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Development of a novel preclinical pig-to-non-human primate model of xenogeneic orthotopic lung transplantation
120
Abstracts
The Journal of Heart and Lung Transplantation January 2002
Methods and Results: 249 cardiac transplant recipients(41 female) between 1989 and 1999 receiving statin therapy in combination with cyclosporin A were assessed from case records for lipid lowering effect, adverse events and survival. Four statins were used simvastatin(n⫽150), pravastatin(49), fluvastatin(40), atorvastatin(10). Mean treatment period(months); simvastatin 115.2, pravastatin 96.1, fluvastatin 59.9, atorvastatin 16.0. Percentage reduction in low density lipoprotein (at mean dose) was as follows; simvastatin(12.4mg) 30.3%, pravastatin(29.3mg) 29.4%, fluvastatin(40mg) 28.9%, atorvastatin(10mg) 32.1%. Adverse events; elevated creatinine kinase and myalgia: simvastatin 18%, pravastatin 9%, fluvastatin 0%, atrovastatin 0%. Elevated hepatic transaminases; simvastatin 6%, pravastatin 0%, fluvastatin 0%, atorvastatin 0%. Overall using the Cox proportional hazards model, percentage reduction in total cholesterol (p⫽0.006) and LDL cholesterol (p⫽0.01) correlated most strongly with improved survival. However there was no significant evidence for a difference in survival time between the four statins used (p⫽0.29). Conclusion: Significant reductions in lipids can be achieved safely using long term statin therapy following cardiac transplantation. Previous evidence has suggested that the benefits of these drugs in this population are immune mediated independent of lipid lowering effect. The results of this study suggest that the lipid lowering effect of these drugs is also important particularly in the long term. 180 RENIN-ANGIOTENSIN-ALDOSTERONE-SYSTEM DIRECTED ANTIHYPERTENSIVE THERAPY ACHIEVES LOWER B-TYPE NATRIURETIC PEPTIDE LEVELS COMPARED WITH CALCIUM CHANNEL BLOCKADE IN HEART TRANSPLANTATION M.H. Park, P.A. Uber, R.L. Scott, L.E. Bourgeois, M.R. Mehra, Ochsner Clinic Foundation, New Orleans, LA Background. The optimal antihypertensive regimen after heart transplantation remains elusive and poorly defined. Whether therapy targeting the renin-antiotensin-aldosterone system (RAAS) evokes benefits beyond blood pressure (BP) lowering in heart transplantation has not been previously determined. We hypothesized that RAAS directed therapy compared with calcium channel blockade (CCB) would be more likely to decrease b-type natriuretic peptide (BNP) levels, a sensitive and specific marker of ventricular overload, at similar blood pressure(BP) control. Methods. In 30 consecutive stable heart transplant recipients, we compared BNP levels between patients receiving a primary RAAS targeted therapy (n⫽14) for hypertension versus CCB therapy (n⫽16). All patients (80% men, 87% whites, age 61⫾4years, tacrolimus, mycophenolate and steroid immunosuppression) achieved BP control to a systolic and diastolic BP⬍140/90 mmHg with stable allograft function (Ejection Fraction 63⫾5%) without ongoing rejection). Results. In the RAAS directed treatment group, 71% received angiotensin receptor blockers while 29% were on angiotensin converting enzyme inhibitors. All patients in the CCB group recieved amlodipine. Findings are depicted in the table below. Systolic BP RAAS Group CCB Group p value
129 ⫾ 11 130 ⫾ 9 0.7
Diastolic BP 84 ⫾ 8 84 ⫾ 6 0.9
BNP (pg/dl) 89 ⫾ 78 174 ⫾ 136 0.05
