FTY720 reduces extracellular matrix expansion associated with ischemia-reperfusion induced injury

BACKGROUND: Ischemia-reperfusion (IR) is one of the strongest nonimmune factors associated with the development of chronic allograft nephropathy (CAN). This effect is often exacerbated by immunosuppressive medications, most notably cyclosporine. Although traditionally the macrophage was thought to stimulate fibroblast activity in CAN, recent evidence supports a role for lymphocytes. FTY720 is a new immunosuppressant that promotes lymphocyte sequestration into lymph nodes and Peyer's patches. This study investigated the effect of FTY720 on renal fibrosis in the rat following an IR insult (IRI). METHODS: A rat model of IRI was used in which male Sprague-Dawley rats (under isoflurane anaesthesia) underwent bilateral flank incision with removal of the right kidney and clamping of the left renal hilum for 45 minutes. Five groups of animals were studied (n=4): nephrectomy only, IRI only, IRI+FTY720 (1 mg/kg/d), IRI+cyclosporine (15 mg/kg/d), and IRI+FTY 720 (1 mg/kg/d) and cyclosporine (15 mg/kg/d). Animals were humanely killed at 30 days. RESULTS: Serum creatinine (SCr) level was significantly reduced in the FTY720-treated animals. IRI alone produced a significant increase in SCr level compared with neprectomized animals (138 micromol/L vs 55 micromol/L; P<.05). This effect was potentiated by treatment with cyclosporine (173 micromol/L vs 55 micromol/L; P<.05). Treatment with FTY720 significantly reduced SCr level in rats following IRI alone (81 micromol/L vs 138 micromol/L; P<.01) and in rats following IRI + cyclosporine (98 micromol/L vs 173 micromol/L; P<.014). Parallel changes were seen in the levels of proteinuria. Fibrosis was assessed using Masson's trichrome (MT) staining. IRI alone produced a significant increase in MT staining compared with nephrectomized animals (0.92 vs 0.03; P<.05). This effect was potentiated by treatment with cyclosporine (1.12 vs 0.92; P=.022). Treatment with FTY720 reduced the level of MT staining in rats following IRI alone (0.34 vs 0.92; P<.05) and in rats following IRI+cyclosporine (70.34 vs 1.12; P<.05). Levels of TGF-beta1 were considerably reduced in FTY720-treated animals (compared with cyclosporine+IRI and IRI only), either alone (196+/-31 pg/mL vs 1105+/-59 pg/mL and 611+/-38; P<.05) or in conjunction with cyclosporine (423+/-26 pg/mL vs 1105+/-59 pg/mL and 611+/-38; P<.05). CONCLUSION: Our study shows that treatment with FTY720 can reduce renal fibrosis as a result of IRI, both alone and in conjunction with cyclosporine. This provides promising evidence for using FTY720 in a calcineurin-free or reduced-dose immunosuppression protocol in an effort to reduce the incidence of CAN.     

Immunosuppression with FTY720 and cyclosporine A inhibits rejection of adult porcine islet xenografts in rats

BACKGROUND: Our aim was to evaluate the effect of FTY720 in discordant islet xenotransplantation. METHODS: Fetal porcine islet-like cell clusters (ICCs) were transplanted into normoglycemic rats that were either left untreated or treated with FTY720 only, with FTY720 plus cyclosporine A (CsA) or with CsA only. Twelve or 24 days after transplantation, graft morphology was evaluated immunohistochemically. Furthermore, adult porcine islets (APIs) were transplanted into diabetic rats immunosuppressed with FTY720 plus CsA. Blood glucose and porcine C-peptide levels were monitored. RESULTS: In untreated rats, the ICC xenografts were completely rejected after 12 days. Treatment with CsA had only a marginal effect on the rejection. In animals given FTY720, only the number of infiltrating cells was somewhat reduced. However, at 12 days, no intact ICCs remained. Immunosuppression with FTY720 plus CsA had a marked inhibitory effect on islet xenograft rejection and plentiful morphologically intact ICCs remained. Twelve days after transplantation, only occasional macrophages and T cells could be detected. At 24 days after transplantation, the findings were similar. Furthermore, diabetic rats transplanted with APIs and immunosuppressed with FTY720 plus CsA remained normoglycemic for 53.0+/-15.8 days. In fact, one animal remained normoglycemic for more than 100 days. Serum levels of porcine C-peptide remained at levels similar to those for human C-peptide in healthy individuals. CONCLUSIONS: Immunosuppression with FTY720 plus CsA inhibited almost all morphological signs of pig-to-rat islet xenograft rejection for up to 24 days after transplantation. Diabetic rats transplanted with APIs and immunosuppressed with FTY720 plus CsA remained normoglycemic for 53.0+/-15.8 days.     

