Immunosuppressive activity of the Chinese medicinal plant Tripterygium wilfordii. II. Prolongation of hamster-to-rat cardiac xenograft survival by combination therapy with the PG27 extract and cyclosporine

BACKGROUND: PG27 is an immunosuppressive fraction purified from an extract of a Chinese medicinal plant Tripterygium wilfordii, which we investigated alone and in combination with cyclosporine (CsA) in a concordant, hamster-to-rat cardiac xenotransplantation model. METHODS: Golden Syrian hamster hearts were heterotopically transplanted into the abdomen of Lewis rat recipients, which were treated intraperitoneally or orally with PG27, CsA, or both. RESULTS: Combination therapy with 30 mg/kg(day of PG27 and CsA at 10 mg/kg/day successfully suppressed acute hamster-to-rat cardiac xenograft rejection. Treatment with PG27 or CsA alone was ineffective. Among several effective combinations, the best regimen involved PG27 at 30 mg/kg/day and CsA at 5 mg/ kg/day from days 8 to 35 and then CsA at 5 mg/kg/day from days 36 to 100, which produced 100% survival beyond 100 days. CsA suppressed the heterospecific lymphocytotoxic antibody response and inhibited IgG but not IgM xenoantibody production (which led to xenograft rejection), whereas PG27 alone did not prevent antibody production. The PG27/CsA combination blocked the lymphocytotoxic antibody response and IgG and IgM xenoantibody production induced by cardiac xenotransplantation. CONCLUSIONS: PG27 combined with CsA substantially prolonged hamster-to-rat cardiac xenograft survival, as well as completely inhibiting xenoantibody production.     

Xenograft survival in two species combinations using total-lymphoid irradiation and cyclosporine

Total lymphoid irradiation (TLI) has profound immunosuppressive actions and has been applied successfully to allotransplantation but not xenotransplantation. Cyclosporine (CsA) has not generally permitted successful xenotransplantation of organs but has not been used in combination with TLI. TLI and CsA were given alone and in combination to rats that were recipients of hamster or rabbit cardiac xenografts. Combined TLI and CsA prolonged survival of hamster-to-rat cardiac xenografts from three days in untreated controls to greater than 100 days in most recipients. TLI alone significantly prolonged rabbit to rat xenograft survival with doubling of survival time. However, combined treatment did not significantly prolong rabbit-to-rat cardiac xenograft survival compared with TLI alone. The hamster and rat are phylogenetically closely related. Transplants from hamsters to rat are concordant xenografts since the time course of unmodified rejection is similar to first-set rejection of allografts. Although the rabbit-to-rat transplant is also between concordant species (average survival of untreated controls: 3.2 days) the rabbit and rat are more distantly related. These results suggest that TLI is an effective immunosuppressant when applied to cardiac xenotransplants in these animal models; that the choice of species critically affects xenograft survival when TLI and/or CsA are used for immunosuppression; and that the closely related species combination tested has markedly prolonged (greater than 100 days) survival using combined TLI and CsA.     

Failure of anti‐Forssman antibodies to induce rejection of mouse heart xenografts

