Journal: The Journal of Biological Chemistry

Article Title: Albumin-based Microbubbles Bind Up-regulated Scavenger Receptors following Vascular Injury *

doi: 10.1074/jbc.M110.134809

Figure Lengend Snippet: Immunohistochemical staining of balloon-injured and control rat aortic tissue for the presence of microbubbles (i.e. human albumin), CD36, TLR-4, and CD68. A, staining for PESDA microbubble proteins at sites of injury demonstrated microbubble retention in a focal pattern in the intimal and media of the aorta. Of note, PESDA shell proteins co-localized with CD36, SRB-1, and TLR-4 (white arrows), but not with CD68. ImageJ co-localization is also presented. B, images are enlarged to better illustrate co-localization of PESDA and TLR-4. However, note that PESDA and TLR-4 were not exclusively co-localized. ImageJ image processing was performed to determine areas of co-localization, which are presented in their respective panels as white pixels. Control tissues illustrate the basal expression of CD36 and were negative for PESDA and CD68. Isotype controls were negative for background staining. Pictures are representative of three infrarenal aortas from control animals and three balloon-injured animals. Scale bars = 50 μm.

Article Snippet: Samples were blocked using 5% donkey serum, washed, and incubated with the following combinations of primary antibodies: rabbit anti-rat CD36 (Novus Biologicals, Littleton, CO), TLR-4 (Rockland, Gilbertsville, PA), mouse anti-rat CD68 (AbD Serotec, Raleigh, NC), and goat anti-human albumin (Bethyl Laboratories, Montgomery, TX).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Albumin-based Microbubbles Bind Up-regulated Scavenger Receptors following Vascular Injury *

doi: 10.1074/jbc.M110.134809

Figure Lengend Snippet: CHO cells were incubated with PESDA only or with PESDA after pretreatment of the CHO cells with the oxidized ligands ox-LDL and MAA-LDL. Pretreatment with these oxidized proteins completely inhibited the binding of PESDA to SRB-1. Note that despite significant inhibition of binding to CD36, SRA, TLR-2, and TLR-4, inhibition was not complete and variable when comparing different receptors and inhibitors. *, p ≤ 0.01 compared with CHO-K1 cell binding; #, p ≤ 0.01 compared PESDA binding to each individual CHO cell expressing the scavenger receptor. Data are expressed as the mean ± S.E. of three separate experiments.

Article Snippet: Samples were blocked using 5% donkey serum, washed, and incubated with the following combinations of primary antibodies: rabbit anti-rat CD36 (Novus Biologicals, Littleton, CO), TLR-4 (Rockland, Gilbertsville, PA), mouse anti-rat CD68 (AbD Serotec, Raleigh, NC), and goat anti-human albumin (Bethyl Laboratories, Montgomery, TX).

Techniques: Incubation, Binding Assay, Inhibition, Expressing

Journal: Arthritis Research & Therapy

Article Title: Between adaptive and innate immunity: TLR4-mediated perforin production by CD28 null T-helper cells in ankylosing spondylitis

doi: 10.1186/ar1840

Figure Lengend Snippet: Messenger RNA expression of TLR2 and TLR4 in CD3 + CD4 + CD28 null and CD3 + CD4 + CD28 + T cells. (a) Fluorescence-activated cell sorting analysis shows the purity of CD3 + CD4 + CD28 null and CD3 + CD4 + CD28 + T cells. (b) mRNA expression of TLR2, TLR4 and β2-microglobulin (β2m, housekeeping gene) in CD3 + CD4 + CD28 null T cells (CD28 - ) and in CD3 + CD4 + CD28 + T cells (CD28 + ). Peripheral blood mononuclear cells were used as positive control (pos co), and a negative control (neg co) was performed in the absence of cDNA. A representative example out of three independent experiments is given.

Article Snippet: To test inhibitory effects of blocking anti-CD14 or anti-TLR4 antibodies, cells were resuspended in RPMI 1640 with 5% autologous serum and were incubated with 10 μg/ml blocking anti-CD14 antibody (R&D Systems), 10 μg/ml anti-TLR4 antibody (Torrey Pines Biolabs, TX, USA) and 10 μg/ml isotype control antibody (Becton Dickinson) for 1 hour before adding LPS at a concentration of 10 μg/ml.

