Cardiac and Pulmonary Histopathology in Baboons Following Genetically-Engineered Pig Orthotopic Heart Transplantation.

BACKGROUND Although improving, survival after pig orthotopic heart transplantation (OHTx) in baboons has been mixed and largely poor. The causes for the high incidence of early failure remain uncertain. MATERIAL AND METHODS We have carried out pig OHTx in 4 baboons. Two died or were euthanized within hours, and 2 survived for 3 and 8 months, respectively. There was evidence of a significant 'cytokine storm' in the immediate post-OHTx period with the elevations in IL-6 correlating closely with the final outcome. RESULTS All 4 baboons demonstrated features suggestive of respiratory dysfunction, including increased airway resistance, hypoxia, and tachypnea. Histopathological observations of pulmonary infiltration by neutrophils and, notably, eosinophils within vessels and in the perivascular and peribronchiolar space, with minimal cardiac pathology, suggested a role for early lung acute inflammation. In one, features suggestive of transfusion-related acute lung injury were present. The 2 longer-term survivors died of (i) a cardiac dysrhythmia with cellular infiltration around the conducting tissue (at 3 months), and (ii) mixed cellular and antibody-mediated rejection (at 8 months). CONCLUSIONS These initial findings indicate a potential role of acute lung injury early after OHTx. If this response can be prevented, increased survival may result, providing an opportunity to evaluate the factors affecting long-term survival.

T and B lymphocyte dynamics after genetically-modified pig-to-baboon kidney xenotransplantation with an anti-CD40mAb-based immunosuppressive regimen.

BACKGROUND: The aim was to monitor recovery of T/B lymphocytes in baboons after depletion by anti-thymocyte globulin (ATG) and anti-CD20mAb (Rituximab), followed by pig kidney transplantation and maintenance therapy with an anti-CD40mAb-based regimen. METHODS: In baboons (n = 14), induction was with ATG and anti-CD20mAb, and maintenance with (i) anti-CD40mAb, (ii) rapamycin, and (iii) methylprednisolone. Follow-up was for 6 months, or until rejection or other complication developed. Baboon blood was collected at intervals to measure T/B cells and subsets by flow cytometry. In a separate study in baboons receiving the same immunosuppressive regimen (n = 10), the populations of T/B lymphocytes in PBMCs, lymph nodes, and spleen were examined. RESULTS: After induction therapy, the total lymphocyte count and the absolute numbers of CD3+, CD4+, and CD8+T cells fell by >80%, and no CD22+B cells remained (all p < 0.001). T cell numbers began to recover early, but no CD22+B cells were present in the blood for 2 months. Recovery of both T and B cells remained at <30% of baseline (p < 0.001), even if rejection developed. At 6 months, effector memory CD8+T cells had increased more than other T cell subsets, but a greater percentage of B cells were naïve. In contrast to blood and spleen, T and B cells were not depleted in lymph nodes. CONCLUSIONS: ATG and anti-CD20mAb effectively decreased T and B lymphocytes in the blood and, in the presence of anti-CD40mAb maintenance therapy, recovery of these cells was inhibited. The recovery of effector memory CD8+T cells may be detrimental to long-term graft survival.

The Genetically Engineered Heart as a Bridge to Allotransplantation in Infants Just Around the Corner?

BACKGROUND: Mortality for infants on the heart transplant waitlist remains unacceptably high, and available mechanical circulatory support is suboptimal. Our goal is to demonstrate the feasibility of utilizing genetically engineered pig (GEP) heart as a bridge to allotransplantation by transplantation of a GEP heart in a baboon. METHODS: Four baboons underwent orthotopic cardiac transplantation from GEP donors. All donor pigs had galactosyl-1,3-galactose knocked out. Two donor pigs had human complement regulatory CD55 transgene and the other 2 had human complement regulatory CD46 and thrombomodulin. Induction immunosuppression included thymoglobulin, and anti-CD20. Maintenance immunosuppression was rapamycin, anti-CD-40, and methylprednisolone. One donor heart was preserved with University of Wisconsin solution and the other three with del Nido solution. RESULTS: All baboons weaned from cardiopulmonary bypass. B217 received a donor heart preserved with University of Wisconsin solution. Ventricular arrhythmias and depressed cardiac function resulted in early death. All recipients of del Nido preserved hearts easily weaned from cardiopulmonary bypass with minimal inotropic support. B15416 and B1917 survived for 90 days and 241 days, respectively. Histopathology in B15416 revealed no significant myocardial rejection but cellular infiltrate around Purkinje fibers. Histopathology in B1917 was consistent with severe rejection. B37367 had uneventful transplant but developed significant respiratory distress with cardiac arrest. CONCLUSIONS: Survival of B15416 and B1917 demonstrates the feasibility of pursuing additional research to document the ability to bridge an infant to cardiac allotransplant with a GEP heart.