The Forssman antigen has been proposed to be a target for the xenograft reaction in selected species combinations, including the rat and mouse, which are Forssman-negative and -positive species respectively. The mouse represents an important experimental model for a variety of immune-mediated disease processes, and the availability of a simple, inexpensive target antigen could provide an important tool for studying a selected portion of the immunologic basis for the rejection of xenografts. We have examined the potential that antibodies directed against mouse Forssman antigen could cause the hyperacute rejection of mouse heart xenografts in naive rat recipients. The Forssman antibodies tested included rat anti-mouse (R-anti-M) antiserum, R-anti-M antiserum depleted of anti-Forssman (anti-F) antibodies, rat anti-sheep red blood cell (SRBC) antiserum containing anti-F antibodies and a rat monoclonal anti-F IgM antibody. Our results demonstrate that the R-anti-M antiserum at day 4 post transplantation displayed significant titers (1:512–4096) of hemagglutinating antibodies for SRBC and mild to moderate levels of IgM that specifically binds to Forssman glycolipid (GalNAcαl–3GalNAcβl–3Galαl–4Galβ1–4Glcβ1–1ceramide) as measured by an enzyme-linked immunosorbent assay (ELISA). Passive transfer of the R-anti-M serum to rats receiving mouse cardiac grafts immediately after transplantation caused hyperacute rejection of the xenografts. Sequential immunoabsorption of R-anti-M sera with SRBCs resulted in total removal of the anti-Forssman activity (as defined by negative hemagglutination titer and minimal binding to Forssman glycolipid in ELISA). The anti-F Ab-depleted R-anti-M antisera, however, retained the capacity to induce hyperacute rejection of the mouse hearts [n = 6, median survival time (MST) 13 min] when passively transferred to rat recipients. Anti-Forssman antibodies induced by immunization of LEW rats with SRBCs or a rat anti-Forssman monoclonal antibody, mAb M.1.22.25, exhibited substantial anti-Forssman activity (hemagglutinating titer 1: 512–4096 and moderate-to-strong binding to Forssman glycolipid in ELISA respectively). These antibodies also failed, however, to trigger hyperacute rejection of mouse cardiac xenografts. In conclusion, our results suggest that the rat anti-Forssman antibodies, including those stimulated by mouse cardiac xenografts, do not appear to play a role in the immediate (hyperacute) rejection of mouse heart xenografts.     

Evidence that deoxyspergualin prevents sensitization and first-set cardiac xenograft rejection in rats by suppression of antibody formation

This study provides evidence of antibodies playing an important role in hamster-to-rat cardiac xenograft rejection and discusses the use of 15-deoxyspergualin (DSG) to suppress this first-set rejection, as well as hyperacute rejection induced by sensitization. The effect of recipient splenectomy (Spx) as an adjuvant to DSG to control first set xenograft rejection was also examined. When hyperimmune serum was taken from control recipients at rejection time and injected i.v. into new recipients of cardiac xenografts, hyperacute graft rejection resulted. Survival depended on the amount of serum injected and ranged from 14.7 +/- 2.5 min with 3 ml of serum to 233.3 +/- 61.1 min with 0.5 ml. Experiments on first-set xenograft rejection revealed that a dose of 2.5 mg/kg/day DSG could prolong xenograft survival from 3.4 +/- 0.5 days in untreated controls to 9.5 +/- 2.6 days (P less than 0.01). A dose of 5 mg/kg/day DSG, though it increased graft survival to 16.4 +/- 5.9 days, proved to be toxic to the recipients. Spx alone prolonged xenograft survival to 5.2 +/- 0.4 days, and, when combined with 2.5 mg/kg/day DSG administration, prolonged graft survival to 22.1 +/- 5.5 days (P less than 0.01 vs. DSG alone). The appearance of cytotoxic antibodies was delayed, and titers decreased from 1:256 in untreated controls to 1:16-1:64 both in the group that underwent Spx only and in the group that received 2.5 mg/kg/day DSG. Combined treatment suppressed antibody response for more than two weeks. Experiments on hyperacute rejection induced by sensitization revealed that 1 ml of hamster whole blood transfused into prospective heart recipients 1 week before grafting resulted in graft loss in 18.2 +/- 6.1 min. Pretransplant transfusion and concomitant daily administration of 5 mg/kg/day DSG until one day after grafting not only prevented hyperacute rejection but prolonged graft survival to 7.0 +/- 0.7 days. This survival was significantly longer than with DSG alone (4.2 +/- 0.8 days, P less than 0.01). We concluded that the marked suppression of antibody formation by DSG plays a major role in preventing first-set xenograft rejection and hyperacute rejection induced by sensitization.     

A crucial effect of splenectomy on prolonging cardiac xenograft survival in combination with cyclosporine

In this study we investigated the effect of splenectomy in combination with cyclosporine (CsA) on survival of heterotopic cardiac xenografts from hamster to rat. A 12-fold prolongation of mean cardiac xenograft survival, to 41 days, was accomplished with the combined therapy. In both untreated controls and CsA-treated recipients rejection occurred in 3 days. Splenectomy by itself prolonged xenograft function to 5 days. Evidence of humoral-mediated rejection in this cross-species combination was given for the extensive thrombosis and hemorrhage in the subepicardial area, the appearance of lymphocytotoxic titers just before graft function ceased, and the presence of IgM deposits in subepicardial vessels of the xenograft. CsA by itself could not modify this pattern of rejection. Splenectomy decreased antibody formation significantly and rejection became more cellular in nature. The regimen of splenectomy in association with CsA suppressed antibody titers and produced a CsA dose-dependent prolongation of xenograft survival. Thus, a complementary or synergistic effect is the result of the immunosuppressive regimen of splenectomy and CsA in hamster-to-rat cardiac xenografts. In this study the effect of splenectomy in controlling the humoral response in concordant xenografts and its role in future clinical xenografting is emphasized.     

