The Effect of Total Blood Exchange with PHP Solution on Cardiac Xenotransplantation

Abstract: Prevention of hyperacute rejection is a difficult and unsolved problem in xenotransplantation. Natural antibodies and complement activation have been known to play an important role in the xenotransplantation between discordant species pairs. In the present study, total blood exchange (TBE) was performed with pyridox-alated-hemoglobin-polyoxyethylene conjugate (PHP) solution (Ajinomoto Co., Inc., Kawasaki, Japan) before cardiac xenotransplantation in order to remove the immunoglobulins and prolong xenograft survival time. Guinea pigs and rats were used as the discordant species combination for donor and recipient. Two groups were established: Group 1, untreated control (n = 8) and Group 2, TBT with PHP solution (n = 8). The exchange blood transfusion was carried out at the rate of 15–20 ml/h utilizing PHP solution using a blood pump. After the blood exchange was processed, hematocrit (Ht) levels dropped to 4 or 5%, and a cardiac xenotransplantation was performed within 24 h. The levels of serum IgA, IgM, and IgG were decreased to less than 25, 25, and 10% of the base line, respectively, after blood exchange. A mean xenograft survival time in Group 2 was prolonged to 472 ± 74 min and to 10.4 ± 1.8 min in Group 1 (p < 0.01). A titer of the anti-guinea pig lymphocytotoxic antibody in rat serum was decreased to almost nil. The data from this study suggest that total blood exchange with PHP solution may be useful in preoperative removal of xenograft antibodies in xenotransplantation.     

Antibody binding to endothelial and epithelial antigens triggers pig-to-rabbit xenograft rejection and its absence results in atypical complement deposition

In pig-to-rabbit kidney xenograft (PRKX), endothelial antigen determinants (EAD) are immediately recognized by IgG and IgA, while IgM does not react with them. The purpose of this study was to investigate the different roles of IgG, IgA, IgM, and complement in the hyperacute rejection of a PRKX model. Nine isolated Landrace pig kidneys were each perfused with 10 ml normal New Zealand rabbit serum. Perfusates (serum A) were collected after discarding the first 0.5 ml. Serum A and rabbit complement were then incubated for 30 min with frozen sections of normal pig kidney. After washing with buffer solution all the specimens were treated for immunohistochemistry. Three frozen sections of normal Landrace pig kidney and three samples of normal New Zealand rabbit serum were used as controls. Immunohistochemical analysis of the nine perfused kidneys demonstrated IgG, IgA and C3 deposition on the peritubular and glomerular vascular endothelium. No IgM reactivity was shown. In the frozen sections exposed to serum A, immunofluorescence showed minimal IgG, IgA and C3 reactivity while IgM deposition was clearly evident on the tubular epithelium. Immunofluorescence of frozen sections exposed to rabbit complement, done by fluorescein-labeled goat anti-rabbit C3 antibodies were positive only in the glomerular endothelium. The same rabbit complement was active in antibody dependent cytotoxicity on human T cells. Our results indicated that in the PRKX model, IgG and IgA acted as preformed antibodies recognizing endothelial EAD. IgM did not bind to any endothelial molecules, but recognized antigens located on the brush border of the tubular epithelium. Furthermore, in this model, absence of antigen-antibody complexes resulted in atypical complement deposition.     

异种器官移植的新进展

目的总结异种器官移植的新进展。方法分析近年来异种器官移植进展的文献报道。结果随着免疫生物学的深入研究,异种器官移植取得了长足的进步,并开始应用于临床,但免疫排斥反应的诸多问题仍在探寻之中。结论异种移植为解决器官衰竭患者移植器官短缺的问题展现了广阔的前景,如何更有效地抑制排斥反应及延长移植物生存期是今后研究的重点。     

异种移植的研究进展

目的总结异种移植的研究现状与进展。方法复习国内外关于异种移植研究的相关文献并进行综述。结果超急性异种排斥反应是异种移植面临的巨大问题,器官或组织的供体经过基因修饰后,在一定程度上可以减轻超急性异种排斥反应。由于不同的器官具有不同的特性,目前异种器官移植移植物的存活时间存在较大差异。结论通过对相关异种移植实验研究的分析,可以全面地了解异种移植的研究背景,为异种移植提供合适的研究方向。将经基因修饰的动物作为异种器官或组织的供体,有望使移植物避免或减轻超急性异种排斥反应。     

