Journal: PLoS Medicine

Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: (A) Sera of patients with active CD contain IgA antibodies directed against MPMR2; such reactivity is not present in patients on GFD. A K562 cell lysate was probed with rabbit antiserum raised against a peptide (VEKIGGASSRGE) of the MPMR2 (Lane 1), with antibodies affinity-purified against the celiac peptide (lane 2), with antibodies affinity-purified against an irrelevant control peptide (Lane 3), with sera from patients with active disease on GCD (Lanes 4, 6, and 8), and with sera from the same patients on GFD (Lanes 5, 7, and 9). A peroxidase-labelled polyvalent anti-human Igs antibody (Lanes 2 and 3) and an anti-human IgA antibody (Lanes 4–9) were used for detection. (B) Cell lysate from untransfected 293T cells was probed with the monoclonal antibody against TLR4 (Lane 1). Cell lysate from 293T cells transfected with the human TLR4 gene was probed with a monoclonal antibody directed against TLR4 (Lane 2), and with antibodies affinity-purified against the celiac peptide (Lane 3). Cell lysate from 293T cells transfected with the TLR4 gene was probed with biotin-labelled anti-TLR4 monoclonal antibody (Lane 4). Cell lysate from 293T cells transfected with the TLR4 gene was probed first with affinity-purified anti-peptide antibodies, followed by an incubation with biotin-labelled anti-TLR4 monoclonal antibody (Lane 5). Cell lysate from human plasmocytoid dendritic cells was probed with the monoclonal antibody against TLR4 (Lane 6). Cell lysate from human monocytes was probed with the monoclonal antibody against TLR4 (Lane 7) and with affinity-purified anti-peptide antibodies (Lane 8). (C) Inhibition of binding of anti-celiac peptide antibodies to solid-phase TLR4 peptide by liquid-phase TLR4 peptide (blue line), by celiac peptide (red line), and VP-7 peptide (red line), but not by an irrelevant control peptide (green line). The y -axis represents percentage of inhibition, and the x -axis indicates inhibitor concentration (μg/ml).

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Affinity Purification, Transfection, Incubation, Inhibition, Binding Assay, Concentration Assay

Journal: PLoS Medicine

Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: Levels of IL-6 (A), IL-12 (B), and TNF-alpha (C) released in the supernatant by monocytes incubated with medium alone (Line 1), with antibodies directed against an irrelevant peptide (Line 2), with LPS (Line 3), with pooled Igs isolated from the 22 patients with active CD (Line 4), with pooled Igs isolated from the same patients on GFD (Line 5), with purified anti-celiac peptide antibodies obtained from ten patients (line 6), with purified anti-celiac peptide antibodies in the presence of an irrelevant mouse IgG2b antibody (20 μg/ml) (Line 7), and with purified anti-celiac peptide antibodies in the presence of the neutralizing mouse monoclonal antibody anti-TLR4, clone HTA 125 (20 μg/ml) (Line 8). The y -axis represents the cytokine concentration expressed as pg/ml. Data represent the mean ±SD of three independently performed experiments.

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Incubation, Isolation, Purification, Concentration Assay

Journal: PLoS Medicine

Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: Activation of NF-κB upon engagement of TLR4. (A) Stimulation of 293T cells transfected with TLR4 by LPS 100 ng/ml (1), 10 ng/ml (2), 1 ng/ml (3), 0.1 ng/ml (4); by affinity-purified anti-celiac peptide antibodies, 4 μg/ml (5), 2 μg/ml (6), 1 μg/ml (7), 0.5 μg/ml (8) by affinity-purified antibodies directed against an irrelevant control peptide, 4 μg/ml (9), 2 μg/ml (10), 1 μg/ml (11), and 0.5 μg/ml (12). (B) Stimulation of 293T cells transfected with TLR4 by LPS 100 ng/ml (1), 10 ng/ml (2), 1 ng/ml (3), 0.1 ng/ml (4), by affinity-purified antibodies against an irrelevant peptide 1 μg/ml (5), by affinity-purified anti-celiac peptide antibodies 1 μg/ml (6), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml anti-TLR4 monoclonal antibody (7), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml celiac peptide (8), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml irrelevant control peptide (9), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml recombinant human tTG (10), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml (11), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml in the presence of 1 μg/ml VP-7 peptide (12), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml in the presence of 1 μg/ml celiac peptide (13), and by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml ovalbumin (14). Results are expressed as percentage of positive control, where the positive control is the OD value obtained upon stimulation of TLR4 transfected cells with 100 ng/ml LPS (maximal concentration used).