One-year monkey heart xenograft survival in cyclosporine-treated baboons. Suppression of the xenoantibody response with total-lymphoid irradiation.

The shortage of cadaveric human organs for transplantation could be alleviated by the use of xenografts. Long-term (> one-year) survival of xenografts in humans or experimental animals has not been achieved with previous immunosuppressive protocols. Poor results in xenotransplantation compared with allotransplantation have been attributed to a more potent antibody response rather than to cell-mediated responses. To investigate these issues a concordant heterotopic cardiac xenograft model was developed in conjunction with cyclosporine and/or total-lymphoid irradiation. Concordant models permit examination of xenoantigen induced antibody and cell-mediated responses since preformed humoral factors (in discordant models) do not cause hyperacute rejection. Four groups of baboon recipients received cervical heart transplants from cynomolgous monkeys. Group I (n = 2), untreated, mean survival (MS) = 6 days; group II (n = 5), CsA and methylprednisolone, MS = 25 days; group III (n = 3), preoperative TLI, MS = 29 days; group IV (n = 6), preoperative TLI and CsA+MP, MS = 255 days (> 77, > 108, > 142, 184, > 480, 540 days). Heart xenografts of CsA-MP-treated recipients appear to be destroyed predominantly by antibody (IgM)-mediated processes whereas cell-mediated rejection is likely the major reaction in TLI-treated recipients. CsA-MP-treated recipients had early immunohistochemical evidence of antibody and complement-mediated rejection (deposition of IgM and complement but not IgG on heart xenografts). In contrast IgM and complement deposits were not detected on heart xenografts in TLI- and TLI-CsA-treated recipients. IgG xenoantibodies were only detected on the two rejected heart grafts of TLI-CsA-treated recipients. CsA-MP-treated recipients rapidly developed high xenoantibody titers (1:256 to 1:512) that immediately preceded rejection. In contrast, TLI-treated animals developed lower levels of xenoantibody (< or = 1:8) and TLI-CsA-treated recipients had no detectable xenoantibody during the initial three months after transplantation (and titers no greater than 1:8 thereafter.) The lack of xenoantibody was likely not due to a generalized inhibitory effect of the immunosuppressants on B cell function since all classes of serum immunoglobulins were in the normal range. Intragraft cytolytic lymphocyte activity was detected in rejecting TLI- and TLI-CsA-treated recipients but could not be detected in xenografts of CsA-MP-treated recipients. One explanation for these data is that TLI (either directly or indirectly) induces a state of specific B cell unresponsiveness to monkey xenoantigens, thereby preventing IgM mediated rejection in the third week after transplantation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tolerance of T-independent xeno-antibody responses in the hamster-to-rat xenotransplantation model is species-restricted but not tissue-specific

