Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Obstructive Sleep Apnea–Induced Hypertension Is Associated With Increased Gut and Neuroinflammation

doi: 10.1161/JAHA.122.029218

Figure Lengend Snippet: Plasma levels of interleukin‐17a ( A ) and interleukin‐10 ( B ). Systolic blood pressure ( C ) in sham and OSA rats treated with a nonimmune IgG or interleukin‐17a neutralizing antibody. n=5 in ( A and B ), n=5–6 in ( C ); * P <0.05 for Sham+IgG vs OSA+IgG, # P <0.05 of OSA+nIL‐17a vs OSA+IgG, *** P <0.001 using two‐way ANOVA ( A , B ) or 2‐way repeated measures ANOVA and multiple comparisons with Tukey's multiple comparisons test ( C ). Error bars represent ±SEM. BP indicates blood pressure; IgG, immunoglobulin G; and OSA, obstructive sleep apnea.

Article Snippet: Plasma was incubated overnight at 4°C with anti–interleukin‐17a rat monoclonal IgG (clone 50 104, R&D Systems, No. MAB421) that was immobilized to wells in a clear round‐bottom 96‐well plate.

Techniques: Clinical Proteomics

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Obstructive Sleep Apnea–Induced Hypertension Is Associated With Increased Gut and Neuroinflammation

doi: 10.1161/JAHA.122.029218

Figure Lengend Snippet: Effects of interleukin‐17a neutralization on Tregs, T H 1, T H 17, TNFα+, and macrophages in the ileum ( A ), cecum ( B ), and brain ( C ) of sham and OSA rats. n=4 in ileum, n=6 in cecum and brain; * P <0.05, ** P <0.005, *** P <0.0005, **** P <0.0001 using 2‐way ANOVA with Tukey's multiple comparisons test. Error bars represent ±SEM. OSA indicates obstructive sleep apnea; T H , T helper cell; and Tregs, T regulatory cells.

Article Snippet: Plasma was incubated overnight at 4°C with anti–interleukin‐17a rat monoclonal IgG (clone 50 104, R&D Systems, No. MAB421) that was immobilized to wells in a clear round‐bottom 96‐well plate.

Techniques: Neutralization

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Obstructive Sleep Apnea–Induced Hypertension Is Associated With Increased Gut and Neuroinflammation

doi: 10.1161/JAHA.122.029218

Figure Lengend Snippet: Plasma levels of interleukin‐17a ( A ) and interleukin‐10 ( B ). Systolic blood pressure ( C ) in sham and OSA rats treated with a nonimmune IgG or interleukin‐17a neutralizing antibody. n=5 in ( A and B ), n=5–6 in ( C ); * P <0.05 for Sham+IgG vs OSA+IgG, # P <0.05 of OSA+nIL‐17a vs OSA+IgG, *** P <0.001 using two‐way ANOVA ( A , B ) or 2‐way repeated measures ANOVA and multiple comparisons with Tukey's multiple comparisons test ( C ). Error bars represent ±SEM. BP indicates blood pressure; IgG, immunoglobulin G; and OSA, obstructive sleep apnea.

Article Snippet: Beginning the day before the onset of sham or OSA, rats were administered 1 μg of purified rat monoclonal anti–interleukin‐17a immunoglobulin G (IgG) (clone 50 104, R&D Systems, No. MAB421) or isotype control purified rat monoclonal IgG2A (clone 54 447, R&D Systems, #MAB006) by intraperitoneal injection.

Techniques: Clinical Proteomics

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Obstructive Sleep Apnea–Induced Hypertension Is Associated With Increased Gut and Neuroinflammation

doi: 10.1161/JAHA.122.029218

Figure Lengend Snippet: Effects of interleukin‐17a neutralization on Tregs, T H 1, T H 17, TNFα+, and macrophages in the ileum ( A ), cecum ( B ), and brain ( C ) of sham and OSA rats. n=4 in ileum, n=6 in cecum and brain; * P <0.05, ** P <0.005, *** P <0.0005, **** P <0.0001 using 2‐way ANOVA with Tukey's multiple comparisons test. Error bars represent ±SEM. OSA indicates obstructive sleep apnea; T H , T helper cell; and Tregs, T regulatory cells.

Article Snippet: Beginning the day before the onset of sham or OSA, rats were administered 1 μg of purified rat monoclonal anti–interleukin‐17a immunoglobulin G (IgG) (clone 50 104, R&D Systems, No. MAB421) or isotype control purified rat monoclonal IgG2A (clone 54 447, R&D Systems, #MAB006) by intraperitoneal injection.