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Activation Assay, Transfection, Affinity Purification, Recombinant, Positive Control, Concentration Assay

Journal: PLoS Medicine

Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: (A) Sequence homology between the celiac peptide and cell junction proteins. The peptide sequence was compared with known protein sequences using the BLASTP via the NCBI BLAST network service (colons indicate identity and asterisks indicate conservative substitutions). (B) Sera of patients with active CD contain IgA antibodies directed against desmoglein 1; such reactivity is not present in patients on GFD. Recombinant desmoglein 1 was probed with antibodies affinity purified against the celiac peptide (Lane 1), with antibodies affinity purified against an irrelevant control peptide (Lane 2), with sera from patients with active disease on GCD (Lanes 3 and 5), and with sera from the same patients on GFD (Lanes 4 and 6). Biotin-labelled primary antibodies followed by peroxidase-labelled avidin (Lanes 1 and 2) and an anti-human IgA antibody (Lanes 3–6) were used for detection. (C) The binding of affinity-purified anti-celiac peptide antibodies to solid-phase desmoglein 1 is inhibited by desmoglein 1 (blue line), tTG (red line), celiac peptide (purple line), VP-7 peptide (yellow line), and TLR4 peptide (green line), but not by the irrelevant control peptide (orange line). The y -axis represents percentage of inhibition, and the x -axis indicates inhibitor concentration (μg/ml).

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Sequencing, Recombinant, Affinity Purification, Avidin-Biotin Assay, Binding Assay, Inhibition, Concentration Assay

Journal: PLoS Medicine

Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: Cumulative [ 3 H] mannitol flux following treatment with pooled antibodies against the celiac peptide (▪) or against the control peptide (♦) purified from ten patients. Purified antibodies were applied to T84 cells in the presence of [ 3 H] mannitol (5 μCi/ml) in the basolateral compartment. Apical buffer was sampled every 30 min over 6 h. * indicates statistically significant difference in mannitol flux at 3 h ( p = 0.01) and at 5 h ( p = 0.004). Representative example of three independently performed experiments. (B) Confluent T84 monolayers were treated for 3 h with pooled Igs from healthy individuals (1), affinity-purified antibodies against the control peptide from five patients (2), pooled antibodies affinity purified against the celiac peptide from ten patients (3), pooled antibodies affinity purified against tTG and negative for anti-desmoglein 1 activity from five patients (4), pooled antibodies affinity purified against the desmoglein peptide from five patients (5), pooled antibodies affinity purified against the VP-7 peptide from ten patients (6), pooled antibodies affinity purified against the TLR4 peptide from seven patients (7), pooled Igs from the 22 patients with active CD (8), pooled Igs from the same patients on GFD (9), and TNF-alpha (250 U/ml) (10). (C) Confluent T84 monolayers were treated for 3 h with pooled Igs from healthy individuals (1), affinity-purified antibodies against the control peptide (2), antibodies affinity purified against the celiac peptide (3), and antibodies affinity purified against the celiac peptide in the presence of: human recombinant tTG (4), celiac peptide (5), recombinant desmoglein (6), and an irrelevant peptide (7). Data are mean percentages of control (untreated sample) ±SD. n = 6 in duplicates.

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Purification, Affinity Purification, Activity Assay, Recombinant

Journal: PLoS Medicine

Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: (A) Binding of rabbit anti-VP-7 antibodies to tTG (blue line). Red line indicates pre-immune rabbit serum. (B) Binding of rabbit anti-VP-7 antibodies to VP-7 peptide (blue line), celiac peptide (red line), desmoglein peptide (yellow line), and TLR4-peptide (green line). Purple line indicates binding to the irrelevant control peptide. (C) TLR4 activation by LPS (100 ng/ml) (1), affinity-purified human anti-celiac peptide antibodies (2), rabbit anti-VP-7 antibodies (3), and pre-immune rabbit serum (4). Results are expressed as percentage of positive control, where the positive control is the OD value obtained upon stimulation of TLR4 transfected cells with 100 ng/ml LPS (maximal concentration used). (D) Confluent T84 monolayers were treated for 3 h with control normal human Ig (1), affinity-purified human antibodies to the irrelevant control peptide (2), affinity-purified human anti-celiac peptide antibodies (3), rabbit anti-VP-7 antibodies (4), and pre-immune rabbit serum (5).

