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.     

Xenotransplantation of hDAF-transgenic swine hearts]

Hearts of transgenic pigs expressing a human regulator of complement activation, decay accelerating factor (hDAF), were transplanted either heterotopically into the abdomen of cynomolgus monkeys or orthotopically into baboons. None of these transgenic hearts was hyperacutely rejected. Immunosuppression with a combination of cyclosporine A, cyclophosphamide and steroids produced a maximum survival of 62 days (median 40 days) in the heterotopic model. Transgenic hearts transplanted into the orthotopic position allowed a maximum survival of 9 days (median 2.5 days). A more effective and less toxic immunosuppressive protocol for the prevention of accelerated xenograft rejection is the subject of ongoing research. The use of organs from transgenic pigs may help to solve the problem of donor shortage in clinical allotransplantation.     

Long-term survival of nonhuman primates receiving life-supporting transgenic porcine kidney xenografts

BACKGROUND: Recently, there has been a resumed interest in clinical xenotransplantation using pig organs. However, no data are available yet regarding the capacity of porcine organs to sustain the life of a primate beyond the first month. We have attempted to obtain long-term survival of nonhuman primates using human decay-accelerating factor (hDAF) transgenic pig organs and an immunosuppressive strategy particularly aimed at neutralizing the humoral component of the immune response. METHODS: hDAF transgenic or control kidneys were transplanted into 14 bilaterally nephrectomized cynomolgus monkeys (Macaca fascicularis) that underwent splenectomy and were immunosuppressed with cyclosporine A, cyclophosphamide, and steroids. All animals also received recombinant erythropoietin. Postoperatively, the primates were monitored daily. Laboratory evaluations included serum biochemistry, hematology, and measurements of hemolytic antipig antibodies. To assess the role of splenectomy in the control of humoral response, historical data were also used from a group of monkeys (n=7) that received the same immunosuppressive regimen and an hDAF transgenic porcine kidney but did not have splenectomy or receive recombinant erythropoietin. RESULTS: This immunosuppressive approach obtained the longest survival time (78 days) described to date of a primate receiving a life-supporting porcine renal xenograft. Furthermore, four of nine animals in this series survived for 50 days or more. Most biochemical measurements in this study (including plasma urea, creatinine, sodium, and potassium concentrations) remained within normal ranges for several weeks in all of the longest-surviving animals. CONCLUSIONS: Normalization of renal function (urea and creatinine) in primate recipients of porcine renal xenografts suggests that pig kidneys may be suitable for future clinical xenotransplantation. Additional immunosuppressive approaches, specifically designed to prevent humorally mediated immunological damage, should be explored to further prolong survival of primates that have received porcine xenografts.     

建立血管内皮细胞组织特异性表达人衰变加速因子的转基因小鼠

目的为了研究异种移植，建立血管内皮细胞组织特异性表达人衰变加速因子(DAF)的转基因小鼠。方法采用受精卵显微注射技术，将含有人内皮细胞粘附分子-2(ICAM-2)基因启动子、人DAF cDNA(插有人DAF基因第一个内含子)、SV40splice／polyA的外源基因导人小鼠受精卵的原核中；选取注射后仍健康的受精卵移植入假孕母鼠的输卵管中待分娩。聚合酶链(PCR)技术及Southern印迹杂交法确定外源基因整合阳性转基因小鼠。RT-PCR方法和流式细胞术分别用于外源基因mRNA及蛋白质水平表达的检测。免疫组织化学方法观察人DAF在转基因小鼠心脏、肝脏、肾脏等器官的表达分布。采用Langendorff心脏灌流装置，用200g／L的稀释人血清灌注转基因小鼠离体心脏，检测其抗超急性排斥反应能力。结果共产仔鼠133只，21只整合有外源基因，整合率16％(21／133)。8只实现mRNA及蛋白质水平表达，蛋白质水平表达强度为人DAF基因在人白细胞表达强度的70％至95％，转基因效率60A(8／133)。免疫组织化学法显示人DAF在转基因小鼠器官组织切片上有较强表达，且表达限于血管内皮细胞。与普通鼠对比，转基因小鼠离体心脏存活时间明显延长，且60min灌注期间内做功仍维持在最大值20％以上。结论成功地建立了血管内皮细胞组织特异性表达人DAF的转基因小鼠。这种小鼠有一定的抗超急性排斥反应能力。     

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.     

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.     

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.     

