转人CRP基因在异种移植中的研究

目的：研究转入补体调节蛋白（CRP）DAF、MCP和CD59基因对抑制人补体激活从而克服超急性排斥反应的作用。方法：利用显微注射建立转人衰变加速因子（hDAF)小鼠和猪的模型和转梁hMCP及hCD59真核表达质粒的猪内皮细胞（EC）,研究小鼠和猪EC表达抑制人补体激活的人补体调节蛋白（CRP）对异种移植超急性排斥反应的抑制作用。结果（1）转人DAF基因小鼠心脏用新鲜人血连续丛外灌注,转基因组心脏搏动时间（174．6min)比对照组（106．5min)明显延延长。（2）转hDAF基因猪心脏异位移植给猕猴、移植心最长存活90h,受者死亡前移植心仍有功能,移植心病理检查未见超急性排斥反应病理改变。（3）转DAF基因基因猪EC死亡率在不同浓度血清时均明显低于对照组,在转hDAF基因猪EC上再分别转染hMCP及hCD59真核表达质粒,转hDAF hMCP或hDAF hCD59在不同血清浓度时EC死亡率较单纯hDAF组明显下降（P＜0．05）。结论,转人DAF及MCP、CD59补体调节蛋白基因能克服人对异种器官或组织的超急性排斥反应。     

Protection from complement-mediated injury in livers and kidneys of transgenic mice expressing human complement regulators

Abstract: The major problem in the use of phylogenetically distant donors is a fast, strong reaction called hyperacute rejection. This reaction mediated by complement is directed against the vascular endothelia of the transplanted organ. Complement activation is tightly controlled by several regulatory proteins which inhibit the formation and function of different complement components. To verify the hypothesis that organs expressing such inhibitory factors could be spared from complement-mediated hyperacute rejection, we have generated mice transgenic for the human complement inhibitor membrane cofactor protein (hMCP) and decay accelerating factor (hDAF). Different levels of hMCP and/or hDAF expression, according to the promoter used, were detected by RNA analysis in the major organs, specifically on the organ vascular endothelia, as revealed by immunohistochemical analysis. The development of an in vivo model of human plasma perfusion allowed the characterization of complement-mediated damage in control animals and the degree of protection due to the presence of hMCP, hDAF, or both in the organs derived from single or double transgenic mice. In this paper we compare the level of expression of complement regulators with the degree of protection in two major organs: liver and kidney.     

Production and characterization of a pig line transgenic for human membrane cofactor protein

A pig line transgenic for human membrane cofactor protein (hMCP) has been established. Offspring from the founder were produced by crossing the founder with pigs heterozygous for the human decay accelerating factor (hDAF) transgene. As a result, pigs transgenic for both hMCP and hDAF have been produced. Ribonuclease protection assay (RPA) indicated that hMCP was expressed in all the tissues analysed. In addition, immunohistochemical results indicated a high level of expression of hMCP on neural tissues and islets where hDAF was absent or weakly expressed. C3 fragment deposition and cytotoxicity assays indicated that hMCP expression alone on pig endothelial cells and peripheral blood lymphocytes (PBLs) provided protection against human complement mediated damage. However, we did not find that porcine endothelial cells expressing both hDAF and hMCP were better protected than those expressing hDAF alone. The expression of hMCP on tissues where hDAF is not expressed could provide these tissues with protection against human complement mediated lysis.     

Coagulation cascade activation triggers early failure of pig hearts expressing human complement regulatory genes

Abstract: Background: Hyperacute rejection (HAR) and early graft failure (EGF) have been described in a minority of pig‐to‐baboon heart transplants using organs transgenic for human complement regulatory proteins (hCRP). Here we investigate the role of coagulation cascade activation in the pathogenesis of HAR and EGF in a consecutive series where a high incidence of these outcomes was observed. Methods: Twenty‐eight naïve wild‐caught Papio anubis baboons received heterotopic heart transplants from pigs transgenic for hDAF (n = 23) or hMCP (n = 5). Immunosuppression consisted of cyclosporine A, cyclophosphamide and MMF (n = 18) or anti‐CD154 mAb (IDEC‐131) and ATG (n = 10). Eleven received anti‐Gal carbohydrates (GAS914, n = 8, or NEX1285, n = 3), of which four also underwent extracorporeal immunoadsorption (EIA), and 12 also received pharmacologic complement inhibitors (C1 INH, n = 9, or APT070, n = 3). Results: Excluding one technical failure, 14 of 27 transplants (11 hDAF, 3 hMCP) exhibited either HAR (n = 10) or EGF (n = 4). Surprisingly, neither complement inhibition (with C1 INH or APT070) nor anti‐Gal antibody depletion with GAS914, NEX1285, or additional EIA consistently prevented HAR or EGF despite low or undetectable complement deposition. Strikingly, most grafts with HAR/EGF exhibited prominent fibrinogen and platelet deposition associated with systemic coagulation cascade activation, consistent with non‐physiologic intravascular coagulation, in many instances despite little evidence for antibody‐mediated complement activation. Conclusion: We conclude that dysregulated coagulation correlates closely with and probably causes primary failure of pig hearts transgenic for hCRP. These data support efforts to define effective strategies to prevent dysregulated coagulation in pig organ xenografts.     

Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay-accelerating factor

BACKGROUND: The expression of human complement regulators has been proved as an effective strategy to overcome hyperacute rejection in discordant xenogeneic organ transplantation. In this study, we tested the hypotheses that expression of triple transgenes for human complement regulators and provide more effective protection to the transplanted pig tissues. METHODS: Pigs transgenic for human complement regulatory proteins, human CD59 (hCD59) and human membrane cofactor protein (hMCP), have been generated using large genomic constructs. Heterozygous human decay-accelerating factor (hDAF) transgenic pigs, from a previously established line, were bred with hCD59 or hCD59 plus hMCP pigs to produce animals that expressed both hCD59 and hDAF, or expressed triple transgenes hCD59, hDAF and hMCP. RESULTS: All three transgenes were widely expressed in most of the tissues analyzed, but the expression of hMCP was at low levels. In cytotoxicity assays on porcine peripheral blood mononuclear cells, the expression of a single transgenic protein, hCD59, or hCD59 in combination with hMCP provided similar protection against human complement-mediated damage as the single expression of hDAF. However, the expression of triple transgenic proteins or double hCD59 and hDAF transgenic proteins provided greater protection than either hCD59 or hDAF alone. CONCLUSIONS: Thus, pigs transgenic for multiple transgenes provide a greater degree of human complement regulation and hence might be more suitable for xenotransplantation.     

Transgenic expression in pig hearts of both human decay-accelerating factor and human membrane cofactor protein does not provide an additional benefit to that of human decay-accelerating factor alone in pig-to-baboon xenotransplantation

This study investigated whether the coexpression of human decay-accelerating factor (hDAF) and human membrane cofactor protein (hMCP) on porcine organs provides an additional benefit to that of hDAF alone to prevent rejection. Heterotopic heart xenotransplantation was performed in baboons with either hDAF (n=5) or hDAF/hMCP (n=5) transgenic pig organs. The only immunosuppression given was GAS914 (a soluble Gal [alpha1-3] Gal polymer) and cyclosporine A. With the exception of one hDAF organ that failed from a left atrium thrombosis, all xenografts developed acute humoral xenograft rejection. Acute humor xenograft rejection occurred at a median time of 152 hr in hDAF hearts and 162 hr in hDAF/hMCP organs. Recipients of hDAF or hDAF/hMCP hearts did not differ in their patterns of serum antiporcine antibodies or in plasma levels of the soluble terminal complement complex sC5b-9. It is concluded that in this pig-to-baboon heterotopic heart transplant, model expression of hDAF/hMCP does not provide an additional benefit in prevention of rejection to that of hDAF alone.     

Reduced fibrin deposition and intravascular thrombosis in hDAF transgenic pig hearts perfused with tirofiban

BACKGROUND: Solid organ xenograft rejection is associated with vascular injury resulting at least in part in platelet activation, and rejected xenografts invariably demonstrate intravascular thrombosis and interstitial hemorrhage. Complement activation plays a prominent role in platelet-endothelial interaction. We tested the effects of platelet GPIIb/IIIa inhibitor tirofiban during perfusion of hDAF pig hearts. METHODS: Using a working-heart model, nontransgenic and hDAF pig hearts were perfused with tirofiban or human blood only. Myocardial damage was determined by hemodynamic parameters (cardiac output, stroke work index) and creatine phosphokinase. Further monitoring included the assessment of complement factors (C3, C4), platelets, fibrinogen, ATIII, and graft histology. RESULTS: Tirofiban increased cardiac output (CO) and stroke work index (SWI) of nontransgenic pig hearts and improved superior CO and SWI of hDAF pig hearts. Although perfusion time of nontransgenic pig hearts was prolonged by tirofiban (196+/-65 min vs. 162+/-122 min), a similar effect in hDAF pig hearts (218+/-116 min vs. 222+/-30 min) could not be demonstrated. Tirofiban reduced consumption of C3 and C4 independently from hDAF. Depletion of fibrinogen was equally diminished by tirofiban and hDAF; the combination of both agents obtained no further reduction. ATIII consumption was most effectively inhibited by this combination. Intravascular fibrin deposition was reduced by tirofiban and hDAF, but particularly by the combination of the two agents. CONCLUSIONS: Improvement of heart performance and reduction of myocardial damage and intravascular thrombosis confirm a role of the GPIIb/IIIa inhibitor tirofiban for the prevention of hDAF pig heart rejection and xenograft function.     