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Binding Assay, Activation Assay, Affinity Purification, Positive Control, Transfection, Concentration Assay

Journal:

Article Title: Interleukin 8 Secretion from Monocytes of Subjects Heterozygous for the ?F508 Cystic Fibrosis Transmembrane Conductance Regulator Gene Mutation Is Altered

doi: 10.1128/CDLI.11.5.819-824.2004

Figure Lengend Snippet: FACS analysis of monocyte CD14 and Toll-like receptor 4 (TLR-4) expression. The levels of expression of both CD14 (A and B) and Toll-like receptor 4 (C and D) were similar in CF and healthy control monocytes under both basal (A and C) and LPS-stimulated (B and D) conditions.

Article Snippet: Antibodies and cytokines were purchased from the following sources: recombinant human IL-8, anti-human IL-8, and IL-1β from R&D Systems (Minneapolis, Minn.); mouse anti-human CD14 from Biosource International (Camarillo, Calif.); mouse anti-human TLR4 from Serotec Inc. (Raleigh, N.C.); and purified mouse immunoglobulin G1 (IgG1) and mouse IgG2a from BD Biosciences (San Jose, Calif.).

Techniques: Expressing

Journal: mBio

Article Title: TLR4 sensing of IsdB of Staphylococcus aureus induces a proinflammatory cytokine response via the NLRP3-caspase-1 inflammasome cascade

doi: 10.1128/mbio.00225-23

Figure Lengend Snippet: IsdB induces proinflammatory cytokine release via the TLR4-MyD88 signaling cascade. ( a ) Human monocytes were left untreated or pre-treated with CLI-095 (1 µM) for 45 min before treatment with either IsdB (10 µg/mL) or LPS (100 ng/mL) for an additional 24 h. n = 5. Cell-free supernatants were analyzed for human IL-6 by ELISA. ( b ) Wild-type (WT) mBMDCs were left untreated or pre-treated with CLI-095 (1 µM, 45 min) prior to treatment with IsdB (10 µg/mL) or LPS (1 ng/mL) for an additional 6 h. n = 4 except for the LPS + inhibitor group ( n = 2). ( c ) WT, TLR4-, and MyD88-KO mBMDCs were left untreated or stimulated with IsdB (10 µg/mL), Lac-IsdB (10 µg/mL), or LPS (1 ng/mL) for 6 h. Cell-free supernatants were analyzed for the release of mouse IL-6 by ELISA. n = 5 WT and n = 3 TLR4-KO and MyD88-KO. ( d ) Molecular docking of hTLR4 and IsdB: red surface on hTLR4 represents the areas of interaction with IsdB. A representative hTLR4-IsdB complex (molecular model 2) demonstrates potential recognition. ( e ) Dim plot analysis represents non-covalent hydrophobic interactions between amino acid residues in the TLR4/IsdB complex. Dotted lines show hydrogen bonds. ( f ) Microscale thermophoresis (MST) analysis between recombinant human TLR4 (rhTLR4) and IsdB or Lac-IsdB or LPS: IsdB and Lac-IsdB shows strong binding toward rhTLR4 with an estimated dissociation constants (Kds) of 98.3 and 153 nM, respectively. In contrast, LPS failed to exhibit high-affinity binding in this assay due to the absence of MD-2. Kd was calculated using NanoTemper Affinity Analysis software. n = 3. ( g ) An ELISA plate was coated overnight at 4°C with recombinant TLR4 (0.5 µg/mL) and incubated with either IsdB (1 µg/mL) or IsdB preincubated with increasing molar ratios of the indicated antibodies, at Room Temperature (RT) for 2 h. The IsdB binding was detected using Avidin-IgG conjugated to Horseradish peroxidase (HRP) and the Tetramethylbenzidine (TMB) substrate, and the optical density (OD) 450 was measured using a microplate reader. The bar graph displays the OD values corresponding to the 1:10 molar IsdB:antibody ratio. n = 3. ( h ) Human peripheral blood mononuclear cells (PBMCs) were left untreated or stimulated with IsdB (10 µg/mL) or IsdB preincubated with increasing molar ratios of a human monoclonal anti-IsdB antibody for 6 h. Data are represented as mean ± SEM of indicated biological replicates (“ n ”) performed in technical duplicate or triplicate. Each point represents one donor (human monocytes) or biological replicate (mBMDCs). One-way ANOVA or two-way ANOVA was utilized to determine statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 represent IsdB or LPS vs untreated cells. ## P < 0.01 IsdB vs IsdB + inhibitor (S). $$ P < 0.01 and $$$$ P < 0.0001 represent LPS vs LPS + inhibitor (S). CLI-095, TLR4 inhibitor; Fnorm, normalized fluorescence; PK, proteinase K. ϕ represents respective controls or unstimulated cells.

