Apoptotic Cell Clearance and Resulting Cytokine Secretion in Pediatric SLE

该试验项目的主要目标是建立一种小血容量测定法， 儿童SLE患者，一个严重缺医少药的人群。我们将建立和验证定量分析 凋亡细胞清除和细胞因子分泌的反应凋亡细胞吞噬使用小 血容量，通过使用流式细胞术定量清除率，和多重细胞因子测定定量 在非常小体积的上清液中分泌几种细胞因子。然后将这些测定应用于 对SLE儿童患者进行试验。 凋亡细胞是SLE中一种潜在的重要抗原，通常被SLE中的吞噬细胞清除， 宽容的方式。这一过程的失调被认为会导致系统性 自身免疫我们认为，在活动性疾病期间，SLE患者的单核细胞减少， (TGF-β和IL-10）和促炎性（TNF-α、IL-1、IFN-α、IL-12）细胞因子增加 响应凋亡细胞的分泌，这有助于SLE传播、疾病发作和组织 损害这些通路在儿童SLE中的相关性尚不清楚。我们已经证明成人SLE 患者对凋亡细胞的抗炎细胞因子分泌显著减少 与正常对照相比。然而，TGF-β分泌的这种缺陷并不反映摄取的缺陷， 表明SLE单核细胞能够吞噬凋亡细胞。 这些测定所需的相对较大的血液体积对直接测定血液中的微量元素提出了重大挑战。 在儿童中解决这些途径。然而，随着小血容量测定和多重ELISA， 在该试验项目中提出，可以从单个250 μ l上清液样品中测定16种细胞因子（8- 上清液体积的倍数减少）。因此，我们应该能够产生必要的上清液 体积，仅2 cc血液，使得该测定可用于儿科SLE人群。 这些技术可能对Sule博士有广泛的适用性，因为她开始在儿科领域发展她的职业生涯。 风湿病学它们还可以在患有风湿性疾病的儿科人群中进行研究，由于后勤问题和缩小样本量的困难，目前尚未进行这些研究。

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.