SAA induces mature IL-1β synthesis from neutrophils.

<p><b>A</b> Neutrophils were stimulated with the indicated concentrations of SAA for 8-1β production using ELISA. Values represent the mean ± SD of two independent experiments. *<i>p</i><0.001 compared to SAA-untreated neutrophils. <b>B</b> Neutrophils were stimulated with the indicated concentrations of SAA for 8 hr. After stimulation, supernatants were analyzed by western blot analysis for the presence of mature IL-1β. Three experiments were performed using different neutrophils and a representative result is shown.</p

SAA-induced IL-1β processing is dependent on caspase-1.

<p><b>A</b> Neutrophils were stimulated with SAA (5 µg/ml) in the presence or absence of Z-YVAD-FMK for 8 h. After stimulation, supernatants were analyzed for IL-1β production using ELISA. Values represent the mean ± SD of two independent experiments. *<i>p</i><0.001 compared to SAA-stimulated neutrophils. <b>B</b> Neutrophils were stimulated with SAA (5 µg/ml) in the presence or absence of Z-YVAD-FMK for 8 h. Supernatants were analyzed by western blot analysis for the presence of mature IL-1β. <b>C</b> Neutrophils were stimulated with SAA (5 µg/ml) in the presence or absence of Z-YVAD-FMK for 8 h. Culture supernatants (SN) and cellular lysates (CL) were analyzed by immunoblot using anti-caspase-1 Ab. Caspase-1 (p20, cleaved subunit; p45, precursor). Two experiments were performed using different neutrophils and a representative result is shown.</p

Effects of R406 on NF-κB p65 phosphorylation in SAA-stimulated neutrophils.

<p>Neutrophils pretreated with or without R406 were stimulated with SAA (5 µg/ml) for 20 min. Cellular lysates were analyzed by western blotting using anti-phospho-specific p65 (A) or anti-p65 (B) antibodies. Two experiments were performed using different neutrophils and a representative result is shown.</p

Effects of R406 on the transcription of pro-IL-1β in SAA-stimulated neutrophils.

<p>Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1(5 µg/ml) for 4 hr. The cells were harvested and analyzed for IL-1β and GAPDH mRNA levels by real-time PCR. Values represent the mean ± SD of three independent experiments. *<i>p</i><0.005 compared to SAA-stimulated neutrophils.</p

Effects of R406 on SAA-induced IL-1β processing in neutrophils.

<p><b>A</b> Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1(5 µg/ml) for 8 hr. After stimulation, supernatants were analyzed by western blot analysis for the presence of mature IL-1β. Three experiments were performed using different neutrophils and a representative result is shown. <b>B</b> Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1 hr. The cells were stimulated with SAA (5 µg/ml) for 8 hr. After stimulation, culture supernatants (SN) and cellular lysates (CL) were analyzed by immunoblot using anti-caspase-1 Ab. Caspase-1 (p20, cleaved subunit; p45, precursor). Three experiments were performed using different neutrophils and a representative result is shown.</p

SAA induces the transcription of pro-IL-1β in human neutrophils.

<p>Neutrophils were incubated with SAA (5 µg/ml) for the indicated periods. The cells were harvested and analyzed for IL-1β and GAPDH mRNA levels by real-time PCR. Values represent the mean ± SD of three independent experiments.</p

Effects of R406 on the transcription of NLRP3 in SAA-stimulated neutrophils.

<p><b>A</b> Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1(5 µg/ml) for the indicated periods. The cells were harvested and analyzed for NLRP3 mRNA by RT-PCR. <b>B</b> Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1 hr. The cells were stimulated with SAA (5 µg/ml) for 4 hr. The cells were harvested and analyzed for NLRP3 mRNA by RT-PCR. Three experiments were performed using different neutrophils and a representative result is shown.</p

Effects of R406 on the protein phosphorylation in SAA-stimulated neutrophils.

<p><b>A</b> Quiescent neutrophils pretreated or untreated with the indicated concentrations of R406 for 1(5 µg/ml) for 20 min. Cellular lysates were subjected to western blotting using phosphotyrosine-specific antibody, 4G10. Three experiments were performed using different neutrophils and a representative result is shown. <b>B</b> Quiescent neutrophils pretreated or untreated with the indicated concentrations of R406 for 1 hr. The cells were stimulated with SAA (5 µg/ml) for 20 min. Syk was immunoprecipitated from each lysates and the immunoprecipitates were subjected to western blotting using phosphotyrosine-specific antibody, 4G10 or anti-Syk antibody. Each lane shows Syk precipitated from 10⁷ cells. Three experiments were performed using different neutrophils and a representative result is shown. <b>C</b> Quiescent neutrophils pretreated or untreated with the indicated concentrations of R406 for 1 hr. The cells were stimulated with SAA (5 µg/ml). Cellular lysates were subjected to western blotting using phospho-specific or pan antibodies against ERK1/2, p38 and JNK1. Three experiments were performed using different neutrophils and a representative result is shown.</p

Circulating microRNA Profiles in Patients with Type-1 Autoimmune Hepatitis

<div><p>Recent studies have demonstrated that micro (mi)RNA molecules can be detected in the circulation and can serve as potential biomarkers of various diseases. This study used microarray analysis to identify aberrantly expressed circulating miRNAs in patients with type 1 autoimmune hepatitis (AIH) compared with healthy controls. Patients with well-documented and untreated AIH were selected from the National Hospital Organization (NHO)-AIH-liver-network database. They underwent blood sampling and liver biopsy with inflammation grading and fibrosis staging before receiving treatment. To further confirm the microarray data, circulating expression levels of miR-21 and miR-122 were quantified by real-time quantitative polymerase chain reaction in 46 AIH patients, 40 patients with chronic hepatitis C (CHC), and 13 healthy controls. Consistent with the microarray data, serum levels of miR-21 were significantly elevated in AIH patients compared with CHC patients and healthy controls. miR-21 and miR-122 serum levels correlated with alanine aminotransferase levels. Circulating levels of miR-21 and miR-122 were significantly reduced in AIH patients with liver cirrhosis, and were inversely correlated with increased stages of fibrosis. By contrast, levels of circulating miR-21 showed a significant correlation with the histological grades of inflammation in AIH. We postulate that aberrantly expressed serum miRNAs are potential biomarkers of AIH and could be implicated in AIH pathogenesis. Alternations of miR-21 and miR-122 serum levels could reflect their putative roles in the mediation of inflammatory processes in AIH.</p></div

Differentially expression miRNAs between control and AIH.

<p>Differentially expression miRNAs between control and AIH.</p