Antiproliferative and overadditive effects of rapamycin and FTY720 in pancreatic cancer cells in vitro

Rapamycin inhibits the growth of several tumors including pancreatic carcinoma both in vitro and in vivo. The antitumor effects of FTY720 were also shown recently. The present study was performed to investigate the in vitro antiproliferative capacity of combined treatment with rapamycin and FTY720 on pancreatic cacinoma cell lines. MATERIALS AND METHODS: The Panc-1 and AsPc-1 cell lines were employed as the pancreatic carcinoma model in vitro. For monotreatment experiments, rapamycin was added in increasing doses from 0.002 micromol/L to 200 micromol/L; FTY720 was used from 1 micromol/L to 15 micromol/L. For combined treatment, two concentrations of rapamycin were combined with seven concentrations of FTY720; or two concentrations of FTY720 with five concentrations of rapamycin. The antiproliferative capacity was assessed by the MTT assay. RESULTS: Rapamycin and FTY720 inhibited MTT incorporation into Panc-1 and AsPc-1 in dose-dependent fashion with or without serum stimulation. In coincubation experiments, great susceptibility to rapamycin was seen when combined with 10 micromol/L FTY720. An effective combination for AsPc-1 was 10 micromol/L FTY720 with 0.002 micromol/L rapamycin, resulting in more than 50% inhibition of MTT incorporation, and for Panc-1, 10 micromol/L FTY720 with 0.002 micromol/L rapamycin and 10 micromol/L FTY720 with 20 micromol/L rapamycin; the corresponding inhibition levels reached about 40% and 60%, respectively. CONCLUSION: Rapamycin and FTY720 showed dose-dependent antiproliferative effects on pancreatic carcinoma cell lines in vitro both alone and in combination. The combined use of rapamycin and FTY720 showed additive and supra-additive antiproliferative effects on pancreatic carcinoma cell lines. The susceptibility of pancreatic carcinoma cells to rapamycin was significantly enhanced when combined with FTY720.     

Dichloroacetate increases skeletal muscle pyruvate dehydrogenase activity during acute limb ischemia

The purpose of this study was to evaluate the effects of dichloroacetate sodium (DCA), a drug that inactivates pyruvate dehydrogenase kinase (PDH-K), on pyruvate dehydrogenase (PDH) activity, lactate level, and function of skeletal muscle in an experimental model of acute limb ischemia. Thirty-two male Sprague-Dawley rats underwent right iliac artery ligation to produce hindlimb ischemia. After 2 hours of ischemia, 16 animals received intravenous DCA (15 mg/100 g body weight) and 16 control animals received an equivalent volume of normal saline. After an additional 1 hour of ischemia (total 3 hours) tibialis anterior muscle from the ischemic limb and contralateral nonischemic limb was excised, rapidly freeze-clamped with Wallenberg tongs cooled in liquid nitrogen, and stored at -70 degrees C. Muscles specimens were subsequently assayed for PDH activity and lactate level by use of spectrophotometric techniques. An additional 16 animals (DCA-treated, n = 8; control, n = 8) underwent ex-vivo gastrocnemius muscle fatigue testing with a 10 g tension preload after 3 hours of limb ischemia. In ischemic hind limbs, DCA treatment significantly (p = 0.025) increased PDH activity (19.6 +/-1.6 micromol/min/g dry weight) compared to controls (13.1 +/-1.3 micromol/min/g dry weight). DCA treatment did not increase (p = 0.13) skeletal muscle PDH activity in the nonischemic limbs (9.6 +/-1.1 micromol/min/g dry weight, controls; 13.2 +/-1.3 micromol/min/g dry weight, DCA group). In DCA-treated animals, hind limb ischemia resulted in no significant increase in muscle lactate levels compared to the nonischemic limb, while control animals demonstrated a significant (p = 0.005) elevation in lactate level in ischemic limbs compared to contralateral nonischemic limb. Ischemia induced a significant decrease in time to muscle fatigue in both DCA-treated and control animals (p = 0.002 and 0.001, respectively). Time to muscle fatigue in DCA-treated animals was increased compared to controls (2.6 +/-0.3 versus 2 +/-0.6 minutes; p < 0.05)in ischemic limbs but was not significantly different in nonischemic limbs (DCA = 3.3 +/-0.5 minutes; control = 3.1 +/-0.6 minutes). Treatment with DCA during acute limb ischemia reduced the depression of PDH activity and lactate level of skeletal muscle. Ischemic muscle function was also improved by DCA treatment. Further investigation of the potential beneficial effects of DCA treatment on muscle injury during ischemia and reperfusion is warranted.     

