The effect of soluble complement receptor type 1 on hyperacute xenograft rejection

In the guinea pig-to-rat model of hyperacute xenograft (Xg) rejection, the effect of complement inhibition using systemically administered soluble complement receptor type 1 (sCR1) on discordant cardiac Xg survival was investigated. In PBS-treated control Xg recipients (n = 13), hyperacute rejection was rapid, with a mean Xg survival of 17 +/- 4 min. Therapy with sCR1 prolonged survival of cardiac Xgs in a dose-dependent manner. A 3 mg/kg bolus of sCR1 (n = 4) prolonged Xg survival to 64 +/- 29 min (not significant). Increasing the sCR1 dose to 5.9 mg/kg (n = 4) significantly delayed Xg rejection to 71 +/- 17 min (P-0.026, log-rank test vs. control). In 10 recipients treated with 15 mg/kg sCR1, mean Xg survival was further prolonged to 189 +/- 36 min (P-0.0004) with no adverse effects. While 2 of 8 recipients receiving 60 mg/kg sCR1 died with functioning Xgs at 30 and 300 min due to anastomotic bleeding, Xg survival averaged over 12 hr (747 +/- 100 min, P-0.0004) in the remaining 6 recipients. sCR1 administration significantly inhibited serum complement activity in a parallel dose-dependent fashion, with the 60 mg/kg dose reducing complement activity by 95 +/- 1 and 96 +/- 1% five and 30 min following Xg reperfusion, respectively. Immunofluorescence microscopy revealed rat IgM bound to all cardiac Xgs in control as well as sCR1-treated recipients. In addition, serial histologic examination of cardiac Xgs harvested within 21 min of graft reperfusion revealed occlusive platelet aggregates within the coronary vessels as well as interstitial hemorrhage and myocardial necrosis in Xgs from control recipients, all of which were only minimally present in Xgs from recipients treated with sCR1. These studies show that complement inhibition with sCR1 significantly delays hyperacute cardiac Xg rejection in this discordant model and may be an important component in a therapeutic protocol for xenotransplantation.     

A soluble chimeric inhibitor of C3 and C5 convertases, complement activation blocker-2, prolongs graft survival in pig-to-rhesus monkey heart transplantation

Complement plays a critical role in many pathologic processes and in xenograft rejection. Therefore, effective complement inhibitors are of great interest. In pig-to-primate organ transplantation, hyperacute rejection results from antibody deposition and complement activation. Complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF) and membrane cofactor protein, inhibits C3 and C5 convertases of both classical and alternative pathways. CAB-2 reduces complement-mediated tissue injury of a pig heart perfused ex vivo with human blood. Therefore, we studied the efficacy of CAB-2 when a pig heart is transplanted heterotopically into rhesus monkeys receiving no immunosuppression. Graft survival in three control monkeys was 1.26 ± 0.2 h; it was markedly prolonged in eight monkeys that received CAB-2. Of the six monkeys that received a single dose of CAB-2 (15 mg/kg i.v.), four had graft survivals of 21, 95, 96, and 108 h, and two died at 7 to11 h post-transplant with a beating graft, as a result of technical complications. The two monkeys given multiple doses of CAB-2 had graft survivals of 95 and 96 h. CAB-2 markedly inhibited complement activation, as shown by a strong reduction in generation of C3a and SC5b-9. At graft rejection, tissue deposition of iC3b, C4 and C9 was similar or slightly reduced from controls, and deposition of IgG, IgM, C1q and fibrin did not change. Thus, complement inhibition with CAB-2 abrogates hyperacute rejection of pig hearts transplanted into rhesus monkeys, but does not prevent delayed/acute vascular rejection. These studies demonstrate that the beneficial effects of complement inhibition on survival of a pig heart xenograft in rhesus monkeys are similar to those in other primate species and that CAB-2 may be useful in xenotransplantation and other complement-mediated conditions.     

