Novel Lymphocyte Surface Markers for Diagnosing Children Predisposed to Rheumatic Fever

用于诊断易患风湿热儿童的新型淋巴细胞表面标志物

• 批准号: 9481797
• 负责人: Chad William Euler
• 金额: $ 15.6万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-08 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9481797
• 关键词: Acute; Acute Pharyngitis; Address; Affect; Affinity; Alleles; Allogenic; Antibiotic Therapy; Antibiotics; Antibodies; Antigen Targeting; Antigens; Autoantibodies; Autoimmune Diseases; Autoimmune Process; B lymphocyte immortalization; B-Lymphocytes; Binding; Biological Response Modifiers; Blood Cells; Cardiovascular system; Cell Culture Techniques; Cell Line; Cell surface; Cells; Cessation of life; Child; Child Mortality; Chronic; Clinical Trials; Complication; Developing Countries; Diagnosis; Diagnostic; Diagnostic Reagent; Diagnostic tests; Disease; Early Intervention; Engineering; Enrollment; Ensure; Epitopes; Ethnic group; Etiology; Fluorescence-Activated Cell Sorting; Future; Genes; Genetic; Genetic Markers; Geographic Locations; Health; Health Care Costs; Heart; Heart Diseases; Heart Valves; Human; Hybridomas; IgG1; Immune; Immune response; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin M; Immunoglobulin Variable Region; Immunoprecipitation; Individual; Infection; Inflammatory; Intervention; Lead; Link; Longevity; Lymphocyte; Mass Spectrum Analysis; Medical; Methods; Molecular Mimicry; Monoclonal Antibodies; Morbidity - disease rate; Mus; Myocardium; Operative Surgical Procedures; Participant; Pathogenesis; Pathology; Patients; Pharyngeal structure; Pharyngitis; Population; Population Group; Populations at Risk; Predisposition; Prevention; Proteomics; Publishing; RNA Sequences; Reagent; Recombinants; Recurrence; Regulation; Research; Research Personnel; Rheumatic Fever; Rheumatic Heart Disease; Risk Factors; Role; Spectrophotometry; Streptococcal Infections; Streptococcus pyogenes; Surface; Techniques; Testing; Tissues; Universities; Vaccines; Western Blotting; antibody engineering; burden of illness; cost; cross reactivity; cytokine; design; diagnostic biomarker; ethnic diversity; experimental study; gene product; global health; health economics; immunoreactivity; insight; mortality; novel; novel diagnostics; pathogen; patient subsets; premature; prospective; seropositive; tool; trait; transcriptome sequencing; young adult

ABSTRACT Acute rheumatic fever (ARF) and rheumatic heart disease are the leading cause of preventable cardiovascular morbidity and mortality in children worldwide. Acute rheumatic fever is a multisystem inflammatory autoimmune disease that occurs in a subset of patients as a delayed complication of an improperly or untreated throat infection with a rheumatogenic strain of S. pyogenes, Group A Streptococcus, (GAS). While the exact triggers and mechanisms of ARF have not been proven, studies indicate that aberrant immune responses develop against host tissues approximately 3 weeks after GAS infection. The most severe pathology of ARF takes place in the heart, where infiltrating immune cells, cytokines and auto-antibodies cause destruction of the heart muscle and permanent damage to the heart valves. While there is no current diagnostic kit available, a method of detecting children who are more susceptible to ARF (3-6% of the total population) would have global health and economic benefits. As such, over the last century investigators have searched for genes related to ARF susceptibility. As ARF is an autoimmune complication of infection, most the research has focused on genetic markers related to different components of the immune response and effects of various immune mediators, but no direct correlations could be made across ARF patients from different ethnic groups and geographical locations. As an alternative, D8/17, a murine monoclonal IgM antibody capable of binding an unknown allogenic epitope on B- cells from ARF patients, was found to bind B lymphocytes in 90-95% of ARF patients from diverse ethnic groups and different geographical locations compared to only 11% in matched controls. While many studies have successfully tested cell culture supernatants of the D8/17 antibody as a marker for rheumatic fever susceptibility across several different population groups, no group to date has identified the specific target antigen that the D8/17 antibody binds to on the surface of B cell lymphocytes. We plan on use cutting edge techniques involving antibody sequencing and recombinant engineering, to elucidate the sequence of the D8/17 antibody variable regions. We then produce new recombinantly expressed mouse IgM and IgG1 versions of the D8/17 antibody. We hypothesize that the IgG1 class- switched antibody will be a better reagent to identify the D8/17 antigen and more amenable for use in diagnostic testing. We will also compare the activity of these newly engineered antibodies and utilize them in fluorescence activated cell sorting (FACS) analysis, immunoprecipitation and mass spectrometry experiments to more specifically derive the D8/17 antigen from our previously immortalized B-cell lines isolated from rheumatic fever patients and controls. By utilizing the class switched D8/17 antibody, we hope to discover novel susceptibility markers in patients predisposed to acute rheumatic fever (ARF), allowing us to derive better diagnostic reagents and gain further insight into the role of the associated genetic factors in pathogenesis. This will be achieved though proteomic and expression analysis of immortalized B lymphocyte lines from rheumatic fever patients to identify the D8/17 marker. We hope that our findings will lead to an ARF specific-diagnostic test for susceptibility to this illness and further elucidate the mechanisms behind this treatable and preventable disease.