Techniques: RNA Expression, Fluorescence, FACS, Expressing, Positive Control, Negative Control

Journal: Arthritis Research & Therapy

Article Title: Between adaptive and innate immunity: TLR4-mediated perforin production by CD28 null T-helper cells in ankylosing spondylitis

doi: 10.1186/ar1840

Figure Lengend Snippet: Surface expression of CD14, TLR4 and TLR2 on CD4 + CD28 + and CD28 null cells in ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. (a) Representative dot plots and histograms show TLR4 expression (filled red curve, black line represents isotype control) on CD4 + CD28 + and CD4 + CD28 null T cells. Gates were set on lymphocytes (forward scatter and sideward scatter) as well as on CD28 + and CD28 null cells expressing high levels of CD4. (b) Box plots summarize the expression of CD14, TLR4 and TLR2 on CD4 + CD28 + and CD28 null T cells in patients as indicated. The Wilcoxon test was used to determine the statistical differences between the groups. *** P < 0.001. SSC, side scatter; FSC, forward scatter; AS, ankylosing spondylitis; PsA, psoriatic arthritis; RA, rheumatoid arthritis.

Article Snippet: To test inhibitory effects of blocking anti-CD14 or anti-TLR4 antibodies, cells were resuspended in RPMI 1640 with 5% autologous serum and were incubated with 10 μg/ml blocking anti-CD14 antibody (R&D Systems), 10 μg/ml anti-TLR4 antibody (Torrey Pines Biolabs, TX, USA) and 10 μg/ml isotype control antibody (Becton Dickinson) for 1 hour before adding LPS at a concentration of 10 μg/ml.

Techniques: Expressing, Control

Journal: Arthritis Research & Therapy

Article Title: Between adaptive and innate immunity: TLR4-mediated perforin production by CD28 null T-helper cells in ankylosing spondylitis

doi: 10.1186/ar1840

Figure Lengend Snippet: CD14 and TLR4-mediated effects. (a) ELISA assays were performed to analyse levels of soluble CD14 (sCD14) in sera from patients with ankylosing spondylitis (AS) ( n = 50) and healthy controls (CO) ( n = 23). The Mann-Whitney test was used to determine the statistical differences between the group of patients and the control group. ** P < 0.01. A blocking antibody (Ab) directed against (b) CD14 and (c) TLR4 or an isotype control were added to peripheral blood mononuclear cells from patients with AS maintained in 5% autologous serum. After 1 hour, lipopolysaccharide (LPS) stimulation at a concentration of 10 μg/ml for 16 hours was started. Box blots show percentages of perforin-producing CD4 + CD28 null T cells from seven independent experiments. Differences were tested for significance using the Wilcoxon test. ** P < 0.01.

Article Snippet: To test inhibitory effects of blocking anti-CD14 or anti-TLR4 antibodies, cells were resuspended in RPMI 1640 with 5% autologous serum and were incubated with 10 μg/ml blocking anti-CD14 antibody (R&D Systems), 10 μg/ml anti-TLR4 antibody (Torrey Pines Biolabs, TX, USA) and 10 μg/ml isotype control antibody (Becton Dickinson) for 1 hour before adding LPS at a concentration of 10 μg/ml.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Control, Blocking Assay, Concentration Assay

Journal: Arthritis Research & Therapy

Article Title: Between adaptive and innate immunity: TLR4-mediated perforin production by CD28 null T-helper cells in ankylosing spondylitis

doi: 10.1186/ar1840

Figure Lengend Snippet: Effects of TNF-α on expression of pattern recognition receptors in vitro and in vivo . (a) Peripheral blood mononuclear cells were stimulated with 20 ng/ml tumour necrosis factor-α (TNF-α) for 24 hours, and CD4 + CD28 null T cells were analysed for expression of CD14, TLR4 and TLR2. Box plots summarize data from seven independent experiments. Medians were compared using the Wilcoxon test. *** P < 0.001, ** P < 0.01. (b) CD4 + CD28 null T cells in patients with active ankylosing spondylitis treated with infliximab at a dosage of 3 mg/kg body weight were tested for the expression of pattern recognition receptors (PRRs) before and 3 weeks after injection ( n = 8). The expression of CD14, TLR4 and TLR2 was detected by flow cytometry. The Wilcoxon test was used to determine differences in expression of PRRs before (pre) and under successful TNF-α blocking treatment (post). ** P < 0.01, * P < 0.05.

Article Snippet: To test inhibitory effects of blocking anti-CD14 or anti-TLR4 antibodies, cells were resuspended in RPMI 1640 with 5% autologous serum and were incubated with 10 μg/ml blocking anti-CD14 antibody (R&D Systems), 10 μg/ml anti-TLR4 antibody (Torrey Pines Biolabs, TX, USA) and 10 μg/ml isotype control antibody (Becton Dickinson) for 1 hour before adding LPS at a concentration of 10 μg/ml.

Techniques: Expressing, In Vitro, In Vivo, Injection, Flow Cytometry, Blocking Assay