cDNA was synthesized using a high-capacity RNA-to-cDNA kit according to the manufacturer’s instructions (Applied Biosystems). Real-time PCR for RORγt, T-bet, Gata3, and AHR expression was performed using power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Primers utilized were: RORγt – 5′-GGCTGTCAAAGTGATCTGGA-3′ forward; 5′-CCTAGGGATACCACCCTTCA-3′ reverse; T-bet – IWR-1 solubility dmso 5′-CTGCCTGCAGTGCTTCTAAC-3′ forward; 5′-GCTGAGTGATCTCTGCGTTC-3′ reverse; Gata3 – 5′-ACTCAGGTGATCGGAAGAGC-3′ forward; 5′-AGAGGAATCCGAGTGTGACC-3′
reverse; AHR – 5′-CACTGACGGATGAAGAAGGA-3′ forward; 5′-TCGTACAACACAGCCTCTCC-3′ reverse. Expression was normalized to glyceraldehydes 3-phosphate dehydrogenase (GAPDH). BALB/c mice were divided into three
groups of 5. Mice were shaved selleck chemicals llc on the dorsum with electric clippers, and injected intradermally with 100 μL of PBS containing 530 pmol VIP, 530 pmol PACAP, or PBS alone. Fifteen minutes after injection, the mice were painted with 10 μL of DNFB (1% in acetone and olive oil (4:1)) epicutanousely at the injection site. Three days after immunization, mice were sacrificed and draining lymph nodes (axillary and inguinal) removed. Lymph nodes were mechanically disrupted and passed through a 70 μm nylon mesh to yield a single cell suspension. CD4+ T cells were isolated as described above. Ninety-six well flat-bottom plates were treated with 10 μg/mL of anti-mouse CD3 mAb in PBS overnight and washed. T cells were cultured (3 × 105 cells/well) in 250 μL of CM containing 2 μg/mL of anti-mouse CD28 mAb. Supernatants were collected 72 h after stimulation and cytokine content determined. Differences in average cytokine levels under different treatments at varying cOVA concentrations were analyzed using ANOVA. Average cytokine levels under each cOVA concentration were then compared between PACAP or VIP treatment and control groups. p-values were adjusted by controlling for the false discovery rate (FDR). For assessment of mRNA levels, effects of intradermal administration
of neuropeptides and effects of anti-IL-6 mAb on Ag presenting cultures, a linear mixed effects model was used to estimate the average level of the biomarkers under different treatments. This model takes into account Montelukast Sodium variations for each treatment both within and between plate and samples. Differences in the average level of the biomarker under pairs of experimental conditions of interest were evaluated using simultaneous tests for general linear hypotheses [[84]]. p-values were again adjusted for multiple comparisons by controlling the FDR. This work was supported by NIH Grant 5R01 AR042429 (R.D.G. and J.A.W.), the Jacob L. and Lillian Holtzmann Foundation (R.D.G.), the Edith C. Blum Foundation (R.D.G.), the Carl and Fay Simons Family Trust (R.D.G.), the Seth Sprague Educational and Charitable Foundation (R.D.G.), the Lewis B. and Dorothy Cullman Foundation (R.D.G.