The Role of Interleukin-6 (IL-6) in the Systemic Inflammatory Response in Xenograft Recipients and in Pig Kidney Xenograft Failure.

Background: In pig-to-baboon transplantation models, there is increasing evidence of systemic inflammation in xenograft recipients (SIXR) associated with pig xenograft failure. We evaluated the relationship between systemic inflammatory factors and pig kidney xenograft failure. Methods: Baboons received kidney transplants from genetically engineered pigs (n=9), and received an anti-CD40mAb-based (n=4) or conventional (n=5) immunosuppressive regimen. The pig kidney grafts were monitored by measurements of serum creatinine, serum amyloid A (SAA), white blood cell (WBC) and platelet counts, plasma fibrinogen, and pro-inflammatory cytokines (baboon and pig IL-6, TNF-α, IL-1ß). Results: Six baboons were euthanized or died from rejection, and 3 were euthanized for infection. Changes in serum creatinine correlated with those of SAA (r=0.56, p<0.01). Serum baboon IL-6 was increased significantly on day 1 after transplantation and at euthanasia (both p<0.05) and correlated with serum creatinine and SAA (r=0.59, p<0.001, r=0.58, p<0.01; respectively). but no difference was observed between rejection and infection. Levels of serum pig IL-6, TNF-α, IL-1ß were also significantly increased on day 1 and at euthanasia, and serum pig IL-6 and IL-1ß correlated with serum creatinine and SAA. The level of serum baboon IL-6 correlated with the expression of IL-6 and amyloid A in the baboon liver (r=0.93, p<0.01, r=0.79, p<0.05; respectively). Conclusion: Early upregulation of SAA and serum IL-6 may indicate the development of rejection or infection, and are associated with impaired kidney graft function. Detection and prevention of systemic inflammation may be required to prevent pig kidney xenograft failure after xenotransplantation.

Histopathology of pig kidney grafts with/without expression of the carbohydrate Neu5Gc in immunosuppressed baboons.

INTRODUCTION: Pigs deficient in three glycosyltransferase enzymes (triple-knockout [TKO] pigs, that is, not expressing the three known carbohydrate xenoantigens) and expressing 'protective' human transgenes are considered a likely source of organs for transplantation into human recipients. Some human sera have no or minimal natural antibody binding to red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs. However, all Old World monkeys exhibit natural antibody binding to TKO pig cells. The xenoantigen targets of Old World monkey natural antibodies are postulated to be carbohydrate moieties exposed when the expression of the carbohydrate N-glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to compare the survival in baboons and histopathology of renal grafts from pigs that either (a) expressed Neu5Gc (GTKO pigs; Group 1) or (b) did not express Neu5Gc (GTKO/CMAHKO [DKO] or TKO pigs; Group 2). METHODS: Life-supporting renal transplants were carried out using GTKO (n = 5) or DKO/TKO (n = 5) pig kidneys under an anti-CD40mAb-based immunosuppressive regimen. RESULTS: Group 1 baboons survived longer than Group 2 baboons (median 237 vs. 35 days; mean 196 vs. 57 days; p < 0.07) and exhibited histopathological features of antibody-mediated rejection in only two kidneys. Group 2 exhibited histopathological features of antibody-mediated rejection in all five grafts, with IgM and IgG binding to renal interstitial arteries and peritubular capillaries. Rejection-free survival was significantly longer in Group 1 (p < 0.05). CONCLUSIONS: The absence of expression of Neu5Gc on pig kidney grafts is associated with increased binding of baboon antibodies to pig endothelium and reduced graft survival.

Evidence that sensitization to triple-knockout pig cells will not be detrimental to subsequent allotransplantation.

