LF 15-0195, a novel immunosuppressive agent prevents rejection and induces operational tolerance in a mouse cardiac allograft model

BACKGROUND: LF 15-0195 (LF) is a new analogue of 15-deoxyspergualin (DSG) that is less toxic and more potent than DSG. The present study was undertaken to determine (1). the dose response of LF monotherapy, (2). its ability to induce tolerance, and (3) its interaction with cyclosporine (CsA), FK 506 (FK), and rapamycin (RAPA). METHODS: Varying doses of LF were administered to determine dose-dependent effects on graft survival in a C57BL/6 to BALB/c heterotopic heart allograft mouse model. Transplanting-donor and third-party skin grafts into long-term survivors were used to assess the tolerance status. CsA, FK, and RAPA were combined with LF to determine their interactive effects on graft survival. RESULTS: The efficacy and toxicity of LF was dose dependent. High-dose LF monotherapy (>2 mg/kg) induced donor-specific operational tolerance, but it was associated with high mortality. Simultaneous administration of high-dose calcineurin inhibitors (CsA FK) prevented tolerance induced by LF. In contrast, a short course of LF combined with a subtherapeutic dose of CsA FK achieved indefinite survival of C57/BL6 cardiac allografts. RAPA and LF had a synergistic effect in induction of tolerance. CONCLUSIONS: The efficacy and toxicity of LF were dose dependent. A short course of LF significantly reduced the requirement of CsA or FK to prevent rejection. RAPA and LF had synergy in induction of tolerance. These data indicate that LF may be a promising agent that warrants further studies in nonhuman primate models of transplantation.     

A short course of high-dose cyclophosphamide induces long-term survival of intestinal allografts in mice

Several transplant programs have recently added cyclophosphamide (CyP) to their immune suppression protocols in an attempt to reduce intestinal graft rejection rates. The present study was undertaken to confirm the benefits of this drug in a murine small bowel transplant model. A short course of monotherapy with CyP 20 mg/kg per dose resulted in a mean survival time (MST) of 17.5 ± 3.6 days, compared with a MST of 7.5 ± 0.7 days in the untreated controls (P < 0.01). Cyclosporin A (CsA) 30 mg/kg per day produced comparable survival rates when used as monotherapy (MST: 14.2 ± 1.3 days) or in combination with CyP 20 mg/kg per dose (MST: 21.3 ± 5.1 days). Treatment with high dose CyP (40 mg/kg per dose) completely prevented graft loss in 8 of 10 animals (MST: 72.5 ± 5.3 days, P < 0.01). However, adding CsA abrogated the induction of long-term survival achieved by CyP alone (MST: 23 ± 0.4 days). These data have important implications for the use of CyP in clinical transplantation. Received: 14 April 2000 Revised: 11 January 2001 Accepted: 9 May 2001     

Evidence that rapamycin rescue therapy delays rejection of major (MHC) plus minor (non-MHC) histoincompatible heart allografts in rats.