Contrast in the efficacy of hDAF mouse hearts between ex vivo perfusion and transplantation into primates

Abstract: In recent experiments, in which we compared hDAF transgenic rat hearts perfused with 15% human serum in the Langendorff device and hDAF rat hearts transplanted into cynomolgus monkeys, we demonstrated that in the ex vivo heart perfusion model both homozygous and heterozygous hDAF hearts survived longer as nontransgenic controls. Surprisingly, we found that only homozygous hDAF hearts were protected against hyperacute rejection in vivo. The first aim of this study was to determine whether perfusion of mouse hearts with higher human serum concentrations or human blood might explain some of the differences found in survival time of the recently performed experiments with rat heart xenografts. Secondly, we investigated whether the observed differences in survival times of rat xenografts between in vivo and ex vivo transplantation would also hold for mouse hearts transgenic for hDAF. An ex vivo model was used to perfuse hDAF mouse hearts and controls with human serum or blood, and hDAF transgenic hearts and controls were transplanted into cynomolgus monkeys. hDAF transgenic mouse hearts survived significantly longer than their controls when perfused with 15% human serum, but no difference was found when 30% human serum was used, or when these hearts were transplanted into cynomolgus monkeys. However, in both the in vivo and ex vivo models the amount of PMNs adhering to the vascular endothelium was significantly lower in hDAF transgenes as compared with their controls. In conclusion, in the ex vivo situation, the efficacy of hDAF transgenesis in preventing HAR is limited by serum complement concentration.     

Effect of complement fragment 1 esterase inhibition on survival of human Decay-Accelerating factor pig lungs perfused with human blood

BACKGROUND: The role of complement in hyperacute lung xenograft rejection has not been fully elucidated. The present study evaluates the effect of complement (C) 1 esterase inhibition on hyperacute rejection of human decay-accelerating factor (hDAF)-positive pig lung by human blood. METHODS: Using a modification of an established ex vivo model, right and left lungs from individual animals were surgically isolated and separately perfused. Pigs homozygous for hDAF were perfused with fresh human blood that was either untreated or treated with complement 1 esterase inhibitor (C1-Inh) at doses of 1 U/ml (n = 5), 5 U/ml (n = 3) or 10 U/ml plasma (n = 5). RESULTS: Only C1-Inh at 10 U/ml prolonged survival time (230 +/- 48.3 minutes) as compared with controls (65.6 +/- 26.5 minutes, p < 0.05) and diminished complement activation (C3a and C5a, p < 0.05). Interestingly, a low concentration of C1-Inh increased the pulmonary vascular resistance (PVR; 1 U/ml: 0.54 +/- 0.3; 10 U/ml: 0.19 +/- 0.08). Sequestration of neutrophils (92 +/- 3%) and platelets (64 +/- 13%) was not prevented by any concentration of C1-Inh. Tissue deposition of C3b and C5b-9 were diminished by hDAF expression, and further blunted by treatment, with 10 U/ml C1-Inh. CONCLUSIONS: Complement plays a critical role in early events of lung hyperacute rejection (HAR). However, even potent inhibition of C1 esterase and C3/C5 convertase, using serum C1-Inh in pig lungs homozygous for hDAF expression, does not prevent rapid lung injury. Our findings implicate innate immune pathways resistant to efficient complement regulation, and suggest a role for neutrophils and platelets in the lung's particular vulnerability.     

