Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock-out pigs.
Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock-out pigs.
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DOI：
10.1111/j.1399-3089.2012.00705.x
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发表时间：
2012-05
影响因子：
3.9
通讯作者：
Seebach JD
中科院分区：
文献类型：
--
作者：
Puga Yung GL;Li Y;Borsig L;Millard AL;Karpova MB;Zhou D;Seebach JD
抗galα 1,3 galβ - r天然抗体是猪-灵长类异种移植超急性排斥反应的原因。虽然缺乏α1,3半乳糖转移酶（GalT）的猪已经克服了超急性排斥反应，但抗体介导的排斥反应仍然是一个问题。有可能是其他酶合成了类似于galα 1,3gal表位的抗原，这些抗原可以被异种反应性抗体识别。糖鞘脂异血红蛋白三己糖神经酰胺（iGb3）代表了这样一种表达另一种gal α1,3 gal表位的候选者。本研究采用几种不同的高灵敏度方法确定了缺乏GalT的浸泡猪是否完全缺失末端galα 1,3 gal二糖。采用流式细胞术和荧光显微镜检测抗体和凝集素的表达；采用酶促法检测GalT活性；采用离子阱质谱法对主动脉内皮中性细胞膜的糖结构进行检测。最后，采用RT-PCR方法检测猪胸腺、脾脏、淋巴结、肾脏、肺和肝组织中iGb3合成酶mRNA的表达。GalT敲除猪的主动脉内皮细胞表面既不表达galα 1,3gal，也不表达iGb3， GalT酶活性也不存在。凝集素染色显示，与野生型猪主动脉内皮细胞（PAEC）相比，GalT敲除细胞中的血型h型糖结构增加。质谱分析未发现GalT敲除PAEC的膜中存在galα 1,3 gal；iGb3也完全不存在，而iGb3的集中形式在两种猪主动脉内皮细胞提取物中都检测到低水平。同种血红蛋白三己糖神经酰胺3合成酶mRNA在所有猪组织中均有表达，无论是来自野生型还是GalT敲除动物。这些结果明确证实了GalT敲除内皮细胞中末端galα 1,3 gal二糖的缺失。未来的工作将集中于异种移植物排斥反应的其他机制，特别是非半乳糖α 1,3gal抗体和细胞反应。
Anti-Galα1,3Galβ-R natural antibodies are responsible for hyperacute rejection in pig-to-primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody-mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb3) represents such a candidate expressing an alternative Galα1,3Gal epitope. The present work determined whether the terminal Galα1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods. The expression of Galα1,3Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally, the presence of iGb3 synthase mRNA was tested by RT-PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples. Aortic endothelial cells derived from GalT knockout pigs expressed neither Galα1,3Gal nor iGb3 on their surface, and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H-type sugar structures present in GalT knockout cells as compared to wild-type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Galα1,3Gal in membranes of GalT knockout PAEC; iGb3 was also totally absent, whereas a fucosylated form of iGb3 was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild-type or GalT knockout animals. These results confirm unequivocally the absence of terminal Galα1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non-Galα1,3Gal antibodies and cellular responses.
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影响因子：
4.4
作者：
Li Y;Thapa P;Hawke D;Kondo Y;Furukawa K;Furukawa K;Hsu FF;Adlercreutz D;Weadge J;Palcic MM;Wang PG;Levery SB;Zhou D
通讯作者：
Zhou D
影响因子：
4.4
作者：
Baumann, BC;Forte, P;Seebach, JD
通讯作者：
Seebach, JD
影响因子：
6.2
作者：
Baumann, BC;Schneider, MKJ;Seebach, JD
通讯作者：
Seebach, JD
DOI：
10.1111/j.1600-6143.2008.02337.x
发表时间：
2008-09
期刊：
American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons
影响因子：
--
作者：
Kiernan K;Harnden I;Gunthart M;Gregory C;Meisner J;Kearns-Jonker M
通讯作者：
Kearns-Jonker M
影响因子：
3.1
作者：
Hara, Hidetaka;Long, Cassandra;Cooper, David K. C.
通讯作者：
Cooper, David K. C.