Journal: PloS one

Article Title: Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation.

doi: 10.1371/journal.pone.0002438

Figure Lengend Snippet: Figure 5. Modified alkane polymers induce activation of TLR-1 and TLR-2 signaling pathways. a) Luciferase activity expressed by human TLR1/2, TLR2, TLR3 and TLR4 stable HEK 3T3 transfectant (pNF-kB-LUC Stratagene). Cells were left untreated or treated for a different time period with 50 mg/ml of unPE, mPE or pre and post implant PE and respective positive controls; PGN(10 mg/ml) for TLR1/2 and TLR2, Poly (I:C)(1 mg/ml) for TLR3 and LPS (10 mg/ml) for TLR4. doi:10.1371/journal.pone.0002438.g005

Article Snippet: Similar experiments were conducted in presence of mouse anti human TLR2 mAb (clone 383936 R&D Systems) which is known to prevent ligand access to the TLR2 binding groove.

Techniques: Modification, Activation Assay, Protein-Protein interactions, Luciferase, Activity Assay, Transfection

Journal: PloS one

Article Title: Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation.

doi: 10.1371/journal.pone.0002438

Figure Lengend Snippet: Figure 6. Direct binding of oxidized alkane polymers to soluble TLR-2 molecules. a) Left panels; fluorescence emission scans collected for free soluble TLR2 and TLR2 in complex with two different concentrations (exponential and plateau) of each analyzed polymer. Central panels; normalized fluorescence data (DF) for each concentration point as a function of the free ligand concentration. Right panels; fluorescence emission scans collected for free soluble TLR2 and TLR2 in complex with each analyzed polymer at two different concentrations (exponential and plateau) in presence of an anti TLR2 mAb known to block the TLR2 binding groove (stoichiometric ratio 2:1 Ab to soluble TLR2 receptor). b) Comparison of the fluorescence emission scans collected for soluble TLR2, mPE and unPE. doi:10.1371/journal.pone.0002438.g006

Article Snippet: Similar experiments were conducted in presence of mouse anti human TLR2 mAb (clone 383936 R&D Systems) which is known to prevent ligand access to the TLR2 binding groove.

Techniques: Binding Assay, Fluorescence, Polymer, Concentration Assay, Blocking Assay, Comparison

Journal: Cancer Immunology, Immunotherapy

Article Title: Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation

doi: 10.1007/s00262-012-1341-2

Figure Lengend Snippet: Mechanisms of LPS-induced A549 proliferation. A549 cells were either sham-incubated ( control ) or exposed to 10 μg/ml of LPS ( n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of neutralizing antibodies targeting TLR2, TLR4, CD14 or EGFR. After 24 h of incubation proliferation was quantified by determining MTS activity. All data are expressed as percentage of unstimulated cells ( control ). Mean ± SEM of six independent experiments are given

Article Snippet: In an additional series of experiments in Fig. , function-blocking antibodies targeting TLR2 (clone TL2.1, e-Bioscience, San Diego, CA, USA), TLR4 (clone HTA 125, e-Bioscience, San Diego, CA, USA), CD14 (MY-4, Coulter Immunotech, Hamburg, Germany), EGFR (Cetuximab, Merck Serono, Germany) or COX inhibitors (indomethacin, Sigma, Deisenhofen, Germany and NS-398, Calbiochem, La Jolla, CA, USA) were applied simultaneously to LPS.

Techniques: Incubation, Control, Activity Assay

Journal: Cancer Immunology, Immunotherapy

Article Title: Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation

doi: 10.1007/s00262-012-1341-2

Figure Lengend Snippet: Mechanisms of LPS-induced PGE 2 synthesis. A549 cells were either sham-incubated ( control ) or exposed to 10 μg/ml of LPS ( n = 3 for LPS 0111:B4 and n = 3 for LPS F515, total n = 6) in the absence or presence of neutralizing antibodies targeting TLR2, TLR4, CD14 and EGFR, or the COX inhibitor indomethacin ( indo ) and the specific COX-2 inhibitor NS-398 for 24 h. 8 h before the end of the incubation period, and AA was added. PGE 2 release into the cell supernatant is given in pg/ml. Data are expressed as mean ± SEM of at least six independent experiments

Article Snippet: In an additional series of experiments in Fig. , function-blocking antibodies targeting TLR2 (clone TL2.1, e-Bioscience, San Diego, CA, USA), TLR4 (clone HTA 125, e-Bioscience, San Diego, CA, USA), CD14 (MY-4, Coulter Immunotech, Hamburg, Germany), EGFR (Cetuximab, Merck Serono, Germany) or COX inhibitors (indomethacin, Sigma, Deisenhofen, Germany and NS-398, Calbiochem, La Jolla, CA, USA) were applied simultaneously to LPS.

