环孢素对共刺激阻断剂延长移植肾存活效应的影响

目的:探讨环孢素(CsA)对共刺激阻断剂CTLA4Ig延长移植肾存活效应的影响。方法:肾移植大鼠分为对照组(第1组)、CTLA4Ig组(第2组)、CsA CTLA4Ig组(第3组)、CTLA4Ig IL-2组(第4组)和CsA CTLA4Ig IL-2组(第5组),观察术后血肌酐(Scr)、移植肾病理改变、移植肾存活时间。结果:与第1组、第4组相比,第2组、第3组、第5组移植肾存活时间显著延长(P<0.01),其中,第3组移植肾存活时间最长(66.1±10.6)d;术后15天,第2组Scr显著低于第3组、第5组(P<0.05);术后30天,第3组、第5组Scr显著低于第2组(P<0.01);术后30天,第3组、第5组移植肾淋巴细胞浸润明显少于第2组。结论:CsA可增强CTLA4Ig延长移植肾存活的效应,对外源性IL-2逆转CTLA4Ig的效应具有抵抗作用。     

阻断第二信号诱导骨移植后对淋巴细胞亚群的抑制作用

目的　研究细胞毒性T淋巴细胞相关抗原(CTLA4Ig)对新鲜同种异体骨移植后血淋巴细胞亚群的影响。方法　在组织相容性抗原完全不同的两组小鼠间进行异位骨移植,于移植后第0、1天,腹腔注射CTLA4Ig100μg,分别于术后2、4、6周取受体外周血,利用流式细胞仪技术测定血淋巴细胞亚群CD     

CTLA4Ig基因和CD40Ig基因转染供肾对异种大鼠移植肾存活的影响

目的 探讨细胞毒性T淋巴细胞相关抗原4融合蛋白(CTLA4Ig)基因和CD40Ig基因转染供肾对异种大鼠移植肾存活的影响.方法 以PcDNA3.1质粒为载体,通过脂质体2000将CTLA4Ig基因和CD40Ig基因转染豚鼠肾脏,再移植(异位肾移植)给SD大鼠.实验分4组进行:第1组供肾以PcDNA3.1空载体脂质体复合物转染(空载体组);第2组供肾转染CD40Ig基因(CD40Ig转染组);第3组供肾转染CTLA4Ig基因(CTLA4Ig转染组);第4组供肾同时转染CTLA4Ig基因和CD40Ig基因(双基因转染组).术后观察各组血清肌酐、移植肾组织病理改变以及移植肾存活时间.结果 空载体组、CD40Ig转染组、CTLA4Ig转染组和双基因转染组受者的存活时间分别为(6.8±1.9)d、(40.7±10.9)d、(49.3±9.5)d和(75.7±8.0)d,3个转染组明显长于空载体组(P＜0.01),其中双基因转染组移植肾存活时间最长,与其他3组比较,差异均有统计学意义(P＜0.01).各组术后血清肌酐水平呈上升趋势,但升高幅度以双基因转染组为最低(P＜0.01).术后第30天,CD40Ig转染组和CTLA4Ig转染组存活大鼠的移植肾组织中可见大量淋巴细胞浸润,而双基因转染组的移植肾组织中仅见少量淋巴细胞浸润.结论 供肾局部同时转染CTLA4Ig基因和CD40Ig基因可明显延长其异种移植后的存活时间.     

