Journal:

Article Title: Induction of tumor necrosis factor-? (TNF-?) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

doi: 10.1046/j.1471-4159.2003.01864.x

Figure Lengend Snippet: (a) BV-2 cells were stimulated with 10 ng/mL of p70, p402 or p40 under serum-free condition. At different times (h) of incubation, supernatants were used for TNF-α assay by ELISA as mentioned in Materials and methods. (b) BV-2 cells were stimulated with different concentrations of p70, p402 or p40 under serum-free condition. After 8 h of incubation, supernatants were used for TNF-α assay. Mouse primary microglia (c) and peritoneal macrophages (d) were stimulated with 10 ng/mL of p70, p402 or p40 under serum-free condition. At different times (h) of incubation, supernatants were used for TNF-α assay by ELISA. Data are expressed as the mean of two separate experiments and duplicates were run within the same experiment.

Article Snippet: Recombinant mouse IL-12 p70 and p40 2 (the p40 homodimer), and recombinant human p40 monomer were obtained from R & D Systems, Minneapolis, MN, USA.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Induction of tumor necrosis factor-? (TNF-?) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

doi: 10.1046/j.1471-4159.2003.01864.x

Figure Lengend Snippet: Cells were stimulated with different concentrations of p70 (a), p402 (b) or p40 (c) under serum-free condition. After 4 h of incubation, cells were taken out directly by adding Ultraspec-II RNA reagent (Biotecx Laboratories Inc.) to the plates for isolation of total RNA, and northern blot analysis for TNF-α mRNA was carried out as described in Materials and methods. (d) Cells were stimulated with 10 ng/mL of p40 under serum-free condition. At different time (h) of incubation, northern blot analysis for TNF-α mRNA was carried out.

Article Snippet: Recombinant mouse IL-12 p70 and p40 2 (the p40 homodimer), and recombinant human p40 monomer were obtained from R & D Systems, Minneapolis, MN, USA.

Techniques: Incubation, Isolation, Northern Blot

Journal:

Article Title: Induction of tumor necrosis factor-? (TNF-?) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

doi: 10.1046/j.1471-4159.2003.01864.x

Figure Lengend Snippet: Cells were stimulated with 10 ng/mL of p40 under serum-free conditions. After different minutes of stimulation, cells were taken out to prepare nuclear extracts, and nuclear proteins were used for EMSA to determine DNA-binding activities of NF-κB (a) and C/EBPβ (c) as described in Materials and methods. The upper and lower arrows in (a) indicate the induced NF-κB band and the unbound probe, respectively. However, the upper, middle and lower arrows in (c) indicate the induced C/EBPβ band, non-specific band, and the unbound probe, respectively. Cells plated at 50–60% confluence in 12-well plates were co-transfected with 0.5 μg of either pBIIX-Luc (b) or pC/EBPβ-Luc (d) and 25 ng of pRL-TK. After 24 h of transfection, cells were stimulated with different concentrations of p40 for 6 h under serum-free condition. Firefly and Renilla luciferase activities were obtained by analyzing total cell extract as described in Materials and methods. Data are mean ± SD of three different experiments.

Article Snippet: Recombinant mouse IL-12 p70 and p40 2 (the p40 homodimer), and recombinant human p40 monomer were obtained from R & D Systems, Minneapolis, MN, USA.

Techniques: Binding Assay, Transfection, Luciferase

Journal:

Article Title: Induction of tumor necrosis factor-? (TNF-?) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

doi: 10.1046/j.1471-4159.2003.01864.x

Figure Lengend Snippet: (a) Cells were transfected with 0.5 μg of either Δp65 or ΔC/EBPβ using Lipofectamine-plus. After 24 h of transfection, cells were stimulated with 10 ng/mL p40 for 12 h under serum-free conditions, and supernatants were used for TNF-α assay. Data are mean ± SD of three different experiments. bp < 0.05 versus p40. (b) Similarly, after 24 h of transfection, cells were stimulated with 10 ng/mL p40 under serum-free condition. After 4 h of incubation, northern blot analysis for TNF-α mRNA was carried out.

Article Snippet: Recombinant mouse IL-12 p70 and p40 2 (the p40 homodimer), and recombinant human p40 monomer were obtained from R & D Systems, Minneapolis, MN, USA.

Techniques: Transfection, Incubation, Northern Blot

Journal:

Article Title: Induction of tumor necrosis factor-? (TNF-?) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

doi: 10.1046/j.1471-4159.2003.01864.x

Figure Lengend Snippet: Cells were stimulated with 10 ng/mL p40 under serum-free condition. At different minutes of incubation, activities of ERK (a) and p38 MAP kinase (b) were assayed as described in Materials and methods.