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。     

Dose-dependent inhibition of complement in baboons by vaccinia virus complement control protein: implications in xenotransplantation

Vaccinia virus complement control protein (VCP) is a potent inhibitor of both the alternative and the classical complement pathways through its binding to activated third and fourth components. In addition to its complement inhibiting abilities, VCP can bind heparan sulfate on cell surfaces, resulting in further functional activities. Altogether, the multiple functions of VCP have been shown to reduce the inflammatory response of the host, helping the vaccinia virus to evade immune destruction. Recently, we reported that VCP is able to block hyperacute xenograft rejection, significantly prolonging graft survival in two separate in vivo heterotopic cervical cardiac xenograft models. Histopathological examination of the transplanted hearts receiving VCP revealed marked VCP deposition on the endothelium, a significant reduction in cardiac tissue damage, and significantly less C3, IgG and IgM deposition in the tissue. It is concluded that VCP may inhibit hyperacute xenorejection by binding to the endothelial surface, blocking complement fixation activation, thereby preventing xenoantibody attachment. In the current study, the level of serum complement inhibition was evaluated following different bolus dosages of VCP in baboons. The results indicated that to achieve a satisfactory level of complement inhibition higher doses of VCP are needed in baboons, than previously observed in rats. The current observations are critical for future assessment of the role of VCP to suppress hyperacute rejection following pig-to-baboon xenotransplantation.     

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.     

Evidence that deoxyspergualin prevents sensitization and first-set cardiac xenograft rejection in rats by suppression of antibody formation

This study provides evidence of antibodies playing an important role in hamster-to-rat cardiac xenograft rejection and discusses the use of 15-deoxyspergualin (DSG) to suppress this first-set rejection, as well as hyperacute rejection induced by sensitization. The effect of recipient splenectomy (Spx) as an adjuvant to DSG to control first set xenograft rejection was also examined. When hyperimmune serum was taken from control recipients at rejection time and injected i.v. into new recipients of cardiac xenografts, hyperacute graft rejection resulted. Survival depended on the amount of serum injected and ranged from 14.7 +/- 2.5 min with 3 ml of serum to 233.3 +/- 61.1 min with 0.5 ml. Experiments on first-set xenograft rejection revealed that a dose of 2.5 mg/kg/day DSG could prolong xenograft survival from 3.4 +/- 0.5 days in untreated controls to 9.5 +/- 2.6 days (P less than 0.01). A dose of 5 mg/kg/day DSG, though it increased graft survival to 16.4 +/- 5.9 days, proved to be toxic to the recipients. Spx alone prolonged xenograft survival to 5.2 +/- 0.4 days, and, when combined with 2.5 mg/kg/day DSG administration, prolonged graft survival to 22.1 +/- 5.5 days (P less than 0.01 vs. DSG alone). The appearance of cytotoxic antibodies was delayed, and titers decreased from 1:256 in untreated controls to 1:16-1:64 both in the group that underwent Spx only and in the group that received 2.5 mg/kg/day DSG. Combined treatment suppressed antibody response for more than two weeks. Experiments on hyperacute rejection induced by sensitization revealed that 1 ml of hamster whole blood transfused into prospective heart recipients 1 week before grafting resulted in graft loss in 18.2 +/- 6.1 min. Pretransplant transfusion and concomitant daily administration of 5 mg/kg/day DSG until one day after grafting not only prevented hyperacute rejection but prolonged graft survival to 7.0 +/- 0.7 days. This survival was significantly longer than with DSG alone (4.2 +/- 0.8 days, P less than 0.01). We concluded that the marked suppression of antibody formation by DSG plays a major role in preventing first-set xenograft rejection and hyperacute rejection induced by sensitization.     