Regression analysis of systolic and diastolic blood pressure with BNPlevels failed to demonstrate any significant correlation. Similarly no significant correlations of renal function and BNP levels were noted in this cohort. Inferences. Despite similar levels of blood pressure control achieved with CCB’s, RAAS directed antihypertensive therapy is associated with lower BNP levels, a sensitive and specific marker of ventricular wall stress. Thus, we suggest that RAAS directed therapy evokes benefits on cardiac allograft adaptation beyond those achieved by blood pressure lowering alone. 181 TACROLIMUS/MYCOPHENOLATE MOFETIL VS CYCLOSPORINE/MYCOPHENOLATE MOFETIL: IMPACT ON INFECTIONS FOLLOWING CARDIAC TRANSPLANTATION J. Groetzner, B. Meiser, J. Schirmer, S. Schenk, M. Muller, F. ¨ berfuhr, B. Reichart, Klinikum Reisch, M. Oberhoffer, F. Kur, P. U Grosshadern, University Hospital, Munich, Germany Background: New combinations of immunosuppressive agents led to a decrease of acute rejection episodes (ARE) after HTx. They might be associated with an increased risk of infections. Therefore we compared the incidence of infections in HTxpatients treated with either a Tacrolimus- (Tac) - or a Cyclosporine- (CsA) based immunosuppression. Methods: In a prospective, randomized clinical trial, 61 HTx patients (n⫽30/31) were included during 1999 and 2001. Immunosuppression consisted of CsA, Mycophenolatmofetil (MMF) and Corticosteroids (Cort) or Tac, MMF and Cort. Cort were stopped as early as 6 month po. Endpoints were incidence of infections and ARE. Infections were defined as positive cultures or laboratory values with the need of antibiotic treatment. Results: Demographic data as well as primary diagnosis and perioperative data were comparable between both groups. Comparison of the infection incidence revealed a trend towards an increase in CsA-patients (0.31 inf./100pat.days vs. 0.27 (Tac); p⫽n.s.). Distribution of the bacterial, viral and fungal infections were similar in both groups: (CsA vs Tac) Bacterial: 45% vs 50%; Viral:27% vs. 29%; Fungal: 28% vs 21%, p⫽n.s.). Incidence of ARE/100 pat.days was significantly lower in the Tac/MMF group when compared to the CsA/MMF group (0.09 and 0.3; p⬍0.003). Nineteen infections in the CsA and 4 in the Tac group occured within 3 weeks after ARE treatment. ( p⬍0.05 ). Especially the incidence of fungal and viral infections following ARE was significantly higher in CsA treated patients (fungal: 7 vs 0; p⫽0.03; Viral: 7 vs 2; p⫽0.05; Bacterial: 6 vs 2; p⫽n.s.). Overall Survival was 90% (CsA) vs. 96% (Tac), the percentage with lethal infections (aspergillus-pneumonia)was comparable (6,7% (CsA) vs. 3,3% (Tac) p⫽n.s.). Conclusion: The immunosuppressive combination of Tac/MMF resulted in a trend to less infections after HTx than CsA/MMF. This tendency was in many cases contributed to augmented immunosuppression during rejection treatment. Since there is a significantly higher incidence of ARE in CsA-treated patients, Tac/MMF proved to be the combination with the favorable safety profile. 182 DEVELOPMENT OF A NOVEL PRECLINICAL PIG-TONON-HUMAN PRIMATE MODEL OF XENOGENEIC ORTHOTOPIC LUNG TRANSPLANTATION
The Journal of Heart and Lung Transplantation Volume 21, Number 1 A.R. Simon,1 S. Fischer,1 R. Tessmann,1 C. Schroeder,1 U. Martin,1 M. Chikobava,2 C. Templin,1 G. Laaf,1 K. Wiebe,1 G. Steinhoff,1 B. Lapin,2 A. Haverich,1 1Division of Cardiothoracic and Vascular Surgery, Hannover Medical School, Hannover, Germany; 2Institute of Primatology, Sochi, Russian Federation Xenogeneic lung transplantation may represent a feasible approach to overcome the relevant shortage of donor organs in clinical lung transplantation. However, rejection processes in xenogeneic pulmonary grafts in primates are especially vicious and more efficient approaches to graft protection are required to ensure organ survival. We report on a novel preclinical pig-toprimate orthotopic single lung transplantation model, which we have developed. The model includes a regimen, which was especially designed to induce peripheral tolerance or immunomodulation for graft protection. Baboons underwent sequential immunoadsorbtion (EIA) and source animal kidney perfusion (EKP). The animals received cyclophosphamide and complement blockade. Subsequently, all animals received 2-3x1010 