Combining the hDAF transgene with the GP IIb/IIIa inhibitor tirofiban improves heart performance and reduces myocardial damage following hyperacute rejection in an ex vivo perfusion model

Xenograft rejection is associated with vascular injury resulting at least in part from platelet activation, and rejected xenografts invariably demonstrate intravascular thrombosis. Assuming that complement activation is a major determinant of humoral immune reactions bringing about platelet-endothelial cell interactions, we tested the effects of the specific platelet glycoprotein IIb/IIIa inhibitor tirofiban in combination with the human decay accelerating factor (hDAF) transgene on hyperacute rejection of pig hearts. Four groups were studied in a working heart-perfusion model. Pig hearts transgenic for hDAF and nontransgenic pig hearts were perfused with human blood containing tirofiban or with unmodified human blood. Cardiac output, stroke work index, and creatine phosphokinases were measured for the evaluation of the extent of myocardial damage. Consumption of complement components was determined. Endothelial deposition of fibrin and intravascular thrombosis were evaluated. Tirofiban improved cardiac output and stroke work index of nontransgenic pig hearts and was able to further increase hemodynamic function of hDAF transgenic pig hearts. Low levels of creatine phosphokinases also revealed a cardioprotective effect of tirofiban. However, a further extension of the survival of hDAF transgenic pig hearts could not be achieved, although tirofiban prolonged beating time of nontransgenic pig hearts. Tirofiban was able to reduce the consumption of complement components independently of hDAF. Intravascular evidence of fibrin and thrombosis tended to be particularly reduced by the combination of tirofiban and hDAF. Thus, the application of tirofiban together with hDAF improves the performance of pig hearts by reducing myocardial damage and intravascular thrombosis.     

Hyperacute lung rejection in the pig-to-human model. 2. Synergy between soluble and membrane complement inhibition

Azimzadeh A, Zorn GL III, Blair KSA, Zhang JP, Pfeiffer S, Harrison RA, Cozzi E, White DJG, Pierson RN III. Hyperacute lung rejection in the pig‐to‐human model. 2. Synergy between soluble and membrane complement inhibition. Xenotransplantation 2003; 10: 120–131. © Blackwell Munksgaard, 2003 Background. The role of complement in hyperacute lung xenograft rejection has not been elucidated. The present study evaluates the effect of complement (C) C3/C5 convertase inhibition on hyperacute rejection of pig lung by human blood. Methods. In an established ex‐vivo model, lungs from pigs heterozygous for human decay accelerating factor (hDAF), non‐transgenic littermate control pigs, or farm‐bred pigs were perfused with fresh human blood that was either unmodified or treated with soluble complement receptor type 1 (sCR1: TP10, 100 μg/ml). Results. Non‐transgenic lungs from littermate controls had a median survival time of 35 min (range 5 to 210; P=0.25 vs. farm‐bred piglets: median 5 min, range 5 to 10). Lungs expressing hDAF survived for a median of 90 min (range 10 to 161; P=0.5 and 0.01 vs. littermate and farm‐bred controls, respectively), with sCR1, whereas hDAF (–) lungs failed by 35 min (range 6 to 307), hDAF (+) lungs survived for 330 min (range 39 to 577) [P=0.002 vs. farm‐bred; P=0.08 vs. hDAF (–); P=0.17 vs. sCR1/hDAF (–)]. The rise in pulmonary vascular resistance (PVR) at 5 min was blunted only by hDAF (+) with sCR1 (0.26 ± 0.2 vs. 0.5 to 0.7 mmHg/ml/min for other groups). Plasma C3a and sC5b‐9 and tissue deposition of C5b‐9 were dramatically diminished using sCR1, and further decreased in association with hDAF. Histamine and thromboxane were produced rapidly in all groups. Conclusion. Complement plays an important role in lung HAR. However, even potent inhibition of C3/C5 convertase, both membrane bound in lung and by a soluble‐phase inhibitor in the blood, does not prevent activation of inflammatory responses known to be particularly injurious to the lung. Our findings implicate a role for innate immune pathways resistant to efficient complement regulation. The role of anti‐species antibody, coagulation pathway dysregulation, and additional environmental or genetic influences remain to be defined.     

