Canine Renal Allograft Survival Research Articles

Bioengineering the Vasculature of Renal Allografts to Prevent Immune Activation: “Immunocloaking”

Introduction: We have previously demonstrated the feasibility of bioengineering the luminal surface within renal allografts without adversely affecting function. The result was prolonged canine renal allograft survival in the absence of systemic immunosuppression or immunologic manipulation of the recipient. Referred to as “immunocloaking” our approach entails coating the vascular surfaces within kidneys with a nano-barrier membrane, NB-LVF4, that is composed of laminin, vitrogen, fibronectin, and type IV collagen. Several integrin complexes are involved in the attachment of NB-LVF4 to the surface of vascular endothelial cells (VEC). NB-LVF4 provides an apical surface that remains non-thrombogenic and non-immunogenic. The goal is to interrupt the normal interface by providing a physical barrier between recipient immune cells in circulation and the renal allograft vasculature. We now report the underlying immunological mechanism involved in immunocloaking. Methods: Proliferation assays (n=5) were performed using responding peripheral blood mononuclear cells (MNCs) stimulated with autologous lymphocytes and untreated confluent VEC (controls) and with confluent immunocloaked VEC (test). Following 72 hours of stimulation, supernatants were collected from each triplicate well and tested for cytokine and chemokine responses using the Luminex xMap platform. Early T cell activation was measured using CD4+, CD69+ detection by flow cytometry. Results: Immunocloaking resulted in statistically significantly inhibition of the cytokines/chemokines: IL-1β, IL-6, γ-IFN, IL-2, TNF-α, CD-69, MIG and MIP-1α (p< 0.5). The inhibition of the cytokines produced by antigen presenting cells, IL-1β, IL-1β, MIP-1α and γ-IFN suggest that antigen presentation was prevented. Similarly, the inhibition of markers of T cell activation, IL-6, IL-2, CD-69 and MIG suggest the blockade of T cell mediated responses.Figure: [Immunocloak Cytokine/APC Analysis]Conclusions: Immunocloaking with the NB-LVF4 provides a mechanism to eliminate the allo-recognition that normally occurs immediately on reperfusion. The lack of allo-responses upon reimplantation could provide a new window of opportunity to introduce adjunct therapies to support tolerance induction.

Pretransplant Kidney-Specific Treatment to Eliminate the Need for Systemic Immunosuppression

Despite significant side effects, chronic systemic immunosuppression remains the backbone of clinical transplantation. We investigated the feasibility of preventing early allorecognition in canine renal allografts using a nonsystemic pretreatment. The renal vasculature was treated with a bioengineered interface consisting of a nano-barrier membrane during 3 hr of ex vivo warm perfusion. Preliminary feasibility of the immunocloaking technology was established by the following criteria: it is possible to achieve approximately 90% coverage of the vasculature with nano-barrier membrane after 3 hr of ex vivo warm perfusion; covering the luminal surfaces prevents allorecognition as determined by mixed lymphocyte-vascular endothelial reaction; covering the luminal surfaces does not negatively affect renal function as determined by autotransplant outcomes; and graft rejection is significantly postponed in canine kidneys treated with the immunocloaking technology. In the absence of systemic immunosuppression, untreated control dogs experienced a mean onset of rejection on day 6, whereas in the treated dogs with modified renal vascular luminal surfaces, the mean onset of rejection was significantly delayed until day 30. The ability to postpone, or eventually eliminate, the allorecognition that occurs immediately on reperfusion could provide a new window of opportunity to introduce adjunct therapies to support tolerance induction. To our knowledge, this is the first time significantly prolonged canine renal allograft survival has been achieved in the absence of systemic immunosuppression or immunologic manipulation of the recipient.

