Background

Aggregating the human leukocyte antigen (HLA) Class I antigens on the endothelial membrane has been known to elicit an activation, an underlying mechanism of chronic rejection in organ transplant recipients. The current study aims at examining the endothelial responses using HLA typed microvascular cultures from human adipose tissues upon exposure to the serum that contain corresponding antibodies collected from mismatched transplant recipients.

Methods

We have successfully cultured 30 microvascular cultures and typed their HLAs. They are functionally competent to respond to inflammatory TNF-α stimulation and the aggregating monoclonal antibody against HLA Class I. The post-transplantation serum was collected either from the recipients with pathologically diagnosed chronic rejection or from the recipients without rejection. We determined their activation either by double-staining the endothelial cells in crude cultures with flow cytometry or by quantifying cytokine releases in purified endothelial cells using ELISA.

Results

Under our current protocol, adipose tissue cultures are functionally intact in regard to its responses to TNF-alpha and anti-HLA Class I antibody. We observed that the post-transplantation serum with rejection contained the pathogenic antibodies and led to proinflammatory activation, as demonstrated by not only increased CD54+/CD31+ and CD106+/CD31+ cell counts but also inflammatory cytokine releases including MCP-1, IL-8 and RANTES.

Conclusion

This methodological study provides the feasibility of examining the pathogenicity of the alloantibodies in mis-transplant serum. Potentially, the endothelial activation elicited as a result of exposure can be used as an alternative readout for chronic rejection.

Significance

We prototype an ex vivo model that enables us to examine whether allogenic antibodies from the recipient can functionally activate microvascular endothelial cells from the donor adipose tissues. This system can be further developed as crossmatch using cellular responses as readouts for chronic rejection for post-transplant surveillance.

中文翻译：

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实体器官移植错配患者血清对脂肪组织微血管培养物活化的影响

背景

已知在内皮膜上聚集人类白细胞抗原 (HLA) I 类抗原会引发激活，这是器官移植受者慢性排斥的潜在机制。目前的研究旨在使用来自人类脂肪组织的 HLA 型微血管培养物在暴露于含有从不匹配的移植受者收集的相应抗体的血清时检查内皮反应。

方法

我们已经成功培养了 30 种微血管培养物并对其 HLA 进行了分型。它们在功能上能够响应炎症性 TNF-α 刺激和针对 HLA I 类的聚集单克隆抗体。移植后血清是从病理诊断为慢性排斥的受体或从无排斥的受体收集的。我们通过使用流式细胞术对粗培养物中的内皮细胞进行双重染色或通过使用 ELISA 量化纯化的内皮细胞中的细胞因子释放来确定它们的激活。

结果

根据我们目前的协议，脂肪组织培养物对 TNF-α 和抗 HLA I 类抗体的反应在功能上是完整的。我们观察到具有排斥反应的移植后血清含有致病性抗体并导致促炎激活，这不仅表现为 CD54+/CD31+ 和 CD106+/CD31+ 细胞计数增加，而且包括 MCP-1、IL-8 和 RANTES 在内的炎性细胞因子释放.

结论

该方法学研究为检查移植错误血清中同种抗体的致病性提供了可行性。潜在地，由于暴露引起的内皮激活可用作慢性排斥的替代读数。

意义

我们对离体模型进行原型设计，使我们能够检查来自受体的同种异体抗体是否可以在功能上激活来自供体脂肪组织的微血管内皮细胞。该系统可以进一步开发为交叉匹配，使用细胞反应作为移植后监测的慢性排斥反应的读数。