Ex vivo and extracorporeal perfusion with hDAF pig kidneys

The present study was undertaken to determine whether human decay accelerating factor (hDAF) transgene would prevent hyperacute rejection (HAR) while perfused with human blood or extracorporeally in baboons. Four hDAF pig kidneys and three non-hDAF pig kidneys were perfused ex vivo with fresh human blood for 6 h. Additionally four hDAF pig kidneys and four non-hDAF pig kidneys were extracorporeally perfused in baboons and pigs, respectively, for 3 h. In ex vivo perfusion, the color of hDAF pig kidneys remained pink at the end of 6-h perfusion and they had normal histology, while non-hDAF kidneys developed HAR. HDAF pig kidneys had superior function over non-transgenic pig kidneys. Urine output was 17.31 +/- 3.70 ml/h for hDAF pig kidneys, and only 5.81 +/- 0.26 ml/h for non-hDAF kidneys (P < 0.05). Creatinine clearance was 1.16 +/- 1.24 ml/min for hDAF kidneys and 0.22 +/- 0.15 ml/min for non-hDAF kidneys (P < 0.05). Other functional data including potassium, urine specific density, and osmolality were normal in the hDAF kidneys, while in non-hDAF kidneys, serum potassium was elevated to over 9 mmol/l by the end of perfusion (P < 0.01). Non-hDAF kidneys also lost more sodium through urine than hDAF kidneys (173.67 +/- 14.05 mmol/l vs. 109 +/- 31 mmol/l, P < 0.05). In the extracorporeal perfusion, all the baboons tolerated the procedure well with normal hemodynamic and hemotologic profiles. These baboons were well until killed 42 to 56 days after perfusion, although their antiporcine antibodies were greatly elevated. We conclude that hDAF transgene protects against HAR, allowing the pig kidney to function normally while perfused with human blood, and that extracorporeal perfusion using hDAF pig kidneys is a safe procedure in baboons.     

HDAF transgenic pig livers are protected from hyperacute rejection during ex vivo perfusion with human blood

The aim of this study was to determine if human decay-accelerating factor (hDAF) protects against hyperacute rejection in an ex vivo liver perfusion system using human blood. Pig livers were perfused ex vivo via the portal vein for an average of 5–6 h using a membrane oxygenator. Three groups were studied. Group I: Wild-type pig livers were alloperfused with fresh pig blood (n=5). Group II: Wild-type pig livers were xenoperfused with fresh human blood (n=5). Group III: hDAF transgenic pig livers were xenoperfused with fresh human blood (n=5). The graft ischemic time, ratio of perfusate volume to liver weight, flow rate, and perfusate hematocrit were similar in each group. The hDAF livers perfused with human blood (Group III) had a lower ALT level, less protein and albumin losses, lower bilirubin levels in the perfusate, less weight gain, and greater bile production than the wild-type livers perfused with human blood. Histology showed classic features of hyperacute rejection in Group II, including massive hemorrhage, severe vasculitits, fibrin and C5b-9 deposition, and endothelial damage within 1 h of perfusion, whereas liver histology studies in Groups I and III were near normal. IgG and IgM deposits were seen in the xenoperfused livers. Electron microscopy (EM) and immuno-EM showed loss of endothelial cells, trapping of white blood cells and platelets, and diffuse fibrin deposits in Group II only. hDAF pig livers perfused with human blood showed superior function and histology when compared with wild-type pig livers. These data suggest that (1) hyperacute rejection may contribute to the inconsistent results using wild-type pig livers for extracorporeal liver support and (2) genetically modified pigs that express hDAF may provide a better donor source than wild-type pigs for extracorporeal liver support.     

The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement

BACKGROUND: It has been proposed that hyperacute rejection (HAR) of pig-to-primate vascularized xenografts is due in large part to ineffective regulation of recipient complement by pig complement regulatory proteins (CRPs), and indeed transgenic expression of human CRPs in pigs can prevent hyperacute rejection. However, at least one pig CRP (CD59) efficiently regulates human complement in vitro, suggesting that it is the level of expression of a particular CRP(s) rather than cross-species incompatibility that explains the HAR of porcine xenografts. We investigated the relative effectiveness of transgenically expressed pig and human CD59 in providing protection of mouse hearts from human complement in an ex vivo setting. METHODS: Transgenic mice expressing pig CD59 or human CD59 under the control of the human ICAM-2 promoter, which restricts expression in tissues to vascular endothelium, were used. Hearts from mice expressing similar levels of pig CD59 or human CD59 were perfused ex vivo with 10% human plasma and heart function was monitored for 60 min. Sections of perfused hearts were examined for deposition of the membrane attack complex (MAC). RESULTS: Control nontransgenic hearts (n=5) were rapidly affected by the addition of human plasma, with mean function falling to less than 10% of the initial level within 15 min. In contrast, hearts expressing either pig CD59 (n=6) or human CD59 (n=8) were protected from plasma-induced injury, maintaining 31 and 35% function, respectively, after 60 min of perfusion. MAC deposition was markedly reduced in both pig CD59 and human CD59 transgenic hearts compared to nontransgenic control hearts. CONCLUSIONS: When highly expressed on endothelium in transgenic mice, pig CD59 provided equivalent protection to human CD59 in a model of human complement-mediated xenograft rejection. Thus supranormal expression of endogenous porcine CRPs may be a feasible alternative to the expression of human CRPs in preventing HAR of pig-to-primate xenografts.     