In Vivo and In Vitro Optimization of Depletion of IgM and IgG Xenoantibodies by Immunoadsorption Using Cell Membrane Proteins

Abstract: A system of immunoadsorption was developed for in vitro depletion of xenoreactive natural antibodies of classes IgG and IgM from monkey and human plasma. Porcine endothelial cell membrane proteins, platelet membrane proteins, and endothelial cells were used as affinity ligands, and cyanogen bromide-activated Sepharose 6 Fast Flow and Sepharose CL4B gels were used for chromatography. Adsorption capacity was evaluated by means of ELISA, immunonephelometry, and cytotoxicity testing. Several consecutive adsorption-desorption cycles were performed. Different parameters influencing immunoadsorption were examined: ligand density on the column gel, adsorbent-plasma contact time, ratio of plasma volume to immunoadsorbent volume, desorption conditions, and temperature. After 2 adsorption-desorption cycles, 99% and 82 to 85% of IgG and IgM antipig antibodies were adsorbed, respectively. Furthermore, there was a 74 to 77% decrease in cytotoxicity. In vivo, we observed that after one adsorption-desorption cycle, 97% of antipig IgG antibodies and 96% of antipig IgM antibodies were adsorbed, and there was an 85% decrease in cytotoxicity. The immunoadsorption method studied and optimized in vitro and in vivo therefore efficiently depleted xenoantibodies and reduced the cytotoxicity. Thus, it can be used in xenotransplantation experiments without eliminating nonspecific antibodies.     

异种移植超急性排斥机理的实验研究

为了研究补体经典和旁路途径在超急性排斥中的作用，选择猪血管内皮细胞为靶，人血清为天然抗体和补体源，用四唑盐法（MTT）行补体依赖的细胞毒反应（CDC）。人血清能溶解（58±5）％的猪血管内皮细胞；加入乙二醇四乙酸阻断经典途径后人血清的溶细胞率降为（51±3）％（P＜0．01）；入血清50℃加热20分钟，阻断旁路途径后，其落细胞率降为（42±5）％（P＜0．01）；将经典途径和旁路途径分别被阻断的人血清混合，血清的细胞毒作用恢复正常。在这一体外超急性排斥模型中，补体经典和旁路两条途径均参与超急性排斥。提示抑制猪-人之间的超急性排斥应考虑补体旁路途径激活的问题。     

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     

Clinical Xenotransplantation: Pigs Might Fly?

Xenotransplantation has the potential to deliver an unlimited supply of organs for transplantation. However, this promise has yet to translate into clinical application, despite substantial research efforts in the last decade. Although increasing numbers of studies are being performed in relevant pre-clinical (pig-to-primate) transplantation models, so far these have highlighted the apparent elusiveness of long-term xenograft survival. Humoral rejection remains the main obstacle to success, but control of T cell-mediated rejection will be a problem in the future and there are major concerns about the possible transmission of porcine endogenous retroviruses (PERV) and other infectious agents. This article reviews recent advances in the understanding of acute vascular rejection (AVR), acute T cell-mediated rejection and PERV transmission and highlights some of the strategies that may prove successful in overcoming these problems. Although progress has been slow, the promise of an inexhaustible supply of organs is sufficient reason to continue research in these areas. Assuming the specific problem of AVR can be ameliorated by one of a number of strategies currently under investigation, there are grounds to believe that xenotransplantation will become a clinical reality. Pig xenografts, currently grounded, might eventually fly!     