Protective effect of CTLA4Ig secreted by transgenic fetal pancreas allografts

BACKGROUND: Pancreas allotransplantation offers a cure for insulin-dependent diabetes mellitus. Systemic immunosuppression used to prevent immune destruction of the graft has side-effects, including increased susceptibility to infection and neoplasia. These unwanted effects may be limited by engineering the graft to secrete immunomodulatory molecules, to achieve local immunosuppression. Several studies have shown that transient local CTLA4Ig results in partial protection of allogeneic grafts. Our intent has been to determine whether sustained secretion of transgenic CTLA4Ig from pancreatic islets is able to protect against allograft rejection. METHODS AND RESULTS: Mouse CTLA4 (test=CTLA4Ig) or CD5 leader sequence (control=CD5LIg) was fused to the Fc of mouse IgG2c, and expressed transgenically under the control of the rat insulin promoter in C57BL/6 mice carrying the bml mutation of H-2K(b) (B6.C-H-2(bm1)). This resulted in expression in pancreatic islets. We used ELISA quantification of transgene products secreted into the supernatants of cultured fetal pancreata to select high (CTLA4Ig(hi)) and low (CTLA4Ig(lo)) expresser transgenic mice. Cultured fetal pancreata were transplanted under the kidney capsule of wholly allogeneic CBA recipient mice. CTLA4Ig(hi) but not CTLA4Ig(lo) expresser grafts showed enhanced survival compared with control CD5LIg grafts at 6 weeks posttransplant, provided the recipient mice were transiently depleted of CD4 T cells (by a single low-dose injection of GK1.5) before transplantation. CONCLUSIONS: Sustained local secretion of CTLA4Ig from transgenic grafts in combination with transient systemic CD4 T-cell depletion can enhance allograft acceptance.     

外用环孢素A联合CTLA4Ig延长异体移植鼠耳存活的研究

目的　探讨局部外用环孢素 A(Cs A)联合细胞毒性淋巴细胞相关抗原 4融合蛋白 (CTL A4 Ig)对异体复合组织移植的免疫抑制及诱导免疫耐受的作用。方法　建立吻合血管的同种异体大鼠耳廓移植模型 ,术后在移植耳皮肤表面外涂 Cs A并联合 CTL A4 Ig腹腔注射治疗 ,观察移植物的排斥反应及存活时间 ,检测移植后受体血清白细胞介素 - 2 (IL- 2 )含量变化。结果　对照组平均存活时间为 (7.8± 1.7)天 ;单纯用 Cs A治疗组为 (15 .2± 1.9)天 ,单纯CTL A4 Ig治疗组为 (16 .6± 2 .1)天 ;Cs A +CTL A4 Ig联合治疗组为 (2 8.8± 3.5 )天 ,与其它各组相比均有统计学意义 (P<0 .0 1) ;且联合治疗组的受体血清 IL - 2含量最低 ,尤以第 5、7天为著 ,与其它各组相比有统计学意义 (P<0 .0 1)。结论　局部外用 Cs A联合 CTL A4 Ig能有效抑制异体复合组织移植排斥反应 ,显著延长移植物存活时间。     

Infusion of stably immature monocyte-derived dendritic cells plus CTLA4Ig modulates alloimmune reactivity in rhesus macaques

BACKGROUND: Immature dendritic cells (DC) can promote long-term transplant survival in rodents. We assessed the impact of stably immature, donor-derived DC on alloimmune reactivity in rhesus macaques. METHODS: CD14 monocytes isolated from leukapheresis products of Macacca mulatta were cultured in granulocyte-macrophage colony stimulating factor plus interleukin (IL)-4+/-vitamin (vit) D3, and IL-10. Major histocompatibility complex class II and cosignaling molecule expression was determined on CD11c cells by flow cytometry. T-cell allostimulatory capacity of the DC, including DC exposed to proinflammatory cytokines, was determined in mixed leukocyte reaction. To test their influence in vivo, purified DC were infused intravenously into allogeneic recipients, either alone or followed by CTLA4Ig, 24 hr later. Proliferative responses of recipient CFSE-labeled T cells to donor or third party DC, cytokine production by stimulated T cells, and circulating alloantibody levels were determined by flow cytometry, up to 100 days postinfusion. RESULTS: VitD3/IL-10-conditioned, monocyte-derived DC were resistant to maturation and failed to induce allogeneic T cell proliferation in vitro. After their infusion, an increase in anti-donor and anti-third party T-cell reactivity was observed, that subsequently subsided to fall significantly below pretreatment levels (by day 56) only in animals also given CTLA4Ig. No increase in circulating immunoglobulin (Ig) M or IgG anti-donor alloantibody titers compared with pretreatment values was detected. With DC+CTLA4Ig infusion, alloreactive IL-10-producing T cells were prevalent in the circulation after day 28. CONCLUSIONS: Maturation-resistant rhesus DC infusion is well-tolerated. DC+CTLA4Ig infusion modulates allogeneic T-cell responses and results in hyporesponsiveness to donor and third party alloantigens.     

