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

Article Title: SHP-1 As a Critical Regulator of Mycoplasma pneumoniae -Induced Inflammation in Human Asthmatic Airway Epithelial Cells

doi: 10.4049/jimmunol.1100573

Figure Lengend Snippet: M. pneumoniae infection induced TLR2–SHP-1 dynamic association. (A) Immunoblot analysis of baseline TLR2 expression in airway epithelial cells from nonasthmatic and asthmatic subjects. The data representing the percentage of densitometry values of nonasthmatic cells are expressed as mean ± SEM (right panel). (B) TLR2-associated SHP-1 levels in airway epithelial cells was measured by coimmunoprecipitation. TLR2 was immunoprecipitated from airway epithelial cell lysates of nonasthmatic and asthmatic subjects without mycoplasma infection (0 h), infected with mycoplasma for 1 and 2 h. TLR2 immunoprecipitates were blotted with anti-TLR2 (top left panel) and anti–SHP-1 (bottom left panel) Ab. TLR2-associated SHP-1 at 2 h after M. pneumoniae infection is expressed as fold change of densitometry values from nonasthmatic cells without mycoplasma treatment (0 h) and was increased in nonasthmatic cells compared with asthmatic cells. The data are presented as mean ± SEM (n = 5). *p < 0.01 compared with asthmatic group.

Article Snippet: The membranes were probed with rabbit anti-human TLR2 Ab (IMGENEX, San Diego, CA), mouse anti-human SHP-1 (Santa Cruz Biotechnology), or a Ser 473 phosphospecific Ab to Akt (Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques: Infection, Western Blot, Expressing, Immunoprecipitation

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

Article Title: SHP-1 As a Critical Regulator of Mycoplasma pneumoniae -Induced Inflammation in Human Asthmatic Airway Epithelial Cells

doi: 10.4049/jimmunol.1100573

Figure Lengend Snippet: Proposed model for the mechanism of SHP-1–mediated inhibition of M. pneumoniae-activated TLR2 signaling in normal and asthmatic airway epithelial cells. The ligation of TLR2 by M. pneumoniae initiates TLR2-mediated proinflammatory signaling pathway, resulting in the production of IL-8. M. pneumoniae binding to TLR2 activates and recruits SHP-1, which inhibits the nuclear translocation of NF-κB directly or through inhibition of PI3K/Akt abrogating NF-κB activation and subsequently prevents IL-8 production. In addition, the nuclear SHP-1 may also inhibit NF-κB function by certain nuclear mediators (left panel). In asthmatic airway epithelial cells, M. pneumoniae-induced SHP-1 activation is defective, which contributes to the increased activation of PI3K/Akt and NF-κB, as well as abundant IL-8 production (right panel).

Article Snippet: The membranes were probed with rabbit anti-human TLR2 Ab (IMGENEX, San Diego, CA), mouse anti-human SHP-1 (Santa Cruz Biotechnology), or a Ser 473 phosphospecific Ab to Akt (Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques: Inhibition, Ligation, Binding Assay, Translocation Assay, Activation Assay

Journal: Gut Microbes

Article Title: ADP-heptose attenuates Helicobacter pylori -induced dendritic cell activation