source animal splenocytes (DS). No further immunosuppression was administered. Finally, the animals underwent orthotopic lung transplantation. Controls included untreated animals and animals not receiving pulmonary grafts. The combination of EIA, EKP and CVF dropped anti-aGAL-Ab levels to the threshold of detection. EKP significantly reduced anti-porcine Ab levels. Serum toxicity was significantly reduced by the treatment. No evidence for PERV infection was obtained. All test animals survived the operation and were extubated immediately following surgery. Lungs remained in the recipients for up to 24 hours, showing no signs of rejection., no petechial hemorrhage, intravascular thrombus or any other pathological finding. We have developed a model of xenogeneic lung transplantation including an attempt at immunomodulation, which is well tolerated. The transplanted xenografts showed satisfying graft function during the observed period and no pathological findings at time of explantation. Accordingly, we are increasing our monitoring period to the attainable maximum. This model is a useful tool, which may help to further overcome the limiting obstacles in xenogeneic lung transplantation. 183 CLASSICAL PATHWAY COMPLEMENT INHIBITION AND SURVIVAL OF h-DAF PIG LUNGS PERFUSED WITH HUMAN BLOOD C. Schroeder,1 S. Pfeiffer,1 D.J.G. White,2 A.M. Azimzadeh,1 R.N. Pierson III,1 1Cardiothoracic Surgery, Vanderbilt University, Nashville, TN; 2Novartis, Pharma AG Company, Basel, Switzerland Introduction: To determine the role of the proximal classical complement pathway in hyperacute pig lung rejection (HALR). Methods: Three doses of soluble C1 esterase inhibitor (sC1Inh) were tested in an established discordant xenogenic lung perfusion model, using side-by-side perfusion of right and left lungs from hDAF transgenic pigs; thus one lung in each pair served as an internal control. In vitro cytotoxicity of human plasma was measured on pig endothelial cells by flow cytometry. sC1Inh was dosed at 1 U/ml, 5 U/ml or 10 U/ml, corresponding to 2-, 6-, and 11-fold the physiological concentration of sC1Inh respectively. Survival endpoints were loss of transpulmonary blood flow, loss of oxygenation, and sequestration of perfusate in the lung. Results: The low dose of C1Inh (1IU/ml) decreased C3a levels and reduced in vitro cytotoxicity by 60 %. High dose sC1 Inh (10
Abstracts
121
IU/ml) reduce in vitro cytotoxicity by ⬎95%. Lung injury occurred rapidly (⬍40 minutes) in hDAF (⫹) lungs despite blood treatment with C1Inh at 1 or 5 IU/ml, but was attenuated (2-4 hour survival) at 10 IU/ml. PVR elevation was not consistently prevented by sC1Inh. Platelet activation (P selectin expression) and platelet loss were decreased but not completely abrogated by C1Inh, even at the highest dose tested. Conclusions: A supra-physiologic dose of 6 IU/ml, which prolongs survival and significantly inhibits platelet activation when pig kidneys are perfused with human blood (Fiane AE, Immunopharmacology, 1999) does not protect pig lungs from HALR by human blood, even in the context of hDAF expression. We conclude that organs vary in their susceptibility to complementmediated injury, and that a supraphysiologic dose of C1Inh is needed to inhibit lung HAR. However, even when in vitro cytotoxicity is prevented by ⬎95 %, platelet activation, PVR elevation, and rapid injury of h-DAF transgenic pig lungs are not completely prevented by high-dose sC1Inh. These observations support the hypothesis that HALR, unlike HAR of other organs, can occur despite effective regulation of the classical pathway of complement activation. 