Development of synthetic immunotoxin that can enable ABC incompatible transplantation and xenotransplantation.

建立一种持久消除B细胞对移植相关碳水化合物的反应的新方法：我们已经证明，携带识别A血型碳水化合物的受体的B细胞仅存在于O型或B型血型人外周血中的SIGM^+CD11b^+CD5^+B-1亚群。一些报道表明，B-1细胞的分离可能是通过胸腺非依赖性2型抗原刺激下的Ig基因重排而发生的。我们已经证明，环孢菌素A(CsA)和他克莫司(Tac)可阻断这种对B-1细胞的分离，尽管CsA对抗体产生细胞或分化的B-1细胞没有影响。基于这些事实，我们推测，在ABO血型不合的器官移植中，特异性地消除具有抗A/B特异性的产生抗体的细胞和分化的B-1细胞，以及随后的CsA/Tac治疗，可能会导致抗-A/B抗体产生的长期抑制。BALB/c小鼠与带有…的人类相似更多的O型或B型血，因为小鼠的血清中有针对人类A型碳水化合物的天然抗体。在小鼠中，识别这些A碳水化合物的B细胞受体也属于CD5^+CD11b^+B-1亚群。本文研究了人工合成的A类碳水化合物(GalNAcα1-3Fucα1-2Gal)与牛血清白蛋白和兔抗牛血清白蛋白抗体偶联后体外清除抗-A免疫球蛋白M产生细胞的能力。Balb/c小鼠先注射A-BSA，6h后再注射抗BSA抗体。注射A-BSA/抗-BSA单抗的小鼠血清抗-A-IgM水平在第14天下降，而人A-RBC在第14天免疫后血清抗-A单抗水平升高。相反，当联合CsA治疗和A-BSA/抗-BSA抗体治疗时，即使在免疫后，小鼠血清中仍检测不到抗-A抗体水平。这些结果与A-BSA/抗-BSA单抗治疗暂时耗尽对A决定簇有反应的B细胞以及CsA/Tac治疗阻止这些细胞的补充的假设是一致的。B细胞对N-羟基神经氨酸反应模型的建立：在人类体内，针对猪血管内皮细胞上N-羟基神经氨酸(NeuGc)表位的天然抗体的存在可能会为异种移植提供另一个障碍，因为抗体-抗原结合会导致排斥反应。到目前为止，还没有动物模型来分析B细胞对NeuGc的反应。我们利用NeuGc缺陷小鼠建立了体内模型。较少

The establishment of a novel method for persistent elimination of B cells responding to transplantation associated carbohydrates : We have demonstrated that B cells bearing receptors that recognize blood group A carbohydrates are found exclusively in the sIgM^+ CD11b^+ CD5^+ B-1 subset in the peripheral blood of humans with blood group O or B. Some reports suggest that the segregation to B-1 cells probably occurs via Ig gene rearrangement on stimulation with thymus-independent type 2 antigens. We have proved that cyclosporin A (CsA) and Tacrolimus (Tac) blocks such segregation to B-1 cells, although CsA has no effect on Ab-producing cells or differentiated B-1 cells. Based on these facts, we hypothesize that the specific elimination of both Ab-producing cells and differentiated B-1 cells with anti-A/B specificity and subsequent CsA/Tac therapy might lead to the long lasting inhibition of anti-A/B Ab production in ABO-incompatible organ transplantation. Balb/c mice resemble humans with … More blood group O or B in that the mice have natural Abs against human blood group A carbohydrates in their sera. B cells bearing receptors that recognize these A carbohydrates in mice also belong to the CD5^+ CD11b^+ B-1 subset. The ability of synthetic A carbohydrates (GalNAcα1-3Fucα1-2Gal) conjugated with BSA and rabbit anti-BSA Abs to deplete anti-A IgM-producing cells was studied in vitro. The Balb/c mice were first injected with A-BSA and then with anti-BSA Abs after 6 hours. In mice that received the injection of A-BSA/anti-BSA Abs, the serum levels of anti-A IgM reduced by day 14. However, immunization with human A RBCs on day 14 resulted in an increase in the serum levels of anti-A Abs. In contrast, when combined with the CsA treatment and treatment with A-BSA/anti-BSA Abs, the serum levels of anti-A Abs were persistently undetectable in the mice even after the immunization. These results are consistent with the hypotheses that treatment with A-BSA/anti-BSA Abs temporally depletes B cells responding to A determinants, and that CsA/Tac treatment prevents the replenishment of these cells. The establishment of a model for analyses of B cells responding to N-glycolylneuraminic acid : The presence in humans of natural antibodies directed against N-glycolylneuraminic acid (NeuGc) epitopes on pig vascular endothelium may provides another barrier in xenotransplantation, as antibody-antigen binding leads to rejection. There has been no animal model for analyses of B cells responding to NeuGcso far. We have established a in vivo model utilizing NeuGc-deficient mice. Less