Combined FTY720/cyclosporine treatment promotes graft survival and lowers the peripheral lymphocyte count in a murine cardiac allotransplantation model

BACKGROUND: FTY720 lowers the peripheral lymphocyte count (PLC) by accelerating the migration of circulating lymphocytes to secondary lymphoid organs. We investigated the efficacy of combined FTY720+cyclosporine (CsA) treatment versus monotherapy on prolonging graft survival and on lowering the PLC. METHODS: BALB/c hearts were heterotopically grafted in C3H mice. FTY720 was administered alone or in combination with CsA. PLC and body weight were determined on day 7, day 28, or the day of rejection. RESULTS: Combining FTY720 with CsA prolonged, dose-dependently and significantly, the allograft survival. FTY720, but not CsA, lowered the PLC dose-dependently. The granulocyte count was not reduced in any group. FTY720 concentrations were not influenced by the CsA co-administration. CONCLUSIONS: Combined FTY720 and CsA treatment was well tolerated, promoted graft survival, and suppressed the inflammatory allo-response. The PLC lowering correlated well with the antirejection effects in the two-drug regimens, suggesting that the PLC might guide FTY720 therapy at low doses.     

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.     

FTY720诱导大鼠心脏移植物长期存活

目的 观察FTY720对大鼠同种异体心脏移植物存活时间的影响。方法 进行SD Wistar大鼠的腹部异位心脏移植，将受者随机分为对照组、甲泼尼龙(MP)组、环孢素A((SsA)组、FTY720组、FTY720与CsA二药联用组和FTY720、CsA及MP三药联用组，各组按分组要求分别于术前3d至术后14d通过灌胃给予FTY720和CsA，术前1d至术后2d腹腔注射给予MP，观察各组动物术后外周血淋巴细胞数量变化和移植物存活时间。结果 FTY720组、二药联用组和三药联用组的大鼠外周血淋巴细胞在给药后3h开始明显下降，停药后开始回升，至停药14d后恢复正常；移植心脏的存活时间，对照组平均为7．8d，CsA组为16．0d，MP组为27．6d，三药联用组为16．8d，而FTY720组和二药联用组分别超过了150d和124d。结论 FTY720可诱导同种异体大鼠心脏移植物长期存活。     

Preoperative administration of FTY720 prolonged renal allograft survival

BACKGROUND: FTY729 is an immunomodulator obtained by chemical modification of Myriocin(ISI-1) which exists in the culture filtrate of an ascomycete, Isaria sinclairii. It has been reported that postoperative administration of FTY720 prolonged survival of various kinds of transplanted organs. In the present study, we evaluated the effect of 2-day preoperative administration of FTY 720 on graft survival. MATERIALS AND METHODS: We used a rat renal transplantation model in which Wistar King Aptekman Hokkaido (WKAH, RT1K) served as the organ donor and Lewis (LEW, RTl) as the recipient. FTY720 was given to the recipients consecutively 2 days (day-2, day-1) before transplantation at the doses of 1, 3 or 5 mg/kg/day. Renal allograft survivals, hematological parameters of recipient blood and phenotypic analysis of recipient splenic cells and graft infiltrate were evaluated. RESULTS: Consecutive 2-day preoperative oral administration of FTY 720 at the doses of 1, 3 or 5 mg/kg/day significantly prolonged WKAH allograft survivals compared with those of the untreated recipients. The number of peripheral blood lymphocytes was markedly decreased in the recipients treated with FTY720 at the doses of 3 mg/kg/day or 5 mg/kg/day on the 5th postoperative day. Preoperative FTY 720 administration significantly decreased the number of CD4 positive cells and the percentage of interleukin 2 receptor (IL-2 R) positive cells infiltrating both spleen and allograft at the dose of 3 mg/kg/day or 5 mg/kg/day. CONCLUSION: FTY 720 could act as a safe and potent immunomodulator by decreasing the number of peripheral lymphocytes, especially CD4 positive cells and IL-2R positive cells when it is given to the recipient preoperatively.     

Long-term islet graft survival in streptozotocin- and autoimmune-induced diabetes models by immunosuppressive and potential insulinotropic agent FTY720