The capacity of delayed onset of rapamycin (RAPA) therapy to block process of destruction was examined in rats undergoing heart allograft rejection. Untreated Wistar Furth (WFu; RT-1u) recipients reject Buffalo (BUF; RT-1b) heart allograft with a mean survival time (MST) of 6.5 +/- 0.5 days. A 14-day i.v.infusion of 0.8 mg/kg RAPA begun on the day of transplantation prolonged the survival to 74.1 +/- 20.2 days (P < 0.001), 0.2 mg/kg to 32.2 +/- 10.0 days (P < 0.001), and 0.08 mg/kg to 36.4 +/- 11.8 days (P < 0.001). When RAPA therapy (0.8 mg/kg) was begun 3 or 4 days after transplantation, the grafts survived 85.2 +/- 31.1 (P < 0.001), and 70.2 +/- 43.3 (P < 0.005) days, respectively. Therapy initiated on day 5 was much less effective; most transplants were rejected within 10 days; one graft survived 32 and two grafts 60 days (MST = 17.6 +/- 20.0, NS). A 0.2 mg/kg RAPA dose prolonged graft survival with initial use on days 3 (31.6 +/- 12.2 days; P < 0.001) or 4 (31.4 +/- 8.1 days; P < 0.001) but not on day 5. The 0.08 mg/kg RAPA prolonged hearts only when started on day 3 (47.2 +/- 2.7 days; P < 0.001) but not on days 4 or 5. WFu recipients treated with a subtherapeutic dose of cyclosporine (1 mg/kg; 9.1 +/- 1.5 days) displayed prolonged heart allograft function when treated subsequently with RAPA (0.8 or 0.08) beginning from days 4, 5, or 6 postgrafting. These in vivo results are supported by in vitro experiments. The frequency of BUF alloreactive elements among normal WFu LN cells (fTc) was 337 +/- 139/10(6) T cells in limiting dilution assay. Addition of RAPA (1 muMol) at the beginning of culture significantly reduced (P < 0.025) the fTc to 17 +/- 6.6/10(6), or alternatively on days 4 or 6 to 37.3 +/- 20.0/10(6) and 58.6 +/- 21.8/10(6), respectively. Thus, both in vivo and in vitro data demonstrate that delayed RAPA therapy may interrupt alloimmune reactions.     

Rapamycin, a potent immunosuppressive drug for vascularized heart, kidney, and small bowel transplantation in the rat

The effectiveness of rapamycin (RAPA) was examined for heart, kidney, and small bowel allografts in rats. Untreated or vehicle only-infused Wistar Furth (RT1u) recipients rejected Buffalo (RT1b) heart allografts within a mean survival time (MST) of 6.5 +/- 0.5 and 6.3 +/- 0.5 days, respectively. In contrast, a 14-day continuous intravenous (i.v.) infusion by an osmotic pump of 0.08 mg/kg/day RAPA to WFu recipients prolonged BUF heart allograft survival to an MST of 34.4 +/- 12.1 days (P = 0.0001). There was a graded dose-response to 0.16 mg/kg (39.0 +/- 8.7 days; P = 0.0001), 0.32 mg/kg (55.7 +/- 3.3 days; P = 0.0001) and 0.8 mg/kg (48.0 +/- 3.6; P = 0.0001). Furthermore, intraarterial/intragraft but not i.v. infusion of 0.02 mg/kg/day prolonged BUF heart allografts--namely, an MST of 14.6 +/- 1.4 days versus 8.6 +/- 2.6 days (P = 0.0001), respectively. Local delivery doses of RAPA were about as effective as the same dose delivered i.v.: 0.08 mg/kg MST 37.0 +/- 18.3 days (P = 0.0001); 0.32 mg/kg, 40.0 +/- 3.9 days (P = 0.0001); and 0.8 mg/kg, 54.8 +/- 8.2 days (P = 0.0001). Systemic i.v. RAPA therapy with 0.08 or 0.8 mg/kg/day prolonged the survival of BUF kidney grafts in WFu recipients--namely, an MST of 52.7 +/- 42.7 (NS) and 90.2 +/- 62.4 (P = 0.001) days, respectively, versus an MST of 11.6 +/- 1.5 days in control WFu recipients only infused with vehicle. While normal WFu rats reject heterotopic BUF small bowel allografts within an MST of 10.0 days, a 14-day course of i.v. RAPA treatment significantly (P = 0.0001) prolonged small bowel allograft survival to an MST of 26.8 +/- 3.7 days.     