Article Snippet: Anti-TLR4 antibody, ATP, Bay 11-0782 (NF-κB signaling inhibitor), CLI-095 (TLR4 inhibitor), Cytochalasin D (phagocytosis inhibitor), LPS-EB ultrapure ( E. coli O111:B4B), MCC950 (NLRP3 inhibitor), MSU, Pepinh-MyD88 (MyD88-inhibitory peptide), Pepinh-TRIF (TRIF inhibitor), YvAD (caspase-1 inhibitor), and ZvAD (pan-caspase inhibitor) were purchased from InvivoGen (San Diego, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Microscale Thermophoresis, Recombinant, Binding Assay, Software, Incubation, Avidin-Biotin Assay, Fluorescence

Journal: mBio

Article Title: TLR4 sensing of IsdB of Staphylococcus aureus induces a proinflammatory cytokine response via the NLRP3-caspase-1 inflammasome cascade

doi: 10.1128/mbio.00225-23

Figure Lengend Snippet: IsdB activates the NLRP3 inflammasome to generate IL-1β. ( a ) mBMDCs were left unprimed or primed with either IsdB (10 µg/mL) or LPS (1 ng/mL) for 3 h followed by treatment with ATP (5 mM) or MSU (200 µg/mL) for additional 6 h, n = 3. ( b ) IsdB- or LPS-primed mBMDCs were incubated with or without MCC950 (5 µM) for 45 min followed by treatment with ATP (5 mM) or MSU (200 µg/mL) for additional 6 h, n = 3. Cell-free supernatants in a and b were analyzed for mouse IL-1β release by ELISA. ( c ) Human monocytes were left unstimulated or stimulated with IsdB (10 µg/mL) or Lac-IsdB (10 µg/mL) or LPS (100 ng/mL) for 24 h, n = 3. The amount of secreted IL-1β in cell culture supernatants was measured by ELISA or visualized by western blot, and the pro-IL-1β in cell lysates was detected by western blot. ( d–i ) Human monocytes were left untreated or pre-treated with the indicated signaling inhibitors for 45 min except for Pepinh-MYD (6 h) ( h ) prior to treatment with IsdB (10 µg/mL) for an additional 24 h. MCC950 (5 µM) n = 4, ZvAD (20 µM) n = 4, YvAD (30 µg/ml) n = 2, CLI-095 (1 µM) n = 3, Pepinh-MYD (50 µM) n = 4, or Bay11-0782 (10 µM) n = 4. In experiments d–f, MSU (200 µg/mL) served as a positive control. In experiments g–h, LPS (100 ng/mL) served as a positive control. IL-1β levels in the supernatants were determined by ELISA. Data are displayed as mean ± SEM of indicated biological replicates (“ n ”) performed in technical duplicate or triplicate. Each point represents one donor (human monocytes) or biological replicate (mBMDCs). In , one-way ANOVA (IsdB) or paired t -test (Lac-IsdB) was utilized to compare the statistics between the groups. Two-way ANOVA was utilized to determine statistical significance between the groups in . * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 represent IsdB or LPS or MSU vs untreated cells or respective controls. # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 represent IsdB vs IsdB + inhibitor (S), IsdB vs IsdB + ATP, or MSU. $ P < 0.05 represents MSU vs MSU + inhibitor (S). ATP, adenosine triphosphate; Bay11-0782, NF-κB inhibitor; CLI-095, TLR4 inhibitor; MCC950, NLRP3 inhibitor; MSU, monosodium urate; Pepin-MYD, MyD88 inhibitor. ϕ represents respective controls or unstimulated cells.

