The synergistic effect of low-dose cyclosporine and fluocinolone acetonide on the survival of rat allogeneic skin grafts

Both CsA and topical FA can prolong the survival of skin allografts under the proper conditions. This study was performed to determine if there is a synergistic effect between these two compounds. Buf (RT1b) rat split-thickness skin grafts were transplanted onto the backs of Lew (RT1l) rats. The MST for the control group was 9.89 +/- 0.35 days. In rats given oral CsA, 2.5 or 5 mg/kg, daily from the second day of grafting, the MST was 16.0 +/- 1.9 and 13.6 +/- 0.4 days (blood CsA levels were 166 +/- 20 and 640 +/- 32 ng/ml at the time of rejection, respectively. Topical FA applied daily beginning 72 hr after grafting resulted in a MST of 24.1 +/- 3.6 days. When both topical FA and 5 mg/kg oral CsA were used, the allograft survival time was more than 100 days in 4 of 7 animals. When oral CsA 2.5 mg/kg was combined with topical CsA and FA, the allograft rejection was delayed until 50 days postgrafting in four of six animals. The synergistic effects of oral CsA and topical FA is significant, and thus allows for the use of a subtherapeutic dosage of each compound and provides a potentially safe means for prolonging skin allograft survival.     

Sex-associated differences in the survival of skin grafts in rats. Enhancement of cyclosporine immunosuppression in male compared with female recipients

Focusing on sex-difference in prolongation of allograft survival time, we have performed skin grafts between fully allogeneic rat strains, AO (RT1u) and DA (RT1a) with an immunosuppressant, cyclosporine. Isografted skins survived indefinitely, whereas allografts were severely rejected at days 7-9 without immunosuppressive treatment. When adult male DA rats received CsA (15 mg/kg/day, i.m., for 14 days postoperatively), allogeneic skin was accepted from either male or female AO rats for 38.8 +/- 20.5 days (mean survival time [MST] +/- SD) and for 44.7 +/- 43.3 days (MST +/- SD), respectively, with normal hair growth at around day 17. Additional CsA administration every 5 days after the initial short course treatment was also effective in preventing chronic rejection. Male AO rat skin grafted onto adult male DA rats survived for over 50 days as long as the treatment was carried out. In contrast, when adult female DA rats were used as recipients, only a few days' prolongation was observed in comparison with a non-treated group. The rejection always occurred, even during the initial course of treatment (MST +/- SD): 10.9 +/- 1.6 days). Younger male DA recipients, 5 and 10 weeks old, rejected AO skin within a shorter time, depending on the age. The maximal graft survival was observed when male adult rats more than 14 weeks old were used as recipients. On the other hand, the CsA serum level of female recipients at day 14 was considerably lower than that of males. However, even when the level of females was adjusted to that of males by the administration of a double dosage (30 mg/kg/day), the female recipients consistently rejected the skins (MST +/- SD: 14.5 +/- 1.9 days). Therefore, these results clearly indicate that this male-associated immunosuppressive effect depends upon the sex and age of the recipient animals.     

Cyclosporine and experimental skin allografts. II. Indefinite survival and development of specific immunologic unresponsiveness

Immunological unresponsiveness toward skin allografts was studied in cyclosporine (CsA)-treated rats. BN skin grafts survive about 22 days and about 34 days in LEW hosts following 7 or 14 days of daily CsA treatment (15 mg/kg/day), respectively; in unmodified hosts grafts are rejected by 9 days. Indefinite (greater than 100 days) survival can, however, be produced by administering maintenance 15 mg/kg CsA every fourth day, following an initial course of the agent for 14 days. Early signs of graft rejection (hair loss, localized epidermal breakdown, and ulcerations) occurring in some animals were reversed by a CsA "pulse" (15 mg/kg/day) for 7 days, reduced gradually to the maintenance dose. CsA was equally effective when started as late as 4 days after grafting, but ineffectual when started after day 4. Once BN grafts were rejected, the agent could not prevent second-set rejection of donor-specific grafts, but significantly prolonged the survival of third-party (WF) skins. Survival of original BN grafts was unchanged by the placement of second BN grafts during both the inductive and maintenance phases; these second grafts survived as long as the original grafts. In contrast, secondary third-party (WF) grafts were promptly rejected; their destruction did not influence survival of the original grafts. Thus, indefinite survival of rat skin allografts is feasible with low maintenance doses of CsA. Graft rejection at later stages can be reversed by resuming daily therapy. Host unresponsiveness is stable and specific both during the early inductive and later maintenance phases.     

