Jan Schmidtko

Acute vascular rejection is associated with systemic complement activation ina pig-to-primate kidney xenograft model

Abstract: The introduction of h‐DAF transgenic porcine organs into pre‐clinical pig‐to‐primate discordant xenotransplantation has led to complete and reliable abrogation of hyperacute xenograft rejection (HAR). Despite additional heavy immunosuppression however, most xenografts are still lost due to acute vascular rejection (AVR), with current treatment protocols being of only limited value. In a life‐supporting model of pig‐to‐primate kidney transplantation, unmodified (n = 8) or h‐DAF‐transgenic (n = 9) porcine kidneys were transplanted into cynomolgus monkeys under cyclophosphamide (CyP), cyclosporine and low‐dose steroid immunosuppression. Longest recipient survival was 11 days in the control group and 68 days in the h‐DAF transgenic group. Stable initial graft function with recipient survival > 4 days was generated in eight animals (two controls and six transgenics). In these animals, plasma complement levels were analyzed during ongoing AVR. Compared with baseline levels, a two‐fold increase in C3a levels and a four‐fold increase in sC5b‐9 levels were measured. In parallel to systemic complement activation, increased deposition of C3 and C5b‐9 along with massive staining for recipient IgM immunoglobulins was detected in the xenografts on immunohistochemistry. We conclude that acute vascular xenograft rejection of porcine kidneys in cynomolgus monkeys is associated with classical pathway complement activation following binding of induced recipient anti‐porcine antibodies. This complement activation can be observed despite membrane bound expression of human complement regulators in the porcine xenografts. Therefore, additional short‐term fluid phase complement inhibition seems necessary for the future development of protocols designed for treatment of AVR in the pig‐to‐primate combination.

Posttransplant lymphoproliferative disorder associated with an Epstein-Barr-related virus in cynomolgus monkeys.

Background. Human posttransplant lymphoproliferative disorder (PTLD) has been shown to be associated with Epstein-Barr virus (EBV) infection. Primate animal models of PTLD and the use of molecular markers in its diagnosis have not been reported. This study was designed to evaluate the frequency, pathology, and molecular characteristics of PTLD in cynomolgus kidney allograft recipients. Methods. Over a 5-year period (January 1995 to November 2000), 160 primate renal transplants were performed at the Massachusetts General Hospital (MGH). Of these, all cases (n=9) that developed PTLD were included. H&E stained paraffin sections of all available tissue samples from the cases were evaluated for the presence of PTLD. Immunoperoxidase staining for T cells (CD3), B cells (CD20), kappa and lambda light chains as well as EBV nuclear antigens (EBNA2) and latent membrane proteins (EBV LMP-1) was done on paraffin sections using standard immunohistochemical (IHC) methods. In situ hybridization for EBV encoded RNA (EBER) was performed in all tissue samples with atypical lymphoid proliferations, using a novel EBER nucleotide probe based on consensus gene sequences from EBV and the related herpes lymphocryptoviruses (LCV) infecting baboons and rhesus macaques. Results. Of 160 consecutive primate renal transplants performed at MGH, 5.6% developed PTLD 28–103 days after transplantation. In all cases, the lymph nodes were involved and effaced by an atypical polymorphous lymphoid proliferation of EBER+ B cells, diagnostic for PTLD. Focal staining for EBNA-2 was noted in tumor cells. In 67% (six of nine) the PTLD infiltrates were present in extra nodal sites, notably liver (56%), lung (44%), heart (44%), renal allograft (44%), and native kidney (22%). The spleen was involved by PTLD in all four animals that had not undergone a pretransplant splenectomy. The PTLD morphology was similar in all cases and predominantly of the polymorphous type, however, some of these showed areas that appeared minimally polymorphous. No cases of monomorphic PTLD were seen. Conclusions. By in situ hybridization, expression of the RNA product, homologous for EBV-encoded RNA (EBER) was identified in the PTLD tumor cells of all cases, indicating latent primate EBV- related infection. This report identifies a novel animal model of EBV associated PTLD in the setting of kidney transplantation, with valuable implications for managing and understanding human PTLD and oncogenesis.

C1-Inhibitor for treatment of acute vascular xenograft rejection in cynomolgus recipients of h-DAF transgenic porcine kidneys

Abstract: At present, the major barrier to successful discordant xenotransplantation of unmodified or complement regulator transgenic porcine xenografts is acute vascular xenograft rejection (AVR). AVR is associated with the intragraft deposition of induced recipient xenoreactive antibodies and subsequent complement activation. In a life‐supporting pig to primate kidney xenotransplantation setting using h‐DAF transgenic donor organs and postoperative immunosuppression, episodes of AVR were either treated with boluses of cyclophosphamide and steroids or with the same regimen supplemented by a three‐day course of C1‐Inhibitor, a multifunctional complement regulator. In 8 out of 10 animals stable initial graft function was achieved; in all animals one or more episodes of AVR were observed. When, in 4 animals, C1‐Inhibitor was added to the standard anti‐rejection treatment regimen, AVR was successfully reversed in 6 out of 7 episodes, while in another group of 4 animals receiving the standard anti‐rejection treatment 0 out of 4 episodes of AVR responded to treatment. Response to anti‐rejection treatment was associated with a significant increase in recipient survival time. We conclude that AVR of h‐DAF transgenic porcine kidneys can be successfully treated by additional short‐term fluid phase complement inhibition.