184 RAPID DECREASE IN ECTO-5’-NUCLEOTIDASE ACITIVITY IN THE PIG HEART AND ENDOTHELIUM IN CONFRONTATION WITH HUMAN BLOOD Z. Khalpey, K.K. Kalsi, Z. Kochan, B. Lama, E. Slominska, M. Forni, M. Bacci, M. Lavitrano, M.H. Yacoub, R.T. Smolenski, 1 Heart Science Centre, Imperial College at Harefield Hospital, harefield, Middlesex, United Kingdom; 2Medical University Gdansk, Gdansk, Poland; 3Universita La Sapienza, Roma, Italy; 4 Universita Bologna, Bologna, Italy Ecto-5’-nucleotidase (e5’N) is a key endothelial enzyme located on the surface of the cell involved in final step extracellular nucleotide breakdown leading to formation of anti-inflammatory and immunosuppressive adenosine. It plays an important role in endothelial-neutrophil interaction and its low expression was found to enhance inflammatory response. We have previously identified that the cardiac activity of e5’N in pigs is lower than in humans. This study was focused to identify whether any further changes occur in the activity in confrontation with human blood. We studied effect of perfusion of pig hearts with fresh human blood and the effect of human plasma in cultured pig endothelial cells. Enzyme activity was measuered in heart or cell homogenates with HPLC based procedure. Pig hearts were perfused with fresh human blood for 4 h using retrograde constant flow perfusion system. In some experiments hearts of transgenic pigs overexpressing human decay accelerating factor (hDAF) were used. Pig aortic endothelial cells were used to study effect of human plasma on e5’N activity in vitro. Cells were exposed to different dilutions of human plasma for up to 3h. Activity of e5’N was 0.46⫾0.02 nmol/min/mg wet wt in normal pig hearts and 0.59⫾0.15 nmol/min/mg wet wt in transgenics overexpressing hDAF. Ex vivo perfusion of pig heart with fresh human blood for 4 h resulted in decrease in e5’N activity to 62⫾3% in normal and to 61⫾7% in transgenic pig hearts, despite attenuation of hyperacute rejection in transgenics. Initial pig aortic endothelial activity was activity of e5’N was 5.5⫾1.2 nmol/min/mg protein. Activity rapidly decreased following exposure to human plasma to 82⫾9% and 63⫾7% after 15 min with heat inactivated and not inactivated respectively. The activity did not decrease any further after 3 h of
Abstracts
The Journal of Heart and Lung Transplantation January 2002
Methods and Results: 249 cardiac transplant recipients(41 female) between 1989 and 1999 receiving statin therapy in combination with cyclosporin A were assessed from case records for lipid lowering effect, adverse events and survival. Four statins were used simvastatin(n⫽150), pravastatin(49), fluvastatin(40), atorvastatin(10). Mean treatment period(months); simvastatin 115.2, pravastatin 96.1, fluvastatin 59.9, atorvastatin 16.0. Percentage reduction in low density lipoprotein (at mean dose) was as follows; simvastatin(12.4mg) 30.3%, pravastatin(29.3mg) 29.4%, fluvastatin(40mg) 28.9%, atorvastatin(10mg) 32.1%. Adverse events; elevated creatinine kinase and myalgia: simvastatin 18%, pravastatin 9%, fluvastatin 0%, atrovastatin 0%. Elevated hepatic transaminases; simvastatin 6%, pravastatin 0%, fluvastatin 0%, atorvastatin 0%. Overall using the Cox proportional hazards model, percentage reduction in total cholesterol (p⫽0.006) and LDL cholesterol (p⫽0.01) correlated most strongly with improved survival. However there was no significant evidence for a difference in survival time between the four statins used (p⫽0.29). Conclusion: Significant reductions in lipids can be achieved safely using long term statin therapy following cardiac transplantation. Previous evidence has suggested that the benefits of these drugs in this population are immune mediated independent of lipid lowering effect. The results of this study suggest that the lipid lowering effect of these drugs is also important particularly in the long term. 180 RENIN-ANGIOTENSIN-ALDOSTERONE-SYSTEM DIRECTED ANTIHYPERTENSIVE THERAPY ACHIEVES LOWER B-TYPE NATRIURETIC PEPTIDE LEVELS COMPARED WITH CALCIUM CHANNEL BLOCKADE IN HEART TRANSPLANTATION M.H. Park, P.A. Uber, R.L. Scott, L.E. Bourgeois, M.R. Mehra, Ochsner