Immunosuppressive activity of FTY720, sphingosine 1-phosphate receptor agonist: I. Prevention of allograft rejection in rats and dogs by FTY720 and FTY720-phosphate

FTY720, a new class of immunomodulator, induces lymphopenia by sequestration of circulating lymphocytes into secondary lymphoid tissues. FTY720 at 0.1 to 1 mg/kg significantly prolonged the allograft survival in a dose-dependent manner and showed a marked synergistic effect in combination with cyclosporine (CsA) in rat skin and cardiac allograft models. In addition, the canine renal allograft survival was significantly prolonged by combination therapy with FTY720 at 0.03 to 1 mg/kg and CsA at 10 mg/kg as compared with monotherapy of FTY720 or CsA. By contrast, the combination therapy with CsA and azathioprine or CsA and mycophenolate mofetil resulted in only an additive effect in rat skin allograft. When FTY720 was administered to rats, FTY720 was metabolized by omega-oxidation of the octyl side chain, and beta-oxidation subsequently, or phosphorylated by sphingosine kinase. Omega- and beta-oxidized 4 metabolities of FTY720 at 10 mg/kg i.v. showed neither lymphopenia nor immunosuppressive activity in rat skin allograft. On the other hand, ( S)-enantiomer of FTY720-phosphate at 0.1 and 1 mg/kg intravenously induced a marked lymphopenia and significantly prolonged the allograft survival in the rat allotransplantation. From these results, it is suggested the lymphopenia and the immunosuppression induced by FTY720 administration is due to the agonistic activity against SIP receptors of the active metabolite, ( S)-FTY720-phosphate.

Prolongation of canine renal allograft survival by combining tacrolimus with antimetabolic agents

The synergistic combination of immunosuppressive drugs is thought to provide a better therapeutic index with fewer side effects than single-drug therapy. Tacrolimus (FK 506), an inhibitor of interleukin-2 (IL-2) synthesis, is a potent immunosuppressive agent that has various adverse effects including nephrotoxicity, neurotoxicity, diabetogenesis, and infectious complications.1 Experimental models have shown that antimetabolic agents are good candidates for combination with tacrolimus.2,3 Several antimetabolic agents, azathioprine (AZA), mizoribine (MIZ), mycophenolate mofetil (RS), and ethyl o-[N-(p-carboxyphenyl)carbamoyl]-mycophenolate (CAM), were tested alone and in combination with tacrolimus to determine if they could improve canine renal allograft survival. while reducing immunosuppression-related adverse events.

Cell migration and chimerism after whole-organ transplantation: The basis of graft acceptance

Improvements in the prevention or control of rejection of the kidney and liver have been largely interchangeable (1, 2) and then applicable, with very little modification, to thoracic and other organs. However, the mechanism by which anti rejection treatment permits any of these grafts to be “accepted” has been an immunological enigma (3, 4). We have proposed recently that the exchange of migratory leukocytes between the transplant and the recipient with consequent long-term cellular chimerism in both is the basis for acceptance of all whole-organ allografts and xenografts (5). Although such chimerism was demonstrated only a few months ago, the observations have increased our insight into transplantation immunology and have encouraged the development of alternative therapeutic strategies (6).

Cell migration and chimerism after whole-organ transplantation: The basis of graft acceptance

Cell migration and chimerism after whole-organ transplantation: The basis of graft acceptance

Effects of combination cyclosporine/mizoribine immunosuppression on canine renal allograft recipients.

Heterotopic renal allografts following bilateral nephrectomies were placed in 21 healthy mongrel dogs. One group of 11 dogs received cyclosporine (5 mg/kg/24 hr, orally), and 1 group of 10 dogs received cyclosporine and mizoribine (5 mg/kg and 3 mg/kg/24 hr, orally). Body weights, blood cell counts, serum chemistry profiles, serum electrolyte levels, urinalysis with cytology and culture, lymphocyte stimulation assays, immunoglobulin levels, whole blood levels of cyclosporine, and serum levels of mizoribine were followed. At the end of each survival period, necropsy and histopathologic examinations were performed. The mean survival time for the cyclosporine group was 12.8 +/- 7 days. The mean survival time for the cyclosporine/mizoribine group was 33.6 +/- 16.4 days, significantly longer (P = .0006) than the cyclosporine group. Death in the cyclosporine/mizoribine group was attributed to the combined effects of renal allograft rejection and development of a mizoribine-dependent enteritis. Serum levels of mizoribine were greater in the last half of the survival period due to compromised renal excretion of the drug. There were no complications due to infection, myelosuppression, or hepatotoxicity. Combination cyclosporine/mizoribine immunosuppression enhanced canine renal allograft survival in this study. Monitoring serum concentrations of mizoribine is imperative to determine toxic (enteritis) levels. Availability of an intravenous form of mizoribine would facilitate immunoregulation during periods of variable intestinal absorption or renal excretion.

