Xenoreactive Antibodies Research Articles

Effect of Human Natural Xenoantibody Depletion and Complement Inactivation on Early Pig Kidney Function

Preformed xenoreactive natural antibodies (XNA) and complement mediate hyperacute xenograft rejection (HXR) in pig-to-human discordant xenotransplantation. In a pig kidney-human blood xenoperfusion model, we investigated whether XNA depletion and/or human complement inactivation preserved early pig kidney function. Pig kidneys were perfused for 180 min with pig blood (AUTO group, n = 8), human blood (HETER group, n = 6), complement-inactivated human blood (COMi group, n = 5), XNA-depleted human blood (ABd group, n = 5) or complement-inactivated and XNA-depleted human blood (ABd&COMi group, n = 5). HETER kidneys were rejected after 15–30 min and showed vascular microthrombi and interstitial hemorrhages. XNA depletion and/or complement inactivation prevented HXR. The glomerular filtration rate in ABd, COMi and ABd&COMi groups was significantly lower than in the AUTO group. Also, beyond 60 min, the COMi group showed a significantly lower glomerular filtration rate than that observed in ABd and ABd&COMi groups. Kidneys from ABd, COMi and ABd&COMi groups displayed endothelial cell edema, as well as higher soluble P-selectin levels and a higher renal myeloperoxidase content than the AUTO group kidneys. COMi and ABd&COMi groups had a significantly lower renal myeloperoxidase level than the HETER group. Also, in contrast to HETER and ABd groups, these complement-inactivated groups failed to show a positive correlation between P-selectin and renal myeloperoxidase. We also investigated platelet-activating factor (PAF) as possible mediator for these functional and pathologic changes. We found that blood PAF levels were similar in HETER, ABd, COMi and ABd&COMi groups and significantly higher than in the AUTO group. Also, when PAF was added to porcine endothelial cell monolayers, morphological changes due to cytoskeleton contraction were observed, and these changes were prevented by preincubation with a PAF receptor antagonist. In conclusion, although depletion of XNA and/or complement inactivation prevent HXR, the pig kidney function is not preserved at the level of the autologous combination. The PAF overproduction observed in the xenogenic combination, which is independent of the presence of XNA and complement, may be, at least in part, responsible for early endothelial cell morphological changes still present when HXR is prevented.

IgY antiporcine endothelial cell antibodies effectively block human antiporcine xenoantibody binding.

Avian IgY antibodies are structurally different from mammalian IgGs and do not fix mammalian complement components or bind human Fc receptors. As these antibody-mediated interactions are believed to play significant roles in both hyperacute rejection (HAR) and acute vascular xenograft rejection (AVXR), IgY antibodies to xenoantigen target epitopes may inhibit these rejection processes. In this report, we show that chicken IgY antibodies to alpha-Gal antigen epitopes and to other porcine aortic endothelial cell (PAEC) antigens block human xenoreactive natural antibody binding to both porcine and rat cardiac tissues and porcine kidney tissues. Chicken IgY antibodies blocked complement-mediated lysis of PAECs by human serum, and inhibited antibody-dependent cell-mediated lysis of PAECs by heat-inactivated human serum plus peripheral blood leukocytes. Binding of IgY to porcine endothelial cells did not affect cell morphology nor expression of E-selectin. These results suggest that avian IgYs could be of potential use in inhibiting pig-to-human xenograft rejection.