Clinic Foundation, New Orleans, LA Background. The optimal antihypertensive regimen after heart transplantation remains elusive and poorly defined. Whether therapy targeting the renin-antiotensin-aldosterone system (RAAS) evokes benefits beyond blood pressure (BP) lowering in heart transplantation has not been previously determined. We hypothesized that RAAS directed therapy compared with calcium channel blockade (CCB) would be more likely to decrease b-type natriuretic peptide (BNP) levels, a sensitive and specific marker of ventricular overload, at similar blood pressure(BP) control. Methods. In 30 consecutive stable heart transplant recipients, we compared BNP levels between patients receiving a primary RAAS targeted therapy (n⫽14) for hypertension versus CCB therapy (n⫽16). All patients (80% men, 87% whites, age 61⫾4years, tacrolimus, mycophenolate and steroid immunosuppression) achieved BP control to a systolic and diastolic BP⬍140/90 mmHg with stable allograft function (Ejection Fraction 63⫾5%) without ongoing rejection). Results. In the RAAS directed treatment group, 71% received angiotensin receptor blockers while 29% were on angiotensin converting enzyme inhibitors. All patients in the CCB group recieved amlodipine. Findings are depicted in the table below. Systolic BP RAAS Group CCB Group p value
129 ⫾ 11 130 ⫾ 9 0.7
Diastolic BP 84 ⫾ 8 84 ⫾ 6 0.9
BNP (pg/dl) 89 ⫾ 78 174 ⫾ 136 0.05
Regression analysis of systolic and diastolic blood pressure with BNPlevels failed to demonstrate any significant correlation. Similarly no significant correlations of renal function and BNP levels were noted in this cohort. Inferences. Despite similar levels of blood pressure control achieved with CCB’s, RAAS directed antihypertensive therapy is associated with lower BNP levels, a sensitive and specific marker of ventricular wall stress. Thus, we suggest that RAAS directed therapy evokes benefits on cardiac allograft adaptation beyond those achieved by blood pressure lowering alone. 181 TACROLIMUS/MYCOPHENOLATE MOFETIL VS CYCLOSPORINE/MYCOPHENOLATE MOFETIL: IMPACT ON INFECTIONS FOLLOWING CARDIAC TRANSPLANTATION J. Groetzner, B. Meiser, J. Schirmer, S. Schenk, M. Muller, F. ¨ berfuhr, B. Reichart, Klinikum Reisch, M. Oberhoffer, F. Kur, P. U Grosshadern, University Hospital, Munich, Germany Background: New combinations of immunosuppressive agents led to a decrease of acute rejection episodes (ARE) after HTx. They might be associated with an increased risk of infections. Therefore we compared the incidence of infections in HTxpatients treated with either a Tacrolimus- (Tac) - or a Cyclosporine- (CsA) based immunosuppression. Methods: In a prospective, randomized clinical trial, 61 HTx patients (n⫽30/31) were included during 1999 and 2001. Immunosuppression consisted of CsA, Mycophenolatmofetil (MMF) and Corticosteroids (Cort) or Tac, MMF and Cort. Cort were stopped as early as 6 month po. Endpoints were incidence of infections and ARE. Infections were defined as positive cultures or laboratory values with the need of antibiotic treatment. Results: Demographic data as well as primary diagnosis and perioperative data were comparable between both groups. Comparison of the infection incidence revealed a trend towards an increase in CsA-patients (0.31 inf./100pat.days vs. 0.27 (Tac); p⫽n.s.). Distribution of the bacterial, viral and fungal infections were similar in both groups: (CsA vs Tac) Bacterial: 45% vs 50%; Viral:27% vs. 29%; Fungal: 28% vs 21%, p⫽n.s.). Incidence of ARE/100 pat.days was significantly lower in the Tac/MMF group when compared to the CsA/MMF group (0.09 and 0.3; p⬍0.003). Nineteen infections in the CsA and 4 in the Tac group occured within 3 weeks after ARE treatment. ( p⬍0.05 ). Especially the incidence of fungal and viral infections following ARE was significantly higher in CsA treated patients (fungal: 7 vs 