Induction of donor-specific unresponsiveness to rat cardiac allograft by donor leukocytes and cyclosporine

Many recent reports have emphasized the importance of donor antigens in the induction of allograft tolerance. This study examines the effect of pretransplant infusion of 10(8) donor leukocytes (DL) combined with peritransplant cyclosporine (CsA) on W/F cardiac allograft survival in Lewis rats. Peritransplant recipient treatment consisted of CsA 20 mg/kg given i.m. on days 0, +1, and +2 relative to heart transplantation. Lewis recipients, 5-8 per group, were pretreated with 10(8) DL with or without peritransplant CsA. A single DL transfusion on day -3 or day -7 prior to transplantation significantly prolonged the mean survival time (MST) of W/F hearts from 7.0 +/- 0.9 days in controls to 12.2 +/- 4.5 days and 12.4 +/- 1.0 days (P less than 0.01), respectively. Two DL infusions on days -7 and -3 or on days -14 and -7 prolonged the MST to 10.6 +/- 1.3 days (P less than 0.02) and 16.4 +/- 2.8 days (P less than 0.001), respectively. The administration of peritransplant CsA alone significantly prolonged W/F heart allograft survival to 43.1 +/- 2.7 days. When pretransplant DL transfusion on day -3 was combined with CsA treatment, 4/8 animals maintained their grafts indefinitely (greater than 100 days). Similarly, DL infusion on day -7 with peritransplant CsA led to indefinite graft survival in 3/5 animals. Administration of DL on days -7 and -3 combined with CsA resulted in indefinite graft survival (greater than 100 days) in 4/6 animals. Transfusion of DL on day -3 alone or in combination with peritransplant CsA, had no effect on a third-party (ACI) heart allograft survival prolongation compared with appropriate controls. To define the underlying mechanisms responsible for donor-specific unresponsiveness in this model, pooled sera and unseparated spleen cells were passively transferred from recipients of long-term cardiac allografts to syngeneic rats receiving donor-type (W/F) or third-party (ACI) cardiac allografts. Transfer of serum (1 ml on days 0, and 1, 0.5 ml on days +2, +3, and +4) from ungrafted recipients of DL on days -14 and -7 led to significant donor graft survival of 9.8 +/- 0.4 days (P less than 0.02) in unmodified hosts. Similarly, passive transfer of serum obtained at 20 and 100 days after transplantation significantly prolonged the MST of donor-type hearts in syngeneic untreated hosts to 11.3 +/- 0.8 and 10.0 +/- 1.1 days, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cyclosporine and skin allografts for the treatment of thermal injury. I. Extensive graft survival with low-level long-term administration and prolongation in a rat burn model

The hypothesis tested in the present and accompanying study is that an effective treatment for severe burns involves early excision of necrotic tissue followed by skin allografting and cyclosporine (CsA) immunosuppressive therapy. LEW (RT1) rats served as recipients of thermal injury and/or skin allografts. BN x LEW F1 (LBN, RT1(l+n)) rats served as skin donors. LEW burn recipients received a hot water (90 degrees C for 10 sec) 30% body surface area (BSA) full-thickness burn. As expected, LEW recipients treated with CsA (25 mg/kg/day for 20 days) demonstrated significant graft prolongation compared with controls (P less than 0.005). Skin graft survival was similarly prolonged in LEW recipients undergoing burn injury, primary wound excision, and CsA administration compared with burn-skin allograft controls (P less than 0.001). Mortality was not increased in the thermal injury-CsA-treated recipients compared with burn controls. A final experiment was initiated to investigate how low-level long-term (greater than 100 days) maintenance CsA treatment influenced skin allograft survival for possible future consideration in burn trauma. Recipients receiving skin allografts plus CsA (20 days, 8mg/kg/day, followed by every other day thereafter) did not reject their grafts. However, a possible early sign of rejection (a single small ulcerative lesion) was noted in five of these long-term CsA-treated animals at a mean of 34 +/- 11 (SD) days. The lesion in these animals did not progress any further during CsA administration. Histopathologic study of selected animals removed from the CsA maintenance regimen for greater than 50 days following long-term administration revealed a number of interesting chronic lesions similar to those previously reported in the skin component of composite tissue (limb) allografts following long-term low-level CsA intervention. In conclusion, CsA was very successful in preventing rejection of skin allografts in a rat burn model without apparent adverse effects.     

