FTY720对大鼠肾脏缺血再灌注损伤中缺氧诱导因子-1表达的影响

目的探讨FTY720对大鼠肾脏在缺血再灌注损伤中的保护作用及对缺氧诱导因子1α(HIF1α)表达水平的影响。方法将125只健康雄性SD大鼠随机平均分为5组。建立大鼠肾脏缺血再灌注模型。FTY720组:缺血再灌注前3d连续用FTY7201mg·kg-1·d-1灌胃;霉酚酸酯(MMF)组:缺血再灌注前3d连续用MMF20mg·kg-1·d-1灌胃;环孢素A(CsA)组:缺血再灌注前3d连续用CsA10mg·kg-1·d-1颈部皮下注射;对照组:术前不给予任何处理;假手术组:不建立肾脏缺血再灌注损伤模型。观察各组大鼠术后0、6、24、72h及7d时的血肌酐水平以及肾组织中HIF1αmRNA和蛋白表达水平。结果(1)大鼠肾脏缺血再灌注后各时间点的血清肌酐值水平均高于假手术组(P<0.05)。但是FTY720组再灌注6、24和72h时血肌酐值明显低于对照组,差异有统计学意义(P<0.05)。而MMF组、CsA组各时间点与对照组比较,差异无统计学意义。(2)再灌注后0、6和24h,FTY720组肾组织中HIF1αmRNA和蛋白水平明显高于同期对照组(P<0.05),而MMF组和CsA组与对照组相比较,差异无统计学意义。结论FTY720可诱导肾脏缺血再灌注早期HIF1αmRNA的快速转录和蛋白表达量的增加,这可能是减轻肾脏缺血再灌注损伤的重要机理之一。     

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。     

腺病毒介导TGF-β 1转基因联合FTY720对大鼠同种异体肺移植缺血-再灌注损伤的保护作用

目的观察腺病毒介导TGF-β₁转基因联合FTY720对大鼠同种异体肺移植缺血-再灌注损伤(IRI)的影响。 方法采用改良的三袖套法吻合技术建立大鼠左肺原位移植模型。以SD大鼠为供受体,随机分为假手术对照组、空白对照组、空载体对照组、TGF-β₁组、FTY720组和TGF-β₁＋FTY720组,每组6只。移植肺再灌注后4 h,检测各项生理指标。 结果腺病毒介导TGF-β₁转基因组荧光现象显著,并确定1×10¹⁰ PFU TGF-β₁基因腺病毒载体为较优转染浓度。肺移植再灌注后4 h,与假手术对照组比较,空白对照组和空载体对照组出现典型的移植肺IRI症状,肺动脉血氧分压(PaO₂)明显降低,二氧化碳分压(PaCO₂)和湿/干重(W/D)比值明显升高,超氧化物歧化酶(SOD)活性明显降低,丙二醛(MDA)含量、髓过氧化物酶(MPO)活性和肿瘤坏死因子-α(TNF-α)含量明显升高。与空白对照组和空载体对照组比较,TGF-β₁组、FTY720组和TGF-β₁＋FTY720组移植肺组织水肿和间质炎症等病理组织学现象明显减轻,PaO₂明显上升,PaCO₂和W/D比值明显下降,SOD活性明显增加,MDA含量、MPO活性和TNF-α含量明显降低。其中,联合处理组病理症状表现出更为明显的减轻趋势。 结论腺病毒介导TGF-β₁转基因联合FTY720能有效减轻大鼠同种异体肺移植IRI。     