INFLUENCE OF COLD ISCHEMIA TIME, PRETRANSPLANT ANTI- PORCINE ANTIBODIES, AND DONOR/RECIPIENT SIZE MATCHING ON HYPERACUTE GRAFT REJECTION AFTER DISCORDANT PORCINE TO CYNOMOLGUS KIDNEY TRANSPLANTATION

Organs transplanted between phylogenetically disparate species, such as from the pig into the primate, are subject to hyperacute rejection (HAR). This form of xenograft rejection is mediated by preformed natural antibodies and is believed to occur invariably in discordant xenografts thus leading to rapid destruction and complete thrombosis of the graft. Recent data, however, have shown that in the porcine to cynomolgus monkey setting, HAR is not inevitably seen after porcine kidney transplantation. The influence of preoperative antiporcine antibody levels in the recipient, cold ischemia time, and donor organ weight on the onset of HAR was investigated by using unmodified large white pigs (aged 3-12 weeks) as organ donors and adult cynomolgus monkeys (aged 1.5-3.5 years) as recipients. Porcine kidney xenotransplantation was performed in either a non-life-supporting model (n=7) or in a life-supporting model (n=8). In both models, no correlation was found between cold ischemia time and HAR. When preoperative anti-porcine antibody levels were investigated, a significant increase in incidence of HAR was observed in animals with elevated anti-porcine IgM (P<0.05) but not IgG levels (P=NS). Interestingly, although 5 of 12 grafts with an organ weight of less than 50 g underwent HAR, none of three grafts with a donor organ weight of more than 70 g showed signs of HAR. In addition, all three larger grafts showed intraoperative and postoperative urine production, although only in 1 (48 g) of the 12 grafts weighing less than 50 g primary graft function was observed. In one animal, a second porcine kidney (23 g) was successfully transplanted (without HAR) immediately after HAR and subsequent removal of a first porcine kidney (20 g). These results indicate that in the porcine to cynomolgus monkey setting anti-porcine IgM rather than IgG anti-porcine antibody levels seem to be of predominant importance for the induction of HAR. By increasing the donor organ size and weight the frequency of the onset of HAR can be at least reduced. This is most likely due to immunoabsorption of the recipients preformed antibodies in the porcine kidney without lethal damage for the graft.

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.

Hemodynamic consequences of porcine kidney xenograft reperfusion in cynomolgus monkeys1,2

Background. After xenograft reperfusion, complement activation may lead to generation of anaphylatoxins and cardiocirculatory instability of the recipient. Methods. In 13 cynomolgus recipients of either unmodified or human decay accelerating factor transgenic porcine kidneys cardiocirculatory parameters were measured by single indicator transpulmonary thermodilution. Results. After graft reperfusion, recipient cardiac output decreased by 25.4% (P <0.05), intrathoracic blood volume by 22.8% (P <0.05), extravascular lung water increased slightly (P =n.s.). The impairment in cardiac output was neither influenced by the graft’s weight or human decay accelerating factor transgenicity. sC3a and sC5b-9 complement levels in the recipient monkeys showed a sharp peak upon reperfusion. Conclusions. After reperfusion a marked and significant cardiodepression accompanied by relative volume depletion were observed. Analysis of volume status ruled out a mere volume shift as the underlying reason for the observed drop in cardiac output. These data may be relevant for the perioperative management of human recipients of discordant xenografts in the future.

Long-term survival of cynomolgus monkeys following pig-to-primate kidney xenotransplantation using h-DAF transgenic donor organs.

KIDNEYS from pigs transgenic for human decay accelerating factor (h-DAF) have been shown not to be hyperacutely rejected when transplanted into nonhuman primates. In the initial reports on the use of h-DAF kidneys profound anemia was the reason for termination of the experiments in most of the monkeys. This was probably due to either species incompatibility of the porcine erythropoietin in the recipient cynomolgus monkeys, the induction of neutralizing anti-pig erythropoietin antibodies, or cyclophosphamide bone marrow toxicity. We have developed a life-supporting kidney xenotransplantation model with bilateral ligature of the recipients’ native ureters but preservation of the native kidneys, thus allowing ongoing recipient erythropoietin production.