Control of early acute xenograft rejection xenoreactions in the hamster-to-Lewis rat xenotransplantation model with cyclosporine (CsA) and leflunomide subdues early T-independent xenoreactivity and uncovers a late immune response that can be controlled by CsA alone. We had attributed this acquired responsiveness to CsA to the induction of tolerance of T-independent xeno-antibody responses in the recipient and recently reported that this tolerance is species-specific. Here we have further characterized the specificity and nature of this tolerant state. Lewis rats transplanted with either hearts, skin, kidney or spleen/pancreas from Golden Syrian hamsters were treated with leflunomide (5 mg/kg/day by gavage) for 14-21 days and CsA (20 mg/kg/day by gavage) continuously from the day of transplant. Some Lewis rats received a second graft of hearts or skin from Golden Syrian hamsters (day 21-30 after first transplant), and a third heart graft from Balb/c mice (day 60 after the first transplant). Serum was harvested and the titers of xenoreactive antibodies were quantified by flow cytometry. All grafts were harvested at the end of each experiment and examined by histological and immunohistochemical methods. The combination of CsA and leflunomide was able to completely inhibit the rejection of kidney, spleen and pancreas xenografts in this hamster-to-rat xenotransplantation model. In addition, only a transient treatment with leflunomide was necessary, and long-term graft survival could subsequently be maintained by CsA alone. Histological examination of these grafts at > 80 days post-transplantation indicated minimal signs of rejection. These immediately vascularized organs induced T-independent B-cell tolerance, so that second grafts of hamster hearts and skin could be maintained with CsA alone. Under the same immunosuppressive regimen, only four out of nine Lewis rats exhibited long-term hamster skin survival, probably reflecting the increased immunogenicity of the skin compared with other vascularized grafts. Nonetheless, all rats that did not reject the hamster skin graft also did not reject the hamster heart while on CsA alone. Finally, we demonstrate that the tolerant state could be maintained for up to 30 days in the absence of xenograft. The vigorous T-independent antibody response that mediates acute xenograft rejection in the hamster-to-rat model can be tolerized by the immunosuppressive regimen of CsA and leflunomide. The lack of organ specificity for the induction of this tolerance suggests that the xenoantigens inducing tolerance may be common endothelial cell antigens. Finally, the presence of the xenograft has been previously shown to be critical for the induction of T-independent B-cell tolerance, however, the tolerant state is relatively stable and persists after the removal of the xenograft.     

Pre-transplant blood transfusion induces tolerance to hamster cardiac xenografts in athymic nude rats

Abstract: The effects of pre‐transplant blood transfusion vary from induction of antibodies and accelerated graft rejection, to prolonged survival and even tolerance. The beneficial ‘transfusion effect’ in allotransplantation is believed to be merely T‐cell mediated. In xenotransplantation, T‐cell independent mechanisms form a major hurdle. In this study we investigated the effects of pre‐transplant hamster blood transfusion on the survival of hamster cardiac xenografts in T‐cell deficient athymic nude rats. Nude rats rejected xenografts after 3.8 ± 0.5 d (n = 8), and immunocompetent Lewis rats after 4.0 ± 0.5 d (n = 8), following a similar IgM response (P = NS). Hamster blood transfusion 3 d before transplantation in nude rats led to an IgM response and long‐term xenograft survival in 17/20. Timing was of importance: blood transfusion 7 d before transplantation resulted in 45% long‐term xenograft survival (n = 20). Injection of purified hamster erythrocytes, leukocytes or minced heart also led to survival of xenografts for > 100 d in nude rats, but not in all cases. Second xenografts transplanted to long‐term survivors did not provoke an IigM response, and were accepted for > 100 d (n = 4). Transfer of serum from long‐term survivors to untreated nude rats resulted in survival of xenografts for > 100 d (n = 4). In Lewis rats pre‐transplant blood transfusion induced hyperacute rejection of xenografts after 158 ± 128 min (n = 8, P < 0.01). We conclude that pre‐transplant hamster blood transfusion can induce long‐term survival of hamster cardiac xenografts in T‐cell deficient athymic nude rats. This blood transfusion effect is mediated by humoral factors and can be transferred by serum. Elucidation of underlying mechanisms might provide some insight into xenotransplantation nonresponsiveness of T‐cell independent immunefactors.     

Heart and liver xenotransplantation under low-dose tacrolimus: graft survival after withdrawal of immunosuppression