Cimetidine, posttransplant peptic ulcer complications, and renal allograft survival: a clinical and investigational perspective.

Upper gastrointestinal (G) tract complications have been a substantial cause of death following renal transplantation. Cimetidine, an H2 receptor antagonist, has been used in the posttransplant period to decrease this hazard. However, H2 receptor antagonists may enhance the immune response and be deleterious for the graft. The magnitude of the hazard of upper GI tract complications after renal transplantation was determined by reviewing 200 renal transplants. The effect of cimetidine treatment on the survival of canine renal allografts was investigated. The upper GI tract complication rate was 1.5% with one related death (0.5%). Treatment of dogs with cimetidine shortened the survival time of renal allografts (18.2 +/- 5.5 [SE] days to 12.5 +/- 2.2 days). Because upper GI tract complications are not a major hazard and H2 receptor antagonist therapy may decrease the survival of dog renal allografts, we believe prophylactic use of cimetidine is not indicated.

EFFECT OF BLOOD TRANSFUSIONS ON CANINE RENAL ALLOGRAFT SURVIVAL

In this study significantly prolonged canine renal allograft survival has been demonstrated after transfusion of 100 ml of third-party whole blood given peroperatively. Peroperative transfusions of third-party leukocyte-free blood or pure lymphocyte cell suspensions did not influence graft survival. Furthermore, no improvement in graft survival has been found after a peroperative transfusion of irradiated whole blood (2500 rad). These data suggest that delayed graft rejection after blood transfusions can only be expected after the administration of whole blood. The role of competent lymphocytes in whole blood is questionable, since a transfusion or irradiated whole blood in combination with nonirradiated lymphocytes did not lead to prolonged graft survival. Immunosuppression of the recipient directly after transfusion seems to be essential to induce the beneficial effect of blood transfusions. This has been demonstrated for a transfusion of whole blood 14 days before transplantation. A single transfusion of 100 ml of whole blood 14 days before transplantation could effectively prolong graft survival if immunosuppression with azathioprine and prednisone was started on the day of transfusion. No improvement in graft survival has been found with such a transfusion if preoperative immunosuppression has been omitted.

Prolongation of canine allograft survival with donor pretreatment

Donor pretreatment of 100 mg/kg each of cyclophosphamide (CY) and methylprednisolone (P) infused 5 hours before nephrectomy invariably prolongs the survival of DLA mismatched, MLC incompatible nonlittermate Beagle renal allografts as well as the survival of mongrel renal allografts. The effect of donor pretreatment appears to be mediated by cyclophosphamide and its metabolites because methylprednisolone pretreatment does not significantly prolong survival. Methylprednisolone is needed, however, because it abolishes cyclophosphamide pretreatment mediated early but transient postoperative renal (allograft) insufficiency. The effect of donor pretreatment appears to be mediated by drugs residing in the graft; mannitol infusions given 1 hour prior to donor nephrectomy or peroperatively into the recipient decrease the renal cortical content of carbon 14 cyclophosphamide and its metabolites and abolishes the prolonged survival. Because donor pretreated kidneys contain less than 0.5% of the infused dose of carbon 14 cyclophosphamide, the drugs appear to exert their effect locally in the transplanted kidney. Donor pretreatment mediating prolonged canine renal allograft survival appears to be an example of influencing a biological process by a localized drug delivery by virtue of unique properties of the drug and because early postoperatively host sensitization occurs mainly at the site of the graft.