doi: 10.1080/19490976.2024.2402543

Figure Lengend Snippet: TLR2 mediated Helicobacter pylori uptake is essential for potent dendritic cell activation. (a) TLR2 mRNA expression upon stimulation with H. pylori wt or mutant (Δ) (MOI 5) at indicated time points. Mean±SD of three individual donors is shown. (b) TLR2 surface expression was monitored by flow cytometry 16 h post-infection (MOI 5) ( n = 4). (c,d) H. pylori strains were stained with eFluor670 proliferation dye prior to infection. One hour post infection with H. pylori wt or the ADP-heptose-deficient mutant (Δ) (MOI 5), DCs were subjected to immunofluorescence and stained for CD45, DAPI and TLR2. Internalization of bacteria as well as TLR2 localization was analyzed by confocal fluorescence microscopy. Orthogonal views of confocal z-stacks of one out of three representative donors are shown. Scale bar: 5 µm. (e) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection. After 1 h of infection with eFluor670 stained H. pylori (MOI 5), DCs were subjected to immunofluorescence and stained for CD45 and DAPI. Maximum intensity projections of confocal z-stacks of one representative out of three donors are shown. Scale bar: 5 µm. (f,g) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection with H. pylori (wt) or the ADP-heptose deficient mutant (Δ) at an MOI of 5. After 16 h CD40 expression (f) and IL-12p70 secretion (g) was monitored by flow cytometry and multiplex assay ( n = 6). For statistical analysis one-way ANOVA with a šídák’s post-hoc test was performed.

Article Snippet: Following primary antibodies were used: mouse anti-human CD45 (ab30470 Abcam, 1:200), rabbit anti-human TLR2 (NB100–56720 Novus Biologicals, 1:400).

Techniques: Activation Assay, Expressing, Mutagenesis, Flow Cytometry, Infection, Staining, Immunofluorescence, Bacteria, Fluorescence, Microscopy, Multiplex Assay

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Pre-incubation with directly labeled TLR2 or TLR4 antibodies does not impact on phagocytosis of carboxyfluorescein succinimidyl ester (CSFE)-labeled E. bovis by polymorphonuclear neutrophils (PMN). Isolated PMN (1 × 10 6 per sample; n = 3) were pre-treated with TLR2 and TLR4 antibodies for 30 min and exposed to carboxyfluorescein succinimidyl ester (CFSE)-labeled E. bovis sporozoites (2.5 µm, 30 min) at a 1:1 ratio for two hours for subsequent flow cytometric analysis. Pre-incubation of PMN with antibodies to bovine TLR2 and TLR4 did not seem to impact on the phagocytosis of CFSE-labeled E. bovis sporozoites. Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc., San Diego, CA, USA).

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Incubation, Labeling, Isolation, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: E. bovis increases TLR2 and TLR4 expression on PMN. PMN (1 × 10 6 per sample; n = 3) were incubated with TLR2 and TLR4 antibodies for 30 min prior to exposure to live E. bovis for two hours for subsequent Flow cytometry analyses (FACS). Incubation of PMN with E. bovis significantly increases TLR2 ( A ) and TLR4 ( B ) expression (**** p < 0.0001). Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc.). p -value notation; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Expressing, Incubation, Flow Cytometry, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: IL-8 production in PMN upon E. bovis exposure. Supernatants of PMN (1 × 10 6 per sample; n = 3) treated with TLR2 and TLR4 antibodies and exposed to live E. bovis sporozoites (1:1 ratio; 2 h) were assessed for the presence of IL-8 by ELISA analysis. TLR2-treated PMN exposed to sporozoites showed a significant increase in IL-8 production (* p < 0.05) when compared to PMN in media. Likewise, a significant increase in IL-8 production (** p < 0.01) was observed in the same experimental condition when compared to the respective control without exposure to E. bovis. Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc., San Diego, CA, USA). p -value notation; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Induction of Toll-like receptor (TLR)-dependent NF-κB activation by E. bovis sporozoites. In order to investigate the activation of TLRs in bovine PMN, we used HEK cells expressing either bovine TLR2 (A) or a combination of bovine TLR4/MD2 (B). Cells were exposed to different E. bovis sporozoite preparations: live, heat killed (HK) or antigen ( Eb Ag) for 24 h and assessed for activation of transcription factor NF-κB using a luciferase reporter assay. ( A ) HK sporozoites and Eb Ag induced substantial TLR2-dependent NF-κB activation compared to media alone (*** p < 0.0001, ** p < 0.01 and * p < 0.05, respectively). TLR2-induced NF-κB significantly increases when exposed to Eb Ag compared to live E. bovis ( p < 0.05). ( B ) HK sporozoites induced a significant NF-κB response when compared to media ( p < 0.05) and when compared to live parasitic stages ( p < 0.01). A significant increase in NF-κB response was observed in Eb Ag when compared to media ( p < 0.05). In both experiments, Pam 3 CSK 4 and Lipopolysaccharides (LPS) served as ligand controls for TLR2 and TLR4/MD2, respectively, and phorbol 12-myristate 13-acetate (PMA) was used as an NF-κB technical control (data not shown for clarity). Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc.).