BACKGROUND: The hamster-to-rat xenotransplantation model is a useful model to investigate the features of extended host response to long-surviving xenografts. Early xenoantibody responses are T-cell independent and resistant to tacrolimus. Treatment with the combination of mofetil mycophenolate plus FK506 avoids acute xenograft rejection completely, but after withdrawal of immunosuppression hamster grafts are rejected by a process called late xenograft rejection (LXR). METHODS: Hamster hearts and livers were transplanted into Lewis rats. Grafted rats were treated with mofetil mycophenolate (25 mg/kg/day) for 8 days and FK506 (0.2 mg/kg/day) for 31 days. Serum IgM and IgG levels were determined by flow cytometry and interferon-gamma levels by ELISA. IgM, IgG, and C3 deposits were measured in tissue by immunofluorescence, and leukocyte infiltration was measured by immunoperoxidase staining. Results. Survival of heart and liver xenografts in the rats was 48+/-4 days and 63+/-8 days, respectively. After cessation of all immunosuppression, hearts were rejected in 18+/-4 days and livers in 33+/-8 days. Production sequences of xenoantibodies in the two organs differed substantially, especially 7 days after transplantation and at the moment of rejection. Quantification of interferon-gamma levels indicated that there were no significant changes after transplantation. Histological and immunohistochemical studies showed signs of humoral mechanism of LXR in rats undergoing heart transplantation and cellular mechanism of LXR in those that received a liver transplant. Conclusions. These observations suggest that rejection in the hamster-to-rat heart xenotransplantation model is mediated by a T cell-independent B-cell response to which a T cell-dependent B-cell response is added in LXR. In the liver xenotransplantation model, our hypothesis is that LXR is mediated by a mixed cell mechanism, involving lymphocytes CD4+ CD45RC+, macrophages, and cytotoxic T lymphocytes. In summary, we have demonstrated and compared the peculiar features of LXR in two different organs.     

Induction of regulatory cells and control of cellular but not vascular rejection by costimulation blockade in hamster-to-rat heart xenotransplantation

BACKGROUND: In heart allograft in the rat, a sustained costimulation blockade with CTLA4Ig prevents alloreactive T-cell activation and promotes a long-term graft survival through the action of tolerogeneic dendritic cells. It is unclear whether similar mechanisms might occur after xenotransplantation. To test that hypothesis, we have analyzed the action of CTLA4Ig in a model of CD4(+)T cell-mediated xenograft rejection. METHODS: Hamster hearts were transplanted into LEW.1A rats receiving an accommodation-inducing treatment consisting of a short course administration of LF15-0195 and a daily administration of cyclosporine A (CSA). To achieve long-term delivery of CTLA4Ig, an intravenous administration of an adenovirus vector coding for mouse CTLA4Ig (Ad-CTLA4Ig) was added to the accommodation induction protocol. On day 40 post-transplantation, rejection was induced by CSA withdrawal. In other xenograft recipients, CD28/B7 costimulation was inhibited at that time only by injections of CTLA4Ig or anti-CD28 antibodies. Graft survival, immunohistology, as well as development of antibodies and regulatory cells were examined. RESULTS: Xenografts survived 6 days after CSA withdrawal in controls and were rejected, as previously described, through the action of CD4(+) xenoreactive T cells. Interfering with CD28/B7 costimulation inhibited this xenoreactive T cell response and delayed rejection to day 10. In recipients that had received Ad-CTLA4Ig, survival was prolonged to day 19 and this was accompanied by the appearance of regulatory cells exhibiting non-donor-specific suppressive activity dependent on IL-2, NO, and IDO. These regulatory cells were different from those previously identified after Ad-CTLA4Ig administration in heart allograft in the rat. In these recipients, rejection occurred as a consequence of an evoked anti-donor IgM response and complement activation and not of a cellular rejection as complement inhibition with cobra venom factor further prolonged xenograft survival. CONCLUSION: CD28/B7 blockade delays CD4(+) T cell-mediated rejection after CSA withdrawal in accommodated recipients of hamster heart xenografts. In addition, a sustained expression of CTLA4Ig has the potential of inducing cellular regulatory mechanisms. However, such treatment does not prevent the development of xenoreactive IgM antibodies that participate in vascular rejection processes in a complement-dependent manner.     

Induction of long-term survival of hamster heart xenografts in rats.

