ABSTRACT
Botulinum toxin type A is a causative agent of human botulism. Due to high toxicity and ease of production it is classified by the Centres for Disease Control and Prevention as a category A bioterrorism agent. The same serotype, BoNT/A, is also the most widely used in pharmaceutical preparations for treatment of a diverse range of neuromuscular disorders. Traditionally, animals are used to confirm the presence and activity of toxin and to establish neutralizing capabilities of countermeasures in toxin neutralization tests. Cell based assays for BoNT/A have been reported as the most viable alternative to animal models, since they are capable of reflecting all key steps (binding, translocation, internalization and cleavage of intracellular substrate) involved in toxin activity. In this paper we report preliminary development of a simple immunochemical method for specifically detecting BoNT/A cleaved intracellular substrate, SNAP-25, in cell lysates of neurons derived from mouse embryonic stem cells. The assay offers sensitivity of better than 0.1LD50/ml (3fM) which is not matched by other functional assays, including the mouse bioassay, and provides serotype specificity for quantitative detection of BoNT/A and anti-BoNT/A antitoxin. Subject to formal validation, the method described here could potentially be used as a substitute for the mouse bioassay to measure potency and consistency of therapeutic products.
Subject(s)
Biological Assay/methods , Botulinum Antitoxin/pharmacology , Botulinum Toxins, Type A/pharmacology , Enzyme-Linked Immunosorbent Assay , Mouse Embryonic Stem Cells/drug effects , Neurogenesis , Neurons/drug effects , Synaptosomal-Associated Protein 25/metabolism , Animals , Biomarkers/blood , Dose-Response Relationship, Drug , Mice , Mouse Embryonic Stem Cells/immunology , Mouse Embryonic Stem Cells/metabolism , Neurons/immunology , Neurons/metabolism , Reproducibility of Results , Synaptosomal-Associated Protein 25/immunology , Time FactorsABSTRACT
Stem cells are multipotent cells that can give rise to a differentiated progeny as well as self-renew. The balanced coordination of these two stem cell fates is essential for embryonic development and tissue homeostasis in the adult. Perturbed stem cell function contributes significantly to a variety of pathological conditions, eg impaired self-renewal capacity due to cellular senescence contributes to ageing, and degenerative diseases or impaired stem cell differentiation by oncogenic mutations contribute to cancer formation. This review focuses on the molecular mechanisms involved in regulating the normal function of neural stem cells in the adult mammalian brain and on the involvement of these cells in brain pathology.
