免疫抑制剂对培养大鼠巨噬细胞和雪旺细胞影响的研究

目的 初步探讨多种免疫抑制剂对培养的大鼠巨噬细胞和雪旺细胞数量的影响。方法 取新生大鼠腹腔巨噬细胞和坐骨神经雪旺细胞进行培养，细胞生长稳定、定量计数后，分别加入不同浓度的环孢素A（ciclosporin A，CsA）、FK506和甲基强地松龙（CsA浓度为10^-5、10^-6、10^-7和10^-8mol／L；FK506浓度为10^-4、10^-7、10^-8和10^-9mol／L；甲基强地松龙浓度为10^-3、10^-4、10^-6和10^-8mol／L），设立不加药物的空白对照组。于培养24、48和72h后，分别取各浓度药物作用的细胞，用MTT法检测细胞数量。结果 对于巨噬细胞，甲基强地松龙仅在10^-4mol／L对其有明显保护作用，而CsA和FK506则分别在10^-6、10^-7、10^-8mol／L和10^-7、10^-8、10^-9mol／L能够较好地保护巨噬细胞。对于雪旺细胞，10^-3mol／L甲基强地松龙对其生长有显著抑制作用，而10^-8mol／L作用72h，则表现为对雪旺细胞明显的保护作用。除了10^-5mol／L CsA作用72h会显著减少培养的雪旺细胞数量外，其他浓度的CsA和FK506对雪旺细胞的数量无显著影响。结论 高浓度的甲基强地松龙以及一定浓度的CsA和FK506能够保护培养的巨噬细胞，但高浓度的甲基强地松龙和CsA可抑制雪旺细胞的生长，长时间、低剂量的甲基强地松龙对雪旺细胞有明显的保护作用。     

环孢素A与他克莫司对人肝癌HepG-2细胞生物学行为的影响

目的探讨环孢素A（CsA）与他克莫司（FK506）对肝癌的增殖、凋亡、复发及转移的影响。方法培养人肝癌细胞HepG-2，分别经5、10、100、500、1000、5000μg／L的CsA、FK506处理24～48h后，采用噻唑蓝（MTT）法检测细胞增殖、流式细胞术测定细胞周期及细胞凋亡、荧光定量聚合酶链反应（PCR）检测转化生长因子（TGF）-β细胞间黏附分子（ICAM）、骨桥蛋白（OPN）。结果肝癌细胞增殖能力48h明显受到抑制，CsA、FK506使G2期显著延长；FK506在小于100μg／L及CSA在小于1000μg／L时，随浓度增加，肝癌凋亡率逐渐增加，当两者分别超过100、1000μg／L时，则出现凋亡率减低；FK506、CSA分别在100、1000μg／L低浓度时对OPN无影响，在1000、5000μg／L高浓度时基因量显著增加。FK506在低浓度时对ICAM及TGF-β无影响，在高浓度时基因量显著增加；而CSA对两者无明显影响。结论CsA、FK506均对肝癌增殖有抑制作用，使G2期阻滞，促凋亡；低浓度时对肝癌转移无影响，高浓度时增加转移危险。     

肝移植患者将他克莫司替换为环孢素A的临床分析

目的分析肝移植后需用环孢素A（CsA）替换他克莫司（FK506）的原因和结果。方法317例肝移植患者术后采用巴昔利单抗、FK506、霉酚酸酯及肾上腺皮质激素预防排斥反应，血FK506浓度谷值，术后0～30d维持在10～15μg／L，30-90d维持在8～12μg／L，90～180d维持在5～8μg／L。术后随访6个月。结果317例患者中，有16例（5．05％）需要将FK506替换为CsA，其中5例（31．25％）主要因为FK506的神经系统不良反应，2例（12．50％）因为血液系统不良反应，1例（6．25％）因为胃肠道不良反应，3例（18．75％）因血FK506浓度始终达不到治疗窗范围，2例（12．50％）因为顽固性高血糖，另有3例（18．75％）因为经济原因。除2例患者因药物替换后发生肾功能损害而再次恢复应用FK506方案外，其余14例患者的不良反应大多在替换为CsA后明显好转，无因换药而死亡的病例，也无与替换药物相关的不良反应。结论肝移植后，当发生FK506的不良反应时，将FK506替换为CsA是安全、有效的。     