Hyperacute lung rejection in the pig-to-human model 4: evidence for complement and antibody independent mechanisms

BACKGROUND: We assessed whether the combination of complement regulation and depletion of xenoreactive antibodies improves the outcome of pulmonary xenografts compared with either strategy alone. METHODS: Lungs from pigs heterozygous (hDAF(+/-)) or homozygous (hDAF(+/+)) for the human decay accelerating factor transgene (hDAF) or their nontransgenic litter mates (hDAF(-/-)) were perfused with heparinized whole human blood. In additional groups, xenoreactive natural antibodies (XNA) were depleted by pig lung perfusion (hDAF(-/-)/AbAbs, hDAF(+/-)/AbAbs) before the experiment. This combined approach was augmented by adding soluble complement receptor 1 (sCR1) to the perfusate in one further group (hDAF(+/-)/AbAbs/sCR1). RESULTS: HDAF(-/-) lungs perfused with unmodified human blood were rejected after 32.5 min (interquartile range, IQR 5 to 210). HDAF(+/-) lungs survived for 90 min (IQR 10 to 161, P = 0.54). Both groups showed a rapid rise in pulmonary vascular resistance (PVR), which is a characteristic feature of hyperacute rejection (HAR). This phenomenon was blunted in the hDAF(+/+) group, although survival (48 min, IQR 14 to 111) was not further prolonged. Antibody depletion (AbAbs) led to a significant increase in survival time (hDAF(-/-)/AbAbs: 315 min, IQR 230 to 427; hDAF(+/-)/AbAbs: 375 min, IQR 154 to 575), reduced PVR and less complement production. Addition of sCR1 reduced complement elaboration but did not further improve survival (200 min, IQR 128 to 580) and surprisingly tended to increase PVR. CONCLUSIONS: Depletion of xenoreactive antibodies is more effective than membrane-bound complement regulation to blunt hyperacute rejection of pulmonary xenografts, but even the combined approach including soluble-phase complement inhibition is not sufficient to reliably prevent organ failure within hours. It therefore seems likely that other factors independent of antibody and complement contribute to HAR in this model.     

Hyperacute rejection of hDAF-transgenic pig organ xenografts in cynomolgus monkeys: influence of pre-existing anti-pig antibodies and prevention by the alpha GAL glycoconjugate GAS914

BACKGROUND: Our introductory pig-to-cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay-accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti-pig antibodies. We evaluated the efficacy of GAS914, a Gal alpha 1-3Gal trisaccharide linked to a poly-l-lysine backbone, in inhibition of HAR. METHODS: hDAF transgenic heterotopic heart (n = 15) or life-supporting kidney (n = 8) transplantation included induction with cyclophosphamide or anti-thymocyte globulin, and maintenance with cyclosporine or tacrolimus, steroids and mycophenolate sodium/mofetil. Four doses of GAS914 were given before transplantation. Rejection was confirmed by graft histology, and anti-pig antibody levels were determined in various assays. RESULTS: Four of six heart transplants without GAS914 treatment showed HAR. Nine subsequent transplants with GAS914 pre-treatment, did not show HAR (chi-square, P < 0.05). Two of four kidney transplants without GAS914 treatment ended with HAR. Four subsequent transplants with GAS914 did not show HAR. Animals with HAR showed extremely high antibody levels. Samples just before transplantation showed significantly higher antibody levels in recipients presenting with HAR. In all assays antibody levels were significantly lowered by GAS914 pre-treatment. CONCLUSIONS: HAR of hDAF solid organs could be ascribed to high levels of anti-pig antibodies. It is hypothesized that the hDAF transgene shows a threshold in efficacy, above which an overwhelming attack by antibodies and complement activation cannot be modulated to prevent HAR. HAR does not occur when animals with lower levels are used, or when antibodies are effectively depleted from the circulation by GAS914 treatment.     