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.     

Donor-specific antigen and cyclosporine in rat islet allografts

Combination therapy with one dose of 3 M KCl extracted donor-soluble antigen (Ag) and a short course of cyclosporine (CsA) has proven to prolong the survival of kidney allografts by enhancing specific T-suppressor populations. This regimen is tested in rat islet allografts in this study (Lewis to ACI). A 3-day perioperative course of 10 mg/kg/day CsA on Days -1, 0, and 1 did not prolong graft survival (MST = 10.7 +/- 2.5 days vs 9.4 +/- 1.2 days in controls). When this course of CsA therapy was combined with a single dose of donor antigen on Day -1, the survival time was prolonged slightly but significantly (MST = 14.0 +/- 5.8 days). Three cycles of a 3-day course of CsA therapy at 7-day intervals, a total of nine doses of 10 mg/kg/day CsA, were effective in delaying rejection of islet allografts (MST = 26.4 +/- 30.3). Moreover, combined therapy with donor antigen and three cycles of a 3-day course of CsA prolonged the survival of islet allografts (MST = 57.7 +/- 51.4 days) with 50% of recipients still normoglycemic at 60 days after transplantation. These findings indicate that the combination therapy of donor antigen with a short course of CsA has a powerful effect to prevent the rejection of islet allografts, as shown in kidney allografts, in rats.     

The synergistic effect of low-dose cyclosporine and fluocinolone acetonide on the survival of rat allogenic skin graft

Either cyclosporine (CsA) or fluocinolone acetonide (FA) may prolong the survival of skin allografts. This study was performed to evaluate the effect of combination of these two treatments. BUF rat skin was transplanted to LEW rat. The mean survival time (MST) of control grafts was 9.9 days. In rats fed low dose CsA (2.5 or 5mg/kg/day, blood CsA 221 or 631 micrograms/ml), the MST were 16.0 days. When FA with or without CsA topically applied only, their MST were 22.7 or 24.1 days. If topical application of CsA + FA in combination with low dose oral CsA, the grafts survived indefinitely when the treatment was continued (100 days). The synergistic effect of CsA and topical FA is significant and provides a potential safe means for prolonging skin allograft survival following burn injury.     

Use of sphingosine-1-phosphate 1 receptor agonist, KRP-203, in combination with a subtherapeutic dose of cyclosporine A for rat renal transplantation

BACKGROUND: We demonstrate the long-term effectiveness of KRP-203 treatment in combination with a subtherapeutic dose of cyclosporine A (CsA) on rat renal allografts. METHODS: We tested the effect of KRP-203 in combination with CsA using a rat skin allograft model. The Pharmacokinetic interaction between CsA and KRP-203 was evaluated. The selectivity of KRP-203 for sphingosine-1-phosphate (S1P)1 and S1P3 receptors were investigated in vitro. Heart rate alteration following bolus injection of phosphorylated KRP-203 (KRP-203-P) or FTY720 (FTY720-P) was also monitored in rats. Finally, the long-term effectiveness of KRP-203 in conjunction with a low dose of CsA was investigated in a rat renal transplantation model. RESULTS: Administration of KRP-203 with CsA prolonged skin allograft survival. KRP-203 and CsA had no effect on the pharmacokinetics of the other. While FTY720-P activated both S1P1 and S1P3 receptors, KRP-203-P selectively activated S1P1, but not the S1P3 receptor (EC50:>1000 nM). Compared to FTY720-P, a tenfold higher dose of KRP-203-P was necessary to induce transient bradycardia. With a low dose of CsA (1 mg/kg/day), KRP-203 (0.3 mg/kg/day) significantly prolonged renal allograft survival (P<0.05, survival time: 9.8 days (CsA) vs. >27.4 days (CsA+KRP)). Although a higher dose of CsA (3 mg/kg/day) alone kept recipients alive, this caused severe renal graft dysfunction. Use of KRP-203 (3 mg/kg/day) in conjunction with CsA markedly improved graft function (P<0.05, creatinine clearance: 0.41+/-0.25 ml/min [CsA] vs. 1.15+/-0.16 ml/min [CsA+KRP]). CONCLUSIONS: The selectivity of KRP-203 for S1P1 reduces the risk of bradycardia, and the combination therapy of KRP-203 with CsA represents a safe and effective strategy for use in renal transplantation.     