肾移植术后他克莫司替换环孢素A的临床观察

目的：探讨他克莫司（FK506）的替换环孢素A（CsA)用于肾移植患者的临床效果。方法：肾移植术后对48例CsA肝中毒、10例牙龈增生、16例多毛、13例高脂血症的患者，用FK506替换CsA治疗，停用CsA 24h后开始给予FK506，初始剂量根据患者体重、肝功能损害程度及术后时间确定，服药后根据全血FK506谷值浓度调整剂量，术后半年内维持在8－10μg/L，年内6－8μg/L，1年以上4－6μg/L，观察替换治疗后患者的临床表现，严密监测肝肾功能、FK506浓度、血糖、血脂等。结果：改用FK506后，48例CsA肝中毒者中47例肝肾功能在10－48d逐渐恢复，1例无明显好转，治疗无效死亡。10例牙龈增生者均得到控制，其中6例增生的牙龈恢复正常。16例因多毛影响外观者均得到明显改善。13例高脂血症者治疗3个月后，总胆固醇水平均明显降低。结论：对应用CsA出现严重的肝功能损害、多毛、牙龈增生及高血脂的患者改用FK506是一种较好的补救方法，不增加移植肾排斥以应的发生率，且副作用少。     

不同免疫抑制剂对大鼠血管平滑肌细胞转化生长因子-β1和Smads信号通路的影响及意义

目的研究不同免疫抑制剂对大鼠血管平滑肌细胞（VSMC）转化生长因子（TGF）-β1和Smacks信号通路的影响，探讨其在慢性移植肾肾病发生、发展中的作用。方法应用大鼠胸主动脉平滑肌细胞体外原代培养技术，分别用CsA（3mg／L）、FK506（1mg／L）、MMF（0．3mg／L）与RPM（10mg／L）处理6、12h。用免疫组织化学和实时荧光定量聚合酶链反应检测TGF—β1、Smacks蛋白和mRNA在平滑肌细胞中的表达及定位。结果CsA组和FK506组TGF—β1、Smad2的蛋白和mRNA表达水平高于对照组、MMF组和RPM组（P〈0．01），Smad7蛋白和mRNA表达低于对照组、MMF组和RPM组（P〈0．01）；MMF组和RAPA组TGF-β1、Smad2蛋白和mRNA表达低于对照组（P〈0．01），而Smad7的表达高于对照组（P〈0．01）。CsA组和FK506组，以及MMF组和RPM组的组间差异无统计学意义（P〉0．05）。结论不同免疫抑制剂可能通过影响血管平滑肌细胞TGF—β1和Smacks信号通路，导致移植物动脉硬化，从而影响慢性移植肾功能丧失的进程。     

胰岛素和睾酮对Ishikawa细胞HOXAIO基因表达的影响

目的观察胰岛素（INS）和睾酮（T）对子宫内膜腺癌细胞HOXA10基因表达的影响。方法免疫细胞化学检测HOXA10蛋白在Ishikawa细胞定位表达;体外培养Ishikawa细胞,予不同浓度INS（90、60、30、3、0．3U／L）或T（10^-3、10^-4、10^-5、10^-5、10^-7mol／L）刺激Ishikawa细胞48h,MTT法检测INS、T对Ishikawa细胞生长的作用;最后分别以30U／LINS和10^-5mol／LT刺激Ishikawa细胞48h,逆转录聚合酶链反应（RT-PCR）方法测定INS和T对Ishikawa细胞HOXA10mRNA表达的影响。结果（1）HOXA10蛋白定位表达于Ishikawa细胞的细胞浆内。（2）不同浓度的INS均可促进Ishikawa细胞的生长,随着浓度的增加作用越强,INS浓度达60、90U／L时,细胞生长状况与浓度为30U／L相似。不同浓度的T均可抑制Ishikawa细胞的生长,随浓度增加抑制作用越明显,浓度达10^-4、10^-3mol／L时,细胞生长抑制状况与浓度为10^-3mol／L相似。（3）RT-PCR检测结果显示INS组和T组Ishikawa细胞中HOXA10mRNA表达均较对照组减弱（P〈0．01或P〈0．05）。结论不同浓度INS和T均可影响Ishikawa细胞生长,高浓度的INS和T降低细胞上HOXA10mRNA的表达。     