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 importance of the spleen for the immunosuppressive action of cyclosporine in transplantation

The spleen plays an important role in the response of the recipient's immune system to a primarily vascularized graft and cyclosporine treatment is known to alter this response. To investigate the interaction between the splenic immune response and CsA's immunosuppressive actions more thoroughly, Lewis recipients of Brown-Norway heterotopic heart grafts were treated i.p. daily with normal saline or with CsA doses of 0.75, 1.5, or 3.0 mg/kg/day from day 1 through day 50 or until rejection. Rats treated with 3 mg/kg were splenectomized intraoperatively (i.o.) or not splenectomized. Rats in subgroups of the other treatment groups were splenectomized i.o., on day 5, not splenectomized, or the recipient's spleen cells were reinfused after i.o. splenectomy. In non-CsA-treated rats, i.o. splenectomy (median survival time, [MST] = 11 days) and day 5 splenectomy (MST = 11 days) prolonged graft survival minimally in comparison with nonsplenectomized animals (MST = 7 days). Reinfusion of the spleen cells reversed this effect (MST = 7 days). Most interestingly, the immunosuppressive efficacy of 1.5 mg/kg of CsA (MST = 91 days) was reduced by day 5 splenectomy (MST = 24 days) and completely abolished by i.o. splenectomy (MST = 11 days). Spleen cell reinfusion partially restored the effect of CsA treatment (MST = 88 days). Since splenectomy resulted in a complete abrogation of the immunosuppressive efficacy of 1.5 mg/kg CsA, our results support the hypothesis that certain spleen cells augment immunosuppression by CsA. These findings provide additional evidence that the immune system's own regulation of its antigraft response can be an important component of the overall suppression of rejection that is associated with the use of certain immunosuppressive drugs.     

15AU81, a prostacyclin analog, enhances donor-specific hepatocytes to prolong the survival of rat heart but not small bowel allografts

Prostacyclin analogs have previously been shown to have not only cytoprotective but also independent immunosuppressive effects. The effect of one such analog, 15AU81, to enhance the immunosuppressive effects of liver was investigated. We have previously demonstrated that cyclosporine (CsA) in conjunction with rapamycin (RAPA) potentiates class I+, class II- donor-specific hepatocytes to prolong rat cardiac and small bowel allograft survival. Brown Norway (BN; RT1n) hepatocytes alone (5 x 10(7)/kg, administered intrasplenically) failed to prolong the survival of BN heart allografts in Wistar Furth (WFu; RT1u) recipients, beyond that of untreated controls (MST = 7.2 +/- 0.8 days). Survival of BN hearts was increased to 11.4 +/- 1.7 days in WFu recipients treated with BN hepatocytes and 50 microg/kg/day 15AU81 administered by continuous s.c. infusion for 14 days using osmotic pumps (p < 0.05). The further addition of RAPA 0.0075 mg/kg/day and CsA 0.375 mg/kg/day delivered for 14 days by continuous i.v. infusion (CIVI) using osmotic pumps (a combination that alone prolonged BN heart allografts in WFu hosts to 18.4 +/- 1.3 days and in conjunction with BN hepatocytes prolonged survival to 27.2 +/- 1.9 days) prolonged allograft survival to 35.2 +/- 5.2 days. In contrast, the survival of small bowel allografts was not enhanced by 15AU81 administration. Survival of BN small bowel transplants in LEW recipients treated with hepatocytes alone (MST = 11.6 +/- 1.5 days) or hepatocytes plus 15AU81 (MST = 10.0 +/- 1.0 days) was similar to controls (MST = 10.2 +/- 1.9 days). Treatment with hepatocytes and RAPA/CsA increased survival to 21.2 +/- 1.5 days. The further addition of 15AU81 failed to augment this (MST = 17.0 +/- 1.9 days). In vitro WFu lymphocyte proliferative responses from animals pretreated with BN hepatocytes, 15AU81, or both treatments, for 2 weeks prior to harvesting, exhibited a reduction of at least 50%, compared to untreated controls upon allostimulation with irradiated BN or ACI spleen cells. These findings demonstrate that 15AU81 interacts favorably with hepatocytes either alone or in conjunction with RAPA and CsA to enhance their immunosuppressive effects on rat heart allograft survival. The failure to enhance small bowel allograft survival may be explained by the inability at this low dosage of 15AU81 to influence the intense graft versus host reaction elicited by small bowel transplants.     