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Activation Assay, Expressing, Luciferase, Reporter Assay, Control, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Immunofluorescence analysis on bovine PMN activation of TLR2 and TLR 4 by E. bovis and concomitant neutrophil extracellular trap (NET) formation. PMN ( n = 3; 5 × 10 5 ) were exposed to vital E. bovis sporozoites (ratio 1:1) on poly- l -lysine-treated coverslips (120 min, 37 °C) and fixed for further antibody exposure (60 min) with anti-TLR2 ( C ) and anti-TLR4 ( D ) antibodies and anti-histone H1, H2A/H2B, H3, H4 antibody ( E , F ). Coverslips were mounted with ProLong Antifade containing DAPI ( A , B ) which was used for observation of PMN nuclei and NET extracellular DNA by fluorescence microscopy analysis. In both cases, expression of TLR2 and TLR4 was observed on the surface of bovine PMN (red) co-localized with NET-derived histones (green) and extracellular DNA (blue), as indicated by yellow arrows (( G , H )—overlay of images collected for nucleic acid, TLR2/TLR and histone staining). Co-localization of TLR-positive signals with early stages of NETosis are indicated by white arrows ( G , H ). TLR-positive signals without NETosis are indicated by orange arrows ( G , H ). Images were visualized by using an inverted Olympus IX81 ® epifluorescence microscope equipped with a digital camera (XM10 ® , Olympus, Tokyo, Japan). Scale bar magnitude: 20 µm.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Immunofluorescence, Activation Assay, Fluorescence, Microscopy, Expressing, Derivative Assay, Staining

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Antibodies.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Recombinant

Journal: American Journal of Physiology - Renal Physiology

Article Title: Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis

doi: 10.1152/ajprenal.00040.2018

Figure Lengend Snippet: Primary antibodies used for immunofluorescence and colocalization studies

Article Snippet: Mouse anti-human TLR2 TLR2 ab16894 5 µg/ml Abcam * 37 , 55 Mouse anti-human TLR4 TLR4 ab22048 5 µg/ml Abcam 14 , 15 Mouse anti-human TLR9 TLR9 ab12121 5 µg/ml Abcam 54 , 65 Rabbit anti-human TLR2 TLR2 sc10739 5 µg/ml Santa Cruz † 3 , 51 Rabbit anti-human TLR4 TLR4 ab13556 1:50 Abcam 44 , 67 Rabbit anti human TLR9 TLR9 ab37154 5 µg/ml Abcam 29 , 30 Goat anti-human TLR9 TLR9 ab53396 1:100 Abcam 52 Rabbit anti-human CD34 Endothelial cells ab81289 1:50 Abcam 16 , 64 Mouse anti-human CD34 Endothelial cells QBend 10 1/50 Dako ‡ 40 , 41 Sheep anti-human nephrin Podocytes LS-C150012 5 µg/ml LifeSpan Bioscience ¶ 38 Sheep anti-human NE Neutrophils LS-B4244 1:100 LifeSpan Biosciences 23 , 38 Mouse anti-human CD68 Macrophages PG-M1 1:40 Dako 38 , 59 Mouse anti human HMGB1 HMGB1 ab184532 § 1:100 Abcam 8 , 34 Goat anti-human fibrinogen Fibrinogen ab6666 § 1:10,000 Abcam 7 , 33 Open in a separate window HMGB1, high-mobility group box 1; NE, neutrophil elastase, TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