甲状旁腺激素促进系膜细胞合成分泌转化生长因子β1

目的：研究甲状旁腺激素（PTH）对大鼠系膜细胞合成与分泌转化生长因子β1（TGF－β1）的影响。方法：（1）分别以10^-12,10^-11,10^-10,10^-9,10^-8mol/l的hPTH1-34刺激大鼠系膜细胞6，12，24，48h后，ELISA方法测定上清中TGF－β1的浓度；（2）分别以10^-12,10^-11,10^-10,10^-9,10^-8mol/L的hPTH1-34刺激大鼠系细胞48h,采用半定量RT－PCR方法检测细胞TGF－β1 mRNA的表达；（3）以10^-8mol/L的hPTH1-34分别刺激大鼠系膜细胞6，12，24，48h，采用半定量RT－PCR方法检测细胞TGF－β1 mRNA的表达，结果：（1）ELISA结果显示，hPTH1-34促进大鼠系膜细胞合成与分泌TGF－β1分泌作用达高峰（P＜0．01），（2）半定量RT－PCR方法结果显示，hPTH1-34促进大鼠系膜细胞TGF－β1 mRNA的表达，并具有浓度依赖和时间依赖性特点（各组与对照组间P＜0．05），结论：hPTH1-34从蛋白和基因水平显著促进系膜细胞TGF－β1的合成与分泌，且呈浓度依赖和时间依赖性特点。     

不同浓度他克莫司在体外对胰岛的毒性作用及致糖尿病作用的临床研究

目的探讨他克莫司（FK506）对胰岛的毒性作用以及其对肾移植患者的致糖尿病作用。方法依据临床肾移植患者术后不同阶段的血FK506浓度，设计体外实验分组，将分离、纯化的人胰岛分别与5μg／L（低浓度组）、15μg／L（中浓度组）和25μg／L（高浓度组）的FK506共培养3d，测定其在2．8mmol／L葡萄糖（低糖）和16．7mmol／L葡萄糖（高糖）刺激下的胰岛素释放量。回顾分析461例肾移植患者的临床资料，患者术后采用FK506（或环孢素A）、硫唑嘌呤（或霉酚酸酯）及激素预防排斥反应，按照空腹血糖（FPG）水平将患者的糖代谢状态分为正常空腹血糖？（FPG〈5．55mmol／L）、空腹血糖调节受损（FPG在5．55～6．88mmol／L）和移植术后糖尿病（PTDM，FPG≥6．89mmol／L）。结果体外实验中，低浓度组的胰岛在葡萄糖的刺激下胰岛素释放量未受影响，高浓度组的胰岛素释放量明显下降（P〈0．05）。肾移植后采用FK506者123例，采用环孢素A（CsA）者338例，二者术后12个月的PTDM累积发病率分别为13．8％和7．7％（P〉0．05），461例术后12个月的PTDM累积发病率为9．3％。肾移植术后并发PTDM的危险因素有年龄、糖尿病家族史、移植前空腹血糖调节受损和抗急性排斥反应治疗。结论FK506对人胰岛的毒性作用呈剂量相关性，且为可逆；肾移植患者避免长期维持较高血FK506浓度、激素用量最小化，对于减少PTDM有重要意义。     

成骨生长肽对大鼠骨髓基质细胞增殖和成骨分化的作用

目的：观察成骨生长肽诱导体外培养的大鼠骨髓基质细胞增殖及向成骨分化的作用。方法：取6周龄SD大鼠，贴壁法分离培养骨髓基质细胞，在不同浓度成骨生长肽的诱导下，观察细胞形态变化，绘制骨髓基质细胞生长曲线，碱性磷酸酶和钙结节组织化学染色。结果：成骨生长肽对骨髓基质细胞增殖和成骨分化的作用呈剂量依赖性：成骨生长肽浓度在10^-10及10^-11mol／L时促进骨髓基质细胞增殖，而在10^-8及10^-9mol／L时对骨髓基质细胞的增殖略有抑制作用；成骨生长肽浓度在10^-10及10^-11mol／L时骨髓基质细胞碱性磷酸酶染色基本呈阴性，与对照组相比差异无统计学意义，而在10^-8及10^-9mol／L浓度下可以显著提高骨髓基质细胞碱性磷酸酶染色的阳性率。结论：成骨生长肽可以明显促进大鼠骨髓基质细胞增殖及向成骨细胞分化，其促成骨活性具有显著的浓度依赖性。     

