Two primer sets, universal: F_338, R_518 [49] and A. muciniphila specific [50], were used in this assay. In order to ensure an optimal specificity with the A. muciniphila genome, the sequence of the forward primer was modified as follow: F-5′-ACWCCTACGGGWGGCAGCAG-3′. The reaction mixture (20 μL) composed of 1× SYBR green PCR Master Mix (Applied Protein Tyrosine Kinase inhibitor Biosystems), 1 μL of each, either

A. muciniphila-specific primers or 16S rRNA universal primers at a final concentration of 0.25 μM, and 5 μL of template DNA adjusted to approximately equal concentration of 20 ng/μL. The PCR temperature profile was as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. The fluorescence acquiring was set in the annealing/extension step. Subsequent to the amplification, a melting curve analysis was performed in order to distinguish putative nonspecific amplification. Serial tenfold dilutions of A. muciniphila pure culture (DSMZ 22959) genomic DNA was used to generate standard curves. A 2 cm part of ileum (starting from Midostaurin mw caecum) was partitioned lengthwise, washed gently in ice-cold PBS and one half stored in tissue homogenate lysis buffer (Ampliqon, Skovlunde, Denmark) at −80°C after immediately frozen

in liquid nitrogen, while the other half was used for flow cytometry analysis. The intestines were homogenized by using a T25 Ultraturrax homeogenizer (IKA, Staufen, Germany) and subsequently centrifuged for 15 min at 10 000 G and 4°C. The supernatant was decanted and centrifugation Resveratrol was repeated twice, the last time in a 5 μm Ultrafree MC-centrifugal filter device (Millipore, Billerica, MA, USA). Samples were kept cold during all steps. Next, the supernatant was analyzed for levels of IFN-γ, TNF-α, IL-1α, IL-1β, IL-2, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-15, and IL-17 by bead-based Milliplex xMAP Luminex technology (Millipore) in accordance with manufacturer’s instructions. Statistical

analysis was performed using GraphPad Prism version 5.02 (GraphPad Software, San Diego, CA, USA). Statistical significance was evaluated by the one-way ANOVA test with Tukey’s posttest when comparing three or more groups. Unpaired two-tailed t-test or Mann–Whitney test was used when comparing two groups. p values less than 0.05 were considered statistically significant. This work was supported by a grant from “Arkitekt Holger Hjortenberg og hustru Dagmar Hjortenbergs fond” and it was carried out as part of “Center for Applied Laboratory Animal Research” (www.calar.dk). The authors declare no financial or commercial conflict of interest. “
“In peripheral lymphocytes, the transcription factors (TFs) NF-κB, NFAT, and AP-1 are the prime targets of signals that emerge from immune receptors. Upon activation, these TFs induce gene networks that orchestrate the growth, expansion, and effector function of peripheral lymphocytes.