The aim of this study was to determine the mechanisms responsible for concordant xenograft rejection using the hamster-to-rat heart graft model. Even though it was known that rat CD4 positive T cells proliferated to hamster stimulators in mixed lymphocyte reactions, the depletion of CD4 positive T cells in rat recipients did not lead to an extension of xenograft survival. Suppression of T cell immunity using other monoclonal antibodies or cyclosporine also failed to improve survival. Only by depleting complement with cobra-venom factor could hamster xenograft survival be prolonged, and long-term survival was achieved by combining CsA with COF. High-antibody titers to hamster cells were found after transplantation of hamster hearts, and evidence is presented that rejection of these "concordant" xenografts is mediated primarily by antibody-complement mechanisms. The antihamster antibodies were produced in the absence of T cell help, which suggests that antibody-mediated graft destruction cannot be inhibited by suppression or depletion of T cells. Pharmacologic depletion of complement for the clinical application of concordant xenografts is a promising avenue of future research.     

Restoration of T cell responsiveness to interleukin-2 in recipients of pancreatic islet xenografts treated with cyclosporine

The immunosuppressive mechanism of cyclosporine (CsA) was studied using pancreatic islet xenotransplantation. A dose of 40 mg/kg/day CsA significantly prolonged survival of hamster islet grafts in BDE rats to 14.0 +/- 7.1 days compared with 2.0 +/- 0.9 days in controls (P less than 0.01), and antihamster lymphocytotoxic antibody was not detected in recipient sera even after rejection. Responses of spleen cells to pokeweed mitogen (PWM) and to phytohemagglutinin (PHA) were inhibited at least 21 days after transplantation in recipients treated with CsA. The addition of exogenous interleukin-2 (IL-2) to spleen cell cultures on day 7 had no effect on the impaired response to PHA. At the time of graft rejection (day 14), the T cell response to PHA recovered with the addition of IL-2. However, significant changes were not observed in the ratio of spleen cell subpopulations, which were studied with monoclonal antibodies of W3/ 13, OX-12, W3/25, and OX-8. From these results we conclude that CsA can significantly prolong islet xenograft survival in closely related species, and inhibit the production of humoral antibodies and T cell responses. At the time of graft rejection, recipient T cell responsiveness to IL-2 was restored. This suggest that islet xenograft rejection is caused by the recovery of cellular immunity in recipients treated with CsA.     

Inhibition of rejection of hamster-to-rat heart xenografts

Prolonged survival of concordant organ xenografts as typified by hamster-to-rat heart transplants is difficult to produce. Studies have revealed that T cells are not primarily involved in rejecting such xenografts and that the rat recipients produce high titres of lytic anti-hamster antibodies. In this study, 200 hamster-to-rat cardiac xenografts performed in 30 different experiments revealed that cyclophosphamide (CyP) and cyclosporin A (CyA) could inhibit this antibody production. CyP alone was relatively ineffective in prolonging graft survival (the median survival time was 14 days versus 3 days in untreated controls). Combining CyP and CyA virtually abolished rejection in this model. Four critically timed doses of CyP combined with continuous CyA resulted in recipients not producing anti-hamster antibodies, despite cessation of CyP therapy, and prolonged graft survival time (median survival time was more than 100 days). Cessation of CyA at 60 and 100 days resulted in the rejection of the xenografts and the appearance of the rat anti-hamster antibodies. Xenografts in recipients given only one or two doses of CyP (and continuous CyA) had a median survival time of 7 and 12 days respectively. However xenograft rejection in rats given only 1 or 2 doses of CyP could be averted by complement depletion using a 3-week course of cobra venom factor (CoF) starting on day 4 or day 7 post-transplantation respectively. Discontinuation of CoF after 3 weeks did not result in graft rejection. These results showed that immunosuppressive therapies directed at inhibiting antibody production may be of value in preventing rejection of concordant xenografts. Short-term complement depletion could rescue xenografts from rejection such that rescued grafts appear to be accommodated.     

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

The role of graft and host accommodation in a hamster-to-rat cardiac transplantation model

