Limb allografts in rats immunosuppressed with FK506. I. Reversal of rejection and indefinite survival

We have tested the effects of FK506 (FK), a new immunosuppressive agent, on a rat limb allograft model. Histoincompatible BN limb allografts were rejected in untreated F344 hosts within 11 +/- 1 days (mean +/- SD) after operation. A single injection of 2 mg/kg, 10 mg/kg, or 50 mg/kg of FK on the day of limb transplantation (day 0) significantly prolonged graft survival in a dose-dependent manner--i.e., mean limb survival times (MST) based on gross signs of skin rejection were 16 +/- 3 days, 51 +/- 6 days, or 104 +/- 17 days, respectively (P less than 0.01). Delayed treatment with a single injection of 10 mg/kg of FK at when early signs of rejection were visible (day 7 or day 10) reversed the ongoing rejection. The MSTs in these groups were comparable to that of those treated with the same dosage of FK on day 0. The FK-induced unresponsiveness toward limb allografts was donor-specific because limb-allografted. FK-protected rats could not accept the skin grafts from a third-party donor. In the next set of experiments, rats were given a single administration of 10 mg/kg of FK on the day of limb allograft, followed by intermittent injections of 3 mg/kg of FK once a week. This regimen produced complete graft survival for more than 200 days, though Pneumocystis carinii pneumonia occurred in most of the recipients. These results represent the unique effects of FK in preventing or reversing the graft rejection and in inducing indefinite survival in this animal model of composite tissue allografts.     

Prolongation of skin allograft survival in rats by a novel immunosuppressive agent, FK506

FK506, an immunosuppresant, was isolated from Streptomyces tsukubaensis. Intramuscular administration of FK506 (0.32 mg/kg or more) 5 days a week for two weeks after grafting prolonged the acceptance time of F344 skin allograft to WKA rats. Similar results were obtained with cyclosporine at 32 mg/kg or more, but other immunosuppressives (i.e., prednisolone, azathioprine, and bredinin) gave only a marginal prolongation. The prolonging effect of FK506 was obtained in various donor-recipient combinations across a major or minor histocompatibility barrier. The agent also prolonged the acceptance time of mouse skin xenografts to rats. Furthermore, maintenance doses of 3.2 or 0.32 mg/kg twice a week after an initial 14-day treatment with the agent at 3.2 mg/kg gave graft survival as long as the treatment was continued for more than 120 days. Our findings show that FK506 has a potent immunosuppressive effect in rats and suggest that the agent merits further investigation.     

The necessity of differential immunosuppression for prevention of immune rejection by FK506 in rat islet allografts transplanted into the liver or beneath the kidney capsule.

The purpose of the present study was to achieve prevention of immune rejection in rat islet allografts by FK506. WKA/Qdj (RT1u) islets were transplanted either into the liver via the portal vein (p.v.) or beneath the kidney capsule (k.c.) of streptozotocin (60 mg/kg) induced diabetic Lewis (RT1(1)) rats. Fresh or cultured (24 degrees C, 1 week) islets were used as donors. A miniosmotic pump (0.2 ml, Alzet 2001) containing 5 mg FK was implanted s.c. at the time of transplantation for continuous delivery of FK506 for 7 days after transplantation. The mean survival time (MST) of the fresh p.v. grafts with a pump was offater than 61.4 +/- 37.2 days (mean +/- SD, n = 17) (control 5.5 +/- 0.6, n = 4). Ten out of 17 were normoglycemic for more than 90 days after transplantation. When low-temperature cultured islets were used and FK506 was delivered for 7 days, all the rats were normoglycemic for more than 90 days after transplantation. The MST of the fresh or cultured k.c. grafts with a pump was 22.0 +/- 14.2 or 24.7 +/- 5.0 days, respectively. Long-term administration of FK506 by repeated implantations (5 times; days 0, 7, 14, 21, and 28) of pumps containing 5 mg FK506 produced marked prolongation of the fresh or cultured k.c. graft survival with an MST of greater than 58.7 +/- 22.1 or greater than 56.9 +/- 18.0 days, respectively. These findings clearly demonstrate that the prevention of immune rejection in the islet allografts transplanted into the liver was achieved by short-term post-transplant administration of FK506 and low-temperature culture of donor islets, and also show that long-term continuous administration of FK506 was needed for the prolongation of the graft survival when the renal subcapsular space was the site for implantation of islets. Thus, the present study indicates that in different transplant sites different immunosuppressive regimes are needed for the control of rejection by FK506 in rat islet allografts.     

