Lozano Ana, Sánchez-Marrugo Carolina, Llinás-Caballero Kevin, Barrios Carlos, Acevedo Nathalie, Zakzuk Josefina, Caraballo Luis
Estudiante, Doctorado en Ciencias Biomédicas, Instituto de Investigaciones Inmunológicas, Universidad de Cartagena.
Médico, Instituto de Investigaciones Inmunológicas, Universidad de Cartagena.
Rev Alerg Mex. 2024 Feb 1;71(1):64-65. doi: 10.29262/ram.v71i1.1365.
To quantify the production of Th1/Th2/Th17 cytokines induced by Ascaris lumbricoides antigens in peripheral blood mononuclear cells (PBMCs) using a multiplex technique.
PBMCs were cultured from individuals with mild A. lumbricoides infection (n = 20) and uninfected individuals (n = 21) and stimulated with A. lumbricoides extract (ExtAscaris), a mix of anti-CD2/CD3/CD28 (CDmix) as a positive control, and only medium (negative control). Cytokines in the supernatants were measured using the BD™ Cytometric Bead Array Human Th1/Th2/Th17 kit, to identify IFN-γ, TNF, IL-10, IL-6, IL-4, IL-2, and IL-17A. Readings were taken on a spectral cytometer (Northern Lights, Cytek, USA), and analysis was performed using R software with packages "tidyverse," "beadplexr," "flowCore," and "arsenal." Cytokine concentrations were calculated using a 5-parameter logistic curve. The t-test was used to compare cases and controls, and statistical significance was set at p < 0.05. The study was approved by the Ethics Committee of the University of Cartagena and the participants provided informed consent. This study was financially supported by the Colombian Sistema General de Regalías under the BPIN2020000100405 - BPIN2020000100364.
Efficient fluorescence intensity extraction for each cytokine was achieved using detection channel R8 and the "mclust" clustering model (Figure 1). No significant differences were found in the levels of the seven cytokines between cases and controls (Figure 2). Although the IFN-γ response to ExtAscaris was higher in cases than in controls (252.5 ng/mL vs. 173.1 ng/mL), the difference was not significant. IL-17A (detection limit: 18.9 pg/mL) was more detectable in cases than controls (5 cases, 23% vs. 2 controls, 9.5%). IL-4 was only detected in the supernatants from CDmix-stimulated cultures but not with the Ascaris extract (Figure 2).
The multiplex technique using spectral flow cytometry combined with open-source analysis proved applicable for quantifying cytokines induced by antigens in PBMCs. However, a more sensitive method is needed to evaluate IL-4 response in the context of ascariasis. The results did not reveal significant differences in cytokine production between cases and controls for the evaluated stimuli.
采用多重技术定量检测蛔虫抗原诱导外周血单个核细胞（PBMCs）产生的Th1/Th2/Th17细胞因子。
从轻度蛔虫感染个体（n = 20）和未感染个体（n = 21）中培养PBMCs，并用蛔虫提取物（ExtAscaris）、抗CD2/CD3/CD28混合物（CDmix）作为阳性对照以及仅用培养基（阴性对照）进行刺激。使用BD™细胞计数微珠阵列人Th1/Th2/Th17试剂盒检测上清液中的细胞因子，以鉴定干扰素-γ（IFN-γ）、肿瘤坏死因子（TNF）、白细胞介素-10（IL-10）、白细胞介素-6（IL-6）、白细胞介素-4（IL-4）、白细胞介素-2（IL-2）和白细胞介素-17A（IL-17A）。在光谱细胞仪（美国Cytek公司的Northern Lights）上进行读数，并使用R软件以及“tidyverse”“beadplexr”“flowCore”和“arsenal”软件包进行分析。使用五参数逻辑曲线计算细胞因子浓度。采用t检验比较病例组和对照组，设定统计学显著性为p < 0.05。本研究经卡塔赫纳大学伦理委员会批准，参与者均提供了知情同意书。本研究由哥伦比亚Sistema General de Regalías根据BPIN2020000100405 - BPIN2020000100364提供资金支持。
使用检测通道R8和“mclust”聚类模型实现了对每种细胞因子的有效荧光强度提取（图1）。病例组和对照组之间七种细胞因子的水平未发现显著差异（图2）。虽然病例组对蛔虫提取物的IFN-γ反应高于对照组（分别为252.5 ng/mL和173.1 ng/mL），但差异不显著。IL-17A（检测限：18.9 pg/mL）在病例组中的可检测性高于对照组（5例，23% 对2例，9.5%）。IL-4仅在CDmix刺激培养的上清液中检测到，而蛔虫提取物刺激的培养上清液中未检测到（图2）。
采用光谱流式细胞术结合开源分析的多重技术被证明适用于定量检测PBMCs中抗原诱导的细胞因子。然而，需要一种更敏感的方法来评估蛔虫病背景下的IL-4反应。对于所评估的刺激物，结果未显示病例组和对照组在细胞因子产生方面存在显著差异。