Techniques: Immunofluorescence

Journal: American Journal of Physiology - Renal Physiology

Article Title: Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis

doi: 10.1152/ajprenal.00040.2018

Figure Lengend Snippet: Toll-like receptor (TLR) 4 is the predominant TLR expressed within glomeruli of patients with antineutrophil cytoplasmic antibody-associated glomerulonephritis. A: semiquantitative assessment and comparison of the intensity of intraglomerular TLR2, TLR4, and TLR9 expression in antibody-associated vasculitis (AAV) patients demonstrates that, compared with TLR2 and TLR9, TLR4 is most highly expressed in glomeruli. AU, arbitrary units; GCS, glomerular cross section. B: semiquantitative assessment and comparison of the proportion of the glomerulus positive for TLR2, TLR4, or TLR9 shows a similar TLR4-dominant pattern. C: representative images showing serial sections stained with anti-TLR2, -TLR4, and -TLR9 antibodies showing that TLR4 is prominently expressed with glomeruli of AAV patients. Data are expressed as means ± SE from 38 biopsies, analyzed by the Kruskal-Wallis test. **P < 0.005 and ****P < 0.0001. Original magnification ×600, scale bar = 20 µm.

Article Snippet: Mouse anti-human TLR2 TLR2 ab16894 5 µg/ml Abcam * 37 , 55 Mouse anti-human TLR4 TLR4 ab22048 5 µg/ml Abcam 14 , 15 Mouse anti-human TLR9 TLR9 ab12121 5 µg/ml Abcam 54 , 65 Rabbit anti-human TLR2 TLR2 sc10739 5 µg/ml Santa Cruz † 3 , 51 Rabbit anti-human TLR4 TLR4 ab13556 1:50 Abcam 44 , 67 Rabbit anti human TLR9 TLR9 ab37154 5 µg/ml Abcam 29 , 30 Goat anti-human TLR9 TLR9 ab53396 1:100 Abcam 52 Rabbit anti-human CD34 Endothelial cells ab81289 1:50 Abcam 16 , 64 Mouse anti-human CD34 Endothelial cells QBend 10 1/50 Dako ‡ 40 , 41 Sheep anti-human nephrin Podocytes LS-C150012 5 µg/ml LifeSpan Bioscience ¶ 38 Sheep anti-human NE Neutrophils LS-B4244 1:100 LifeSpan Biosciences 23 , 38 Mouse anti-human CD68 Macrophages PG-M1 1:40 Dako 38 , 59 Mouse anti human HMGB1 HMGB1 ab184532 § 1:100 Abcam 8 , 34 Goat anti-human fibrinogen Fibrinogen ab6666 § 1:10,000 Abcam 7 , 33 Open in a separate window HMGB1, high-mobility group box 1; NE, neutrophil elastase, TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

Techniques: Expressing, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis

doi: 10.1152/ajprenal.00040.2018

Figure Lengend Snippet: Glomerular Toll-like receptor (TLR) 4 expression is associated with more severe glomerular disease, and glomerular infiltrating myeloid cells express TLR2, TLR4, and TLR9. The proportion of glomeruli with the most active glomerular lesions containing both cellular crescents and segmental necrosis correlates with both the intensity of TLR4 staining (A) and the percentage area of the glomeruli positive for staining (B). Both the intensity (C) and area (D) of TLR4 staining correlate inversely with estimated glomerular filtration rate (eGFR) at presentation. AU, arbitrary units; GCS, glomerular cross section. A–D: data are means ± SE from the 38 antibody-associated vasculitis patients’ biopsies analyzed by the nonparametric Spearman’s correlation. Kidney biopsies from n = 10 patients, stained for TLR2, TLR4, or TLR9 (green), 4′,6-diamidino-2-phenylindole (DAPI, blue, nuclear marker), and CD68 (macrophages, red, E) or neutrophil elastase (neutrophils, red, F). White arrows indicate double-positive cells for TLR and macrophages/neutrophils. Areas with asterisks denote nonmacrophage/neutrophil-positive cells staining positive for TLRs. Original magnification ×600, scale bar = 20 µm.