芬戈莫德对血管紧张素Ⅱ介导的慢性肾脏病的影响

目的通过建立血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)灌注的大鼠肾损害模型,使用药物芬戈莫德(Fingolimod,FTY720)进行干预,来评估该药物对大鼠肾功能的影响,并探讨FTY720对AngⅡ灌注大鼠肾脏炎症改变的作用及可能涉及的机制,从而为肾脏疾病的治疗提供新的策略。方法 36只SPF级雄性SD大鼠随机分为3组:(1)模型组12只,即AngⅡ灌注组,皮下植入含AngⅡ的渗透性微量泵,持续灌注28 d(AngⅡ:400 ng·kg~(-1)·min~(-1));(2)干预组12只,即AngⅡ+FTY720干预组,在模型组的基础上,FTY720以0.5 g·kg~(-1)·d~(-1)灌胃28 d;(3)对照组12只,以等量的生理盐水灌胃28 d。每周称体质量,留24 h尿液,并于第28天处死各组大鼠,经心脏采血,并留取肾脏标本。放射免疫法检测各组大鼠血液中AngⅡ及肾匀浆组织中AngⅡ的浓度。对尿液及血液样本进行尿蛋白与血生化分析。光镜下观察各组大鼠肾组织病理学改变并行Jablonski评分。免疫印迹法检测炎症因子TNF-α、IL-6在肾脏中的表达。结果 (1)AngⅡ可导致模型组大鼠血肌酐、尿素氮水平升高,尿蛋白排泄明显增加,炎症因子TNF-α、IL-6在肾脏组织中的表达水平升高;(2)干预组的肾脏病理损伤较模型组减轻,且Jablonski评分明显低于模型组,血肌酐、尿素氮水平明显降低,尿蛋白明显减少,肾脏组织中炎症因子TNF-α、IL-6水平明显降低,差异具有统计学意义(P0.05)。结论 FTY720可减轻AngⅡ诱导的大鼠肾脏的病理损伤,可能是通过减少炎症因子的表达发挥了减轻尿蛋白,改善肾功能的作用。     

淫羊藿苷减轻大鼠肾脏缺血再灌注损伤的作用及机制研究

目的 探讨淫羊藿苷对大鼠肾脏缺血再灌注损伤的影响及其机制.方法 实验分为3组.实验组采用SD大鼠制备单侧肾脏缺血再灌注损伤模型,从术前2d开始至术后12d采用淫羊藿苷灌胃,剂量为100 mg·kg-1·d-1;对照组也采用模型大鼠,用溶剂灌胃;假手术组仅游离肾蒂,不用任何药物.检测术后14d内各组大鼠的体重和肾功能变化.术后24 h取大鼠肾组织,检测丙二醛和氧自由基代谢酶的水平.术后3d和14 l行肾组织病理学检查.结果 术后3、7、11和14d,实验组大鼠肌酐清除率均高于对照组(P＜0.01).术后3d,实验组大鼠肾小管损伤Paller评分低于对照组(P＜0.01).术后24 h,与对照组相比较,实验组丙二醛含量降低(P＜0.05),谷胱甘肽还原酶水平和还原型谷胱甘肽含量升高(P＜0.05),过氧化氢酶和双氧化物歧化酶水平升高(P＜0.05).结论 淫羊藿苷可减轻大鼠肾脏缺血再灌注损伤,其机制可能是通过影响氧自由基代谢酶系统而减轻氧化应激损伤.     

FTY720在鼠类异种胰岛移植排斥反应中的作用

目的 在小鼠异种胰岛细胞移植模型上，研究FTY720在控制异种胰岛移植排斥反应中的作用。方法 使用胰管内胶原酶注射和不连续密度梯度纯化法获取大鼠胰岛，建立小鼠肾被膜下移植模型。受体小鼠随机分为3组：对照组，末使用任何免疫抑制药物；实验1组，自移植当日起每日单独喂服FTY720(1.0mg／kg)；实验2组，亦于移植当日起每日联合喂服FTY720(1.0mg／kg)和环孢霉素A(CsA，15mg／kg)，均连续喂饲14d。分别于术后第3，5，7，14天切取移植物，观察并分析排斥反应。结果 对照组和实验1组，植入。肾被膜下的胰岛组织多在1周内完全被排斥，术后第7天胰岛轮廓消失，仅见大量淋巴细胞浸润。实验2组于移植后第7天及第14天肾被膜下仍可见大量完整的胰岛细胞，几乎未见或偶尔可见淋巴细咆浸润。结论 FTY720单独作为免疫抑制剂并不能抑制鼠类异种胰岛移植的排斥反应；联合应用FTY720及CsA则可有效地抑制鼠类异种胰岛移植排斥反应的发生。     

