2, Fig. 3 and Fig. 4). Alexafluor 488 labeled BSA as the control culture did not bind to any of these cell lines (data not shown). Our binding data for pure BoNT/A confirmed previously published research in which the purified BoNT/A bound to cell lines of neuronal origin, but not to those of non-neuronal origin (Kurokawa et al., 1987). But it has not been reported before that in addition binding to human neuronal cells, both learn more BoNT/A complex and NAPs can also bind to non-neuronal cells such as lymphoblasts, skeletal muscle cells, and fibroblasts. Although BoNT/A in its purified and complex forms all bind to

SH-SY5Y, the intracellular responses of the SH-SY5Y cells to these BoNT/A components have not been well studied. Among all the 28 human inflammatory cytokines tested, there were three categories of cytokine release responses: (1) no detectable release, (2) release but no significant differences between BoNT/A, BoNT/A complex or NAPs treatment, and (3) significantly different release induced by BoNT/A, BoNT/A complex or NAPs. The release of the following thirteen Tanespimycin solubility dmso cytokines was below the limit of detection after exposure to different components of BoNT/A associated proteins: IL-1β, MIG, IL-1ra, IL-2, IL-5, IL-17, Eotaxin, basic FGF, G-CSF, GM-CSF, MIP-1α, MIP-1β, and PDFF-BB (Supplementary Table

S1). For the following seven cytokines positive releases were detected, but there were no significant changes after the treatment with BoNT/A, BoNT/A complex, Axenfeld syndrome or NAPs: IL-4, IL-7, IL-9, IL-10, IL-12, IL-13, and IFN-γ (Τable S1). The cytokines

which were significantly induced by different components of BoNT/A and its associated proteins are listed in Table 1. Pure 150 kDa BoNT/A did not significantly increase the release of any inflammatory cytokines from SH-SY5Y cells, compared to BSA control. Exposure to NAPs or BoNT/A complex, however, increased the release of multiple inflammatory cytokines. The release of IL-6, MCP-1, and VEGF were all significantly increased after exposure to BoNT/A complex and NAPs compared with control. In addition, BoNT/A complex induced a significant increase of MCP-1 release compared with NAPs. BoNT/A complex, but not NAPs or BoNT/A, also induced dramatic increase in IP-10, IL-8, TNF-α, and RANTES compared with the control. These results suggest the possibility of NAPs may contribute to local and systemic inflammatory process after the administration of NAPs-containing BoNT/A drugs in patients. Over five million patients are being treated with botulinum neurotoxins globally (Singh et al., 2010), and because of the safety concerns of this being the most toxic substance known to mankind, the United States Food and Drug Administration (US FDA) has designated all botulinum neurotoxin based drugs for black box label (Kuehn, 2009). There have been reports of side effects such as cognition issues and flu-like symptoms from BoNT-based therapeutics (Alam et al., 2002, Costa et al., 2005 and Cote et al.