Mouse-to-rabbit xenotransplantation: a new small animal model of hyperacute rejection mediated by the classical complement pathway

BACKGROUND: Hyperacute rejection of porcine organs transplanted into primate recipients is initiated by the binding of preformed xenoreactive natural antibodies to the vascular endothelium of the graft and activation of the classical complement pathway. Several small animal models are currently employed to study various aspects of xenograft rejection; however, none has been shown to manifest hyperacute rejection mediated by the classical pathway of complement activation. METHODS: We performed heterotopic mouse heart transplants into weanling rabbits, adult rabbits, and C6-deficient rabbits. The recipients received no immunosuppression. Rejected grafts were subjected to histologic analysis and immunofluorescence staining for rabbit IgG, IgM, and C3. Levels of preexisting cytotoxic antibodies as well as classical and alternative complement pathway activities were determined in rabbit serum using mouse red cells as targets. RESULTS: Mean graft survival was 37+/-9.6 min for mouse-to-weanling rabbit transplants (n=10), and 40+/-11.1 min for mouse-to-adult rabbit transplants (n=5). Rejected grafts showed diffuse interstitial hemorrhage, endothelial cell damage, myocyte necrosis, moderate diffuse deposition of rabbit IgG, and dense deposition of rabbit IgM and C3 on the vascular endothelium of the graft, consistent with hyperacute rejection. One mouse-to-C6-deficient rabbit transplant was rejected at 21 hr with severe interstitial hemorrhage, cellular necrosis and a moderate cellular infiltrate consisting primarily of neutrophils and some mononuclear cells. A second transplant in a C6-deficient rabbit was functioning when the recipient died at 6.5 hr as a result of complications of surgery; the graft had normal myocytes and vasculature with minimal spotty interstitial hemorrhage. Both weanling and adult rabbit serum were found to have high titers of cytotoxic IgM anti-mouse antibodies and strong classical complement pathway activity with minimal alternative pathway activity towards mouse red cells. CONCLUSIONS: The mouse-to-rabbit species combination manifests hyperacute xenograft rejection. In vitro studies suggest that this process is mediated by IgM anti-mouse natural antibodies and activation of the classical pathway of complement.     

中华眼镜蛇蛇毒因子在豚鼠—大鼠异种心脏移植中的作用

目的 应用中眼镜蛇蛇毒因子（CVF）消耗补体,观察豚鼠心脏在移植入Wistar大鼠腹腔内后对超急性排斥反应的变化。方法 按0．2μg/g体重CVF大鼠腹腔内分两次间隔6小时注射,18小时后进行心脏移植。脾切除及腹腔内注射环磷酰胺（Cy）均在移植前一天进行。设计分为四组：A组为对照组,不用任何药物;B组仅用CVF;C组应用CVF＋Cy＋脾切除;D组Cy＋脾切除。Cy的用量为60mg/kg体重腹腔内注射。检测各组受体的供心存活时间,并在供心停跳后取出行光镜、电镜检查。结果 A、B、C、D各组供心存活时间分别为15－3120分钟。供心存活时间的统计学分析：A组与B、C两组比较P值＜0．01,A组与D组比较,B组与C组比较P值＞0．05,B组与D组比较,C组与D组比较P值＜0．01。光镜、电镜结果提示：B、C组与A、D组有明显不同。结论 CVF能明显抑制补体活性,减轻或延缓超急性排斥反应的发生,使供体器官存活时间延长。CVF具有异种器官移植的基础研究和临床开发意义。     

Antibody-mediated activation of the classical complement pathway in xenograft rejection

Transplant rejection is a multifactorial process involving complex interactions between components of the innate and the acquired immune system. In view of the shortage of donor organs available for transplantation, xenotransplantation of pig organs into man has been considered as a potential solution. However, in comparison to allografts, xenografts are subject to extremely potent rejection processes that are currently incompletely defined. Consequently, an appropriate and safe treatment protocol ensuring long-term graft survival is not yet available. The first barrier that has to be taken for a xenograft is hyperacute rejection, a rapid process induced by the binding of pre-formed antibodies from the host to the graft endothelium, followed by activation of the classical complement pathway. The present review concentrates on the role of antibodies and complement in xenograft rejection as well as on the approaches for treatment that target these components. The first part focuses on porcine xenoantigens that are recognized by human xenoreactive antibodies and the different treatment strategies that aim on interference in antibody binding. The second part of the review deals with complement activation by xenoreactive antibodies, and summarizes the role of complement in the induction of endothelial cell damage and cell activation. Finally, various options that are currently under development for complement inhibition are discussed, with special reference to the specific inhibition of the classical complement pathway by soluble complement inhibitors.     