Piceatannol in combination with low doses of cyclosporine A prolongs kidney allograft survival in a stringent rat transplantation model

BACKGROUND: The discovery of new immunosuppressive agents has enhanced short-term graft survival. However, current immunosuppressants often induce toxicities that limit their clinical use. Thus, there is a need for new immunosuppressants for use in clinical transplantation. Piceatannol blocks Syk and ZAP-70, tyrosine kinases involved in immune cell activation. We examined whether piceatannol prolongs kidney allograft survival in the stringent ACI-to-Lewis rat model. METHODS: Kidney recipients were divided into four groups. Group 1 (n=8) received piceatannol 30 mg/kg per day intravenously and cyclosporine A (CsA) 2 mg/kg per day intramuscularly from day -3 to day 7 after transplantation. At day 8, piceatannol was reduced to 10 mg/kg per day and the combined treatment continued until day 60. Group 2 (n=9) received 2 mg/kg per day CsA alone from day -3 to day 60. Group 3 (n=4) received piceatannol alone as in group 1. Group 4 (n=2) received only the vehicle dimethyl sulfoxide from day -3 to day 60. Graft rejection was defined as either a serum creatinine level more than 2 mg/dL or animal death. RESULTS: Group 1 animals survived for at least 115 days (n=8, P<0.05), with several animals maintaining their grafts for more than 200 days. In contrast, 8 of 9 animals in group 2 rejected their grafts within 10 days of transplantation; one animal survived for 71 days. Excellent graft function was maintained in group 1 animals despite withdrawal of immunosuppression. CONCLUSIONS: These results are the first to show that piceatannol, when combined with subtherapeutic dosages of CsA, prevents graft rejection, suggesting that targeting Syk and Zap could be useful for preventing graft rejection.     

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.     

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.     

Treatment with a short course of LF 15-0195 and continuous cyclosporin A attenuates acute xenograft rejection in a rat-to-mouse cardiac transplantation model

Searching for a novel immunosuppressive agent to effectively prevent acute vascular rejection (AVR) is essential for success in clinical xenotransplantation. We previously reported that Lewis rat hearts transplanted into BALB/c mice developed typical AVR in 6 days. The present study was undertaken to determine the efficacy of LF 15-0195, a new immunosuppressive analog of 15-deoxyspergualin in the prevention of AVR in a rat-to-mouse cardiac xenograft model. We transplanted 2-week old Lewis rat hearts into BALB/c mice. Four groups were included in this study: untreated recipients and cyclosporin A (CsA) treated recipients were controls; LF 15-0195 treated recipients or LF 15-0195 combined with CsA treated recipients were experimental groups. Mouse recipients received either LF 15-0195 2 mg/kg subcutaneously from day-1 to post-operative day 14, or CsA 15 mg/kg subcutaneously daily, from day 0 to endpoint rejection, or the two drugs in combination. We observed that high dose CsA did not inhibit AVR and the graft was rejected in 11.3 +/- 1.9 days. Graft histology and immunohistology showed typical AVR, characterized by interstitial hemorrhage, intravascular fibrin deposition, thrombosis, and massive deposition of anti-rat immunoglobulin G (IgG) and immunoglobulin M (IgM). Serum xenoreactive antibodies (xAbs) were markedly elevated in these animals as well. In contrast, we observed that treatment with LF 15-0195 alone significantly prolonged graft survival to 19.3 +/- 0.7 days. Notably, xAbs were significantly decreased and the rejection pattern of these grafts was cell-mediated rejection (CMR), instead of AVR. When CsA was combined with LF 15-0195, the graft mean survival time was further increased to 58.5 +/- 17.3 days. Antibody production and T-cell infiltration were significantly inhibited at the terminal stages of graft survival and pathology showed striking attenuation of both AVR and CMR. Sequential studies on days 6 and 14 demonstrated that LF 15-0195 either alone or combined with CsA completely inhibited antibody production. However, intragraft infiltration by Mac-1 positive cells including natural killer cells, macrophages and granulocytes in LF 15-0195 treated recipients was similar to that of untreated recipients. We conclude that LF 15-0195 effectively prevented AVR by markedly inhibiting the production of anti-donor IgG xAbs. Also, treatment with short course LF 15-0195 and continuous CsA significantly reduced T-cell infiltration. Studies to test this therapy in inhibiting AVR in a pig-to-non-human primate xenotransplantation model are underway.     

