Journal: PLoS ONE

Article Title: Porphyromonas Gingivalis and E-coli Induce Different Cytokine Production Patterns in Pregnant Women

doi: 10.1371/journal.pone.0086355

Figure Lengend Snippet: Leukocytes were selected in the forward-sidescatter plot (fig. 1A) and copied to a sidescatter-CD14 plot. Monocytes (CD14 positive cells), granulocytes (CD14 negative cells with high SSC) and lymphocytes (CD14 negative cells with low SSC) were gated (fig. 1B). CD14 positive cells were copied to a TLR2/TLR4 plot. Using the isotype control sample, gates were set in the TLR2/TLR4 plot so that at least 99% of the isotype controls were negative for TLR2/TLR4 expression (fig. 1C). This gate was then used to identify the percentages of TLR4/TLR2 double positive, TLR2 single and TLR4 single positive monocytes as well as their mean fluorescence intensity (MFI), in the antibody incubated samples (fig. 1D).

Article Snippet: Immediately after sampling, 500 µl of whole blood was mixed with 500 µl of RPMI and incubated with PerCp–labeled mouse-anti-human-CD14 (clone TüK4; Invitrogen Corporation, Breda, The Netherlands) together with FITC-labeled mouse-anti-human-TLR2 (clone TL2.1; eBioscience, Breda, The Netherlands) and PE-labeled mouse-anti-human-TLR4 (clone HTA 125; eBiosciences), or with anti-CD14 together with TLR2 and TLR4 isotype controls for 30 minutes at room temperature (RT) in the dark.

Techniques: Expressing, Fluorescence, Incubation

Journal: PLoS ONE

Article Title: Porphyromonas Gingivalis and E-coli Induce Different Cytokine Production Patterns in Pregnant Women

doi: 10.1371/journal.pone.0086355

Figure Lengend Snippet: Expression of TLR2 and TLR4 on monocytes of pregnant (open squares) and non-pregnant women (black squares). A: Percentage of TLR2 positive monocytes (left graph) and mean fluorescent intensity of TLR2 staining of monocytes (right graph). B: Percentage of TLR4 positive monocytes (left graph) and mean fluorescent intensity of TLR4 staining of monocytes (right graph). *: significantly increased vs pregnant women (student’s T test, p<0.05).

Article Snippet: Immediately after sampling, 500 µl of whole blood was mixed with 500 µl of RPMI and incubated with PerCp–labeled mouse-anti-human-CD14 (clone TüK4; Invitrogen Corporation, Breda, The Netherlands) together with FITC-labeled mouse-anti-human-TLR2 (clone TL2.1; eBioscience, Breda, The Netherlands) and PE-labeled mouse-anti-human-TLR4 (clone HTA 125; eBiosciences), or with anti-CD14 together with TLR2 and TLR4 isotype controls for 30 minutes at room temperature (RT) in the dark.

Techniques: Expressing, Staining

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 Hematology & Oncology

Article Title: Genetically modified "obligate" anaerobic Salmonella typhimurium as a therapeutic strategy for neuroblastoma

doi: 10.1186/s13045-015-0196-3

Figure Lengend Snippet: Sal-YB1 could upregulate TLR4 expression and downregulate IRAK and IκBα in tumor tissues. a Flow cytometry of TLR4. Dashed line , gray filled histogram, continuous line represented isotype control, YB1-untreated and -treated mice, respectively. Based on the isotype control, TLR4 expression in the YB1-treated mice was gated as 50.9 %. b IRAK and IκBα Western blotting. There was a significant difference for IRAK ( P < 0.05) and IκBα ( P < 0.001) between the YB1-treated and -untreated groups. The band density of IRAK and IκBα were normalized to β-actin. The error bars showed means ± SEM for three nude mice in each group. * P < 0.05 and *** P < 0.001 by an unpaired two-tailed t test

Article Snippet: The TLR4 expression induced by Sal-YB1 was detected by anti-human CD284 (TLR4) PE (HTA 125), using mouse IgG2a K isotype control PE (eBM2a) (Ebioscience, San Diego, USA).

Techniques: Expressing, Flow Cytometry, Control, Western Blot, Two Tailed Test

Journal: Journal of Hematology & Oncology

Article Title: Genetically modified "obligate" anaerobic Salmonella typhimurium as a therapeutic strategy for neuroblastoma

doi: 10.1186/s13045-015-0196-3

Figure Lengend Snippet: Possible mechanisms of Sal-YB1-induced cytotoxic effects on neuroblastoma. Bio-engineered Salmonella with the assistance of macrophages should contribute to the neuroblastoma reducing progression. TNFα release from macrophages is likely to be one of the cytotoxic components. Abbreviation: TLR4 toll-like receptor 4, MyD88 myeloid differentiation primary response gene 88, IRAK interleukin 1 receptor-associated kinase, TAK1 TGF-β-activated kinase 1, IκBα nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, IKK IκB kinase, NF-kB nuclear factor kappa B, TNFα tumor necrosis factor alpha, TNFR tumor necrosis factor receptor, TNFAIP6 tumor necrosis factor alpha-induced protein 6, TSG6 TNF-stimulated gene 6 protein, Casp . caspase

Article Snippet: The TLR4 expression induced by Sal-YB1 was detected by anti-human CD284 (TLR4) PE (HTA 125), using mouse IgG2a K isotype control PE (eBM2a) (Ebioscience, San Diego, USA).

Techniques: