Introduction. In vitro experimentation is intentionally contrived to isolate specific phenomena in the context of profound biological complexity. Mycoplasmas in the upper airway likely contribute to this complexity and play a largely unknown role in both health and disease. Similarly, the presence and role of mycoplasma in in vitro investigation are largely unknown. Hypothesis. We hypothesize mycoplasma in human vocal fold fibroblasts (VFF) will affect both basal gene-expression patterns as well as the cell response to exogenous stimuli. Aim. We sought to determine mycoplasma presence across vocal fold fibroblast cultures, basal transcriptional changes as a function of mycoplasma, and responsiveness to exogenous glucocorticoids in mycoplasma-positive and -negative VFF. Methodology. PCR-based mycoplasma detection was performed in an immortalized human VFF line as well as rat and rabbit primary VFF cultures and extracted rat laryngeal tissue. RNA sequencing was performed in mycoplasma-positive and -negative human cells at baseline and in response to dexamethasone. Results. Mycoplasma was identified in the human cell line as well as primary culture from rabbits. Mycoplasma was not detected in tissue or primary culture from rat vocal folds. Basal mRNA expression in human VFF differed significantly following mycoplasma treatment. In addition, differential responses to dexamethasone were observed across multiple pathways as a function of mycoplasma presence in these cells. Pathways including apoptosis, DNA damage repair, and G1 to S cell cycle signalling were significantly enriched in mycoplasma-positive cells. Conclusion. Variability of mycoplasma presence across culture conditions and differential responses to exogenous stimuli as a function of mycoplasma presence are potentially problematic for the translation of in vitro experimentation in the upper aerodigestive tract. It remains unclear if these findings represent contamination or the baseline state of this specialized tissue.

中文翻译：

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支原体影响声带成纤维细胞中的基线基因表达和对糖皮质激素的反应

介绍。 在深奥的生物学复杂性背景下，有意进行体外实验以分离特定现象。上呼吸道支原体可能导致这种复杂性，并且在健康和疾病中起着未知的作用。同样，支原体在体外研究中的存在和作用在很大程度上是未知的。假设。我们假设人类声带成纤维细胞（VFF）中的支原体会影响基础基因表达模式以及细胞对外源刺激的反应。目标。我们试图确定声带成纤维细胞培养物中支原体的存在，支原体功能的基础转录变化以及支原体阳性和阴性VFF对外源糖皮质激素的反应性。方法。在永生化的人VFF系以及大鼠和兔原代VFF培养物和提取的大鼠喉组织中进行基于PCR的支原体检测。在基线和对地塞米松的反应中，在支原体阳性和阴性人类细胞中进行RNA测序。结果。在人细胞系以及兔的原代培养物中鉴定出支原体。在组织或原代培养物中未从大鼠声带中检测到支原体。支原体治疗后，人VFF中的基础mRNA表达有显着差异。另外，在这些细胞中，通过支原体的存在，跨多种途径观察到了对地塞米松的不同反应。支原体阳性细胞中包括凋亡，DNA损伤修复和G1到S细胞周期信号转导的途径显着丰富。结论。支原体在整个培养条件下的变异性和对外源性刺激的差异反应作为支原体存在的函数可能对体外翻译产生问题在上消化道进行实验。尚不清楚这些发现是否代表该特殊组织的污染或基线状态。