Hypodermin A,a new inhibitor of human complement for the prevention of xenogeneic hyperacute rejection

Abstract: Background: Hyperacute rejection (HAR) of discordant xenografts in the pig‐to‐human combination can be prevented using tranplants expressing transgenic molecules that inhibit human complement. Hypodermin A (HA), a serine esterase that degrades C3, was tested in the guinea‐pig‐to‐rat and in the pig‐to‐human combinations. Methods: Hypodermin A was tested in vitro, ex vivo, and in vivo models of HAR in the guinea‐pig‐to‐rat combination. Hamster ovary cells (CHO) and a line of porcine aortic endothelial cells (PAEC₁₁) were transfected with HA complementary DNA (cDNA). Results: The pattern of degradation of rat and human C3 by HA was different (multiple bands lower than 40 kDa) from the physiologic pattern observed after spontaneous degradation of rat C3 or physiologic activation of human C3. The CH50 activity in serum was significantly lower in rats treated with 3.2 mg HA/kg than in untreated rats (45 ± 16 U/ml vs. 700 ± 63 U/ml, P < 0.05). Sera from rats injected with 3.2 mg/kg of HA were less effective in lysing guinea‐pig endothelial cells (12 ± 7%) than normal rat sera (79 ± 3%; P < 0.001). Ex vivo, guinea‐pig hearts perfused by rat serum supplemented with HA survived longer than those perfused by non‐treated serum (210 ± 34 and 154 ± 71 min, respectively; P < 0.05). In vivo, guinea‐pig hearts transplanted into HA treated rats survived longer than in non‐treated rats (27 ± 5 min vs. 13 ± 4 min; P < 0.001). In the presence of human serum, smaller amounts of C6 and C5b‐9 were deposited onto HA‐transfected CHO cells than onto control cells. The mHA‐PAEC₁₁ cells were significantly more resistant to lysis by human C than control PAEC₁₁ cells. Conclusions: These data suggest that transgenic HA could be used to prevent hyperacute xenogeneic rejection.     

Study of the microcirculation in hDAF transgenic rat livers xenoperfused with human blood

Abstract: Background: The microcirculation was assessed in the livers of human decay accelerating factors (hDAF) and wild‐type transgenic rats by fluorescent intravital microscopy, histology and histomorphology to determine the benefits of hDAF expression for the microcirculation of a rat liver xenograft perfused with human blood. Methods: Male hDAF transgenic rats (group A; n = 20) and wild‐type Sprague–Dawley rats (group B; n = 20) were xenoperfused with human blood, while other male wild‐type Sprague–Dawley rats (group C; n = 10) were perfused with allogeneic blood. Following plasma and leukocyte staining with fluorescein sodium, and platelet staining with rhodamine, the right lobe of the liver was assessed by intravital microscopy, counting the numbers of perfused sinusoids and leukocytes adhering to the endothelium per mm², and calculating the acinar perfusion index (Pi). The liver underwent histological assessment at the end of each experiment. Mean ± SEM values were calculated and the Mann–Whitney U‐test was used for statistical analysis. Results: The number of perfused sinusoids was higher in the group of hDAF rat livers (group A) and controls (group C) than in the group of non‐transgenic rat livers perfused with human blood (group B) (P < 0.05), although only group C still had a significantly more perfused sinusoids than the other groups after 90 min of perfusion (P < 0.05). The acinar perfusion index was higher in groups A and C than in group B (P < 0.05); here again, only group C still had a significantly higher Pi than group B after 90 min of perfusion (P < 0.05). There was a massive accumulation of leukocytes that peaked after 5 min and persisted throughout the perfusion in all three groups. Histology showed portal and subendothelial hepatic vein hemorrhage, necrosis and inflammatory reaction, which were particularly evident in group B. Conclusion: In our study, rat livers transgenic for hDAF were better protected against early tissue damage by perfusion with human blood, but this did not result in a longer xenograft survival.     

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.     

Soluble Galalpha(1,3)Gal conjugate combined with hDAF preserves morphology and improves function of cardiac xenografts