Article Snippet: Mouse anti-human TLR2 TLR2 ab16894 5 µg/ml Abcam * 37 , 55 Mouse anti-human TLR4 TLR4 ab22048 5 µg/ml Abcam 14 , 15 Mouse anti-human TLR9 TLR9 ab12121 5 µg/ml Abcam 54 , 65 Rabbit anti-human TLR2 TLR2 sc10739 5 µg/ml Santa Cruz † 3 , 51 Rabbit anti-human TLR4 TLR4 ab13556 1:50 Abcam 44 , 67 Rabbit anti human TLR9 TLR9 ab37154 5 µg/ml Abcam 29 , 30 Goat anti-human TLR9 TLR9 ab53396 1:100 Abcam 52 Rabbit anti-human CD34 Endothelial cells ab81289 1:50 Abcam 16 , 64 Mouse anti-human CD34 Endothelial cells QBend 10 1/50 Dako ‡ 40 , 41 Sheep anti-human nephrin Podocytes LS-C150012 5 µg/ml LifeSpan Bioscience ¶ 38 Sheep anti-human NE Neutrophils LS-B4244 1:100 LifeSpan Biosciences 23 , 38 Mouse anti-human CD68 Macrophages PG-M1 1:40 Dako 38 , 59 Mouse anti human HMGB1 HMGB1 ab184532 § 1:100 Abcam 8 , 34 Goat anti-human fibrinogen Fibrinogen ab6666 § 1:10,000 Abcam 7 , 33 Open in a separate window HMGB1, high-mobility group box 1; NE, neutrophil elastase, TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

Techniques: Expressing, Staining, Filtration, Marker

Journal: American Journal of Physiology - Renal Physiology

Article Title: Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis

doi: 10.1152/ajprenal.00040.2018

Figure Lengend Snippet: Correlation of glomerular expression of TLR2, TLR4, and TLR9 with glomeruli injury and renal function

Article Snippet: Mouse anti-human TLR2 TLR2 ab16894 5 µg/ml Abcam * 37 , 55 Mouse anti-human TLR4 TLR4 ab22048 5 µg/ml Abcam 14 , 15 Mouse anti-human TLR9 TLR9 ab12121 5 µg/ml Abcam 54 , 65 Rabbit anti-human TLR2 TLR2 sc10739 5 µg/ml Santa Cruz † 3 , 51 Rabbit anti-human TLR4 TLR4 ab13556 1:50 Abcam 44 , 67 Rabbit anti human TLR9 TLR9 ab37154 5 µg/ml Abcam 29 , 30 Goat anti-human TLR9 TLR9 ab53396 1:100 Abcam 52 Rabbit anti-human CD34 Endothelial cells ab81289 1:50 Abcam 16 , 64 Mouse anti-human CD34 Endothelial cells QBend 10 1/50 Dako ‡ 40 , 41 Sheep anti-human nephrin Podocytes LS-C150012 5 µg/ml LifeSpan Bioscience ¶ 38 Sheep anti-human NE Neutrophils LS-B4244 1:100 LifeSpan Biosciences 23 , 38 Mouse anti-human CD68 Macrophages PG-M1 1:40 Dako 38 , 59 Mouse anti human HMGB1 HMGB1 ab184532 § 1:100 Abcam 8 , 34 Goat anti-human fibrinogen Fibrinogen ab6666 § 1:10,000 Abcam 7 , 33 Open in a separate window HMGB1, high-mobility group box 1; NE, neutrophil elastase, TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