Oral efficacy of the new immunomodulator FTY720 in cynomolgus monkey kidney allotransplantation,given alone or in combination with cyclosporine or RAD

BACKGROUND: FTY720 is a novel immunomodulator with a unique mechanism of action, i.e. chemokine-dependent lymphocyte homing into secondary lymphoid organs associated with profound lymphocyte depletion in blood. We investigated its efficacy, either FTY720 alone or together with cyclosporine or the rapamycin derivative rapamycin derivative (RAD), in cynomolgus monkey kidney allotransplantation. METHODS: Life-supporting allotransplantation was performed in bilaterally nephrectomized hosts. Compounds were given once daily by oral gavage. Monitoring was done by serum creatinine and urea, and rejection was concluded when values exceeded 500 micromol/L and 50 mmol/L, respectively (5-6 times the upper limit of reference values). Rejection was confirmed by graft histology. The termination point was set to 100 days after transplantation. In addition, animals were monitored for 24 hr drug concentrations and thorough inspection of potential adverse side effects. RESULTS: FTY720 given alone at 3.0 mg/kg per day prolonged rejection-free survival (33-85 days, mean 24 hr concentration between 54 and 66 ng/mL [n=3]), but it was not efficacious at a 0.3 mg/kg per day dose. For cyclosporine alone, 30 mg/kg per day during maintenance was efficacious (average concentration above 100 ng/mL, historical data from our group), and for RAD alone 0.75 mg/kg per day (concentration above 10 ng/mL). Efficacious FTY720-cyclosporine-A (CsA) or FTY720-RAD combinations were established using 0.1-0.3 mg/kg per day FTY720, 10-30 mg/kg per day cyclosporine, and/or 0.25-0.50 mg/kg per day RAD. Compared with single-compound treatment, FTY720 effective doses and 24 hr trough concentrations were at least tenfold lower in combination treatment and those of cyclosporine and RAD about twofold lower, indicative of effective synergy between the compounds. Already at the lowest FTY720 dose tested (0.03 mg/kg per day), there was a profound lymphocyte depletion down to about 30% of pretransplant values, which further increased at the highest dose (3.0 mg/kg per day, to about 14% of pretransplant values). Lymphocyte depletion was reflected by a decrease in T and B subpopulations. CONCLUSION: FTY720 is an effective immunosuppressant in prevention of acute kidney allograft rejection in cynomolgus monkeys and synergizes with cyclosporine and/or RAD in yielding rejection-free allograft survival.     