The current evidence is that sensitization to a pig xenograft does not result in the development of antibodies that cross-react with alloantigens, and therefore, sensitization to a pig xenograft would not be detrimental to the outcome of a subsequent allograft. This evidence relates almost entirely to the transplantation of cells or organs from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. However, it is not known whether recipients of triple-knockout (TKO) pig grafts who become sensitized to TKO pig antigens develop antibodies that cross-react with alloantigens and thus be detrimental to a subsequent organ allotransplant. We identified a single baboon (B1317) in which no (or minimal) serum anti-TKO pig antibodies could be measured-in our experience unique among baboons. We sensitized it by repeated subcutaneous injections of TKO pig peripheral blood mononuclear cells (PBMCs) in the absence of any immunosuppressive therapy. After TKO pig PBMC injection, there was a transient increase in anti-TKO pig IgM, followed by a sustained increase in IgG binding to TKO cells. In contrast, there was no serum IgM or IgG binding to PBMCs from any of a panel of baboon PBMCs (n = 8). We conclude that sensitization to TKO pig PBMCs in the baboon did not result in the development of antibodies that also bound to baboon cells, suggesting that there would be no detrimental effect of sensitization on a subsequent organ allotransplant.

Evidence suggesting that deletion of expression of N-glycolylneuraminic acid (Neu5Gc) in the organ-source pig is associated with increased antibody-mediated rejection of kidney transplants in baboons.

Pigs deficient in three glycosyltransferase enzymes (triple-knockout [TKO] pigs) and expressing "protective" human transgenes are likely sources of organs for transplantation into human recipients. Testing of human sera against red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs has revealed minimal evidence of natural antibody binding. However, unlike humans, baboons exhibit natural antibody binding to TKO pig cells. The xenoantigen specificities of these natural antibodies are postulated to be one or more carbohydrate moieties exposed when N-glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to compare the survival of renal grafts in baboons from pigs that either expressed Neu5Gc (GTKO pigs; Group1, n = 5) or did not express Neu5Gc (GTKO/CMAHKO [DKO] or TKO pigs; Group2, n = 5). An anti-CD40mAb-based immunosuppressive regimen was administered in both groups. Group1 kidneys functioned for 90-260 days (median 237, mean 196 days), with histopathological features of antibody-mediated rejection in two kidneys. Group2 kidneys functioned for 0-183 days (median 35, mean 57), with all of the grafts exhibiting histologic features of antibody-mediated rejection. These findings suggest that the absence of expression of Neu5Gc on pig kidneys impacts graft survival in baboon recipients.

A perspective on the potential detrimental role of inflammation in pig orthotopic heart xenotransplantation.

There is a critical shortage of deceased human donor organs for transplantation. The need is perhaps most acute in neonates and infants with life-threatening congenital heart disease, in whom mechanical support devices are largely unsuccessful. If orthotopic (life-supporting) heart transplantation (OHTx) were consistently successful in the genetically engineered pig-to-nonhuman primate (NHP) model, a clinical trial of bridging with a pig heart in such patients might be justified. However, the results of pig OHTx in NHPs have been mixed and largely poor. We hypothesise that a factor is the detrimental effects of the inflammatory response that is known to develop (a) during any surgical procedure that requires cardiopulmonary bypass, and (b) immediately after an NHP recipient is exposed to a pig xenograft. We suggest that the combination of these two inflammatory responses has a direct detrimental effect on pig heart graft function, but also, and possibly of more importance, on recipient baboon pulmonary function, which further impacts survival of the pig heart graft. In addition, the inflammatory response almost certainly adversely impacts the immune response to the graft. If our hypothesis is correct, the potential steps that could be taken to reduce the inflammatory response or its effects (with varying degrees of efficacy) include (a) white blood cell filtration, (b) complement depletion or inactivation, (c) immunosuppressive therapy, (d) high-dose corticosteroid therapy, (e) cytokine/chemokine-targeted therapy, (f) ultrafiltration or CytoSorb hemoperfusion, (g) reduction in the levels of endogenous catecholamines, (h) triiodothyronine therapy and (i) genetic engineering of the organ-source pig. Prevention of the inflammatory response, or attenuation of its effects, by judicious anti-inflammatory therapy may contribute not only to early survival of the recipient of a genetically engineered pig OHTx, but also to improved long-term pig heart graft survival. This would open the possibility of initiating a clinical trial of genetically engineered pig OHTx as a bridge to allotransplantation.

