Characterization of B Lymphocyte Deficiency in Pediatric Sickle Cell Disease

项目总结 感染是镰状细胞病(SCD)的常见并发症，高达45%的患者会发生感染。在非洲， 高达90%的SCD儿童在5岁前死亡，其中许多死亡归因于感染。在……里面 SCD，脾在婴儿期就受到损害，导致边缘B区细胞缺乏。其临床意义 B细胞缺陷在SCD中的作用尚不完全清楚，需要研究疾病固有的和- B细胞亚群的外在修饰物。在这项研究中，我将量化患有AND儿童的B细胞亚群 在德克萨斯州和坦桑尼亚没有SCD的情况下，研究B细胞缺陷的机制，并将它们与 重要的临床终点。 目的1：探讨MZB缺乏症的发病机制。假设：两者之间存在差异 SCD患者和非SCD患者B细胞的发育、存活和功能受B细胞内在因素的影响。 B细胞群体的差异可能是由于分化和增殖动力学和/或 生死存亡。为了研究分化和增殖，我将测量分化的效率 从SCD和非SCD供者到MZB的过渡B细胞，以及MZB到浆细胞的体外B细胞 细胞共培养体系。为了检查存活率，我将测量凋亡率和标记物的表达 细胞凋亡(如Annexin V、caspase 3/9、bcl2)在培养的原生和培养的MZB中。 目的2：探讨慢性萎缩性胃炎脾微环境对B细胞的影响。假设： 外在因素，如SCD的炎症环境，会损害B细胞的发育、存活和 功能。为了检测微环境对B细胞培养的影响，我将非SCD B细胞培养在 培养液中添加SCD血清或脾提取液。要调查提取影响的哪些组件 B细胞，我将利用多重ELISA建立提取液和血清的细胞因子谱。 目的3：确定低MZB是否与感染和炎症性并发症有关 SCD。假设：SCD中危及生命的感染在患有严重MZB的儿童中更为常见 缺乏症。为了确定嗜B细胞病毒对MZB数量是否有混淆作用，我将 使用定制的多重ELISA板测量对EBV和疟疾的免疫反应。以确定是否 感染性并发症与SCD中更严重的MZB缺乏有关，我将比较MZB在 有或没有并发症的儿童，如急性胸腔、骨髓炎或菌血症。 尽管有共同的驱动突变，但SCD的临床表型具有很高的变异性。总而言之，这一领域的研究 提案将克服对感染和炎症性并发症患者进行风险分层的关键障碍 SCD.对SCD如何催化B细胞缺陷的深入了解可能有助于找到新的治疗方法 目标或生物标志物，以减轻感染对SCD的影响。我的中心假设是B细胞 异常会导致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.