Control of intestinal allograft rejection by FTY720 and costimulation blockade

INTRODUCTION: The clinical application of small bowel transplantation (SBTx) is hampered by its pronounced immunogenicity. In this study we examined the effects of the novel immunosuppressant FTY720 and costimulation blockade by an anti-CD40L mAb (MR-1) in a stringent mouse model of SBTx. METHODS: SBTx was performed in mice with a full MHC mismatch (donors: C3H=H-2(k); recipients: C57BL/6=H-2(b)). Recipients were divided into four groups: 1, untreated group; 2, MR1 monotherapy (500 microg IV on days 0, 2, 4, and 7); 3, FTY720 monotherapy (1 mg/kg body weight PO for 14 consecutive days after transplantation); 4, FTY720 plus MR1-treated group. Graft rejection grades were assessed by H&E staining. Graft mesenteric lymph nodes (MLNs), Peyer's patches (PPs), as well as intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were analyzed by flow cytometry and three-color immunofluorescence staining. RESULTS: Neither FTY720 nor MR1 monotherapy was efficient in preventing the rejection of mouse intestinal allografts, whereas FTY720 plus MR1 profoundly inhibited the rejection response at the 14th posttransplant day. The infiltration of host lymphocytes was reduced in graft MLNs, PPs, IELs, and LPLs by FTY720 therapy. FTY720 plus MR1 inhibited host CD8(+) T-cell infiltration in graft LPLs when compared with grafts treated with FTY720 only. Additionally, two subpopulations, CD11b(+high) Gr1(-) and CD11b(+intermediate) Gr1(+) cells, were decreased in FTY720-treated grafts. CONCLUSIONS: FTY720 plus MR1 efficiently delayed intestinal allograft rejection in a mouse model by preventing the infiltration of host lymphocytes, particularly of CD8(+) cells.     

FTY720 Pretreatment Reduces Warm Hepatic Ischemia Reperfusion Injury Through Inhibition of T-Lymphocyte Infiltration

Ischemia and reperfusion (IR) injury remains a significant problem in clinical liver transplantation. We investigated the effects of lymphocyte depletion with FTY720 in models of warm hepatic IR. Using 60-min partial warm hepatic IR, three groups of rats were studied: Sham--laparotomy alone; Control--water p.o. x 3 d before ischemia; Treatment--FTY720 p.o. x 3 d before ischemia. Animals were sacrificed for analysis at 6 h and 24 h post reperfusion. The effect of FTY720 pretreatment on survival was also studied using 150 min total hepatic IR with portojugular shunt. FTY720 treatment significantly reduced serum glutamic pyruvic transaminase and peripheral blood lymphocytes compared to controls at 6h and 24h (p < 0.0005). Histological grade was significantly improved in treated livers vs. controls (p < 0.05). CD3 immunocytochemical analysis revealed a significant reduction in T-cell infiltration in FTY720-treated livers (p < 0.0002). No difference in tissue myeloperoxidase levels was observed. Seven-day survival was significantly improved in treated rats vs. controls following total hepatic ischemia (p < 0.05). In conclusion, FTY720 ameliorates the biochemical and histological manifestations of hepatic IR by preventing T-lymphocyte infiltration and prolongs survival following a more severe ischemic insult. Myeloperoxidase data suggest this mechanism is independent of neutrophil activation. These results indicate that T lymphocytes are pivotal mediators in hepatic IR and may have important implications in liver transplantation.     

Randomized controlled trial of FTY720 versus MMF in de novo renal transplantation

BACKGROUND: Phase II trials of FTY720, a novel immunomodulator, have shown promise in preventing rejection with both standard and reduced cyclosporine exposure. This study was designed to confirm those findings. METHODS: This one-year, multicenter, randomized, phase III study in 696 de novo renal transplant patients compared FTY720 5 mg plus reduced-dose cyclosporine (RDC) or FTY720 2.5 mg plus full-dose cyclosporine (FDC) with mycophenolate mofetil (MMF) plus FDC. All patients received concomitant corticosteroid therapy without antibody induction. The primary efficacy composite endpoint was the incidence of first treated biopsy-proven acute rejection (treated BPAR), graft loss, death or premature study discontinuation at month 12. RESULTS: FTY720 2.5 mg plus FDC was demonstrated to be non-inferior to MMF plus FDC as the primary efficacy endpoint (30.8% and 30.6%) was comparable. The FTY720 5 mg plus RDC treatment regimen was discontinued due to an increased incidence of acute rejection episodes (primary endpoint 43.3%). FTY720 was associated with significantly lower creatinine clearance with a mean difference at 12 months between FTY720 2.5 mg plus FDC and MMF plus FDC of 8 ml/min. CONCLUSIONS: While FTY720 2.5 mg plus FDC yielded similar efficacy to MMF plus FDC, the FTY720 5 mg plus RDC did not allow a 50% reduction in cyclosporine exposure. The associated lower creatinine clearance indicated that FTY720 combined with cyclosporine provided no benefit over standard care.     