Synergistic interaction of 3 M KCl-extracted donor antigens (e-HAg) with cyclosporine or cyclosporine/sirolimus for prolongation of rat heart allograft survival

Extracted donor histocompatibility antigens (e-HAg) may potentiate the effects of drugs to protect organ allografts from rejection. We examined the capacity of e-HAg when combined with cyclosporine (CsA) alone, sirolimus (rapamycin, RAPA) alone, or CsA/RAPA combinations to prolong heart allograft survival in rats. Wistar-Furth (WF: RT1^u) rats that received CsA (10 mg/kg/day) by oral gavage for 3 (days 0, 1 and 2) or 7 (days 0, 1, 2, 3, 4, 5 and 6) consecutive days displayed modest prolongation of Brown Norway (BN; RT1ⁿ) heart allograft survival from a mean survival time of 7.2 ± 0.8 days in untreated controls to 12.2 ± 1.1 days and 18.6 ± 2.7 days, respectively (p < 0.01). Although administration on the day of transplantation (day 0) of a single intravenous (i.v.) dose of BN e-HAg (5 mg/kg) failed to affect allograft survival, both three (days 0, 1 and 2) and five (days 0, 1, 2, 3 and 4) injections significantly potentiated the effect of a 3-day course of oral CsA (18.6 ± 1.3 days (p < 0.01) and 20.0 ± 1.4 days (p < 0.01), respectively) and of a 7-day course of oral CsA (25.3 ± 4.4 days (p < 0.05) and 33.5 ± 9.3 days (p < 0.01), respectively). Median-effect analysis confirmed a synergistic interaction between CsA (0.5 mg/kg × 7 days, i.v.) and e-HAg with combination index (CI) values less than 0.7 (CI = 1 shows additive interactions, CI < 1 synergistic, and CI> 1 antagonistic, interactions). In contrast, e-HAg failed to affect the immunosuppressive effect of RAPA. However, e-HAg (5.0 mg/kg × 3 days) significantly potentiated the effects of a 7-day or 14-day course of RAPA (0.01 mg/kg)/CsA (0.5 mg/kg) combination therapy, namely from 26.0 ± 4.8 days with a 7-day treatment of CsA/RAPA alone to 32.6 ± 3.6 days (p < 0.01) and from 28.2 ± 2.7 days with a 14-day course of CsA/RAPA alone to 42.0 ± 4.9 days (p < 0.05), respectively (CI = 0.2–0.5). Thus, e-HAg potentiates the immunosuppressive effects of CsA alone and of the CsA/RAPA combination, but not of sirolimus alone.     

Efficacy of SDZ RAD compared with CsA monotherapyand combined Rad/FTY720 treatment in a murine cardiac allotransplantation model.

OBJECTIVE: The macrolide immunosuppressant RAD and the immunomodulator FTY720 have distinct mechanisms ofaction. We investigated the efficacy of RAD (everolimus, certican) alone or in combination with FTY720 on graft survival (GS)and histology in comparison with CsA, using mouse strains with strong MHC disparity. METHODS: Heterotopic cardiac grafting was performed using the C57B1/6 to C3H strain combination. Osmotic mini-pumps filled with CsA or RAD were implanted subcutaneously. IFTY720 was administered as a single daily dose by gavage. Peripheral lymphocyte count (PLC) was determined at 1, 4 and 8 weeks or on the day of sacrifice. Body weight was recorded on the day of surgery and weekly. Grafts were histologically evaluated. MAIN FINDINGS: In placebo-treated mice the allografts were rejected after 7 days. Monotherapy with 10 and 30 mg/kg/day CsA achieved 10 and 22.5 days median survival time (MST), while 0.1, 0.3, 1 and 3 mg/kg/day RAD resulted in 10.5, 20, > 56 and > 56 days MST, respectively. FTY720 lowered the PLC significantly, while the lower CsA dose and RAD did not influence the PLC. Adding FTY720 to the 0.6 mg/kg/day dose of RAD extended GS modestly but reduced significantly the perivascular infiltration and endothelialitis in the grafts compared with RAD monotherapy. CONCLUSIONS: Underthe conditions of the present experiment RAD was more potent than CsA in extending the GS. Combining FTY720 and RADwas well tolerated with respect to weight gain and lack of clinically detectable infections in the mice. The 2-drug regimens suppressed the inflammatory allo-response better than RAD monotherapy.     

