Development of cell based assays as replacement assays for Botulinum toxins and antitoxins

肉毒杆菌毒素，一些最有毒的天然物质是由肉毒梭菌产生的蛋白质。它们是神经毒性的，对神经细胞有毒，并导致肉毒杆菌中毒，通常与食用含有毒素的食物或严重吸毒者有关。毒素通过阻断神经功能导致呼吸和肌肉麻痹，甚至死亡。只要及时给予患者，抗毒素治疗是可能的。尽管极少量的毒素具有很高的毒性，但可以安全地用于治疗疼痛的肌肉痉挛和不自主的肌肉收缩。它越来越多地应用于许多新的医疗条件和美容目的。肉毒中毒的诊断是通过向动物注射病人的体液和疑似食物样本来确认毒素的存在。在制药工业中，治疗性毒素和抗毒素的生产商需要许多动物进行严重的致死性试验，以确认安全性、效力和稳定性。迄今为止开发的替代方法具有局限性，因为仍然依赖于动物或提供组织或仅反映有助于毒素在动物中作用模式的几个因素中的一个。避免这些限制的一种方法是开发体外功能测定，其提供了整个动物替代的可能性，因为涉及肉毒杆菌中毒的所有关键步骤。由于肉毒杆菌毒素通过阻断神经肌肉神经末梢处的化学物质（神经递质，如乙酰胆碱）的释放来诱导麻痹，因此相关的体外测定必须集中于该活性。已建立的人神经元细胞系和人干细胞尚未提供所需的敏感性，并且迄今为止的研究已经证实，在这些细胞可以与神经毒素一起使用之前，需要分化成神经元样结构。在这项工作中，我们将分化的细胞应用到微电极阵列（MEA），以执行原位细胞功能的测量。我们将通过染色参与神经递质释放的成分来观察细胞回收活性，并通过选择性检测细胞内被毒素攻击的关键蛋白质来支持这些研究，这些蛋白质也与神经递质释放有关。这种新方法反映了毒素的全部功能，但它完全不以动物为导向，也不依赖于其他应用和策略中使用的任何动物实验。

Botulinum toxins, some of the most poisonous naturally occurring substances are proteins produced by the bacterinum Clostridium botulinum. They are neurotoxic, that is toxic to the nerve cells and responsible for causing botulism, most often associated with eating food containing toxin or in hard drug users. The toxins cause respiratory and muscle paralysis, and even death, by blocking the nerve function. Treatment is possible with antitoxins, provided they are given to patients on time. Although highly toxic in extremely small quantities toxin can be administered safety to treat painful muscle spasms and involuntary muscle contractions. It is increasingly applied to many new medical conditions and for cosmetic purposes. Botulism is diagnosed by injecting animals with body fluids from patients and in suspected food samples to confirm presence of toxins. In pharmaceutical industries manufacturers of therapeutic toxins and antitoxins require many animals in severe lethality assay to confirm safety, potency and stability. Alternative methods developed to date have limitations by either still relying on animal or for provision of tissues or for reflecting only one of several factors that contribute to toxin mode of action in animals. One approach to avoid these limitations is to develop in vitro functional assay which offers the potential for entire animal replacement because of involving all key steps of botulinum intoxication. Because botulinum toxin induces paralysis by blocking the release of chemicals (neurotransmitters such as acetylcholine) at the neuromuscular nerve endings, relevant in vitro assays must focus on this activity. Established human neuronal cell lines and human stem cells have not offered the required sensitivity and studies to date have confirmed need for differentiation into neuronal like structures before these cells can be used with neurotoxins. In this work we will apply differentiated cells onto micro-electrode arrays (MEAs) in order to perform measurement of cell function in situ. We will support these studied by also looking at cell recycling activity by staining of the components involved in neurotransmitter release and by selectively detecting key proteins within the cells which are attacked by toxins and are also associated with neurotransmitter release. This new approach reflects the full function of the toxin yet it is entirely non animal orientated and will not rely on any animal experimentation used in other applications and strategies.