Effect of FTY720 on apoptosis of smooth muscle cells

BACKGROUND: Hyperproliferation of smooth muscle cells (SMCs) plays a key role in allograft arteriosclerosis. This prompted us to investigate the effect of the novel immune modulator and synthetic sphingolipid FTY720 on apoptosis of SMCs. METHODS: Rabbit SMC cultures were treated with FTY720 and apoptosis and necrosis were detected by fluorescence microscopy. RESULTS: We investigated dose- and time-dependent effects of FTY720 and found that clinically relevant low doses of FTY720 (<1 micromol/L) did not induce apoptosis, whereas 10 micromol/L FTY720 induced apoptosis after 48 hours incubation. CONCLUSION: At doses of FTY720 used in clinics for treatment of renal allografts and multiple sclerosis. FTY720 did not induce SMC apoptosis.     

The impact of FTY720 (fingolimod) on vasodilatory function and arterial elasticity in renal transplant patients.

BACKGROUND: FTY720 has recently demonstrated a similar efficacy in prevention of acute graft rejection compared with mycophenolate mofetil (MMF) in a large phase III trial of de novo renal transplant recipients. Creatinine clearance, however, was significantly lower in FTY720-treated patients. In the present study, we examined the impact of FTY720 on vascular function in a subgroup of patients of this trial. METHODS: Eighteen patients (12 FTY720, 6 MMF) agreed to be enrolled for an analysis of vascular function. Vascular measurements were performed 1.5 years post-transplant and were repeated 3 months after conversion of the patients from FTY720 to MMF. Arterial stiffness was assessed as augmentation index (AI(75)); endothelium-dependent and -independent vasodilation were measured sonographically as flow-mediated dilation (FMD) and as vasodilation after application of glyceroltrinitrate (GTN). RESULTS: Conversion from 2.5 mg FTY720 to MMF led to a significant improvement of FMD (5.40 +/- 1.84 vs 7.77 +/- 3.36%, P 0.02). AI(75) and GTN tended to be higher after conversion without reaching significance (83 +/- 20.43 vs 78.69 +/- 15.39%, P 0.06; 13.76 +/- 4.52 vs 17.39 +/- 3.76%, P 0.07). In the MMF group, AI(75), FMD and GTN did not significantly change during the observation period. CONCLUSION: The present study constitutes the first analysis of the impact of FTY720 on vascular function in humans and reveals an improvement of arterial vasodilatory function after discontinuation of FTY720 in de novo renal transplant recipients on cyclosporine.     