TLR4／NF—κB信号通路与肾脏缺血后处理的肾脏保护作用的关系

目的探讨小鼠。肾脏缺血后处理（ischemic postconditioning,IPC）对缺血再灌注损伤（ischemia-reperfusion injury,IRI）的保护作用及与Toll样受体（toll—like receptor,TLR）4／核因子（nuclear factor,NF）-κB信号通路的关系。方法BALB／c小鼠42只随机分组：假手术组6只、IRI组18只与IPC组18只,IRI组与IPC组均设术后1d、3d、5d亚组,每个亚组各6只小鼠。建立肾脏IRI及IPC模型;于术后1d、3d、5d取血检测血清肌酐（Scr）、血尿素氮（BUN）,取肾组织行病理学检查,计算术后1d急性肾小管损伤的Jablonski评分,采用蛋白质印迹（Western blot）法检测术后1d、3d、5d的TLR4、NF—κB表达水平。结果术后各时点,IRI组的Scr、BUN水平均明显高于假手术组（均为P〈0．05）;术后1d,IPC组的Scr、BUN水平均明显低于IRI组（均为P〈0．05）,且于术后5d两指标明显下降并恢复至正常范围。与假手术组比较,IRI组和IPC组肾小管损伤程度较为严重（均为P〈0．05）;与IRI组比较,IPC组的肾小管损伤程度显著减轻（P〈0．05）。术后1d、3d,IPC组的TLR4表达水平较IRI组明显降低（均为P〈0．05）。术后1d、3d、5d,IPC组的NF—κB表达水平较IRI组明显降低（均为P〈0．05）。结论IPC可减轻缺血再灌注对肾脏的损伤,其保护作用可能通过抑制TLR4／NF—κB信号通路实现。     

纤维蛋白肽Bβ15～42对大鼠缺血再灌注损伤肾脏局部炎性反应的影响

目的 观察纤维蛋白肽Bβ15～42(the fibrin-derived peptide Bβ15-42,FgBβ15～42肽)对大鼠肾脏缺血再灌注损伤(IRI)后肾脏局部炎性反应的影响并探讨其机制.方法 将SD大鼠随机分成假手术组(Sham组)、IRI组、阴性治疗组和FgBβ15 ～ 42肽治疗组.Sham组:分离肾动脉后关闭腹腔;IRI组:采用双侧肾动脉夹闭的方法制作肾脏IRI模型;阴性治疗组:于肾脏再灌注后立即尾静脉注射随机肽段3.6 mg/kg; FgBβ15～42肽治疗组:于肾脏再灌注后立即尾静脉注射FgBβ15～ 42肽3.6 mg/kg.后3组按照再灌注24h、48 h分为两个亚组,Sham组与各亚组均为8只大鼠.常规生化法检测肾功能;HE、PAS染色观察肾脏组织学改变;免疫组化、实时荧光定量PCR法及Western印迹检测肾组织白细胞介素1β(IL-1β)、细胞间黏附分子1(ICAM-1)的mRNA及蛋白表达.结果 与Sham组相比,IRI组的Scr和BUN水平均显著增加(均P ＜0.05),肾小管及间质病理损伤显著,以再灌注48 h更为明显;与IRI组相比,FgBβ15～ 42肽治疗组Scr和BUN显著下降(均P＜0.05),小管间质损伤程度明显减轻(P＜0.05).与Sham组相比,IRI组IL-1β和ICA M-1的mRNA和蛋白水平于再灌注24h显著上升,48 h稍微下降,但仍维持在较高水平;FgBβ15～ 42肽治疗组大鼠肾组织IL-1β和ICAM-1的表达于再灌注24h、48 h显著低于同时间点的IRI组(均P＜0.05),但仍明显高于Sham组.上述各指标在阴性治疗组和IRI组之间的表达差异无统计学意义.结论 FgBβ15～42肽对肾脏IRI具有保护作用,其作用机制可能与其减少炎性因子IL-1β、黏附分子ICAM-1的表达有关.     