Local expression of IDO, either alone or in combination with CD40Ig, IL10 or CTLA4Ig, inhibits indirect xenorejection responses

Abstract: Background: To overcome cell‐mediated xenorejection by transgenic expression of immunomodulatory molecules by a graft, it is likely that expression of multiple molecules will be required. Previous studies support the use of the immunomodulatory agents indoleamine 2,3‐dioxygenase (IDO), CD40Ig, interleukin 10 (IL10), and CTLA4Ig for suppression of rejection responses. We examined the effects of local expression of these molecules by a porcine cell line (PIEC) on indirect murine xenorejection responses in vitro and in vivo. Methods: The PIEC stable lines expressing IDO, CD40Ig, and IL10 as single molecules were generated. In addition, PIEC lines expressing IDO with either CD40Ig, IL10 or CTLA4Ig were generated to produce cell lines expressing two molecules. BALB/c mice were primed with wild type PIEC, followed by harvesting splenocytes used as responder cells and PIEC expressing immunomodulatory molecules as stimulators, in proliferation and cytokine assays. In vivo effects of modified PIEC were examined by transplantation of PIEC lines expressing the immunomodulatory molecules under the renal capsule of naïve mice. PIEC grafts were harvested for histological evaluation at days 7 and 14. Results: Proliferation of primed BALB/c splenocytes was inhibited most significantly by IDO compared with control cells (49%, P = 0.02). In addition both Th1 (interferon‐gamma) and Th2 (IL4 and IL10) cytokines were markedly inhibited in vitro by IDO expression. IL10 expressing cells did not inhibit proliferation as potently (37%, P = 0.03) whilst CD40Ig lead to an increase in proliferative responses (59%, P = 0.02). Co‐expression of CD40Ig, IL10, and CTLA4Ig with IDO resulted in further modest reductions in proliferation compared with IDO expression alone. When transplanted under the renal capsule of BALB/c mice, those grafts expressing IDO demonstrated significantly lower levels of lymphocyte infiltration at days 7 and 14 than control grafts and those expressing CD40Ig, CTLA4Ig or IL10 alone. Grafts co‐expressing IDO and a second molecule were no better protected than those expressing IDO alone. Graft cell viability (PIECs) was reduced in some IDO expressing grafts suggesting high levels of IDO expression may inhibit PIEC viability, however, grafts co‐expressing IDO‐CTLA4Ig and IDO‐IL10 were not affected in this way. Conclusion: Indoleamine 2,3‐dioxygenase appears to be a potent molecule for protecting xenografts from cell‐mediated rejection responses activated via the indirect pathway. Co‐expression of IDO with both CTLA4Ig and IL10 warrants further investigation. Overall these findings support pursuing further studies, in larger animal models, to determine whether increased IDO activity within the graft itself can attenuate xenorejection responses.     

Adenovirus‐mediated gene transfer of CTLA4lg gene results in prolonged survival of heart allograft

Abstract It has been demonstrated that the administration of CTLA4Ig protein can induce the suppression of allograft and xenograft rejection. The purpose of this study is to determine the effect of adenovirus‐mediated gene transfer with CTLA4Ig gene on the transgene expression and suppression of alloimmune response in allogeneic cardiac transplantation of rats. Adenoviral vectors with β galactosidase or human CTLA4Ig cDNA (Adex/LacZ, Adex/hCTLA4Ig) were constructed. These vectors were transduced to the liver of Lewis (LEW) rats by intravenous injection. Then LEW rats received heterotopic cardiac transplantation from Dark Agouti rats. Experiments were performed using both CTLA4Ig gene transduction and/or immunosuppressant FK506. The transgene expression after adenovirus‐mediated transfer with CTLA4Ig cDNA lasted for several months, compared with several weeks after that in controls, which was proportional to the blood concentration of CTLA4Ig protein. By the administration of 1 × 10⁹ PFU of Adex/hCTLA4Ig, the survival of cardiac grafts was significantly prolonged, compared with controls or the use of 1 × 10⁸ PFU of Adex/hCTLA4Ig. In the rats with beating grafts over 100 days, the blood concentrations of CTLA4Ig were undetectable. The combination therapy using a low titer Adex/hCTLA4Ig and low‐dose of FK506 was synergistically effective on this cardiac transplantation model. In conclusion, adenovirus‐mediated gene transfer with CTLA4Ig gene was efficient for the prolongation of both transgene expression and allograft survival.     

