Wang Lei, Sun Yi, Yang Weiping, Lindo Paul, Singh Bal Ram
Prime Bio Inc., Dartmouth, MA 02747, USA.
Prime Bio Inc., Dartmouth, MA 02747, USA.
Toxicon. 2014 May;82:52-60. doi: 10.1016/j.toxicon.2014.02.004. Epub 2014 Feb 21.
Type A Botulinum neurotoxin (BoNT/A), the most potent poison known to mankind, is produced by Clostridium botulinum type A as a complex with neurotoxin-associated proteins (NAPs). Currently BoNT/A in purified and complex forms are both available in therapeutic and cosmetic applications to treat neuromuscular disorders. Whereas Xeomin(®) (incobotulinumtoxin A, Merz Pharmaceuticals, Germany) is free from complexing proteins, Botox(®) (onabotulinumtoxin A, Allergan, USA) contains NAPs, which by themselves have no known role in the intracellular biochemical process involved in the blockade of neurotransmitter release. Since the fate and possible interactions of NAPs with patient tissues after intramuscular injection are not known, it was the aim of this study to evaluate the binding of BoNT/A and/or the respective NAPs to cells derived from neuronal and non-neuronal human tissues, and to further explore neuronal cell responses to different components of BoNT/A. BoNT/A alone, the complete BoNT/A complex, and the NAPs alone, all bind to neuronal SH-SY5Y cells. The BoNT/A complex and NAPs additionally bind to RMS13 skeletal muscle cells, TIB-152 lymphoblasts, Detroit 551 fibroblasts besides the SH-SY5Y cells. However, no binding to these non-neuronal cells was observed with pure BoNT/A. Although BoNT/A, both in its purified and complex forms, bind to SH-SY5Y, the intracellular responses of the SH-SY5Y cells to these BoNT/A components are not clearly understood. Examination of inflammatory cytokine released from SH-SY5Y cells revealed that BoNT/A did not increase the release of inflammatory cytokines, whereas exposure to NAPs significantly increased release of IL-6, and MCP-1, and exposure to BoNT/A complex significantly increased release of IL-6, MCP-1, IL-8, TNF-α, and RANTES vs. control, suggesting that different components of BoNT/A complex induce significantly differential host response in human neuronal cells. Results suggest that host response to different compositions of BoNT/A based therapeutics may play important role in local and systemic symptoms in patients.
A型肉毒杆菌神经毒素（BoNT/A）是人类已知的毒性最强的毒素，由A型肉毒杆菌产生，以与神经毒素相关蛋白（NAPs）形成复合物的形式存在。目前，纯化形式和复合形式的BoNT/A均可用于治疗和美容应用，以治疗神经肌肉疾病。其中，Xeomin（®）（incobotulinumtoxin A，德国默克制药公司）不含复合蛋白，而Botox（®）（onabotulinumtoxin A，美国艾尔建公司）含有NAPs，而NAPs本身在涉及神经递质释放阻断的细胞内生化过程中没有已知作用。由于肌肉注射后NAPs与患者组织的命运及可能的相互作用尚不清楚，本研究的目的是评估BoNT/A和/或各自的NAPs与源自人类神经组织和非神经组织的细胞的结合，并进一步探索神经元细胞对BoNT/A不同成分的反应。单独的BoNT/A、完整的BoNT/A复合物以及单独的NAPs均能与神经元SH-SY5Y细胞结合。除了SH-SY5Y细胞外，BoNT/A复合物和NAPs还能与RMS13骨骼肌细胞、TIB-152淋巴母细胞、底特律551成纤维细胞结合。然而，纯BoNT/A未观察到与这些非神经元细胞的结合。尽管纯化形式和复合形式的BoNT/A均能与SH-SY5Y细胞结合，但SH-SY5Y细胞对这些BoNT/A成分的细胞内反应尚不清楚。对SH-SY5Y细胞释放的炎性细胞因子的检测表明，BoNT/A不会增加炎性细胞因子的释放，而暴露于NAPs会显著增加IL-6和MCP-1的释放，暴露于BoNT/A复合物会显著增加IL-6、MCP-1、IL-8、TNF-α和RANTES的释放，与对照组相比，这表明BoNT/A复合物的不同成分在人类神经元细胞中诱导出显著不同的宿主反应。结果表明，宿主对基于BoNT/A的不同组成的治疗药物的反应可能在患者的局部和全身症状中起重要作用。
