Kobayashi Takaaki, Liu Dage, Ogawa Haruko, Miwa Yuko, Nagasaka Takaharu, Maruyama Shoichi, Li Yu-Teh, Onishi Akira, Iwamoto Masaki, Kuzuya Takafumi, Kadomatsu Kenji, Uchida Kazuharu, Nakao Akimasa
Department of Applied Immunology, Nagoya University School of Medicine, Nagoya, Japan.
Transpl Immunol. 2009 Jan;20(3):132-8. doi: 10.1016/j.trim.2008.09.007. Epub 2008 Oct 7.
ABO incompatibility in organ transplantation is still a high risk factor for antibody-mediated rejection, despite the progress in effective treatments. We have explored the possibility of using the enzyme to remove the blood type A/B antigen in organs.
Recombinant endo-beta-galactosidase (ABase), which releases A/B antigen, was produced in E. coli BL-21. Human A/B red blood cells (RBC) were digested with ABase, and subjected to flow cytometric analysis after incubation with human sera. Purified recombinant ABase was intravenously administered to a baboon. Biopsies were taken from kidney and liver before and 1, 4 and 24 h after in vivo administration. Excised baboon kidneys were perfused with cold UW solution+/-purified recombinant ABase and preserved at 4 degrees C. Biopsies were taken before and 1 and 4 h after ex vivo perfusion. The change in A/B antigen expression was analyzed by immunohistochemical study.
ABase removed 82% of A antigen and 95% of B antigen in human A/B red blood cells, and suppressed anti-A/B antibody binding and complement activation effectively. ABase was also found to remain active at 4 degrees C. In vivo infusion of ABase into a blood type A baboon demonstrated a marked reduction of A antigen expression in the glomeruli of kidney (85% at 1 h, 9% at 4 h and 13% at 24 h) and the sinusoids of liver (47% at 1 h, 1% at 4 h and 3% at 24 h) without serious adverse effects. After ex vivo perfusion and cold storage of excised baboon kidney (blood type B) with ABase, the expression levels of B antigen in glomeruli were reduced to 49% at 1 h and 6% at 4 h.
This alternative approach might be useful for minimizing antibody removal and anti-B cell immunosuppression as an adjuvant therapy in ABO-incompatible kidney, liver and possibly heart transplantation.
尽管在有效治疗方面取得了进展，但器官移植中的ABO血型不相容性仍然是抗体介导排斥反应的高风险因素。我们探索了使用酶去除器官中A/B血型抗原的可能性。
在大肠杆菌BL-21中生产释放A/B抗原的重组内切β-半乳糖苷酶（ABase）。用ABase消化人A/B红细胞（RBC），与人血清孵育后进行流式细胞术分析。将纯化的重组ABase静脉注射给一只狒狒。在体内给药前以及给药后1、4和24小时，从肾脏和肝脏获取活检组织。用冷UW溶液±纯化的重组ABase灌注切除的狒狒肾脏，并在4℃下保存。在体外灌注前以及灌注后1和4小时获取活检组织。通过免疫组织化学研究分析A/B抗原表达的变化。
ABase去除了人A/B红细胞中82%的A抗原和95%的B抗原，并有效抑制了抗A/B抗体结合和补体激活。还发现ABase在4℃时仍保持活性。将ABase体内注入一只A型狒狒体内，结果显示肾脏肾小球中A抗原表达显著降低（1小时时为85%，4小时时为9%，24小时时为13%），肝脏血窦中A抗原表达也显著降低（1小时时为47%，4小时时为1%，24小时时为3%），且无严重不良反应。用ABase对切除的狒狒肾脏（B型）进行体外灌注和冷藏后，肾小球中B抗原的表达水平在1小时时降至49%，4小时时降至6%。
作为ABO血型不相容的肾脏、肝脏以及可能的心脏移植的辅助治疗，这种替代方法可能有助于减少抗体清除和抗B细胞免疫抑制。