Article Snippet: Recombinant mouse IL-12 p70 and p40 2 (the p40 homodimer), and recombinant human p40 monomer were obtained from R & D Systems, Minneapolis, MN, USA.

Techniques: Incubation

Journal:

Article Title: Induction of tumor necrosis factor-? (TNF-?) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

doi: 10.1046/j.1471-4159.2003.01864.x

Figure Lengend Snippet: Cells pre-incubated with different concentrations of PD98059 and SB203580 for 1 h received 10 ng/mL p40 under serum-free condition. (a) After 12 h of stimulation, supernatants were used for TNF-α assay by ELISA. Data are mean ± SD of three different experiments. ap < 0.05 versus p40; bp < 0.005 versus p40; cp < 0.001 versus p40. (b) After 4 h of stimulation, northern blot analysis for TNF-α mRNA was carried out.

Article Snippet: Recombinant mouse IL-12 p70 and p40 2 (the p40 homodimer), and recombinant human p40 monomer were obtained from R & D Systems, Minneapolis, MN, USA.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Northern Blot

Journal:

Article Title: Induction of tumor necrosis factor-? (TNF-?) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

doi: 10.1046/j.1471-4159.2003.01864.x

Figure Lengend Snippet: Cells were co-transfected with 25 ng of pRL-TK and 0.5 μg of either pBIIX-Luc (a) or pC/EBPβ-Luc (b). After 24 h of transfection, cells were incubated with different concentrations of PD98059 and SB203580 for 1 h followed by the stimulation with 10 ng/mL of p40 for 6 h under serum-free condition. Firefly and Renilla luciferase activities were obtained by analyzing total cell extract. Data are mean ± SD of three different experiments. ap < 0.001 versus control; bp < 0.005 versus p40; cp < 0.001 versus p40.

Article Snippet: Recombinant mouse IL-12 p70 and p40 2 (the p40 homodimer), and recombinant human p40 monomer were obtained from R & D Systems, Minneapolis, MN, USA.

Techniques: Transfection, Incubation, Luciferase, Control

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

Article Title: IFN-beta-1b inhibits IL-12 production in peripheral blood mononuclear cells in an IL-10-dependent mechanism: relevance to IFN-beta-1b therapeutic effects in multiple sclerosis.

doi: 10.4049/jimmunol.165.1.548

Figure Lengend Snippet: FIGURE 1. The effect of serial IFN-b-1b doses (10–5000 IU/ml) on LPS- plus IFN-g-induced IL-12 p40 in normal PBMC. The graph shows the means and SDs for six donors. The asterisk indicate a statistically significant p value (p , 0.001) for LPS plus IFN-g plus IFN-b compared with LPS plus IFN-g treatment.

Article Snippet: Recombinant human IL-12 p40, p70, and human IL-10 were purchased from R&D Systems (Minneapolis, MN) to be used as standards in the ELISA.

Techniques:

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

Article Title: IFN-beta-1b inhibits IL-12 production in peripheral blood mononuclear cells in an IL-10-dependent mechanism: relevance to IFN-beta-1b therapeutic effects in multiple sclerosis.

doi: 10.4049/jimmunol.165.1.548

Figure Lengend Snippet: FIGURE 2. The effect of serial doses of IFN-b-1b (10–5000 IU/ml) on SAC- plus IFN-g-induced IL-12 in normal PBMC. The graph represents means and SDs for six donors. The effect of IFN-b plus SAC plus IFN-g was compared statistically with that of SAC plus IFN-g. The asterisk in- dicate a statistically significant p value. A, IL-12 p40 levels. B, IL-12 p70 levels.

Article Snippet: Recombinant human IL-12 p40, p70, and human IL-10 were purchased from R&D Systems (Minneapolis, MN) to be used as standards in the ELISA.

Techniques:

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

Article Title: IFN-beta-1b inhibits IL-12 production in peripheral blood mononuclear cells in an IL-10-dependent mechanism: relevance to IFN-beta-1b therapeutic effects in multiple sclerosis.

doi: 10.4049/jimmunol.165.1.548

Figure Lengend Snippet: FIGURE 4. The inhibitory effect of IFN-b on SAC- plus IFN-g-induc- ible IL-12 p40 (A and B) and p70 (C and D) in PBMC from six MS patients. E and F, SAC- plus IFN-g-induced IL-10 levels in the six MS patients. A, C, and E, Absolute cytokine levels; B, D, and F, mean percent change in cytokine levels. p, p , 0.05; p , 0.01; ppp, p , 0.001.

Article Snippet: Recombinant human IL-12 p40, p70, and human IL-10 were purchased from R&D Systems (Minneapolis, MN) to be used as standards in the ELISA.