Transplantation tolerance by a combined therapy with sulfatide, anti-LFA-1/ICAM-1 monoclonal antibodies and FK506 in rat cardiac transplantation

Abstract Selectins promote a rolling phenomenon of leukocytes along activated endothelial surfaces, which is the first step in the events that ultimately lead to leukocyte transmigration at acute inflammatory sites. Our previous study revealed that sulfatide, which is one of the selectin inhibitors, prolonged graft survival in rat cardiac allografts. In the present study, to obtain a longer graft acceptance, we examanined a combination treatment of sulfatide, monoclonal antibodies (mAbs) against LFA-1/ICAM-1, and FK506 in a Fischer 344 (F344, RT1^lvl) to Lewis (LEW, RT1¹) rat heart transplantation. FK506 alone ( n = 6) and FK506/sulfatide-treated LEW rats ( n = 6) rejected F344 heart grafts with an MST of 49 and 55.2 days, respectively. Otherwise, four out of six heart grafts treated with sulfatide, mAbs against LFA-1/ ICAM-1, and FK506 ( n = 6) survived for over 100 days after transplantation. The proliferative response of alloreactive T cells obtained from tolerant rats against both F344 alloantigen and thirD-party alloantigen on day 104 post-grafting was significantly suppressed as compared to that from naive LEW rats. On light microscopic examination, specimens of tolerant rat on day 104 postgrafting showed an almost normal appearance. Our results suggested that blocking both each step of leukocyte entry and recognition of alloantigens by a combination treatment of sulfatide, mAbs against LFA-1/ICAM-1, and FK506 could lead to tolerance.     

Development of donor-specific immunoregulatory T-cells after local CTLA4Ig gene transfer to pancreatic allograft

BACKGROUND: CTLA4Ig gene transfer directly to graft tissue might have the potential to avoid the need for systemic immunosuppression. In our previous studies of bio-breeding (BB) rats, local adenovirus-mediated CTLA4Ig gene transfer protected the pancreas from autoimmune and alloimmune responses. This study investigated the potency of local CD28/B7 costimulatory blockade for induction of donor-specific tolerance and further examined the existing mechanisms. METHODS: Brown Norway (BN; RT1)-pancreaticoduodenal grafts transfected with Ad.CTLA4Ig via intraarterial ex vivo perfusion were transplanted into streptozotocin-induced diabetic Lewis (LEW; RT1) rats. RESULTS: Ad.CTLA4Ig transduced grafts combined with a short course of FK506 resulted in indefinitely prolonged survival (>156 days vs. 19.5 days with FK506 alone). CTLA4Ig was predominantly expressed in grafts on day 4. The expression was gradually diminished and was only slightly detectable at day >100. The proliferative responses against BN antigen were remarkably enhanced among recipients with rejected grafts, but the T-cells from tolerant recipients (>100 days) showed poor cytotoxic responses. On adoptive transfer assay, the splenic T-cells of tolerant recipients were able to suppress the rejection of BN, but not third-party Wistar Furth (WF; RT1) hearts in irradiated (480 cGy) LEW recipients. The percentage of CD4CD25 splenic T-cells was significantly increased in tolerant recipients (13.53 +/- 4.06% vs. 6.06 +/- 0.56% in naive rats). CONCLUSION: CTLA4Ig gene transfer to the pancreaticoduodenal allograft combined with a short course of FK506 induces donor-specific tolerance. The mechanism of maintaining tolerance could be explained by development of splenic T suppressor cells.     