PIG-TO-PRIMATE ISLET XENOGRAFTS REVERSE DIABETES BEFORE UNDERGOING ACUTE CELLULAR REJECTION

31 The functional response and immunobiology of primarily non-vascularized islet cell xenografts remain poorly defined in relevant pre-clinical models. This project was therefore aimed to delineate the initial functional response and basic mechanisms operative in the rejection process of adult porcine islets transplanted to a clinically applicable site in non-immunosuppressed non-human primates. Six streptozotocin-diabetic and two non-diabetic female Rhesus monkeys were used as recipients and adult pigs served as donors. After pretreatment in culture for two days, 20,000 islet equivalents/kg body weight were transplanted into the portal vein of heparinized, non-immunosuppressed recipient animals. Plasma glucose, fasting serum porcine C-peptide, complement activation products (C3a, sC5b-9), xenoreactive natural antibodies (XNAb), and serum nitrite were measured pre-transplant and at intervals post-transplant. Two monkeys each were sacrificed at 12, 24, 48, and 72 hrs after transplantation. Livers bearing islet xenografts were examined by routine histology and immunohistochemistry. Mean fasting plasma glucose levels in diabetic recipients were 75, 42, 85, and 157 mg/dl at 12, 24, 48 hrs, and 72 hrs posttransplant. Porcine C-peptide, negative in all animals pretransplant, was present in 8/8, 5/6, 4/4, and 1/2 recipients at 12, 24, 48, and 72 hrs posttransplant, respectively. Levels of C3a and sC5b-9 showed no evidence of complement activation. Pretransplant IgM XNAb serum levels varied between animals. On day 1, 2, and 3 after xenograft, IgM XNAb levels remained unchanged compared to pretransplant levels in the same primates. Nitrite levels increased significantly (38 mM) in 2 primates on day 2. Histopathological findings varied considerably between recipient animals. Embolized islets were discernable at each time point. Insulin-positivity was however significantly reduced at 48 and 72 hrs. At 12 and 24 hrs, islets were found associated with thrombi. Strong immunostaining for IgG, IgM, C3, C5, and C9 was seen at 12 and 24 hrs, and minimal at 72 hrs. Neutrophils and few T cells were adherent to the islets as early as 12 hrs. At 48 hrs, neutrophils had disappeared, and macrophages became increasingly discernable. However, T cells were the predominant cells from 24 to 72 hrs posttransplant. Only few B cells were noted, whereas NK cells remained absent in the developing granulomatous lesions. Pig-to-Rhesus monkey intraportal islet xenografts normalized blood glucose for 48 hrs. Antibody and complement deposition may not be solely due to the intravascular location of the graft. Islet xenografts succumbed to acute cellular rejection, initially dominated by T cells followed by granulomatous lesions.

INHIBITION OF COMPLEMENT-MEDIATED XENOGENEIC INJURY BY THE MEMBRANE ATTACK COMPLEX IS SUFFICIENT TO ALLOW INDUCTION OF ACCOMMODATION IN THE HAMSTER-TO-RAT MODEL

55 Accommodation is long-term graft survival in the presence of xenoreactive antibodies and complement. Accommodation can be obtained in the hamster-to-rat cardiac model by a combination of complement inactivation with cobra venom factor (CVF) and daily administration of cyclosporin A (CSA). Since CVF inactivates both C3 and subsequent components following C3, experiments with CVF suggest that several complement activation fragments, in addition to the membrane attack complex (MAC), may play a role in preventing accommodation in this model. To define which complement-mediated process needs to be inhibited to allow induction of accommodation, we investigated accommodation of a hamster heart xenograft in C6-deficient (C6D) PVG rats and in normocomplementemic PVG rats treated with antibodies against C6. Golden Syrian hamster hearts were transplanted heterotopically into normal or C6D rats. C6D rats had complete deficiency of C6 hemolytic activity; addition of purified C6 to the serum fully restored CH50 total complement activity, confirming that other complement components were intact. Graft survival in untreated rats was 3-4 days (n=4) and in untreated C6D rats was 5 days (n=5). Experimental groups received either CVF (60 U/Kg at day-1 pre-tx. and 20U/day × 9 days) or polyclonal goat anti-C6 antiserum (Cappel) (140 mg IgG/kg/day × 5 days) with or without CSA. The table shows the number of grafts performed in each group and grafts surviving >100 days. C6D rats bearing hamster hearts had no reconstitution of hemolytically active C6. Histopathologic examination was performed on rejected hearts (<100 day survivors) or after sacrifice (>100 day survivors).TableC6D rats treated with CSA or CSA and CVF had 90% long-term graft survival. Normal rats treated with CSA and CVF, or CSA and anti-C6 antibodies had >60% long-term graft survival. Histologically, rejected hearts had myofiber necrosis, interstitial hemorrhage, acute inflammation and vascular damage. In contrast, hearts from >100 day survivors lacked these findings and had preservation of normal architecture; however, a mononuclear cell infiltrate was present. These features are characteristic of accommodated hearts in the hamster-to-rat model. In conclusion, the finding that C6D rats and normal rats treated with anti-C6 antibodies develop accommodation without CVF treatment to inactivate C3 and C5 demonstrates that xenogeneic injury due to the MAC is responsible for preventing the development of accommodation in the hamster-to-rat model. Accommodation occurred in the face of unabated formation of fragments derived from C3 and C5.

