水苏糖对模拟异种心脏移植超急性排斥反应的抑制作用

目的观察水苏糖对猪到人异种心脏移植超急性排斥反应的抑制作用。方法以人血液体外灌注猪游离心脏为基础，模拟猪到人异种心脏移植的超急性排斥反应模型。实验分为A、B两组：即分别用人血液灌注及人血液加水苏糖灌注猪离体心脏。观察灌注后两组心脏的跳动时间；体外灌注1h后，对两组灌注心脏进行免疫组织化学（测定IgG及IgM的沉积）及病理学分析。结果A组灌注心脏平均跳动时间为（9．5±2．5）min；B组灌注心脏平均跳动时间为（46．8±8．1）min，其中有1个心脏在灌注的1h内一直跳动；两组心脏跳动时间比较，差异有统计学意义（P〈0．01）。A组心肌间质呈弥漫性出血、水肿，血管扩张，内皮细胞肿胀、坏死；免疫组织化学检查显示心肌血管内皮组织中有IgG及IgM沉积。B组心肌间质未见出、凝血和坏死，血管内皮细胞未见肿胀；免疫组织化学检测未见IgG及IgM沉积。结论水苏糖对异种心脏移植超急性排斥反应具有抑制作用。     

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。     

水苏糖在猪到猴异种心脏移植超急性排斥反应中的作用（附视频）

目的观察水苏糖在猪到猕猴异位心脏移植超急性排斥反应中的作用。方法采用猪到猕猴腹腔内心脏异位移植模型。根据是否经水苏糖处理分为实验组及对照组,每组3只猕猴,观察移植心脏移植后24h内持续搏动时间和组织形态学变化。结果对照组移植心脏持续搏动时间分别为5、15、20min;心肌间质弥漫性出血、水肿,血管扩张,内皮细胞肿胀、坏死。实验组移植心脏持续搏动时间分别为50min、97min和24h;心肌间质未见出血、坏死,血管内皮细胞未见肿胀。结论水苏糖可能对猪到猕猴异位心脏移植的超急性排斥反应有抑制作用。     

猪—猴异种血管移植免疫反应初探

目的 探讨猪－猴异种血管移植超急性排斥反应（HAR）的机理。方法 猪股静脉原位异种移植于恒河猴,发生HAR后通过免疫组化检测移植血管IgG、IgG、C3及C4的沉积。结果 大量IgM、C3和C4沉积于移植静脉内皮, 未发现IgG沉积于移植血管内皮。结论 猪－猴异种移植HAR是由异种自然抗体IgM与异抗原特异结合启动,进而以经典途径激活补体系统而发生。     

猪血管内皮细胞与人血清反应早期PGI2和TXA2的变化

目的 探讨在猪与人异种移植超急性排斥反应中血管内皮细胞的作用。方法 用体外培养的猪血管内皮细胞和不同人的血清共同反应,建立猪与人异种移植超急性排斥反应的体外实验模型。用放射免疫法检测上清液中前列环素（ＰＧＩ２）和血栓素Ａ２（ＴＸＡ２）的稳定代谢产物（ＰＧＦ１α和ＴＸＢ２）含量,将其作为评定血管内皮细胞激活和损伤的标志。结果 各组血清与猪血管骨皮细胞反应０．５ｈ后,正常人血清组ＰＧＦ１α／ＴＸＢ２比     

人血清对猪血管内皮细胞的细胞毒效应研究

目的 探讨异种(猪/人）超急性排斥的发生机制。方法 人血清为天然抗体和补体源。用四唑盐法行补体依赖的细胞毒反应，建立体外超急性排斥模型。结果 正常人血清能溶解猪血管内皮细胞；而Clg缺乏及B因子缺乏的人血清对猪血管内皮细胞的溶解率较低(P＜0．01)；同种猪血清及灭活补体的人血清不溶解猪血管内皮细胞；将经典或旁路途径有缺陷的人血清等体积混合，其细胞毒作用恢复正常。结论 治疗猪/人之同的超急性排斥应考虑补体旁路途径激活的问题。     