Effects of immunosuppressant FTY720 on renal and hepatic hemodynamics in the rat

BACKGROUND: FTY720 is a novel immunomodulator that may provide an opportunity for a reduction in calcineurin inhibitor dosage in transplant recipients with renal/hepatic side effects. However, the effects of FTY720 on renal or hepatic hemodynamics are unknown. The aim of this study was to establish the hemodynamic and renal actions of FTY720 at therapeutically relevant dosages. METHODS: The effects of acute and repeat oral administration of FTY720 on systemic, renal, and hepatic hemodynamics were investigated in the anesthetized male Lewis rat. Renal function and renal tubular parameters were examined in animals that received repeat high dosage of FTY720. RESULTS: Seven-day oral administration of FTY720 did not cause any significant changes in markers of hepatocyte injury, nor did it cause any reduction in renal function (elevated urea and creatinine). Histological examination of liver and kidney from animals treated with repeat FTY720 for 1 or 3 weeks did not reveal any sclerosis, tubular changes, infiltrates, or fibrosis. Hepatocyte, vascular, and biliary structures were normal. Compared with the vehicle (saline), oral administration of FTY720 at dosages up to 5 mg/kg/day for 1 week did not have any significant effects on systemic, hepatic, or renal hemodynamics. Five min after intravenous FTY720 administration (1 mg/kg), mean arterial pressure (MAP) rose to 114+/-3.3% of baseline (P <0.01) before returning to the normal range within 30-45 min. Lower doses of FTY720 (0.3 and 0.5 mg/kg, i.v.) did not affect MAP. Renal cortical perfusion, renal artery blood flow, and renal vascular resistance were not altered by FTY720 at i.v. doses up to 1 mg/kg. Animals that received FTY720 (5 mg/kg/day) for 3 weeks showed a significant reduction in body weight (-4.8+/-1% of baseline at 3 weeks, P <0.001); however, weight-adjusted creatinine clearance, 24 h urine production, and urine osmolality were not different from those in control animals (0.71+/-0.1 vs. 0.74+/-0.1 ml/min/100 g, 2.63+/-0.2 vs. 3.12+/-0.2 ml/100 g, and 2003+/-33 vs. 1966+/-56 mOsm/kg, respectively). FTY720 at the same repeat oral dosage was, nevertheless, associated with a significantly lower 24 h sodium excretion and a significantly lower fractional excretion of sodium compared with those in control animals (223.4+/-35 vs. 304.5+/-50 micromol/100 g and 1.75+/-0.3 vs. 2.23+/-0.3%, respectively; P <0.05). CONCLUSIONS: Our data indicate that, at least in the short term, oral FTY720 does not cause any significant adverse effects on renal or hepatic hemodynamics, nor does it cause any reduction in glomerular perfusion and thus may provide reasonable rescue/add-on therapy in calcineurin-inhibitor treated transplant recipients. At high repeat oral dosages, however, FTY720 may alter renal handling of sodium.     

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.     

Effect of FTY720 in rat small bowel transplantation: apoptosis of crypt cells and lymphocytes in gut-associated lymphoid tissues

AIM: We investigated the extent of apoptosis in crypt cells and Peyer's patches (PPs) during small bowel allograft rejection in rats to examine the effect of FTY720 during rejection. METHODS: Orthotopic small bowel transplantations (SBTs) were performed from BN to LEW rats. Isografted animals served as controls. Three groups of SBT animals were studied on days 3, 5, and 7 after operation: isograft, untreated allograft, allograft with FTY720. FTY720 was orally administered by gavage (1 mg/kg/d) to allograft recipients on 7 consecutive days. Cryostat sections were prepared from grafts, including PPs. An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonal antibodies against rat Fas/Fas-L. RESULTS: Graft survival was prolonged in the FTY720-treated group. The number of ISEL-positive enterocytes in the allografts increased significantly on days 3, 5, and 7 compared with the isograft group. In the FTY720-treated group, the number of ISEL-positive enterocytes in the allografts was down-regulated significantly on days 3, 5, and 7 compared with untreated allograft group. In the PPs, the number of ISEL-positive mononuclear cells increased significantly in the allografts compared with the isograft group. In the FTY720-treated groups, the number of ISEL-positive mononuclear cells were down-regulated significantly in the allografts compared with the untreated allograft group. The number of Fas/FasL-positive enterocytes were increased significantly in allografts compared with isograft group. In FTY720-treated groups, the number of Fas/FasL-positive enterocytes were down-regulated significantly on day 7 compared with the untreated allograft group. In the PPs, Fas/FasL-positive mononuclear cells also increased significantly on day 7 in the allografts compared with isografts. In the FTY720-treated groups, Fas/FasL-positive mononuclear cells were down-regulated significantly in the allografts compared with the untreated allograft group. CONCLUSIONS: The number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes increased during small bowel graft rejection. FTY720 prevented up-regulation of the number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes while also prolonging small bowel allograft survival.     