利塞膦酸钠对人成骨样细胞MG63 OPG及TNFα表达的影响

目的研究利塞膦酸钠对人成骨样细胞MG 63护骨素（OPG）及肿瘤坏死因子α（TNFα）表达的影响，以阐明利塞膦酸钠发挥骨保护作用的机制。方法以不同浓度（10^-10mol／L；10^-9mol／L；10^-8mol／L；10^-7mol／L）的利塞膦酸钠干预MG 63细胞72h；ELISA法测定细胞条件培养液中OPG及TNFα的含量。结果利塞膦酸钠可下词MG 63细胞TNFα的表达；上调OPG的表达；并提高碱性磷酸酶（ALP）活性。结论利塞膦酸钠可通过调节成骨样细胞MG 63 OPG、TNFα的表达，从而发挥其抗骨吸收的作用．     

Everolimus and mycophenolate mofetil are potent inhibitors of fibroblast proliferation after lung transplantation

BACKGROUND: Dysregulated fibroblast proliferation is thought to play an important role in the progression of bronchiolitis obliterans (BO) after lung transplantation. Augmented immunosuppression is often used to treat BO. We investigated the effect of methylprednisolone (mPRED), cyclosporine A (CsA), tacrolimus (FK506), azathioprine (AZA), mycophenolate mofetil (MMF), and everolimus (rapamycin derivative [RAD]) on the proliferative capacity of fibroblasts cultured from transbronchial biopsies of lung transplant recipients. METHODS: Primary cultures of human lung fibroblasts were obtained from 14 transbronchial biopsies of lung transplant recipients. Subconfluent cells were serum starved for 24 hr followed by growth stimulation in the presence or absence of the respective drug in six concentrations ranging as follows: 0.01 to 100 mg/L for mPRED; 0.01 to 50 mg/L for CsA and AZA; 0.001 to 5 mg/L for FK506 and MMF; and 0.00001 to 1 mg/L for RAD. Proliferation was quantified by [3H]thymidine incorporation and direct cell count. A toxic drug effect was excluded by trypan blue. RESULTS: Drug concentrations (mg/L) causing a 50% inhibition of fibroblast proliferation were mPRED 4; CsA 20; FK506 0.3; AZA 7; MMF 0.3; and RAD 0.0006. Drug concentrations (mg/L) causing inhibition of fetal bovine serum-induced proliferation were mPRED 60; CsA 45; FK506 3; AZA 35; MMF 1; and RAD 0.003. CONCLUSIONS: RAD and MMF were the most potent antifibroproliferative drugs and were effective at concentrations achieved clinically, supporting their use for the treatment of patients with early BO. Our method holds promise as an in vitro model to assess the likely in vivo responses of human lung fibroblasts to specific immunosuppressive drugs.     

不同免疫抑制剂所致肾移植受者血脂水平变化的比较

目的 探讨不同免疫抑制剂对肾移植患者术后血脂变化的影响. 方法 肾移植术后患者283例,分别选择他克莫司(FK506),环孢素(CsA)和西罗莫司(SRL)免疫抑制剂治疗方案,比较不同免疫抑制剂患者移植前及术后不同时段血清总胆固醇(TC)和三酰甘油(TG)浓度差异. 结果 FK506组93例患者服药前与服药后96周血清TC和TG浓度分别为(4.9±1.1)、(1.45=0.8)mmol/L与(4.9±1.1)、(1.4±1.0)mmol/L,差异无统计学意义(P>0.05).CsA组106例患者分别为(4.8±1.0)、(1.6±0.8)mmol/L与(6.6±1.7)、(3.2±1.0)mmol/L,差异有统计学意义(P<0.01).CsA组和SRL组患者血清TC和TG浓度一般于服药后12～24周时开始升高.51例服用12周CsA后改为FK506,患者改药前及改药后72周血清TC和TG浓度分别为(6.7±1.1)、(2.8±1.0)mmol/L与(4.7±1.7)、(1.5±1.1)mmol/L,差异有统计学意义(P<0.01). 结论 脂质紊乱是肾移植患者非免疫因素引起慢性排斥反应和慢性移植物失功的重要原因,CsA和SRL是引起肾移植患者术后血脂增高的主要因素之一.对于高脂血症肾移植患者免疫抑制剂应用可优先考虑FK506,避免SRL、CsA合用而加剧血脂升高.     