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.     

Combined FTY720/cyclosporine A treatment promotes graft survival and lowers the peripheral lymphocyte count in DA to lewis heart and skin transplantation models

OBJECTIVE: The immunomodulator, FTY720, lowers the peripheral lymphocyte count (PLC) by inducing migration of circulating lymphocytes to secondary lymphoid organs. We investigated the efficacy of mono- vs. combined-FTY720/CsA therapy on graft survival (GS) and on lowering the PLC in a solid organ and a skin graft model, using strains with strong MHC disparity. Methods: Heterotopic cardiac or tail skin grafting was performed using the DA (RT1a) to Lewis (RT1(1)) rat strain combination. FTY720 was administered as a single daily dose by gavage alone or in combination with subcutaneously delivered CsA. PLC, body weight and drug concentrations were determined on day 7, 28, or the day of rejection. MAIN FINDINGS: In placebo-treated animals the heart and skin allografts rejected after 6 and 8 days. FTY720 delayed rejection of both the solid organ and skin grafts. The maximal effect was achieved at 1 mg x kg(-l) x day(-1) FTY720, resulting in a median survival time (MST) of 14 days for both allotransplants comparable to the effect achieved by 1 mg x kg x day(-1) CsA in both models. In the cardiac graft experiment with CsA co-administration, doses of 0.3 and 1 mg/kg were used. Under these conditions very small doses of FTY720 were effective in maintaining grafts throughout the treatment period. Adding higher FTY720 doses to the 1 mg x kg(-1) x day(-1) CsA was needed to effectively extend the skin GS, e.g. 0.3 mg x kg(-l) x day(-1) FTY720 prolonged GS from 13 to 47.5 days MST, i.e. well beyond the 28 day-treatment period. CsA did not influence the PLC at clinically relevant doses. FTY720 lowered the PLC significantly and dose-dependently, at doses lower than those needed for the prolongation of both cardiac and skin GS with FTY720 monotherapy. In rats with skin grafts the PLC was markedly lowered up to 1 mg x kg(-1) x day(-1) FTY720, whereas, in the heart model, it was lowered up to 0.1 mg x kg(-1) x day(-1). Independently of the graft type, within the combination regimens 0.3 mg x kg(-1) x day(-1) FTY720 achieved a maximal PLC depletion. CONCLUSIONS: Combining FTY720 and CsA was very well tolerated with respect to weight gain and lack of any clinically detectable infections. In the strain combination used FTY720 monotherapy was less effective than previously reported in maintaining grafts. The two-drug regimens extended strikingly the GS for both models. However, the prolongation of the heart GS was smoothly dose-related with FTY720 doses ranging from 0.01 to 1 mg x kg(-1) x day(-1) , whereas, the skin graft prolongation was modest at doses up to 0.1 mg x kg(-1) x day(-1) and remarkably enhanced at 0.3 and 1 mg x kg(-1) x day(-1) FTY720.     