The effect of transfusions and donor pretreatment on canine renal allograft survival

Allograft survival is influenced by a complexity of known and unknown factors. The effect of several types of transfusion on renal allograft survival has been studied in mongrel dogs. Nontreated and drug-pretreated kidneys were transplanted. Preoperative and peroperative cell suspensions of unrelated dogs were transfused to graft recipients. All recipients received low-dose postoperative immunosuppression. One peroperative transfusion of 100 ml third-party blood enhanced graft survival of nontreated kidneys significantly. A transfusion of 100 ml blood 14 days before transplantation led to variable results, whereas a transfusion of cells obtained from a third party spleen, given 14 days before transplantation, did not prolong graft survival of nontreated kidneys at all. The administration of procarbazine hydrochloride and methylprednisolone to the donor before procuring the graft increased survival of canine renal allografts significantly. A combination of donor drug pretreatment and 100-ml peroperative third-party blood led to graft survival comparable to that obtained by either treatment. Pretreatment of the recipient with spleen cells 14 days before transplantation even abrogated the effect of donor drug pretreatment completely. These results show that preoperative transfusions cannot successfully be combined with donor drug pretreatment as commonly practiced in man. This is a possible explanation for the conflicting data on donor pretreatment in man. Furthermore this study gives evidence for the effectiveness of a peroperative blood transfusion on kidney graft survival.

A study on the mechanism of donor pretreatment: effect of procarbazine hydrochloride and methylprednisolone in immunocompetent cells.

Significantly prolonged canine renal allograft survival can be obtained by donor pretreatment with procarbazine hydrochloride and methylprednisolone. This is thought to be caused either by a reduced antigenicity of the graft or by a local immunosuppressive effect by drugs transplanted with the graft. In this study a decrease in the number of peripheral donor T and B lymphocytes was observed at the time of procuring. Leukocytes harvested from dogs pretreated with a combination of procarbazine hydrochloride and methylprednisolone showed a decrease in their ability either to stimulate or respond to mixed leukocyte cultures (MLCs). Complete restoration of MLC responses was obtained however by purification and washing of these leukocytes. Sera of pretreated animals were not able to reduce MLC responses. It was concluded that drug metabolites in or on the cells were apparently responsible. A local inhibition of the immunocompetence of host lymphocytes by small amounts of transplanted drug metabolites in or on the graft cells might be responsible for the beneficial effect of donor pretreatment with procarbazine hydrochloride and methylprednisolone. Furthermore, this postulation explains the abrogation of prolonged survival of pretreated grafts after systemic administration of nontreated donor blood or donor leukocyte-free blood, as we reported earlier.

The effect of two different renal preservation methods on canine renal allograft survival

Preservation of human cadaver kidneys for transplantation has been achieved primarily by two methods, hypothermic pulsatile perfusion with cryoprecipitated plasma and cold storage with an electrolyte solution. It has been suggested that pulsatile perfusion results in an increased antigenicity of the transplanted kidney. To investigate the possibility that pulsatile perfusion causes changes which may accelerate allograft rejection, machine preservation was compared with simple cold storage. The kidneys were preserved by either one of the two methods for 6 or 24 hours followed by allotransplantation in nephrectomised dogs. No immunosuppressive drugs were given. Kidneys which were allografted without undergoing any preservation (0 hrs) had a mean survival time of 10.4 +/- 1.7 days (n =5). Kidneys preserved by machine perfusion for 6 and 24 hours survival for 9.6 +/- 1.4 (n = 5) and 10.9 +/- 1.3 (n =9) days respectively. The mean survival time for simple cold storage for 6 and 24 hours was 9.3 +/- 1.3 (n =7) and 12.0 +/- 1.9 (n =6) days. Our findings suggest that in kidneys exposed to minimal warm ischaemia there is no significant difference between the two methods of preservation on renal allograft survival for the time intervals tested.