Techniques: Neutralization

Journal: Arthritis Research & Therapy

Article Title: Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

doi: 10.1186/ar2406

Figure Lengend Snippet: IL-17 expression on T cells within rheumatoid synovium and in synovial fluid CD4 + CD3 + T cells . (a) Rheumatoid synovial tissue was examined by immunohistochemistry. IL-17 (red) was found to colocalise with CD3 + T cells (blue) in perivascular cuffs (purple). Blood vessels were localised with von Willebrand factor (vWF) (green). Nuclear staining is shown in grey. (b) IL-17 (red) expression is associated with CD4 + T cells (blue) but not CD8 + (green) T cells. Nuclear staining is shown in grey. (c) flow cytometric analysis of peripheral blood (PB) and synovial fluid (SF) CD3 + T cells demonstrates that IL-17 is expressed in SF CD4 + T cells. PE, phycoerythrin; FITC, fluorescein isothyocyanate.

Article Snippet: The sections were rehydrated with PBS pH 7.4 with 5% foetal calf serum (Biosera Ltd, Ringmer, UK), and indirect immunofluorescence was performed using the following primary antibody combinations: IgG 2b mouse anti-human CD3 (UCHT-1, 17 μg/ml; gift from Peter Beverley, University College Hospital, London, UK) with rabbit anti-human von Willibrand factor (A0082, 6.2 μg/ml; Dako, Glostrup, Denmark) and IgG 1 mouse anti-human IL-17 (12-7179, 1.25 μg/ml; eBiosciences); or IgG 2b mouse anti-human CD4 (OKT4) with IgG 2a mouse anti-human CD8 (OKT8, both OKT clones used as ascitic fluid 1/100; American Type Culture Collection, Middlesex, UK) and rabbit anti-human IL-17 (AHP455G, 20 μg/ml; AbD Serotec, Oxfordshire, UK).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Arthritis Research & Therapy

Article Title: Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

doi: 10.1186/ar2406

Figure Lengend Snippet: Rheumatoid arthritis synovial fibroblasts stimulated with TNFα and IL-17 efficiently extend neutrophil survival . (a) Peripheral blood neutrophils were cocultured with rheumatoid arthritis synovial fibroblasts (RASF) pretreated for 24 hours with the indicated concentrations of cytokines. Recombinant human (rh)TNFα concentrations: open circle, 0 pg/ml; open square, 1 pg/ml; open triangle, 10 pg/ml; open inverted triangle, 100 pg/ml; filled circle, 1,000 pg/ml; filled square, 10,000 pg/ml. * P < 0.05 versus rhTNFα = 0 pg/ml. (b) Peripheral blood neutrophils were cultured alone or cocultured with RASF pretreated for 24 hours with the indicated cytokines both at a concentration of 10 ng/ml. ** P < 0.01. Data represent mean ± standard deviation from at least five independent experiments. Absolute neutrophil survival was determined by flow cytometry using fixed volume dumping, with exclusion of apoptotic cells by gating on cells with a maintained mitochondrial membrane potential as assessed by 3,3'-dihexyloxacarbocyanine iodide staining. Neutrophil morphology was examined on cytospins after 24 hours of coculture with (c) untreated RASF or (d) RASF stimulated with TNFα and IL-17.

Article Snippet: The sections were rehydrated with PBS pH 7.4 with 5% foetal calf serum (Biosera Ltd, Ringmer, UK), and indirect immunofluorescence was performed using the following primary antibody combinations: IgG 2b mouse anti-human CD3 (UCHT-1, 17 μg/ml; gift from Peter Beverley, University College Hospital, London, UK) with rabbit anti-human von Willibrand factor (A0082, 6.2 μg/ml; Dako, Glostrup, Denmark) and IgG 1 mouse anti-human IL-17 (12-7179, 1.25 μg/ml; eBiosciences); or IgG 2b mouse anti-human CD4 (OKT4) with IgG 2a mouse anti-human CD8 (OKT8, both OKT clones used as ascitic fluid 1/100; American Type Culture Collection, Middlesex, UK) and rabbit anti-human IL-17 (AHP455G, 20 μg/ml; AbD Serotec, Oxfordshire, UK).