What Therapeutic Regimen Will Be Optimal for Initial Clinical Trials of Pig Organ Transplantation?

We discuss what therapeutic regimen might be acceptable/successful in the first clinical trial of genetically engineered pig kidney or heart transplantation. As regimens based on a calcineurin inhibitor or CTLA4-Ig have proved unsuccessful, the regimen we administer to baboons is based on induction therapy with antithymocyte globulin, an anti-CD20 mAb (Rituximab), and cobra venom factor, with maintenance therapy based on blockade of the CD40/CD154 costimulation pathway (with an anti-CD40 mAb), with rapamycin, and a corticosteroid. An anti-inflammatory agent (etanercept) is administered for the first 2 wk, and adjuvant therapy includes prophylaxis against thrombotic complications, anemia, cytomegalovirus, and pneumocystis. Using this regimen, although antibody-mediated rejection certainly can occur, we have documented no definite evidence of an adaptive immune response to the pig xenograft. This regimen could also form the basis for the first clinical trial, except that cobra venom factor will be replaced by a clinically approved agent, for example, a C1-esterase inhibitor. However, none of the agents that block the CD40/CD154 pathway are yet approved for clinical use, and so this hurdle remains to be overcome. The role of anti-inflammatory agents remains unproven. The major difference between this suggested regimen and those used in allotransplantation is the replacement of a calcineurin inhibitor with a costimulation blockade agent, but this does not appear to increase the complications of the regimen.

Pig kidney xenotransplantation: Progress toward clinical trials.

Pig organ xenotransplantation offers a solution to the shortage of deceased human organs for transplantation. The pathobiological response to a pig xenograft is complex, involving antibody, complement, coagulation, inflammatory, and cellular responses. To overcome these barriers, genetic manipulation of the organ-source pigs has largely been directed to two major aims-(a) deletion of expression of the known carbohydrate xenoantigens against which humans have natural (preformed) antibodies, and (b) transgenic expression of human protective proteins, for example, complement- and coagulation-regulatory proteins. Conventional (FDA-approved) immunosuppressive therapy is unsuccessful in preventing an adaptive immune response to pig cells, but blockade of the CD40:CD154 costimulation pathway is successful. Survival of genetically engineered pig kidneys in immunosuppressed nonhuman primates can now be measured in months. Non-immunological aspects, for example, pig renal function, a hypovolemia syndrome, and rapid growth of the pig kidney after transplantation, are briefly discussed. We suggest that patients on the wait-list for a deceased human kidney graft who are unlikely to receive one due to long waiting times are those for whom kidney xenotransplantation might first be considered. The potential risk of infection, public attitudes to xenotransplantation, and ethical, regulatory, and financial aspects are briefly addressed.

Does expression of a human complement-regulatory protein on xenograft cells protect them from systemic complement activation?

BACKGROUND: There are many causes of systemic complement activation, which may have detrimental effects on a pig xenograft. Transgenic expression of one or more human complement-regulatory proteins (hCRPs), e.g., hCD46, provides some protection to the xenograft, but it is not known whether it protects the xenograft from the effects of systemic complement activation. We used wild-type (WT) pig aortic endothelial cells (pAECs) to activate complement, and determined whether the expression of hCD46 on a1,3galactosyltransferase gene-knockout (GTKO) pAECs protected them from injury. METHODS: CFSE-labeled and non-labeled pAECs from a WT, a GTKO, or a GTKO/hCD46 pig were separately incubated with heat-inactivated pooled human serum in vitro. Antibody pre-bonded CFSE-labeled and non-labeled pAECs were mixed, and then incubated with rabbit complement. The complement-dependent cytotoxicity was measured by flow cytometry. RESULTS: There was significantly less lysis of GTKO/CD46 pAECs (6%) by 50% human serum compared to that of WT (91%, p<0.001) or GTKO (32%, p<0.01) pAECs. The lysis of GTKO pAECs was significantly increased when mixed with WT pAECs (p<0.05). In contrast, there was no significant change in cytotoxicity of GTKO/CD46 pAECs when mixed with WT pAECs. CONCLUSIONS: The expression of hCD46 protected pAECs from systemic complement activation.