Expression of complement regulatory proteins on islets of Langerhans: a comparison between human islets and islets isolated from normal and hDAF transgenic pigs

BACKGROUND: The expression of regulators of complement activity (RCAs) on islet cells may be of great importance for protecting them against complement-mediated lysis in the immediate posttransplant period after intraportal islet transplantation. We examined porcine and human islet cells for expression of RCA. We also examined to what extent human decay accelerating factor (hDAF) is expressed on adult and fetal islet cells isolated from hDAF transgenic (TG) pigs having a high transgene expression on endothelial cells. Moreover, the susceptibility of the various types of cells to lysis in human serum and blood was investigated. METHODS: Adult human islets (n=5), normal adult and fetal porcine islets (n=9 and n=8, respectively), and islets from adult and fetal hDAF TG pigs (n=5 and n=6, respectively) were examined. With islet single-cell suspensions and flow cytometry, adult human islet cells were examined for expression of hDAF (CD55), hCD59, and human membrane cofactor protein (hMCP; CD46), while porcine islet cells were examined for expression of pCD59 and pMCP. Islet cells from hDAF TG pigs were also examined for hDAF expression. Porcine peripheral blood lymphocytes, normal and hDAF TG porcine endothelial cell lines, a human endothelial cell line, and the human cell line U937 served as controls. Islet cytotoxicity was assayed after incubation of the islet cells with fresh human serum. Furthermore, adult islets from normal control pigs and hDAF TG pigs were exposed to fresh human blood in vitro for 60 min, and the inflammatory reaction elicited was compared between the different types of islets. RESULTS: All human islet cell preparations expressed hCD59, two of five expressed hMCP, but none expressed hDAF. Porcine islet cells expressed both pCD59 and pMCP. Normal adult porcine islet cells exposed to fresh human serum resulted in 74+/-5.4% cell lysis (mean+/-SEM, n=16). In comparison, only 1.3+/-2.8% (n=20, P<0.001) of human islet cells were lysed in the human serum. One islet cell preparation from an hDAF TG pig expressed small amounts of hDAF. This preparation from hDAF TG pigs bound significantly less C3c than did normal control islets (mean fluorescence ratio 16+/-2.2 and 58+/-4.3, respectively; P=0.046) and were partially protected from cell lysis in fresh human serum (47+/-10% and 78+/-18% cell lysis, respectively; P=0.046). The other four preparations from hDAF TG pigs were negative for hDAF and were equally susceptible to lysis as normal control islets. All fetal pancreatic islet cells from hDAF TG pigs analyzed were negative for hDAF expression. When exposed to fresh human blood in vitro, adult and fetal islets from hDAF TG pigs elicited equally strong inflammatory changes as did the normal control islets. The inflammatory changes were characterized by activation of the complement and coagulation systems, resulting in islet damage with "dumping" of insulin into the blood. CONCLUSIONS: Porcine and human islet cells express species-restricted complement regulatory proteins, with the human islet cells expressing mainly hCD59. A low expression of hDAF was detected on islet cells from one of five hDAF TG pigs. These islet cells displayed reduced islet cell cytotoxicity in fresh human serum. We conclude that protection from complement-mediated lysis will be important in the context of intraportal pig-to-human islet transplantation, and expression of a human RCA on islet cells should be beneficial in this context.     

Human decay-accelerating factor expressed on rat hearts inhibits leukocyte adhesion

In xenotransplantation the use of donors transgenic for recipient-type complement regulatory protein decay-accelerating factor (DAF/CD55) or membrane co-factor protein (MCP/CD46) protects grafts against hyperacute rejection (HAR), which is primarily mediated by xenoreactive natural antibodies and complement. In the Langendorff model, we previously demonstrated that rat hearts transgenic for human CD55 (hCD55), perfused with human serum, were protected against HAR. However, ex vivo, these hearts were found to be destroyed by a process occurring after the period of HAR. The question arose as to whether hearts transgenic for hCD55 are also protected against adhesion and infiltration by cells implicated in the early phases of xenograft rejection. The aim of the present study was to analyze this process in the ex vivo heart perfusion model. hCD55-transgenic rat hearts and their controls were perfused with either heat-inactivated or normal human blood solutions for 60 min. Although most of the hearts had stopped beating within the 60-min perfusion period, the perfusion was not stopped to enable adhesion of cells during a fixed period identical for all groups. Independent of the presence of complement, H&E-stained tissues of hCD55-transgenic hearts revealed fewer PMN leukocytes adhering to the endothelium than the controls (mean: 31% vs 60%). Standard histology and immunohistochemistry showed that hCD55-transgenic hearts exhibited less interstitial edema, hemorrhage, microthrombosis, fibrin deposition, and leukocyte infiltration than did the controls. All hearts showed mild to moderate levels of P-selectin and similar levels of ICAM-1, C3c, C9, IgA, IgG, and IgM deposition. hCD55 expressed on rat hearts not only inhibits complement activation, but also human leukocyte adhesion and apparently functions as an anti-adhesion molecule. hCD55 is an efficient factor in protecting grafts against HAR and protects the graft against adhesion of leukocytes as well.     