Hyperacute lung rejection in the pig-to-human model. 2. Synergy between soluble and membrane complement inhibition

Azimzadeh A, Zorn GL III, Blair KSA, Zhang JP, Pfeiffer S, Harrison RA, Cozzi E, White DJG, Pierson RN III. Hyperacute lung rejection in the pig‐to‐human model. 2. Synergy between soluble and membrane complement inhibition. Xenotransplantation 2003; 10: 120–131. © Blackwell Munksgaard, 2003 Background. The role of complement in hyperacute lung xenograft rejection has not been elucidated. The present study evaluates the effect of complement (C) C3/C5 convertase inhibition on hyperacute rejection of pig lung by human blood. Methods. In an established ex‐vivo model, lungs from pigs heterozygous for human decay accelerating factor (hDAF), non‐transgenic littermate control pigs, or farm‐bred pigs were perfused with fresh human blood that was either unmodified or treated with soluble complement receptor type 1 (sCR1: TP10, 100 μg/ml). Results. Non‐transgenic lungs from littermate controls had a median survival time of 35 min (range 5 to 210; P=0.25 vs. farm‐bred piglets: median 5 min, range 5 to 10). Lungs expressing hDAF survived for a median of 90 min (range 10 to 161; P=0.5 and 0.01 vs. littermate and farm‐bred controls, respectively), with sCR1, whereas hDAF (–) lungs failed by 35 min (range 6 to 307), hDAF (+) lungs survived for 330 min (range 39 to 577) [P=0.002 vs. farm‐bred; P=0.08 vs. hDAF (–); P=0.17 vs. sCR1/hDAF (–)]. The rise in pulmonary vascular resistance (PVR) at 5 min was blunted only by hDAF (+) with sCR1 (0.26 ± 0.2 vs. 0.5 to 0.7 mmHg/ml/min for other groups). Plasma C3a and sC5b‐9 and tissue deposition of C5b‐9 were dramatically diminished using sCR1, and further decreased in association with hDAF. Histamine and thromboxane were produced rapidly in all groups. Conclusion. Complement plays an important role in lung HAR. However, even potent inhibition of C3/C5 convertase, both membrane bound in lung and by a soluble‐phase inhibitor in the blood, does not prevent activation of inflammatory responses known to be particularly injurious to the lung. Our findings implicate a role for innate immune pathways resistant to efficient complement regulation. The role of anti‐species antibody, coagulation pathway dysregulation, and additional environmental or genetic influences remain to be defined.     

Targeting complement activation in brain-dead donors improves renal function after transplantation

Kidneys recovered from brain-dead donors have inferior outcomes after transplantation compared to kidneys from living donors. Since complement activation plays an important role in renal transplant related injury, targeting complement activation in brain-dead donors might improve renal function after transplantation. Brain death (BD) was induced in Fisher rats by inflation of an epidurally placed balloon catheter and ventilated for 6h. BD animals were treated with soluble complement receptor 1 (sCR1) 1h before or 1h after BD. Kidney transplantation was performed and 7 days after transplantation animals were sacrificed. Plasma creatinine and urea were measured at days 0, 1, 3, 5 and 7 after transplantation. Renal function was significantly better at day 1 after transplantation in recipients receiving a sCR1 pre-treated donor kidney compared to recipients of a non-treated donor graft. Also treatment with sCR1, 1h after the diagnosis of BD, resulted in a better renal function after transplantation. Gene expression of IL-6, IL-1beta and TGF-beta were significantly lower in renal allografts recovered from treated donors. This study shows that targeting complement activation, during BD in the donor, leads to an improved renal function after transplantation in the recipient.     