Prevention and cure of autoimmune diabetes in nonobese diabetic mice by continuous administration of FTY720

BACKGROUND: Treatment of nonobese diabetic (NOD) mice with FTY720 before the development of insulitis prevents the onset of diabetes. In this study, the authors investigated whether FTY720 treatment of NOD mice with established insulitis prevents the development of diabetes. METHODS: FTY720 (1 mg/kg) was administered continuously to euglycemic NOD mice starting at 14 or 23 weeks of age. A group of untreated, age-matched NOD mice served as controls. Mice with more than 300 mg/dL blood glucose on three consecutive measurements were considered diabetic. RESULTS: Diabetes developed in control mice starting at 13 weeks of age and reached 78% by 33 weeks of age. Mice at 14 and 23 weeks of age exhibited extensive insulitis that progressed with age. Continuous oral administration of FTY720 starting at either age completely prevented the development of diabetes. However, its withdrawal at 37 weeks of age led to abrupt diabetes onset. Pancreases of FTY720-treated diabetes-free mice showed peripheral insulitis, with strong insulin staining. The protection from diabetes was also achieved by intraperitoneal injection of FTY720 or sirolimus (1.5 mg/kg). Unlike FTY720, withdrawal of sirolimus did not induce diabetes. Continuous oral FTY720 (3 mg/kg) treatment in overtly diabetic NOD mice led to complete reversal of diabetes in 6 of 11 mice. The standard adoptive transfer study in NOD-severe combined immunodeficient mice showed that peripheral lymphoid organs of FTY720-treated mice contained diabetogenic cells but not dominant immunoregulatory cells. CONCLUSIONS: FTY720, which does not cause generalized immunosuppression, may be a safe and benign therapeutic agent for chronic use to prevent or cure type 1 diabetes.     

Effect of cyclosporine on renal ischemic injury

To determine whether cyclosporine exacerbated renal ischemic injury and whether or not the timing of cyclosporine administration was important, rats were subjected to 30 or 45 min of ischemia. Cyclosporine was administered either before or after the renal ischemic insult. A single intravenous dose of cyclosporine, 20 mg/kg, before ischemia had no additional deleterious effect on inulin clearance compared with rats subjected to ischemia alone. In contrast, a significant exacerbation of the diminished glomerular filtration rate (GFR) produced by ischemia occurred when a low dose of cyclosporine (5 mg/kg) was given after ischemia. With 30 min of ischemia, GFR was 160 +/- 40 microliter/min/100 g in rats receiving cyclosporine (5 mg/kg) after ischemia compared with 280 +/- 40 microliter/min/100 g in rats subjected to ischemia alone. After 45 min of ischemia, cyclosporine (5 mg/kg) markedly reduced GFR to 20 +/- 10 microliter/min/100 g, a value significantly lower (P less than 0.05) than that observed in rats subjected to 45 min of ischemia alone (290 +/- 100 microliter/min/100 g). Plasma potassium concentrations tended to be higher and urinary potassium and sodium excretion lower in rats subjected to ischemia plus cyclosporine compared with ischemia alone. These findings indicate that even a single low dose of parenteral cyclosporine can exacerbate renal ischemic injury if given immediately after the ischemic insult. This interaction may contribute to the acute renal failure observed with cyclosporine use. In contrast, the kidney appears to be relatively resistant to a single dose of cyclosporine injury when the drug is administered prior to ischemia. These data suggest that the administration of parenteral cyclosporine immediately after transplantation could have deleterious effects and should probably be avoided.     

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.     

FTY720-induced lymphocyte homing modulates post-transplant preservation/reperfusion injury