Testicular sertoli cells protect islet beta-cells from autoimmune destruction in NOD mice by a transforming growth factor-beta1-dependent mechanism

Testicular Sertoli cells protect pancreatic islet grafts from allo- and autoimmune destruction; however, the mechanism(s) of protection is unclear. The aim of this study was to determine whether Fas ligand (FasL) and/or transforming growth factor (TGF)-beta, immunoregulatory proteins produced by Sertoli cells, might mediate the protective effects of these cells against autoimmune destruction of islet beta-cells. Sertoli cells were purified from testes of NOD mice and implanted under the right renal capsule of diabetic NOD mice, whereas NOD islets were implanted under the left renal capsule. Of the mice that received islet and Sertoli cells grafts, 64% (9 of 14) remained normoglycemic at 60 days posttransplantation compared with 0% (0 of 6) of the mice that received islet grafts alone. Immunohistochemical examination of Sertoli cell grafts in normoglycemic mice revealed that TGF-beta1 expression by Sertoli cells remained high, whereas FasL expression by Sertoli cells decreased progressively posttransplantation. Also, plasma levels of TGF-beta1 were significantly elevated in mice that received Sertoli cells and islet grafts, and anti-TGF-beta1 antibody administration completely abrogated the protective effect of Sertoli cells on islet graft survival, whereas anti-FasL antibody did not. Islet graft destruction in anti-TGF-beta1-treated mice was associated with increases in interferon (IFN)-gamma-producing cells and decreases in interleukin (IL)-4-producing cells in the islet grafts. We conclude that 1) Sertoli cell production of TGF-beta1, not FasL, protects islet beta-cells from autoimmune destruction and 2) TGF-beta1 diverts islet-infiltrating cells from a beta-cell-destructive (IFN-gamma+) phenotype to a nondestructive (IL-4+) phenotype.     

干预协同刺激信号诱导不明原因早期复发性流产患者外周血Th1/Th2转换的体外研究

目的观察体外干预细胞毒性T细胞相关抗原4(CTLA-4)对不明原因早期复发性流产(URSA)患者外周血辅助性T细胞(Th)1/Th2转换的影响。方法用人绒毛膜癌细胞株JEG-3制备的滋养细胞抗原刺激30例URSA患者外周血单核细胞,分为实验组A:加重组蛋白CTLA-4Ig(10和1 mg/L)组,实验组B:加CTLA-4抗体(10和1 mg/L)组;对照组:加IgG(10和1 mg/L)组。采用酶联免疫吸附试验(ELISIA)测上清液中白细胞介素(IL)-2、干扰素γ(IFN-γ)和IL-4水平。结果与相应浓度的对照组相比,CTLA-4Ig组IL-4水平显著升高(P<0.05),IL-2显著降低(P<0.05),IFN-γ两组间无显著性差异(P>0.05);而CTLA-4抗体组IL-4水平均显著低于对照组(P<0.01),而IFN-γ、IL-2水平均明显高于对照组(P<0.05)。CTLA-4Ig 10 mg/L组与CTLA-4Ig 1mg/L组比较,IL-4显著升高(P<0.05),IL-2显著降低(P<0.05),IFN-γ两组间无显著性差异(P>0.05)。与同浓度的对照组及CTLA-4抗体1 mg/L组相比,CTLA-4抗体10 mg/L组IL-4较低,而IFN-γ和IL-2稍高,但两组间亦无显著性差异(P>0.05)。结论CTLA-4Ig使URSA患者Th1/Th2型细胞因子向Th2型细胞因子偏移,有利于URSA的治疗;而CTLA-4抗体可以上调Th1型细胞因子,下调Th2型细胞因子,不利于妊娠。     

Immunotherapy with nondepleting anti-CD4 monoclonal antibodies but not CD28 antagonists protects islet graft in spontaneously diabetic nod mice from autoimmune destruction and allogeneic and xenogeneic graft rejection.