Long-term kidney graft survival by delayed T cell ablative treatment in rhesus monkeys

BACKGROUND: Tolerance to organ allografts in primates including man has been elusive, although in rodents and pigs tolerance can be achieved to organ allografts with relatively short courses of immunosuppressive treatment. In all varieties of graft acceptance that do not require full-dose maintenance immunosuppression, immunological engagement of donor and recipient and an early unstable period have been observed. On the basis of the hypothesis that elimination of aggressive T cell function should tip the balance in favor of an operationally tolerant state, experiments have been performed in monkeys allowing recipient-donor interaction before T-cell ablation and a short course of immunosuppression. METHODS: Rhesus monkeys received an allogeneic kidney graft from a MHC-mismatched donor. The animals either received anti-CD3 immunotoxin (FN18-CRM9) alone, started 2 days after transplantation, or in combination with a short course of cyclosporine (CsA) and/or rapamycin (RAPA), started at 5 days after transplantation. Kidney function was followed by monitoring serum creatinine levels and regular biopsies. Humoral and cellular antidonor immunity was tested in vitro before and at several time points after transplantation. RESULTS: Graft survival of monkeys that received CsA alone (mean survival time (MST)=29.3) was significantly prolonged compared with the controls (MST=6). FN18-CRM9 treatment alone also resulted in prolonged graft survival (MST=29.4). The combined treatment of FN18-CRM9 and CsA and/or RAPA resulted in prolonged graft survival after all immunosuppression was stopped (MST=207.8). CONCLUSIONS: It seems feasible to postpone immunosuppression posttransplantation and yet prevent allograft rejection without the need of permanent immunosuppression.     

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.     

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 mitigates graft coronary artery disease in murine cardiac allografts: description and validation of a novel fully allogeneic model.

BACKGROUND: The effect of cyclosporine (CsA) on the development of graft coronary artery disease (GCAD) is controversial. We developed a novel allogeneic mouse model of heart transplantation and investigated the effect of CsA on acute rejection and GCAD. METHODS: Hearts of FVB mice (H-2(q)) were heterotopically transplanted into 60 C57BL/6 mice (H-2(b)). CsA was administered to recipients at 10, 20 or 30 mg/kg/day for 10 or 30 days after transplantation. Untreated recipients as well as isograft recipients served as controls. Viability of the grafts was assessed daily by palpation. Parenchymal rejection was scored in grafts surviving 30 days in the 30-day treatment groups. GCAD was evaluated by the percentage of luminal narrowing, intima/media ratio and percentage of diseased vessels. Blood CsA and creatinine levels were also evaluated. Results were evaluated statistically. RESULTS: All groups except the untreated control group and the allograft groups treated with 10 or 20 mg for 10 days showed significant graft survival (>/=33% survival for 30 days). An inverse correlation was observed between CsA treatment dose, parenchymal rejection score and degree of GCAD in the 30-day treatment groups. However, graft survival in the 20-mg/kg/day group was significantly better than that in the 30-mg/kg/day group. Serum creatine levels showed no nephrotoxicity. CONCLUSIONS: Relatively high-dose CsA mitigated parenchymal rejection and GCAD of the mouse cardiac allografts. In addition, a valuable mouse model mimicking the clinical course of GCAD was achieved with CsA treatment of 20 mg/kg/day for 30 days.     

Macrophage depletion prevents anti-graft antibody production and results in long-term survival in xenotransplantation

Liposome-encapsulated dichloromethylene diphosphonate (clodronate) is known to deplete macrophages. We examined the effect of clodronate on xenoreactive antibody production and xenograft rejection. Hamster cardiac grafts were transplanted into Lewis rats. Clodronate (4 mL/kg) was injected intravenously on the day before transplantation. In some groups, cyclosporine A (CsA) at a dose of 15 mg/kg was given daily intramuscularly until the end of each experiment. Untreated Lewis rats rejected the grafts at 2 and 3 days after transplantation. Neither CsA treatment alone nor clodronate treatment alone prolonged graft survival. Five of 7 Lewis recipients treated with clodronate and CsA did not reject hamster hearts for 100 days. Antibody production in the CsA plus clodronate-treated group was suppressed compared with control groups.