Intragraft delivery of 16, 16-dimethyl PGE2 induces donor-specific tolerance in rat cardiac allograft recipients

The stable prostaglandin E2 analogue, 16,16-dimethyl PGE2 (di-M-PGE2) was continuously infused by osmotic pump directly into rat heterotopic cardiac allografts. Intragraft delivery of 20 micrograms/kg/day di-M-PGE2 for 2 weeks completely prevented graft rejection for more than 150 days (n = 10), while untreated Buffalo recipients rejected Lewis cardiac allografts within 8 days after transplantation (mean survival time = 7.4 +/- 0.5 days, n = 5). When given for only 1 week, 20 micrograms/kg/day had a partial effect, since 60% of recipients accepted grafts long-term and 40% experienced rejection by day 14 (n = 5). In contrast, systemic intravenous administration of 20 micrograms/kg/day di-M-PGE2 for 2 weeks could not prolong graft survival (MST = 7.0 +/- 0.0 days, n = 3), and the higher dose of 200 micrograms/kg/day resulted in death by day 2 (n = 5). Long-term BUF recipients of LEW cardiac allografts accepted LEW donor strain skin grafts for more than 35 days while rejecting third-party Wistar Furth skin grafts in a normal fashion (MST = 7.3 +/- 0.5 days, n = 3), indicating the induction of donor-specific tolerance. Long-surviving LEW cardiac allografts retransplanted into naive BUF recipients were rejected within 7 days (MST = 6.7 +/- 0.5 days, n = 3), indicating no change in graft immunogenicity. Therefore, a 14-day infusion of di-M-PGE2 directly into a strongly MHC-mismatched cardiac allograft uniformly has resulted in long-term engraftment and the development of recipient donor-specific tolerance.     

Evidence that cyclosporine prevents rejection and recurrent diabetes in pancreatic transplants in the BB rat

Cyclosporine prevents the development of diabetes in spontaneously diabetic BB rats and NOD mice. However, islet transplants have been shown to be subject to immunologic destruction in hosts treated with CsA and anti-CD4 antibody or those rendered tolerant to donor antigens. This study determines whether the minimum dose of CsA necessary to prevent rejection of pancreatic transplants will also prevent recurrent diabetes in pancreas transplants in BB rats. Lewis recipients promptly reject BN (n = 13, MST 9.2 +/- 0.7 days) and Fisher (n = 6, MST = 11.3 +/- 0.8 days) pancreatic transplants. Treatment with CsA 5 mg/kg/day 0-50 days posttransplant and 2 mg/kg/day 51-100 days (low-dose CsA) produced indefinite survival of BN (n = 5) but not Fisher (n = 4) allografts. Fisher allografts were uniformly successful if maintained on 5 mg/kg/day (n = 4). Treatment with CsA 5 mg/kg/day for 14 days had no deleterious effect on glucose tolerance in Lewis isografts (control [K = 1.81 +/- 0.24, n = 8] vs. CsA treated [K = 1.76 +/- 0.20, n = 8, P = NS]). Low-dose CsA ensured permanent survival of MHC-compatible WF (n = 7, MST greater than 115 days) and MHC-incompatible BN (n = 9, MST greater than 117 days, P = NS) pancreatic transplants in spontaneously diabetic BB/Wor hosts. Three of 11 recipients surviving greater than 100 days enjoyed seemingly permanent acceptance of their allografts after discontinuation of CsA. Immunocompetence was demonstrated by rejection of their pancreas transplants when challenged with donor skin. Vascularized pancreatic allografts treated with CsA appear to be less vulnerable to recurrent diabetes than are islet transplants. Low-dose CsA protects vascularized pancreatic allografts in BB rats from both rejection and recurrent diabetes.     

