Identifying Specific Antigenic Targets of Kawasaki Disease

项目摘要/摘要 川崎病(KD)是发达国家儿童获得性心脏病的主要原因。科威特第纳尔 可能导致冠状动脉动脉瘤，从而导致终生心脏病、心肌梗死和 死亡。临床和流行病学特征支持遗传易感儿童的感染病原学， 但50多年来，人们一直没有对这个原因进行研究。延误或漏诊会增加罹患癌症的风险。 冠状动脉动脉瘤。迫切需要的诊断测试和改进的治疗方法的发展 取决于确定病因。在初步研究中，我们分析了外周血浆母细胞 单细胞RT-PCR检测KD发热发病后1~3周的反应并制备60株单抗 (MAb)来自这些浆母细胞。我们用这些单抗来确定它们的靶抗原。我们发现32/60 来源于9/11 KD患者的单抗在纤毛虫中鉴定胞浆内病毒样包涵体(ICI) KD患儿的支气管上皮细胞，而不是婴幼儿对照组。使用多肽阵列鉴定结合表位(S)， 氨基酸替代阵列和ELISA，我们发现32个单抗中有5个(16%)来自3名KD患者 对于冠状动脉动脉瘤，识别KD多肽表位1。KD多肽1完全阻断结合 这些单抗到KD ICI，表明ICI中存在具有结合表位序列的蛋白质。 此外，5/8 KD患者在发烧后第8天的治疗前血清，但0/17婴儿对照血清有。 多肽表位的免疫球蛋白抗体。我们最近使用这种方法确定了另外两个表位 需要进一步研究。这些发现支持我们的假设，即至少有一部分KD病例是由 感染一种目前未知的、普遍存在的含有KD多肽表位1的病毒病原体 序列。在这个提议中，我们假设识别以浆母细胞反应为靶标的抗原 到KD可导致病原学的鉴定和KD血清学试验的发展。为了测试这些 假设，我们将：1)确定我们的KD单抗小组识别的与KD结合的特定蛋白质 组织，2)筛选由KD免疫反应识别的额外抗原靶点，以及3)评估 KD和对照血清对KD抗原的抗体反应。我们的发现将为KD的发病机制提供信息， 长期目标是改善KD的诊断和治疗，使之能够预防和减少 儿童时期出现的冠状动脉瘤的长期后果造成的医疗费用。

PROJECT SUMMARY/ABSTRACT Kawasaki Disease (KD) is the leading cause of acquired heart disease in children in developed nations. KD can result in coronary artery aneurysms that can lead to lifelong heart disease, myocardial infarction, and death. The clinical and epidemiologic features support an infectious etiology in genetically susceptible children, but the cause has eluded more than 50 years of study. Delayed and missed diagnoses increase the risk of coronary artery aneurysms. The development of urgently needed diagnostic tests and improved therapies are dependent upon identifying the etiology. In preliminary studies, we analyzed the peripheral blood plasmablast response at 1-3 weeks after KD fever onset using single cell RT-PCR and made 60 monoclonal antibodies (Mab) from these plasmablasts. We used these Mab to determine their target antigens. We found that 32/60 Mab, derived from 9/11 KD patients, identify intracytoplasmic virus-like inclusion bodies (ICI) in ciliated bronchial epithelium of KD children but not infant controls. Using peptide arrays to identify binding epitope(s), amino acid substitution arrays, and ELISA, we found that 5 of the 32 (16%) Mab, derived from 3 KD patients with coronary artery aneurysms, recognize KD peptide epitope 1. KD peptide 1 completely blocks binding of these Mab to KD ICI, indicating that a protein with the binding epitope sequence is present in the ICI. Moreover, pre-treatment sera from 5/8 KD patients >day 8 after fever onset but 0/17 infant control sera have IgG antibody to the peptide epitope. We recently identified two additional epitopes using this approach that require further study. These findings support our hypothesis that at least a subset of KD cases results from infection with a presently unidentified, ubiquitous viral causative agent containing the KD peptide epitope 1 sequence. In this proposal, we hypothesize that identification of antigens targeted by the plasmablast response to KD can lead to identification of the etiology(ies) and development of KD serologic tests. To test these hypotheses, we will: 1) identify the specific proteins recognized by our panel of KD Mab that bind to KD tissues, 2) screen for additional antigenic targets recognized by the immune response to KD, and 3) evaluate the antibody responses of KD and control sera to KD antigens. Our findings will inform pathogenesis of KD, with the long-term goals of improving diagnosis and treatment of KD, enabling prevention, and reducing healthcare costs from the long-term consequences of coronary artery aneurysms arising in young childhood.