INTERACTIONS BETWEEN HUMAN NATURAL KILLER CELLS AND B CELLS CAUSE AUGMENTATION OF IGG HUMORAL RESPONSES

53 Background: Delayed vascular rejection of pig-to-primate xenografts involves NK cells, macrophages and deposition of induced IgG antibodies. Since we and others have shown previously that human NK cells bind to the same major porcine carbohydrate xenoantigen recognized by human xenoreactive antibodies, gal alpha 1,3-gal (α Gal), we investigated the role of human NK cells in augmentation of the human anti-porcine B cell antibody response. Methods and Results: Human B cells isolated by immunomagnetic beads to > 95% purity demonstrated minimal proliferation above background when exposed to porcine aortic endothelial cells (PAEC). Following addition of irradiated NK cells (> 90% purity), B cell proliferation increased by over 6-fold. This effect required NK cell pre-activation with IL-2 or IL-12, suggesting a role for inducible co-stimulatory molecules. Incubation with Mabs against either CD40 ligand (on activated NK cells) or CD40 (on B cells) reduced B cell proliferation by 52% and 44%, respectively, whereas isotype control antibody had no effect. In the absence of NK cells, stimulation of B cells with anti-Ig plus anti-CD40 had minimal effect on proliferation, whereas the addition of IFN-gamma or GM-CSF, but not other cytokines such as IL-10, increased proliferation to levels comparable following culture with activated NK cells. Pre-incubation of NK cells with IB4, a lectin with specific binding affinity for α Gal, reduced NK-driven B cell proliferation by 80-90%, whereas control had no effect. In an ELISA using porcine endothelial cell monolayers as targets, addition of NK cells to B cells induced anti-pig antibody isotype switching, with the ratio of anti-pig IgM/IgG decreasing from 1.6 for B cells alone to 0.67 for B cells co-cultured with NK cells. Conclusions: These results demonstrate that human NK cells augment B cell anti-α Gal IgG responses, that these effects involve CD40L/CD40 and specific cytokines, and that NK-B cell interactions may be critical to the delayed vascular process of pig-to-primate xenograft rejection.

Target Cell Susceptibility to Lysis by Human Natural Killer Cells Is Augmented by α(1,3)-Galactosyltransferase and Reduced by α(1,2)-Fucosyltransferase

Susceptibility of porcine endothelial cells to human natural killer (NK) cell lysis was found to reflect surface expression of ligands containing Gal alpha(1,3)Gal beta(1,4)GlcNAc [corrected], the principal antigen on porcine endothelium recognized by xenoreactive human antibodies. Genetically modifying expression of this epitope on porcine endothelium by transfection with the alpha(1,2)-fucosyltransferase gene reduced susceptibility to human NK lysis. These results indicate that surface carbohydrate remodeling profoundly affects target cell susceptibility to NK lysis, and suggest that successful transgenic strategies to limit xenograft rejection by NK cells and xenoreactive antibodies will need to incorporate carbohydrate remodeling.

XENOREACTIVE ANTIBODY RECOGNITION OF RELEVANT ANTIGENS IN SWINE KIDNEY AND LUNG

535 Background: The role of antibody in swine-to-baboon lung xenograft injury is different from that in hyperacute rejection of heart and kidney xenografts, which involves the binding of xenoreactive antibody to Galα(1-3)Gal epitopes on the endothelium. Removal of xenoantibody by pretransplant ex-vivo column absorption or kidney perfusion is not beneficial to subsequent pulmonary graft survival; however, the use of pretransplant lung perfusion does have a beneficial effect on subsequent pulmonary graft outcome. One possible explanation for this finding is that pretransplant lung perfusion removes additional antibodies that are specific for unique lung antigens. The purpose of this study was to determine if these relevant unique antigens exist on the pulmonary microvasculature. Methods: (1)Xenoreactive Antibody Depletion: Human plasma was perfused through porcine lungs and kidneys harvested by standard technique in order to absorb out xenoantibody. (2)Antigen Extraction: Antigens from porcine lungs and kidneys were extracted by organ perfusion with a solution of Triton-X 100 and protease inhibitors. Antigens from porcine aortic endothelial cells (PAEC) were obtained from culture for comparison. (3)Western Blot Analysis: Membrane antigens were separated by SDS page, transferred to membranes, and stained with normal human serum, human serum perfused through porcine lung, or human serum perfused through porcine kidney, and labeled with anti-IgM. Results: Perfusion of serum through both the lung and through the kidney removed higher molecular weight (HMW) bands, between 97.4kD and 220 kD, which were recognized by normal IgM in human serum; this removal was consistent across all three cell extract types (PAEC, lung microvascular, and kidney microvascular). These HMW bands were previously found to correspond to antigens that possess Galα(1-3)Gal epitopes. Normal human serum also stained numerous lower molecular weight (LMW) antigens, corresponding to bands between 30kD and 46kD, in kidney and lung extracts; these were not present in PAEC extracts. However, antibodies recognizing these LMW antigens were not significantly depleted from human serum by lung or kidney perfusion. Conclusions: Porcine lung perfusion did not remove any additional xenoreactive antibody from human serum as compared to kidney perfusion. This suggests that there are no lung-specific antigens that are recognized by human xenoreactive antibodies, and therefore that antibody removal does not explain the beneficial effects of pretransplant porcine lung perfusion on pulmonary xenograft survival.