FK506和供体特异性输血在大鼠异位心脏移植中的作用

目的探讨FK506和供体特异性输血在大鼠异位心脏移植中的作用。方法利用大鼠异位心脏移植模型以了解在移植当天或移植术后第4日进行供者特异性输注（DST）的基础上,应用FK506能否延长移植物的存活。结果在移植当天行DST或单独用FK5061mg／kg连续10天,可将移植心中位存活时间从对照组的5天分别有效延长至7天和11天,而FK506与DST联合应用时并不产生增强效应。结论FK506和DST单独应用时虽均能延长大鼠同种移植心存活,但是它们没有协同作用。     

Transplantation tolerance by a combined therapy with sulfatide,anti-LFA-1/ICAM-1 monoclonal antibodies and FK506 in rat cardiac transplantation

Selectins promote a rolling phenomenon of leukocytes along activated endothelial surfaces, which is the first step in the events that ultimately lead to leukocyte transmigration at acute inflammatory sites. Our previous study revealed that sulfatide, which is one of the selectin inhibitors, prolonged graft survival in rat cardiac allografts. In the present study, to obtain a longer graft acceptance, we examanined a combination treatment of sulfatide, monoclonal antibodies (mAbs) against LFA-1/ICAM-1, and FK506 in a Fischer 344 (F344, RT1^lvl) to Lewis (LEW, RT1^l) rat heart transplantation. FK506 alone (n = 6) and FK506/sulfatide-treated LEW rats (n = 6) rejected F344 heart grafts with an MST of 49 and 55.2 days, respectively. Otherwise, four out of six heart grafts treated with sulfatide, mAbs against LFA-1/ICAM-1, and FK506 (n = 6) survived for over 100 days after transplantation. The proliferative response of alloreactive T cells obtained from tolerant rats against both F344 alloantigen and third-party alloantigen on day 104 postgrafting was significantly suppressed as compared to that from naive LEW rats. On light microscopic examination, specimens of tolerant rat on day 104 postgrafting showed an almost normal appearance. Our results suggested that blocking both each step of leukocyte entry and recognition of alloantigens by a combination treatment of sulfatide, mAbs against LFA-1/ICAM-1, and FK506 could lead to tolerance.     

Specific unresponsiveness to fully allogeneic kidney allografts in rats induced by procarbazine hydrochloride and antilymphocyte serum

A short course of procarbazine hydrochloride (PCH; 50 mg/kg) and antilymphocyte serum (ALS; 5 ml/kg), administered to Lewis (LEW;RT1(1] rats in the first week following transplantation of Brown Norway (BN;RT1n) kidneys, substantially prolonged allograft survival and induced long-term survival in 62% of the grafts. The two agents acted synergistically, in that neither of them administered alone had much effect. Graft recipients did not produce detectable cytotoxic antibodies and antigen-reactive cells injected i.v. were not diverted to the liver, thus showing that neither antibodies nor immune complexes are likely to mediate the unresponsiveness. Spleen cells from graft-bearing recipients failed to cause graft-versus-host responses (GVHR) in both (LEW X BN)F1 and (LEW X DA)F1 hybrids, but they specifically suppressed the GVHR given by normal syngeneic cells to donor strain (BN) antigens. This suppression was specific because the response against third-party antigens (DA; RT1a) was unaffected. Adoptive transfer of spleen and thymus cells from PCH-ALS-treated LEW rats bearing healthy BN kidneys caused a profound prolongation of BN graft survival in sublethally irradiated LEW recipients. This transfer was specific and mediated by W3/13+ (T) lymphocytes. It is concluded that a limited regimen of PCH and ALS given in the first postoperative week incites the generation of specific suppressor T lymphocytes and that this form of immunosuppression, even without preoperative donor antigen, is an effective way of prolonging kidney allograft survival.     

FK506 as the sole immunosuppressive agent for prolongation of islet allograft survival in the rat.