Techniques:

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

Article Title: IFN-beta-1b inhibits IL-12 production in peripheral blood mononuclear cells in an IL-10-dependent mechanism: relevance to IFN-beta-1b therapeutic effects in multiple sclerosis.

doi: 10.4049/jimmunol.165.1.548

Figure Lengend Snippet: FIGURE 5. Anti-IL-10 Ab (10 mg/ml), but not control Ab (Con Ab), blocked the inhibitory effect of IFN-b on LPS- plus IFN-g-induced IL-12 p40 in PBMC from normal donors. The results represent the means and SEs for the four donors presented in Table IV. The numbers in parentheses represent the mean percent inhibitory effect on IL-12 p40 production for the four donors at each IFN-b-1b dose compared with no IFN-b-1b treat- ment (point 0 on the x-axis).

Article Snippet: Recombinant human IL-12 p40, p70, and human IL-10 were purchased from R&D Systems (Minneapolis, MN) to be used as standards in the ELISA.

Techniques: Control

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

Article Title: IFN-beta-1b inhibits IL-12 production in peripheral blood mononuclear cells in an IL-10-dependent mechanism: relevance to IFN-beta-1b therapeutic effects in multiple sclerosis.

doi: 10.4049/jimmunol.165.1.548

Figure Lengend Snippet: FIGURE 6. Blocking of the inhibitory effect of IFN-b-1b on SAC- plus IFN-g-induced IL-12 p40 by anti-IL-10, but not control Ab, obtained at serial IFN-b-doses (donor RC 798). The numbers in parentheses indicate the percent inhibition of IL-12 p40 by IFN-b-1b compared with no IFN-b treatment (point 0 on the x-axis).

Article Snippet: Recombinant human IL-12 p40, p70, and human IL-10 were purchased from R&D Systems (Minneapolis, MN) to be used as standards in the ELISA.

Techniques: Blocking Assay, Control, Inhibition

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

Article Title: IFN-beta-1b inhibits IL-12 production in peripheral blood mononuclear cells in an IL-10-dependent mechanism: relevance to IFN-beta-1b therapeutic effects in multiple sclerosis.

doi: 10.4049/jimmunol.165.1.548

Figure Lengend Snippet: FIGURE 7. Blocking of the inhibitory effect of IFN-b-1b on IL-12 p40 (A) and p70 (C) production by increasing concentrations of anti-IL-10 Ab. The dotted lines represent IL-12 levels induced by SAC plus IFN-g with or without IFN-b-1b in the absence of Ab. Anti-IL-10 Ab treatment resulted in superinduction of IL-12 p40 and p70 compared with levels induced by SAC plus IFN-g without Ab. Anti-IL-10 Ab treatment reversed the inhibitory effect of IFN-b-1b on IL-12 p40 and p70 production in an Ab dose-dependent fashion up to 85% of the superinduced p40-levels (A) and 100% of the superinduced p70 levels (B). Neither control Ab (Con Ab; B and D) nor anti-IL-4 Ab (not shown) had this effect.

Article Snippet: Recombinant human IL-12 p40, p70, and human IL-10 were purchased from R&D Systems (Minneapolis, MN) to be used as standards in the ELISA.

Techniques: Blocking Assay, Control

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

Article Title: IFN-beta-1b inhibits IL-12 production in peripheral blood mononuclear cells in an IL-10-dependent mechanism: relevance to IFN-beta-1b therapeutic effects in multiple sclerosis.

doi: 10.4049/jimmunol.165.1.548

Figure Lengend Snippet: FIGURE 8. Effect of IFN-b-1b (100 IU/ml) treatment on IL-12 p40 production in two MBP-specific TCL from two MS patients (MDPF3 and RSMD2).

Article Snippet: Recombinant human IL-12 p40, p70, and human IL-10 were purchased from R&D Systems (Minneapolis, MN) to be used as standards in the ELISA.

Techniques:

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

Article Title: IFN-beta-1b inhibits IL-12 production in peripheral blood mononuclear cells in an IL-10-dependent mechanism: relevance to IFN-beta-1b therapeutic effects in multiple sclerosis.

doi: 10.4049/jimmunol.165.1.548

Figure Lengend Snippet: FIGURE 9. Effect of IFN-b-1b (100 and 1000 IU/ml) on relative IL-12 p40 (A) and IL-10 (B) mRNA levels in the MBP-specific TCC. C, Effect of anti-IL-10 Ab (2.5 mg/ml) on IFN-b inhibition of IL-12 p40. Anti-IL-10 Ab in the absence of IFN-b resulted in superinduction of IL-12 (first three lanes). Anti-IL-10 Ab also abolished the inhibitory effect of IFN-b-1b (100 IU/ml) on IL-12 production (last three lanes).

Article Snippet: Recombinant human IL-12 p40, p70, and human IL-10 were purchased from R&D Systems (Minneapolis, MN) to be used as standards in the ELISA.

Techniques: Inhibition