Cystic Fibrosis (CF) is primarily a progressive lung disease, characterized by chronic pulmonary infections with opportunistic pathogens. Such infections typically commence early in life, producing an inflammatory response marked by IL-8 chemokine production and neutrophilic infiltration, major contributory factors in CF progression. Studying this inflammation, especially early in life, is critical for developing new strategies for preventing or slowing disruption to the structural integrity of the CF airways. However, evaluating the immune responses of bronchoalveolar lavage (BAL) cells from children with CF faces technical challenges, including contamination carried from the lung due to pre-existing infections and low cell number availability. Here, we describe a technique for preparing BAL cells from young children with CF and using those cells in a bacterial stimulation assay. Initial antibiotic treatment proved essential for preventing resident bacteria from overgrowing BAL cell cultures, or non-specifically activating the cells. ACTB, identified as an optimal reference gene, was validated for accurate analysis of gene expression in these cells. Pseudomonas aeruginosa and Staphylococcus aureus were used as bacterial stimulants to evaluate the immune response of BAL cells from young children with CF. Addition of gentamicin prevented bacterial overgrowth, although if added after 3 hours of culture an extremely variable response resulted, with the bacteria causing a suppressive effect in some cultures. Addition of gentamicin after 1 hour of culture completely prevented this suppressive effect. This technique was then able to reproducibly measure the IL-8 response to stimulation with S. aureus and P. aeruginosa, including co-stimulation with both bacteria.

中文翻译：

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囊性纤维化幼儿支气管肺泡灌洗免疫细胞的细菌刺激试验

囊性纤维化 (CF) 主要是一种进行性肺部疾病，其特征是机会性病原体的慢性肺部感染。这种感染通常在生命早期开始，产生以 IL-8 趋化因子产生和中性粒细胞浸润为标志的炎症反应，这是 CF 进展的主要促成因素。研究这种炎症，尤其是在生命早期，对于开发预防或减缓 CF 气道结构完整性破坏的新策略至关重要。然而，评估来自 CF 儿童的支气管肺泡灌洗 (BAL) 细胞的免疫反应面临技术挑战，包括由于预先存在的感染和低细胞数量可用性而从肺部携带的污染。这里，我们描述了一种从患有 CF 的幼儿中制备 BAL 细胞并在细菌刺激试验中使用这些细胞的技术。最初的抗生素治疗证明对于防止常驻细菌过度生长 BAL 细胞培养物或非特异性激活细胞至关重要。ACTB被确定为最佳参考基因，经验证可准确分析这些细胞中的基因表达。铜绿假单胞菌和金黄色葡萄球菌被用作细菌刺激剂来评估来自 CF 幼儿的 BAL 细胞的免疫反应。添加庆大霉素可防止细菌过度生长，但如果在培养 3 小时后添加，则会产生极其多变的反应，细菌会在某些培养物中产生抑制作用。在培养 1 小时后加入庆大霉素完全阻止了这种抑制作用。然后，该技术能够可重复地测量 IL-8 对金黄色葡萄球菌和铜绿假单胞菌刺激的反应，包括与两种细菌的​​共同刺激。