肾移植术后肝功能异常患者用他克莫司替换环孢素A疗效的初步观察

目的　观察他克莫司 (FK5 0 6 )替换环孢素A(CsA)并联合应用霉酚酸酯 (MMF)及泼尼松 (Pred)防治肾移植术后肝功能异常患者的有效性及安全性。方法　肾移植术后 8例肝功能异常患者 (男性 5例 ,女性 3例 ,平均 38.2 3岁 ) ,用FK5 0 6替换CsA治疗 ,停用CsA 2 4h后 ,开始给予FK5 0 6。FK5 0 6初始剂量根据患者体重、肝功能损害程度及术后时间确定 ,服药 1周后 ,根据全血FK5 0 6谷值浓度调整剂量 ,使其谷值浓度维持于 5～ 15 μg/L。结果　用FK5 0 6替换CsA ,1个月后患者血中直接胆红素从替换前的 (2 2 .6 6± 17.19) μmol/L下降至 (7.0 5± 2 .32 ) μmol/L ,P <0 .0 5 ;间接胆红素从替换前的 (4 2 .15± 34.15 ) μmol/L下降至 (14.5 4± 2 .5 9) μmol/L ,P <0 .0 5 ;血清丙氨酸转氨酶从替换前的 (83 .0 0± 93 .14)IU/L下降至 1个月后的 (2 9.5 0± 15 .41)IU/L ,P >0 .0 5 ;血清肌酐从 (177.91±86 .41) μmol/L下降至 (135 .92± 34.0 5 ) μmol/L ,P >0 .0 5。 3例腹水的患者均于药物替换 1个月后完全消失。仅有 1例患者出现便秘、食欲下降伴上肢颤抖。结论　用FK5 0 6替换CsA并联合应用MMF及Pred对防治肾移植术后肝功能异常是安全和有效的措施     

Different effects of cyclosporine a and FK506 on potassium transport systems in MDCK cells

BACKGROUND: Hyperkalemia and metabolic acidosis are common manifestations in patients receiving the immunosuppressive agent cyclosporine A (CsA) and the recently introduced FK506. We compared the acute toxic and antiproliferative effects as well as the effects on the transport activity of Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter of CsA and FK506 in an established cell line of distal/collecting tubule origin (MDCK cells). METHODS: MDCK cells were exposed to various concentrations of CsA or FK506 and the effects on cell viability (MTT test and neutral red uptake), plasma membrane integrity (lactate dehydrogenase (LDH) release) and cell proliferation (bromodeoxyuridine (BrdU) incorporation) were compared. For transport studies, after confluence, MDCK cells were exposed to CsA or FK506 for 48 h in the presence and absence of aldosterone. Ouabain- and bumetanide-sensitive (86)Rubidium uptake measurements were used to study the activity of the Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter at the surface of intact cells. Results: After 24 h of exposure CsA reduced the number of viable cells to 50% at 30 microM, whereas for FK506 2-3 times higher concentrations had to be employed. Similarly, LDH release was stimulated tenfold by 30 microM CsA but only fourfold by 70 microM FK506. In contrast, DNA synthesis was affected at lower concentrations of FK506 than of CsA. In cells treated for 24 h BrdU incorporation was significantly inhibited by 3 microM FK506, whereas a similar inhibition required 10 microM CsA. The transport activity of Na(+)/K(+)-ATPase and of Na(+)/K(+)/2Cl(-) cotransporter were significantly decreased (37 and 63%, respectively) on CsA administration (8 microM). In CsA-treated cells the K(+) channel blockers barium (1 mM), TEA (10 mM) and quinine (1 mM) did not further inhibit the transport activities suggesting that CsA might also act via inhibition of K(+) channels. FK506 at 8 microM had no effect on Na(+)/K(+)-ATPase transport activity but stimulated Na(+)/K(+)/2Cl(-) cotransporter activity by 59%. The stimulatory effect was abolished by K(+) channel blockers indicating that recycling of K(+) might increase by FK506. The simultaneous presence of aldosterone (5 microM) protected the cells from the inhibitory effect of CsA on Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter activity. The stimulatory effect of FK506 on the Na(+)/K(+)/2Cl(-)cotransporter activity was completely abolished in the presence of aldosterone. Conclusions: Both CsA and FK506 showed acute toxicity in MDCK cells in vitro with the effects of FK506 being less pronounced. CsA and FK506 had different effects on the in vivo transport rates of the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl(-) cotransporter; CsA inhibited the activity of the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl(-) cotransporter whereas FK506 stimulated the activity of Na(+)/K(+)/2Cl(-) cotransporter. These effects were abolished by the application of aldosterone.     