The purpose of the present study was to determine immunosuppressive effects of a new immunosuppressive agent, FK506, on rat islet allografts and also whether FK is toxic to the islet grafts since the diabetogenic effects of FK is controversial. Hand-picked clean fresh islets (WKA/Qdj:RT1u) were transplanted either beneath the renal capsule or into the liver via the portal vein of the diabetic (STZ, 60 mg/kg) rats (Lewis:RT1(1)). FK506 was administered s.c. for 7 days after transplantation. The mean survival times (MST)* of the renal subcapsular grafts receiving 0 (control), 0.32 or 1.0 mg/kg FK were 7.2 +/- 1.1 (mean +/- SD, n = 5), 13.8 +/- 4.8 (n = 4), and 20.2 +/- 8.0 days (n = 5), respectively. The MST of the intrahepatic grafts receiving 0, 0.1, 0.32, or 1.0 mg/kg FK were 4.4 +/- 1.1 (n = 5), 7.2 +/- 0.8 (n = 5), greater than 45.3 +/- 23.1 (n = 6) or greater than 54.4 +/- 8.8 days (n = 5), respectively. Histologically, islets were found easily in the liver of normoglycemic recipients for more than 60 days after transplantation and appeared intact, with well-granulated beta cells. Foci of mononuclear cells were occasionally seen adjacent to the islet cells. The plasma glucose of the recipients with 1.0 mg/kg FK fluctuated between 150 and 350 mg/dl without rejection. In the recipients treated with 3.2 mg/kg FK the plasma glucose of all the recipients (n = 3) returned to pretransplant levels by 21 days after transplantation. However, islet cells were present in the liver of all these recipients without mononuclear cell infiltration. Immunohistochemically islet grafts stained weakly for insulin, but to the same extent as the controls for glucagon and somatostatin. These findings clearly demonstrate the immunosuppressive effect of FK506 on islet allografts and the importance of the transplant site for prolongation of graft survival by FK, and also suggest that FK has toxic effects on the islet grafts (B cells) when used in high dosages.     

Synergistic effect of vincristine with tacrolimus or sirolimus in prevention of acute heart allograft rejection in the rat

The application of multiple immunosuppressive therapy for organ transplantation could enhance therapeutic efficacy, while minimizing the toxicity of individual drugs used in the regimen. In this study, the effect of the combined therapy of vincristine (VCR) with tacrolimus (FK506) or sirolimus (rapamycin, RAPA) was tested in prevention of acute heart allograft rejection in the rat. A Brown Norway (BN, RT 1(n)) to Lewis (LEW, RT 1(1)) rat combination was used in a heart allografting model. VCR was administered intraperitoneally once daily, while FK506 and RAPA were given by gavage once daily for 14 days after transplantation. There were dose-related prolongations of mean survival time (MST) to monotherapy of VCR, FK506, or RAPA. The MST in combination therapy indicated that a synergistic interaction was produced when compared with the respective monotherapies: VCR 0.05 mg/kg/day + FK506 0.5 mg/kg/day (16.00 +/- 1.79 days, P = 0.001; combination index (CI) = 0.557); VCR 0.05 mg/kg/day + FK506 1.0 mg/kg/day (29.00 +/- 10.54 days, P = 0.001; CI = 0.598); VCR 0.05 mg/kg/day + RAPA 0.2 mg/kg/day (17.33 +/- 1.97 days, P = 0.001; CI = 0.500); and VCR 0.05 mg/kg/day + RAPA 0.4 mg/kg/day (21.17 +/- 3.19 days, P = 0.001; CI = 0.838). Combination therapy of VCR and FK506 or RAPA produced synergistic effects in prevention of acute heart allograft rejection in the rat.     

