转染人补体调节基因对猪血管内皮细胞的保护作用

供者血管内皮细胞(EC)是猪到人异种移植时引发超急性排斥反应的靶抗原的主要分布部位。利用转基因技术让猪的内皮细胞表达人补体调节基因(hDAF)，可能会增强其血管内皮细胞的防御能力，借以克服超急性排斥反应的发生，联合转染一种以上人补体调节蛋白基因可能效果更好。为此，我们进行了如下实验。     

补体系统在异种移植中作用的研究进展

补体系统在异种移植超急性排斥反应中的作用已基本被阐明。通过转基因方法使猪血管内皮细胞表达人补体调节蛋白,可以避免发生异种移植超急性排斥反应。但移植过程中的缺血再灌注损伤却不可避免,补体介导的血管内皮细胞激活可以引起急性血管性排斥反应,并影响移植物的长期存活。本文就补体系统在异种移植领域的研究进展作一综述。     

小鼠血管内皮细胞表达人DAF和CD59对其心脏在人血清灌注下做功的影响

目的探讨联合转人衰变加速因子（DAF）和CD59基因对小鼠心脏在人血清灌注下做功及组织结构的影响。方法采用受精卵显微注射技术，将人DAF和CD59基因注入昆明小鼠受精卵的原核中，选取注射后仍健康的受精卵植入假孕母鼠的输卵管中待分娩。提取足月产小鼠（G0代）基因组DNA，以聚合酶链反应（PCR）及Southern印迹杂交确定外源基因整合情况，应用流式细胞术检测人DAF和CD59在转基因小鼠白细胞上的表达，免疫组织化学染色检测人DAF和CD59在转基因小鼠心脏、肝脏及肾脏组织中的表达。取出转基因成功小鼠的心脏，在体外以人血清进行灌注，测定其做功能力，HE染色及免疫组化染色观察灌注后心脏的组织学变化以及补体C3c的沉积情况。以单一转DAF基因的小鼠为对照。结果共获得G0代小鼠135只，经PCR及Southern印迹杂交分析，11只（8．1%，11／135）整合有人DAF和CD59外源基因，其中6只的白细胞膜表面人DAF和CD59表达阳性，强度分别为（86±6）％和（85±8）%，单转人DAF基因者（87±7）%，差异无统计学意义。人DAF及CD59仅表达于转基因小鼠心脏、肝脏及肾脏组织中的血管内皮细胞上。在人血清灌注后60min内，普通小鼠的心脏做功急剧下降，接近40min时心脏停止搏动；转DAF基因小鼠的心脏做功也下降，但仍维持在最大值的20%以上；转DAF和CD59双基因小鼠的心脏做功维持在最大值的40％以上。在人血清灌注的10～60min，转基因小鼠心脏做功能力明显强于普通小鼠心脏（P〈0．05）；灌注20-60min时，转双基因小鼠的心脏做功能力明显强于转DAF基因小鼠心脏（P〈0．05）。普通小鼠心脏在人血清灌注后组织裂解较多见，肿胀严重，而转基因小鼠心脏组织的病理改变明显轻于普通小鼠，转双基因者的病理变化又明显轻于单一转DAF者，心肌血管中人C3c的沉积也明显少于单一转DAF者。结论血管内皮细胞特异性表达人DAF和CD59，可明显阻抑其心脏在异种血清灌注下的做功能力下降及C3c在血管中的沉积，这可能对抵御超急性排斥反应有一定帮助。     

补体系统在异种移植中作用的研究进展

补体系统在异种移植超急性排斥反应中的作用已基本被阐明。通过转基因方法使猪血管内皮细胞表达人补体调节蛋白，可以避免发生异种移植超急性排斥反应。但移植过程中的缺血再灌注损伤却不可避免，补体介导的血管内皮细胞激活可以引起急性血管性排斥反应，并影响移植物的长期存活。本文就补体系统在异种移植领域的研究进展作一综述。     

联合转移人HT和DAF基因对模拟异种移植超急性排斥反应的抑制作用

目的探讨联合转移人α1，2-岩藻糖苷转移酶（HT）和衰变加速因子（DAF）基因对模拟异种移植超急性排斥反应的抑制作用。方法通过显微注射制备转人HT及DAF基因的子代小鼠，以聚合酶链反应和流式细胞仪筛选出人HT和（或）DAF基因整合与表达阳性的子代转基因小鼠。以同时表达人HT和DAF基因的小鼠为实验组，单一表达人HT或DAF基因的小鼠为HT对照组和DAF对照组，取其心脏，采用改良的Langendorff心脏灌注装置灌注心脏，先以KH液逆向灌注，30min后再以含15％（体积分数）人AB血清的Krebs—Henseleit液（KH液）灌注。记录不同灌注时间的心率、左心室收缩压及左心室舒张末压，计算左心室获得压，以心率与左心室获得压的乘积作为心脏左心室收缩功能的指标，免疫组化法观察灌注后的心脏血管内皮细胞和组织中IgM与C3c的沉积情况。结果KH液灌注过程中，各组间心脏做功能力的差异无统计学意义；KH液中加入人血清后，HT对照组和DAF对照组的心脏做功能力明显下降，在灌注60min时心脏做功分别为最大值的27％和23％，平均做功时间分别为118min和89min，普通小鼠心脏在灌注后50min时停止搏动，而实验组的心脏做功能力下降缓慢，至60min时心脏做功维持在最大值的67％以上，平均做功时间为225min，其心脏做功能力与搏动时间高于HT对照组和DAF对照组（P〈0．05）。免疫组化染色显示，实验组小鼠心脏血管内皮细胞与组织中未见IgM和C3c沉积，HT对照组可见C3c少量沉积，DAF对照组可见IgM沉积和C3c少量沉积，普通小鼠心脏可见IgM和C3c沉积。结论小鼠联合转移人HT和DAF基因后，其心脏在人血清灌注下的免疫损伤明显减轻，这可能对抵御异种移植超急性排斥反应有一定帮助。     

