After blocking with 5% fat-free dried milk for 1 h at room temperature, the membranes were incubated overnight with Abs raised against pro-IL-1��, cleaved IL-1��, caspase-1, and caspase-1p10. The membranes were revealed by HRP-conjugated secondary Ab (Cell Signaling Technology, Danvers, MA, SKI-606 USA) using the West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA). Western blot signals were detected using the LAS-1000 (Luminiscence Analyzing System; Fuji, Tokyo, Japan) and processed with AIDA 1000/1D image Analyzer software, version 3.28 (Raytest Isotopenmessgeraete, Straubenhardt, Germany). After stripping, the membranes were reprobed with anti-actin Ab (Abcam, Cambridge, USA). DNA Extraction Genomic DNA was extracted from the whole blood samples of CD patients and healthy donors using a standard salting out protocol [25].

Finally, total genomic DNA was quantified with a spectrophotometer, diluted at a final concentration 20 ng/��l and stored at ?20��C. Genotyping of NALP1 and NALP3 Polymorphisms A single nucleotide polymorphism (SNP) in NALP1 (rs12150220) and NALP3 (rs10754558) was selected. Genotyping was performed with the 5�� nuclease assay technology for allelic discrimination using fluorogenic Taqman probes on a 7500 Fast Real Time system (Applied Biosystems, Foster City, CA, USA). Each SNP was analyzed by a specific Taqman SNP genotyping assay (Applied Biosystems). Briefly, the polymerase chain reaction (PCR) in 12 ��l final volume with 20 ng of genomic DNA was performed using the ABI 7500 Thermal Cycler.

The thermal cycle-sequencing conditions were as follows: initial denaturation at 95��C for 10 min, followed by 45 cycles of denaturing at 95��C for 15 s and annealing and extension at 60��C for 1 min. Data were measured and analyzed using the Applied Biosystems Software Package SDS 2.1. Statistical Analyses Data were expressed as mean �� SD. Statistical analysis was performed using two-tailed Mann-Whitney U test from Graph Pad (PRISM 5.0). The statistical differences between the patient and the control groups for the genotyping of NALP1 and NALP3 polymorphism were analyzed by the Fisher��s exact two-tailed test. Relative risk was estimated by calculating the odds ratio (OR) and 95% confidence interval (95% CI). We applied a Bonferroni correction for multiple comparisons in the analysis of variant allele distributions at each SNP.

A value of p<0.05 was considered to be statistically significant. Results PDWGF Induces IL-1�� and IL-1�� Release from Carfilzomib Celiac Monocytes and PBMC Gliadin digest, but not ��-gliadin synthetic peptides, was found to stimulate IL-1�� production in celiac PBMC and its monocytes fraction [7]. To investigate the underlying mechanism, we first determined the production of IL-1��, together with other members of IL-1 family, IL-18 and IL-1��.