Limited effectiveness of FK506 administration for ongoing rejection in heterotopic rat heart transplantation

A comparison was made of the histological findings for myocardial tissue of heterotopic transplanted rat hearts administered with FK506. ACI rats were used as donors and Lewis rats as recipients. FK506 was used for 6 days except for group I (control group). Group II received 0.32 mg/kg/day of FK506 from the day of operation while group III was given the same dosage from the 4th day after transplantation. Group IV was given 1.28 mg/kg/day of the agent from the day of grafting and group V received the same dose from the 4th postoperative day. The graft survival time was longer for all groups given FK506, but was significantly longer only for groups administered with FK506 from the day of operation. Histological studies performed 10 and 20 days after transplantation showed that a moderate rejection was seen in about half of the grafts receiving FK506 from the 4th day after grafting. An ultra-structural study of these cases showed that infiltrating large lymphocytes still remained in the interstitial tissues and that the cytoplasmic organelles of the myocytes had been focally destroyed. These results suggest that, although FK506 suppressed any further rejection, the effect might be limited and the myocardial changes of the cardiac graft might persist even after administration for ongoing rejection.This work was supported by a Ministry of Education Grant (A) 03770873.     

Gemcitabine with cyclosporine or with tacrolimus exerts a synergistic effect and induces tolerance in the rat

BACKGROUND: This study investigated the effect of the antineoplastic agent gemcitabine (dFdC) in combination with cyclosporine (CsA) or with FK506 on acute heart allograft rejection in a rat model. METHODS: Transplantations were performed in the fully allogeneic Lewis-to-Brown Norway strain combination. dFdC, CsA, and FK506 single-drug therapy and combinations of dFdC with CsA and FK506 were administered at various dosages starting on day 1 to prevent and on day 4 to treat acute rejection until day 20. Animals who did not reject their graft were intraperitoneally injected with 108 splenic donor-type lymphocytes. In addition, Lewis and third-party skin grafts were transplanted to these animals. RESULTS: Mean graft survival times under CsA, FK506, and dFdC monotherapy were 18.3/63.7 days (1 mg/5 mg per kg), 41.7 days, and 24.7/38.7 days (100 microg/150 microg per kg), respectively. CsA and FK506 in combination with dFdC prolonged graft survival to more than 100 days (CsA) and more than 95.2 days (FK506). Graft survival after treatment of an ongoing rejection was 21.5/38.3 days for CsA (1 mg/5 mg per kg) and 17.7/59.2 days for dFdC (100 microg/150 microg per kg). The combination of CsA+dFdC prompted indefinite survival of five of six hearts. Lymphocyte inoculation did not induce graft rejection. Notably, none of the Lewis, but all third-party, skin grafts were rejected immediately. Histomorphologic analysis of grafted hearts, however, demonstrated typical features of chronic rejection. CONCLUSIONS: The combination of CsA and FK506 with low-dose dFdC exerts a synergistic effect in the prevention and treatment of acute allograft rejection in this model. Although chronic rejection could not be prevented, strain-specific tolerance was achieved. Therefore, combining standard immunosuppressants with dFdC is a novel, promising strategy for prevention and treatment of acute allograft rejection.     

Enforcement of specific unresponsiveness by the long-term presence of allogeneic heart grafts

Mechanisms underlying the maintenance of long-term heart allografts were analyzed in rats treated with Cyclosporine. It was shown that acceptance of allografts after cyclosporine did not involve the attenuation of immunogenicity of the grafts. This conclusion was drawn from two observations: (1) Pretreatment of the donors with cyclosporine did not cause prolongation of graft survival time; and (2) cyclosporine stabilized allografts were normally rejected by secondary recipients when retransplanted on day 30. Studies of the acceptance of skin grafts from the heart donor strain indicated the existence of a mechanism to maintain donor-specific unresponsiveness in the presence of a stable allograft in a time-dependent manner. Thus, the mean rejection time of F344 skin grafts on WKA rats bearing F344 hearts was more than 80 days when transplanted on day 210, but it was 32 days when they were transplanted on day 30. Active participation of specific suppressor cells in the maintenance of unresponsiveness was suggested because data obtained in the cell transfer experiments was statistically significant. Transfer of lymphocytes from rats bearing long-term grafts showed a tendency to delay rejection of skin grafts by sublethally irradiated hosts, but it failed to delay the rapid rejection by normal lymphocytes cotransferred to the same recipients.     

