Characterization of B Lymphocyte Deficiency in Pediatric Sickle Cell Disease

项目摘要 感染是镰状细胞病（SCD）的常见并发症，发生在高达45%的患者中。在非洲， 其中高达90%的患有SCD的儿童在5岁之前死亡，这些死亡中的许多人归因于感染。在 SCD，脾脏在婴儿期受损，导致边缘区B细胞缺乏。及其临床意义 B细胞缺乏症的发病机制尚不完全清楚，需要研究疾病内在因素和 B细胞亚群的外源性修饰剂。在这项研究中，我将量化儿童的B细胞亚群， 在德克萨斯州和坦桑尼亚，研究B细胞缺乏的机制，并将其与 重要的临床终点。 目的1：研究SCD患者MZB缺乏的机制。假设：存在差异， 来自SCD和非SCD受试者的B细胞由于B细胞内在因子的发育、存活和功能。 B细胞群的差异可能是由于分化和增殖动力学的差异和/或 生存为了检查分化和增殖，我将测量细胞的分化效率。 使用体外B转化系统，将SCD和非SCD供体的过渡B细胞转化为MZB，并将MZB转化为浆细胞 细胞共培养系统。为了检测存活率，我将测量细胞凋亡率和 在培养物中的天然和培养物衍生的MZB中的细胞凋亡（例如膜联蛋白V、半胱天冬酶3/9、BCL 2）。 目的2：探讨SCD时脾脏微环境对B细胞的影响。假设： 外源性因素如SCD中的炎性环境损害B细胞发育、存活， 功能为了检测微环境对培养物中B细胞的影响，我将在培养液中培养非SCD B细胞。 补充SCD血清或脾提取物的培养基。研究浸提液的哪些成分会影响 B细胞，我将使用多重ELISA开发提取物和血清的细胞因子谱。 目的3：确定低MZB是否与感染和炎症并发症有关。 SCD。假设：SCD中危及生命的感染在重度MZB儿童中更常见 缺陷为了确定嗜B细胞病毒是否对MZB数量具有混杂效应，我将 使用定制的多重ELISA板测量对EBV和疟疾的免疫应答。以确定是否 感染性并发症与SCD中更严重的MZB缺乏相关，我将比较MZB与 有或无并发症的儿童，如急性胸部、骨髓炎或菌血症。 尽管存在共同的驱动突变，但SCD的临床表型高度可变。总之，这方面的研究 一项提案将克服对感染和炎症并发症患者进行风险分层的关键障碍 的SCD。对SCD如何催化B细胞缺陷的深入了解可能有助于确定新的治疗方法 靶点或生物标志物，以减轻感染对SCD的影响。我的主要假设是B细胞 异常导致SCD儿童的发病率和死亡率。

PROJECT SUMMARY Infections are frequent complications in sickle cell disease (SCD), occurring in up to 45% of patients. In Africa, where up to 90% of children with SCD die by age 5 years, many of these deaths are attributed to infection. In SCD, the spleen is damaged in infancy and results in marginal zone B cell deficiency. The clinical implications of B cell deficiency in SCD are not fully understood and requires investigating both disease-intrinsic and - extrinsic modifiers of B cell subsets. In this investigation, I will quantify B cell subsets in children with and without SCD in Texas and in Tanzania, investigate mechanisms of B cell deficiency, and compare them to important clinical endpoints. Aim 1: Characterize mechanisms of MZB deficiency in SCD. Hypotheses: There is a difference in the development, survival, and function of B cells from SCD and non-SCD subjects due to B-cell intrinsic factors. Differences in B cell populations may be due to differences in kinetics of differentiation and proliferation and/or survival. To examine differentiation and proliferation, I will measure the efficiency of differentiation of transitional B cells from SCD and non-SCD donors into MZBs, and MZBs into plasma cells using an in vitro B cell co-culture system. To examine survival, I will measure the rate of apoptosis and expression of markers of apoptosis (e.g. annexin V, caspase 3/9, BCL2) in native and culture-derived MZBs in culture. Aim 2: Determine the effect of the microenvironment of the spleen in SCD on B cells. Hypothesis: Extrinsic factors such as the inflammatory environment in SCD impair B cell development, survival, and function. To examine the effect of the microenvironment on B cells in culture, I will culture non-SCD B cells in media supplemented with SCD serum or spleen extract. To investigate which components of the extract impact B cells, I will develop a cytokine profile for the extract and serum using multiplex ELISA. Aim 3: Determine whether low MZB is associated with infectious and inflammatory complications of SCD. Hypothesis: Life-threatening infections in SCD are more common among children with severe MZB deficiency. To determine whether B-cell tropic viruses have a confounding effect on MZB number, I will measure immune response to EBV and malaria using a custom multiplex ELISA panel. To determine whether infectious complications are associated with more severe MZB deficiency in SCD, I will compare MZB among children with and without complications such as acute chest, osteomyelitis, or bacteremia. Despite a shared driver mutation, the clinical phenotype in SCD is highly variable. Together, the studies in this proposal will overcome critical barriers to risk-stratifying patients for infectious and inflammatory complications of SCD. Increased insights into how SCD catalyzes B cell deficiency may help identify novel therapeutic targets or biomarkers to mitigate the impact of infections on SCD. My central hypothesis is that B cell abnormalities contribute to morbidity and mortality in children with SCD.