BACKGROUND: T-cell activation and the subsequent induction of effector functions require not only the recognition of antigen peptides bound to MHC molecules by T-cell receptor (TCR) for antigen but also a costimulatory signal provided by antigen presenting cells. CD4 T-cell activation and function require the CD4 molecule as a coreceptor of TCR. The CD28/B7 pathway is a major costimulatory signal for T-cell activation and differentiation. METHODS: The effect of targeting CD4 by nondepleting anti-CD4 monoclonal antibodies (mAbs) versus blocking CD28/B7 by CTLA4Ig, anti-CD80 mAbs, and anti-CD86 mAbs on the prevention of recurrence of autoimmune diabetes after MHC-matched nonobese diabetes-resistant (NOR) islet transplantation in nonobese diabetic (NOD) mice were compared. Whether nondepleting anti-CD4 mAbs prolong allogeneic islet graft survival and xenogeneic pig islet graft survival in diabetic NOD mice were studied. Furthermore, the effect of nondepleting anti-CD4 mAbs combined with CTLA4Ig on allogeneic islet graft survival in NOD mice was investigated. RESULTS: Recurrence of autoimmune diabetes can be prevented by nondepleting anti-CD4 mAbs. Blocking the CD28/B7 costimulatory pathway by CTLA4Ig or by anti-CD80 mAbs and anti-CD86 mAbs cannot prevent recurrence of autoimmune diabetes after islet transplantation. Short-term treatment with nondepleting anti-CD4 mAbs significantly prolongs allogeneic islet graft survival and xenogeneic pig islet graft survival in diabetic NOD mice. But nondepleting anti-CD4 mAbs combined with CTLA4Ig decreased allogeneic islet graft survival. CONCLUSIONS: Nondepleting anti-CD4 mAbs but not CD28 antagonists protect islet grafts in diabetic NOD mice from autoimmune destruction and allogeneic and xenogeneic graft rejection. The efficacy of nondepleting anti-CD4 mAbs is compromised when it combines with CTLA4Ig.     

激活型Akt1转染大鼠胰岛联合应用细胞毒T淋巴细胞相关抗原4免疫球蛋白延长异种胰岛移植存活

目的 观察激活型Akt1基因转染大鼠胰岛对异种胰岛移植功能和存活的影响.方法 提取大鼠胰岛培养转染,AO/EB和Tunel染色检测存活和凋亡;BALB/C糖尿病小鼠肾被膜下胰岛移植.分4组,实验组:Akt1转染胰岛联合应用细胞毒T淋巴细胞相关抗原4免疫球蛋白(CTLA-4Ig);Akt1组:移植Akt1转染的胰岛;未转染组:单纯胰岛移植,CTLA-4Ig组:单纯胰岛联合应用CTLA-4Ig;观察胰岛移植后功能存活时间和平均生存时间.结果 Akt1转染胰岛体外细胞抗凋亡生存率提高了25%;实验组移植物功能存活(32.5±6.6)d,平均生存时间(39.6±5.9)d比Akt1组(15.4±3.5)、(22.5±1.7)d和CTLA-4Ig组(18.7±4.5)、(21.7±3.5)d未转染组(4.5±2.8)、(6.8±3.1)d明显延长(P<0.05).结论 激活型Akt1转染大鼠胰岛联合应用CTLA-4Ig,可减少移植胰岛凋亡,延长异种胰岛移植物存活时间.     