Article Snippet: Anti-TLR4 antibody, ATP, Bay 11-0782 (NF-κB signaling inhibitor), CLI-095 (TLR4 inhibitor), Cytochalasin D (phagocytosis inhibitor), LPS-EB ultrapure ( E. coli O111:B4B), MCC950 (NLRP3 inhibitor), MSU, Pepinh-MyD88 (MyD88-inhibitory peptide), Pepinh-TRIF (TRIF inhibitor), YvAD (caspase-1 inhibitor), and ZvAD (pan-caspase inhibitor) were purchased from InvivoGen (San Diego, USA).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The PPE18 of Mycobacterium tuberculosis interacts with TLR2 and activates IL-10 induction in macrophage.

doi: 10.4049/jimmunol.0901367

Figure Lengend Snippet: FIGURE 2. The recombinant PPE18 protein specifically binds to TLR2 receptor on macrophage to stimulate IL-10 production. A, PMA-differentiated THP-1 macrophages were either left untreated or incubated for 10–15 min with increasing concentrations of either rPPE18-biotin or rPPE18-biotin preincubated with F(ab)2 of either anti-PPE18 Ab or NMS. Cells were washed and incubated with streptavidin-FITC. The amount of bound rPPE18 was measured by flow cytometry. B, Binding efficiency of rPPE18 at a concentration of 3 g/ml was compared between undifferentiated and PMA-differentiated THP-1 macrophages by flow cytometry. C, IL-10 induction by rPPE18 at concentration of 3 g/ml was compared between undifferentiated and PMA- differentiated THP-1 macrophages. D, IL-10 production in PMA-differentiated THP-1 macrophages stimulated with rPPE18 (3 g/ml) in the presence of titrating concentrations of anti-TLR2 or anti-TLR4 or anti-CD14 or isotype (IgG2a) control Ab. Results are mean SD of three different experiments.

Article Snippet: For TLR2 and TLR4 staining, cells were treated (30 min at 4°C) with anti-human TLR2 mAb (Imgenex) or anti-human TLR4 mAb HTA125 (mouse IgG2a) (Imgenex) or isotype control IgG2a and then incubated with FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich) at 4°C for a further 30 min. To examine PPE18 binding, cells (0.5–1 106) were incubated at room temperature with increasing concentrations of rPPE18 conjugated to biotin (PPE18-biotin) for 10–15 min. For competition assay for binding of rPPE18, cells were preincubated with 100 and 10-fold excess of Pam3CSK4 (Calbiochem) for 15 min on ice followed by incubation with rPPE18-biotin.

Techniques: Recombinant, Incubation, Cytometry, Binding Assay, Concentration Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The PPE18 of Mycobacterium tuberculosis interacts with TLR2 and activates IL-10 induction in macrophage.

doi: 10.4049/jimmunol.0901367

Figure Lengend Snippet: FIGURE 3. PPE18 interacts with TLR2 but not with other TLRs. A, Cell surface expression of TLR2 (i) and TLR4 (ii) in HEK293 cells transfected with either WT-TLR2 or WT-TLR4 as measured by flow cytometry. In parallel experiment, binding of rPPE18 to TLR2- (iii) or TLR4- (iv) transfected HEK293 cells was assessed by flow cytometry. B, Lysates from HEK293 cells transfected with either the WT-TLR2 or the backbone vector (pcDNA3.1) were incubated with rPPE18 immobilized with TALON resin. The bound protein was extracted and loaded along with the whole cell extracts from HEK293 cells transfected with either WT-TLR2 or pcDNA3.1 vector alone and immunoblotted with 1–2 g/ml anti-TLR2 Ab. C, Cell lysates from PMA-differentiated THP-1 macrophages were incubated with rPPE18 immobilized with TALON. The bound protein (lane 1) was extracted and loaded along with the whole cell extracts from THP-1 macrophages as positive control (lane 2) and immunoblotted with 1–2 g/ml Ab to either TLR1 or TLR2 or TLR4 or TLR6. The membranes were incubated with anti-rabbit IgG-HRP and the blots were visualized by chemiluminescence. D, Human MDM were pretreated with either anti-TLR2 blocking Ab (20 g/ml) or isotype control Ab (20 g/ml) followed by incubation (10–15 min) with biotinlyated rPPE18 (3 g/ml). The cells were washed and incubated with steptavidin-FITC. The amount of bound rPPE18 was measured by flow cytometry. Data are representative of three to four independent experiments.