Techniques: Incubation, Control

Journal: Cells

Article Title: Role of TLR4 Receptor Complex in the Regulation of the Innate Immune Response by Fibronectin

doi: 10.3390/cells9010216

Figure Lengend Snippet: TLR4 mediates IL-8 expression in response to FnIII-1c and LPS in dermal fibroblasts. Monolayers of human dermal fibroblasts in 10% FBS/DMEM were treated for 24 h with ( A ) FnIII-1c or FnIII-13 (1-20 µg/mL), ( B ) LPS (1-100 ng/mL), ( C ) LPS (100 ng/mL) or FnIII-1c (10 µM) in the presence of the designated amounts of blocking antibody to TLR4 or TLR2. IgG served as control. ( D ) TNF-α (25 ng/mL), LPS (100 ng/mL) or FnIII-1c (10 µM) in the presence of increasing amounts of the TLR4 inhibitor, TAK-242. The wells without antibodies ( C ) or inhibitors ( D ) were set as 100%. IL-8 concentration in conditioned medium was determined by ELISA. The data represent the mean ± S.E. of triplicate assays from three separate experiments.

Article Snippet: Recombinant human CD14, human TNF-α, human IL-1α, anti-human MD-2 antibody, and neutralizing antibodies: anti-human CD14, anti-human TLR2 and anti-human TLR4 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Blocking Assay, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Soluble CEACAM8 Interacts with CEACAM1 Inhibiting TLR2-Triggered Immune Responses

doi: 10.1371/journal.pone.0094106

Figure Lengend Snippet: (a) FACS analysis of CEACAM1 and TLR2 (black lines) expression on the cell surface of NHBE cells. Grey lines represent isotype-matched control mAb staining. (b, c and d) ELISA for IL8 and IL6, respectively, in supernatants of NHBE cells incubated with Pam 3 Cys alone, with Pam 3 Cys plus soluble CEACAM8-Fc and rat-CEACAM1-Fc, respectively (100 ng/ml each) or left untreated for 16 h. (e) 50 ng purified protein of CEACAM8-Fc and ratCEACAM1-Fc were analyzed by SDS-PAGE and stained by Coomassie blue to demonstrate equal amount and integrity of the used fusion proteins. (f) FACS analysis CEACAM1 and TLR2 expression (black lines) on the surface of A549 cells. Grey lines represent the isotype-matched control. (g) IL-8 released into the supernatants of A549 cells pre-incubated for 1 h with anti-CEACAM1 (mAb 18/20) or left untreated followed by stimulation with Pam 3 Cys alone or with Pam 3 Cys and CEACAM8-Fc and ratCEACAM1-Fc, respectively (100 ng/ml) for 16 h. Samples were measured by commercial ELISA. IL8 secretion following treatment of the cells with Pam3Cys served as positive control. (h) A549 cells were challenged either with CEACAM8-Fc or with mAb 18/20 (20 ng/ml). The IgG antibody was used as control. Cell lysates were assessed after CEACAM1 immunoprecipitation with anti-phospho tyrosine mAb 4G10 (upper panel). Then membrane was stripped and re-probed with anti-CEACAM1 mAb (lower panel) to measure the amount of precipitated CEACAM1. Data presented in (a, e, f and h) are from one experiment representative of three independent experiments, and the data presented in (b, c, d and g) are mean ± s.e.m. of three different experiments performed in triplicates, * P<0.05.

Article Snippet: After blocking with 10% FCS in PBS for 10 min, 0.5×10 6 cells were incubated with 10 µg/ml mouse anti-TLR2 monoclonal antibody (mAb) (Acris Antibodies GmbH, Herford, Germany) or 3.5 µg/ml anti-CEACAM1 mAb 18/20 (B.B.

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Incubation, Purification, SDS Page, Positive Control, Immunoprecipitation

Journal: Innate Immunity

Article Title: Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses

doi: 10.1177/1753425911426591

Figure Lengend Snippet: Mycobacteria increase alveolar TLR expression. TLR2 and TLR4 expression on alveolar epithelial cells. (A) Alveolar epithelial cells were infected with mycobacteria for 3 d and stained with monoclonal anti-TLR2 or anti-TLR4 Ab and expression was analyzed with flow cytometry (A,B) and confocal microscopy (C). Unstimulated alveolar epithelial cells have low basal level expression of both receptors, with TLR4 more abundant than the TLR2. The mean fluorescence intensity (MFI) was calculated as an increase from negative control cells. Mycobacterial infection significantly increased epithelial TLR2 expression as detected by confocal microscopy and quantified by flow cytometry, but TLR4 expression was less affected. Confocal images showing alveolar epithelial TLR2 or TLR4 expression after 3 d of mycobacterial infection (green). Cellular DNA was counter-stained with propidium iodide (red). Original magnification x 300. (D) Alveolar epithelial TLR2 and TLR4 expression upon mycobacterial infection for up to 5 d was analyzed by Western blot. Epithelial cells express continuous TLR2 that was further increased in response to mycobacterial infection, while TLR4 expression was not affected. Data are presented as mean ± SEM of four separate experiments; ** P < 0.01, *** P < 0.001, TLR2 expression compared to TLR4 expression.