异种肝移植中补体作用及抑制策略

近 40年来 ,随着同种肝移植的巨大成功 ,供肝短缺的问题日益突出。异种移植作为较有希望的解决途径之一 ,得到越来越多的重视。但阻碍异种肝移植临床实施的首要障碍即是超急性排斥反应 (ＨＡＲ)。大量的研究表明 ,补体在ＨＡＲ发生中起着重要作用 ;而抑制补体激活可以阻止ＨＡＲ ,尤其是转人补体调节蛋白 (ｈＣＲＰ)基因的动物的出现 ,更使人们看到了征服ＨＡＲ的光明前景。这里 ,将近年补体在异种肝移植中超急性排斥反应中的作用及其防治策略的进展作一综述。一、补体在超急性排斥反应发生中的作用及激活途径1.补体在超急性排斥反应发生中…     

清除补体避免猪到猕猴异种心脏移植超急性排斥反应的实验研究

目的 观察纯化的眼镜蛇毒因子(CVF)对猪到狱猴异种心脏移植超急性排斥反应的影响。方法 以幼猪为供者，施行猪到狱猴腹腔内异位心脏移植，实验组(n＝4)使用CVF完全清除受者体内补体，对照组(n＝5)不使用CVF，两个组术后均采用环抱素A、甲泼尼龙和环磷酰胺抑制排斥反应，通过检测血清C3、C4水平及总补体活性验证CVF的效果，移植心停跳时切取移植心进行病理检查。结果 在使用CVF后，实验组血清C3降为0，总补体活性CH50值也几乎为0，末发现明显毒副反应，移植猪心存活时间平均为lld，最长达13d，病理学提示均发生了延迟性异种排斥反应；对照组3个移植心在移植后60min内发生超急性排斥反应，另2个分别存活22h及6d。结论 纯化的CVF有良好的清除补体的作用，且末见明显副作用；使用CVF可克服猪到狱猴异种心脏移植超急性排斥反应的发生。     

猪到猕猴异种心脏移植超急性排斥反应的免疫学及病理学变化

目的观察猪到猕猴异种心脏移植超急性排斥反应时的免疫学及病理学变化。方法采用猪到猕猴腹腔内异位心脏移植模型,检测发生超急性排斥反应者的血液中补体、天然抗体及T淋巴细胞亚群的变化,并对移植心脏进行免疫组化(测定C3、C4、C5b9、IgG及IgM的沉积)及病理学分析。结果发生超急性排斥反应时,血清补体C3、C4的含量、总补体活性及抗猪内皮细胞天然抗体均有一定程度的下降;CD4 /CD8 T淋巴细胞的比率也有所下降;移植心脏中均有补体C3、C4、C5b9的沉积,IgG及IgM也均有沉积,但IgG和IgM沉积强度的差异无统计学意义;病理学改变主要为心肌间质弥漫性出血、水肿,毛细血管内普遍淤血。结论补体通过经典途径激活参与猪到猕猴异种心脏移植超急性排斥反应;超急性排斥反应时受者血中天然抗体水平明显下降;CD4 T淋巴细胞可能参与异种移植超急性排斥反应过程并有所消耗;发生超急性排斥反应的移植物突出病理表现为间质出血。     

转人α-1,2岩藻糖苷转移酶基因在异种移植中的研究

目的 研究转人α-1，2岩藻糖苷转移酶基因（HT）在克服超急性排斥反应（HAR）中的作用。方法 通过脂质体转染将人HT基因转入猪血管内皮细胞（PIECs）中，利用显微注射建立转HT基因小鼠模型，经流式细胞仪检测转HT基因阳性的PIECs表面抗原及小鼠外周血单核细胞表面抗原的变化。结果 转HT基因的PIECs表面的α-1，3半乳糖（α-Gal）比阴性对照下降了50％～60％，H抗原由无表达到44％～78％表达阳性；转HT基因小鼠白细胞表面的α-Gal比阴性对照下降了63％左右，H抗原阳性表达率为95％以上。结论 HT基因在细胞和小鼠体内均有较好的表达，可减少异种抗原与异种抗体的反应，在克服超急性排斥反应中能发挥一定的作用。     

转人CD59基因的猪胚胎成纤维细胞的建立

补体介导的超急性排斥反应(HAR)是异种移植成功应用的主要障碍。CD59 是一种补体活化调控蛋白,具有良好的抗超急性排斥反应作用。通过对猪胚胎成纤维细胞进行基因修饰,体细胞克隆,有希望克服上述问题。材料与方法一、猪胚胎的获取孕30 d实验用猪(购自中国农业科学院实验站)     

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.     

