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: Scientific Reports

Article Title: Discovery of new MD2 inhibitor from chalcone derivatives with anti-inflammatory effects in LPS-induced acute lung injury

doi: 10.1038/srep25130

Figure Lengend Snippet: ( A ) Chemical structure of compound 20. ( B ) Surface plasmon resonance (SPR) analysis shows the direct interaction between compound 20 and rhMD2 protein. ( C ) Fluorescence measurements show that compound 20 inhibited bis-ANS binding to rhMD2 in a dose-dependent manner. ( D ) Flow cytometry analysis as used to detect the effects of compound 20 on FITC-LPS binding to cell surface. ( E ) Compound 20 displaces the biotin-LPS binding to rhMD2 determined by MD2 cell-free ELISA assay. ( F ) Immunoprecipitation assay shows that compound 20 pretreatment at 10 μM significantly reduced the formation of TLR4-MD2 complex induced by 0.5 μg/mL LPS. The gels were run under the same experimental conditions and were shown as cropped gels/blots (Cropped gels/blots with indicated cropping lines are shown in ). ( G ) RAW264.7 macrophages after pretreatment with vehicle, compound 20 (5 or 10 μM), or TLR2 inhibitor (5 or 10 μM) for 30 min followed by incubation with Pam3CK (0.1 μg/mL) for 12 h. The protein level of TNF-α and IL-6 in the media was measured by ELISA and normalized to the respective total protein amount. ( H ) SPR analysis shows no direct interaction between compound 20 and rhTLR4 protein.

Article Snippet: Recombinant human MD2 (rhMD2) protein, Recombinant human TLR4 (rhTLR4) protein, and TLR2 inhibitor CU-CPT22 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: SPR Assay, Fluorescence, Binding Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Incubation

Journal: Scientific Reports

Article Title: Discovery of new MD2 inhibitor from chalcone derivatives with anti-inflammatory effects in LPS-induced acute lung injury

doi: 10.1038/srep25130

Figure Lengend Snippet: ( A ) Molecular docking of compound 20 with MD2 protein was carried out with the Sybyl-2.0 molecular modeling software from Tripos. Compound 20 did not inhibit biotin-LPS binding to ( B ) rhMD2 R90A/Y102A , ( C ) rhMD2 R90A and ( D ) rhMD2 Y102A determined by ELISA. SPR analysis showed no interaction between compound 20 and ( E ) rhMD2 R90A/Y102A , ( F ) rhMD2 R90A and ( G ) rhMD2 Y102A respectively. Data are mean values (±SEM) of at least three separate experiments.

Article Snippet: Recombinant human MD2 (rhMD2) protein, Recombinant human TLR4 (rhTLR4) protein, and TLR2 inhibitor CU-CPT22 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Software, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Discovery of new MD2 inhibitor from chalcone derivatives with anti-inflammatory effects in LPS-induced acute lung injury

doi: 10.1038/srep25130

Figure Lengend Snippet: SD rats were treated intragastrically with compound 20 at a dosage of 20 mg/kg b.wt for one week, then challenged with 5 mg/kg LPS. Rats were euthanized with ketamine after 6 h of LPS induction. ( A ) Protein concentration in BALF. ( B ) Lung Wet/Dry ratio. ( C ) Histopathological changes in lung tissues determined by H&E staining. ( D , E ) Number of total cells and neutrophils in BALF. ( F ) Macrophage infiltration in lung tissue measured by CD68 immunohistochemical staining. ( G ) Neutrophils activity in lung tissue determined by MPO activity. ( H – J ) Expression of inflammatory genes in lung tissue using QPCR. ( K ) Immunoprecipitation assay shows that compound 20 significantly reduced the formation of TLR4-MD2 complex in lung tissue induced by LPS. Data are mean values (±SEM) of 3–5 separate experiments. *p < 0.05 and **p < 0.01 vs. only-LPS stimulated group. The gels were run under the same experimental conditions. Shown are cropped gels/blots (Cropped gels/blots of 5 K with indicated cropping lines are also shown in ).

Article Snippet: Recombinant human MD2 (rhMD2) protein, Recombinant human TLR4 (rhTLR4) protein, and TLR2 inhibitor CU-CPT22 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Protein Concentration, Staining, Immunohistochemical staining, Activity Assay, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: Simultaneous Control of Infection and Inflammation by Keratin-Derived Antibacterial Peptides (KAMPs) Targeting TLRs and Co-Receptors

doi: 10.1101/2021.01.18.427180

Figure Lengend Snippet: ( A-C ) Molecular docking of KAMP-10 and KAMP-18C (ribbon representation) to TLRs and their co-receptors (ribbon and surface representations). Top five models of the peptides (1 to 5: yellow, green, red, magenta, cyan) docked to the receptors are shown. (A) KAMP-10 was predicted to bind to the hydrophobic cavity of MD-2 that is in complex with TLR4 (top) or in the soluble form (bottom). (B-C) KAMP-18C was predicted to bind to hydrophobic pocket at the central domain of TLR2 (B) and at the N-terminal domain of CD14 (C). ( D ) Representative FACS plots (left) and quantification (right) showing the extent of LPS binding to TLR4/MD-2-conjugated beads. Latex beads with human TLR4-MD-2 complex conjugated on the surface were incubated with the peptides (KAMP-10 or SC-10 at 48 μg/ml, or KAMP-18C at 96 μg/ml) in the absence or presence of soluble MD-2 (1 μg/ml), washed, then incubated with FITC-LPS (1 μg/ml). Bead surface TLR4 was labeled with antibody followed by flow cytometry. Percent of TLR4-MD-2 + beads that were FITC-LPS + is shown. ( E-F ) Representative FACS plots (top) and quantification (bottom) showing the extent of LTA binding to TLR2-conjugated beads. (E) Human TLR2-conjugated beads were incubated with the peptides (48 or 96 μg/ml), washed, then incubated with LTA (1 μg/ml) and CD14 (0.1 μM). (F) CD14 (0.1 μM) was preincubated with equal molar of peptides (0.1 μM) before TLR2-conjugated beads and LTA (1 μg/ml) were added. In comparison, KAMP-10 alone (0.1 μM) was preincubated with TLR2-conjugated beads, followed by bead washing then incubation with LTA (1 μg/ml) and CD14 (0.1 μM). TLR2 and LTA were labeled with antibody for flow cytometric analysis. Percent of TLR2 + beads that were FITC-LTA + is shown. Mean (n=3 replicates) ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (ANOVA with Dunnett’s post hoc test).

Article Snippet: To assess KAMPs binding to TLR4/MD-2, TLR4/MD-2 complex-conjugated beads (5,000 in FACS buffer) were preincubated with KAMP-10 (48 μg/ml), KAMP-18C (96 μg/ml) or SC-10 (48 μg/ml) in the presence or absence of recombinant human MD-2 (R&D) (1 μg/ml) for 1 h on ice.

Techniques: Binding Assay, Incubation, Labeling, Flow Cytometry