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。     

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。     

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。     

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。     

A recombinant soluble chimeric complement inhibitor composed of human CD46 and CD55 reduces acute cardiac tissue injury in models of pig-to-human heart transplantation

BACKGROUND: Inasmuch as complement plays a critical role in many pathological processes and in xenograft rejection, efficient complement inhibitors are of great interest. Because the membrane-associated complement inhibitors are very effective, recombinant soluble molecules have been generated. METHODS: We tested the efficacy of complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), in two models of pig-to-human xenotransplantation in which tissue injury is complement mediated. The in vitro model consisted of porcine aortic endothelial cells and human serum, and the ex vivo model consisted of a porcine heart perfused with human blood. RESULTS: In vitro, addition of CAB-2 to serum inhibited cytotoxicity and the deposition of C4b and iC3b on the endothelial cells. Ex vivo, addition of CAB-2 to human blood prolonged organ survival from 17.3 +/- 6.4 min in controls to 108 +/- 55.6 min with 910 nM (100 microg/ml) CAB-2 and 219.8 +/- 62.7 min with 1820 nM (200 microg/ml) CAB-2. CAB-2 also retarded the onset of increased coronary vascular resistance. The complement activity of the perfusate was reduced by CAB-2, as was the generation of C3a and SC5b-9. The myocardial tissues had similar deposition of IgG, IgM, and Clq; however, CAB-2 reduced the deposition of C3, C4, and C9. Hearts surviving >240 min demonstrated trace to no deposition of C9 and normal histologic architecture. CONCLUSION: These results indicate that CAB-2 can function as an inhibitor of complement activation and markedly reduce tissue injury in models of pig-to-human xenotransplantation and thus may represent a useful therapeutic agent for xenotransplantation and other complement-mediated conditions.     

Anatomical study on the surgical technique used for xenotransplantation: porcine hearts into humans

BACKGROUND: The pig heart is an ideal graft for orthotopic cardiac xenotransplantation regarding its physiological attributes and ready availability. Although single clinical attempts have been performed since the 1960s, details concerning the surgical technique of pig-to-human transplantation have never been reported. The present investigation should verify which anatomical differences between humans and pigs require special care in cardiac xenotransplantation. MATERIAL AND METHODS: We transplanted four pig hearts into human thoraces after autopsy. Implantation was performed using both the biatrial (modified Shumway) and bicaval techniques. The implanted hearts were not perfused. RESULTS: The four-legged walk of the pig implies a more transverse heart position and therefore a different outflow-angle of the great vessels. Accordingly, the thin-walled pulmonary artery and the superior vena cava (in bicaval technique) tend to kink and narrow. A special feature of porcine anatomy is the left azygous vein that empties into the coronary sinus. It must be ligated before the implantation. CONCLUSIONS: Keeping the porcine anatomical particularities in mind, technical problems in pig-to-human heart transplantation can be avoided. The anastomosis of the pulmonary artery requires special care. By using the biatrial technique surgeons can prevent imminent stenoses of the caval vein anastomoses.     

补体在猪到猴异种心脏移植中的作用及机理

目的 探讨补体在异种大动物猪到猴心脏移植排斥反应中的作用及机理.方法 以梅山猪为供者,中国猕猴为受者,行异种腹腔异位心脏移植.随机将受者分为3组.A组(5只):为空白对照组,受者心脏移植后不作任何处理.B组(5只):为照射预处理组,受者于心脏移植前28 d、即1.5个月龄时接受60Coγ3 Gy全身剂量照射,其余同A组.C组(8只):为照射+胸腺注射预处理组,心脏移植前21 d,将供者的脾细胞(按照5×107个/只的数量)注入受者的两侧胸腺内,其余同B组.观察心脏移植术后各组移植心的存活时间;猪对猴单向混合淋巴细胞培养的刺激效应;采用双抗体夹心法检测补体C3和CD46的血清浓度;通过流式细胞术检测受者外周血细胞表面IgM、IgG阳性细胞百分比水平.结果 A、B、C三组移植心的存活时间分别为:(36.6±5.8)h、(65.6±6.5)h和(91.1±22.8)h,C组移植心的存活时间明显延长,与A组比较,P＜0.01,与B组比较,P＜0.05.C组在猪对猴单向混合淋巴细胞反应中的刺激效应较A、B组明显下降(P＜0.01).B、C组移植前补体水平(C3)无明显变化,但随着IgM、IgG水平的上升,发生排斥反应时C3和CD46水平显著降低.C组猕猴特异性抗猪抗体IgM及IgG的上升速度均较A、B组明显延缓.结论 对受者进行异种胸腺注射联合全身照射预处理在抑制T淋巴细胞免疫及体液免疫方面有重要作用,但无法抑制异种排斥反应中补体的激活,补体通过经典途径参与了延迟性异种排斥反应的发生.     