BACKGROUND: Toxicity of current immunosuppressive agents to islet grafts is one of the major obstacles to clinical islet transplantation (Tx). This study was designed to assess the efficacy of FTY720, a novel immunomodulator with a unique mechanism of action, on islet graft survival and function in streptozotocin (STZ)- and autoimmune-induced diabetic recipients. METHODS: Islet allograft from BALB/C mice or islet isografts were transplanted into STZ-induced diabetic CBA mice and autoimmune nonobese diabetic (NOD) mice. FTY720 was administered orally at 0.5 mg/kg per day in STZ diabetic recipients or 3 mg/kg per day in NOD recipients after Tx. Functional status of the islet graft was monitored by measuring blood glucose daily. Insulin secretion from mouse islets was measured with an insulin scintillation proximity assay. RESULTS: Under the treatment of FTY720, long-term normoglycemia (>100 days) was achieved in 100% of STZ diabetic recipients and 50% of diabetic NOD recipients compared with a respective 11 and 7 days in untreated animals after allogeneic islet Tx. Normoglycemia persisted only temporarily (<4 weeks) in untreated NOD recipients of NOD islets, but was maintained for >100days with FTY720 treatment. Histologically, leukocyte infiltration observed in untreated animals was largely inhibited in FTY720-treated ones. Additionally, FTY720 stimulated insulin secretion from isolated islets by approximately twofold under both normoglycemic and hyperglycemic conditions. CONCLUSIONS: FTY720 is highly effective in protecting allo- and autoimmune response-mediated islet graft destruction without direct toxicity to the islets. The effect is likely attributable to its action in preventing effector lymphocyte infiltration into the grafted tissue.     

Significant prolongation of orthotopic corneal-graft survival in FTY720-treated mice

BACKGROUND: The novel immunomodulator, FTY720, mainly acts through sequestering of lymphocytes to secondary lymphatic tissue, thereby suppressing their infiltration into grafted organs. This study aimed to investigate its influence on corneal-graft survival. METHODS: Sixteen BALB/c mice (H-2d) received corneal transplants from C3H (H-2k) mice. Eight mice were treated with FTY720 (10 mg/kg per day) orally from day -1 to day 11, and all animals received 0.1% dexamethasone eye drops for the same time. In addition, eyes and regional lymph nodes from similarly treated animals were subjected to immunohistochemistry and proliferation assays. RESULTS: FTY720 significantly prolonged graft survival from 28+/-8.1 to 36.5+/-7.1 days (P=0.021). In treated animals, corneal infiltration by CD4+ and F4/80+ cells was reduced from 70.8+/-60.3 to 7.0+/-9.0 (P=0.004) and from 97.5+/-30.7 to 44.8+/-24.9 (P=0.01) cells, respectively, and allogeneic T-cell proliferation was decreased. CONCLUSIONS: FTY720 treatment substantially protects corneal allografts and may provide an immunomodulatory strategy in clinical corneal transplantation.     

Macrophage colony-stimulating factor inhibits tumor necrosis factor production and prolongs skin graft survival

BACKGROUND: Despite the availability of a variety of immunosuppressive agents, acute rejection and infection after organ transplantation remain serious problems. METHODS AND RESULTS: We examined the effect of macrophage colony-stimulating factor (M-CSF) on the production of tumor necrosis factor (TNF) in a Bacille de Calmette Guérin-lipopolysaccharide-challenged mouse model. Both serial and repeated injections of M-CSF inhibited TNF production in a dose-dependent manner. Electrophoretic mobility shift assay showed that M-CSF-induced inhibition of TNF production was a result of suppression of nuclear factor-kappaB. High-dose M-CSF significantly prolonged skin graft survival in mice with orthotopic transplantation compared with the control and low-dose M-CSF groups. The combined administration of low-dose M-CSF and cyclosporine also significantly prolonged graft survival compared with the control and low-dose single agent-treated groups. CONCLUSIONS: Our results indicate that M-CSF at a high dose is a potent inhibitor of cytokine production and can potentially be used as an immunosuppressive agent for allograft rejection.     

Skin allograft survival and analysis of renal parameters after FTY720 + tacrolimus treatment in mice

Calcineurin inhibitors such as cyclosporine (CsA) and tacrolimus (FK506) show similar efficacy to prevent rejection within the first year after organ transplantation. However, their use is limited by side effects, such as kidney damage, hypertension, new-onset diabetes, and hyperlipidemia. The consensus opinion suggests that compared with CsA, FK506 has fewer negative effects on blood pressure, serum lipids, and renal function. Nevertheless, FK506 use is associated with a higher incidence of posttransplantation diabetes mellitus. FTY720 is a new compound that has shown beneficial effects in animal models of rejection in transplantation, ischemia/reperfusion injury, autoimmune diseases, and tumor development. Our aim was to investigate whether FTY720 + tacrolimus association could provide additional immunosuppression without causing renal toxicity. FTY720 as a monotherapy or in association with FK506 was administered to C57BL/6 mice for 21 days to prevent skin graft rejection and to evaluate renal function and structure. Increased skin allograft survival in the FTY720 + FK506 group was associated with decreased cell numbers in the spleen, blood, and axillary lymph nodes. Changes in major histocompatibility complex (MHC) class II and intercellular adhesion molecule-1 (ICAM-1) expressions in splenocytes were also found in this group. The major effects already described for FK506 (diabetes) or FTY720 (lymphopenia) were observed after 21 days administration even when the drugs were associated. FTY720 associated with FK506 caused fewer changes in kidney structure, and blood glucose levels were lower than in FK506 monotherapy.     