Soluble antigen and cyclosporine-induced specific unresponsiveness in rats. Frequency of alloantigen-specific T cytotoxic cells in normal, sensitized, and unresponsive rats

The cellular mechanisms of unresponsiveness induced with a combined KCl-extracted donor antigen (HAg) and CsA regimen were dissected by limiting-dilution (LD) assay. While untreated Wistar-Furth (WFu, RT1u) rats reject Buffalo (BUF, RT1b) heart allografts within a mean survival time of 6.6 +/- 0.5 days, recipients treated with 3 cycles of CsA alone (-1,0,1; 7,8,9; 15,16,17) maintained BUF heart allografts up to an MST of 22.5 +/- 8.9 days. When CsA was combined with BUF HAg (-1), BUF heart survival was further prolonged up to an MST of 34.2 +/- 6.6 days, while third-party BN HAg was ineffective (MST of 21.5 +/- 2.1 days). On day 10 postgrafting, the frequency of T cytotoxic cells (fTc) within the splenic pan-T-cell population was 1:1437 +/- 301 in CsA and 1:1087 +/- 438 in CsA/HAg treated recipients. In contrast, on day 30 postgrafting, both CsA and CsA/HAg treated WFu rats bearing functional BUF hearts showed within their splenic pan-T-cell populations a profound decrease in fTc to 1:2966 +/- 824 with CsA alone and to 1:4946 +/- 938 with CsA/HAg treatment. In contrast, both untreated WFu rats who rejected BUF heart allografts and CsA-treated WFu recipients who had rejected their BUF heart allografts on day 20 displayed an increased fTc to 696 +/- 243 and to 1:1169, respectively, when examined at day 30 postgrafting. Additionally, both the W3/25+ and OX8+ T cell subsets specifically suppressed the proliferative response of normal WFu T cells against BUF and, to a lesser degree, third-party BN irradiated stimulators. Thus, CsA-treated animals develop a potent specific-suppressor mechanism that is augmented by pretreatment with donor soluble antigen. This suppressor activity may decrease the frequency of alloantigen-specific Tc cells and thereby prolong the survival of BUF heart allografts.     

1alpha,25-dihydroxyvitamin D3 shows strong and additive immunomodulatory effects with cyclosporine A in rat renal allotransplants.

BACKGROUND: Vitamin D3 and its metabolites have long been found to exert immunosuppressive effects both in vivo and in vitro. The present study investigated the effect of 1alpha,25-dihydroxycholecalciferol (1,25DHC) on vascularized renal allografts in rats. METHODS: Three days prior to transplantation, two groups of animals were subjected to 1,25DHC (1 microg/kg/day IP) and a low calcium diet, which was continued until the end of the experiments. Recipient organs were removed and single allografts were transplanted in a high responder strain combination (ACI --> Lewis). Following transplantation, low-dose cyclosporine A (3.2 mg/kg/day CsA) administration was started in two experimental groups of recipients (one group receiving 1,25 DHC additionally) whereas the control allograft recipients received no immunosuppression (control III). Graft survival and renal function was monitored until death or the end of experiments and allograft rejection was assessed histologically using the Banff classification. RESULTS: 1,25DHC significantly prolonged allograft survival in comparison to control III (9.6 +/- 1 vs. 5.7 +/- 0.2 days; P=0.009). In addition, a combination of 1,25DHC and low-dose CsA increased allograft survival compared to CsA administration alone (24 +/- 0.9 vs. 13 +/- 0.3 days; P=0.008). 1,25DHC preserved renal creatinine clearance and decreased proteinuria in comparison to control III, and the combination of 1,25DHC and low-dose CsA again showed an additive effect on preservation of renal function. 1,25DHC and low-dose CsA both decreased interleukin (IL)-2 and IL-12 expression levels in serum and allografts, and a combination treatment produced the strongest attenuation of IL-2 and IL-12 expression. In addition, 1,25DHC increased IL-4 and IL-10 expression levels in allografts, whereas CsA alone did not alter IL-4 and IL-10 expression. In contrast, combination of 1,25DHC and low-dose CsA showed a significant increase in IL-10 expression levels whereas IL-4 expression was not elevated. CONCLUSION: Monotherapy with 1,25DHC significantly prolongs survival of renal allografts and preserves graft function in rats. A combination of 1,25DHC and CsA caused an additive effect on graft survival with differential regulation of pro- and anti-inflammatory cytokines, as compared to 1,25DHC administration alone.     