BACKGROUND: A novel immunomodulator, FTY720, modulates lymphocyte migration to injured tissues via enhanced lymphocyte sequestration to secondary lymphoid organs. We tested whether or not single-dose FTY720 (0.5 mg/kg) pretreatment rescues renal grafts from post-transplant preservation/reperfusion injury. METHODS: Rat renal grafts were cold-preserved in University of Wisconsin (UW) solution for 4 hours and then transplanted into syngeneic or allogeneic recipients that received a single dose of FTY720 24 hours before transplantation. Flow cytometry analysis of peripheral blood and lymph nodes was performed to confirm the biologic effect of FTY720. Grafts were harvested after 24 hours. Renal sections were examined histologically and stained for intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), platelet endothelial cellular adhesion molecule-1 (PECAM-1), major histocompatibility complex (MHC) class II, and inflammatory cells. Interleukin-1 (IL-1) production was determined in renal protein extracts. RESULTS: FTY720 pretreatment significantly increased CD3+ T-cell sequestration to lymph nodes in the face of peripheral lymphopenia. Isografts and allografts from the FTY720-treated groups did not develop increased creatinine (0.55 +/- 0.12 in isografts and 0.62 +/- 0.08 mg/dL in allografts), compared with vehicle controls (2.28 +/- 0.20 in isografts and 2.24 +/- 0.18 mg/dL in allografts). Kidneys from FTY720-treated groups also showed lower acute tubular damage scores. Furthermore, FTY720 decreased neutrophil influx, although circulating neutrophils were unchanged. FTY720 also prevented postischemic IL-1 intragraft production not affecting infiltration with recipient ED-1+ macrophages and MHC class II-positive cells. Expression of ICAM-1, VCAM-1, and PECAM did not differ among groups. CONCLUSION: FTY720 ameliorated morphologic and functional consequences of post-transplant reperfusion injury. Thus, FTY720-induced peripheral T-cell absence may influence intragraft IL-1 production and neutrophil infiltration, despite proadhesive endothelial properties. FTY720 may broaden the utility in renal transplantation as a pretreatment strategy against preservation/reperfusion injury.     

Prolongation of canine liver allograft survival by a novel immunosuppressant, FTY720: effect of monotherapy and combined treatment with conventional drugs

BACKGROUND: The immunosuppressive effect and other properties of a novel immunosuppressant, FTY720, have been studied mostly in the experimental transplantation of various extrahepatic organs. In this experiment, we evaluated the antirejection potency and adverse effects of this agent on liver grafts using a canine liver transplantation model. METHODS: Forty-eight orthotopic liver transplantations were performed by the standard technique under a veno-venous bypass. Liver recipients were divided into two studies: a single-dose study with FTY720 at various doses and a combined dose study with conventional immunosuppressants (cyclosporine or tacrolimus) alone and combined with FTY720. Survival, biochemical and hematological tests, blood levels of immunosuppressants, and postmortem histology were determined. RESULTS: The median survival of untreated control animals was 9 days, whereas treatment with FTY720 at a dose of 0.1 mg/kg/day prolonged graft survival to 49.5 days. FTY720 at 1 mg/kg/day showed a slight but insignificant prolongation to 16 days, but when the dose was increased to 5 mg/kg/day, the graft was rejected at 10 days. The combination of FTY720, 0.1 mg/kg/day, with a subtherapeutic dose of cyclosporine, 5 mg/kg/ day, prolonged median animal survival from 40 days with cyclosporine alone to 74 days. A combination of FTY720 (0.1 mg/kg/day) with tacrolimus (0.5 mg/kg/ day) compromised animal survival, reducing survival from 83.5 days with tacrolimus alone to 30.5 days due to infectious complication and emaciation by overimmunosuppression. No evident drug-induced side effects were observed. CONCLUSIONS: FTY720 has a potent immunosuppressive effect when used alone at 0.1 mg/kg/day in canine liver transplantation. FTY720 is a promising candidate for future clinical application in orthotopic liver transplantation.     

FTY720预防大鼠肾脏缺血再灌注损伤的实验研究

目的:研究新型免疫抑制剂FTY720对大鼠肾脏缺血再灌注损伤(IRI)的预防作用.方法:制备大鼠肾脏IRI模型,从下腔静脉注入不同剂量的FTY720,观察术后第1、2、3、5、7 d血清肌酐值(Scr)和术后第2、7 d外周血淋巴细胞数(PLC)的变化,并在术后第2 d取肾脏作组织学检查观察急性肾小管坏死的情况.结果:FTY720处理组动物术后Scr水平显著低于对照组并呈剂量依赖性;FTY720处理组术后第2 d的PLC显著低于对照组;组织学检查显示FTY720处理组肾脏的缺血性损伤轻于对照组.结论:FTY720可以减少PLC,对大鼠肾脏IRI有预防作用.