Ralf Lorenz

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P <0.05) over nontransgenic controls (207.5±54.6 vs. 57.5±64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.

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.

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.

Effect of semi-invasive cardiovascular monitoring on the perioperative outcome of kidney or lung xenotransplantation in cynomolgus monkeys

Summary >Background: Cynomolgus monkeys (Macaca fascicularis) are one of the predominant primate models in experimental transplantation. Particularly in xenotransplantation experiments, they can be subjected to profound cardiovascular instability by surgical trauma, volume shifts and inflammatory mediator release. Meticulous perioperative management including deliberate intraoperative monitoring is therefore required. Methods: 26 porcine kidneys (group A) and 3 single lung lobes (group B), obtained from unmodified pigs or from pigs transgenic for human complement regulators, were transplanted into 29 cynomolgus monkeys. In group A 1 (n = 11) the monkeys were monitored intraoperatively by ECG, non-invasive arterial, and central venous blood pressure (standard cardiovascular monitoring, SCVM). In group A 2 (n = 15) measurements of cardiac output (CO), intrathoracic blood volume and extravascular lung water (extended cardiovascular monitoring, XCVM) were added to the intraoperative monitoring regime. XCVM plus continuous pulse contour CO was used in the 3 monkeys undergoing porcine single lung xenotransplantation. Results: In group A 2 significantly more animals had initial graft function if compared to group A 1 . 40% of the monkeys of group A 2 but 73% of group A 1 died within the first postoperative week. Group A 2 displayed a trend towards greater overall survival rates, but this difference did not reach statistical significance (p = 0,07). In group B stable lung xenograft reperfusion was achieved in all 3 recipients despite recurrent phases of severe cardiovascular instability. Two lung recipients could be weaned from the ventilator, and 1 monkey survived more than 24 hours postoperatively. Conclusions: The application of XCVM in small primates undergoing kidney xenotransplantation led to significantly better early postoperative results and longer survival times. In single lung xenotransplantation XCVM plus continuous pulse contour CO showed to be indispensable for the therapeutic management of the profound cardiovascular instabilities observed. Our data indicate that comprehensive anaesthesiologic monitoring can contribute substantially to the advancement of experimental organ transplantation in small monkeys.