BACKGROUND: We evaluated the importance and mechanism of graft and host accommodation in hamster-to-rat cardiac xenotransplantation models. METHODS: To evaluate graft accommodation, accommodated hamster grafts (Group 2) were transplanted to na?ve host rats treated with FK506, and compared with na?ve hamster grafts (Group 1). To evaluate host accommodation, three groups were evaluated: naive hamster hearts were transplanted to na?ve hosts treated with FK506 (Group 3: 0.5 mg/kg, Group 4: 1.0 mg/kg) and splenectomy, and compared with accommodating hosts (Group 5) with FK506 0.5 mg/kg and splenectomy. We examined graft survival, histopathology, antihamster antibodies and B-1 cells in blood. RESULTS: Graft survival in Group 2 (3.4+/-0.9 days) was not significantly different from that in Group 1 (2.8+/-0.4 days). Graft survival in Groups 4 and 5 (>30 days) was significantly prolonged compared with that in Group 3 (6.0+/-0.7 days). Histopathology of Groups 1-3 showed humoral rejection, whereas Groups 4 and 5 showed normal histology and expression of protective genes. In Groups 1-3, antihamster immunoglobulin (Ig) M and B-1 cells increased significantly compared to Groups 4 and 5, where IgM and B-1 cells remained low or were reduced. CONCLUSIONS: Host accommodation was more important than graft accommodation. Accommodating grafts expressing protective genes were rejected with an elevation of both IgM and B-1 cells. In accommodated hosts, both IgM and B-1 cells decreased, suggesting that B-1 cells may be responsible for the production of antihamster antibodies. These results suggest that sufficient suppression of B-1 cells, resulting in decreased titers of antihamster antibodies, may play an important role in host accommodation.     

Hamster cardiac xenografts are protected against antibody mediated damage, early after transplantation to Lewis rats

Abstract: Antibodies play a crucial role in the rejection of xenografts. We tested the hypothesis that xenografts are protected against antibody-mediated attack early after transplantation in a concordant model. We investigated the role of xenoreactive antibodies as a stimulus for protection and the effects of a total blockade of the antibody response by the leflunomide analog malononitrilamide 279. Hamster cardiac xenografts were transplanted to Lewis rat recipients. Second transplants and retransplants of xenografts were performed to untreated rats that had a xenograft in place for 3 d. Untreated rats rejected hamster cardiac xenografts after 4.0 ± 0.0 d. Significant levels of anti-donor IgM, as measured by flowcytometry, were present on day 3 after transplantation (11.2% ± 2.8 vs. 1.2% ± 0.0 on day 0, P  < 0.001). 'Fresh' second xenografts transplanted to rats that had a first xenograft in place for 3 d and had anti-hamster antibodies, underwent hyperacute rejection. The first xenografts remained functioning. Xenografts that were removed on day 3 from untreated rats and then retransplanted remained functioning. Xenografts that were removed on d 3 from rats that had been treated with malononitrilamide 279, 15 mg/kg/d and were retransplanted underwent hyperacute rejection. IgM levels at the time of removal were 1.1% ± 0.5 in these rats and not different from baseline ( P  = 0.96). We conclude that xenografts are protected against antibody-mediated damage early after transplantation. The presence of anti-donor antibodies might be an essential stimulus for the induction of protection. There seems to be a delicate balance between the injurious and protective effects of antibodies. Treatment strategies that are designed to block antibody formation completely might prevent the induction of protection.     

Functional cooperation of xenoproteins after hamster-to-rat liver transplantation: With particular reference to hamster C3 and secretory component for rat IgA

Abstract: Long-term survival after hamster-to-rat liver xenotransplantation has provided the opportunity to study the posttransplantation source of major serum proteins and the functional consequences of several different receptor-ligand interactions, where one or the other is a xenogeneic protein. We report here that serum albumin, α-1-antitrypsin, complement component 3, and other acute phase reactants switch from recipient to donor origin during the first week after transplantation while serum immunoglobulins remain largely that of recipient. Despite the disparate source of complement (hamster) and immunoglobulins (rat), these two proteins were able to cooperate effectively to produce lysis of sheep red blood cells. Moreover, rat IgA was successfully processed by hamster hepatocytes and biliary epithelial cells, being present in the bile of successful liver xenograft recipients within one day after transplantation. The ability of these liver xenograft recipients to survive long-term in conventional and viral-free animal facilities without grossly obvious morbidity or unusual susceptibility to stress, suggests that xenogeneic proteins are able to successfully interact with several different physiologic I systems in the hamster-to-rat combination.     