Article Snippet: For TLR2 and TLR4 staining, cells were treated (30 min at 4°C) with anti-human TLR2 mAb (Imgenex) or anti-human TLR4 mAb HTA125 (mouse IgG2a) (Imgenex) or isotype control IgG2a and then incubated with FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich) at 4°C for a further 30 min. To examine PPE18 binding, cells (0.5–1 106) were incubated at room temperature with increasing concentrations of rPPE18 conjugated to biotin (PPE18-biotin) for 10–15 min. For competition assay for binding of rPPE18, cells were preincubated with 100 and 10-fold excess of Pam3CSK4 (Calbiochem) for 15 min on ice followed by incubation with rPPE18-biotin.

Techniques: Expressing, Transfection, Cytometry, Binding Assay, Plasmid Preparation, Incubation, Positive Control, Blocking Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Resistin binding to human leucocytes and epithelial cells. (A) Human leucocytes obtained from peripheral blood were incubated for 30 min with human recombinant resistin (500 ng/ml), washed and cell bound resistin was visualized by anti-resistin antibodies using flow cytometry. Pronounced binding of resistin to lymphocyte and monocyte cell population was observed. ( filled area ). In contrast, neutrophils display significant expression of resistin on their cell surface prior to incubation with exogenous resistin. (B) THP1 monocytic cells bind exogenous resistin in a dose-dependent manner (B 1 ). This resistin binding ( filled area , B 2 and B 3 ) was abolished by pre-incubation of THP1 cells with TLR4 antibodies (5 μg/ml) ( white line , B 2 ), but not with isotype-matched mouse IgG ( white line , B 3 ). (C) HEK293 epithelial cells transfected with TLR4 display significantly higher binding capacity for exogenous resistin ( bold , C 1 ) as compared to HEK-null cells transfected with empty vector ( bold , C 4 ). Binding of resistin to HEK-TLR4 cells is decreased by pre-treatment of cells with antibodies against TLR4 (C 3 ) and CD14 (C 2 ) prior to exposure to resistin. This is not observed in HEK-null cells treated with the same antibodies (C 5 , C 6 ).

Article Snippet: Mouse anti-TLR2 (clone TL2.1) were from Alexis Biochemicals (Lausen, Switzerland) whereas anti-TLR4 (clone HTA125), anti-CD14 (clone UCHM1) antibodies and mouse IgG 2a isotype were from Serotec.

Techniques: Binding Assay, Incubation, Recombinant, Flow Cytometry, Expressing, Transfection, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Western blot analysis of the membrane fraction of THP1 cells (1 × 10 7 ) incubated with resistin (500 ng/ml). The equal protein amounts of membrane extracts were precipitated using mouse anti-resistin antibodies (7.5 μg), anti-TLR4 antibodies (10 μg) or non-specific mouse IgG (10 μg), followed by incubation with protein A/protein G sepharose beads. Immunoprecipitates obtained by precipitation with either TLR4 ab or resistin ab were subjected to electrophoresis and immunoblotting using TLR4 ab (A). Immunoprecipitates obtained by precipitation with either TLR4 ab, resistin ab or non-specific control ab were subjected to electrophoresis and immunoblotting using resistin antibodies (B). Co-precipitation of TLR4 in the resistin bound membrane fraction is clearly visible. Sensitivity of the system for resistin was tested using various concentrations of human recombinant resistin (C).

Article Snippet: Mouse anti-TLR2 (clone TL2.1) were from Alexis Biochemicals (Lausen, Switzerland) whereas anti-TLR4 (clone HTA125), anti-CD14 (clone UCHM1) antibodies and mouse IgG 2a isotype were from Serotec.