Evidence that cyclosporine does not inhibit allograft rejection by IL-2-treated sensitized splenocytes

Splenocytes sensitized in vitro to the H-2 allotype of a skin allograft have been shown to cause accelerated rejection of the skin allograft after adoptive transfer of the splenocytes. Treatment of the host with splenectomy or sublethal radiation did not alter the accelerated rejection. In the present study, cyclosporine (CsA) given subcutaneously to mice bearing 1 day old skin grafts prevented the rejection of the graft despite the adoptive transfer of sensitized cells. If the CsA was given for 14 days at 50 mg/kg every other day, the grafts were rejected an average of 6 days after the cessation of CsA. If the CsA was given for 20 days 50 mg/kg every other day, the grafts were not rejected even after cessation of CsA. When no sensitized cells were given, the same pattern resulted; that is, when a 14 day course of CsA was given the grafts were rejected after cessation of the CsA but when a 20-d course was given, the grafts were not rejected even after the CsA was stopped. If splenocytes were sensitized in the presence of interleukin-2 (IL-2), they caused rejection of the skin allografts in animals even on treatment with CsA. We concluded that CsA can prevent skin allograft rejections in the murine system. Moreover, the dose of CsA was critical, in that a longer course of CsA was necessary for tolerance. CsA further prevented the accelerated rejection of skin allografts by adoptive transfer of specifically sensitized splenocytes. Donor irradiation did not alter the effect of the CsA or of the adoptively transferred cells. CsA could not prevent the rejection of skin allografts when the adoptively transferred cells were sensitized to antigen in the presence of IL-2.     

Successful intestinal transplantation in pigs treated with cyclosporine

To date, it has not been possible to reliably prevent intestinal allograft rejection in large animals. This study was undertaken to determine if continuous i.v. cyclosporine (CsA) followed by p.o. CsA would prevent rejection in outbred piglets with orthotopic, in-continuity intestinal allografts. Untreated recipients (n = 7) died of rejection (2), interstitial pnuemontitis (3), or technical complications (2) at 5.3 +/- 1 days. Intestinal recipients treated with i.v. CsA 8 mg/kg/day and i.v. steroids (n = 3) died of rejection (mean survival 11.3 +/- 3.2 days). CsA 20 mg/kg/day i.v. plus i.v. steroids for 21 days, followed by p.o. CsA 25 mg/kg/day (n = 6) prevented rejection; however, most of the recipients developed fatal infections (mean survival 28 +/- 8 days). Intravenous CsA 15 mg/kg/day for 7-10 days (n = 16), followed by p.o. CsA 30 mg/kg/day in tapering doses reliably prevented graft rejection, permitting long-term survival (mean survival 121 +/- 32 days). Rejection did not occur in 7 animals when CsA was discontinued at 97 +/- 11 days. Seven animals surviving more than 100 days maintained normal nutritional indices and gained weight at the same rate as control animals. This study provides a rationale for further experimentation to determine the feasibility of intestinal transplantation in man.     

Experimental pancreatic allotransplantation in large animals. The role of donor kidney and cyclosporine in modifying rejection

Experiments were carried out in outbred dogs and pigs to evaluate the relative immunogenicity of pancreatic islets and segmental pancreas grafts, and whether these could be ameliorated by transplanting a kidney simultaneously from the same donor animal. Various immunosuppressive regimens were also studied. Pancreatic islet allografts never normalized blood glucose in totally pancreatectomized recipients despite the use of cyclosporine (CsA) in high doses (40 mg/kg/day) and the simultaneous transplantation of a kidney from the same donor. These grafts which never "took" contrast sharply with the experience of pancreatic islet autografts prepared in the same way and inoculated into the spleen, which in all nine instances normalized blood glucose in pancreatectomized recipients. Segmental transplants were performed in swine with duct drainage into the jejunum. Totally pancreatectomized pigs died at 7.8 +/- 1.0 days. In recipients suppressed with low-dose azathioprine (Az) and prednisone (Pred) pancreas grafts alone were rejected in 12.9 +/- 10 days. Synchronous pancreas and kidney transplants treated similarly extended the mean survival of pancreatic grafts to 20 +/- 10 days--which, however, was not significant (P less than 0.1 greater than 0.05). Mean survival time of pancreatic grafts in recipients receiving CsA at 20 mg/kg/day and prednisone 1 mg/kg/day was 14 +/- 6.3 days. The combination of CsA 20 mg/kg/day, Az 2 mg/kg/day, and Pred 1 mg/kg/day prolonged the mean survival time to 39.8 +/- 22 days. These results allow us to conclude that: crude preparations of islet tissue invariably capable of normalizing blood sugar at day 4 when used as autografts failed to "take" despite the existence of alternative sources of antigen present in a well vascularized kidney from the same donor, and despite very high dosages of CsA; triple immunosuppressive therapy had synergistic effects on pancreatic allograft survival; and simultaneous transplantation of kidney and pancreas had little effect on survival times of the pancreas or the kidney.     

