Journal: Mediators of Inflammation

Article Title: Leptomeningeal Cells Transduce Peripheral Macrophages Inflammatory Signal to Microglia in Reponse to Porphyromonas gingivalis LPS

doi: 10.1155/2013/407562

Figure Lengend Snippet: Characterization of macrophages in gingiva of chronic periodontitis patients and cultured macrophages after P.g. LPS stimulation. Immunofluorescent CLMS images of TLR2, TLR4, TNF- α , and iNOS in gingiva of chronic periodontitis patients (a), scale bar = 20 μ m. The mean mRNA levels of TNF- α , IL-10, iNOS, and Arg I of THP-1 human monocyte-like cell line after P.g. LPS (LPS, 1 μ g/mL) treatment for 24 h (b). Data are presented by mean ± SEM ( n = 3), *** P < 0.001 versus nontreated cells (none). Time course of TNF- α and IL-1 β release in RAW264.7 mouse macrophages after P.g. LPS treatment (c). Data are presented by mean ± SEM ( n = 4, each), *** P < 0.001, * P < 0.05 versus nontreated cells (none). The LPS-induced TNF- α secretion from RAW264.7 mouse macrophages at 48 h with the neutralizing antibodies against TLR2 (10 μ g/mL), TLR4 (10 μ g/mL), or a specific NF- κ B inhibitor, Bay 11-7082 (Bay, 20 μ M) (d). Data are presented by mean ± SEM ( n = 4, each), *** P < 0.001 versus nontreated cells (none), and ††† P < 0.001 versus P.g. LPS alone.

Article Snippet: Antibodies of mouse anti-TLR2 (T2.5), mouse anti-TLR4 (HTA-125) were purchased from eBioscience (San Diego, CA, USA).

Techniques: Cell Culture

Journal: Mediators of Inflammation

Article Title: Leptomeningeal Cells Transduce Peripheral Macrophages Inflammatory Signal to Microglia in Reponse to Porphyromonas gingivalis LPS

doi: 10.1155/2013/407562

Figure Lengend Snippet: Secretion of proinflammatory mediators by leptomeningeal cell after treatment with the conditioned medium from P.g. LPS-treated macrophages and P.g. LPS. The mean mRNA levels of TNF- α and IL-1 β of leptomeningeal cells at 4 h incubated with P.g. LPS (LPS) and conditioned medium of P.g. LPS-treated RAW264.7 mouse macrophages (MCM) (a). Data are presented by mean ± SEM ( n = 3), *** P < 0.001 versus nontreated cells (none). ††† P < 0.001 versus P.g. LPS alone. Time course of TNF- α and IL-1 β secreted by leptomeningeal cells after treatment with P.g. LPS (100 ng/mL) (b). Data are presented by mean ± SEM ( n = 4, each), *** P < 0.001 versus nontreated cells (none). The P.g. LPS-induced TNF- α secretion by leptomeningeal cells at 6 h with the neutralizing antibodies against TLR2 (10 μ g/mL), TLR4 (10 μ g/mL), or Bay 11-7082 (Bay, 20 μ M) (c). Data are presented by mean ± SEM ( n = 4, each), *** P < 0.001 versus nontreated cells (none), and ††† P < 0.001 versus P.g. LPS alone.

Article Snippet: Antibodies of mouse anti-TLR2 (T2.5), mouse anti-TLR4 (HTA-125) were purchased from eBioscience (San Diego, CA, USA).

Techniques: Incubation

Journal: Mediators of Inflammation

Article Title: Leptomeningeal Cells Transduce Peripheral Macrophages Inflammatory Signal to Microglia in Reponse to Porphyromonas gingivalis LPS

doi: 10.1155/2013/407562

Figure Lengend Snippet: Secretion of proinflammatory mediators by microglia after treatment with the conditioned medium from P.g. LPS-treated leptomeningeal cells and P.g. LPS. The mean mRNA levels of TNF- α and IL-1 β of MG6 microglia at 24 h incubated with P.g. LPS (LPS) and the conditioned medium of leptomeningeal cells (LCM) after treatment with P.g. LPS (a). Data are presented by mean ± SEM ( n = 3), * P < 0.05, *** P < 0.001 versus nontreated cells (none). ††† P < 0.001 versus P.g. LPS alone. The P.g. LPS-induced TNF- α secretion in MG6 microglia at 48 h with the neutralizing antibodies against TLR2 (10 μ g/mL), TLR4 (10 μ g/mL), or Bay 11-7082 (Bay, 20 μ M) (b). Data are presented by mean ± SEM ( n = 4, each), *** P < 0.001 versus nontreated cells (none), and ††† P < 0.001 versus P.g. LPS alone.