Pathogenesis and pathology of delayed xenograft rejection in pig-to-rhesus monkey cardiac transplantation

It has been recognized that delayed xenograft rejection (DXR) is the major barrier to the acceptance of xenotransplantation after overcoming hyperacute rejection. OBJECTIVES: This study sought to investigate the pathogenesis and pathology of delayed xenograft rejection following pig-to-rhesus monkey heart xenotransplantation. METHODS: Heterotopic xenogeneic heart transplants in the abdominal cavity were performed using piglet donors to four monkey recipients. Complete complement depletion was achieved in the recipients with repetitive doses of high-activity cobra venom factor (Y-CVF). The recipients were immunosuppressed with a combination of cyclosporine, cyclophosphamide, and steroids. Sera were analyzed for C3 and C4 levels and complement activity and anti-pig endothelial xenoantibody. The grafts were examined histopathologically and immunohistochemically for C3, C4;C5b-9, IgM, IgG, tumor necrosis factor-alpha (TNF-alpha), intercellular adhesion molecule-1(ICAM-1), CD57(NK cells), CD68 (macrophages), CD4, and CD8. RESULTS: Xenografts survived 8, 10, 13, and 13 days respectively, all developing DXR. Venous thrombosis was the outstanding feature within DXR xenografts, complicated by interstitial edema, local hemorrhage, myocardial necrosis, and mild to moderate cellular infiltration. The serum C3 levels and complement activity decreased to almost 0 from the day of transplantation due to treatment with Y-CVF. The C4 level began to decrease 2 to 4 days before the cardiac xenografts lost their function. Anti-pig endothelial xenoantibody also decreased after transplantation, slightly increasing during DXR. All rejected xenografts showed C3, C4, C5b-9, IgG, and IgM deposits to various degrees. Large numbers of macrophages (50% of total leukocytes) infiltrated the entire xenograft with a few natural killer cells (8% to 10%), as well as some CD4+ T cells (15%) and CD8+ T cells (25%). Upregulation of ICAM-1 on graft endothelial cells and TNF-alpha in the interstitium were also demonstrated in the rejected heart. CONCLUSION: Both humoral and cell-mediated immunologic reactions may play important roles in the pathogenesis of DXR. Besides C3, C4, C5b-9, IgM, and IgG destroying the xenograft, NK cells, macrophages, and CD4+ and CD8+ T cells may further aggravate the development of DXR.     

Importance of the Gal alpha1-3 Gal antigen in discordant islet xenotransplantation: immunosuppression, which inhibits porcine islet xenograft rejection in ordinary mice, is equally effective in Gal-knockout mice

BACKGROUND: Islet xenotransplantation will most likely be performed in diabetic patients treated with immunosuppressive drugs. The importance of the galactosyl alpha(1-3) galactose (Galalpha1-3Gal) antigen in immunosuppressed islet xenograft recipients has not been studied. METHODS: Fetal porcine islet-like cell clusters (ICCs) were transplanted into the renal subcapsular space of both Gal-knockout mice and ordinary mice. Transplantations were performed in untreated mice and mice immunosuppressed with cyclosporine A (CsA) plus 15-deoxyspergualin (DSG). Studies were also performed in immunosuppressed Gal-knockout mice that had been actively immunized against Galalpha1-3Gal. Evaluation was performed 12 days after transplantation using morphologic techniques. The levels of serum immunoglobulin (Ig)G and IgM to the Galalpha1-3Gal antigen or to the ICCs were determined. RESULTS: No difference in the morphologic appearance could be seen between ordinary mice and Gal-knockout mice. No deposits of IgG, IgM, or C3 could be detected. Almost no difference could be seen between immunosuppressed Gal-knockout mice and immunosuppressed ordinary mice. In immunosuppressed, immunized Gal-knockout mice, the results were similar. In ordinary mice treated with CsA+DSG, the levels of anti-Gal IgM were lower than they were in untreated mice, whereas the levels of anti-Gal IgG were similar. In Gal-knockout mice (including immunized animals) treated with CsA+DSG, the levels of anti-Gal IgG and IgM were lower than they were in untreated Gal-knockout mice. CONCLUSIONS: After renal subcapsular transplantation, antibodies against Galalpha1-3Gal have no major influence on islet xenograft rejection in the pig-to-mouse model. Immunosuppression, which inhibits rejection in the pig-to-mouse model, is equally effective when transplantation is performed across the Galalpha1-3Gal barrier.