Human Xenoreactive Natural Antibodies Up regulate IL-8 in Porcine Endothelial Cells and Invoke Migration of Human Neutrophils in vivo

Human Xenoreactive Natural Antibodies Up regulate IL-8 in Porcine Endothelial Cells and Invoke Migration of Human Neutrophils in vivo

XENOGENEIC HUMORAL GRAFT-VS-HOST DISEASE FOLLOWING HAMSTER TO RAT BONE MARROW TRANSPLANTATION

1005 We have reported that xenogeneic chimeras can be created in rats by bone marrow transplantation (BMTx) from hamsters. However, all recipients with successful engraftment succumbed to GVHD, which can be either cellular and/or humoral in origin. LEW rats underwent splenectomy (Spx) on day -7 and TBI of 10 Gy 3 d before BMTx. 300×106 unfractionated hamster BM cells were infused intravenously. Recipient treatment groups were divided as shown in the table. Rat recipients were observed for clinical signs of GVHD. Chimerism was detected by flow cytometry using a rat anti-hamster Ab. Sera from these chimeras were taken and anti-rat and anti-hamster antibody titers were measured by complement dependent cytotoxicity (CDC). Histopathological studies were donc with H&E. Serum transfer experiments were performed using a hamster anti-rat spleen cell Ab that was injected into the portal vein of naive rats who were sacrificed 18 h later for histological study of the liver. Results were as follows:TableGroup 1 allowed engraftment in 2/8 rats. While only one recipient developed the classical dermatitis of allogeneic GVHD, both rats had extensive infarction of the liver with focal hepatocyte necrosis in zones 1 and 2, areas that were infiltrated by polymorphonuclear leukocytes which was characteristic of humoral injury. The central vein and pericontral hepatocytes (zone 3) were not compromised, ruling out venoocclusive disease. In Group 2, 90% of the rats were engrafted; 6/9 animals showed dermatitis and 3/9 showed pure humoral injury. In Group 3, although tacrolimus prevented dermatitis, all animals developed the fatal liver injury that resulted in decreased survival when compared to Group 2. CDC test showed that xenoreactive natural antibodies (XNA) were present in both species against each other. XNA against hamster lymphocyte disappeared in BMTx recipients. However, cytotoxic antibody against rat lymphocytes had appeared in some of the rats suffering from humoral injury to the liver. This kind of injury could be reproduced in naive rats by intraportal injection of hamster anti-rat spleen cell antibody. In conclusion, xenogeneic GVHD may have a dual presentation: a classical cellular GVHD not distinct to the allogeneic one, and a humoral GVHD affecting solely the recipient liver. The degree of humoral injury is potentiated by T cell suppressants such as tacrolimus. This humoral injury may be the result of mature hamster B lymphocytes present in the donor BM.

Delayed Xenograft Rejection is Initiated by Elicited IgM Anti-Graft Antibodies.