猪到人异种心脏移植超急性排斥反应实验模型的建立：人血体外灌注猪冠状动脉系统

目的建立一种猪到人异种心脏移植超急性排斥反应实验模型。方法 8只广西巴马猪正中切口锯开胸骨进胸,肝素化,完整剪取猪心脏,用4℃生理盐水反复冲洗各心腔及心脏表面,用改良圣托马斯液经主动脉灌注,冲洗冠状动脉系统后,建立人血液体外灌注猪游离心脏冠状动脉系统,记录各模型心脏搏动的持续时间,1h后对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 8个人血液体外灌注猪心脏冠状动脉系统实验模型均成功建立,灌注心脏平均搏动时间为（9.25±1.90）min;心肌间质弥漫性出血、水肿,血管扩张,内皮细胞肿胀、坏死;心肌血管内皮细胞有IgG及IgM沉积。结论通过人血液体外灌注猪心脏冠状动脉系统建立的猪到人异种心脏移植实验模型能模拟超急性排斥反应,操作简单,容易复制。     

Anti-lymphocyte antibodies late in the course of pediatric renal transplantation

 Beyond the immediate post-transplant period, physicians are often reluctant to use anti-lymphocyte preparations to treat episodes of acute renal functional deterioration attributable to acute rejection. This is due to the perception that such episodes are less likely to be reversible, and to concern regarding the potential adverse effects of anti-lymphocyte antibodies, including opportunistic infections, lymphoproliferative disorders, and the development of human anti-mouse antibodies. Records were reviewed for all 365 renal transplants performed in 267 patients at our center from 1971 to 1996. Anti-lymphocyte antibodies were used in an attempt to reverse 6 episodes of corticosteroid-resistant acute rejection in 5 children at a mean interval of 24.5 months following transplantation. The mean serum creatinine at initiation of therapy with the anti-lymphocyte agents was 2.9 mg/dl. Following treatment, the mean serum creatinine decreased to 1.3 mg/dl (P=0.03, Student’s t-test). Two patients developed uncomplicated opportunistic infections after completion of anti-lymphocyte therapy; none have developed lymphoproliferative disorders or antibodies to OKT3. We conclude that in the correct clinical setting with corticosteroid-resistant acute rejection, the use of anti-lymphocyte antibodies should not be withheld solely on the basis of length of time since transplantation. Received: 27 January 1998 / Revised: 19 May 1998 / Accepted: 6 July 1998     

Inhibition of platelet aggregation by the GPIIb/IIIa antagonist Reopro does not significantly prolong xenograft survival in an ex vivo model

Effects on hyperacute rejection were studied in a discordant model with the platelet GPIIb/IIIa antagonist Reopro. Pig kidneys perfused with human blood survived median 118 min in the Reopro group and 103 min in the controls (P = 0.22). Platelet and leukocyte counts decreased, whereas plasma thrombospondin and soluble as well as platelet membrane P-selectin increased significantly in both groups without significant intergroup differences. beta-Thromboglobulin and myeloperoxidase increased significantly more in the control group than in the Reopro group (P = 0.009 and P = 0.02, respectively). The classical complement pathway was substantially and similarly activated in both groups. Light and electron microscopy revealed arterial thrombi and numerous glomerular platelet aggregates in the control group in contrast to the Reopro group. In conclusion, Reopro reduced platelet aggregation, and platelet and leukocyte activation to some extent, but had no effect on complement activation and did not significantly prolong xenograft survival, even though better preservation of morphology was shown.     

Eighteen years experience in pediatric acute dialysis: analysis of predictors of outcome

This study reviewed the 18-year experience of acute dialysis in the pediatric intensive care unit, in order to identify factors that could predict outcome, and to determine whether newer modalities of acute dialysis have influenced this outcome. Sixty-six children (ages 1 day to 19 years) received acute dialysis from May 1980 to April 1998. Factors predicting outcome were analyzed using univariate and Cox regression analysis. Modality of dialysis in the first 15 years was exclusively peritoneal dialysis, with a mortality of 63.9%. However, in the last 3 years, with increasing patient numbers, continuous hemodiafiltration (CHDF) was the modality of choice (56.7%), with a mortality of 73.3%. Univariate analysis showed that age <1 year, coma, acute tubular necrosis, disseminated intravascular coagulopathy, assisted ventilation, and hypotension were associated significantly with poor outcome (P<0.05). Cox regression analysis revealed that mortality was significantly higher in patients on mechanical ventilation (RR 5.96, 95% CI 1.82–19.50), or with age <1 year (RR 2.00, 95% CI 1.08–3.73). In conclusion, despite the increasing use of CHDF over the last 3 years, there was no significant improvement in mortality, probably related to the fact that more critically ill patients were dialyzed. Received: 21 March 2000 / Revised: 12 October 2000 / Accepted: 19 October 2000     