Inhibition of the membrane attack complex of complement for induction of accommodation in the hamster-to-rat heart transplant model

BACKGROUND: To induce accommodation in the hamster-to-rat cardiac transplantation model, in addition to cyclosporin A (CSA) to inhibit T-cell-mediated graft rejection, cobra venom factor (CVF) is often used to prevent complement-mediated graft rejection. Although it is generally assumed that CVF makes accommodation possible because it inactivates the complement membrane attack complex (MAC), it is not known which complement components must be inactivated and whether complement activation products generated by CVF are also involved in the induction of accommodation. Therefore, to investigate mechanisms by which CVF contributes to accommodation, we studied induction of accommodation of hamster hearts grafted into rats with complement deficiencies of C6; these rats cannot assemble the MAC but, in contrast to CVF, retain in their native state all complement proteins that precede the MAC. METHODS: Golden Syrian hamster hearts were transplanted heterotopically into the abdomen of normocomplementemic and C6-deficient (C6D) PVG rats. Graft rejection was determined by cessation of palpable cardiac contractions. CSA, 10 mg/kg, was administered daily to all rats. Graft survival was compared in rats given CVF (60 U/kg 1-day pre-transplant and 20 U/kg/day for the next 9 days), C6D rats given no CVF, normocomplementemic rats given anti-C6 IgG or non-immune IgG but no CVF, and C6D rats reconstituted with normocomplementemic rat serum. Total complement and C6 serum levels were measured using hemolytic assays in rat peripheral blood. RESULTS: We found that hamster hearts transplanted into C6D rats receiving CSA but no CVF survived long-term, with histology typical of an accommodated heart. The accommodated hamster heart did not reconstitute C6 levels of the C6D recipient rats. Moreover, in normocomplementemic rats given anti-C6 antibodies (abs) to induce partial C6 deficiency, accommodation also developed without administration of CVF. Accommodation of the hamster heart failed to develop in C6D rats whose complement was reconstituted by administration of normocomplementemic rat serum given before and following transplantation. CONCLUSIONS: These studies demonstrate that, in this model, inhibition of MAC-mediated graft rejection is sufficient to allow the development of accommodation. Inactivation of C3 or other complement proteins of the alternate pathway, or the presence of complement-derived biologically active fragments, is not needed for development of accommodation.     

Pre-treatment of donor with 1-deamino-8-d-arginine vasopressin could alleviate early failure of porcine xenograft in a cobra venom factor treated canine recipient.

OBJECTIVE: Unlike cardiac or renal xenotransplants, the depletion of complement using cobra venom factor (CVF) does not improve pulmonary xenograft survival. Several cases suggest that the swine von Willebrand factor (vWF) may play a major role in presenting a different pathogenesis of pulmonary xenograft dysfunction from other organs. To evaluate the role of vWF and the complement system in mediating hyperacute vascular injury of pulmonary xenografts and elucidate pathogenesis of the injury, we performed swine-to-canine orthotropic single lung xenotransplantation after pre-treatment of 1-deamino-8-d-arginine vasopressin (DDAVP) and CVF. METHODS: We set up three groups for lung xenotransplantation: group I served as the control group; group II, recipients pre-treated with CVF; group III, donors pre-treated with DDAVP (9 mg/kg, 3 days)/recipients pre-treated with CVF (60 u/kg). Hemodynamic data, coagulation and complement system parameters, and grafted lung pathologies were examined serially for 3h after transplantation. RESULTS: DDAVP infusion reduced the vWF content in swine lung tissue in vivo (7.7+/-2.4 AU/mg vs 16.0+/-5.6 AU/mg, P < 0.0001). Infusion of CVF 24 h prior to transplantation effectively depleted the recipient's serum C3 and complement hemolytic activity below the detectable range. Regardless of the use of CVF, both groups I and II transplanted with unmodified grafts showed an immediate drop in leukocytes and platelet counts after transplantation. However, in group III, in recipients transplanted with DDAVP pre-treated swine lung, the platelet count did not decrease after transplantation (P = 0.0295). The decrease of plasma antithrombin and fibrinogen tended to be attenuated in group III. Light microscopic examination revealed extensive vascular thromboses in both capillary and larger vessels, as well as early pulmonary parenchymal damage in groups I and II, but were rarely observed in group III. CONCLUSIONS: Complement inhibition alone was not enough to alleviate intravascular thrombosis, the main pathology in pulmonary xenotransplantation. Pre-infusion of DDAVP to the donor animal was effective in preventing platelet sequestration and attenuated intravascular thrombosis. It is suggested that the strategies targeting vWF would be promising for successful pulmonary xenotransplantation.     

