Journal:

Article Title: Tolerance induced by inhaled antigen involves CD4 + T cells expressing membrane-bound TGF-? and FOXP3

doi: 10.1172/JCI200420509

Figure Lengend Snippet: Cell contact and TGF-β–dependent inhibition of proliferation by CD4+ T cells from tolerized mice. (A) Mice were first exposed to PBS (inflammation group) or 1% OVA (tolerance group) daily for 10 days and then were immunized with OVA/alum on days 21 and 27. Splenic CD4+ T cells isolated on day 34 were stimulated in vitro with different concentrations of OVA (10–200 ∝g/ml) and APCs at equivalent cell numbers (105 cells per well). Cells were mixed as described in the legend to Figure ​Figure22 or separated by transwell. In the transwell experiments, cells from the inflammation group were plated in the wells, and cells from tolerized mice on the insert and thymidine incorporation in the former group was measured. *P < 0.05 versus proliferation of cells in the inflammation group. (B) Chicken IgY anti–TGF-β1 (100 ng/ml) or isotype control (chicken IgY) was added to mixed cultures. **P < 0.05 of mixed cultures incubated with anti–TGF-β1 compared with mixed cultures incubated without Ab. (C) Anti–IL-10 (1 mg/ml) or isotype control was added to mixed cultures. All assays were incubated for 72 hours, after which the cells were pulsed for measurement of [3H]-thymidine incorporation. Each data point represents the mean plus or minus SEM of triplicate wells. Shown is a representative experiment of three experiments.

Article Snippet: In some experiments, neutralizing Ab or relevant, matching isotype controls were added to the mixture of CD4 + T cells from the inflammation and tolerance groups as follows: anti–TGF-β1 at 50 ng/ml and 100 ng/ml (R&D Systems Inc.); isotype control: normal chicken IgY (R&D Systems Inc.) at 100 ng/ml (shown); monoclonal anti-mouse IL-10 Ab (R&D Systems Inc.) at 0.1 ∝g/ml and 1 ∝g/ml (shown); and rat IgG1 isotype control (R&D Systems Inc.) at 1 ∝g/ml.

Techniques: Inhibition, Isolation, In Vitro, Incubation

Journal:

Article Title: Tolerance induced by inhaled antigen involves CD4 + T cells expressing membrane-bound TGF-? and FOXP3

doi: 10.1172/JCI200420509

Figure Lengend Snippet: Expression of membrane-bound TGF-β on freshly isolated cells from both groups of mice and their similar cytokine secretion profile. (A) Cell surface TGF-β and CD25 expression on freshly isolated CD4+ T cells from inflammation and tolerance groups. The boxed area denotes cells expressing high levels of CD25. (B) CD4+ T cells from the two groups’ cells were cultured with OVA/APCs for two rounds of stimulation (maintaining equal numbers of cells during restimulation). Cells expressing cell surface TGF-β were isolated using PE-labeled anti–TGF-β Ab, anti-PE microbeads, and separation on magnetic columns. Equal numbers of positively selected cells were restimulated with OVA/APCs for 72 hours, and the indicated cytokines in the culture supernatants were measured by ELISA. Cells expressing IL-10 were isolated using the MACS IL-10 secretion assay, stimulated, and assessed for cytokine production as described above. All data are representative of two independent experiments.

Article Snippet: In some experiments, neutralizing Ab or relevant, matching isotype controls were added to the mixture of CD4 + T cells from the inflammation and tolerance groups as follows: anti–TGF-β1 at 50 ng/ml and 100 ng/ml (R&D Systems Inc.); isotype control: normal chicken IgY (R&D Systems Inc.) at 100 ng/ml (shown); monoclonal anti-mouse IL-10 Ab (R&D Systems Inc.) at 0.1 ∝g/ml and 1 ∝g/ml (shown); and rat IgG1 isotype control (R&D Systems Inc.) at 1 ∝g/ml.

Techniques: Expressing, Membrane, Isolation, Cell Culture, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Real-time PCR probes and details of primers

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Real-time Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Examples of imunohistochemical staining for the IL-1 family. IL-1β (row A), IL-1Ra (row B), and IL-1 receptor, type I (row C) in grade-1 non-degenerate discs (column 1) and grade-12 degenerate discs (column 2), IgG controls (row D) were all negative. Immunopositivity is revealed by brown staining. N.B In non-degenerate discs, no cell clusters were seen and little immunopositivity was observed in the single cells. In degenerate discs, a large number of cell clusters were observed, which were predominately immunopositive. Bars = 570 μm.

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Immunopositive staining for the IL-1 family in human intervertebral discs. Numbers of cells with immunopositivity for IL-1α (a) , IL-1β (b) , IL-1 receptor antagonist (c) , IL-1 receptor, type I (d) , and IL-1β-converting enzyme (e) , according to place of origin in the disc and grade of intervertebral disc degeneration ( n = 30). Data are presented as means ± 2 standard errors (as a representative of 95%CI). * P < 0.1,; ** P < 0.05

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Effects of IL-1β on gene expression in cells from non- degenerate or degenerate intervertebral discs

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Expressing

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Effect of IL-1 treatment on the IL-1 family gene expression in human intervertebral disc cells. Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene and untreated controls (hence control is graphed at 1 on the log scale) for IL-1α (a) , IL-1β (b) , IL-1 receptor antagonist (IL-1Ra) (c) , and IL-1 receptor, type I (IL-RI) (d) following IL-1β treatment of disc cells from two regions of non-degenerate (non-deg) ( n = 6) and degenerate ( n = 24) discs. ** P < 0.05.

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Expressing, Control