Heart Xenotransplantation Research Articles

Prevention of hyperacute xenograft rejection in orthotopic xenotransplantation of pig hearts into baboons using immunoadsorption of antibodies and complement factors.

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 antipig 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.

Induction of anti-Forssman antibodies in the hamster-to-rat xenotransplantation model.

In the hamster-to-rat heart xenotransplantation model, the serum response of the host contributes to determine whether the xenograft is accommodated or rejected. To further characterize the serum response in this model, we compared anti-hamster antibodies found in naive LEW-1A rats, or in LEW-1A rats rejecting or accommodating a hamster heart, using a combination of cobra venom factor (CVF) and cyclosporin A (CsA) given for 10 days, and then CsA alone. Hamster hearts grafted into rat recipients contained IgG and IgA deposits to the same extent whether the xenograft was rejected or accommodated. Only immunoglobulins of the IgM isotype were found to be more abundant in recipients rejecting their graft. A significant part of this IgM response was directed toward the Forssman antigen, a sphingolipid present in the hamster but not in the rat. However, although anti-Forssman antibodies bind in situ to hamster tissues, this binding was not able to induce hyperacute rejection after antibody transfer. Furthermore, depletion of anti-Forssman antibodies from a rejecting serum did not modify its rejection properties. Unlike the pig-to-primate discordant xenotransplantation model, in which preexisting anti-carbohydrate antibodies are directly responsible for hyperacute rejection, in the concordant hamster-to-rat situation, the evoked IgM anti-Forssman carbohydrate antibodies do not appear to be the main cause of the vascular rejection.

IMMUNOPATHOLOGY OF PIG LIVERS USED IN CLINICAL EX-VIVO XENOPERFUSIONS: ACUTE VASCULAR REJECTION.

214 Introduction: The pathogenesis of pig livers used in clinical xenoperfusions has been described as different than classical hyperacute rejection. Discordant xenotransplantation in the face of antibody or complement depletion results in acute vascular rejection (AVR). AVR is characterized by xenoantibody binding to graft endothelium with fibrin deposition, and upregulation of inflammatory mediators and vasoactive enzymes including endothelin-1 (ET-1), and inducible nitric oxide synthase (iNOS). Patients with liver failure have diminished levels of complement and are less able to initiate hyperacute rejection than healthy subjects. The purpose of this study was to describe the immunopathology of AVR in pig-to-human-liver xenotransplantation. Methods: Pig livers (n=3) that were used in 3 separate clinical xenoperfusions were studied. CH50 levels as an indicator of serum complement activity were measured prior to perfusion for each patient (n=3). Biopsies of perfused livers were stained using hematoxylin and eosin, and immunostained for the presence of human IgM, IgG, and fibrin. Sections were also immunostained for vasoactive mediators: ET-1, endothelin converting enzyme (ECE-1), and iNOS. Results: Pig livers (n=3) were perfused for t=4.5, 6 and 11 hours for three different patients with liver failure. Complement activity was severely diminished preperfusion in each of the patients (CH50 26 ± 19 U, normal range 120-300 U). Histology revealed sinusoidal dilatation with parenchymal hemorrhage and neutrophil infiltration. The longest perfused liver (11 hours) had extensive macrovesicular steatosis. Immunostaining revealed diffuse sinusoidal binding of IgM and IgG to the portal and sinusoidal endothelium in all three livers. There was fibrin deposition in small and medium sized portal venules. ET-1 and ECE-1 stained prominently in the sinusoids in each of the livers. Staining for iNOS was also prominent in the sinusoidal endothelium as well. Conclusions: Injury of pig livers used in xenoperfusion in the absence of complement is consistent with AVR as described in pig-to-primate models of kidney and heart xenotransplantation.

Development of xenogeneic microchimerism correlated with graft outcome in hamster-to-rat heart xenotransplantation

Development of xenogeneic microchimerism correlated with graft outcome in hamster-to-rat heart xenotransplantation

Effects of Various Routes of Donor-Specific Antigen Administration on Graft Survival in a Model of Discordant Heart Xenotransplantation