Techniques: Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis

doi: 10.1152/ajprenal.00040.2018

Figure Lengend Snippet: Correlations between glomerular and tubulointerstitial TLR expression and presenting eGFR

Article Snippet: Mouse anti-human TLR2 TLR2 ab16894 5 µg/ml Abcam * 37 , 55 Mouse anti-human TLR4 TLR4 ab22048 5 µg/ml Abcam 14 , 15 Mouse anti-human TLR9 TLR9 ab12121 5 µg/ml Abcam 54 , 65 Rabbit anti-human TLR2 TLR2 sc10739 5 µg/ml Santa Cruz † 3 , 51 Rabbit anti-human TLR4 TLR4 ab13556 1:50 Abcam 44 , 67 Rabbit anti human TLR9 TLR9 ab37154 5 µg/ml Abcam 29 , 30 Goat anti-human TLR9 TLR9 ab53396 1:100 Abcam 52 Rabbit anti-human CD34 Endothelial cells ab81289 1:50 Abcam 16 , 64 Mouse anti-human CD34 Endothelial cells QBend 10 1/50 Dako ‡ 40 , 41 Sheep anti-human nephrin Podocytes LS-C150012 5 µg/ml LifeSpan Bioscience ¶ 38 Sheep anti-human NE Neutrophils LS-B4244 1:100 LifeSpan Biosciences 23 , 38 Mouse anti-human CD68 Macrophages PG-M1 1:40 Dako 38 , 59 Mouse anti human HMGB1 HMGB1 ab184532 § 1:100 Abcam 8 , 34 Goat anti-human fibrinogen Fibrinogen ab6666 § 1:10,000 Abcam 7 , 33 Open in a separate window HMGB1, high-mobility group box 1; NE, neutrophil elastase, TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

Techniques: Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis

doi: 10.1152/ajprenal.00040.2018

Figure Lengend Snippet: Colocalization of the endogenous Toll-like receptor (TLR) ligands high-mobility group box 1 (HMGB1) and fibrinogen with TLRs in glomeruli of patients with antibody-associated vasculitis (AAV). A: TLR2, TLR4, or TLR9 (green) is colocalized with the endogenous TLR ligand HMGB1 (red) within glomeruli of patient biopsies with AAV. Insets show higher-power magnification of cells positive for both HMGB1 and the relevant TLR. DAPI, 4′,6-diamidino-2-phenylindole. B: TLR2, TLR4, or TLR9 (green) is colocalized with the endogenous TLR2 and TLR4 ligand fibrinogen (red) within glomeruli of patient biopsies with AAV. Insets show higher-power magnification of colocalization of fibrinogen and the relevant TLR. Original magnification ×600, scale bar = 20 µm.

Article Snippet: Mouse anti-human TLR2 TLR2 ab16894 5 µg/ml Abcam * 37 , 55 Mouse anti-human TLR4 TLR4 ab22048 5 µg/ml Abcam 14 , 15 Mouse anti-human TLR9 TLR9 ab12121 5 µg/ml Abcam 54 , 65 Rabbit anti-human TLR2 TLR2 sc10739 5 µg/ml Santa Cruz † 3 , 51 Rabbit anti-human TLR4 TLR4 ab13556 1:50 Abcam 44 , 67 Rabbit anti human TLR9 TLR9 ab37154 5 µg/ml Abcam 29 , 30 Goat anti-human TLR9 TLR9 ab53396 1:100 Abcam 52 Rabbit anti-human CD34 Endothelial cells ab81289 1:50 Abcam 16 , 64 Mouse anti-human CD34 Endothelial cells QBend 10 1/50 Dako ‡ 40 , 41 Sheep anti-human nephrin Podocytes LS-C150012 5 µg/ml LifeSpan Bioscience ¶ 38 Sheep anti-human NE Neutrophils LS-B4244 1:100 LifeSpan Biosciences 23 , 38 Mouse anti-human CD68 Macrophages PG-M1 1:40 Dako 38 , 59 Mouse anti human HMGB1 HMGB1 ab184532 § 1:100 Abcam 8 , 34 Goat anti-human fibrinogen Fibrinogen ab6666 § 1:10,000 Abcam 7 , 33 Open in a separate window HMGB1, high-mobility group box 1; NE, neutrophil elastase, TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