Tacrolimus versus cyclosporine induction therapy in pulmonary transplantation in miniature swine.

OBJECTIVE: Tacrolimus has been shown to provide superior immunosuppression in various solid organ transplant settings. The purpose of our study was to compare the survival of porcine lung allografts after induction with either cyclosporine A (CsA) or tacrolimus. METHODS: Single lung transplantation from MHC mismatched donors was performed in 10 minipigs. Immunosuppression included 1.5 mg/kg per day methylprednisolone and 1.0 mg/kg per day azathioprine. CsA (n=5) was adjusted to trough levels of 300-500 ng/ml, tacrolimus (n=5) was adjusted to 16-26 ng/ml. All immunosuppressive drugs were discontinued on postoperative day (POD) 28. Allograft survival was monitored by sequential chest radiographs, bronchoscopy and transbronchial biopsy histology. Peripheral blood leukocytes were scanned for donor chimerism and CD3, CD4, CD8 and CD25 expression. RESULTS: The animals survived a 4-week course of immunosuppression without radiological or histological signs of rejection on POD 28. Median allograft survival in CsA-treated animals was 55+/-15 days and all animals rejected their grafts within 42 days after withdrawal of immunosuppression. In tacrolimus-treated animals, median survival was 152+/-65 days with the longest survivor being electively sacrificed on POD 390 (P=0.0064). The degree of donor leukocyte chimerism and the frequency of CD4+CD25+ T-cells were higher in the tacrolimus group, however, these differences were not statistically significant. CONCLUSION: The results of our study show that primary immunosuppression with tacrolimus is superior to cyclosporine after pulmonary allotransplantation in a large animal model.     

Long-term prolongation of cardiac allografts by subtherapeutic levels of cyclosporine in rats conditioned with pretransplant blood transfusions and cyclosporine

This study was aimed at ascertaining whether long-term graft survival was achievable with short term cyclosporine (CsA) therapy or with subtherapeutic doses of CsA in rats conditioned with blood transfusions (BT) combined with CsA. Previous studies had shown that donor-specific transfusions combined with a short course of CsA interacted synergistically, resulting in considerable prolongations of ACI and BUF grafts in LEW hosts receiving no postoperative treatment. The donor-specific depression of alloreactivity was confirmed in the present study by showing a depression of mixed-lymphocyte reaction (MLR) reactivity as well as of humoral antidonor responses in BT-CsA conditioned rats. The effects of postoperative CsA were then studied in recipients conditioned with BT-CsA or BT alone. ACI and BUF cardiac graft survival in LEW hosts conditioned with BT and treated with a five-day postoperative course of CsA (20 mg/kg/day) were indistinguishable from graft survival in untransfused hosts (ACI: 35.6 +/- 15.5 vs. 38.8 +/- 7.4; BUF: 58.4 +/- 39.8 vs. 48.0 +/- 21.7) indicating no interaction between BT and CsA under these conditions. In contrast, the effect of a post-operative five-day course of CsA (10 mg/kg/day) was extended by conditioning the recipients with donor-specific BT and CsA (ACI:41.7 +/- 7.0 vs. 27.4 +/- 11.6; P less than 0.05). More remarkably, a thirty-day course of subtherapeutic doses of CsA (2.5 mg/kg/day) resulted in long-term prolongation (greater than 100 days) of ACI grafts in a large proportion of hosts conditioned with donor-specific BT and CsA, while the majority of controls conditioned with nonspecific BT and CsA or CsA alone rejected their grafts within three weeks (P less than 0.01). The possible mechanisms of this phenomenon are discussed.     