FTY720对大鼠急性脊髓损伤后RhoA表达的影响

目的观察大鼠急性脊髓损伤(spinal cord injury,SCI)后芬戈莫德(fingolimod,FTY720)对RhoA表达的影响,进一步探讨FTY720治疗SCI的可能机制。方法采用改良Allen’s法建立大鼠急性SCI模型。雌性SD大鼠75只,随机分为假手术组(n=15)、损伤组(n=30)和FTY720组(n=30)。FTY720组伤后30min一次性腹腔注射FTY7200.5mg/kg,假手术组和损伤组以相同方法给予浓度为0.9%的生理盐水。分别于伤后1天、3天、7天、14天及21天,应用RT-PCR和Western印迹法分别检测RhoA mRNA和RhoA蛋白的表达。结果损伤组RhoA mRNA和蛋白的表达于伤后3天开始明显增加,7天达到高峰,21天仍维持高水平表达;在同一时间点,与FTY720组和假手术组比较,差异有统计学意义(P0.05)。FTY720组伤后1天、3天和7天的RhoA表达高于假手术组(P0.05)。假手术组各时间点RhoA表达维持在较恒定水平。结论大鼠急性SCI后RhoA表达增加,FTY720可能通过抑制SCI后RhoA的表达,以达到减轻继发性SCI、促进脊髓神经功能恢复的目的。     

外源性硫化氢在大鼠肾脏缺血再灌注损伤中的保护作用

目的 观察不同剂量外源性硫化氢(H2S)供体硫氢化钠对大鼠肾脏缺血再灌注损伤( IRI)的保护作用.方法 健康雄性Wistar大鼠28只随机分为4组,即假手术组( Sham)、肾缺血再灌注(IR)组、硫氢化钠(NaHS)高剂量组、硫氢化钠低剂量组.大鼠右肾切除后,以NaHS作为硫化氢的供体,NaHS高、低剂量组分别经左肾动脉插管,按照1.5 μmol/min、300 nmol/min的剂量连续15 min给药,假手术组及IR组给予同体积生理盐水.停药5 min 后,NaHS组和IR组用无损伤微动脉夹夹闭左侧肾蒂45 min后解除阻断,建立大鼠急性IRI模型,假手术组不夹闭左肾动脉,其他操作同模型组.于肾脏恢复血流24h时留取血和肾组织标本,检测血清尿素氮(BUN)、血肌酐(Scr);半定量分析肾脏病理损伤;检测肾组织H2S生成率;采用实时定量PCR法检测胱硫醚-β-合成酶(CBS)、胱硫醚-γ-裂解酶(CSE )mRNA表达.结果 与假手术组相比,IR组H2S生成率显著降低(P＜0.01);CBS、CSE mRNA表达显著下降(P＜0.01 );Scr、BUN显著升高(P＜0.01);肾脏病理表现为急性肾小管坏死,且最严重.与IR组相比,NaHS预处理组H2S生成率升高(P＜0.05);CBS、CSE mRNA表达升高(P＜0.01 );Scr、BUN降低(P＜0.01);病理损伤明显减轻.NaHS两个剂量组之间差异无统计学意义.结论 外源性H2S对大鼠IRI具有保护作用.     

转化生长因子-β1转基因联合FTY720对大鼠心脏移植物缺血-再灌注损伤的影响

目的 探讨经冠脉灌注转化生长因子(TGF)-β1基因联合FTY720对移植心脏缺血-再灌注损伤(IRI)的影响及其机制.方法 实验分为空白对照组、空载体组、FTY720组、转基因组和转基因+FTY720组.心脏移植8 h后切取移植心脏,免疫组织化学检测mTGF-β1、ICAM-1、核转录因子(NF)-κB表达;逆转录-聚合酶链反应(RT-PCR)检测mTGF-β1 mRNA转录强度;测定超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、髓过氧化物酶(MPO)活性.结果 mTGF-β1成功转到心肌细胞,转基因组和转基因+FIY720组mTGF-β1的表达强度分别是(1.08±0.24)和(1.16±0.22),明显高于其他3组(P<0.01);而两组ICAM-1的表达积分分别是(2.43±0.46)和(1.90±0.20),NF-κB的表达积分分别是(9.80±1.85)和(10.10±2.27),较其他3组显著下调(P<0.01);FRY720组、转基因组和转基因+FTY720组心肌细胞凋亡指数分别是(9.20±1.12)、(5.90±1.09)和(5.40±0.77),显著减少(P<0.01);FTY720组、转基因组和转基因+FTY720组SOD活性分别是(51.03±5.54)、(55.91±6.66)和(73.42±6.42)U/mg,明显升高(P<0.01),MDA含量(10.90±1.93)、(11.02±2.45)和(9.28±1.64)U/g,明显下降(P<0.01);而MPO活性分别是(4.38±1.43)、(4.63±1.04)和(3.16±0.64)U/g,亦明显下降(P<0.01);转基因和FTY720两因素对减轻心肌细胞凋亡以及对SOD、MDA和MPO的影响存在交互作用(P<0.05).结论 TGF-β1和FTY720均具有减轻移植心脏缺血-再灌注损伤的作用,机制可能与抑制心肌细胞凋亡、下调ICAM-1、NF-κB表达、增高SOD活性等因素有关;两者联合应用在减轻缺血-再灌注损伤方面有协同作用.     

