Neuron-derived CP 868596 TNF-α may maintain the activity of neurons by sustaining physiologic levels of neurotransmitter release through regulation of adrenergic autoreceptor activity (Ignatowski et al., 1997). Flu like symptoms have been reported in between 1.7 and 20% of patients treated with various preparations of BoNT/A (Baizabal-Carvallo et al., 2011). It has been reported that neurons and glial cells produce cytokines in cell culture, in particular after addition of inflammatory stimuli (Schobitz et al.,

1994). A recent published study evaluated blood cytokines of patients following treatment with BoNT and discovered that inflammatory cytokines are increased in many patients following BoNT injection (Baizabal-Carvallo et al., 2013), although clear role of NAPs was not deciphered. Our study suggested that the observed flu-like symptoms after BoNT/A application may also be the result of inflammation resulting from the inflammatory mediators released in response to some components in the BoNT/A complexing proteins. Due to the fact that in the current study, we utilized laboratory grade pure BoNT/A, BoNT/A Complex, and NAPs instead of commercially available products, and the in vitro concentrations of BoNT/A and associated protein

in the current study are higher than the therapeutical use, further research needs to be done for this aspect. For the comparison of current commercially available Thiamet G Epacadostat BoNT/A products, the primary challenge is that the lack of standardized measurement. The units of different BoNT/A products are not interchangeable due to the differences of LD50 protocols in house (Sesardic, 2010). We plan to compare the host response of cells to different currently available products and will need to determine a relative equipotency point for these agents during the treatment. It is very important to utilize the concentration in the range

of nM in cellular model to avoid false negative results. We will also include fM to pM concentration range, which is therapeutic dose, to facilitate our understanding of clinical observations. It is concluded that BoNT/A in the complex form with the presence of NAPs protein induced significant inflammatory cytokine release. The presence of NAPs has also been shown to accentuate the immune response to the BoNT/A injections (Siatkowski et al., 1993 and Goschel et al., 1997). As the presence of associated proteins within the therapeutic formulation of BoNT/A complex increases the protein load, and may accentuate the immune response or inflammatory process, further experiments to investigate effects of BoNT/A components are warranted. It may help to clarify different physical effects caused by BoNT/A in its purified or complex forms.