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.

摘要 急性风湿热（ARF）和风湿性心脏病是可预防的心血管疾病的主要原因 和儿童死亡率。急性风湿热是一种多系统炎性自身免疫性疾病， 发生在一个子集的患者作为一个不适当的或未经治疗的咽喉感染的延迟并发症， 致流变性菌株S.化脓性链球菌、A组链球菌（GAS）。虽然急性肾衰的确切诱因和机制 尚未得到证实，研究表明，针对宿主组织的异常免疫应答大约在3周后发生， GAS感染后ARF最严重的病理发生在心脏，浸润的免疫细胞， 细胞因子和自身抗体导致心肌的破坏和心脏瓣膜的永久性损伤。而 目前没有可用的诊断试剂盒，这是一种检测更易患ARF的儿童的方法（3-6%的 总人口）将产生全球健康和经济效益。因此，在上个世纪， 寻找与ARF易感性相关的基因。由于ARF是感染的自身免疫并发症，大多数研究 一直专注于与免疫反应的不同组成部分和各种免疫反应的影响相关的遗传标记。 介质，但在不同种族和地理的ARF患者中没有直接相关性 地点作为替代方案，D8/17，能够结合未知同种异体表位的鼠单克隆IgM抗体 ARF患者的B细胞上，发现在90-95%来自不同种族的ARF患者中结合B淋巴细胞 和不同的地理位置相比，只有11%的匹配对照。虽然许多研究成功地 测试了D8/17抗体的细胞培养上清液作为风湿热易感性的标志物， 由于不同的人群群体，迄今为止还没有一个群体鉴定出D8/17抗体结合的特异性靶抗原， B细胞淋巴细胞的表面。我们计划使用包括抗体测序在内的尖端技术， 重组工程，以阐明D8/17抗体可变区的序列。然后我们生产新的 重组表达的D8/17抗体的小鼠IgM和IgG 1形式。我们假设IgG 1类- 转换抗体将是鉴定D8/17抗原的更好的试剂，并且更适合用于诊断测试。 我们还将比较这些新工程抗体的活性，并将其用于荧光激活的细胞， 分选（FACS）分析、免疫沉淀和质谱分析实验，以更特异性地获得D8/17 抗原来自我们以前从风湿热患者和对照中分离的永生化B细胞系。利用 类转换的D8/17抗体，我们希望发现新的易感性标志物，在患者易患急性 风湿热（ARF），使我们能够获得更好的诊断试剂，并进一步了解的作用， 发病机制中的相关遗传因素。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化B淋巴细胞系以鉴定D8/17标记物。我们希望我们的发现 将导致ARF特异性诊断测试对这种疾病的易感性，并进一步阐明背后的机制， 这种疾病是可以治疗和预防的。