0; p⫽0.03; Viral: 7 vs 2; p⫽0.05; Bacterial: 6 vs 2; p⫽n.s.). Overall Survival was 90% (CsA) vs. 96% (Tac), the percentage with lethal infections (aspergillus-pneumonia)was comparable (6,7% (CsA) vs. 3,3% (Tac) p⫽n.s.). Conclusion: The immunosuppressive combination of Tac/MMF resulted in a trend to less infections after HTx than CsA/MMF. This tendency was in many cases contributed to augmented immunosuppression during rejection treatment. Since there is a significantly higher incidence of ARE in CsA-treated patients, Tac/MMF proved to be the combination with the favorable safety profile. 182 DEVELOPMENT OF A NOVEL PRECLINICAL PIG-TONON-HUMAN PRIMATE MODEL OF XENOGENEIC ORTHOTOPIC LUNG TRANSPLANTATION
The Journal of Heart and Lung Transplantation Volume 21, Number 1 A.R. Simon,1 S. Fischer,1 R. Tessmann,1 C. Schroeder,1 U. Martin,1 M. Chikobava,2 C. Templin,1 G. Laaf,1 K. Wiebe,1 G. Steinhoff,1 B. Lapin,2 A. Haverich,1 1Division of Cardiothoracic and Vascular Surgery, Hannover Medical School, Hannover, Germany; 2Institute of Primatology, Sochi, Russian Federation Xenogeneic lung transplantation may represent a feasible approach to overcome the relevant shortage of donor organs in clinical lung transplantation. However, rejection processes in xenogeneic pulmonary grafts in primates are especially vicious and more efficient approaches to graft protection are required to ensure organ survival. We report on a novel preclinical pig-toprimate orthotopic single lung transplantation model, which we have developed. The model includes a regimen, which was especially designed to induce peripheral tolerance or immunomodulation for graft protection. Baboons underwent sequential immunoadsorbtion (EIA) and source animal kidney perfusion (EKP). The animals received cyclophosphamide and complement blockade. Subsequently, all animals received 2-3x1010 source animal splenocytes (DS). No further immunosuppression was administered. Finally, the animals underwent orthotopic lung transplantation. Controls included untreated animals and animals not receiving pulmonary grafts. The combination of EIA, EKP and CVF dropped anti-aGAL-Ab levels to the threshold of detection. EKP significantly reduced anti-porcine Ab levels. Serum toxicity was significantly reduced by the treatment. No evidence for PERV infection was obtained. All test animals survived the operation and were extubated immediately following surgery. Lungs remained in the recipients for up to 24 hours, showing no signs of rejection., no petechial hemorrhage, intravascular thrombus or any other pathological finding. We have developed a model of xenogeneic lung transplantation including an attempt at immunomodulation, which is well tolerated. The transplanted xenografts showed satisfying graft function during the observed period and no pathological findings at time of explantation. Accordingly, we are increasing our monitoring period to the attainable maximum. This model is a useful tool, which may help to further overcome the limiting obstacles in xenogeneic lung transplantation. 183 CLASSICAL PATHWAY COMPLEMENT INHIBITION AND SURVIVAL OF h-DAF PIG LUNGS PERFUSED WITH HUMAN BLOOD C. Schroeder,1 S. Pfeiffer,1 D.J.G. White,2 A.M. Azimzadeh,1 R.N. Pierson III,1 1Cardiothoracic Surgery, Vanderbilt University, Nashville, TN; 2Novartis, Pharma AG Company, Basel, Switzerland Introduction: To determine the role of the proximal classical complement pathway in hyperacute pig lung rejection (HALR). Methods: Three doses of soluble C1 esterase inhibitor (sC1Inh) were tested in an established discordant xenogenic lung perfusion model, using side-by-side perfusion of right and left lungs from hDAF transgenic pigs; thus one lung in each pair served as an internal control. In vitro cytotoxicity of human plasma was measured on pig endothelial cells by flow cytometry. sC1Inh was dosed at 1 U/ml, 5 U/ml or 10 U/ml, corresponding to 2-, 6-, and 11-fold the physiological concentration of sC1Inh respectively. Survival endpoints were loss of transpulmonary blood flow, loss of oxygenation, and sequestration of perfusate in the lung. Results: The low dose of C1Inh (1IU/ml) decreased C3a levels and reduced in vitro cytotoxicity by 60 %. High dose sC1 Inh (10
