VIRUS-MEDIATED MYELOSUPPRESSION

病毒介导的骨髓抑制

• 批准号: 3793584
• 负责人: BEVERLY TOROK-STORK
• 金额: --
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3793584
• 关键词: Parvoviridae; antibody formation; bioassay; bone marrow transplantation; cell type; colony stimulating factor; complementary DNA; cytokine; cytomegalovirus; diagnosis design /evaluation; disease /disorder model; enzyme linked immunosorbent assay; hematopoiesis; immunogenetics; immunosuppression; myelogenous leukemia; myeloid stem cell; polymerase chain reaction; radioimmunoassay; serotyping; stromal cells; tissue /cell culture; virus DNA; virus genetics; virus infection mechanism; virus protein

Our previous studies have shown that cytomegalovirus (CMV) can infect long- term marrow cultures (LTMC) and cause significant myelosuppression. The mechanism of suppression differed among the various virus isolates used in the study. In some cases direct infection of myeloid precursors could be documented. In other cases various stromal components were infected resulting in alterations of cytokine production. In this application we propose to further dissect the LTMC model, identify the cytokines produced by the major cellular components and determine which of these may be compromised by virus infection. This involves establishing LTMC from normal bone marrow, exposing it to virus, and then assessing the extent of myelosuppression by quantitating changes in the number of myeloid cells produced. Condition media harvested from the LTMC will be evaluated for cytokines using a combination of bioassays, RIA, and ELISA. Cytokine production by distinct cell types will be determined by immunomagnetic selection of cells followed by amplification of cytokine cDNA using polymerase chain reaction (pcr). Standard dual label immune cytochemistry on LTMC will associate cell type with the presence or absence of viral protein, and PCR amplification of viral DNA will identify the presence of viral genome. In order to extrapolate our in vitro LTMC observations to myelosuppression in vivo we will determine if infection of the same cell types occur in vivo. Immunomagnetic techniques will be used to isolate distinct cells from blood and bone marrow obtained from infected patients and isolated cell types will be evaluated for CMV genome by PCR amplification of viral DNA. The presence of CMV in distinct cells types will then be evaluated in relation to clinical outcome. Isolation and identification of cells containing CMV DNA will also be determined for marrow obtained from healthy normal CMV seropositive marrow donors with the eventual goal being to eliminate these cells from the marrow inoculum. The studies proposed in this project will provide an experimental model that should be applicable for studying the hematopoietic suppressive effect of any virus. Initially they will be extended to evaluate the effects of AAV and B19 parvoviruses should these be shown to infect patients posttransplant. Information obtained from this project in conjunction with project 9 should help us to prevent and/or treat viral infections that complicate hematopoietic recovery.

我们以前的研究表明，巨细胞病毒（CMV）可以感染长期 术语骨髓培养物（LTMC）并引起明显的骨髓抑制。 这 抑制机制在用于用于 研究。 在某些情况下，直接感染髓样前体可能是 记录。 在其他情况下，各种基质成分被感染 导致细胞因子产生的改变。 在此应用中，我们 建议进一步剖析LTMC模型，识别产生的细胞因子 通过主要的细胞成分，并确定其中哪一个可能是 因病毒感染而损害。 这涉及从 正常的骨髓，将其暴露于病毒，然后评估 通过定量髓样细胞数量的变化来骨髓抑制 生产。 将评估从LTMC收获的条件介质 使用生物测定，RIA和ELISA的组合的细胞因子。 细胞因子 通过不同的细胞类型的生产将由免疫磁性确定 选择细胞，然后使用细胞因子cDNA扩增 聚合酶链反应（PCR）。 标准双标签免疫细胞化学 在LTMC上，将使细胞类型与病毒的存在或不存在 蛋白质和病毒DNA的PCR扩增将确定存在 病毒基因组。 为了推断我们的体外LTMC观测值 骨髓抑制在体内，我们将确定同一细胞的感染是否感染 类型发生在体内。 免疫磁技术将用于分离 从感染患者获得的血液和骨髓的不同细胞 PCR将评估孤立的细胞类型的CMV基因组 病毒DNA的扩增。 不同细胞类型中CMV的存在 然后将对临床结果进行评估。 隔离和 还将确定识别含有CMV DNA的细胞的细胞 从健康的正常CMV血清阳性骨髓供体获得的骨髓与 最终的目标是从骨髓接种物中消除这些细胞。 这 该项目提出的研究将提供一个实验模型 应该适用于研究的造血抑制作用 任何病毒。 最初，它们将被扩展以评估AAV的影响 和B19细节性病毒应显示为感染患者 移植后。 从该项目获得的信息以及 项目9应该帮助我们预防和/或治疗病毒感染 复杂的造血恢复。