不同浓度他克莫司对新生大鼠背根神经元生长的影响

目的 观察并寻找适宜新生大鼠背根神经元生长的最佳他克莫司(FK506)浓度.方法 取8只新生24 h SD大鼠的背根神经节,剥除神经外膜,剪成约0.1 mm~3大小的碎块,消化后进行纯化培养,建立体外新生大鼠脊髓背根神经元培养体系.培养96 h后,将背根神经元分为:空白对照组(A组)、1×10~(-10) mol/L FK506组(B组)、1×10~(-9)mol/L FK506组(C组)和1×10~(-8)mol/L FK506组(D组).继续培养48 h后,应用甲基噻唑基四唑(MTT)比色法检测细胞活力及逆转录酶-聚合酶链式反应法(RT-PCR)检测生长相关蛋白-43(GAP-43)mRNA的表达情况.比较4组神经元活力和GAP-43 mRNA的表达情况.结果 A、B、C、D组的OD值分别为0.472±0.030、0.481±0.013、0.573±0.016、0.342±0.004,4组之间比较差异有统计学意义(F=26.753,P<0.01),C、D两组之间比较及C、D组分别与A、B组比较,差异均有统计学意义(P<0.05).A、B、C、D组的GAP-43 mRNA的表达值分别为0.375±0.016、0.388±0.009、0.490±0.003、0.283±0.009,4组之间比较差异有统计学意义(F=72.374,P<0.01),C、D两组之间比较及C、D组分别与A、B组比较,差异均有统计学意义(P<0.05).结论 1×10~(-9)mol/L浓度的FKS06对大鼠脊髓背根神经元生长的促进和保护作用最好,1×10~(-10)mol/L浓度的FK506对大鼠脊髓背根神经元生长具有促进和保护作用,1×10~(-8)mol/L浓度的FK506对神经元的生长具有抑制作用.     

Differences in Type 1 and Type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuppression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients

Recent multicenter, randomized clinical trials have shown that in renal transplant patients tacrolimus (FK506) was more efficient than cyclosporine A (CsA) at preventing acute rejection. In order to try and evaluate whether this difference was related to a different in vivo T-cell suppression we assessed, in a prospective study, the frequencies of interleukin (IL)-2-, IL-4-, IL-5-, IL-6-, IL-10-, interferon-gamma (IFN-gamma)- and double-positive IL-2/IFN-gamma-producing whole T cells, CD4 + and CD8 + T-cell subsets by means of cytokine flow cytometry. This was performed after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol myristate acetate (PMA) and ionomycin, in the presence of monensin, in 14 healthy volunteers (controls) and in 14 renal transplant patients. The immunosuppression of the latter was based either on CsA (n = 7) or on FK506 (n = 7). Cytokine-expressing T-cell frequencies were assessed immediately pretransplantation (DO), and subsequently 3 months (M3) and 6 months (M6) afterwards in fasting patients prior to the morning intake of the immunosuppressive drug. We found that at DO the frequencies of IL-2-(22 +/- 2% vs. 22.2 +/- 2%), IFN-gamma-(26 +/- 3% vs. 29 + 3.4%) and IL-4-(0.8 +/- 0.2% vs. 1.4 +/- 0.2%)-expressing T lymphocytes were not significantly different between the controls and the patients, respectively. Conversely, the frequency of IL-2/IFN-gamma double positive cells was higher in the latter (9.3 +/- 1.6%) than in the controls (5.6 +/- 0.8); p = 0.06. Finally, on D0 the frequencies of IL-5-, IL-6-, and IL-10-producing T lymphocytes were lower than 1%, in both groups, as well as after grafting, i.e. on M3 and M6. As compared to baseline (DO): (a) chronic immunosuppression significantly decreased the frequencies of IL-2-, IL-4- and IL-2/IFN-gamma-expressing T cells, whereas those of IFN-gamma, IL-5, IL-6, and IL-10 were not significantly affected; (b) the frequencies of cytokine-expressing T cells were not statistically different between M3 and M6; (c) the decrease in the frequencies of IL-2- and IL-2/IFN-gamma-expressing T cells affected CD4 + and CD8 + cells equally; (d) there was a marginal decrease in the frequency of IFN-gamma-expressing cells only in the CD4 + subset but not in the CD8 population; and (e) for CsA, but not for FK506, the frequency of the IL-2-expressing T cells was negatively correlated with the whole blood trough levels. When we compared the frequencies of cytokine-expressing cells in FK506- and CsA-treated patients, we found that the frequency of IL-2-expressing T cells was significantly lower with FK506 (10.9+/-1.61%) than with CsA (16.3 +/- 1.8%; p = 0.03), whereas the frequencies of the other cytokine-expressing cells were not statistically different between the two groups. In conclusion, our study clearly demonstrated that studied ex vivo, FK506 and CsA decrease the frequencies of cells expressing IL-2, IL-4 and IL-2/IFN-gamma in vivo but do not affect those expressing IFN-gamma. Meanwhile, the frequency of IL-2-producing T cells was more affected with FK506 than with CsA and was negatively correlated with the CsA trough level. Finally, our results regarding IL-2 might explain to some extent the higher efficiency of FK506 in vivo than CsA.     

Immunophilin ligands FK506 and cyclosporine A improve neurologic and histopathologic outcome after transient spinal cord ischemia in rabbits

BACKGROUND: We comparatively evaluated the protective effect of the immunophilin ligands cyclosporine A (INN: ciclosporin), FK506, and rapamycin on the spinal cord in a rabbit model of transient ischemia. Both cyclosporine A and FK506 inhibit calcineurin, whereas rapamycin does not. METHODS: Thirty-six male New Zealand White rabbits were divided into the following 6 groups: group C, 15 minutes of spinal cord ischemia; group FK, FK506 (1 mg/kg) administered 30 minutes before ischemia; group CsA, cyclosporine A (30 mg/kg) administered 30 minutes before ischemia; group CsA-C, chronic administration of cyclosporine A (20 mg/kg) for 9 days before ischemia; group R, rapamycin (1 mg/kg) administered 30 minutes before ischemia; and group R+FK, rapamycin (1 mg/kg) administered 20 minutes before FK506 pretreatment (1 mg/kg). Group CsA-C was added because the drug does not readily cross the blood-brain barrier. Neurologic function was evaluated by Johnson's 5-point scale at 8, 24, and 48 hours after ischemia, and histopathology was assessed 48 hours after ischemia. RESULTS: At 24 and 48 hours after ischemia, the Johnson score was better in groups FK (4.0 +/- 1.1), R+FK (3 +/- 1.1), and CsA-C (2.7 +/- 1.2) than in group C (0.8 +/- 1.2). Numbers of morphologically intact anterior horn cells were higher in groups FK (31.3 +/- 9.9), R+FK (23.2 +/- 4.5), and CsA-C (18.3 +/- 6.8) than in group C (6.3 +/- 4.3). CONCLUSIONS: FK506 and chronic administration of cyclosporine A, but not rapamycin, protect the spinal cord from transient ischemia. Although these results are compatible with inhibition of calcineurin in the mechanism of neuroprotective action of these drugs, other effects through different pathways cannot be excluded before further study.     