Article Snippet: Antibodies of mouse anti-TLR2 (T2.5), mouse anti-TLR4 (HTA-125) were purchased from eBioscience (San Diego, CA, USA).

Techniques: Incubation

Journal: Journal of Inflammation (London, England)

Article Title: Low level bacterial endotoxin activates two distinct signaling pathways in human peripheral blood mononuclear cells

doi: 10.1186/1476-9255-8-4

Figure Lengend Snippet: Effect of blocking antibodies on endotoxin-dependent IL-8 release by PBMC . Freshly isolated human PBMC (~1.0 × 10 6 total cells/well, 100,000 monocytes) were prepared as described in Methods in 100 μl of incubation buffer containing 100 ng/ml LBP, and (A) 1 μg/well of non-immune mouse IgG or MEM-18 (anti-CD14), or (B) 2 μg/well non-immune mouse IgG or HTA-125 (anti-TLR4). Cultures were then pre-incubated at 37°C for 30 min, and 100 μl of 2× the final endotoxin concentration in incubation buffer was added to the wells. Cultures were incubated at 37°C for 6 h. At the end of the incubation period, samples were collected and frozen for subsequent IL-8 analysis by ELISA. Data are expressed as the mean ± S.E.M. of triplicate samples; error bars for some conditions were so small they are obscured by the symbols. (A) *p < 0.05, **p < 0.001 for mouse IgG values relative to MEM-18 values for the same LPS concentration. (B) *p < 0.05, **p < 0.001 for mouse IgG values relative to HTA-125 values for the same LPS concentration. Similar results were seen in 3 independent experiments.

Article Snippet: Blocking antibodies to TLR4 (HTA-125) and to CD11, CD11b, and CD18 were purchased from eBioscience (San Diego, CA).

Techniques: Blocking Assay, Isolation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation (London, England)

Article Title: Low level bacterial endotoxin activates two distinct signaling pathways in human peripheral blood mononuclear cells

doi: 10.1186/1476-9255-8-4

Figure Lengend Snippet: Effect of anti-TLR4 blocking antibody on endotoxin-induced superoxide production by PBMC . Human PBMC (~1.0 × 10 6 total cells/well, 100,000 monocytes) were prepared in 100 μl of incubation buffer containing 2 μg/well of mouse non-immune IgG or of HTA-125, and were placed in individual wells of a 96-well white Optiplate. Plates were incubated at 37°C for 30 min. Plates were then cooled on ice, and 100 μl of incubation buffer containing 0.2 mM lucigenin and 2× the final indicated concentration of endotoxin was added to the wells. The plates were then placed in the luminometer and two-sec readings were taken at 37°C every 2 min for 180 min. The resulting readings in relative light units (LU) were summed to provide a measure of activity. Data are expressed as total LU (in thousands) per 100,000 monocytes and represent the mean of duplicate samples. There were no significant differences between the mouse IgG concentration curve and the HTA-125 concentration curve.

Article Snippet: Blocking antibodies to TLR4 (HTA-125) and to CD11, CD11b, and CD18 were purchased from eBioscience (San Diego, CA).

Techniques: Blocking Assay, Incubation, Concentration Assay, Activity Assay

Journal: Journal of Inflammation (London, England)

Article Title: Low level bacterial endotoxin activates two distinct signaling pathways in human peripheral blood mononuclear cells

doi: 10.1186/1476-9255-8-4

Figure Lengend Snippet: Endotoxin-dependent IL-8 release and ROS formation by freshly isolated human monocytes is regulated by distinct signaling pathways.

Article Snippet: Blocking antibodies to TLR4 (HTA-125) and to CD11, CD11b, and CD18 were purchased from eBioscience (San Diego, CA).

Techniques: Isolation