Monoclonal anti-tumor necrosis factor-alpha antibody treatment of rat cardiac allografts: synergism with low-dose cyclosporine and immunohistological studies

Tumor necrosis factor (TNF) levels have been reported to be elevated during episodes of human renal, hepatic, and cardiac transplant rejection. In addition, we have shown polyclonal anti-TNF antibodies to have immunosuppressive effects. The present study was performed to evaluate the efficacy of a monoclonal anti-TNF-alpha antibody in rat cardiac transplantation as the sole immunosuppressant and in conjunction with low-dose cyclosporine (CsA). We also performed immunohistological studies to localize intragraft TNF and evaluate graft infiltrating cells (GICs), and we measured serum TNF levels by an ELISA. Untreated Buffalo to Lewis heterotopic rat cardiac transplants reject in 10.5 +/- 0.4 days. A 10-day induction course of CsA (2 mg/kg/day, po) prolonged survival to 16.7 +/- 2.7 days (P less than 0.05 vs control), and 10 days of anti-TNF (2000 U/day, ip) prolonged survival to 22.6 +/- 0.8 days (P less than 0.05 vs control). Combination of anti-TNF plus CsA synergistically prolonged graft survival to 40.7 +/- 1.8 days. Three-day courses of anti-TNF were moderately effective (13.7 +/- 0.5 days, P less than 0.05 vs control) and were also synergistic with CsA (27.8 +/- 2.2). Intragraft TNF localization using immunoperoxidase showed extensive perivascular and mononuclear cell staining in control hearts vs minimal staining in anti-TNF-treated groups. Likewise, serum TNF levels were significantly lowered for treated groups vs control (83.1 +/- 14.0 pg/ml for control; 39.5 +/- 13.8 for anti-TNF; and 13.4 +/- 5.4 for anti-TNF + CsA; P less than 0.05 vs control for all groups).(ABSTRACT TRUNCATED AT 250 WORDS)     

Antilymphoblast globulin, cyclosporine, and steroids in cadaveric renal transplantation

In order to avoid cyclosporine (CsA) nephrotoxicity and rejection, especially during the early posttransplant periods, different immunosuppression regimens have been adopted. A prospective trial was conducted to evaluate the benefits of initially low CsA doses associated with antilymphoblast globulin and steroids in the first days after transplant, in comparison with higher doses of CsA and steroids. Between 1/86 and 1/88, two groups of first-cadaver renal transplant recipients were documented based on the immunosuppression regimen used. In group A (n = 50), oral CsA was started at 8 mg/kg/day and subsequent doses adjusted to maintain CsA whole-blood levels between 300 and 600 ng/ml. Horse ALG at 10 mg/kg was given the day after transplant and on alternate days to a maximum of 6 doses. After 3 doses, ALG was stopped if CsA blood levels were equal to or greater than 400 ng/ml. ALG dosage modifications were made in order to maintain peripheral CD3+ cells between 10 and 20%. Prednisone was given at 0.25 mg/kg/day. In group B (n = 50), oral CsA was started at 15 mg/kg/day. The CsA whole-blood levels were maintained between 300 and 800 ng/ml. Prednisone was administered at 0.5 mg/kg/day. The incidence of postransplant renal failure was the same in both groups (16%), but the duration of oliguria was lower in group A than in group B (3.3 +/- 2 vs. 16.2 +/- 10.7 days, P less than 0.05), as well as the incidence of acute rejection during the first 3 months (18% vs. 40%, P = 0.01. The cumulative doses of CsA and steroids were significantly lower in group A than in group B. Mean serum creatinine at 6 and 12 months remained similar in both groups. There was no difference between the 2 groups in the incidence of infection. There was no mortality in either group. The actuarial graft survival was significantly higher in group A than in group B at one (100% vs. 94%), two (97% vs. 87%), and three years (89% vs. 73%), respectively (P = 0.041). In summary, the triple regimen using simultaneously low-dose CsA, ALG, and steroids minimizes early graft dysfunction, provides efficient immunosuppression without severe infections, and gives good long-term patient and graft survival.