ppar agonist Our results indicate that the antibody response

Our results indicate that the antibody response to flavivirus NS1 is more virus-type specific than to the E protein. These results encourage future studies to identify NS1 epitopes (linear and conformational) that could be used for virus-specific serologic diagnosis. If the virus-specific epitopes are linear, synthetic peptides representing the epitopes could be directly employed by a diagnostic assay; if the virus type-specific epitopes are conformational, the epitopes need to be displayed in a correct structural conformation. Employment of these virus-specific epitopes in the absence of cross-reactive epitopes of NS1 will further improve assay specificity. The same approach could also be applied to identify virus-specific epitopes in NS5 and even E protein. In accordance with this notion, recent studies have shown that domain III of ZIKV E protein contains virus-type specific conformational epitopes (Stettler et al., 2016; Zhao et al., 2016). To further improve the differentiation power of the current assay (i.e., virus-type specificity), DENV NS5 could be added to the current multiplex platform. Since the number of patient specimens was limited in the current study, more well characterized samples are needed to further verify the assay as well as to compare the specificity of antibody responses to NS1 and NS5.
Funding Sources P.Y.S. was supported by University of Texas Medical Branch (UTMB) startup award, UTMB Innovation and Commercialization award, University of Texas STARs Award, NIH grant R01AI087856, Pan American Health Organization grant SCON2016-01353, and UTMB CTSAUL1TR-001439.
Conflict of Interest Statement
Author Contributions
Acknowledgements
Introduction Nevertheless, we already showed that genetic ablation of PKCθ in mdx does not lead to apparent adverse effects, while it significantly ameliorates the progression of the disease, preventing a robust inflammatory response (Madaro et al., 2012). In this study, we show that in vivo pharmacological inhibition of PKCθ in mdx mice significantly ameliorates the dystrophic phenotype, at both morphological and functional levels.
Materials and Methods
Results
Discussion This study demonstrates that pharmacological inhibition of PKCθ in 4-week-old mdx mice, significantly prevents muscle wasting and inflammatory response, while maintains muscle regeneration and improves function and performance. We used the C20, one of the previously identified potent and selective PKCθ inhibitors (Cywin et al., 2007), and show that in vivo C20 treatment prevented ConA-induced T ppar agonist activation, as expected for a PKCθ inhibitor (Bermejo et al., 2015; Cywin et al., 2007; Zanin-Zhorov et al., 2010). Although its eventual clinical application should be further investigated, our data provide proof of principle that pharmacological targeting of PKCθ in muscular dystrophy would be effective in ameliorating the disease, as our previous genetic studies suggested. We show that two week C20 treatment of 4-week-old mdx mice significantly reduced histo-pathological features of dystrophic muscle. We decided to treat mdx mice at 4week of age, when immune cells are infiltrating muscle, thus, if our hypothesis was correct, we would expect that C20 treatment should prevent/reduce inflammatory response. On the other hand, previous studies have demonstrated that PKCθ expression/activity is required for complete histogenesis, differentiation and homeostasis of skeletal muscle, as well as for cardiomyocyte survival and remodeling (Camerino et al., 2014; Madaro et al., 2011; Messina et al., 2010; Paoletti et al., 2010; Tokugawa et al., 2009; Zappelli et al., 1996). We already showed that in a context of chronic inflammation, where immune cells activity is a key determinant, as in muscular dystrophy, lack of PKCθ predominantly affects immune response ameliorating the disease, and no obvious adverse effects were observed in other tissues (i.e. skeletal and cardiac muscle) (Madaro et al., 2012). We show here that C20 treatment of 4-week-old WT mice, did not result in deleterious effects on either skeletal or cardiac muscle phenotype, nor on the ability of skeletal muscle to respond to external stimuli, as to fasting; however, increased skeletal and cardiac muscle mass was observed. In TA muscle this is possibly due to a rise in the number of myofibers, as we observed, which might be beneficial in a context of muscle wasting; although the underlying mechanism(s) is not clear yet, it may explain the boosted muscle regenerative ability, observed in C20-treated dystrophic mice. In fact, we did not observe myofiber number increase in TA derived from C20-treated mdx mice, and the percentage of centrally and peripherally nucleated myofiber was unaltered. However, since myofiber damage was reduced, compared to muscle from untreated mdx mice, as discussed below, the maintenance of muscle regeneration might be due to increase in satellite cells activation and myogenesis, in line with the C20-induced fiber number increase in WT muscle. Besides, we showed that 2week C20 treatment of 4-week-old mdx mice, significantly reduced muscle fiber degeneration, immune cells infiltration, as well as inflammatory pathways activation, such as NFκB. Indeed, NFκB is activated very early in muscle from mdx mice, probably because of contraction-induced muscle injury. NFκB is known to regulate the expression of many inflammatory genes, including cytokines and chemokines in both immune cells and muscle fibers, thus maintaining an inflammatory environment.