Effect of intravenous immunoglobulin (IVIg) on primate complement-dependent cytotoxicity of genetically engineered pig cells: relevance to clinical xenotransplantation.

Triple-knockout (TKO) pigs may be ideal sources of organs for clinical xenotransplantation because many humans have no preformed antibody to TKO pig cells. Intravenous immunoglobulin (IVIg) is widely used for severe infection or the treatment/prevention of antibody-mediated rejection in allotransplantation. Anti-pig antibodies in IVIg could be harmful in clinical xenotransplantation. It is unknown whether anti-TKO pig antibodies are present in IVIg. The main aim of this study was to investigate in vitro whether IVIg contains anti-TKO pig antibodies with cytotoxic effect to pig cells. Undiluted pooled human serum (HS) and five different commercial preparations of IVIg were tested for IgM and IgG binding to red blood cells (RBCs) from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and TKO pigs by flow cytometry. Complement-dependent lysis of IVIg against these pig pRBCs was measured by hemolytic assay. Pooled HS and 4 of 5 IVIg commercial preparations contained anti-pig IgG that bound to WT and GTKO pRBCs, but not to TKO pRBCs. One preparation of IVIg contained antibodies that bound to TKO pRBCs, but there was no cytotoxicity of IVIg to TKO pRBCs. The results suggest that IVIg administration to human recipients of TKO pig grafts would be safe. However, the specific preparation of IVIg would need to be screened before its administration.

Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (Triple-Knockout).

Triple-knockout (TKO) pigs (with added protective human transgenes) are likely to be optimal sources of organs for clinical organ xenotransplantation because many humans have minimal or no natural antibody to TKO pig cells. However, Old World monkeys (OWMs) have naturally-existing antibodies directed to TKO cells. We measured anti-pig IgM/IgG binding, and complement-dependent cytotoxicity to wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and TKO pig peripheral blood mononuclear cells (PBMCs) using sera from humans, several OWMs, and two New World monkeys (NWMs). Furthermore, we compared survival of GTKO (n = 5) and TKO (n = 3) pig kidneys in baboons. OWMs had significantly greater IgM binding and cytotoxicity to TKO PBMCs than humans or NWMs. Mean anti-TKO IgM was significantly higher in OWMs and significantly lower in NWMs than in humans. Cytotoxicity of OWM sera to TKO PBMCs was significantly greater than of human serum, but there was no significant difference between human and NWM sera. The median survival of TKO pig kidneys (4 days) in baboons was significantly shorter than that of GTKO kidneys (136 days) (p < 0.05). Even though considered ideal for clinical xenotransplantation, the presence of naturally-existing antibodies to TKO pig cells in OWMs complicates the transplantation of TKO pig kidneys in OWMs.

The final obstacle to successful pre-clinical xenotransplantation?

Genetically engineered pigs are now available for xenotransplantation in which all three known carbohydrate xenoantigens, against which humans have natural antibodies, have been deleted (triple-knockout [TKO] pigs). Furthermore, multiple human transgenes have been expressed in the TKO pigs, all of which are aimed at protecting the cells from the human immune response. Many human sera demonstrate no or minimal antibody binding to, and little or no cytotoxicity of, cells from these pigs, and this is associated with a relatively low T-cell proliferative response. Unfortunately, baboons and other Old World NHPs have antibodies against TKO pig cells, apparently directed to a fourth xenoantigen that appears to be exposed after TKO. In our experience, most, if not all, humans do not have natural antibodies against this fourth xenoantigen. This discrepancy between NHPs and humans is providing a hurdle to successful translation of pig organ transplantation into the clinic, and making it difficult to provide pre-clinical data that support initiation of a clinical trial. The potential methods by which this obstacle might be overcome are discussed. We conclude that, whatever currently available genetically engineered pig is selected for the final pre-clinical studies, this may not be the optimal pig for clinical trials.

Clinical Pig Kidney Xenotransplantation: How Close Are We?