Prolonged survival of canine renal allografts by pretreatment with viable lymphoid cells: J. Y. Rivard, J. G. Beaudoin, and R. D. Guttmann ( 10: 88, 1973)

Prolonged survival of canine renal allografts by pretreatment with viable lymphoid cells: J. Y. Rivard, J. G. Beaudoin, and R. D. Guttmann ( 10: 88, 1973)

Prolonged survival of canine renal allografts by pretreatment with viable lymphoid cells.

SUMMARY Donor lymphocyte pretreatment carried out to improve allograft survival has been studied in dose-time response fashion. The effect of various doses of viable lymphocytes administered at different pregrafting intervals is reported in this study of 57 canine renal allografts. The use of 107 cells given 11 days prior to transplantation seemed to be optimal. Accelerated rejection was not observed. Survival of allograft recipients was prolonged to a mean of 15.5 days with cell pretreatment and up to a mean of 22.3 days when small doses of immunosuppressive drugs were added, suggesting an additive effect. Based on prior experiments in an inbred rat system the mechanism for these results would appear to be immunological enhancement induced by active immunization.

ACTIVE ENHANCEMENT OF CANINE RENAL ALLOGRAFTS USING SOLUBILIZED LYMPHOCYTE ANTIGEN

SUMMARY Subcellular solubilized antigen derived from donor lymphocytes has been found to prolong canine renal allograft survival when administered i.v. before transplantation and when combined with postoperative azathioprine and methylprednisolone in small doses. The prolongation was found to be dependent upon the combination of the total dosage of antigen and the duration of pretreatment. In two groups of animals, significant prolongation was found to be associated with the presence of specific antidonor antibodies. In one other group, pretreated for only 2 weeks, survival was prolonged to a significant degree, but no antibodies were detected. Enhancement was the mechanism thought most likely to explain the data when antibody was present, but tolerance might have been achieved in one group of dogs. The pattern of antibody production was related to the duration of pretreatment, as well as to antigen dosage, and only cytotoxic antibody was measured. Cell-mediated immunity also was found to be related to these two factors, but it did not appear to play a key role in the prolongation of allograft survival.

Treatment of Renal Allograft Rejection in Dogs

A previous study in this laboratory suggested that an external thoracic duct fistula could prolong the survival of canine renal allografts. 1 The mechanism for this apparent interference with the transplant immunity might be the depletion in the population of immunologically competent lymphocytes resulting from uninterrupted drainage of the thoracic duct. Gowans has shown that lymphopenia can be brought about by prolonged drainage of the thoracic duct in rats. 2 Others have reported similar findings in dogs. 3 Although the role of the lymphocytes in transplant immunity has not been ascertained, it seems only reasonable to implicate them because of their consistent involvement in the antigen-antibody reaction. Their well documented presence at the site of the tissue graft rejection marks them teleologically as an intermediary in this phenomenon, specifically as carriers of antibodies. For this reason immunosuppressive drugs have been tested according to their ability to produce lymphopenia. Even some

Antiserum to lymphocytes: prolonged survival of canine renal allografts.

A horse immunized with dog lynmphocytes produced an antiserum which agglutinated canine lymphocytes in vitro and caused prolonged lymphopenia in dogs in vivo. Renal transplants in dogs treated with this antiserum survived for long periods, two of the grafts surviving beyond 350 days with normal function and histologic appearance.

Prolongation of canine renal allograft survival by epsilon-aminocaproic acid, an in vitro inhibitor of complement

Prolongation of canine renal allograft survival by epsilon-aminocaproic acid, an in vitro inhibitor of complement

1965. Prolongation of canine renal allograft survival by epsilon-aminocaproic acid, an in vitro inhibitor of complement

1965. Prolongation of canine renal allograft survival by epsilon-aminocaproic acid, an in vitro inhibitor of complement