Prevention of renal ischemic reperfusion injury using FTY 720 and ICAM-1 antisense oligonucleotides

BACKGROUND: Renal damage secondary to ischemia-reperfusion injuries (I-R) is frequent in organ transplantation and adversely affects the graft survival. An important component of this damage is caused by initial adhesion of neutrophils and lymphocytes to endothelial cells. FTY 720, which induces lymphopenia, has previously been shown to display protective effects in models of I-R. The purpose of the present study was to evaluate the combination of FTY 720 and intracellular adhesion molecule and ICAM-1 antisense oligonucleotides (AS-oligos), an agent designed to block the adhesion process. METHODS: Sprague-Dawley rats underwent syngenic kidney transplantation after donor kidneys had been preserved in cold solution for 2 hours. The treatment groups included: (1) FTY 720 (1 mg/kg) before reperfusion, (2) AS-oligos (2 mg/kg) during kidney perfusion, and (3) the combination of FTY 720 and AS-oligos. All animals were followed daily after transplantation; some were sacrificed on the second day for histologic analysis. RESULTS: All treated groups showed a maximal serum creatinine that was significantly less than the control (group 1: 2.76 +/- 1.4, group 2: 2.44 +/- 2.05, group 3: 1.51 +/- 0.42, and control: 4.04 +/- 0.5; P <.01) and returned to the basal value earlier. Also, treated animals showed less histologic stigmata of acute tubular damage. FTY 720 and AS-oligos used in combination showed a mild additive effect. CONCLUSIONS: The use of FTY 720 and/or AS-oligos significantly prevents functional renal damage secondary to I-R, displaying a mild additive effect in this model. Both agents offer the advantage of use during the donor and the graft operations.     

Reduction of ischemia-reperfusion injury in the rat kidney by FTY720, a synthetic derivative of sphingosine

BACKGROUND: The current shortage of organ donors has led many centers to use marginal and nonheart-beating donors (NHBDs). Recent research has implicated the infiltration of lymphocytes as an important mediator of ischemia-reperfusion injury (IRI). FTY720 is an immunosuppressant that promotes lymphocyte sequestration into lymph nodes. The purpose of this study was to examine the potential for FTY720 to abrogate IRI when subjected to increasing ischemic times. METHODS: Male Sprague-Dawley rats underwent bilateral flank incision with removal of the right kidney and clamping of the left hilum. Groups were divided into ischemia times of 45, 55, and 65min; each group was further divided into a control group (IRI only), IRI+FTY720 (1 mg/kg/d), and IRI+cyclosporine (15 mg/kg/d), n=4 per group. RESULTS: Thre days after 45 min of ischemia, serum creatinine in the ischemia only (477+/-37 micromol/L) and cyclosporine groups (698+/-32 micromol/L) was significantly increased compared with the FTY720-treated animals (194+/-66 micromol/L). The beneficial effect of FTY720 was also observed at 55 and 65 min; indeed, FTY720-treated animals demonstrated signs of recovery from 65 min of ischemia whereas control and cyclosporine-treated animals required sacrifice between days 3 and 5. Treatment with FTY720 reduced renal damage assessed histologically and also reduced apoptosis and increased cell proliferation. CONCLUSION: Treatment with FTY720 reduced IRI and prevented unrecoverable acute renal failure after significant ischemic injury. This study suggests that FTY720 may help improve the quality of grafts from NHBD and marginal donors by abrogating the IRI insult.     

