Journal: PLoS ONE

Article Title: Decrease in Blood Pressure and Regression of Cardiovascular Complications by Angiotensin II Vaccine in Mice

doi: 10.1371/journal.pone.0060493

Figure Lengend Snippet: (A) T cell proliferation was determined by analyzing [ 3 H]-thymidine incorporation. Splenocytes from mice on day 42 were stimulated for 36 hours with Ang II-KLH, KLH, Ang II or angiotensinogen (AGT) at a concentration of 10 µg/ml. The stimulation index is expressed as the ratio of stimulation to no stimulation. The data are expressed as the mean stimulation index ± the standard error of the mean per 10 6 splenocytes. * P <0.001 vs. no stimulation. (B) A representative photograph from the ELISPOT assay. The ELISPOT assay detected splenocytes that produced IL-4 and/or IFN-γ? Splenocytes from mice on day 42 were stimulated for 48 hours with 10 µg/ml Ang II-KLH, KLH, Ang II or angiotensinogen (AGT). (C) The quantification of spots in the ELISPOT assay. The data are expressed as the mean number of spots ± SEM per 10 6 splenocytes. * P <0.001 vs. saline. (a,c) The results are from 6 control mice (saline) and 6 experimental mice (1,000 ng Ang II-KLH with adjuvant).

Article Snippet: Briefly, 96-well ELISPOT plates (Millipore, Tokyo, Japan) were coated with anti-mouse IFN-γ or IL-4 antibody, as recommended by the manufacturer (R&D Systems, Tokyo, Japan).

Techniques: Concentration Assay, Enzyme-linked Immunospot, Produced, Saline, Adjuvant

Journal: Arthritis Research & Therapy

Article Title: Inhibition of Th17 differentiation by anti-TNF-alpha therapy in uveitis patients with Behçet's disease

doi: 10.1186/ar3824

Figure Lengend Snippet: Cytokine concentrations in ocular fluids from uveitis patients with Behçet's disease during infliximab treatment . The levels of cytokines such as IFN-γ, IL-2, IL-4, IL-10, TNF-α, IL-6, and IL-17 were evaluated by ELISA or CBA. Ocular fluid samples from patients who had Behçet's disease (BD) with active uveitis (n = 6) or inactive uveitis (n = 4; no signs or symptoms of uveitis at the remission stage without treatment) were used for the assay. We also collected ocular fluid samples from BD patients receiving infliximab (IFX, n = 8). The controls consisted of the aqueous humor of patients with age-related cataracts (n = 3) and the vitreous fluid of patients with idiopathic macular holes (n = 3). P value indicates active uveitis group (active UVE) vs. infliximab group (IFX treatment). CBA, cytometric beads array; n.s., not significant.

Article Snippet: For the induction of human Th17 cells, purified CD4 + T cells from BD patients or healthy donors were co-cultured with anti-human CD3 antibody (2 μg/ml, BD PharMingen, San Diego, CA, USA), anti-human CD28 antibody (2 μg/ml, BD PharMingen), anti-human IFN-γ antibody (5 μg/ml, R&D Systems, Minneapolis, MN, USA), anti-human IL-4 antibody (5 μg/ml, R&D Systems), and recombinant human proteins such as IL-1β (20 ng/ml, Peprotech, Rocky Hill, NJ, USA), IL-6 (20 ng/ml, R&D Systems), IL-23 (20 ng/ml, R&D Systems), and TNF-α (20 ng/ml, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Inhibition of Th17 differentiation by anti-TNF-alpha therapy in uveitis patients with Behçet's disease

doi: 10.1186/ar3824

Figure Lengend Snippet: In vitro effects of infliximab on CD4 + T cells from uveitis patients with Behçet's disease . CD4 + T cells from Behçet's disease (BD) patients (black bars) with active uveitis or healthy donors (HD, open bars) were co-cultured with rIL-2 and anti-human CD3/CD28 abs in the presence of infliximab (IFX). As the control abs for infliximab, anti-human TNF-α monoclonal antibody (αTNF-α) or anti-human IL-6 monoclonal antibody (αIL-6) were used. The levels of cytokines such as IFN-γ (A) , IL-4 (B) , IL-10 (C) , TNF-α (D) , IL-6 (E) , and IL-17 (F) in supernatants of T cells were evaluated by ELISA or CBA. Bars, mean ± SEM. Cytokine production by T cells. Asterisks mean values significantly higher than medium only (Abs (-)): * P < 0.05, ** P < 0.005, *** P < 0.0005. Abs, antibodies; CBA, cytometric beads array; n.s., not significant; SEM, standard error of the mean.

Article Snippet: For the induction of human Th17 cells, purified CD4 + T cells from BD patients or healthy donors were co-cultured with anti-human CD3 antibody (2 μg/ml, BD PharMingen, San Diego, CA, USA), anti-human CD28 antibody (2 μg/ml, BD PharMingen), anti-human IFN-γ antibody (5 μg/ml, R&D Systems, Minneapolis, MN, USA), anti-human IL-4 antibody (5 μg/ml, R&D Systems), and recombinant human proteins such as IL-1β (20 ng/ml, Peprotech, Rocky Hill, NJ, USA), IL-6 (20 ng/ml, R&D Systems), IL-23 (20 ng/ml, R&D Systems), and TNF-α (20 ng/ml, R&D Systems).

Techniques: In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Inhibition of Th17 differentiation by anti-TNF-alpha therapy in uveitis patients with Behçet's disease

doi: 10.1186/ar3824

Figure Lengend Snippet: Establishment of IL-17-producing Th17 cells from uveitis patients with Behçet's disease . (A) Purified CD4 + T cells from a Behçet's disease (BD) patient or a sarcoidosis (SAR) patient (black bars) with active uveitis or a healthy donor (HD, open bars) in the presence of anti-CD3/CD28 antibodies, anti-IFN-γ antibody, anti-IL-4 antibody, rIL-1β, rIL-6, rIL-23, and rTNF-α. For ELISA analysis, supernatants of polarized Th17 cell lines or control CD4 + T cell lines in the presence of only anti-CD3/CD28 antibodies were harvested. The graph indicates the amount of IL-17 determined by ELISA (pg/ml). (B) For flow cytometric analysis, harvested Th17 cell lines from BD or HD were stained with anti-IL-17 abs and anti-CD4 Abs after permeabilization. The numbers in the histograms indicate the percentages of cells that were double-positive for IL-17/CD4. Abs, antibodies.

Article Snippet: For the induction of human Th17 cells, purified CD4 + T cells from BD patients or healthy donors were co-cultured with anti-human CD3 antibody (2 μg/ml, BD PharMingen, San Diego, CA, USA), anti-human CD28 antibody (2 μg/ml, BD PharMingen), anti-human IFN-γ antibody (5 μg/ml, R&D Systems, Minneapolis, MN, USA), anti-human IL-4 antibody (5 μg/ml, R&D Systems), and recombinant human proteins such as IL-1β (20 ng/ml, Peprotech, Rocky Hill, NJ, USA), IL-6 (20 ng/ml, R&D Systems), IL-23 (20 ng/ml, R&D Systems), and TNF-α (20 ng/ml, R&D Systems).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Staining