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）：本拨款申请的目的是开发一种新的方法来更好地表征老年受试者的eb病毒感染细胞。EBV是一种重要的病原体，给卫生保健系统带来了相当大的成本。EBV是传染性单核细胞增多症以及鼻咽癌、伯基特淋巴瘤和其他免疫抑制个体b细胞淋巴瘤的病原体。尽管从体外转化的b淋巴母细胞样细胞系的研究中已经了解了很多关于EBV的知识，但关于EBV体内发病机制的关键空白仍有待回答，包括EBV如何在持续免疫监视下持续存在。我们有新的数据显示老年受试者有血浆病毒血症（EBV DNA）。此外，分子分析显示ebv感染的b细胞中早期和晚期结构基因的频繁转录。因此，我们迫切需要开发更好地表征ebv感染细胞的检测方法。我们有令人鼓舞的初步数据，证明我们能够使用荧光原位杂交（FISH）结合流式细胞术在单细胞水平检测ebv感染的B细胞。这一应用本身并不是假设驱动的，而是支持了一种研究技术的发展，该技术将用于表征由PCR确定的高病毒载量的老年受试者存档标本中ebv感染的b细胞。具体目标是：(1)设计和测试针对EBERs的多个反义探针（MAPs）；(2)应用map表征老年受试者ebv感染b细胞。本课题的成功完成将提供一种检测外周血中EBV感染B细胞的新方法，并将该方法纳入更全面的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：eb病毒（EBV）是一种重要的病原体，对卫生保健系统造成了相当大的损失。EBV是传染性单核细胞增多症以及鼻咽癌、伯基特淋巴瘤和其他免疫抑制个体b细胞淋巴瘤的病原体。尽管从体外转化的b淋巴母细胞样细胞系的研究中已经了解了很多关于EBV的知识，但关于EBV体内发病机制的关键空白仍有待回答，包括EBV如何在持续免疫监视下持续存在。我们将开发一种新的基于fish的检测外周血中EBV感染细胞的单细胞水平的方法，这可以更好地用于了解EBV在衰老中的免疫学作用。