胰岛与Fas配体阳性睾丸细胞共同移植产生免疫豁免

目的通过胰岛与睾丸细胞共同移植诱导胰岛移植物长期生存。方法胶原酶、胰蛋白酶、Ｄｎａｓ酶消化制备睾丸ｓｅｒｔｏｌｉ细胞培养４８小时;纯化的供体（Ｗｉｓｔａｒ大鼠）胰岛５００个培养后的睾丸ｓｅｒｔｏｌｉ细胞共同移植,移植前后不用全身性免疫抑制剂,ＴＵＮＥＬ法标记胰岛移植物周围凋亡的淋巴细胞,ＳＡＢＣ法测定共同移植物ＦａｓＬ稳定表达情况。结果胰岛与１×１０７个睾丸ｓｅｒｔｏｌｉ细胞共同移植可以纠正链脲酶素所至的糖尿病高血糖状态达６０天（有效率达１００％,６／６）,单纯的胰岛移植或与１×１０５个睾丸ｓｅｒｔｏｌｉ细胞共同移植,移植物仅能存活５～６天。移植物周围可见明显的凋亡的淋巴细胞存在。结论胰岛与Ｆａｓ配体阳性睾丸细胞共同移植可以诱导局部免疫豁免,使移植物长时间存活。     

阻断B7/CD28共刺激通路抑制大鼠肝移植急性排斥反应的研究

目的:建立大鼠原位肝移植(ROLT)急性排斥反应模型,观察细胞毒淋巴细胞抗原4免疫球蛋白(CTLA-4Ig)在大鼠肝移植术后对共刺激分子B7-1/B7-2的影响及其抗排斥反应的作用。方法:采用"二袖套管"法先行建立DA-Lewis大鼠组合肝移植急性排斥反应模型,随机分为对照组(A组)与实验组(B组)两组,于肝移植术后48h每只受体大鼠腹腔内一次性注射CTLA-4Ig 75μg,分别于术后3、5、7和10d采用RT-PCR检测B7-1和B7-2 mRNA在两组肝脏组织中的表达情况,并同时观察其肝功能变化。结果:1)B7-1和B7-2 mRNA在A组高水平表达,而在B组表达明显降低(P<0.01);2)B组动物术后未见明显排斥反应,血清ALT、TBIL和DBIL水平明显低于对照组(P<0.01)。结论:移植术后应用CTLA-4Ig可以降低肝组织中B7-1和B7-2的表达;动态检测B7分子的表达有助于观察肝移植排斥反应的进程。     

Alloantigen-driven T cell death mediated by Fas ligand and tumor necrosis factor-alpha is not essential for the induction of allograft acceptance

BACKGROUND: Fas ligand (FasL)-Fas and tumor necrosis factor alpha (TNFalpha)-tumor necrosis factor receptor (TNFR) interactions regulate immune responses and contribute to self-tolerance by mediating antigen-driven T cell apoptosis. It is not known whether FasL and TNFalpha, expressed by the recipient's lymphoid or nonlymphoid cells, are essential for the apoptosis of alloreactive T lymphocytes and the induction of allograft acceptance. METHODS: We compared the survival of fully allogeneic vascularized cardiac allografts between wild-type (wt) and FasL-mutant (gld) recipient mice. In addition, we studied cardiac allograft survival in gld mice injected with TNFalpha-neutralizing antibody. Allograft acceptance (graft survival >100 days) was induced by treating the recipients with CTLA4Ig, a recombinant fusion protein that blocks B7-CD28 T cell costimulation. In vivo alloantigen-driven apoptosis of mature CD4+ and CD8+ T lymphocytes was analyzed in mice repeatedly stimulated with allogeneic splenocytes. RESULTS: We found that CTLA4Ig induces 100% long-term acceptance of cardiac allografts in wt and gld mice. Similarly, CTLA4Ig induced 100% allograft acceptance in gld recipients injected with TNFalpha-neutralizing antibody. In vivo alloantigen-driven apoptosis of mature CD4+ and CD8+ T cells was significantly reduced in gld mice and in wt mice treated with anti-TNFalpha antibody. However, neutralizing TNFalpha activity in gld mice failed to abrogate alloantigen-driven T cell apoptosis. CONCLUSIONS: These data indicate that: (1) FasL and TNFalpha expression are not obligatory for the induction of long-term allograft acceptance by CTLA4Ig and (2) FasL- and TNFalpha-independent death pathways contribute to alloantigen-driven T cell apoptosis.     

