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: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: Immunofluorescence (IF) microphotographs in the dentate gyrus (DG) of the DG in sham and TBI groups at different time points. (a) BrdU-labelled NSCs (red fluor), expression of TLR2 (green fluor), cell nuclei (blue fluor), and BrdU + /TLR2 + /DAPI + cells indicated that TLR2 expression in labelled proliferating cells was possible NSCs; (b) BrdU + cells showed the proliferation of NSCs in the DG during different time points posttrauma. BrdU + cells were more in the TBI group than that in the sham group ( ∗ p < 0.05), and numbers of these cells were significantly different among different time points posttrauma ( # p < 0.05); (c) numbers of BrdU + /TLR2 + cells indicated that the expression of TLR2 was quite different in proliferating cells of the DG among different time points posttrauma. There were more BrdU + cells in the TBI group than that in the sham group ( ∗ p < 0.05), and the numbers of these cells are obviously different among different time points posttrauma ( # p < 0.05). Scale bar: 50 μ m; data is shown as mean ± SEM.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Immunofluorescence, Expressing

Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: IF in the DG of the TBI group (mouse brain got from 3 days posttrauma). (a) BrdU (red), nestin (green), and DAPI (blue), respectively, exhibited proliferating cells, NSCs, and cell nuclei in the DG. Merged pictures of BrdU + /nestin + /DAPI + showed NSCs (the percentage of NSCs in proliferating cells was 84.30% ± 6.54%); scale bar: 50 μ m; data were expressed as mean ± SEM. (b) Nestin (green), TLR2 (red), and DAPI (blue), respectively, exhibited NSCs, TLR2 expression, and cell nuclei in the DG. Merged pictures of nestin + /TLR2 + /DAPI + showed the expression of TLR2 on NSCs. Scale bar: 50 μ m; data were expressed as mean ± SEM.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Expressing

Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: Expression of TLR2 protein and mRNA in the DG (western blotting and PCR). (a) Western blotting: electrophoresis bands of TLR2 protein controlled with β -actin; (b) western blotting: the optical density of TLR2 electrophoresis; (c) real-time PCR: the expression of TLR2 mRNA and GAPDH was used as the endogenous reference gene. The TLR2 expression in the protein and mRNA level was significantly higher in the TBI group than that in the sham group ( ∗ p < 0.05), and the TLR2 expression was significantly different among various time points ( # p < 0.05). Data were expressed as mean ± SEM.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Expressing, Western Blot, Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: Gene sequences for primer synthesis.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Sequencing