Abstracts
121
IU/ml) reduce in vitro cytotoxicity by ⬎95%. Lung injury occurred rapidly (⬍40 minutes) in hDAF (⫹) lungs despite blood treatment with C1Inh at 1 or 5 IU/ml, but was attenuated (2-4 hour survival) at 10 IU/ml. PVR elevation was not consistently prevented by sC1Inh. Platelet activation (P selectin expression) and platelet loss were decreased but not completely abrogated by C1Inh, even at the highest dose tested. Conclusions: A supra-physiologic dose of 6 IU/ml, which prolongs survival and significantly inhibits platelet activation when pig kidneys are perfused with human blood (Fiane AE, Immunopharmacology, 1999) does not protect pig lungs from HALR by human blood, even in the context of hDAF expression. We conclude that organs vary in their susceptibility to complementmediated injury, and that a supraphysiologic dose of C1Inh is needed to inhibit lung HAR. However, even when in vitro cytotoxicity is prevented by ⬎95 %, platelet activation, PVR elevation, and rapid injury of h-DAF transgenic pig lungs are not completely prevented by high-dose sC1Inh. These observations support the hypothesis that HALR, unlike HAR of other organs, can occur despite effective regulation of the classical pathway of complement activation. 184 RAPID DECREASE IN ECTO-5’-NUCLEOTIDASE ACITIVITY IN THE PIG HEART AND ENDOTHELIUM IN CONFRONTATION WITH HUMAN BLOOD Z. Khalpey, K.K. Kalsi, Z. Kochan, B. Lama, E. Slominska, M. Forni, M. Bacci, M. Lavitrano, M.H. Yacoub, R.T. Smolenski, 1 Heart Science Centre, Imperial College at Harefield Hospital, harefield, Middlesex, United Kingdom; 2Medical University Gdansk, Gdansk, Poland; 3Universita La Sapienza, Roma, Italy; 4 Universita Bologna, Bologna, Italy Ecto-5’-nucleotidase (e5’N) is a key endothelial enzyme located on the surface of the cell involved in final step extracellular nucleotide breakdown leading to formation of anti-inflammatory and immunosuppressive adenosine. It plays an important role in endothelial-neutrophil interaction and its low expression was found to enhance inflammatory response. We have previously identified that the cardiac activity of e5’N in pigs is lower than in humans. This study was focused to identify whether any further changes occur in the activity in confrontation with human blood. We studied effect of perfusion of pig hearts with fresh human blood and the effect of human plasma in cultured pig endothelial cells. Enzyme activity was measuered in heart or cell homogenates with HPLC based procedure. Pig hearts were perfused with fresh human blood for 4 h using retrograde constant flow perfusion system. In some experiments hearts of transgenic pigs overexpressing human decay accelerating factor (hDAF) were used. Pig aortic endothelial cells were used to study effect of human plasma on e5’N activity in vitro. Cells were exposed to different dilutions of human plasma for up to 3h. Activity of e5’N was 0.46⫾0.02 nmol/min/mg wet wt in normal pig hearts and 0.59⫾0.15 nmol/min/mg wet wt in transgenics overexpressing hDAF. Ex vivo perfusion of pig heart with fresh human blood for 4 h resulted in decrease in e5’N activity to 62⫾3% in normal and to 61⫾7% in transgenic pig hearts, despite attenuation of hyperacute rejection in transgenics. Initial pig aortic endothelial activity was activity of e5’N was 5.5⫾1.2 nmol/min/mg protein. Activity rapidly decreased following exposure to human plasma to 82⫾9% and 63⫾7% after 15 min with heat inactivated and not inactivated respectively. The activity did not decrease any further after 3 h of