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 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.     

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.     

Complement regulatory proteins are expressed at low levels in embryonic human, wild type and transgenic porcine neural tissue

Allotransplantation of human foetal neural tissue for neurodegenerative disorders has been shown to provide clinical benefit but is limited by a number of issues including donor supply. The use of porcine foetal tissue as an alternative source of cells is being investigated but xenotransplants survive poorly as a result of immunological rejection, which may involve complement. In this study we investigated the expression of the membrane-bound complement regulatory proteins--decay accelerating factor (DAF), membrane co-factor protein (MCP) and CD59 in embryonic neural tissue. Cells were derived from human foetuses, wild-type porcine foetuses and porcine foetuses transgenic for human complement regulatory proteins and analysed using flow cytometry and immunocytochemistry. Functional assessment of human complement regulatory protein expression in transgenic porcine tissue was assessed by C3b deposition and cell survival on exposure to human complement. Human and wild-type porcine foetal neural tissue expressed moderate levels of MCP and CD59 but low or no levels of DAF. Neural tissue from porcine foetuses transgenic for human MCP (E174) expressed the transgene but failed to significantly inhibit human C3b deposition compared with non-transgenic tissue. In contrast, foetal neural tissue from two different human DAF transgenic pig lines (A74 and E71) known to express high levels of human DAF on endothelial cells, failed to express significant levels of human DAF in foetal neural tissue. Complement regulatory proteins such as MCP and CD59 are expressed in the human and wild-type embryonic brain but in contrast, DAF is expressed at very low levels. Pigs transgenic for human DAF express very low levels of human DAF on embryonic neural tissue. In pigs transgenic for human MCP, the transgene is expressed at similar levels to that in human embryonic neural tissue but at an insufficient level to prevent activation of the complement cascade. Thus alternative approaches to reducing complement activation by xenografted neural foetal tissue will be required if this process proves to be important in the rejection process.     

KIDNEYS DERIVED FROM MICE TRANSGENIC FOR HUMAN COMPLEMENT BLOCKERS ARE PROTECTED IN AN IN VIVO MODEL OF HYPERACUTE REJECTION

Purpose

The major obstacle to successful discordant kidney xenotransplantation is hyperacute rejection (HAR). Complement plays a key role in the induction of HRA, defined by endothelial cell activation, loss of vascular integrity, hemorrhage and thrombosis. The activation of complement is tightly controlled by a number of species-specific regulatory proteins which inhibit, at different points, the cascade of events leading to the formation of the membrane attack complex (MAC). We have tested the hypothesis that kidneys derived from transgenic mice expressing two human complement inhibitors, Decay Accelerating Factor (hDAF) and Membrane Cofactor Protein (MCP), could be protected from human complement-mediated damage.

Materials and Methods

Control and transgenic mice were perfused with human plasma by cannulation of the right jugular vein, at a perfusion rate of 10 micro L./min. for two hours. Complement C3 deposition was detected on kidney sections by immunohistochemistry using specific FITC antibody. Complement-induced tissue damage was evaluated by histopathological examination.

Results

Heavy deposition of complement C3 was observed on kidneys derived from perfused control mice. This was associated with a characteristic HAR pathology of severe interstitial hemorrhage, inflammatory reaction, loss of glomerula and tubuli structure. Kidneys derived from mice transgenic for hDAF or hMCP were partially protected from both complement C3 deposition and tissue damage. The expression of both dDAF and hMCP in double transgenic mice significantly increases the protection from human complement-mediated damage.

Conclusions

A novel model of in vivo perfusion with human plasma has been adopted to recreate the initial event of HAR. Our data show that this murine model could be very valuable to determine the effect of transgenic human molecules in protecting vascularized organs from human complement attack.     

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.     

Characterization of human CD55 and CD59 transgenic pigs and kidney xenotransplantation in the pig-to-baboon combination

New transgenic pigs expressing combinations of regulators of complement activation and other molecules are needed to resist xenograft hyperacute rejection (HAR) and to further analyze and treat xenograft rejection. Double transgenic pigs for human CD55 (hCD55) and human CD59 (hCD59) using the promoter of the human elongation factor 1 alpha gene were generated, and their kidneys were transplanted into nonimmunosuppressed baboons. hCD55 and hCD59 were mainly expressed by the endothelial cells, and these cells showed increased resistance to complement-mediated lysis. Baboons receiving kidneys from hCD55hCD59 pigs survived for 5 and 6 days, and displayed alterations in coagulation. Thrombocytopenia and platelet microthrombi were present within the kidneys. Nontransgenic kidneys showed HAR in less than 2 days. Kidneys from pigs expressing hCD55hCD59 displayed protection against HAR in the absence of immunosuppression. Rejection was associated with coagulopathy leukocyte infiltration and a rebound of anti-alpha Gal antibodies.