513 Introduction. Delayed xenograft rejection (DXR) is a vascular type of rejection that can occur in a T cell-independent manner. The immunological mechanisms leading to DXR, however, remain unclear. In this study we tested whether the pathogenesis of DXR was initiated by the generation of elicited anti-graft IgM xenoreactive antibodies (Xabs) following transplantation. While generally assumed to be the case, this has never been shown. Methods. Hamster hearts were transplanted into adult rats depleted of circulating IgM by the administration of an anti-μ monoclonal antibody (Mab) 1 day prior to transplantation (30 mg/kg), and 1 and 3 days after transplantation (15 mg/kg). Cyclosporin A (CyA) was administered at the time of transplantation and daily thereafter (15 mg/kg). Results. Anti-μ administration, without further treatment, resulted in depletion of circulating IgM to undetectable levels, and had no effect on complement, NK cells or monocyte/macrophages. Depletion of IgM suppressed DXR, leading to rejection 5-7 days post-transplantation (Table) Rejection was associated with the generation of T cell-dependent IgG antibodies. When IgM depletion was combined with CyA treatment, anti-graft circulating IgG antibodies where no longer detected and xenografts survived long-term (> 50 days). IgM Xabs returned to the circulation 20-30 days after transplantation, which correlated with the clearance of circulating anti-μ Mab, but was not associated with xenograft rejection. Continued survival of the xenograft in the presence of IgM and complement indicated that the xenograft had accommodated. Accommodation was associated with the expression in the xenograft of the protective genes A20, Heme oxygenase-1 and bcl-xL as early as 3 days after transplantation.TableConclusion. DXR of hamster to rat cardiac xenografts can occur through the rapid generation of T cell-independent elicited Xabs of the IgM isotype. Temporary depletion of IgM antibodies and continuing T cell immunosuppression leads to xenograft accommodation. Expression of the protective genes does not require the presence of anti-graft IgM antibodies. Xenograft accommodation is associated with the expression of heme oxygenase-1, which we have previously shown to be functionally associated with xenograft survival.

Fetal porcine islet-like cell clusters transplanted to cynomolgus monkeys: an immunohistochemical study.

The mechanism(s) involved in acute cellular xenograft rejection have hitherto been generated in vitro or in different experimental models, with pig tissue being transplanted to rodents. There is an urgent need to validate these results in a clinically more relevant combination of species. Fetal porcine islet-like cell clusters (ICC) were transplanted under the kidney capsule in cynomolgus monkeys, either untreated or given immunosuppression with cyclosporine (CsA; 10 mg/kg body weight, intramuscularly) and 15-deoxyspergualin (DSG; 5 mg/kg body weight, intramuscularly). ICC xenografts were examined at 1, 3, 6, or 10-12 days after transplantation, using immunohistochemical techniques. Serum levels of xenoreactive antibodies were measured with ELISA. No deposits of IgM, IgG, Clq, or C3 were detected within the ICC xenograft in any of the monkeys. Likewise, no significant increase in the levels of xenoreactive antibodies were found after transplantation. In untreated animals, a few N-Elastase-positive cells (neutrophil granulocytes) were seen in the xenograft at day 1. A few mononuclear cells were present in the adjacent renal parenchyma, but they did not infiltrate the xenograft. At this time (day 1), early signs of necrosis were observed in the central parts of the graft. On day 3, the graft had a large, central necrotic area that contained polymorphonuclear cells; the remaining parts of the xenograft showed severe infiltration with CD8+ T cells. Occasional CD68+ cells (macrophages) were seen on days 1 and 3. On day 6, large numbers of macrophages were found infiltrating the entire graft. A few CD20+ B cells, accumulated as small clusters, were also found. Only a few natural killer cells (CD56+) were detected. The CsA/DSG-treated monkeys showed markedly fewer CD2+/CD8+ T cells on day 6 than the untreated monkeys, and the ICC graft was clearly better preserved. However, the number of CD8+ and CD68+ cells had increased considerably at 12 days after transplantation and diffusely infiltrated the whole ICC xenograft. Porcine ICC transplanted under the kidney capsule in cynomolgus monkeys were rejected by an acute cell-mediated rejection progressing during the first 6 days after transplantation. The process was not dependent on host Ig or C3 binding to the graft. Although the rejection of porcine ICC was significantly delayed in CsA/DSG-treated monkeys, the ICC xenografts were almost completely destroyed 12 days after transplantation.

Significance of histochemical expression of hanganutziu–deicher antigens in pig, baboon and human tissues