BACKGROUND: Cytotoxic anti-Galalpha(1,3)Gal antibodies play a key role in the rejection of pig organs transplanted into primates. Regimens reducing anti-Galalpha(1,3)Gal antibodies were associated with severe side effects unable to prevent antibody rebound until soluble synthetic oligosaccharides with terminal Galalpha(1,3)Gal inhibiting antigen binding became available. We displayed kinetics of anti-pig and anti-Galalpha(1,3)Gal IgM and IgG antibody levels using GAS914, a Galalpha(1,3)Gal trisaccharide conjugated to poly-l-lysine, and investigated corresponding changes of parameters of heart function. METHODS: Using a working heart model, hDAF pig hearts were perfused with human blood containing GAS914 (group 1). As controls hDAF pig hearts (group 2) and landrace pig hearts (group 3) were perfused with human blood only. Levels of anti-Galalpha(1,3)Gal (IgM, IgG) and anti-pig antibodies were assessed to prove the effectiveness of GAS914. As parameters of heart function, cardiac output (CO), stroke work index (SWI), coronary blood flow (CBF) and coronary resistance were measured. Creatine phosphokinases, lactate dehydrogenase and aspartate aminotransferase were evaluated as markers of myocardial damage. Histological and immunohistochemical investigations were performed at the end of perfusion. RESULTS: In group 1 an immediate and extensive reduction in both IgM and IgG anti-Galalpha(1,3)Gal was found. Anti-pig antibodies were eliminated accordingly. Antibody binding to GAS914 was complete before the start of organ perfusion. Corresponding to rapid antibody elimination in group 1 GAS914 not only was able to significantly prolong the beating time of the heart in hDAF pigs, but also to clearly improve functional parameters. When switching to the working heart mode hDAF pig hearts perfused with human blood containing GAS914 (group 1) revealed a CO starting at a significantly higher level than hDAF (group 2) and non-transgenic pig hearts (group 3) perfused with human blood only. Similarly, in group 1 SWI was significantly increased at the beginning of perfusion compared to that of group 2 and group 3. The increase in CBF during perfusion and the corresponding fall of coronary resistance occurred without significant differences between the groups revealing the independence of hDAF and GAS914. CONCLUSIONS: Due to an immediate and profound reduction in Galalpha(1,3)Gal-specific antibodies, soluble Galalpha(1,3)Gal conjugates not only prolong survival, but also improve the hemodynamic performance of the heart in DAF pigs.     

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.     

转人CD46基因抑制人补体在转基因小鼠心脏组织中的沉积

目的 观察转人源性膜辅助蛋白(CD46)基因对转基因小鼠心脏中人补体沉积的抑制作用.方法 以近交系昆明小鼠为对象,采用显微注射法制备转人CD46基因小鼠,用逆转录聚合酶链法(RT-PCR法)检测外源基因的整合情况.以Fo代转基因小鼠11只为实验组,同窝非转基因小鼠10只为对照,快速切取小鼠心脏,用改良的Langendorff法经小鼠主动脉逆行灌注预先制备的含补体的B型血人血浆.观察并记录小鼠心脏搏动时间;采用免疫荧光和免疫组织化学法检测小鼠心脏组织中补体C3c及C9的沉积情况.结果 Fo代转基因小鼠外源基因的整合率为33.3%.实验组小鼠心脏搏动时间为(42.6±20.6)min(15～77 min),长于对照组的(20.2±12.5)min(7～40 min),差异有统计学意义(P＜0.01).实验组小鼠心脏组织中补体C3c及C9的沉积均少于对照组.结论 通过显微注射法制备的转人CD46基因小鼠,其外源基因可稳定表达;转人CD46基因可以抑制人补体C3c及C9在转基因小鼠心脏组织中的沉积.     

No functional benefit for hDAF-transgenic rat livers despite protection from tissue damage following perfusion with human serum

Currently, xenogeneic extracorporeal liver perfusion is used in the treatment of acute liver failure. In order to determine whether transgeneity for human regulatory proteins could improve the functional outcome of the ex-vivo liver in relation to the histopathological changes, we studied the effect of the humoral mechanism in xenogeneic isolated rat liver perfusion in normal and transgenic rat livers. Isolated rat liver perfusion was performed for 2 h in normal rat livers with Krebs Henseleit (KH) and human serum (HS), and in livers transgenic for human decay accelerating factor (hDAF; Tg HS). Function of the liver was established by measurement of liver enzymes and bile production, and clearance of bromosulphophthalein (BSP). Tissue specimens taken after perfusion were analysed by routine histology and immunofluorescence staining for C3c deposition. No change in release of liver enzymes could be established throughout the perfusion period. In the 2nd hour, a higher level of bile production was seen for the transgenic group than for the HS group. The transgenic rat livers outperformed the normal livers perfused with HS, when BSP concentration in the bile was measured; however, clearance of BSP from the perfusate was not significantly different. Haematoxylin-eosin (HE) staining of the liver tissue showed evidence of hyperacute rejection in the HS group. There was only mild tissue injury in the transgenic liver. High-intensity fluorescent staining for C3c deposition was seen in the normal livers perfused with HS, and significantly less in the transgenic livers. Although histologically less tissue damage and less C3c deposition was shown, no significantly improved function of the livers transgenic for hDAF was established. These results suggest that for short-term extracorporeal liver perfusion transgenesis offers no functional benefit.     

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.     

Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4⁺T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.     

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.     

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.     

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。     

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.