Techniques: Western Blot, Incubation, Electrophoresis, Recombinant

Journal: Journal of Cellular and Molecular Medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Resistin-induced production of cytokines in human PBMC is dependent on TLR4 expression. Secretion of (A) IL-6 and (B) IL-8 in human PBMC (1 × 10 6 /ml, n = 9) following resistin stimulation was significantly decreased in the PBMC cultures pre-treated with TLR4 antibodies (* P < 0.001). This decrease of cytokine expression was not observed in the PBMC cultures pre-treated with TLR2 antibodies or isotype matched mouse IgG. (C) HEK293 cells transfected with TLR4 display significantly higher secretion of IL-8 following stimulation with resistin (** P < 0.01) as compared to HEK293 cells transfected with TLR2 or those transfected with an empty vector, HEK-null cells. Ligands specific for TLR2 (PamCys, 2 ng/ml) and TLR4 (LPS, 100 ng/ml) were used as positive controls.

Article Snippet: Mouse anti-TLR2 (clone TL2.1) were from Alexis Biochemicals (Lausen, Switzerland) whereas anti-TLR4 (clone HTA125), anti-CD14 (clone UCHM1) antibodies and mouse IgG 2a isotype were from Serotec.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Resistin competes with LPS for binding to TLR4. (A) Treatment of human PBMC ( n = 3) with resistin (250 and 1000 ng/ml) significantly diminished secretion of IL-6 in response to LPS. (B) Analogous reduction of LPS-induced production of IL-8 was observed in HEK-TLR4 cells cultured in the presence of resistin.

Article Snippet: Mouse anti-TLR2 (clone TL2.1) were from Alexis Biochemicals (Lausen, Switzerland) whereas anti-TLR4 (clone HTA125), anti-CD14 (clone UCHM1) antibodies and mouse IgG 2a isotype were from Serotec.

Techniques: Binding Assay, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: TLR4 is required for the induction of IL-8 expression in HEK293 cells in response to stimulation with resistin. (A) Down-regulation of TLR4 and MyD88 synthesis was achieved by transfecting HEK-TLR4 cells with siRNA sequences specific for these genes. This resulted with a decrease of IL-8 production in response to resistin as well as LPS stimulation in the siRNA transfected cell cultures (* P < 0.05). (B) Down-regulation of MyD88 synthesis in HEK-TLR4 cells transfected with MyD88-specific siRNA and not non-targeting siRNA sequence is shown following 48 and 72 hrs incubation time. (C) Resistin-induced secretion of IL-8 in HEK-TLR4 cells is significantly reduced following incubation with anti-CD14 and anti-resistin antibodies. In contrast, transfection of HEK293 cells with MD2/CD14 in the absence of TLR4 did not lead to resistin-induced activation of HEK293 cells.

Article Snippet: Mouse anti-TLR2 (clone TL2.1) were from Alexis Biochemicals (Lausen, Switzerland) whereas anti-TLR4 (clone HTA125), anti-CD14 (clone UCHM1) antibodies and mouse IgG 2a isotype were from Serotec.

Techniques: Expressing, Transfection, Sequencing, Incubation, Activation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Structural requirements of resistin-TLR4 interaction. HEK293 cells transfected with TLR4, TLR2 or empty vector were stimulated with equimolar concentrations of resistin and resistin peptides, assigned as aa23–42, aa43–64, aa51–108, aa64–88 (50 nM) (A). Localization of the peptides in the whole resistin sequence is schematically shown above the graphs. Differences of IL-8 production in comparison to HEK-null cells are indicated by asterisk (* P < 0.05 and ** P < 0.01). Inhibition of IL-8 production by antibodies against resistin and CD14 are evaluated in HEK-TLR4 cells (B). Differences of IL-8 production in HEK-TLR4 cells in the presence of antibodies are indicated by asterisk (* P < 0.05 and ** P < 0.01).

Article Snippet: Mouse anti-TLR2 (clone TL2.1) were from Alexis Biochemicals (Lausen, Switzerland) whereas anti-TLR4 (clone HTA125), anti-CD14 (clone UCHM1) antibodies and mouse IgG 2a isotype were from Serotec.

Techniques: Transfection, Plasmid Preparation, Sequencing, Inhibition

Journal: Journal of Cellular and Molecular Medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Structural requirements for the pro-inflammatory effects (IL-8 secretion) of resistin on HEK293 cells transfected with TLR-4 or TLR-2

Article Snippet: Mouse anti-TLR2 (clone TL2.1) were from Alexis Biochemicals (Lausen, Switzerland) whereas anti-TLR4 (clone HTA125), anti-CD14 (clone UCHM1) antibodies and mouse IgG 2a isotype were from Serotec.