THERE is increasing interest in the clinical application of xenotransplantation. This interest has been sparked predominantly by the severe shortage of donors. In addition, xenotransplantation might be useful for avoiding the recurrence of viral disease in the transplanted organ. The humoral barrier leading to the immune response is well known for clinical practice of xenotransplantation; that is, xenoreactive natural antibodies to the graft endothelium. Human xenoreactive natural antibodies are directed predominantly against Gala1–3Gal (a-Gal antigen), a saccharide expressed in cells of lower mammals and New World monkeys, but not in cells of Old World monkeys, apes, or humans. Anti–a-Gal antibody plays a major role in hyperacute rejection (HAR) after pig-to-human xenotransplantation. Antibody binding to this sugar, expressed as a modification of endothelial cell membrane glycoproteins and glycolipids, activates complement by the classical pathway. Many investigators believe that pig-to-human xenotransplantation will become a common clinical application and that the pig-to-baboon model represents a good experimental design for study. Hanganutziu–Deicher (HD) antibodies have been known as antibodies detectable in patients with serum sickness. In 1924, the Romanian pathologist Hanganutziu noticed that a serum taken from a patient who received antitetanus horse serum abnormally and strongly agglutinated sheep red blood cells. This agglutination was not sheep-specific, as red blood cells from the horse, guinea-pig, rabbit, calf, and pig expressed similar titers. In 1926, Deicher replicated these results. The antibodies reacting broadly with heterologous antigens were called heterophilic antibodies. HD antibodies are heterophilic antibodies as Forssman antibodies and Paul–Bunnell antibodies. Higashi et al and Merrick et al found that antigens recognized by HD antibodies are gangliosides containing N-glucolylneuramic acid (NeuGc). Gangliosides are sialic acid–containing glycophospholipids present on the surface of all mammalian cells. Sialic acids are a group of nine carbohydrates of two main types: N-acetylneuraminic acid (NeuAc) and N-glucolylneuramic acid. NeuAc is expressed ubiquitously, whereas, NeuGc, the hydroxylated form of NeuAc, is not present in birds or humans. HD antigen is widely distributed in mammalian species with the exception of humans. Some antipig antibodies are directed against epitopes other than the a-Gal antigen. Grafting animals into human patients should be followed by an increase in HD antibodies. Therefore, it is possible that non–a-Gal antibody can cause rejection, even if a-Gal epitopes are successfully removed from donor pig organs. The purpose of this study is to elucidate the significance of HD antigen as non–a-Gal epitopes in clinical xenotransplantation.

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

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. 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. 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. 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.

Use of von Willebrand diseased kidney as donor in a pig-to-primate model of xenotransplantation.

The coagulation process in hyperacute and delayed xenograft rejection is essential and depends upon platelet adhesion and aggregation. The initial binding of platelets to the damaged endothelium is due to the interaction of the platelet receptor glycoprotein Ib with von Willebrand factor (vWF), which is present on activated endothelial cells and bound to the subendothelial matrix. We hypothesized that the use of organs from animals with homozygous von Willebrand disease (vWD), severely deficient in vWF, might prevent the thrombosis encountered in delayed xenograft rejection. Ten baboons were treated by extracorporeal immunoadsorption of xenoreactive natural antibodies (XNA) through the donor pig liver to inhibit hyperacute rejection and received heterotopic vWD or control pig kidney xenografts. XNA levels, coagulation, and platelet activation markers were studied, and specimens of rejected kidneys were analyzed histologically. Although XNA depletion was comparable in both groups, neither kidney function nor survival times of control (n=5) or vWD (n=5) porcine kidneys showed any difference. Platelet and coagulation activation was evidenced in both groups after surgery and at rejection time. Immunohistochemical analysis revealed a weak endothelial vWF immunostaining in the rejected vWD kidneys, whereas it was undetectable in the nongrafted vWD kidneys, suggesting the deposition of baboon plasma vWF on the porcine vessels. The use of vWD organs did not improve the survival time of grafted kidneys in this xenotransplantation model. Further studies on the use of vWD organs, in association with other therapeutic approaches, such as complement inhibition, are nevertheless necessary to evaluate the usefulness of vWF deficiency as an adjunctive therapy to decrease the coagulation process during xenograft rejection.

The role of antibodies in dysfunction of pig-to-baboon pulmonary transplants

Abstract Objective: Pulmonary transplantation has become the preferred treatment for end-stage lung disease, but application of the procedure is limited because of a paucity of donors. One way to solve donor limitations is to use animal organs as a donor source or xenotransplantation. The current barrier to pulmonary xenotransplantation is the rapid failure of the pulmonary xenograft. Although antibodies are known to play a role in heart and kidney xenograft rejection, their involvement in lung dysfunction is less defined. This project was designed to define the role of antibodies in pulmonary graft rejection in a pig-to-baboon model. Methods: Orthotopic transgenic swine left lung transplants were performed in baboons depleted of antibodies by one of three techniques before transplantation: (1) ex vivo swine kidney perfusion, (2) total immunoglobulin-depleting column perfusion, and (3) ex vivo swine lung perfusion. Results were compared with those of transgenic swine lung transplants in unmodified baboons. Results: All three techniques of antibody removal resulted in depletion of xenoreactive antibodies. Only pretransplantation lung perfusion improved pulmonary xenograft function compared with lung transplantation in unmodified baboons. Conclusions: The pathogenesis of pulmonary injury in a swine-to-primate transplant model is different from that in renal and cardiac xenografts. Depletion of antibodies alone does not have a beneficial effect and may actually be detrimental. (J Thorac Cardiovasc Surg 2000;120:29-38)

Disseminated intravascular coagulation in association with the delayed rejection of pig-to-baboon renal xenografts.

Intravascular fibrin deposition and platelet sequestration occur with porcine xenograft rejection by baboons. Disseminated intravascular coagulopathy may arise either as a direct consequence of the failure to fully deplete xenoreactive natural antibodies and block complement, or because of putative cross-species molecular incompatibilities in this discordant species combination. Three baboons were conditioned with retrovirally transduced autologous bone marrow to induce tolerance to swine antigens. Xenoreactive natural antibodies and complement were depleted by plasmapheresis and the use of Gal alpha1-3Gal column adsorptions; baboons were then splenectomized and underwent renal xenografting from inbred, miniature pigs. Soluble complement receptor type-1 with protocol immunosuppression (mycophenolate mofetil, 15-deoxyspergualin, steroids, and cyclosporine) was administered. A bleeding diathesis was clinically evident from days 5 to 12 after transplantation in two baboons. Low levels of circulating C3a, C3d, and iC3b were measured despite the absence of functional circulating complement components. Profound thrombocytopenia with abnormalities in keeping with disseminated intravascular coagulopathy were observed. Prolongation of prothrombin and partial thromboplastin times was accompanied by evidence for tissue factor-mediated coagulation pathways, high levels of thrombin generation (prothrombin fragment F(1+2) production and thrombin-antithrombin complex formation), fibrinogen depletion, and production of high levels of the fibrin degradation product D-dimer. Importantly, these disturbances resolved rapidly after the excision of the rejected xenografts in two surviving animals. Histopathological examination of the rejected xenografts confirmed vascular injury, fibrin deposition, platelet deposition, and localized complement activation. Systemic coagulation disturbances are associated with delayed xenograft rejection.

Intact pancreatic islet function despite humoral xenorecognition in the pig-to-monkey combination.

The aim of this study was to analyze humoral xenoreactivity of various Old World primate species sera against pig islets and the effects of these sera on pig islet viability and function after culture. Freshly isolated or cultured adult pig islets were analyzed by immunohistology or by cytofluorimetry for Old World primate xenoreactive natural antibody (XNA) binding and complement deposition. Complement-mediated cytotoxicity was evaluated by 51Cr release assays. After 4 days of culture in 50% sera from Old World primates, the morphology and in vitro metabolic function of pig islets were also analyzed. Chimpanzee, Macaca mulatta (rhesus), or baboon XNA binding was detectable only on intra-islet endothelial cells (ECs). Incubation of pig islets with sera from all Old World primate species tested showed C3 and C4 deposition on ECs and on some surrounding endocrine cells. However, membrane attack complex (MAC) showed a pattern of positivity similar to XNA binding, i.e., restricted to ECs only. No deposition of factor B was detected. Although complement cascade was activated, no cytotoxicity was observed after incubation of islets with chimpanzee serum, whereas between 10% and 35% 51Cr specific release was obtained with rhesus, baboon, or Macaca fascicularis sera. Despite this cytotoxic effect, purified pig islets showed a normal morphology and a well-preserved insulin release in response to an acute glucose stimulus, after prolonged culture with 50% serum obtained from all primate species considered. Despite the fact that pig beta-cell function was not affected by the serum of any of the primate species tested, some of them yielded significant lysis of islet cells, presumably as a result of a cytotoxic effect on intra-islet ECs. These data show that Old World primate sera from different species do not have equivalent effect on pig islets; these differences should be taken into account in preclinical trials of pig islet xenotransplantation.

Intracellular expression in pig cells of anti-alpha1,3galactosyltransferase single-chain FV antibodies reduces Gal alpha1,3Gal expression and inhibits cytotoxicity mediated by anti-Gal xenoantibodies.

