It is, however, notable that NAPs PARP inhibitor drugs by themselves do not exert as much influence as the BoNT/A complex, except for IL-6 which showed equal response (Table 1). MCP-1 and VEGF were two other cytokines which were induced by NAPs alone, albeit not as strongly as BoNT/A complex. BoNT/A complex and NAPs both contain associated proteins for BoNT/A. However, exposition to BoNT/A complex, but not to NAPs,

resulted in significant increase of IP-10, IL-8, IL-15, TNF-α, and RANTES. For the current research, cytokine release was examined after 48 h of incubation. The kinetics of cytokine release have been studied for 24 h to up to one week in lymphocyte (Arva and Andersson, 1999) and kinetics of TNF, IL-6, and IL-8 gene expression after inflammatory stimuli BAY 80-6946 supplier have been shown to have multiple peak at 2–4 h and 24 h (DeForge and Remick, 1991). Although no cytokine release was induced by pure BoNT/A in the current experimental setting, further investigation with different incubation time on complex patterns of cytokine gene expression and production with pure BoNT/A as well as other components of BoNT/A complex is needed. Higher effect of BoNT/A

complex could arise from one or more of the following reasons. One, there is higher level of binding of the BoNT/A in the BoNT/A complex allowing more NAPs to enter the cell. Two, interaction between BoNT/A and NAPs introduce conformational changes which are more critical for triggering cytokine response. Three, there is a physiological link between the effects of BoNT/A and NAPs intracellularly, leading to synergistic host cell response. A previous study on the co-culture of microglia and SH-SY5Y

cells has shown the expression of IL-6, IL-8, and MCP-1 with borrelia burgdorferi stimulation, a spirochete that causes lyme disease, and it is known to potently induce the production of inflammatory mediators in a variety of cells (Myers et al., 2009). Release of MCP-1 from SH-SY5Y has also been reported during the neuroinflammation process (Mitchell et al., 2009). Physiological Chlormezanone role of cytokine release in neuronal cells can be manifold. The presence and activity of pro-inflammatory cytokines IL-1β and TNF-α were first reported in human and rat brain a decade ago (Breder et al., 1988 and Plata-Salaman et al., 1988). Cytokine release studies enable us to identify cytokines that are produced specifically upon BoNT/A, its complex, or NAPs stimulation. The SH-SY5Y cell line has been proven to be a useful in vitro model for TNF production from neurons and the regulation of that production by alpha2-adrenergic receptor activation (Renauld and Spengler, 2002). Additionally, TNF-α has been shown not only play the critical roles in pathological development and inflammatory induction, but on modulating cell proliferation of neural progenitors in CNS inflammation (Downen et al., 1999 and Wu et al., 2000).