TREATMENT STRATEGIES FOR THE ANTI PHOSPHOLIPID SYNDROME

抗磷脂综合征(APS)，又称休斯综合征，是一种与心肌梗死、卒中血栓形成和复发性胎儿丢失相关的多器官血管疾病。在50岁以下的中风患者中，45%的人也表现出磷脂抗体水平升高。我们的假设是，循环中存在抗磷脂抗体会导致血管紊乱，不断升级的组织损伤可以通过结合破坏性抗体的试剂来减轻。为了验证这一假设，有必要创造一种基因定义的APS小鼠，它能将抗磷脂抗体分泌到血液中。我们已经制备并鉴定了一种能特异性识别磷脂酰丝氨酸的鼠单抗。初步数据表明，我们的单抗结合特异性类似于APS患者中存在的抗体；因此，这种单抗适合开发一种治疗模式。我们的单抗抗磷脂抗体的重链和轻链将通过RT-PCR进行克隆。这些cDNA将包括内源性分泌肽信号序列。Biggen小鼠将被用来直接在肝脏中诱导配体的表达。每个单抗免疫球蛋白链将被不同的HA或FLAG表位标记。转基因动物将被表征并与正常动物比较APS的标志性症状，包括体外凝血时间延长、反复发生的胎儿丧失和血管疾病。这项提案的另一个目标是制定APS的治疗策略。我们的方法是中和致病抗体。在体外结合和阻断单抗磷脂抗体的多肽可以从随机多肽的组合噬菌体展示文库中选择。分离的多肽将被评估是否有能力减轻这只转基因小鼠表现出的APS症状。这项研究将产生一种遗传定义的APS小鼠模型，这将被证明对开发治疗策略以改善与APS相关的症状是有用的。

Antiphospholipid Syndrome (APS), also known as Hughes Syndrome, is a multiorgan vascular disease associated with myocardial infarction, stroke thrombosis and recurrent fetal loss. Forty- five percent of the people under the age of fifty who suffer strokes also demonstrate elevated levels of phospholipid antibody. Our hypothesis is that the presence of antiphospholipid antibodies in the circulation leads to vascular disorders, and that the escalating tissue damage can be abated with agents that bind the destructive antibodies. In order to test this hypothesis, it is necessary to create a genetically defined APS mouse that secretes antiphospholipid antibodies into the blood. We have produced and characterized a mouse monoclonal antibody which specifically recognizes phosphatidylserine. Preliminary data demonstrates that our monoclonal antibody binding specificity is similar to antibodies present in APS patients; this monoclonal antibody is therefore appropriate to develop a treatment model. The heavy and light chain of our monoclonal antiphospholipid antibody will be cloned by RT-PCR. These cDNAs will include the endogenous secretory peptide signal sequence. Bigenic mice will be used to direct ligand induced expression in the liver. Each monoclonal immunoglobulin chain will be differentially tagged with HA or FLAG epitopes. The transgenic animals will be characterized and compared with normal animals for the hallmark symptoms of APS, including prolonged in vitro coagulation times, recurrent fetal loss and vascular disease. An additional goal of this proposal is to develop treatment strategies for APS. Our approach is to neutralize the disease-causing antibodies. Peptides which bind and block the monoclonal antiphospholipid antibody in vitro can be selected from a combinatorial phage-display library of random peptides. Isolated peptides will be assessed for the ability to reduce the symptoms of APS displayed by this transgenic mouse. This study will produce a genetically-defined mouse model of APS which will prove useful for developing treatment strategies to ameliorate the symptoms associated with APS.