lumen area (pⱕ0.031) and 45% reduction of stenosis (pⱕ0.0008). Conclusions: LDE is able to concentrate in the graft, which enables the targeting of paclitaxel. Treatment with LDE-paclitaxel markedly reduced CAV, which may open a new perspective to achieve longer survival after HT. 97 Autoimmunity to Cardiac Myosin Is Associated with Acute and Chronic Rejection of Cardiac Allografts in Non-Human Primates A.M. Azimzadeh,1 A. Newton,1 X. Cheng,1 K. Tahir,1 A. Ward,1 T. Zhang,1 S. Kelishadi,1 D. Fairweather,2 M. Cunningham,3 R.N. Pierson III.1 1Surgery, University of Maryland School of Medicine, Baltimore, MD; 2Environmental Health Sciences, Johns Hopkins University, Baltimore, MD; 3Microbiology & Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK. Purpose: Immune responses to autoantigens have been described in animal and human allograft recipients. We previously reported immunity to vi-
mentin with development of cardiac allograft vasculopathy (CAV) in non-human primates. Here we report immunity to cardiac myosin, a heartspecific autoantigen, after transplantation in this model. Methods and Materials: Cynomolgus monkeys received a heterotopic heart allograft and were separated into 3 groups based on graft outcome: 1) Acute rejection (AR, median survival time [MST] ⬍20 days), after antiCD28 costimulation blockade, subtherapeutic Cyclosporine A (CsA), or anti-CD20 monotherapy; 2) chronic rejection (MST ⬍90 days), seen with therapeutic CsA (trough 400-600 ng/ml); and 3) graft acceptance (MST ⬎90 days), observed with CsA⫹anti-CD20. Antibodies (IgM and IgG) against human and monkey CM were assayed by ELISA until the time of graft explant. Positive samples were assayed for ␣CM IgG by western-blot. Results: AR was associated with increased ␣CM IgM (9/15) and/or IgG (7/15) levels, detected within 30 days post transplantation. CsA was associated with moderate to severe CAV (CAV score ⬎2); CM Abs were detected in 83% (5/6). When AR and CAV were prevented with CsA⫹Bcell depletion (CAV score ⬍0.4), CM Ab were not detected. Detection of immunity to CM correlated closely with detection of both serum alloantibody and C4d deposition in the graft. By western-blot, reactivity to the CM heavy chain was detected. Conclusions: Detection of humoral immunity to cardiac myosin is closely associated with both acute and chronic rejection of cardiac allografts, but is not detected when CAV is prevented. We conclude that increasing titer of antibody against CM is a biologic marker for chronic cardiac allograft injury. Whether loss of immunoregulation (tolerance) to the autologous CM protein is an epiphenomenon or pathogenic remains to be determined. If immunity against CM contributes significantly to allograft injury, inducing robust tolerance to CM may help attenuate heart allograft injury. 98 Role of Antibodies to HLA, Collagen-V and K-␣1-Tubulin in Antibody Mediated Rejection and Cardiac Allograft Vasculopathy V. Tiriveedhi,1 D.S. Nath,2 D. Phelan,3 N. Moazami,4 G. Ewald,5 T. Mohanakumar.1,6 1Department of Surgery, Washington University School of Medicine, St. Louis, MO; 2Department of Surgery, Children’s National Medical Center, Washington, DC; 3HLA Laboratory, BarnesJewish Hospital, St. Louis, MO; 4Surgery, Minneapolis Cardiothoracic Surgery Consultants, Minneapolis, MN; 5Cardiovascular Division, Washington University School of Medicine, St. Louis, MO; 6Depatment of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO. Purpose: Donor specific antibodies (DSA) to mismatched HLA and cardiac self antigens have been implicated in the pathogenesis of acute and chronic allograft rejection following human adult heart transplantation (HTx). We determined the role of DSA and antibodies (Abs) to self antigens, collagen-V (Col-V) and K-␣1-Tubulin (KAT) in the pathogenesis of acute antibody mediated rejection (AMR) and cardiac allograft vasculopathy (CAV) following HTx. Methods and Materials: 137 HTx recipients - 60 early period (EP, ⬍12 months) and 77 late period (LP, ⬎12 months) patients were enrolled. Circulating DSA was determined using LUMINEX. Abs against Col-I, II, IV, V and KAT were measured using ELISA. Frequency of CD4⫹ T helper cells (CD4⫹Th) secreting IFN-␥, IL-5, IL-10 or IL-17 specific to Col-V or KAT were determined using ELISPOT. Results: A significant association between AMR and DSA (AMR(⫹): 78%, AMR(-):18%, p⫽0.03) and Abs to Col-V (AMR(⫹): 383⫾72g/ mL, AMR(-):172⫾49g/mL, p⫽0.033) and KAT (AMR(⫹): 252⫾49g/ mL, AMR(-): 61⫾21g/mL, p⫽0.014) was demonstrated. AMR(⫹) patients demonstrated increased frequencies of CD4⫹Th cells secreting IFN-␥ and IL-5 with reduction in IL-10 specific for Col-V and KAT. CAV(⫹) patients developed DSA (CAV(⫹):79%, CAV(-):25%, p⫽0.03) and Abs to Col-V (CAV(⫹):835⫾142g/mL, CAV(-): 242⫾68g/mL, p⫽0.025) and KAT (CAV(⫹): 768⫾206g/mL, CAV(-): 196⫾72g/mL p⫽0.001) with increased frequencies of CD4⫹Th cells IL-17 with reduction in IL-10 specific for Col-V and KAT. Conclusions: We conclude that development of Abs to HLA and self antigens were associated with marked increases in CD4 T cells secreting IFN-␥ and IL-5 specific for self antigens in AMR and IL-17 in CAV with reduction in T cells secreting IL-10 in both AMR and CAV.