CTLA4Ig和西罗莫司短期联合使用延长异种胰岛移植物的存活

目的 观察细胞毒性T淋巴细胞相关抗原4融合蛋白(CTLA4Ig)与西罗莫司(SRL)联用阻断共刺激通路对异种胰岛移植物存活的影响.方法 取C57BL/6小鼠,腹腔注射链佐星,制成糖尿病模型.采用随机单位组设计分组法将糖尿病小鼠分为7组,各组均于小鼠左肾包膜下移植SD大鼠胰岛300胰岛当量.CTLA4Ig组分别于移植当天及移植后第2、4、6天腹腔注射CTLA4Ig 0.5 mg/d;SRL组分别于移植当天及移植后第1、2天给予SRL灌胃,0.2 mg·kg-1·d-1,其后隔天用药1次,共用2周;MRI组分别于移植当天及移植后第2、4天腹腔注射仓鼠抗小鼠CD154单克隆抗体(MR1)0.5 mg/d;CTLA4Ig和SRL联用组(SRL联用组)、CTLA4Ig和MRl联用组(MR1联用组)以及CTLA4Ig、MR1和SRL联用组(三药联用组)各药物的剂量与用法同上述各组;对照组仅行胰岛移植,不予以药物.观察至移植后200 d,通过监测受者血糖水平来判断排斥反应的发生情况.记录各组移植物的存活(即无排斥反应)时间.发生排斥反应者,或未发生排斥反应、移植物存活时间＞200 d者,取移植胰岛,行HE染色及免疫荧光染色,进行组织学观察.结果 对照组移植物存活时间中位数为17 d,该组最终均发生排斥反应.SRL组、MRl组和CTLA4Ig组移植物存活时间中位数分别为34 d、98 d和77 d.均明显长于对照组(P＜0.05),三组中分别有90%(9/10)、62.5%(5/8)和83.3%(5/6)的小鼠发生排斥反应.SRL联用组移植物存活时间中位数为130 d,明显长于上述4组(P＜0.01),有50%(3/6)的小鼠发生排斥反应.MR1联用组以及三药联用组移植物存活时间中位数均＞200 d,分别有42.9%(3/7)和25%(2/8)的小鼠发生排斥反应.组织学检查结果显示,对照组发生排斥反应时,其移植胰岛破坏严重,可见大量CD4+和CD8+淋巴细胞及巨噬细胞浸润,并可见IgG、IgM和补体C3沉积.其它组发生排斥反应者的组织学改变与对照组相似.SRL联用组存活200 d的小鼠,其移植胰岛组织中未见或仅有少量炎症细胞浸润,胰岛素和胰高血糖素染色阳性,未见IgG、IgM和补体C3沉积.结论 短期联合使用CTLA4Ig和SRL能显著延长小鼠体内大鼠来源的胰岛的存活时间.     

Anti-B7-1 blocks mononuclear cell adherence in vasa recta after ischemia

BACKGROUND: Blocking the costimulatory pathway by CTLA-4 Ig, reactive with both B7-1 and B7-2 costimulatory molecules, protects the kidney during acute ischemia/reperfusion injury. This study investigated whether and how B7-1 and/or B7-2 proteins are involved in renal ischemia/reperfusion injury (IRI). METHODS: Uninephrectomized rats were submitted to warm renal ischemia (30 min) and received control monoclonal antibody (mAb; 17E3), anti-B7-1 (3H5), anti-B7-2 (24F), a combination of anti-B7-1/B7-2, or CTLA-4 Ig. Renal function, morphology, and the kinetics of inflammatory cells were studied for a ten-day period. Binding sites of the injected antibodies were detected by secondary staining with anti-mouse Ab. RESULTS: Compared with controls, acute renal failure (ARF) in the anti-B7-1 group was attenuated both functionally and morphologically. Anti-B7-1/B7-2 and CTLA-4 Ig also were protective in IRI. ARF was not altered by anti-B7-2 treatment. Two hours after reperfusion, B7-1 was expressed along the endothelial cells of the ascending vasa recta. Expression of B7-1 increased over time during the first 24 hours and decreased thereafter. Two hours after reperfusion, adherence/accumulation of T cells and monocytes/macrophages was found in the vasa recta of the ischemic kidney. Anti-B7-1-treated animals had fewer T cells and monocytes/macrophages in the vasa recta compared with controls. Leukocyte accumulation in these vessels after anti-B7-2 treatment was not different from IRI controls. CONCLUSION: These observations strongly support the key role of the B7-1 protein in the protection of renal IRI through inhibition of T cell and monocyte adherence at the level of the ascending vasa recta.     