Patients with ESKD who would benefit from a kidney transplant face a critical and continuing shortage of kidneys from deceased human donors. As a result, such patients wait a median of 3.9 years to receive a donor kidney, by which time approximately 35% of transplant candidates have died while waiting or have been removed from the waiting list. Those of blood group B or O may experience a significantly longer waiting period. This problem could be resolved if kidneys from genetically engineered pigs offered an alternative with an acceptable clinical outcome. Attempts to accomplish this have followed two major paths: deletion of pig xenoantigens, as well as insertion of "protective" human transgenes to counter the human immune response. Pigs with up to nine genetic manipulations are now available. In nonhuman primates, administering novel agents that block the CD40/CD154 costimulation pathway, such as an anti-CD40 mAb, suppresses the adaptive immune response, leading to pig kidney graft survival of many months without features of rejection (experiments were terminated for infectious complications). In the absence of innate and adaptive immune responses, the transplanted pig kidneys have generally displayed excellent function. A clinical trial is anticipated within 2 years. We suggest that it would be ethical to offer a pig kidney transplant to selected patients who have a life expectancy shorter than the time it would take for them to obtain a kidney from a deceased human donor. In the future, the pigs will also be genetically engineered to control the adaptive immune response, thus enabling exogenous immunosuppressive therapy to be significantly reduced or eliminated.

Is interleukin-6 receptor blockade (tocilizumab) beneficial or detrimental to pig-to-baboon organ xenotransplantation?

The interleukin (IL)-6/IL-6 receptor-α (IL-6Rα)/signal transduction and activation of the transcription 3 (STAT3) pathway plays an important role in inflammation. Anti-human IL-6Rα blockade by tocilizumab (TCZ) has been used in pig-to-baboon organ xenotransplant models, but whether it is beneficial remains uncertain. After xenotransplant, there were significant increases in both baboon and pig IL-6 in the baboon serum, especially in baboons that received TCZ before xenotransplant. In vitro observations demonstrated that human, baboon, and pig IL-6 can activate the IL-6/IL-6Rα/STAT3 pathway in human, baboon, and pig cells, respectively. Activation of the IL-6/IL-6Rα/STAT3 pathway was blocked by TCZ in human and baboon cells but not in pig cells (ie, pig IL-6R). Siltuximab (human IL-6 inhibitor) bound to both human and baboon, but not pig, IL-6 and suppressed activation of the IL-6/IL-6Rα/STAT3 pathway. These results indicate that TCZ and siltuximab do not cross-react with pig IL-6R and pig IL-6, respectively. Rapamycin partially inhibited human, baboon, and pig IL-6/IL-6Rα/STAT3 pathways and suppressed inflammatory gene expression. TCZ treatment increased serum IL-6 because it could no longer bind to baboon IL-6Rα. We suggest that increased serum IL-6 may be detrimental to the pig xenograft because it is likely to bind to pig IL-6R, resulting in activation of pig cells.

Indicators of impending pig kidney and heart xenograft failure: Relevance to clinical organ xenotransplantation - Review article.

In pig-to-baboon organ xenotransplantation, coagulation dysfunction and inflammation have been suggested to be associated with acute humoral xenograft rejection. We have evaluated platelet counts, plasma fibrinogen, and parameters of inflammation as indicators of xenograft failure in baboons with kidney and heart grafts from genetically-engineered pigs. Blood chemistry, hematologic, immune, and inflammatory parameters were measured in recipient baboons (n = 16) with organs from α1,3-galactosyltransferase gene-knockout pigs expressing human complement- and coagulation-regulatory proteins. Thrombocytopenia and reduction of plasma fibrinogen level were observed in baboons developing graft failure, and these correlated with histopathologic findings of glomerular and interstitial thrombosis, and vasculitis in the graft. Not infrequently, in baboons with pig kidney grafts, a consumptive coagulopathy developed prior to a rise in serum creatinine. In contrast, when kidney graft survival was prolonged, no changes were observed in platelet count or fibrinogen. Indicators of the inflammatory response, particularly the serum amyloid A (SAA) assay, increased when graft failure was developing. There were no changes in cellular immune parameters, e.g., T or B cell counts or phenotypes that indicated graft failure. Therefore, in clinical xenotransplantation, noninvasive parameters (e.g., platelet count, fibrinogen level, SAA) might provide more reliable indicators of impending xenograft failure than measurements of immune parameters or even of serum creatinine.