Techniques: Recombinant, Cell Culture, Concentration Assay, Standard Deviation, Flow Cytometry, Staining

Journal: Arthritis Research & Therapy

Article Title: Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

doi: 10.1186/ar2406

Figure Lengend Snippet: Coculture with stimulated rheumatoid arthritis synovial fibroblasts doubles the functional lifespan of peripheral blood neutrophils . (a) Peripheral blood neutrophils were cocultured with untreated rheumatoid arthritis synovial fibroblasts (RASF) (open squares) or with RASF stimulated with IL-17 and TNFα (RASF IL-17/TNF ) (closed squares), and their survival was assessed every 24 hours by flow cytometry. *** P < 0.001. (b) The ability to produce superoxide radical in response to f-Met-Leu-Phe was determined in freshly isolated neutrophils (open bars) and neutrophils cocultured with RASF IL-17/TNF for 24 hours (filled bars). ** P < 0.01 versus unstimulated cells.

Article Snippet: The sections were rehydrated with PBS pH 7.4 with 5% foetal calf serum (Biosera Ltd, Ringmer, UK), and indirect immunofluorescence was performed using the following primary antibody combinations: IgG 2b mouse anti-human CD3 (UCHT-1, 17 μg/ml; gift from Peter Beverley, University College Hospital, London, UK) with rabbit anti-human von Willibrand factor (A0082, 6.2 μg/ml; Dako, Glostrup, Denmark) and IgG 1 mouse anti-human IL-17 (12-7179, 1.25 μg/ml; eBiosciences); or IgG 2b mouse anti-human CD4 (OKT4) with IgG 2a mouse anti-human CD8 (OKT8, both OKT clones used as ascitic fluid 1/100; American Type Culture Collection, Middlesex, UK) and rabbit anti-human IL-17 (AHP455G, 20 μg/ml; AbD Serotec, Oxfordshire, UK).

Techniques: Functional Assay, Flow Cytometry, Isolation

Journal: Arthritis Research & Therapy

Article Title: Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

doi: 10.1186/ar2406

Figure Lengend Snippet: Soluble, temperature-sensitive factors released by stimulated rheumatoid arthritis synovial fibroblasts extend neutrophil survival . (a) Peripheral blood neutrophils were either cocultured with fibroblasts (Fb), with conditioned medium from IL-17 and TNFα pretreated fibroblasts (FCM), or on a transwell filter suspended above fibroblasts (Tw) for 24 hours. Error bars show the mean ± standard deviation from three independent experiments. ** P < 0.01, * P < 0.05; ns, nonsignificant. (b) Culture supernatant from rheumatoid arthritis synovial fibroblasts stimulated with IL-17 and TNFα was heated to 92°C for the times indicted before culture with neutrophils for 24 hours, and neutrophil survival was measured. Error bars show the mean ± standard deviation from three independent experiments

Article Snippet: The sections were rehydrated with PBS pH 7.4 with 5% foetal calf serum (Biosera Ltd, Ringmer, UK), and indirect immunofluorescence was performed using the following primary antibody combinations: IgG 2b mouse anti-human CD3 (UCHT-1, 17 μg/ml; gift from Peter Beverley, University College Hospital, London, UK) with rabbit anti-human von Willibrand factor (A0082, 6.2 μg/ml; Dako, Glostrup, Denmark) and IgG 1 mouse anti-human IL-17 (12-7179, 1.25 μg/ml; eBiosciences); or IgG 2b mouse anti-human CD4 (OKT4) with IgG 2a mouse anti-human CD8 (OKT8, both OKT clones used as ascitic fluid 1/100; American Type Culture Collection, Middlesex, UK) and rabbit anti-human IL-17 (AHP455G, 20 μg/ml; AbD Serotec, Oxfordshire, UK).

