Apoptotic Cell Clearance and Resulting Cytokine Secretion in Pediatric SLE

儿童 SLE 中的凋亡细胞清除和由此产生的细胞因子分泌

• 批准号: 7480334
• 负责人: SANGEETA DILEEP SULE
• 金额: $ 2.83万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7480334
• 关键词: Address; Adult; Affect; Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Antigens; Apoptosis; Apoptotic; Area; Arthritis; Attention; Autoimmunity; Award; Biological Assay; Blood; Blood Volume; Cell Death; Cells; Child; Childhood; Complement 1q; Data; Defect; Development; Disease; Enzyme-Linked Immunosorbent Assay; Flare; Flow Cytometry; Foundations; Funding; Genes; Genetic; Genomics; Genotype; Goals; Human; Inflammatory; Interleukin-1; Interleukin-10; Interleukin-12; Investigation; Lead; Logistics; Lupus; Mus; Mutation; Pathogenesis; Pathway interactions; Patients; Pattern; Phagocytes; Phagocytosis; Physicians; Pilot Projects; Population; Positioning Attribute; Prevalence; Process; Research Personnel; Rheumatism; Rheumatology; Role; Sampling; Scientist; Systemic Lupus Erythematosus; Techniques; Thinking; Tissues; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Underserved Population; United States; career; cytokine; design; experience; human TNF protein; monocyte; mouse model; response; uptake

The primary objective of this pilot project is to establish a small blood volume assay that can be used in pediatric SLE patients, a critically underserved population. We will establish and validate quantitative assays of apoptotic cell clearance and cytokine secretion in response to apoptotic cell phagocytosis using small blood volumes, by using flow cytometry to quantify clearance, and multiplex cytokine assays to quantify secretion of several cytokines in very small volumes of supernatant. These assays will then be applied in a pilot way to a population of pediatric patients with SLE. Apoptotic cells, a potentially important antigen in SLE, are normally cleared by phagocytes in an antiinflammatory, tolerance-inducing way. Dysregulation of this process is thought to lead to systemic autoimmunity. We propose that during active disease, monocytes from SLE patients have diminished antiinflammatory (TGF- beta and IL-10) and increased pro-inflammatory (TNF-alpha, IL-1, IFN-a, IL-12) cytokine secretion in response to apoptotic cells, which contributes to SLE propagation, disease flares, and tissue damage. The relevance of such pathways in pediatric SLE is unknown. We have shown that adult SLE patients have strikingly decreased anti-inflammatory cytokine secretion in response to apoptotic cells compared to normal controls. However, this defect in TGF-beta secretion did not reflect a defect in uptake, indicating that the SLE monocytes are capable of phagocytosing the apoptotic cells. The relatively large blood volumes required for these assays present a significant challenge to directly addressing these pathways in children. However, with the small blood volume assays and multiplex ELISA proposed in this pilot project, 16 cytokines can be assayed from a single 250 mu l supernatant sample (an 8- fold reduction in supernatant volume). Thus, we should be able to generate the necessary supernatant volume with only 2 cc of blood, making this assay accessible to the pediatric SLE population. These techniques may have broad applicability for Dr. Sule, as she begins to grow her career in pediatric rheumatology. They may also enable studies in the pediatric population with rheumatic diseases which are currently not pursued due to logistic issues and difficulties with scaling down sample volumes.

这个试点项目的主要目标是建立一种小容量血液分析，可以用于 儿童系统性红斑狼疮患者，服务严重不足的人群。我们将建立和验证定量分析。 小分子细胞吞噬作用对细胞凋亡清除和细胞因子分泌的影响 血容量，通过使用流式细胞术来量化清除率，并通过多重细胞因子分析来量化 在极少量的上清液中分泌几种细胞因子。然后，这些分析将被应用于 向儿童SLE患者人群试点的方法。 凋亡细胞是系统性红斑狼疮潜在的重要抗原，通常由吞噬细胞在抗炎、 耐受性诱导方式。这一过程的失调被认为会导致系统性 自身免疫力。我们认为，在疾病活动期，SLE患者的单核细胞抗炎作用减弱。 (转化生长因子-β和IL-10)和增加的促炎细胞因子(肿瘤坏死因子-α、IL-1、干扰素-α、IL-12) 对凋亡细胞作出反应的分泌，这有助于系统性红斑狼疮的传播、疾病发作和组织 损坏。这些通路在儿童系统性红斑狼疮中的相关性尚不清楚。我们已经证明成人系统性红斑狼疮 患者对凋亡细胞的反应显著减少了抗炎细胞因子的分泌 与正常对照组相比。然而，这种转化生长因子-β分泌的缺陷并不反映摄取的缺陷， 提示SLE单核细胞具有吞噬凋亡细胞的能力。 这些检测所需的相对较大的血量对直接 在儿童中解决这些途径。然而，随着小血容量检测和多重酶联免疫吸附试验 在这一试点项目中，可以从单个250亩L的培养上清液中检测到16种细胞因子(8份)。 上清液体积的折叠减少)。因此，我们应该能够产生必要的上清液 体积只有2毫升的血液，使这种检测方法对儿童系统性红斑狼疮患者来说是可行的。 这些技术可能对Sule医生有广泛的适用性，因为她开始在儿科发展自己的职业生涯 风湿病。它们还可以在患有风湿病的儿科人群中进行研究，目前由于后勤问题和缩小样本量的困难而没有进行研究。