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首页> 外文期刊>Transplant immunology >Effects of interleukin-12p40 gene transfer on rat corneal allograft survival.
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Effects of interleukin-12p40 gene transfer on rat corneal allograft survival.

机译：白细胞介素12p40基因转移对大鼠角膜同种异体移植存活的影响。

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PURPOSE: Despite the immunologically privileged nature of the cornea, graft rejection remains the major cause of human corneal allograft failure. Gene therapy is an interesting approach to introduce immunoregulatory molecules into the graft or the recipient to prevent rejection. In this study we investigated the immmunomodulatory effects of adenovirus-mediated gene transfer of a Th1 antagonist, interleukin-12p40 (IL-12p40), in vitro and on allogeneic graft survival in a rat experimental keratoplasty model. METHODS: Donor corneas were transduced with an E1/E3 deleted adenoviral (Ad) vector encoding the IL-12p40 gene (AdIL-12p40) and assayed for the expression of the therapeutic gene. Cell culture supernatants containing IL-12p40 protein were generated by transducing human corneal endothelial cells with AdIL-12p40 and analysed for their capacity to inhibit production of IFN-gamma by naive T cells. The effect of both local (ex vivo Ad-mediated gene transfer) and systemic (i.p.-injection) over-expression of IL-12p40 was investigated by analysing the survival of corneal allografts transplanted from Wistar-Furth rats to fully MHC-class I/II incompatible Lewis rats. Moreover, the intra-graft mRNA-expression profile of cytokines and T cell markers was investigated at different time points after gene transfer. RESULTS: Adenovirus-mediated gene transfer in cultured corneas led to significant IL-12p40 protein expression as determined by specific ELISA. Moreover we could show that IL-12p40 protein containing supernatants significantly inhibited the production of IFN-gamma by alloreactive naive T cells. Interestingly, neither ex vivo genetic modification of cultured corneas before transplantation nor systemic AdIL-12p40 treatment of recipients receiving allogeneic corneas did improve corneal allograft survival. Real-time RT-PCR analysis of ex vivo modified cornea allografts on day 7 after transplantation showed significantly higher IL-4 mRNA-expression levels in the AdIL-12p40 group compared to the control group. Other significant differences in mRNA-expression levels of intra-graft CD3, CD25, IFN-gamma, TNF-alpha, and IL-10 could not be detected, neither on day 7 nor on the day of rejection. CONCLUSIONS: Despite the capacity of IL-12p40 protein to inhibit the production of IFN-gamma of naive T cells in vitro and some Th1/Th2 shift in vivo, no prolongation of allogeneic graft survival of both AdIL-12p40 modified rat corneas and systemically treated rats could be obtained after transplantation. The possible binding of Ad-mediated IL-12p40 with ubiquitously expressed IL-12p35 in vivo might therefore limit the application of IL-12p40 for the prevention of transplant rejection.
机译：目的：尽管角膜具有免疫学上的特权，但移植排斥仍是人角膜同种异体移植失败的主要原因。基因疗法是将免疫调节分子引入移植物或受体以防止排斥反应的有趣方法。在这项研究中，我们研究了腺病毒介导的Th1拮抗剂白细胞介素12p40（IL-12p40）的基因转移在体外和在大鼠实验性角膜移植模型中的同种异体移植存活率的免疫调节作用。方法：用编码IL-12p40基因（AdIL-12p40）的E1 / E3缺失的腺病毒（Ad）载体转导供体角膜，并测定其治疗基因的表达。通过用AdIL-12p40转导人角膜内皮细胞来生成含有IL-12p40蛋白的细胞培养上清液，并分析其抑制幼稚T细胞产生IFN-γ的能力。通过分析从Wistar-Furth大鼠移植至完全MHC I类/ II不相容的Lewis大鼠。此外，在基因转移后的不同时间点，研究了细胞因子和T细胞标志物在移植物中的mRNA表达谱。结果：通过特异性ELISA测定，腺病毒介导的在培养的角膜中的基因转移导致IL-12p40蛋白的显着表达。此外，我们可以证明，含有IL-12p40的上清液能显着抑制同种反应性幼稚T细胞产生的IFN-γ。有趣的是，在移植前对培养的角膜进行离体遗传修饰或对接受异体角膜的受体进行全身性AdIL-12p40处理均不能改善同种异体角膜移植的存活率。移植后第7天对体外修饰的角膜同种异体移植进行实时RT-PCR分析，与对照组相比，AdIL-12p40组的IL-4 mRNA表达水平明显更高。在移植后的第7天或排斥日均无法检测到移植物中CD3，CD25，IFN-γ，TNF-α和IL-10的mRNA表达水平的其他显着差异。结论：尽管IL-12p40蛋白具有抑制幼稚T细胞IFN-γ产生的能力，并且在体内具有Th1 / Th2的转移，但AdIL-12p40修饰的大鼠角膜和系统治疗的同种异体移植物存活时间均未延长移植后即可获得大鼠。因此，Ad介导的IL-12p40与体内普遍表达的IL-12p35的可能结合可能因此限制了IL-12p40在预防移植排斥中的应用。

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