Techniques: Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Overall structure of TLR4-resistin complex. Side view of the complex (A) on the cell surface TLR4 is associated with MD-2 and adopts the horseshoe-like shape forming an interface for LPS binding. CD14 delivers LPS molecules to the TLR4-MD-2 complex. Dimerization of TLR4-MD-2 complex is followed by recruitment of adaptor proteins MyD88 and IRAK initiating intracellular signalling. We show that interaction between resistin and TLR4 reduces LPS effects, potentially by interfering with its binding to TLR4-MD-2 complex. Top view (B) shows potential binding sites of resistin. R1, resistin occupies LPS binding site in the convex of TLR4 associated to MD-2; R2, resistin binds to the upper central part of the TLR4 convex facilitating dimerization and recruitment of adaptor proteins; R3, resistin stretches through the central part of TLR4 forming several interactions by identical C-terminal loops of its hexamer.

Article Snippet: Mouse anti-TLR2 (clone TL2.1) were from Alexis Biochemicals (Lausen, Switzerland) whereas anti-TLR4 (clone HTA125), anti-CD14 (clone UCHM1) antibodies and mouse IgG 2a isotype were from Serotec.

Techniques: Binding Assay

Journal: Journal of nephrology

Article Title: Does the Renal Expression of Toll-like Receptors Play a Role in Patients with IgA Nephropathy?

doi: 10.1007/s40620-019-00640-z

Figure Lengend Snippet: Linear discriminant analysis (LDA) showed that the intensity of staining of the kidney biopsy tissue for TLR 4, 7, 8, and 9 differentiated all diagnoses (four groups of patients with IgA nephropathy, ANCA-associated vasculitis and healthy controls). The accuracy of the discrimination was 100% according to confusion matrix. Groups of patients with IgA nephropathy: 1. normal renal function and proteinuria <1 g/day at the time of renal biopsy, 2. normal renal function and proteinuria >1 g/day at the time of renal biopsy, 3. renal insufficiency (serum creatinine >150 μmol/l) and proteinuria >1 g/day at the time of renal biopsy, 4. renal insufficiency (serum creatinine >150 μmol/l) and proteinuria <1 g/day at the time of renal biopsy. Control groups: 5 – patients with ANCA-associated vasculitis, 6 – healthy controls.

Article Snippet: Separate sets of sections were incubated with polyclonal TLR 7 rabbit IgG (LifeSpan Biosciences, Inc, Seattle, WA, USA), TLR 8 rabbit IgG (Sigma Aldrich, Darmstadt, Germany), TLR 9 rabbit IgG (Sigma Aldrich, Darmstadt, German) and monoclonal TLR 4 mouse IgG (Novus Biologicals, LLC, Littleton, CO,USA).

Techniques: Staining

Journal: Journal of nephrology

Article Title: Does the Renal Expression of Toll-like Receptors Play a Role in Patients with IgA Nephropathy?

doi: 10.1007/s40620-019-00640-z

Figure Lengend Snippet: Linear discriminant analysis (LDA) confirmed that the intensity of staining of the kidney biopsy tissue for TLR 4, 7, 8, and 9 discriminated four groups of patients with IgA nephropathy. The accuracy of the discrimination was 100 % according to confusion matrix. Groups of patients with IgA nephropathy: 1. normal renal function and proteinuria <1 g/day at the time of renal biopsy, 2. normal renal function and proteinuria >1 g/day at the time of renal biopsy, 3. renal insufficiency (serum creatinine >150 μmol/l) and proteinuria >1 g/day at the time of renal biopsy, 4. renal insufficiency (serum creatinine >150 μmol/l) and proteinuria <1 g/day at the time of renal biopsy. Centroid is a vector of parameters mean values for each group in two-dimensional space of principal-component coordinates (F1 and F2).

Article Snippet: Separate sets of sections were incubated with polyclonal TLR 7 rabbit IgG (LifeSpan Biosciences, Inc, Seattle, WA, USA), TLR 8 rabbit IgG (Sigma Aldrich, Darmstadt, Germany), TLR 9 rabbit IgG (Sigma Aldrich, Darmstadt, German) and monoclonal TLR 4 mouse IgG (Novus Biologicals, LLC, Littleton, CO,USA).

Techniques: Staining, Plasmid Preparation