Prolonged survival of rat whole-limb allografts treated with cyclophosphamide, granulocyte colony-stimulation factor and FK506

We evaluated the efficacy of a new protocol using cyclophosphamide (CYP), granulocyte colony-stimulation factor (G-CSF) and FK506 to induce high level chimerism following rat whole-limb allotransplantation. The present study investigated the dose requirement and toxicity of CYP monotherapy in inducing stable bone marrow chimerism. Fifty-six whole-limb allotransplants from LacZ transgenic rats to LEW rats were performed. CYP at a dose of 100 mg to 200 mg/kg was injected 2 days before transplantation and G-CSF of 25 microg/kg/day was given for 4 days. FK506 was used for 28 days at 1 mg/kg/day. The level of chimerism was evaluated by semi-quantitative polymerase chain reaction. The survival of limb allografts in recipients treated with CYP of 150 mg/kg was significantly prolonged to 107 days. The onset of rejection was more prolonged to 158 days in recipients with CYP of 200 mg/kg, with two of eight grafts surviving >1 year and three recipients (38%) showed chronic, nonlethal GVHD with a high level of bone marrow chimerism. Limb allografting could contribute to chimerism in the recipient. Pretreatment with CYP had the dose-dependent effects of prolonging the survival of limb allografts. A CYP dose of 200 mg/kg appears to significantly prolong limb graft survival but frequently causes chronic nonlethal GVHD in the longer surviving recipients.     

Efficacy of transient treatment with FK506 in the early phase on cyclophosphamide-induced bone marrow chimerism and transplant tolerance across MHC barriers

BACKGROUND: The use of mixed allogeneic bone marrow chimerism to induce donor-specific transplantation tolerance has been extensively demonstrated. In the present study, we assessed the effect of combined use of a short course of FK506 and a single-dose cyclophosphamide (CYP) on the induction of tolerance and development of GVHD after allogeneic BMT. MATERIALS AND METHODS: Lewis rat (RT1(l)) recipients received BMT from Brown Norway (RT1(n)) donors on the next day after injection of CYP at a dose of 200 mg/kg. The recipients were further treated with no FK506 (n = 8), 0.3 mg/kg/day FK506 on days 10-16 (n = 6), or the same dose of FK506 on days 0-6 (n = 6). In a subgroup of animals, heterotopic heart transplantation was performed to investigate transplantation tolerance. RESULTS: Six of eight recipient rats that did not receive FK506 died of severe GVHD, while high levels of chimerism were induced. Recipients of FK506 in the later phase developed mild transient GVHD around 2 to 3 weeks after BMT and recovered thereafter; however, the level of chimerism was significantly decreased (2.8 +/- 2.3% on day 100). Treatment with FK506 in the early phase completely prevented the development of GVHD and induced stable allogeneic chimerism in the long-term (13.8 +/- 8.3% on day 100). These recipients with stable chimerism accepted subsequent BN heart allografts indefinitely (>200 days x 5), while rejecting third-party (BUF) heart allografts by day 12. CONCLUSIONS: Early transient FK506 promotes the induction of stable bone marrow chimerism without GVHD after BMT with CYP pretreatment. The timing of treatment with FK506 is critical with a view to preventing GVHD and inducing stable long-lasting chimerism.     

Immunosuppression with a combination of pg490-88 and a subtherapeutic dose of FK506 in a canine renal allograft model