The function of transgenic human DAF-expressing porcine livers during hemoperfusion with human blood

Abstract  Extracorporal pig liver perfusion could bridge the deadly problem of acute human liver failure. However, preformed natural antibodies and complement activation (CA) are the predominant mechanisms of hyperacute xenoge-neic rejection. The blockade of both pathways of CA in the xenograft, using transgenic livers expressing human decay accelerating factor on the endothelial surface results in prolonged graft survival and lower release of mediators.     

Transplantation of renal precursor cells: a new therapeutic approach

The number of kidney transplantations performed per year is limited due to availability of donor organs. One possible solution to the organ shortage is the use of renal xenografts. However, the transplantation of xenografts is complicated by hyperacute and acute rejection. It has been postulated that the host immune response might be attenuated following the transplantation of renal precursor cells or embryonic kidneys (metanephroi) instead of developed (adult) kidneys. Transplanted metanephroi become chimeric organs in that their blood supply originates, at least in part, from the host. It is possible to transplant a developing metanephros, without the use of immunosuppression, from one rat to another. Transplanted metanephroi grow, develop, become vascularized, and function in host rats. Transplantation of metanephroi may be a promising novel therapeutic approach for the treatment of chronic renal failure. Received: 29 December 1999 / Revised: 13 March 2000 / Accepted: 13 March 2000     

Expression of complement regulatory proteins in the developing human kidney

Complement homologous restriction factor CD59 and complement receptor CD35 are typically involved in the regulation of the host defense system. Recent observations in the human fetal kidney suggest a further role for complement cell surface regulators CD35 and CD59 in kidney development and maturation. We investigated this possible role by localizing CD35 and CD59 protein and mRNA in the developing and adult kidney. Adult tissue and fetal tissue ontogeny were analyzed using immunohistochemistry and in situ hybridization. CD35 protein and mRNA were localized to the podocyte of the glomerulus in the human fetal and adult kidney. Expression was initiated after vascularization of the early developing glomerulus. CD59 protein and mRNA were observed as early as 8 weeks’ gestation and were localized primarily to the ureteric duct epithelium in the fetal kidney and predominantly to the collecting duct in the adult. Interestingly, CD59 expression was translocated from the basolateral surface in the fetal kidney to the apical surface in the adult kidney. The specific spatial and temporal expression of CD35 and CD59 suggests a possible role for these complement regulatory proteins in renal cell differentiation. Received: 29 October 1999 / Revised: 21 March 2000 / Accepted: 22 March 2000     

Influence of ischemic time on hyperacute xenograft rejection of pig hearts in a working heart perfusion model with human blood

Abstract In xenotransplantation long ischemic time of grafts is supposed to have a marked influence on hyperacute rejection (HXR). We investigated the influence of different cold ischemic times on HXR of ex vivo “working pig hearts” perfused with human blood. Xenoreactive natural antibodies (XNAb) as a trigger of HXR were eliminated by Ig‐Therasorb immunoadsorption (IA). Explanted Landrace pig hearts of group Gl and group G3 (with additional IA) underwent 4 h of cold ischemia prior to xenoperfusion. Control groups G2 and G4 (with IA) were kept ischemic for only 46.6 ± 15.8 and 51.2 ± 4.2 min, respectively. Ischemic time prolonged the perfusion time in our working heart model (G1: 356 ± 46.1 min; G2: 125 ± 31 min; P < 0.05). IA had no additional impact on perfusion time but was effective by itself. The heart weight increased fourfold more in G2 as compared to the other groups. IA without ischemia significantly improved cardiac output in G4 (G3: 198.8 ± 15.4 mL/min; G4: 338.5 ± 16.0 mL/min). Coronary flow in G2 was significantly lower than in Gl (Gl: 157.9 ± 9.15 mL/min; G2: 59.4 ± 20.1 mL/min). Histological signs of HXR (light and electron microscopy) could be found in G2 in contrast to the other groups. Parameters of serological damage showed a minimum in G4 and the maximum in G2. In G1 1XNAb were nearly equally eliminated immediately after the start of xenoperfusion as in IA groups G4 and G3. Four hours of ischemic time showed beneficial effects in preventing HXR, possibly caused by changes of the endothelial cell surface (for example, glycosylation or loss of α1‐3Gal epitopes with a hapten effect).     