Induction of operational tolerance to discordant dopaminergic porcine xenografts

BACKGROUND: Porcine embryonic neural tissue transplanted intracerebrally could potentially relieve the symptoms of Parkinson's disease if the immune response toward the graft could be overcome. However, conventional immunosuppressive treatments have proven inefficient in preventing rejection. An alternative is blocking the costimulatory signals for lymphocyte activation. Treatment with cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4Ig) and anti-CD40L has been successful in preventing rejection of xenografts in some experimental studies, but not all. Lymphocyte function antigen (LFA)-1 is an important costimulatory molecule for CD8+ T cells, and we hypothesize that blockade with anti-LFA-1 may enhance the efficacy of CTLA4Ig and anti-CD40L therapy. METHODS: C57BL/6 mice received intracerebral transplants of ventral mesencephalic tissue from embryonic porcine donors. CTLA4Ig, anti-CD40L, and anti-LFA-1 were administered every other day on days 0 to 8, and the transplants were studied after 4 to 6 weeks. Grafts were histologically analyzed for size, survival of dopaminergic nerve cells, and immune responses. Recipients were challenged with cultured glia cells of donor origin or an allogeneic skin graft to evaluate tolerance induction. RESULTS: Mice treated with all three substances had large grafts containing high amounts of dopamine cells but a low degree of immune response. Grafts in recipients challenged with glial cells showed an increased immunologic activity but were not rejected. Triple-treated mice showed a normal rejection process of the allogeneic skin grafts. CONCLUSION: After a short course of costimulation blocking therapy, discordant neural xenografts demonstrate long-term survival, withstand immunologic challenge, yet maintain host-versus-graft reactivity. Anti-LFA-1 complements CTLA4Ig and anti-CD40L in the induction of operational tolerance to these xenografts.     

Effects of Gene Transfer CTLA4Ig and Anti-CD40L Monoclonal Antibody on Islet Xenograft Rejection in Mice

Blockade of a costimulatory pathway by adenovirus-mediated cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) gene transfer and anti-CD40L mAb(MR1) have been reported to enhance graft survival in several experimental transplantation models. In this study, we investigated the effects of gene transfer of CTLA4Ig and MR1 on islet xenograft rejection in mice. Recombinant adenovirus AdCTLA4Ig was constructed to express CTLA4Ig. Islet grafts from adult male DA rats transferred with AdCTLA4Ig were transplanted to streptozocin-induced diabetic Balb/c mice. The diabetic mice were treated with MR1 after transplantation. We evaluated the islet xenograft mean survival time as well as changes in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) levels in transplanted mice. The mean survival of islet xenografts in the MR1 treatment group was 34.9 ± 5.62 days, in the AdCTLA4Ig treatment group it was 56.5 ± 10.64 days, and in the AdCTLA4Ig plus MR1 treatment group it was 112.9 ± 19.26 days, all significantly prolonged compared with an untreated group (8.1 ± 0.83 days). Within 1 week after transplantation the levels of IL-2 and TNF-α showed sharp increases in the untreated group, being significantly higher than those observed prior to transplantation. In conclusion, using both AdCTLA4Ig and MR1 can improve the islet xenograft survival. The beneficial effects of the combined use of the 2 reagents were superior to either 1 alone, possibly related to down-regulated expression of Th1 cell-related cytokines.