Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Basal TLR4 expression by (A) primary adult baboon lung DCs and (B) KG-1-derived DCs under steady-state condition. Cell-surface expression of TLR4 was detected by flow cytometry after staining the cells with TLR4-specific antibody. The percent number and MFI values of cells stained with TLR4-specific antibody (TLR4) are compared with isotype control antibody-stained cells (I) in M1 region. (C): Western blot showing undetectable expression of TLR4 in 5 μg cell lysate protein of KG-1-derived DCs (KG1-DC). An equal amount of cell lysate protein of HEK293 cells stably transfected with TLR4 (HEK-TLR4) served as positive control.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Staining, Western Blot, Stable Transfection, Transfection, Positive Control

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Localization of exogenously-added recombinant TLR4-MD2 protein by confocal microscopy and flow-cytometry. Confocal microscopic images of KG-1-derived DCs pulsed with Alexa-fluor 594-conjugated recombinant TLR4-MD2 protein for (A) 1h and (B) 4h. Vybrant DiO (green) dye stains the cytoplasm, and Hoechst 33342 (blue) dye stains the nucleus of the cell. The images were acquired using 63 X objective. (C) Flow-cytometric charts of KG-1-derived DCs pulsed with Alexa-fluor 495-conjugated recombinant TLR4-MD2 protein after 1h (dark line) and 4h (dotted line). The histogram chart of non-pulsed cells (negative control) is shown under the black area. Cells were gated in M region. Percent number of cells (and MFI values) positive for fluorescence are shown within the chart. Results are representative of two experiments.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Recombinant, Confocal Microscopy, Flow Cytometry, Derivative Assay, Negative Control, Fluorescence

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A, recombinant TLR4-MD2 protein and MD2 protein on phagocytic function of KG-1-derived DCs. (A): Confocal microscopic images of KG-1-derived DCs incubated with pHrodo-labeled E. coli bioparticles for 3h. Phagocytosed bioparticles fluoresce red. Cells without phagocytosed particles and extracellular bacteria do not fluoresce. Enlarged images of a cell (shown as circle) are also shown in the figure, at different z-stack slices. (B): The extracellular bacteria that are either settled at the bottom or lie towards the top do not emit any fluorescence. These images confirm that fluorescence is of phagocytosed bioparticles. Next, KG-1-derived DCs were incubated with (C): purified baboon lung SP-A (0.2 and 2 μM), (D): recombinant TLR4-MD2 protein (0.06–0.6 μM) and functional-grade anti-human TLR4 antibody (HTA125 clone, Imgenex, CA; control reaction), (E): recombinant MD2 protein (0.02–0.2 μM), and (F): purified baboon lung SP-A (2 μM) and TLR4-MD2 protein (0.6 μM), for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically at 550 nm excitation and 600 nm emission wavelengths. Results are mean (SEM) of three different experiments. * p<0.05 or ns: not significant as compared to basal phagocytosis.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Derivative Assay, Incubation, Labeling, Bacteria, Fluorescence, Functional Assay

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of simultaneous addition of purified SP-A and recombinant TLR4-MD2 protein on phagocytic function of primary (A) adult baboon lung DCs and (B) fetal baboon lung DC-precursor cells. The DCs were incubated with respective proteins for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically. * p<0.05, ns: not significant or otherwise indicated. Results are mean (SEM) of three different experiments performed at different times.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Incubation, Labeling

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A and recombinant TLR4-MD2 proteins on TNF-α secretion by DCs against E. coli. (A) Primary adult baboon lung DCs or (B) fetal baboon lung DC-precursor cells were incubated with effector molecules for an hour prior to addition of pHrodo-labeled E. coli bioparticles. After 3h incubation at 37°C in 5% CO2 incubator, the cell-free supernatants were collected and subjected to ELISA for measurement of TNF-α. The results are representative of two experiments performed separately in triplicate. * p<0.05, ** p<0.001, ns: not significant.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Incubation, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: The ginsenoside metabolite compound K, a novel agonist of glucocorticoid receptor, induces tolerance to endotoxin-induced lethal shock

doi: 10.1111/j.1582-4934.2007.00181.x

Figure Lengend Snippet: Regulatory effect of C-K on the TLR4/LPS-induced secretion of proinflammatory cytokines in human monocytes. ( A ) Structure of the ginseno-side metabolite C-K. ( B ) Human monocytes were treated with increasing concentrations of C-K or a solvent control for 45 min before LPS stimulation (100 ng/ml). The supernatants were harvested after 18 hr and assessed for cytokine production by ELISA. The data shown are the mean ± SE of three experiments. Statistical differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001) compared to cell cultures without C-K pre-treatment are indicated. D, solvent control (0.1% Dimethyl sulfoxide [DMSO]); M, media control.

Article Snippet: Human embryonic kidney (HEK) 293 cells stably transfected with human TLR4, MD2 and CD14 (HEK/TLR4/MD2/CD14) or HEK 293 cells stably trans-fected with human TLR1/TLR2 (HEK/TLR1/TLR2) were purchased from InvivoGen (San Diego, CA).

Techniques: Solvent, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: The ginsenoside metabolite compound K, a novel agonist of glucocorticoid receptor, induces tolerance to endotoxin-induced lethal shock

doi: 10.1111/j.1582-4934.2007.00181.x

Figure Lengend Snippet: Regulatory effect of C-K on the TLR4/LPS-induced activation of MAPK and NF-κB signalling in murine macrophages. ( A ) Murine Bone marrow-derived macrophages (BMDMs) were stimulated with lipopolysac-charide (LPS) (100 ng/ml) at times indicated. ( B and C ) Murine BMDMs were treated with increasing concentrations of C-K or a solvent control for 45 min before LPS stimulation (100 ng/ml). The cells were harvested after 30 min (for Panel B) and 9 hr (for Panel C) of LPS stimulation, lysed with ice-cold lysis buffer, and subjected to Western blot analysis to detect the activation of mitogen-activated protein kinases (MAPKs) (p38 and ERK1/2) and NF-κB. The blots were stripped and re-probed for MAPKs, IαBβ, and phospho-IκB kinase-α/β. The data are representative of three separate experiments. D, solvent control (0.1% DMSO); M, media control.

Article Snippet: Human embryonic kidney (HEK) 293 cells stably transfected with human TLR4, MD2 and CD14 (HEK/TLR4/MD2/CD14) or HEK 293 cells stably trans-fected with human TLR1/TLR2 (HEK/TLR1/TLR2) were purchased from InvivoGen (San Diego, CA).

Techniques: Activation Assay, Derivative Assay, Solvent, Lysis, Western Blot