Techniques:

Journal: American Journal of Physiology - Renal Physiology

Article Title: Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis

doi: 10.1152/ajprenal.00040.2018

Figure Lengend Snippet: Tubulointerstitial Toll-like receptor (TLR) 2, TLR4, and TLR9 expression in antibody-associated vasculitis (AAV) patients. A: representative TLR2, TLR4, and TLR9 staining often in and around the same tubules and infiltrating cells. AU, arbitrary units; GCS, glomerular cross section. Semiquantitative assessment and comparison of the intensity (B) and the proportion (C) of tubulointerstitial high-powered field (mean of 10) positive for TLR2, TLR4, or TLR9 expression in AAV patients demonstrating a TLR4-dominant pattern. Tubulointerstitial TLR4 intensity (D) and extent (E) are negatively correlated with presenting estimated glomerular filtration rate (eGFR). Original magnification ×200, scale bar = 50 µm. *P < 0.05, **P < 0.005, and ****P < 0.0001. Data are means ± SE from 38 biopsies in each group analyzed by the Kruskal-Wallis test. AU, arbitrary units.

Article Snippet: Mouse anti-human TLR2 TLR2 ab16894 5 µg/ml Abcam * 37 , 55 Mouse anti-human TLR4 TLR4 ab22048 5 µg/ml Abcam 14 , 15 Mouse anti-human TLR9 TLR9 ab12121 5 µg/ml Abcam 54 , 65 Rabbit anti-human TLR2 TLR2 sc10739 5 µg/ml Santa Cruz † 3 , 51 Rabbit anti-human TLR4 TLR4 ab13556 1:50 Abcam 44 , 67 Rabbit anti human TLR9 TLR9 ab37154 5 µg/ml Abcam 29 , 30 Goat anti-human TLR9 TLR9 ab53396 1:100 Abcam 52 Rabbit anti-human CD34 Endothelial cells ab81289 1:50 Abcam 16 , 64 Mouse anti-human CD34 Endothelial cells QBend 10 1/50 Dako ‡ 40 , 41 Sheep anti-human nephrin Podocytes LS-C150012 5 µg/ml LifeSpan Bioscience ¶ 38 Sheep anti-human NE Neutrophils LS-B4244 1:100 LifeSpan Biosciences 23 , 38 Mouse anti-human CD68 Macrophages PG-M1 1:40 Dako 38 , 59 Mouse anti human HMGB1 HMGB1 ab184532 § 1:100 Abcam 8 , 34 Goat anti-human fibrinogen Fibrinogen ab6666 § 1:10,000 Abcam 7 , 33 Open in a separate window HMGB1, high-mobility group box 1; NE, neutrophil elastase, TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

Techniques: Expressing, Staining, Filtration

Journal: American Journal of Physiology - Renal Physiology

Article Title: Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis

doi: 10.1152/ajprenal.00040.2018

Figure Lengend Snippet: Comparison of intrarenal TLR expression in MPO-AAV and PR3-AAV patients

