Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Synovial fibroblasts directly induce Th17 pathogenicity via the cyclooxygenase/prostaglandin E2 pathway, independent of IL-23.

doi: 10.4049/jimmunol.1300274

Figure Lengend Snippet: FIGURE 1. Limited contribution of IL-1b and IL-6 to the perseverance of the proinflammatory loop induced upon Th17–RASF interaction. Sorted primary memory CCR62 T cells and/or CCR6+ T cells (Th17) from healthy individuals were stimulated with anti-CD3/anti-CD28 and cultured with RASF for 2 d. (A) IL-1b, IL-6, and RORC gene transcription levels were detected by quantitative real-time PCR, and IL-6 and IL-17A protein levels were detected by ELISA in CCR62 T cell–RASF cultures (filled bars) or in Th17–RASF cultures (open bars). Gene transcription levels were normalized against HPRT values. (B) Th17–RASF cultures were stim- ulated with anti-CD3/anti-CD28 and cultured for 2 d in the presence or absence of anti–IL-1b (2.5 mg/ml) and/or anti–IL-6R (25 mg/ml). Ex- pression of indicated cytokines and MMPs was determined by ELISA in supernatant of Th17–RASF cultures in absence (black bars) or presence of anti–IL-1b (dark gray bars), anti–IL-6R (light gray bars), or both (white bars). (C) IL-23p19 and IL-12/23p40 gene transcription levels were detected by quantitative real-time PCR and normalized against HPRT gene expression values. Means and SEM are given for six healthy donors. Results are representative of at least three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. n.d., Not detectable.

Article Snippet: IL-23 expression was detected by ELISA using the combination of biotin-conjugated anti-human IL-12/23p40 (eBioscience) and anti-human IL-23p19 (R&D Systems).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Microbes and infection

Article Title: TLR3 and TLR7 are involved in expression of IL-23 subunits while TLR3 but not TLR7 is involved in expression of IFN-beta by Theiler's virus-infected RAW264.7 cells.

doi: 10.1016/j.micinf.2007.07.001

Figure Lengend Snippet: Fig. 2. Temporal pattern of p19, p40, and IFN-b expression in response to TMEV infection. Real-time PCR of p19 (A), p40 (B), and IFN-b (C) mRNA in RAW264.7 cells. RAW264.7 cells (1 106) were uninfected (con- trol) or infected with 1 106 PFU of TMEV for 3, 6, and 24 h. Bar graphs represent mean SE of relative levels of 3 samples and 3 independent exper- iments. * Indicates that the mean is significantly different than control at a ¼ 0.05 confidence level.

Article Snippet: Ninety-six-well plates were coated with 2.0 mg/mL antimouse IL-12/23 p40 (clone C15.6) from BD-Pharmingen (San Diego, CA) or 1.0 mg/mL anti-mouse IL-23 p19 (Clone 320229; R & D Systems, Minneapolis, MN) in bicarbonate buffer (pH 9.6) at 4 C overnight.

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Control

Journal: Microbes and infection

Article Title: TLR3 and TLR7 are involved in expression of IL-23 subunits while TLR3 but not TLR7 is involved in expression of IFN-beta by Theiler's virus-infected RAW264.7 cells.

doi: 10.1016/j.micinf.2007.07.001

Figure Lengend Snippet: Fig. 3. Temporal pattern of p19, p40, and IFN-b expression in response to TLRs agonists. Real-time PCR of p19 (A), p40 (B), and IFN-b (C) mRNA in RAW264.7 cells, unstimulated (control) or stimulated with poly (I:C) (50 mg/mL), loxoribine (200 mM), or LPS (500 ng/mL) for 3, 6, and 24 h. Data are means SE of relative levels of p19, p40, and IFN-b mRNA of 3 samples from 3 independent experiments. * Indicates that the mean is signif- icantly different than control at a ¼ 0.05 confidence level.

Article Snippet: Ninety-six-well plates were coated with 2.0 mg/mL antimouse IL-12/23 p40 (clone C15.6) from BD-Pharmingen (San Diego, CA) or 1.0 mg/mL anti-mouse IL-23 p19 (Clone 320229; R & D Systems, Minneapolis, MN) in bicarbonate buffer (pH 9.6) at 4 C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Microbes and infection

Article Title: TLR3 and TLR7 are involved in expression of IL-23 subunits while TLR3 but not TLR7 is involved in expression of IFN-beta by Theiler's virus-infected RAW264.7 cells.

doi: 10.1016/j.micinf.2007.07.001

Figure Lengend Snippet: Fig. 6. Expression of IL-23 p19, p40 subunit and IFN-b from RAW264.7 cells after TLR3 or TLR7 knockdown. Real-time PCR of p19 mRNA (A), p40 mRNA (B), IFN-b mRNA (C), immunoblot of p19 protein (D), ELISAs of p40 protein (E), and IL-23 p19/p40 (F) from 1 106 RAW264.7 cells that were transfected with empty vector, shTLR3, shTLR7, shTLR3 plus shTLR7, or sh expression vectors producing scrambled (scr) RNA sequences. Cells were then either uninfected (control) or infected with 1 106 PFU of TMEV for 24 h. Bar graphs (AeC) represent means SE of p19, p40 and IFN-b mRNA relative to GAPDH mRNA of 5 samples and 2 independent experiments. * Indicates that the mean is significantly different than controls at a ¼ 0.05 confidence level.

Article Snippet: Ninety-six-well plates were coated with 2.0 mg/mL antimouse IL-12/23 p40 (clone C15.6) from BD-Pharmingen (San Diego, CA) or 1.0 mg/mL anti-mouse IL-23 p19 (Clone 320229; R & D Systems, Minneapolis, MN) in bicarbonate buffer (pH 9.6) at 4 C overnight.

Techniques: Expressing, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Control, Infection