Rapid FISH Detection of EBV-infected Cells

EBV 感染细胞的快速 FISH 检测

• 批准号: 7660561
• 负责人: Raymond Preston Stowe
• 金额: $ 7.75万
• 依托单位: MICROGEN, LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7660561
• 关键词: Age; Aging; Anti-Sense Probes; Antigens; Applications Grants; Archives; B-Cell Lymphomas; B-Lymphocytes; Biological Assay; Blood; Burkitt Lymphoma; Cell Aging; Cell Line; Cell surface; Cells; Coupled; Data; Detection; Development; Epstein-Barr pathogenesis; Epstein-Barr virus encoded RNA 1; Epstein-Barr virus encoded RNA 2; Flow Cytometry; Fluorescence; Fluorescent in Situ Hybridization; Genetic Transcription; Healthcare Systems; Human Herpesvirus 4; Immunobiology; Immunologic Surveillance; Immunology; In Vitro; Individual; Infectious Mononucleosis; Knowledge; Label; Learning; Methanol; Methodology; Methods; Molecular Analysis; Nasopharynx Carcinoma; Phenotype; Plasma; Population; Proteins; RNA Sequences; Research; Reverse Transcriptase Polymerase Chain Reaction; Sorting - Cell Movement; Specimen; Structural Genes; Technology; Testing; Viral Load result; Viremia; Virus; aged; base; cell age; cost; design; immunosuppressed; in vivo; infected B cell; lymphoblastoid cell line; magnetic beads; novel; paraform; pathogen; peripheral blood; public health relevance; sample fixation; viral DNA

DESCRIPTION (provided by applicant): The objective of this grant application is to develop a new method to better characterize Epstein-Barr virus (EBV)-infected cells from aged subjects. EBV is an important pathogen and results in considerable cost to the health care system. EBV is the causative agent of infectious mononucleosis as well as nasopharyngeal carcinoma, Burkitt's lymphoma, and other B-cell lymphomas in immunosuppressed individuals. Although much has been learned about EBV from studies of in vitro transformed B-lymphoblastoid cell lines, critical gaps in knowledge regarding EBV pathogenesis in vivo remain to be answered, including how EBV persists in spite of the continuous immune surveillance. We have new data which shows that aged subjects have plasma viremia (EBV DNA). In addition, molecular analysis revealed frequent transcription of early and late structural genes in EBV-infected B-cells. Thus, we have an immediate need to develop assays to better characterize EBV-infected cells. We have encouraging preliminary data that demonstrates our ability to detect EBV-infected B cells at the single-cell level using fluorescent in situ hybridization (FISH) combined with flow cytometry. This application is not hypothesis driven per se but supports the development of a research technology that will be used to characterize EBV-infected B-cells in archived specimens from aged subjects that have high viral load as determined by PCR. The specific aims are: (1) Design and test multiple antisense probes (MAPs) that target EBERs; and (2) Application of MAPs to characterize EBV-infected B-cells from aged subjects. The successful completion of this project will yield a novel method to detect EBV-infected B cells in peripheral blood, and this methodology will be incorporated into a more comprehensive R01 application to investigate the immunobiology of EBV in aging. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus (EBV) is an important pathogen and results in considerable cost to the health care system. EBV is the causative agent of infectious mononucleosis as well as nasopharyngeal carcinoma, Burkitt's lymphoma, and other B-cell lymphomas in immunosuppressed individuals. Although much has been learned about EBV from studies of in vitro transformed B-lymphoblastoid cell lines, critical gaps in knowledge regarding EBV pathogenesis in vivo remain to be answered, including how EBV persists in spite of the continuous immune surveillance. We will develop a novel FISH-based assay to detect EBV-infected cells in peripheral blood at the single-cell level which can be used to better understand the immunology of EBV in aging.

描述（由申请人提供）：本赠款应用的目的是开发一种新方法，以更好地表征来自老年受试者的Epstein-Barr病毒（EBV）感染的细胞。 EBV是一个重要的病原体，导致医疗保健系统的成本巨大。 EBV是感染性单核细胞增多症以及鼻咽癌，伯基特淋巴瘤和其他B细胞淋巴瘤的病因。尽管从体外转化的B淋巴细胞细胞系的研究中了解了EBV，但有关体内EBV发病机理的知识的关键差距仍有待解决，包括EBV仍在持续的免疫监测，包括如何持续。我们有新数据表明，老年受试者患有血浆病毒血症（EBV DNA）。此外，分子分析表明，在EBV感染的B细胞中，早期和晚期结构基因的经常转录。因此，我们立即需要开发测定，以更好地表征感染EBV感染的细胞。我们具有令人鼓舞的初步数据，该数据证明了我们使用荧光原位杂交（FISH）结合流式细胞仪在单细胞水平上检测EBV感染的B细胞的能力。该应用本身不是假设驱动的，而是支持研究技术的发展，该技术将用于表征来自老年受试者的存档标本中EBV感染的B细胞，这些标本具有高病毒负荷，由PCR确定。具体目的是：（1）设计和测试针对Ebers的多个反义探针（地图）； （2）应用地图以表征来自老年受试者的EBV感染的B细胞。该项目的成功完成将产生一种新的方法来检测外周血中EBV感染的B细胞，并且该方法将纳入更全面的R01应用中，以研究EBV在衰老中的免疫生物学。公共卫生相关性：Epstein-Barr病毒（EBV）是重要的病原体，其造成了巨大的卫生保健系统。 EBV是感染性单核细胞增多症以及鼻咽癌，伯基特淋巴瘤和其他B细胞淋巴瘤的病因。尽管从体外转化的B淋巴细胞细胞系的研究中了解了EBV，但有关体内EBV发病机理的知识的关键差距仍有待解决，包括EBV仍在持续的免疫监测，包括如何持续。我们将开发一种新型的基于鱼类的测定法，以检测单细胞水平的外周血中的EBV感染细胞，该细胞可用于更好地了解衰老中EBV的免疫学。