抽象的 急性风湿热（ARF）和风湿性心脏病是可预防心血管发病率的主要原因 和全世界儿童的死亡率。急性风湿热是一种多系统炎症自身免疫性疾病， 发生在患者的一部分中，是不适当或未经治疗的喉咙感染的延迟并发症 链球菌的风湿性菌株，A组链球菌，（气体）。而ARF的确切触发器和机制 尚未得到证明，研究表明，大约3周对宿主组织的异常免疫反应发展 气感染后。最严重的ARF病理发生在心脏中，浸润的免疫细胞， 细胞因子和自身抗体会导致心肌破坏和对心脏瓣膜的永久损害。尽管 目前没有可用的诊断套件，一种检测更容易受到ARF的儿童的方法（占3-6％的儿童 总人口将具有全球健康和经济利益。因此，在上个世纪，调查人员有 搜索与ARF敏感性有关的基因。由于ARF是感染的自身免疫并发症，因此大多数研究 已重点关注与免疫反应的不同成分相关的遗传标记和各种免疫的影响 调解人，但在不同种族和地理的ARF患者之间没有直接相关性 位置。作为替代方案，D8/17，一种鼠单克隆IgM抗体，能够结合未知的同种表位 在来自ARF患者的B细胞上，发现90-95％的ARF患者的B淋巴细胞结合了来自不同族裔的ARF患者 和不同的地理位置，而匹配的对​​照中只有11％。虽然许多研究已经成功 D8/17抗体的测试细胞培养上清液是几种风湿热敏感性的标记 不同的人群群体，迄今为止尚无人群确定D8/17抗体与ON结合的特定靶抗原 B细胞淋巴细胞的表面。我们计划使用涉及抗体测序和 重组工程，以阐明D8/17抗体变量区域的序列。然后我们生产新的 D8/17抗体的重组表达的小鼠IgM和IgG1版本。我们假设IgG1类 - 开关抗体将是一种更好的试剂，可以识别D8/17抗原，并且更适合用于诊断测试。 我们还将比较这些新设计的抗体的活性，并将其用于荧光激活细胞 分类（FACS）分析，免疫沉淀和质谱实验，以更具体地得出D8/17 从风湿热患者和对照组中分离出的先前永生的B细胞系的抗原。通过利用 类切换D8/17抗体，我们希望发现易感急性的患者的新型敏感性标记 风湿热（ARF），使我们能够获得更好的诊断试剂，并进一步了解 发病机理中的相关遗传因素。这将通过蛋白质组学和表达分析来实现 来自风湿热患者的永生B淋巴细胞系以鉴定D8/17标记。我们希望我们的发现 将导致ARF特异性诊断测试，以实现对这种疾病的敏感性，并进一步阐明背后的机制 这种可治疗且可预防的疾病。