Correale Jorge, Equiza Tomas Rivero
Department of Neurology, Institute for Neurological Research Dr. Raúl Carrea, FLENI, Montañeses 2325, Buenos Aires, 1428, Argentina,
Methods Mol Biol. 2014;1190:257-69. doi: 10.1007/978-1-4939-1161-5_18.
Multiple sclerosis (MS) is an inflammatory demyelinating disease affecting the central nervous system. Autoimmunity appears to play a key role in both susceptibility to MS and development of disease, and pathogenesis has been linked to defects in distinct regulatory cell subsets. B cells are known for their capacity to produce antibodies. Recent advances in B cell biology, however, have demonstrated that regulatory B cells, a functional subset of B cells, contribute to tolerance development. Regulatory B cells were originally described in mouse autoimmunity and inflammation models where they dampen inflammation, but have also been found in several helminth infection models. We recently demonstrated that helminth-infected MS patients show a significantly lower clinical and radiological disease activity. Parasite-driven protection was associated with regulatory T cell induction and secretion of suppressive cytokines such as IL-10 and TGF-β. In addition, helminth infections in MS patients induced regulatory B cell populations producing high levels of IL-10, dampening harmful immune responses through a mechanism mediated, at least in part, by the ICOS-B/RP-1 pathway. More importantly, production of IL-10 by B cells in this study was restricted to helminth-infected individuals exclusively.The first part of this chapter will detail the criteria used in this study for selection of helminth-infected MS patients, MS patients without infection, and patients infected with Trypanosoma cruzi. Methods for isolation of peripheral blood CD19(+) cells and in particular for their stimulation with heat-inactivated Staphylococcus aureus Cowan strain, CDw32L cells, and CD40 antibody will also be described in detail. Finally, we will illustrate the procedures used to analyze phenotypic surface markers on these cells and characterize them in terms of IL-4, IL-6, IL-10, TNF-α, lymphotoxin, and TGF-β secretion.
多发性硬化症（MS）是一种影响中枢神经系统的炎性脱髓鞘疾病。自身免疫似乎在MS易感性和疾病发展中均起关键作用，其发病机制与不同调节性细胞亚群的缺陷有关。B细胞以其产生抗体的能力而闻名。然而，B细胞生物学的最新进展表明，调节性B细胞作为B细胞的一个功能亚群，有助于免疫耐受的形成。调节性B细胞最初在小鼠自身免疫和炎症模型中被描述，它们可减轻炎症，在几种蠕虫感染模型中也有发现。我们最近证明，感染蠕虫的MS患者临床和影像学疾病活动度显著降低。寄生虫驱动的保护作用与调节性T细胞的诱导以及抑制性细胞因子如IL-10和TGF-β的分泌有关。此外，MS患者的蠕虫感染诱导了产生高水平IL-10的调节性B细胞群体，通过至少部分由ICOS-B/RP-1途径介导的机制减轻有害免疫反应。更重要的是，本研究中B细胞产生IL-10仅限于感染蠕虫的个体。本章第一部分将详细介绍本研究中用于选择感染蠕虫的MS患者、未感染的MS患者以及感染克氏锥虫患者的标准。还将详细描述外周血CD19(+)细胞的分离方法，特别是用热灭活的金黄色葡萄球菌考恩菌株、CDw32L细胞和CD40抗体对其进行刺激的方法。最后，我们将说明用于分析这些细胞表面表型标志物并根据IL-4、IL-6、IL-10、TNF-α、淋巴毒素和TGF-β分泌对其进行表征的程序。