Effect of FTY720 treatment on postischemic pancreatic microhemodynamics

CD4+ T cells contribute to disturbances of pancreatic microcirculation after cold and even after warm ischemia/reperfusion (I/R). The aim of this study was to investigate a possible protective role of FTY720 (fingolimod) in this setting. In an in vivo model (42 Wistar rats), ischemia of the pancreas was induced for 60 minutes under anesthesia with xylazin/ketanest. Sham-operated (SO) (I), untreated ischemic (II), and treatment group with FTY720 pre-treatment (1 mg/kg body weight IV) (III) were investigated. The effect of FTY720 on I/R injury was assessed by in vivo microscopy 30–90 minutes after reperfusion and by measurement of serum lipase. In the untreated ischemic group (II), capillary constriction to 85.3 ± 6.3% of SO diameters and a reduction of functional capillary density to 67% was found. After 30 minutes of reperfusion, the number of T cells in capillaries was increased (165.7%; P < .05 vs I). FTY720 pretreatment reduced this number to 54.2% of SO (P < .05 vs II). Likewise, the number of adherent leukocytes in capillaries (145.4 ± 11.2% of SO) was reduced in group III (109.3 ± 11.4%; P < .05 vs II), leading to an improvement in functional capillary density in the treatment group (98.2 ± 2% of SO; P < .05 vs II). According to improved microcirculation, lipase values were reduced in the therapy group (P < .05). In conclusion, FTY720 ameliorates the microcirculatory and biochemical manifestations of pancreatic I/R injury by preventing T-cell infiltration.     

Prevention of graft vessel disease by combined FTY720/cyclosporine. A treatment in a rat carotid artery transplantation model

BACKGROUND: Graft vessel disease (GVD) is an important problem often responsible for late graft loss. The effects of FTY720, an immunomodulator with a novel mechanism of action were investigated in combination with cyclosporine A (CsA) in a carotid artery allograft model. METHODS: A segment of the carotid artery of Lewis rats was replaced by a DA allograft. Seven groups of eight rats were treated for 8 weeks with vehicle (P), CsA 0.3 (C0.3), 1 (C1) or 3 (C3) mg x kg(-1).day(-1) or a combination of CsA 1 with FTY 0.01 (C1F0.01), 0.03 (C1F0.03), and 0.1 (C1F0.1) mg x kg(-1).day(-1). Lumen area was estimated by magnetic resonance imaging, peripheral lymphocyte count and drug concentrations were determined at 1 and 8 weeks. Neointima, media, and lumen area were measured morphometrically. Intimal and adventitial infiltration of mononuclear cells, and medial smooth muscle cells number was assessed using a score. RESULTS: FTY720 did not influence CsA blood concentrations. FTY720 but not CsA decreased the PLC dose dependently. Magnetic resonance imaging revealed that treatment groups have larger lumen size than group P. Histological and morphometric evaluation showed that all aspects of GVD were dose dependently suppressed by treatment and lumen narrowing was prevented. CONCLUSIONS: CsA, at clinically relevant blood levels, suppressed GVD only partly. The addition of FTY720 was well tolerated and completely suppressed GVD development. In vivo lumen size did not correlate with the histologically estimated neointimal thickness.     

FTY720 in corneal concordant xenotransplantation

BACKGROUND.: Currently, there are no effective treatments for the control of corneal xenograft rejection. We evaluated the efficacy and mode of action of a novel immunosuppressant, FTY720, in a model of corneal xenograft transplantation. METHODS.: Rat-to-mouse corneal xenografts were performed and the effects of treatment with daily intraperitoneal injections of FTY720 (0.5 or 3.0 mg/kg/day) or saline from 2 days pretransplantation were assessed clinically. Immunohistochemical studies of the grafts and flow cytometry of the draining lymph node subpopulations were performed at the time of clinical rejection. RESULTS.: Treatment with FTY720 delayed the onset of corneal rejection, from 8 days postgraft in saline-treated mice to 12.0 +/- 0.89 days for low-dose FTY720 treatment and 15.6 +/- 3.1 days for high-dose FTY720 treatment (both P<0.001). Histologically, FTY-treated animals had a markedly reduced inflammatory response in the anterior chamber and cornea after replacement of the xenograft epithelium with normal healthy host epithelium. In contrast, saline-treated xenografts had persisting corneal epithelial defects and ulceration. In the draining lymph nodes, FTY720 not only inhibited the increase in the cell number observed in saline-treated recipients of xenografts, but also reduced the expression of activation markers on B cells (MHC class II and CD86). CONCLUSIONS.: FTY720 treatment significantly delayed rejection and decreased its severity in a dose-dependent manner in a rat-to-mouse model of corneal xenotransplantation. Since corneal xenograft rejection is mediated not by natural antibodies or CD8+ T cells directly, but by CD4+ T cells, the data from these experiments imply that FTY720 mediated its effect via CD4+ T cells.