Clinical impact of complement deposition findings on biopsies in acute rejection episodes of pediatric renal transplant patients

IntroductionRejection is the most important problem for renal graft function and survival. Complement system plays a key role in immune responses from host to graft. It was demonstrated that complement system activation is related with renal fibrosis. We evaluate clinical impact of complement deposition findings on biopsies in acute rejection episodes of pediatric renal transplant patients.MethodDemographics of the patients, graft functions, acute rejection episodes and graft loss were recorded from data files of 165 pediatric renal transplant patients. Findings of 98 renal biopsies were retrospectively evaluated.ResultsThirty three patients with kidney transplant had 44 acute rejection episodes (32 pure cellular acute rejection episodes / 1 pure humoral acute rejection episode / 11 combined acute cellular and acute humoral rejection episodes) proven by biopsy. C1q staining was positive in 7 biopsies, C3 staining in 15 biopsies and, C4d staining in 15 biopsies. 26 patients had graft fibrosis. All patients with a rejection history had a significant decrease in GFR value during follow-up. Patients who did not have fibrotic changes in first biopsy had same level of deterioration of GFR when compared with patients who had fibrotic changes in first biopsy.ConclusionWe could not demonstrate a significant relation between complement deposition and renal fibrosis, and between complement deposition and GFR values. Our data demonstrated that graft outcomes and graft loss after acute rejection episodes cannot be predicted only with complement deposition on graft or only with graft fibrosis.     

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.     

sCR1sLe(X) reduces lung allograft ischemia-reperfusion injury but does not ameliorate acute rejection.

BACKGROUND: Combined inhibition of complement and leukocyte adhesion by sCR1sLe(X) reduces lung allograft dysfunction up to 24 h. In the present study its effect on graft function and acute rejection was evaluated up to 5 days after experimental transplantation. METHODS: Orthotopic single left lung transplantation was performed in 35 male rats (Brown Norway to Fischer 344) after a total ischemic time of 20 h. Two groups were assessed after 1, 3, and 5 days post-transplant, respectively (n=5 per group and time point): controls vs. recipients which received 10 mg/kg sCR1sLe(X) 15 min prior to reperfusion. In addition, five animals received 10 mg/kg per day sCR1sLe(X) for 5 days. For blood gas analysis of the graft, the contralateral lung was occluded for 5 min to assess graft function. Lung grafts were flushed, and histological grading was performed in blinded fashion according to the International Society for Heart and Lung Transplantation criteria. RESULTS: Graft PaO(2) in recipients treated with sCR1sLe(X) was superior on day 1 (383+/-118 vs. 56+/-15 mmHg; P<0.0001) and day 3 (446+/-48 vs. 231+/-108 mmHg; P<0.0001). Five days after transplantation, no difference in PaO(2) was found (61+/-28 vs. 83+/-31 mmHg; P=0.59). Repeated treatment with sCR1sLe(X) for 5 days did not improve PaO(2) (64+/-5 mmHg; P=0.65 vs. control; P=0.93 vs. sCR1sLe(X)). At any time point, there was no difference in the degree of rejection between groups. CONCLUSIONS: In this model sCR1sLe(X) provided marked improvement of graft function up to 3 days, but inhibition of both complement system and selectin dependent leukocyte adhesion failed to protect against acute rejection.     

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.     

