Journal:

Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

doi: 10.1128/IAI.00004-09

Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P < 0.002 compared to control (Mann-Whitney U test, n = 6).

Article Snippet: For specificity control, some supernatants were preincubated with either 50 μg/ml LPS ( Salmonella enterica serovar Minnesota; Sigma), which has a high binding activity for TLR4/MD-2, or 10 μg/ml anti-mouse TLR4/MD-2 MTS510 MAb (Hycult Biotechnology, Uden, The Netherlands) for 1 h at room temperature.

Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Biochemical and biophysical research communications

Article Title: Platelet-derived high-mobility group box 1 promotes recruitment and suppresses apoptosis of monocytes

doi: 10.1016/j.bbrc.2016.07.078

Figure Lengend Snippet: HMGB1 inhibits monocyte apoptosis induced by ABT-737 (A,C) or staurosporine (B,D), which is reversed when monocytes are pretreated with a blocking TLR4 antibody, as evaluated by Annexin V (A,B) and TMRE (C,D) stainings. (E) U0126, a specific MEK/ERK inhibitor, inhibits the effect of rHMGB1 on TMRE fluorescence in monocytes. (F) rHMGB1 (100 ng/ml) induces phosphorylation of ERK in monocytes, which does not occur when monocytes are pretreated with a blocking TLR4 antibody. Data are presented as mean ± SD for N≥4 and at least three separate experiments in all studies. * p<0.05, # p<0.05, ** p<0.01 (Student’s t test).

Article Snippet: When indicated, HMGB1 receptors were blocked on monocytes with anti-human RAGE polyclonal antibody (20 μg/ml, goat IgG), anti-human TLR2 monoclonal antibody (2 μg/ml, mouse IgG2b) or anti-human TLR4 polyclonal antibody (10 μg/ml, goat IgG) (R&D Systems, Wiesbaden, Germany).

Techniques: Blocking Assay, Fluorescence, Phospho-proteomics

Journal: Kidney international

Article Title: Reduced expression of Toll-like receptor 4 contributes to impaired cytokine response of monocytes in uremic patients.

doi: 10.1038/sj.ki.5001548

Figure Lengend Snippet: Figure 2 | Flow cytometric analysis of TLR4 in peripheral monocytes. The light scatter and fluorescence channels were set at a logarithmic gain to identify monocyte population by cell size and granularity. Cells that were positive for CD14 FITC mAb were gated on the SSC-FL1 plot as described in Materials and Methods. The cells labeled with anti-TLR4 PE mAb and anti-CD14 FITC mAb were profiled on the FL1–FL2 plot and compared to the samples that had been treated with anti-CD14 FITC mAb and isotype irrelevant IgG PE ((a) control IgG and (b) TLR4 mAb). Cutoff quadrant markers based on the negative control were set individually for each measurement. The percentage of cells that were positive for anti-TLR4 mAb (frequency) and their MFI were calculated on the histogram for the assessment ((c) control IgG and (d) anti-TLR4 mAb).

Article Snippet: PE-labeled anti-human TLR4 mAb (HTA-125, mouse IgG2a) was obtained from Santa Cruz Biotechnology (CA, USA).

Techniques: Fluorescence, Labeling, Control, Negative Control

Journal: Scientific Reports

Article Title: TLR4 antagonist FP7 inhibits LPS-induced cytokine production and glycolytic reprogramming in dendritic cells, and protects mice from lethal influenza infection

doi: 10.1038/srep40791

Figure Lengend Snippet: DCs were seeded at 1 × 10 6 cells/ml in fresh medium and stimulated for 24 h with increasing amounts of LPS, in the absence or presence of 10 μM FP7 ( a , b ). Control cells (Ctl) received solvent instead of FP7. Glucose consumption and lactate production were monitored using enzymatic detection kits. Means ± SEM from 5 or 6 experiments are shown. ( c ) DCs were incubated with 10 μg/ml TLR4-blocking antibody or isotype control antibody 1 h prior to LPS addition. Glucose consumption was normalized to non-stimulated control DCs and means ± SEM from 3 experiments are shown. *P < 0.05; # P < 0.01 FP7 compared to Ctl cells.

Article Snippet: Neutralizing mouse IgG monoclonal anti-human TLR4 and non-blocking control antibodies were kindly provided by Dr. F. Neumann, Innaxon Biosciences.

Techniques: Control, Solvent, Incubation, Blocking Assay

Journal: Critical Care

Article Title: Development of a point-of-care-testing system for procalcitonin

doi: 10.1186/cc11776

Figure Lengend Snippet: Figure 1(abstract P80) Influence of antibodies against TLR4, CD14 and CD11b on neutrophil phagocytic activity in whole blood. Control cells without any incentives (C), cells activated by S-LPS (C1) and cells activated by Re-LPS (C2).

Article Snippet: Fluorescein-labeled bioparticles E. coli K12 (Molecular Probes), mouse antihuman TLR4 mAbs HTA125 (IgG2a isotype; Serotec), anti-human CD14 clone UCHM-1 mAbs (IgG2a isotype; Sigma), and anti-human CD11b mAbs clone 44 (IgG1 isotype; Sigma).

Techniques: Activity Assay, Control

Journal: PLoS ONE

Article Title: Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-κB Pathway

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: (A-B) High pulsatility flow (HPF), due to the use of a “stiff” tube upstream to cell culture, upregulated proinflammatory molecule mRNA (ICAM-1, VCAM-1, MCP-1 and E-selectin) and protein (MCP-1) in healthy PAECs, compared to low pulsatility flow (LPF) or to static conditions. (C-D) The mRNA and protein expression of TLR2 but not TLR4 in PAECs was highly upregulated by HPF. *: p<0.05 versus Static, †: p<0.05 versus LPF.

Article Snippet: Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO).

Techniques: Cell Culture, Expressing

Journal: PLoS ONE

Article Title: Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-κB Pathway

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: (A, C) At the mRNA level, TLR2/4 inhibitor OxPAPC or TLR2 siRNA but not TLR4 inhibitor CLI-095, decreased PAEC expression of ICAM-1, VCAM-1, MCP-1 and E-selectin mRNAs under HPF; TLR4 siRNA decreased ICAM-1 and E-selectin but not VCAM-1 and MCP-1 mRNAs. “*”: p<0.05 versus LPF, “†”: p<0.05 versus HPF. (B, D) At the protein level, the MCP-1 expression in PAECs exposed to HPF was inhibited by OxPAPC or TLR2 siRNA treatment but not CLI-095 or TLR4 siRNA. The black line in the blot images (D, right) shows separated lanes obtained on the same gel.

Article Snippet: Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-κB Pathway

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: (A) PAECs from calves with hypoxia-induced pulmonary hypertension (PH) show elevated expression of TLR2 and TLR4, compared to control (CO). *:p<0.05. (B) Both immunostaining and western blotting results show elevated MCP-1 expression by PH-ECs compared to CO-ECs from calves. “PA” indicates the lumen of a pulmonary artery. *:p<0.05. (C) Enhanced TLR2 expression in the pulmonary arterial endothelium of human with pulmonary arterial hypertension (PAH). Cryosections of human intra-lobar pulmonary arteries were immunostained with TLR2 (red fluorescence) and counterstained with DAPI (cell nuclei, blue). Elastic lamellae showed green auto-fluorescence.

Article Snippet: Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO).

Techniques: Expressing, Immunostaining, Western Blot, Fluorescence