BACKGROUND: PG490-88 is a water soluble, semisynthetic derivative of a novel compound PG490 (triptolide) purified from the Chinese herb Tripterygium Wilfordii Hook F. In this study, we evaluated the immunosuppressive effect of PG490-88 alone or combined with FK506 in a dog renal transplantation model. METHODS: Recipient and donor male beagle dogs were obtained from different breeders to ensure MHC mismatching. PG490-88 and/or FK506 were administered orally based on protocol design. RESULTS: All dogs in the untreated group developed acute vascular rejection with a median survival time of 6 days. The grafts from this group presented with massive hemorrhage, IgM, IgG, and C4c deposition. Administration of PG490-88 0.06 mg/kg/day significantly prolonged graft survival to a median survival time of 11 days (P=0.038, vs. control). Treatment with FK506 0.3 mg/kg/day did not prolong graft survival with a median survival time of 9 days. Although FK506 0.6 mg/kg/day significantly prolonged survival, this dose was not tolerated by the dogs. The combination of PG 0.06 mg/kg/day and FK506 0.3 mg/kg/day significantly prolonged survival to a median survival time of 15 days (P=0.017, vs. control). Compared to the untreated control group, the pattern of acute humoral rejection was attenuated in renal allografts treated with PG490-88 and/or FK506. C4c deposition was significantly decreased in renal allografts treated with PG490-88 monotherapy and combination therapy. CONCLUSIONS: PG490-88 alone and combined with low dose FK506 significantly prolonged renal allograft survival in a dog model. This agent attenuated acute humoral rejection by inhibiting complement activation and T-cell infiltration.     

Mucosal glutamine utilization after small-bowel transplantation: an electrophysiologic study.

A short course of FK 506 after small bowel transplantation averts rejection in the rat and achieves indefinite survival of the recipient whose nutritional status is dependent on the function of the intestinal graft. Ex vivo electrophysiologic studies using the Ussing Cell were conducted to delineate functional competence of the graft by evaluating mucosal ion transport and glutamine utilization. Orthotopic small-bowel transplantation was performed in Lewis (LEW) rats as recipients of either Brown-Norway (BN) allografts or LEW syngeneic grafts. Allograft recipients received FK 506 either as a short course (2 mg/kg on Day 0-4 after transplantation) or continuously (2 mg/kg Day 0-4, then 0.5 mg/kg weekly). Ileal mucosa was harvested from small bowel grafts 9 and 60 days after transplantation and mounted in the Ussing Cell containing Hanks' balanced salt solution with/without L-glutamine (20 mM). Transmembrane potential difference (PD), which represents mucosal active ion transport, and mucosal resistance, an index of membrane integrity, were recorded. Nine days after transplantation, mucosal PD was the same in the ileum from syngeneic grafts, allografts treated with FK 506 and normal LEW and BN rats, and the addition of glutamine increased PD equally in all groups. In comparison, PD was markedly decreased in allografts undergoing rejection, and the glutamine response was blunted. Sixty days after transplantation, mucosal PD was reduced in allografts treated with a short course of FK 506, but normal in allografts receiving continuous immunosuppression with FK 506 and in syngeneic grafts. A decrease of mucosal resistance was not a feature of rejection nor a sequel of limited FK 506 therapy. Our data indicate that allograft rejection results in a significant decrease in mucosal PD and a poor response to glutamine. Control of rejection by FK 506 preserves normal electrophysiologic responses of the allograft mucosa.     

Immunological unresponsiveness to hepatic allografts in rats. Immunological reactivities of the recipient to donor antigens

Wistar (RT1bv1) rats transplanted orthotopically with ACI (RT1a) livers survive indefinitely without any immunosuppression, while heterotopic heart grafts or skin grafts are rejected acutely in this combination. Levels of alkaline phosphatase after liver allografting remain significantly higher than those found in controls receiving syngeneic grafts. We studied changes in immune responsiveness in rats receiving liver grafts. Local graft-versus-host reactivity was present at all times assayed. Delayed type hypersensitivity reactions were already positive 2 weeks after liver transplantation and increased in strength. Liver graft-bearing rats were subsequently grafted with donor or third-party skin. Third-party skin grafts survived significantly longer on liver-grafted rats than on untreated controls when grafted within the first week after grafting. Donor-type skin grafts survived longer than controls when grafted within the first 4 weeks after liver grafting, although the skin grafts were eventually rejected. Donor-type skin grafted more than 8 weeks after liver grafting was rejected acutely. In an adoptive transfer assay, ACI hearts survived significantly longer in Wistar rats given serum from Wistar donors 2-4 weeks after ACI liver grafts than in untreated controls. On the other hand, spleen cells obtained at any period after liver grafting were not capable of prolonging cardiac allograft survival after transfer to syngeneic recipients. Thus cellular responses to ACI antigen are not changed during the life-span of liver-grafted animals. Evidence suggests that a serum "enhancing" factor protects the donor liver from rejection in the initial period after liver transplantation. The long-term acceptance of liver grafts is discussed.     

Ex vivo and systemic transfer of adenovirus-mediated CTLA4Ig gene combined with a short course of FK506 therapy prolongs islet graft survival

Adenovirus-mediated CTLA4Ig gene transfer has been reported to enhance graft survival in several rodent transplantation models. In this study, we investigated the efficacy of ex vivo and systemic transfer of the CTLA4Ig gene by adenoviral vectors in pancreatic islet allo-transplantation. Islet grafts from BN rats were transplanted to chemically induced diabetic LEW rats. First, ex vivo CTLA4Ig gene transfer into isolated islets was performed prior to transplantation. Survival of transduced grafts under the kidney capsule was slightly prolonged (8.6+/-1.3 days) compared with survival of untransduced grafts (6.7+/-1.2 days); when combined with a short course of FK506, graft survival was further extended (32.6+/-10.7 days vs. 13.7+/-1.0 days with FK506 alone). Secondly, systemic gene transfer was accomplished by intravenous administration immediately after the transplantation procedure. In these animals, islet grafts under the kidney capsule survived longer (15.2+/-3.3 days) than in controls (6.7+/-1.2 days), and when FK506 was administered perioperatively, all the islet grafts survived for more than 100 days. In systemically transduced recipients, the survival of islet grafts transplanted into the liver was not significantly different from that of the grafts placed under the kidney capsule. In order to examine organ-specific immunogenicity, heterotopic BN cardiac grafts were transplanted to LEW rats intra-abdominally, with the virus transferred systemically as in the islet model. In contrast to the islet grafts, all the cardiac grafts were accepted for longer than 100 days, even without FK506 therapy. Finally, the LEW recipients with long-surviving islet or cardiac grafts were re-transplanted with islet grafts from the same donor strain (BN) on day 100. The second islet grafts survived longer than 100 days in half of the cardiac recipients, but consistently failed in the islet recipients. We conclude that in this transplant model, CTLA4Ig gene transfer and FK506 treatment synergistically improved islet graft survival, systemic transfer of the gene was more effective than ex vivo transfer to the islets, and donor-specific tolerance could not be achieved for islet transplantation but was achieved for cardiac transplantation.     

Long‐term small bowel allograft function induced by short‐term FK 506 application is associated with split tolerance

Abstract Functional long‐term allograft survival after experimental small bowel transplantation (SBT) is limited by chronic rejection. Initial application of high‐dose FK 506 has been shown to induce stable long‐term graft function. In order to examine whether this long‐term function is associated with donor‐specific tolerance, we analyzed the functional status of recipient T cells in vivo and in vitro. One‐step orthotopic SBT was performed in the allogeneic Brown Norway (BN)‐to‐Lewis rat strain combination. FK 506 was given daily at a dose of 2 mg/kg from days 0‐5 in the rejection model and from days 0‐9 in the long‐term functional model. Mean survival time in the rejection model was 98 ± 2.8 days. Histological examination of these small bowel allografts disclosed signs of chronic rejection. In contrast, all animals of the long‐term functional model survived long term (> 250 days) without clinical signs of chronic rejection. The latter model, furthermore, produced evidence of donor‐specific tolerance. Whereas heterotopic Dark Agouti (DA) hearts were rejected regularly within 7 days, BN hearts survived indefinitely (> 70 days). In vitro, mixed leukocyte reactivity of CD4 + T cells was similarly strong against donor (BN) antigens as against third‐party (DA) antigens. The split tolerance revealed by our in vivo and in vitro results enabled acceptance of both the small bowel allograft without signs of chronic rejection and of donor‐specific heart allografts.