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。     

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.     

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.     

Efficacy of double filtration plasmapheresis in removing xenoantibodies and prolonging xenograft survival in an ex vivo swine perfusion model

Introduction

In xenotransplantation, antibodies mediate humoral rejection, resulting in organ dysfunction. Removal of xenoantibodies is likely a first step for successful transplantation. Double filtration plasmapheresis (DFPP) selectively removes large molecular weight pathogenic substances, such as immunoglobulins (Ig), without other plasma proteins. The antibodies fractions removed and the changes in blood biochemistry are unanswered questions after DFPP in addressed this ex vivo swine heart perfusion model.

Materials and Methods

Twelve swine hearts were perfused with human blood in a modified Langendorff's apparatus. The perfusate containing human blood was divided into 2 groups: controls (N = 6) and DFPP-treated group (N = 4). Blood counts, biochemistry data, and immunological profiles were compared at 3 time points: before and after DFPP and after heart perfusion.

Result

Perfusion times of control and DFPP groups were 5.43 ± 1.81 vs 9.25 ± 3.00 hours, respectively. Only the values of albumin and total protein showed difference. The immunologic profile revealved complete removal of IgM and most IgG, IgA, C3, and C4, namely, 79.95%, 88.58%, 83.15%, and 87.97%, respectively.

Conclusion

DFPP showed excellent efficacy to remove xenoantiboidies and prolong xenograft survival in an ex vivo perfusion model.     

Prevention of hyperacute xenograft rejection in orthotopic xenotransplantation of pig hearts into baboons using immunoadsorption of antibodies and complement factors

Abstract To prevent hyperacute xenograft rejection (HXR) caused by preformed natural antibodies (XNAb) after orthotopic heart xenotransplantation (oXHTx) of landrace pig hearts into baboons, we used immunoadsorption of immunoglobulins IgG, IgM and IgA and complement with the reusable Ig‐Therasorb column. In addition to functional data, tissue was sampled for histological, immunohistochemical and electron microscopical analysis. We performed three oXHTx of landrace pig hearts to baboons using extracorporeal circulation (ECC) connected to the immunoadsorption unit. Intraoperative treatment consisted of four cycles of immunoabsorption (IA). One oXHTx of a baboon without IA served as a control. A mismatch of donor and recipient heart size was prevented by selecting a 30‐40% lower body weight of donor pigs than recipients. Four cycles of IA removed more than 80% of IgG, IgM and IgA, 86% of anti‐pig antibodies and 66% of complement factors C3 and C4 from plasma. The graft of the control animal failed after 29 min. Orthotopic xenotransplantation with IA was selectively terminated after 100 min, 11 h and 21 h, respectively without any histological signs of HXR in light and electron microscopy. After weaning off from ECC these donor xenografts showed sufficient function with normal ECG and excellent cardiac output in echocardiography and invasive measurement (1.93 ± 0.035 l/min). The myocardium of the control xenograft demonstrated more deposits of Ig and complement components (C3, C4) than in the IA group. Baboons survive HXR after orthotopic pig heart xenotransplantation due to antibody depletion by reusable Ig‐Therasorb column treatment. Long‐term survival in an orthotopic baboon xenotransplantation model after IA, especially in combination with transgenic pig organs, could be a reliable preclinical trial for future clinical xenotransplantation programs.