Reversible encephalopathy associated with tacrolimus in pediatric renal transplants

Neurological complications post transplant have been described with the use of calcineurin inhibitors. Although tacrolimus may be a better immunosuppressant than cyclosporine, its neurological side effects may be worse. Two children, living-related kidney transplant recipients, were treated with antibody induction, mycophenolate mofetil, prednisone, and tacrolimus. Soon after transplant, they each developed an encephalopathy, which when visualized by magnetic resonance imaging showed that it affected both white and grey matter of the brain. Although the encephalopathy was associated with the use of tacrolimus, there was a complete neurological recovery without cessation of the drug. Received: 7 September 2000 / Revised: 2 January 2001 / Accepted: 30 January 2001     

Chronic anemia as a complication of parvovirus B19 infection in a pediatric kidney transplant patient

. This is a report of unexplained anemia that persisted for 4 months in an adolescent renal transplant patient receiving immunosuppression that included prednisone, tacrolimus, and mycophenolate mofetil. This patient required monthly blood transfusions for fatigue, palpitations, and hematocrit levels between 15% and 17%. In addition, his posttransplant course was notable for the development of insulin-dependent diabetes mellitus. While receiving low-dose prednisone, he was switched from tacrolimus to cyclosporin and tapered off insulin injections over the next 2 months. At 4.5 months post-transplantation, further diagnostic evaluation was suggestive of parvovirus B19 infection as the cause for our patient’s chronic anemia. After testing negative for serum-specific parvovirus B19 IgM and IgG antibodies, parvovirus B19 infection was detected in blood by the polymerase chain reaction. Treatment with intravenous immunoglobulin (1 g/kg per day × 2 days) resulted in normalization of both his reticulocyte count and hematocrit within 6 weeks. At 4 months after receiving the immunoglobulin infusion, he has maintained a normal hematocrit level and stable renal function without requiring further blood transfusions. Received August 23, 1996; received in revised form and accepted November 20, 1996     

Pretransplant blood transfusions with cyclosporine in pediatric renal transplantation

Pretransplant transfusions were repeatedly shown to be associated with improved graft survival in the ”pre-cyclosporine era,” and have recently been shown to be beneficial in patients on modern immunosuppressive regimes. In an attempt to improve this transfusion effect and minimize the potential development of cytotoxic antibodies, we have given these transfusions, with concomitant cyclosporine cover, prior to transplantation. Ninety-two renal transplantations were performed in 91 children in the study group (group 1) and all received pretransplant transfusions with cyclosporine cover. Results were compared with a preceding group of 102 children (104 transplantations) who had received pretransplant transfusions without cyclosporine cover (group 2). There were 70 cadaver and 22 living-related donor (LRD) transplants in group 1, and 88 cadaver and 16 LRD transplants in group 2. Graft survival rates (1- and 5-year) for cadaver transplantation were 96% and 90% in group 1 compared with 78% and 64% in group 2 (P=0.001). For LRD transplantation, these figures were 95% and 87% in group 1 and 81% and 69% in group 2. There was no difference between the two groups in terms of age at transplantation, sex, donor age, HLA-A, -B, -DR mismatches, or cold and warm ischemia times. All cadaver graft recipients received quadruple, sequential immunosuppression post transplant. However, 9 patients in group 1 were changed to tacrolimus for recurrent rejection episodes. No patient developed persistent lymphocytotoxic antibodies post transfusion or side effects of cyclosporine. Cyclosporine can be safely given with whole blood prior to transplantation with no adverse effect and no sensitization. Graft survival was significantly improved in this group of patients and graft loss due to rejection was exceptional. This effect should be further evaluated in prospective studies. Received: 10 June 1999 / Revised: 9 March 2000 / Accepted: 10 March 2000