Article Snippet: Slides were incubated with anti-TLR2, anti-TLR4 or control IgG Ab (10 µg/ml; mouse IgG isotype control, R&D Systems) for 40 min at room temperature (23°C).

Techniques: Expressing, Infection, Staining, Flow Cytometry, Confocal Microscopy, Fluorescence, Negative Control, Western Blot

Journal: Innate Immunity

Article Title: Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses

doi: 10.1177/1753425911426591

Figure Lengend Snippet: TLR2 is important for neutrophil diapedesis. The impact of TLR2 and TLR4 on neutrophil diapedesis was investigated with the Transwell model. Alveolar epithelial cells were infected for 3 d with mycobacteria. After infection, epithelial cells were combined with endothelial cells and human neutrophils in the Transwell model and neutrophil diapedesis was recorded in the lower compartment after 3 h. Blocking with anti-TLR2 Abs or with both TLR2 and TLR4 Abs induced a significant decrease of mycobacteria-induced neutrophil diapedesis. Blocking of TLR4 did not have any impact. Data are presented as mean ± SEM of four separate experiments; ** P < 0.01, *** P < 0.001, compared with infected control.

Article Snippet: Slides were incubated with anti-TLR2, anti-TLR4 or control IgG Ab (10 µg/ml; mouse IgG isotype control, R&D Systems) for 40 min at room temperature (23°C).

Techniques: Infection, Blocking Assay

Journal: Innate Immunity

Article Title: Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses

doi: 10.1177/1753425911426591

Figure Lengend Snippet: TLR2 control cytokine secretion. Influence of TLR2 and TLR4 on mycobacteria induced cytokine secretion from neutrophils, monocytes and alveolar epithelial cells. The cells were treated with Abs against TLR2 and/or TLR4 before infection with live BCG. Secretion of CXCL8, IL-6, CCL2 and TNF-α was measured after 3 h for leukocytes and after 3 d for epithelial cells. Blocking of TLR2 significantly reduced the cytokine secretion of all the infected cells, while blocking of TLR4 had the highest inhibitory function on epithelial and monocyte cytokine secretion. Blocking of both receptors significantly reduced cytokine secretion of all the infected cells. Data are presented as mean ± SEM of four separate experiments; * P < 0.05, ** P < 0.01, *** P < 0.001, compared with infected control.

Article Snippet: Slides were incubated with anti-TLR2, anti-TLR4 or control IgG Ab (10 µg/ml; mouse IgG isotype control, R&D Systems) for 40 min at room temperature (23°C).

Techniques: Infection, Blocking Assay

Journal: Applied and Environmental Microbiology

Article Title: Piliation of Lactobacillus rhamnosus GG Promotes Adhesion, Phagocytosis, and Cytokine Modulation in Macrophages

doi: 10.1128/AEM.03949-14

Figure Lengend Snippet: Role of CD11b in adhesion and uptake of L. rhamnosus GG by RAW 264.7 cells and effect of TLR2 in the subsequent immunomodulatory response. (A) Role of CD11b in adhesion of the mutants to macrophages (left) and phagocytosis of the mutants by macrophages (right) relative to the results for the wild-type strain, which were set equal to 1. Bacterial strains (5 × 107 CFU/ml) were coincubated for 1 h with 1 × 106 RAW 264.7 cells that had previously been treated with anti-mouse CD11b monoclonal antibody (5 μg/ml) for 30 min. The data are presented as the mean relative adhesion or phagocytosis ± standard deviation. These experiments were done in triplicate. Asterisks represent statistically significant differences compared to the results for the controls (no antibody treatment). (B) The effect of TLR2 on the relative levels of IL-10 and IL-6 mRNA expression by RAW 264.7 macrophages was analyzed by pretreatment of macrophages with antibodies against TLR2 (5 to 10 μg/ml) for 30 min and then coincubation with L. rhamnosus GG strains for 3 h. Bacteria were administered at a ratio of 1:50 of cells to bacteria. The results are mean values ± standard deviations from three separate experiments. The values are normalized against those for GAPDH. Asterisks denote statistically significant differences from the results for the control (no antibody treatment).

Article Snippet: The blocking monoclonal anti-human/mouse CD282 TLR2 purified antibody (clones T2.5 and 6C2) and monoclonal anti-mouse CD11b purified antibody (clone M1/70) were obtained from eBioscience (San Diego, CA, USA).

Techniques: Standard Deviation, Expressing