The effect of soluble complement receptor type 1 on hyperacute allograft rejection.

A major obstacle to successful organ transplantation in sensitized recipients is antibody-mediated hyperacute rejection. We hypothesized that human recombinant soluble complement receptor type 1 (sCR1), which inhibits activation of the complement cascade at multiple stages, would delay this process. Using a well-established model of hyperacute rejection, 21 Lewis rats each received three successive ACI rat skin grafts which resulted in high serum titers of ACI-specific antibodies. These hypersensitized Lewis rats then received heterotopic ACI cardiac allografts. Immediately prior to allograft reperfusion, sCR1 at 3 mg/kg (n = 11) or an equivalent volume of phosphate-buffered saline (PBS) (n = 10) was administered intravenously. Five minutes following allograft reperfusion, hemolytic complement activity was reduced by 63 +/- 2% (SEM) in the sCR1 group vs 25 +/- 3% in the PBS group (P less than 0.0001, Wilcoxon rank sum test (WRST)). Graft survival in the sCR1 group was prolonged to 32.0 +/- 4.47 hr vs 3.25 +/- 0.81 hr in the PBS group (P less than 0.0001, WRST). Serial histologic examination of allografts showed that sCR1 therapy prevented the early development of luminal platelet thrombi in the allograft coronary vessels. This study demonstrates that a single 3 mg/kg dose of sCR1 significantly prolongs ACI cardiac allograft survival in the hypersensitized Lewis rat recipient. Complement inactivation, mediated by sCR1, may prove useful for transplantation in sensitized recipients.     

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts

BACKGROUND: In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). METHODS: Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. Results: Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. CONCLUSIONS: The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.     

The pathogenesis of hyperacute xenograft rejection.

A critical shortage of donor organs has driven many in the transplantation community to consider the use of animals as organ donors for humans, that is, xenotransplantation. While successful xenotransplantation of primate kidneys was achieved 25 years ago, most now advocate use of nonprimates as donors because of the risk of disease transmission and ethical concerns attendant to the use of primates. The major hurdle to xenotransplantation of organs between phylogenetically disparate species is the hyperacute rejection reaction that invariably destroys organ xenografts placed in unmodified recipients. Here we review recent insights concerning the pathogenesis of hyperacute rejection reactions. We focus particular attention on the endothelial cell, which serves not only as the target of xenoreactive antibodies and complement, but also, as a consequence of endothelial cell activation, as an instrument of tissue injury. We discuss a phenomenon called "accommodation" in which an organ graft acquires resistance to humoral-mediated injury.     

Perfusion of rabbit hearts with pig blood results in complement mediated hyperacute xenograft rejection

Investigation of hyperacute rejection of discordant xenografts has been hampered by the lack of a model for the study of rapid time course events. In vivo models are unsuitable for observation of early rejection processes and, along with most ex-vivo perfusion preparations, are insensitive since no functional demand is placed on the organ which may have undergone extensive damage whilst still appearing viable. For this reason a blood perfused isolated working heart preparation has been developed [1]. With left atrial and left ventricular loading the heart performs measurable work as it ejects into a mock circulation with both afterload and compliance components. When cardiac function is compromised the heart is no longer able to eject against the fixed afterload and both cardiac output and coronary circulation cease with resultant organ failure. The model is thus highly sensitive to minimal organ damage and has an easily identifiable endpoint. In the present study, we used this preparation to study the discordant species combination of rabbit hearts perfused with pig blood.     

Mechanisms of hyperacute rejection of discordant liver xenograft

Abstract: Use of discordant liver xenografts is a possible means of overcoming the ever-increasing organ shortage. Immunological barriers, however, remain the major obstacle to the application of this therapeutic option. The aim of the study was to establish the model and to further investigate mechanisms of hitherto assumed liver xenograft rejection in the discordant guinea pig to rat combination and to determine the role of complement activation in this model.
Male guinea pigs served as donors, and all recipient Lewis rats underwent splenectomy prior to orthotopic liver transplantation with a modified cuff technique. Animals were grouped according to their pharmacologic therapy: group 1: control group, no further therapy; group 2: plasmapheresis immediately prior to transplantation; group 3: Cobra venom factor; group 4: plasmapheresis and Cobra venom factor; group 5: Cyclosporin A; and group 6: RS 61443 with three animals in each group. Median survival was 62 (52–68) minutes in group 1, 150 (129–190) in group 2, 745 (260–1034) in group 3, 45 (18–60) in group 4, 91 (76–118) in group 5, and 212 (170–245) in group 6. Histologically, portal and sinusoidal congestion, interstitial haemorrhages, intragraft granulocyte deposits, and parenchymal damage were found to be most pronounced in groups 1 and 3. In all groups endothelial cells stained positive for IgM and complement component C3 compatible with classical pathway activation. From our findings where deposition of Factor H was noted it is concluded that in this discordant model the complement system is activated by the alternative pathway. Intragraft IgM deposits, however, suggest that the classical activation pathway may also be involved.     

异种肾移植超急性排斥反应时血液流变学的变化

采用猪-犬肾移植建立超急性排斥反应模型，以研究此时的血液流变学变化。分别在移植前、移植肾超急性排斥发生时和排斥肾切除后10～15分钟检测外周血的血液流变学参数变化。结果异种肾移植发生超急性排斥时，血小板聚集显著增高，移植肾切除后血中的血小板数和纤维蛋白原明显下降，血小板聚集也恢复原有水平。提示血小板、纤维蛋白原参与了异种移植超急性排斥反应的发生过程，而血小板聚集可能是诊断异种移植超急性排斥反应一个有价值的指标。     

Immunopathology of hyperacute xenograft rejection in a swine-to-primate model

Hyperacute rejection is the inevitable consequence of the transplantation of vascularized organs between phylogenetically distant species. The nature of the incompatibility and the pathogenetic mechanisms that lead to hyperacute xenograft rejection are incompletely understood. We investigated these issues by the immunopathological analysis of tissues from swine renal and cardiac xenografts placed in rhesus monkeys. Hyperacute rejection was associated with deposition of recipient IgM and classic but not alternative complement pathway components along endothelial surfaces, the formation of platelet and fibrin thrombi, and the infiltration of neutrophils. In animals from which natural antibody was temporarily depleted by organ perfusion, rejection was observed at 3 days to 5 days posttransplant. The immunopathology of rejection in these tissues revealed focal vascular changes similar to those observed in hyperacute rejection. A xenograft functioning for a prolonged period in a recipient temporarily depleted of circulating natural antibody contained recipient IgM along endothelial surfaces but no evidence for significant deposition of complement, formation of platelet and fibrin thrombi, or infiltration of neutrophils. These results suggest that rhesus IgM contributes significantly to the development of hyperacute rejection in the swine to Rhesus model and that the fixation of complement is a critical factor in the recruitment of the coagulation cascade and platelet aggregation--and possibly in the adherence and infiltration of PMN.     

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.     

Delayed hyperacute xenograft rejection in porcine to canine fetal liver transplantation.

Fetal tissues are generally considered to express weaker antigenic cell-surface molecules than adult tissues. We have reported that transplantation of porcine fetal liver tissue (fragments) is useful for acute and chronic hepatic failure in rats. We further investigated, in the present study, whether transplantation of a porcine fetal liver has the advantage of delayed hyperacute xenograft rejection (HAR) in comparison with that of an adult liver. Porcine fetal liver heterotopically transplanted into dogs was compared. Haematoxylin-eosin (HE) and immunohistochemical studies using IgM, C3, IgG antibodies were performed in serial biopsies of the liver grafts. Lectin binding to target antigen epitopes on pig and dog tissues was studied by flow cytometry. Carbohydrate expression on the liver was also studied by immunohistochemistry. The macroscopic and HE section findings indicate that HAR started 15 min postgraft in fetal and adult liver grafts. Thereafter, vascular changes and parenchymal damage progressed more rapidly in the adult grafts. The final HAR time in adult liver transplantation was determined to be 60 min, while it was determined to be 180 min in fetal liver transplantation. IgM, C3 and IgG were deposited more strongly in the adult grafts than in the fetal grafts up until 60 min after xenografting. Phaseolus vulgaris erythroagglutinin lectin competitively blocked dog sera binding to porcine PBLs. The fetal liver expressed oligosaccharide at a significantly lower level than the adult liver. We conclude that porcine fetal liver xenografts had a significantly delayed HAR.