Incidence of hyperacute rejection in pig-to-primate transplantation using organs from hDAF-transgenic donors

BACKGROUND: Cynomolgus monkeys or baboons received under immunosuppression kidney or heart grafts from pigs transgenic for human decay-accelerating factor (hDAF) or from control pigs. Hyperacute rejection (HAR) is often difficult to differentiate from nonimmunological causes of organ or recipient dysfunction (NIC), and therefore, a thorough pathology review of all cases with 0-4 days survival (inclusive) was conducted. METHODS: Pathology slides were blinded and together with limited clinical data reviewed by two pathologists. After unblinding, data were compared with the original diagnosis made during the course of the program, and a final diagnosis was reached considering the complete clinical dataset. RESULTS: Life-supporting kidney transplantation was performed in 245 cynomolgus monkeys (234 hDAF, 11 controls), of which 102 cases had 0-4 day survival. None of the hDAF cases showed HAR, whereas this occurred in 27% of controls (P<10-6). Heterotopic heart transplantation was performed in 65 monkeys (57 hDAF, 8 controls), of which 41 cases had 0-4 day survival. HAR was observed in 7% of hDAF cases and in 57% of controls (P=0.002). Heterotopic heart transplantation in baboons was performed in 33 animals (28 hDAF, 5 controls), of which 15 cases had 0-4 day survival. HAR was observed in 11% of hDAF cases and in 20% of controls. Sixteen baboons were subjected to orthotopic heart transplantation, all from hDAF donors, out of which eight survived 0-4 days. The incidence of HAR was 6%. CONCLUSIONS: In the largest series of pig-to-primate solid organ transplants performed thus far, the presence of the hDAF transgene fully prevents HAR of cynomolgus monkey kidney transplants and partially inhibits HAR of heart grafts in cynomolgus monkeys or baboons. The incidence of HAR in control grafts is significantly higher.     

Life-supporting human complement regulator decay accelerating factor transgenic pig liver xenograft maintains the metabolic function and coagulation in the nonhuman primate for up to 8 days

BACKGROUND: It is not known whether the pig liver is capable of functioning efficiently when transplanted into a primate, neither is there experience in transplanting a liver from a transgenic pigs expressing the human complement regulator human complement regulator decay accelerating factor (h-DAF) into a baboon. The objective of this study was to determine whether the porcine liver would support the metabolic functions of non-human primates and to establish the effect of hDAF expression in the prevention of hyperacute rejection of porcine livers transplanted into primates. METHODS: Five orthotopic liver xenotransplants from pig to baboon were carried out: three from unmodified pigs and two using livers from h-DAF transgenic pigs. FINDINGS: The three control animals transplanted with livers from unmodified pigs survived for less than 12 hr. Baboons transplanted with livers from h-DAF transgenic pigs survived for 4 and 8 days. Hyperacute rejection was not detected in the baboons transplanted with hDAF transgenic pig livers; however, it was demonstrated in the three transplants from unmodified pigs. Baboons transplanted with livers from h-DAF transgenic pigs were extubated at postoperative day 1 and were awake and able to eat and drink. In the recipients of hDAF transgenic pig livers the clotting parameters reached nearly normal levels at day 2 after transplantation and remained normal up to the end of the experiments. In these hDAF liver recipients, porcine fibrinogen was first detected in the baboon plasma 2 hr postreperfusion, and was present up to the end of the experiments. One animal was euthanized at day 8 after development of sepsis and coagulopathy, the other animal arrested at day 4, after an episode of vomiting and aspiration. The postmortem examination of the hDAF transgenic liver xenografts did not demonstrate rejection. INTERPRETATION: The livers from h-DAF transgenic pigs did not undergo hyperacute rejection after orthotopic xenotransplantation in baboons. When HAR is abrogated, the porcine liver maintains sufficient coagulation and protein levels in the baboon up to 8 days after OLT.