The carbohydrate structure Gal alpha1,3Gal expressed on pig cells is the major antigen recognized by xenoreactive natural antibodies in the higher primates. In xenotransplantation, natural antibodies binding to that structure initiate hyperacute rejection, and the anti-Gal alpha1,3Gal antibodies that are elicited probably take part in later phases of vascularized graft rejection. This epitope also appears to be involved in innate cellular responses. Inactivation of alpha1,3 galactosyltransferase in transgenic pigs would certainly lead to the success of xenotransplantation, but gene knockout in pigs is not feasible yet. As a novel strategy to inhibit alpha1,3 galactosylation, we generated recombinant single-chain Fv (ScFv) antibodies directed against pig alpha1,3-galactosyltransferase and evaluated the effect of their intracellular expression on enzyme activity and Gal alpha1,3Gal expression. After in vitro transfection in pig cells, the scFv antibody anti-pig alpha1,3-galactosyltransferase reduced the amount or function of enzyme by up to 70% as evidenced by immunofluorescence and measurement of cell-associated activity. Consequently, Gal alpha1,3Gal on cell membranes was reduced to the same extent. This led to a profound (more than 90%) reduction in the cytotoxicity involving anti-Gal antibodies and complement. Although not sufficient to knock out the overall human anti-pig natural xenoreactivity, intracellular expression of the scFv antibody anti-alpha1,3-galactosyltransferase in pig cells significantly decreases the amount of Gal alpha1,3Gal and could be important to protect cells from elicited antibodies as well as from innate effectors.

Prevention of initial perfusion failure during xenogeneic ex vivo liver perfusion by selectin inhibition.

Endothelial cell activation triggered by xenoreactive antibodies and complement products is the main feature of discordant xenograft rejection. The contribution of early cell-mediated mechanisms to this rejection process is poorly understood, and the function of adhesion molecules in xenogeneic cell interactions in vivo is unclear. The aim of the study was to investigate the role of selectins in mediating cell-dependent initial perfusion failure and functional restrictions in xenoperfused guinea pig (GP) livers. Isolated GP livers were hemoperfused in a flow-constant, recirculating perfusion system via the portal vein. Microhemodynamic parameters such as sinusoidal perfusion rate and leukocyte flux were analyzed using intravital fluorescence microscopy. Hepatic oxygen consumption and bile production, as well as liver enzymes, potassium level, and numbers of white blood cells and platelets in the perfusate, were determined. The GP livers were perfused either with GP blood (control perfusion), with unmodified rat blood (xenoperfusion), or with rat blood treated with the selectin-blocking polysaccharide Fucoidin. A significant sinusoidal perfusion failure was observed in the xenoperfusion group, which was accompanied by distinct signs of a functional restriction-like reduced oxygen consumption, bile production, and increased perfusion pressure. However, there were significantly fewer impairments in the Fucoidin group. Furthermore, fewer platelets were trapped and a smaller number of stagnant leukocytes were observed in this group. Fucoidin did not suppress complement activation during xenoperfusion. Considering that Fucoidin inhibits the selectin-dependent interactions among white blood cells, platelets, and sulfate-containing proteoglycans on the surface of vascular endothelium, these findings suggest an important role for early cellular interactions in the development of organ failure during xenogeneic rejection.

Chemical and lectin-gold electron microscopical studies of the expression of the Galalpha1-determinant in the pig aorta.

In the xenotransplantation research field, pig aortic endothelial cells are frequently used in different model systems, e.g., for the study of the xenoantibody-antigen reaction. The Gal(alpha1),3Gal determinant is the major target for human xenoreactive antibodies in pig tissue. Characterisation of the Gal(alpha1)- distribution in pig aortic endothelial cells is thus important for understanding the reaction occurring at the endothelial cells during the xenorejection. We have determined the complete structure of the major Gal(alpha1),3Gal terminated glycolipid, Gal(alpha1),3nLc4Cer. Structural studies were performed on isolated glycosphingolipids by mass spectrometry and NMR spectroscopy. The results show a predominance of the pentasaccharide among the Gal(alpha1)-terminated glycolipids but also the presence of several Gal(alpha1)-terminated glycolipids with extended carbohydrate core chains. Ultrastructural localisation of the Galalpha1-antigen in pig aorta was done by lectin-gold electron microscopic studies of aortic wall sections using the Griffonia simplicifolia isolectin B4. Gal(alpha1)-determinants are predominantly localised on the luminal surface of pig aortic endothelial cells and endothelial cells of vasa vasorum and, to a lesser extent, vascular subendothelium.