C1 Inhibitor for Prophylaxis of Xenograft Rejection After Pig to Cynomolgus Monkey Kidney Transplantation. Transplantation 2002: 73: 688.

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.

A Primate Model for Discordant Pig to Primate Kidney Xenotransplantation Without Hyperacute Graft Rejection

Organs transplanted between phylogenetically disparate species, such as from the pig into the primate, are subject to intragraft deposition of preformed recipient immunoglobulin M (IgM) antibodies with subsequent complement activation finally leading to complete and rapid destruction of the xenograft (hyperacute graft rejection, HAR). Current therapeutic strategies for abrogation of HAR include pretransplant antibody absorption by specific or nonspecific extracorporeal column perfusion, ex vivo donor organ perfusion, the administration of substances interfering with complement activation, or even the genetic alteration of the donor. Here, in the pig to cynomolgus monkey species combination, we are describing an experimental model for abrogation of HAR by using large, relative to the recipient weight, oversized donor kidneys as xenotransplants. Porcine kidney xenotransplantation (n = 15) was performed using large white pigs of different weights and ages as organ donors and cynomolgus monkeys as recipients. In grafts with an organ weight below 50 g (20 to 48 g, median 25 g), primary nonfunction (PNF) of the porcine kidney was observed in 11 out of 12 cases and complete HAR in 5 out of 12 experiments. In contrast, none of three grafts with a donor organ weight >70 g showed signs of HAR or PNF. In one animal, a second porcine kidney from the same donor (23 g) was successfully transplanted immediately after HAR and subsequent removal of a first porcine kidney (20 g). By using appropriate immunohistochemistry stainings of reperfusion biopsies, profound deposition of recipient natural antibodies in both small and large xenografts was shown, with only scarce deposition of C3 and C5b-9 in the latter, indicating only incomplete intragraft activation of the complement cascade in these organs. Intraoperative cardiac output (CO) measurements performed in 7 experiments demonstrated a 20 to 50% decrease in CO following reperfusion in 6 out of 7 grafts irrespective of the donor organ weight. The intraoperative decrease in CO was not associated with perioperative morbidity or mortality. The use of oversized doner kidneys can enable the study of a variety of immunologic and physiologic sequela beyond HAR associated with life-supporting discordant primate kidney transplantation.Organs transplanted between phylogenetically disparate species, such as from the pig into the primate, are subject to intragraft deposition of preformed recipient immunoglobulin M (IgM) antibodies with subsequent complement activation finally leading to complete and rapid destruction of the xenograft (hyperacute graft rejection, HAR). Current therapeutic strategies for abrogation of HAR include pretransplant antibody absorption by specific or nonspecific extracorporeal column perfusion, ex vivo donor organ perfusion, the administration of substances interfering with complement activation, or even the genetic alteration of the donor. Here, in the pig to cynomolgus monkey species combination, we are describing an experimental model for abrogation of HAR by using large, relative to the recipient weight, oversized donor kidneys as xenotransplants. Porcine kidney xenotransplantation (n = 15) was performed using large white pigs of different weights and ages as organ donors and cynomolgus monkeys as recipients. In grafts with an organ weight below 50 g (20 to 48 g, median 25 g), primary nonfunction (PNF) of the porcine kidney was observed in 11 out of 12 cases and complete HAR in 5 out of 12 experiments. In contrast, none of three grafts with a donor organ weight >70 g showed signs of HAR or PNF. In one animal, a second porcine kidney from the same donor (23 g) was successfully transplanted immediately after HAR and subsequent removal of a first porcine kidney (20 g). By using appropriate immunohistochemistry stainings of reperfusion biopsies, profound deposition of recipient natural antibodies in both small and large xenografts was shown, with only scarce deposition of C3 and C5b-9 in the latter, indicating only incomplete intragraft activation of the complement cascade in these organs. Intraoperative cardiac output (CO) measurements performed in 7 experiments demonstrated a 20 to 50% decrease in CO following reperfusion in 6 out of 7 grafts irrespective of the donor organ weight. The intraoperative decrease in CO was not associated with perioperative morbidity or mortality. The use of oversized doner kidneys can enable the study of a variety of immunologic and physiologic sequela beyond HAR associated with life-supporting discordant primate kidney transplantation.

Langzeittransplantatüberleben nach diskordanter Xeno-Nierentransplantation im präklinischen Modell

Durch die Verwendung von transgenen Spendertieren mit Expression humaner Komple-mentregulatoren kann die nach diskordanter Xenotransplantation auftretende hyperakute Abstosungsreaktion (HAR) inhibiert werden. In den hier vorgestellten Untersuchungen analysierten wir den Einflus der Expression von h-DAF im Spenderorgan auf das Xeno-transplantatuberleben in einem Primatenmodell.