C1 inhibitor for prophylaxis of xenograft rejection after pig to cynomolgus monkey kidney transplantation

BACKGROUND: Early rejection of discordant porcine xenografts in primate recipients is initiated by the intragraft binding of either preformed (hyperacute xenograft rejection) or induced (acute vascular rejection) antiporcine recipient antibodies with subsequent complement activation via the classical pathway. We have investigated the efficacy of the supplemental administration of C1-inhibitor (C1-INH), a specific inhibitor of the classical complement activation pathway, for prophylaxis of xenograft rejection in a pig to primate kidney xenotransplantation setting. METHODS: Based on the results of pharmacokinetic studies performed in two nontransplanted monkeys, supplemental C1-INH therapy was administered daily to three Cynomolgus monkeys receiving a life-supporting porcine kidney transplant together with cyclophosphamide-induction/cyclosporine A/mycophenolat-mofetil/steroid immunosuppressive therapy. RESULTS: In the three monkeys receiving porcine kidney xenografts and continuous C1-INH treatment none of the grafts underwent hyperacute rejection; all xenografts showed initial function. Recipient survival was 13, 15, and 5 days. No graft was lost due to acute vascular rejection. All animals died with a functioning graft (latest creatinine 96, 112, and 96 micromol/liter) due to bacterial septicemia. CONCLUSION: We conclude that, in our model, supplemental C1-INH therapy together with a standard immunosuppressive regimen can be helpful for prevention of xenograft rejection in a pig to primate kidney xenotransplantation setting. The optimal dose and duration of C1-INH treatment, however, has yet to be determined.     

延长非协调性异种心脏移植存活时间的研究

目的 寻找延长非协调性异种心脏移植存活时间的方法。方法 实验分Ａ、Ｂ、Ｃ、Ｄ４组,将异种心脏（豚鼠－大鼠）移植于颈部。Ａ组：移植前受体用眼镜蛇蛇毒因子（ＣＶＦ）１５０ｕｇ．ｋｇ－１．ｄ＾－１,腹膜腔内注射（ｉ．ｐ）,每天分２次,相隔３ｄ,共４次;Ｂ组：在Ａ组的基础上,移植前１ｈ加用己酮可可碱（ＰＴＸ）５０ｍｇ／ｋｇ（ｉ．ｐ）,然后每隔６ｈ按２５ｍｇ／ｋｇ（ｉ．ｐ）至发生排斥反应为止;Ｃ组：在Ａ组的     

Synthetic peptides which inhibit the interaction between C1q and immunoglobulin and prolong xenograft survival

BACKGROUND: Acute vascular xenograft rejection (AVXR), also termed delayed xenograft rejection (DXR), occurs when hyperacute rejection (HAR) is prevented by strategies directed at xenoreactive natural antibodies and/or complement activation. We have hypothesized that AVXR/DXR is initiated in part by early components of the complement cascade, notably C1q. We have developed synthetic peptides (termed CBP2 and WY) that interfere with the interaction between C1q and antibody. METHODS: CBP2 and the WY-conjugates were used as inhibitors of immunoglobulin aggregate binding to solid phase C1q. Inhibition of complement activation by the peptides of the classical system was determined using lysis assays with sensitized sheep red blood cells or porcine aortic endothelial cells as targets and of the alternate complement pathway using guinea pig red blood cells as targets. Two transplant models were used to study the effects of administering peptides to recipients: rat heart transplant to presensitized mouse, and guinea heart transplant to PVG C6-deficient rats. RESULTS: CBP2 and WY-conjugates inhibited immunoglobulin aggregate binding to C1q. The peptides also inhibited human complement-mediated lysis of sensitized sheep red blood cells and porcine aortic endothelial cells in a dose-dependent manner and the WY-conjugates prevented activation of the alternate complement pathway as shown by inhibition of guinea pig red blood cells lysis with human serum. In addition, the use of the peptides and conjugates resulted in significant prolongation of xenograft survival. CONCLUSIONS: The CBP2 and WY peptides exhibit the functional activity of inhibition of complement activation. These peptides also prolong xenograft survival and thus provide reagents for the study of the importance of C1q and other complement components in transplant rejection mechanisms.