The effects of different immunosuppressants on chronic allograft nephropathy by affecting the transforming growth factor-beta and Smads signal pathways

OBJECTIVE: This study investigated the effects of various immunosuppressants on chronic allograft nephropathy (CAN) by affecting transforming growth factor-beta (TGF-beta) and Smads signal pathway. METHODS: Vascular smooth muscle cells (VMSC) from rat aorta were incubated for 6 or 12 hours with various immunosuppressants. Cyclosporine (CsA) (3 microg/mL), FK506 (1 microg/mL), mycophenolate mofetil (MMF) (0.3 microg/mL), rapamycine (Rapa) (10 microg/mL), CsA (1 microg/mL/MMF 0.3 microg/mL). We used the Sprague-Dawley Wistar rat accelerated kidney sclerosis model. Before transplantation, the kidney was preserved 1 hour in 0 degrees C to 4 degrees C heparin sodium chloride solution to reinforce the cold ischemia injury. The rats were divided into eight groups (each group n = 8): group A, pseudo-OP; group B, isotransplantation; group C, CsA 6 mg/kg . d; group D, FK506 0.15 mg/kg . d; group E, MMF 20 mg/kg . d; group F, Rapa 0.8 mg/kg. d; group G, CsA 3 mg/kg . d + MMF 20 mg/kg . d. The serum creatinine levels and pathological changes, according to the Banff scheme, were observed at 2, 4, 6, 8 and 12 weeks posttransplantation. Immunohistochemistry and quantitative fluorescence polymerase chain reactions were used to end localize and quantitate the expression of TGF-beta1 and Smad 2, 3, 7 in VMSC and in the transplanted kidney. RESULTS: CsA and FK506 stimulated gene expression and protein production of TGF-beta1, smad2, and smad3, but inhibited expression of smad7 both in VSMC and in the transplanted kidney. In contrast, MMF and Rapa down-regulated gene expression and protein production of TGF-beta1, smad2, 3 while up-regulating expression of smad7. There was no significant difference between the CsA group and the FK506 group, as well as the MMF group and the Rapa group. The group treated with CsA + MMF was similar to the MMF and the Rapa groups. CONCLUSION: Our study suggested that various immunosuppressants affected differentially TGF-beta1 and Smads signal pathways in rat VSMC and kidney grafts. CsA and FK506 can cause CAN, owing to up-regulated expression of smad2 and smad3, and down-regulation of smad7 expression. MMF and Rapa can prevent the CAN progression, because of down-regulation of the expression of smad2 and smad3, with increased smad7 production.     

Human T cell responses to human and porcine endothelial cells are highly sensitive to cyclosporin A and FK506 in vitro

BACKGROUND: Human T cells proliferate in response to both human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) via the second signals LFA-3/CD2 and B7-2 (CD86), respectively. Previous studies have shown that stimulation of T cells via CD28 or phorbol myristate acetate (PMA) activation is highly resistant to inhibition by cyclosporine A (CsA) and tacrolimus (FK506), as is the response of T cells to phytohemmaglutinin in the presence of endothelial cells. We have investigated the inhibitory effects of CsA and FK506 on the direct response of human CD4+ T cells to HUVEC and PAEC and the effect of adding B7-1 transfectants. METHODS: T cell proliferation, interleukin-2 release bioassays and a multiple cytokine bioassay employing the TF-1 cell line were used as indicators of T cell responses to HUVEC and PAEC either in the presence or absence of CsA and FK506. In some experiments, B7-1 transfectants were also added. RESULTS: Proliferative responses and interleukin-2 release were highly sensitive to CsA, the ID50 being significantly less for HUVEC (6.5 ng/ml) than PAEC (15 ng/ml). The ID50 of CsA for the mixed lymphocyte response (MLR) was similar to PAEC (18.6 ng/ml), all these values being significantly less than the T cell activation by phytohemmaglutinin (PHA) (227 ng/ml). Addition of B7-1 transfectants significantly increased interleukin-2 production by T cells/HUVEC and resistance to CsA was greatly increased to an ID50 of > 1000 ng/ml. In contrast, addition of B7-1 transfectants to T cells/PAEC had no effect either on T cell proliferation, IL-2 production, or CsA resistance. Similar results were obtained with FK506. Using the TF-1 cell line, it was determined that cytokines other than IL-2 are released during CD4+ T cell/EC interactions, with similar sensitivity to CsA and FK506. CONCLUSIONS: It is concluded that both allogeneic and xenogeneic T cell/endothelial responses should be inhibited by therapeutic levels of CsA in vivo, assuming the absence of trans-stimulation by B7 molecules.