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Development of in vitro functional replacement assays for Botulinum toxin and antitoxin preparations

Abstract : Botulinum neurotoxins are lethal toxins that induce prolonged muscle paralysis by blocking the release of neuronal transmitters from peripheral cholinergic nerve endings. Although highly toxic, botulinum toxins are available as licensed drugs for the treatment of a variety of medical disorders and increasingly applied for cosmetic purposes which results in substantial increase of animal use in the lethality assays worldwide. Recent review of existing alternative methods by international experts recommended research on cellular neurotoxicity mechanisms for novel test assays as essential for progress. The mouse phrenic nerve-diaphragm assay is an in vitro assay that closely mimics in vivo respiratory paralysis. Improved reproducibility was found with in-bred mice and excellent agreement with in vivo lethality assay, indicating the suitability of hemidiaphragm assay as a highly sensitive and accurate alternative to the mouse lethality test. In addition, human neuroblastoma cell lines and murine stem cells were established in culture and differentiated into a mature neuronal phenotype. Radiolabelled neurotransmitter release technique was optimised and the presence of specific neuronal markers assessed with labelled antibodies. Differentiated SH-SY5Y cells showed higher sensitivity to Botulinum type A than undifferentiated cells with evoked neurotransmitter release dose-dependently inhibited after exposure to picomolar amounts of type A toxin. Multi-electrode-array (MEAs) platform was implemented in parallel with vesicle trafficking and neurotransmitter release measurements. Reproducible and robust differentiation protocols generated stable neuronal cultures forming extensive networks over the multi-electrode culture dish surface and exhibiting spontaneous activity until up to 6 weeks after seeding indicating that networks of cultured neuronal cells represent suitable basis for the development of a cell based assays for antitoxin countermeasures. Investigating the effects of botulinum on neuronal communication will reveal fresh insights needed to revive cell based assay developments and to develop improved therapeutic countermeasures.
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Submitted on : Wednesday, July 1, 2020 - 4:48:12 PM
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  • HAL Id : pasteur-02886669, version 1



Christine Rasetti-Escargueil. Development of in vitro functional replacement assays for Botulinum toxin and antitoxin preparations. Bioassays 2011: Scientific Approaches & Regulatory Strategies, Oct 2011, Bethesda, United States. ⟨pasteur-02886669⟩



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