Article Snippet: Mouse anti-human TLR2 TLR2 ab16894 5 µg/ml Abcam * 37 , 55 Mouse anti-human TLR4 TLR4 ab22048 5 µg/ml Abcam 14 , 15 Mouse anti-human TLR9 TLR9 ab12121 5 µg/ml Abcam 54 , 65 Rabbit anti-human TLR2 TLR2 sc10739 5 µg/ml Santa Cruz † 3 , 51 Rabbit anti-human TLR4 TLR4 ab13556 1:50 Abcam 44 , 67 Rabbit anti human TLR9 TLR9 ab37154 5 µg/ml Abcam 29 , 30 Goat anti-human TLR9 TLR9 ab53396 1:100 Abcam 52 Rabbit anti-human CD34 Endothelial cells ab81289 1:50 Abcam 16 , 64 Mouse anti-human CD34 Endothelial cells QBend 10 1/50 Dako ‡ 40 , 41 Sheep anti-human nephrin Podocytes LS-C150012 5 µg/ml LifeSpan Bioscience ¶ 38 Sheep anti-human NE Neutrophils LS-B4244 1:100 LifeSpan Biosciences 23 , 38 Mouse anti-human CD68 Macrophages PG-M1 1:40 Dako 38 , 59 Mouse anti human HMGB1 HMGB1 ab184532 § 1:100 Abcam 8 , 34 Goat anti-human fibrinogen Fibrinogen ab6666 § 1:10,000 Abcam 7 , 33 Open in a separate window HMGB1, high-mobility group box 1; NE, neutrophil elastase, TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

Techniques: Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis

doi: 10.1152/ajprenal.00040.2018

Figure Lengend Snippet: Comparison of Toll-like receptor (TLR) expression in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis and control disease tissues, minimal change disease, and Class IV International Society of Nephrology/Renal Pathology Society lupus nephritis. TLR2, TLR4, and TLR9 expression both in the intensity of the signal (A–C) and percentage area covered (D–F) is significantly increased within glomeruli of antibody-associated vasculitis (AAV) patients compared with those with minimal change disease. The intensity and extent of TLR2 expression in glomeruli were significantly increased compared with those with lupus nephritis. In the tubulointerstitial compartment, TLR2, TLR4, and TLR9 expression was significantly increased both in intensity of signal (G–I) and the area of the tubulointerstitium expressing the relevant TLR (J–L), in the AAV patient cohort compared with minimal change disease (G–L). *P < 0.05, **P < 0.05, ***P < 0.0005, and ****P < 0.0001. Data are means ± SE from 8 minimal change disease, 10 class IV lupus nephritis, and 38 AAV biopsies, in each group analyzed by the Kruskal-Wallis test. AU, arbitrary units; GCS, glomerular cross section; HPF, high-powered field.

Article Snippet: Mouse anti-human TLR2 TLR2 ab16894 5 µg/ml Abcam * 37 , 55 Mouse anti-human TLR4 TLR4 ab22048 5 µg/ml Abcam 14 , 15 Mouse anti-human TLR9 TLR9 ab12121 5 µg/ml Abcam 54 , 65 Rabbit anti-human TLR2 TLR2 sc10739 5 µg/ml Santa Cruz † 3 , 51 Rabbit anti-human TLR4 TLR4 ab13556 1:50 Abcam 44 , 67 Rabbit anti human TLR9 TLR9 ab37154 5 µg/ml Abcam 29 , 30 Goat anti-human TLR9 TLR9 ab53396 1:100 Abcam 52 Rabbit anti-human CD34 Endothelial cells ab81289 1:50 Abcam 16 , 64 Mouse anti-human CD34 Endothelial cells QBend 10 1/50 Dako ‡ 40 , 41 Sheep anti-human nephrin Podocytes LS-C150012 5 µg/ml LifeSpan Bioscience ¶ 38 Sheep anti-human NE Neutrophils LS-B4244 1:100 LifeSpan Biosciences 23 , 38 Mouse anti-human CD68 Macrophages PG-M1 1:40 Dako 38 , 59 Mouse anti human HMGB1 HMGB1 ab184532 § 1:100 Abcam 8 , 34 Goat anti-human fibrinogen Fibrinogen ab6666 § 1:10,000 Abcam 7 , 33 Open in a separate window HMGB1, high-mobility group box 1; NE, neutrophil elastase, TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

Techniques: Expressing