CURRENTLY, transplantation is the best treatment for patients with end-stage organ disease. However, organ shortage severely limits the number of patients who can be treated this way and leads to increased mortality on organ waiting lists. On the other hand, appropriate animals are considered limitless alternative organ sources for such patients. Accordingly, intensive research is underway at many centers to overcome the biological barriers which currently stand in the way of organ acceptance between species. Hyperacute rejection is invariably seen after discordant xenotransplantations with immediate vascular reconstruction. Various strategies have been proposed to solve this problem. Apart from the considerable success achieved with transfection of recipient complement regulatory genes to the donor, no satisfactory progress has been made in overcoming or eliminating this type of rejection. Immunologic tolerance induction against nonself tissues is an attractive means of achieving total or partial acceptance of transplanted organs. The best-known attempts regarding tolerance induction have involved the administration of donor-specific antigens (DSA) (ie, donor-specific cells [DSCs] or, rarely, donor-specific cell-free extracts [DCFE] to the recipient through various routes, with or without concomitant immunosuppression. It has been demonstrated that donor-specific transfusion (DST), or the administration of DSA via various routes to the recipient prior to allotransplantations, generally evokes a certain tolerance status and prolongs allograft survival. Similar attempts at DSA administration have been made recently in xenotransplantation models, but, contrary to the outcome in allogenic models, these resulted in increased sensitivity against, not tolerance to, donor organs. As has been demonstrated repeatedly, the response of the immune system varies according to the antigen administration route. The immune response to antigen administered via the portal vein or oral route is less vigorous than that resulting from the systemic venous route. In order to study xenotolerance, we planned to administer either DCFE or DSCs in a discordant xenotransplant combination. This initial study reports the results of guinea pig-specific DCFE administration to recipient rats without any additional pretreatment. We have chosen DCFE because, as has been suggested, this option is free of the undesirable possibility of graft-vs-host disease and is easier to handle and obtain than DSCs.

PREVENTION OF HYPERACUTE REJECTION BY HUMAN DECAY ACCELERATING FACTOR IN XENOGENEIC PERFUSED WORKING HEARTS

As a potential source of organs for xenotransplantation, pigs that are transgenic for human decay accelerating factor (DAF) have been bred in order to overcome hyperacute rejection. We investigated the protective effect of human DAF in a porcine working heart model perfused by human blood. Hearts of normal landrace pits served as controls. The following parameters were measured: stroke work index, coronary flow and arteriovenous oxygen consumption, 6-keto prostaglandin F1alpha and prostaglandin E2 as markers of endothelial cell activation; creatine phosphokinase and lactate dehydrogenase for evaluation of the extent of myocardial damage; TNFalpha and IL-6 as markers of mononuclear cell activation. Histological and ultrastructural investigations from myocardial tissue sections were done at the end of perfusion. Human (h) DAF appeared to inhibit complement-mediated endothelial cell activation of transgenic pig hearts successfully. This was in contrast to landrace pig hearts, which had a sixfold increase of prostaglandin levels during perfusion with human blood. The cardiac weight increase during perfusion time due to interstitial edema tended to be less in the hDAF group. Myocardial damage was minimal in transgenic hearts, whereas normal pig hearts produced a threefold increase of creatine phosphokinase and lactate dehydrogenase levels. In these hearts, electron microscopy revealed single cell necrosis of myocytes and vacuolization of mitochondria with cristae rupture. According to the results obtained in the working heart model, the breeding of pigs that are transgenic for hDAF represents a promising step to making heart xenotransplantation a clinical reality in the future.

Inhibition of chronic vascular rejection in primate cardiac xenografts using mycophenolate mofetil

Similar to human allografts, cardiac xenografts also appear susceptible to chronic vascular rejection. The study described here evaluated the influence of mycophenolate mofetil on the incidence and severity of vascular rejection in a primate model of heart xenotransplantation. Nine baboons received heterotopic cardiac xenografts from donor cynomolgus monkeys. All baboons were placed on a cyclosporine and methylprednisolone-based immunosuppressive regimen. In addition, group 1 baboons received azathioprine (4 mg · kg −1 · day −1) and group 2 baboons received mycophenolate mofetil (70 mg · kg −1 · day −1). Biopsy specimens were obtained at regular intervals and reviewed blindly by a pathologist. A total of 50 biopsy specimens, 29 from group 1 and 21 from group 2, were reviewed. Histologic evidence of vascular rejection was present in 16 of the 29 biopsy specimens from the group 1 animals and in only 2 of the 21 specimens from the group 2 animals ( p < 0.005). The mean graft survival was 3 months in group 1 versus 10 months in group 2. At 1-year follow-up, profound intimal proliferation in the coronary vasculature was noted in the biopsy specimens from group 1, whereas the coronary vessels were found to be normal in the specimens from group 2. The use of mycophenolate mofetil, in combination with cyclosporine and steroids, resulted in reduced vascular rejection and prolonged xenograft survival.