The primary objective of this pilot project is to establish a small blood volume assay that can be used inpediatric SLE patients, a critically underserved population. We will establish and validate quantitative assaysof apoptotic cell clearance and cytokine secretion in response to apoptotic cell phagocytosis using smallblood volumes, by using flow cytometry to quantify clearance, and multiplex cytokine assays to quantifysecretion of several cytokines in very small volumes of supernatant. These assays will then be applied in apilot 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 systemicautoimmunity. 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) cytokinesecretion in response to apoptotic cells, which contributes to SLE propagation, disease flares, and tissuedamage. The relevance of such pathways in pediatric SLE is unknown. We have shown that adult SLEpatients have strikingly decreased anti-inflammatory cytokine secretion in response to apoptotic cellscompared 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 directlyaddressing these pathways in children. However, with the small blood volume assays and multiplex ELISAproposed 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 supernatantvolume 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 pediatricrheumatology. 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.
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