Techniques: Standard Deviation

Journal: Arthritis Research & Therapy

Article Title: Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

doi: 10.1186/ar2406

Figure Lengend Snippet: GM-CSF in conditioned medium from stimulated rheumatoid arthritis synovial fibroblasts maintains neutrophil viability . Conditioned medium from rheumatoid arthritis synovial fibroblasts stimulated with IL-17 and TNFα (RASF IL-17/TNF ) maintains neutrophil viability in part through the release of granulocyte–macrophage colony-stimulating factor (GM-CSF) and via phosphatidylinositol-3-kinase-dependent and NF-κB-dependent pathways. (a) Using either an irrelevant control antibody (open bars) or specific GM-CSF antibodies (filled bars) conjugated to agarose beads, serum-free conditioned medium (unstimulated or IL-17A/TNFα stimulated) was depleted of GM-CSF and added to freshly isolated peripheral blood neutrophils for 24 hours. Error bars show the mean ± standard deviation from three independent experiments. * P < 0.05. (b) The degree of depletion of GM-CSF was determined by ELISA in fibroblast-conditioned medium (FCM) from unstimulated or IL-17A/TNFα-stimulated FCM, before (open bars) and after (filled bars) depletion with anti-GM-CSF antibodies/agarose beads. A fixed dose of 100 pg/ml recombinant human (rh)GM-CSF was used as a positive control for the ELISA and to check the efficiency of GM-CSF depletion (filled bars). ND, not detectable. (c) Freshly isolated neutrophils were pretreated with vehicle control (open bars), 20 μM Ly294002 (filled bars) or 1 μM Bay 11-7085 (filled bars) before being cultured for 24 hours in FCM from unstimulated or IL-17/TNFα-stimulated fibroblasts. * P < 0.05. (d) Neutrophils that had been exposed to medium alone (cont), TNFα (as a positive control), or IL-17/TNFα-stimulated FCM were subjected to western blotting and were labelled using primary antibodies to inhibitor of NF-κB (IκB) and, as a loading control, β-actin.

Article Snippet: The sections were rehydrated with PBS pH 7.4 with 5% foetal calf serum (Biosera Ltd, Ringmer, UK), and indirect immunofluorescence was performed using the following primary antibody combinations: IgG 2b mouse anti-human CD3 (UCHT-1, 17 μg/ml; gift from Peter Beverley, University College Hospital, London, UK) with rabbit anti-human von Willibrand factor (A0082, 6.2 μg/ml; Dako, Glostrup, Denmark) and IgG 1 mouse anti-human IL-17 (12-7179, 1.25 μg/ml; eBiosciences); or IgG 2b mouse anti-human CD4 (OKT4) with IgG 2a mouse anti-human CD8 (OKT8, both OKT clones used as ascitic fluid 1/100; American Type Culture Collection, Middlesex, UK) and rabbit anti-human IL-17 (AHP455G, 20 μg/ml; AbD Serotec, Oxfordshire, UK).

Techniques: Isolation, Standard Deviation, Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control, Cell Culture, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

doi: 10.1186/ar2406

Figure Lengend Snippet: Lack of contribution of IFNβ, TNFα, IL-6 and lipopolysaccharide to survival induced by conditioned medium . (a) Recombinant IFNβ or fibroblast-conditioned medium (FCM) from rheumatoid arthritis synovial fibroblasts stimulated with TNFα and IL-17 (RASF IL-17/TNF ) were added to neutrophils in the presence or absence of an anti-CD118 (type I interferon receptor) blocking antibody (filled bars) or irrelevant control. (b) Using either irrelevant control antibodies, specific granulocyte–macrophage colony-stimulating factor (GM-CSF) and/or TNFα antibodies conjugated to agarose beads, serum-free conditioned medium (unstimulated or IL-17A/TNFα stimulated) was depleted of GM-CSF and/or TNFα and added to freshly isolated peripheral blood neutrophils for 24 hours. In some experiments, additional blockade of IFNβ receptors (CD118) and IL-6 receptors was employed after depletion steps. Error bars show the mean ± standard deviation from three independent experiments. (c) Lipopolysaccharide (10 ng/ml) or FCM from RASF IL-17/TNF was added to neutrophils in the presence or absence of polymyxin B (50 μg/ml, filled bars). ** P < 0.01, * P < 0.05; ns, nonsignificant.

Article Snippet: The sections were rehydrated with PBS pH 7.4 with 5% foetal calf serum (Biosera Ltd, Ringmer, UK), and indirect immunofluorescence was performed using the following primary antibody combinations: IgG 2b mouse anti-human CD3 (UCHT-1, 17 μg/ml; gift from Peter Beverley, University College Hospital, London, UK) with rabbit anti-human von Willibrand factor (A0082, 6.2 μg/ml; Dako, Glostrup, Denmark) and IgG 1 mouse anti-human IL-17 (12-7179, 1.25 μg/ml; eBiosciences); or IgG 2b mouse anti-human CD4 (OKT4) with IgG 2a mouse anti-human CD8 (OKT8, both OKT clones used as ascitic fluid 1/100; American Type Culture Collection, Middlesex, UK) and rabbit anti-human IL-17 (AHP455G, 20 μg/ml; AbD Serotec, Oxfordshire, UK).

Techniques: Recombinant, Blocking Assay, Isolation, Standard Deviation