Use of FTY 720 and ICAM-1 antisense oligonucleotides for attenuating chronic renal damage secondary to ischemia-reperfusion injury

INTRODUCTION: Chronic allograft nephropathy is the main cause of graft loss. Although many factors are involved in its development, ischemia-reperfusion injury has received increasing attention as a risk determinant. In a previous study of syngeneic renal transplantation and ischemia, we demonstrated a protective effect of acute damage by FTY 720 and antisense oligonucleotides of ICAM-1 (Oligos). The purpose of the current study was to evaluate the impact of these agents on the development of chronic graft damage in the same model. METHODS: Lewis rat were used as donors and recipients. The harvested left kidney was kept in Collins solution for 2 hours. Recipient animals received treatment with FTY 720 or Oligos or saline. At 12 and 36 weeks after transplantation, creatinine clearance, GFR, proteinuria, and arterial blood pressure were recorded. Tissue from some animals were submitted for histological studies and quantification of mRNA TGF-beta1. RESULTS: All groups showed decreased levels of GFR and creatinine clearence, but only the untreated animals showed significant deterioration compared to the pretransplant values (0.53 +/- 0.24 versus 0.21 +/- 0.24 at 36 weeks respectively; P < .05). Proteinuria was also significant in control animals at 36 weeks. Blood pressure showed a moderate increase in all groups. Histological analysis showed that treated animals had fewer signs of chronic damage according to the Banff score. All groups displayed slight increases in TGF-beta1 without differences among them. CONCLUSIONS: In this model the use of FTY or antisense oligonucleotides of ICAM-1 were associated with less functional and morphological evidence of chronic graft damage secondary to an ischemia-reperfusion injury.     

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.     

Effect of FTY720 and ex vivo graft irradiation in rat small bowel transplantation: apoptosis of crypt cells and lymphocytes

OBJECTIVE: 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 and ex vivo graft irradiation during rejection. MATERIALS AND METHODS: Orthotopic small bowel transplantations (SBT) were performed from Brown Norway (BN) rats to Lewis (LEW) rats. Four groups of SBT animals were studied on days 3, 5, and 7 after operations: untreated allograft, allograft with FTY720, allograft with irradiation, and allograft with FTY720+irradiation. Cryostat sections were prepared from the 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/FasL. RESULTS: The graft survival was prolonged in the FTY720-treated groups. In the FTY720-treated group, the number of ISEL-positive enterocytes was significantly down-regulated on days 3, 5, and 7 compared with the untreated allograft group. The number of ISEL-positive mononuclear cells was also significantly down-regulated compared with the untreated allograft group. The FTY720 the radiation and the FTY720+irradiation treated groups showed significantly down-regulated numbers of Fas/FasL-positive enterocytes on day 7 compared with the untreated allograft group. Fas/FasL-positive mononuclear cells were also significantly down-regulated in the allograft compared with the untreated allograft group. CONCLUSIONS: FTY720 and ex vivo graft irradiation prevented up-regulation of the number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes, and also prolonged small bowel allograft survival. Combination FTY720 and ex vivo graft irradiation did not affect graft survival and apoptotic cell expression compared with the FTY720 only group. These findings suggest that FTY720 may prevent both rejection-associated and sepsis-induced apoptosis during the late phase of small bowel graft rejection.     

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.     

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.     

Effect of FTY720 and Ex Vivo Graft Irradiation in Rat Small Bowel Transplantation: Expression of Mucosal Addressin Cell Adhesion Molecule-1

Purpose We performed a semiquantitative analysis of the expression of Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and gut-associated tissues (GALT) during small bowel graft rejection in the rat to confirm the effect of FTY720 and ex vivo graft irradiation. Methods Small bowel transplantations (SBT) were performed from BN rats to LEW rats. Four groups of SBT animals were studied on days 3, 5, and 7 after operations (untreated, FTY720, ex vivo graft irradiation, FTY720+ex vivo graft irradiation). Indirect immunoperoxidase staining was performed against CD4 and MAdCAM-1. The number of CD4-positive cells in the allografts was also analyzed by flow cytometry. Results The graft survival was prolonged only in the FTY720-treated groups. The SBT allografts treated by FTY720 demonstrated less infiltration, but the ex vivo graft irradiation group did not show any effect on its expression. In the FTY720-treated groups, MAdCAM-1 expression on high endothelial venules (HEVs) in Peyer's patches (PPs) was upregulated and its expression on endothelial cells of vessels in the lamina propria was downregulated in comparison with the allograft group without FTY720. Conclusions It is important to prevent the infiltration of CD4-positive cells, the downregulation of MAdCAM-1 expression on HEVs in PPs and the upregulation of MAdCAM-1 expression on endothelial cells of vessels in the lamina propria for the prolongation of graft survival.