Journal: PLOS ONE

Article Title: Toll-like receptors 1–9 in small bowel neuroendocrine tumors–Clinical significance and prognosis

doi: 10.1371/journal.pone.0302813

Figure Lengend Snippet: Baseline characteristics and comparison stratified by Toll-like receptor (TLR) 2 cytoplasmic intensity in primary tumors and lymph node metastases.

Article Snippet: After this, sections were incubated with rabbit polyclonal antibodies (TLRs 1, 2, 6, 7) and mouse monoclonal antibodies (TLRs 3, 4, 5, 8, 9) in dilute solution (Dako S2022); TLR1 for 60 minutes (diluted 1:300, Abcam ab189337), TLR2 for 60 minutes (diluted 1:500, Rockland 600-401-956), TLR3 for 120 minutes (diluted 1:30, Novus NBP2-24875), TLR4 for 60 minutes (diluted 1:1000, Abnova H00007099-M02), TLR5 for overnight in +4°C (diluted 1:75, Novus NBP2-24787), TLR6 for 60 minutes (diluted 1:750, Abnova PAB 3555), TLR7 for 60 minutes (diluted 1:500, Novus NB100-56682), TLR8 for 60 minutes (diluted 1:850, Novus NBP2-24917) and TLR9 for 60 minutes (diluted 1:300, Novus NBP2-24729).

Techniques: Comparison

Journal: PLOS ONE

Article Title: Toll-like receptors 1–9 in small bowel neuroendocrine tumors–Clinical significance and prognosis

doi: 10.1371/journal.pone.0302813

Figure Lengend Snippet: Immunohistochemical staining examples of Toll-like receptors (TLRs) 1, 2, 4, 5, 6, 7, 8 and 9 in representative small bowel neuroendocrine tumor samples showing ( A ) High TLR1 cytoplasmic intensity, ( B ) intermediate TLR2 cytoplasmic intensity, ( C ) intermediate TLR4 cytoplasmic intensity, ( D ) high TLR5 cytoplasmic and nucleic intensity, ( E ) Intermediate TLR6 cytoplasmic intensity, ( F ) intermediate TLR7 cytoplasmic intensity, ( G ) high TLR8 cytoplasmic intensity and ( H ) intermediate TLR9 cytoplasmic intensity in x20 magnification. The scale bar length is 50 μm (bottom left corner). Arrows indicate TLR-positive tumor cell islets.

Article Snippet: After this, sections were incubated with rabbit polyclonal antibodies (TLRs 1, 2, 6, 7) and mouse monoclonal antibodies (TLRs 3, 4, 5, 8, 9) in dilute solution (Dako S2022); TLR1 for 60 minutes (diluted 1:300, Abcam ab189337), TLR2 for 60 minutes (diluted 1:500, Rockland 600-401-956), TLR3 for 120 minutes (diluted 1:30, Novus NBP2-24875), TLR4 for 60 minutes (diluted 1:1000, Abnova H00007099-M02), TLR5 for overnight in +4°C (diluted 1:75, Novus NBP2-24787), TLR6 for 60 minutes (diluted 1:750, Abnova PAB 3555), TLR7 for 60 minutes (diluted 1:500, Novus NB100-56682), TLR8 for 60 minutes (diluted 1:850, Novus NBP2-24917) and TLR9 for 60 minutes (diluted 1:300, Novus NBP2-24729).

Techniques: Immunohistochemical staining, Staining

Journal: PLOS ONE

Article Title: Toll-like receptors 1–9 in small bowel neuroendocrine tumors–Clinical significance and prognosis

doi: 10.1371/journal.pone.0302813

Figure Lengend Snippet: Median Toll-like receptor (TLR) staining intensity in primary small bowel neuroendocrine tumors and lymph node metastases.

Article Snippet: After this, sections were incubated with rabbit polyclonal antibodies (TLRs 1, 2, 6, 7) and mouse monoclonal antibodies (TLRs 3, 4, 5, 8, 9) in dilute solution (Dako S2022); TLR1 for 60 minutes (diluted 1:300, Abcam ab189337), TLR2 for 60 minutes (diluted 1:500, Rockland 600-401-956), TLR3 for 120 minutes (diluted 1:30, Novus NBP2-24875), TLR4 for 60 minutes (diluted 1:1000, Abnova H00007099-M02), TLR5 for overnight in +4°C (diluted 1:75, Novus NBP2-24787), TLR6 for 60 minutes (diluted 1:750, Abnova PAB 3555), TLR7 for 60 minutes (diluted 1:500, Novus NB100-56682), TLR8 for 60 minutes (diluted 1:850, Novus NBP2-24917) and TLR9 for 60 minutes (diluted 1:300, Novus NBP2-24729).

Techniques: Staining

Journal: PLOS ONE

Article Title: Toll-like receptors 1–9 in small bowel neuroendocrine tumors–Clinical significance and prognosis

doi: 10.1371/journal.pone.0302813

Figure Lengend Snippet: Disease-specific survival rates based on Toll-like receptor (TLR) 1, 2, 4, 5, 6, 7, 8, and 9 cytoplasmic staining intensity and TLR5 nucleic staining intensity in both primary tumors and lymph node metastases.

Article Snippet: After this, sections were incubated with rabbit polyclonal antibodies (TLRs 1, 2, 6, 7) and mouse monoclonal antibodies (TLRs 3, 4, 5, 8, 9) in dilute solution (Dako S2022); TLR1 for 60 minutes (diluted 1:300, Abcam ab189337), TLR2 for 60 minutes (diluted 1:500, Rockland 600-401-956), TLR3 for 120 minutes (diluted 1:30, Novus NBP2-24875), TLR4 for 60 minutes (diluted 1:1000, Abnova H00007099-M02), TLR5 for overnight in +4°C (diluted 1:75, Novus NBP2-24787), TLR6 for 60 minutes (diluted 1:750, Abnova PAB 3555), TLR7 for 60 minutes (diluted 1:500, Novus NB100-56682), TLR8 for 60 minutes (diluted 1:850, Novus NBP2-24917) and TLR9 for 60 minutes (diluted 1:300, Novus NBP2-24729).

Techniques: Staining

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: Genetics and Molecular Research

Article Title: Protective effects of bifidobacteria on intestines in newborn rats with necrotizing enterocolitis and its regulation on TLR2 and TLR4

doi: 10.4238/2015.september.28.2

Figure Lengend Snippet: Figure 2. Immunohistochemistry can detect the expression level of TLR2 in neonatal intestinal tissues of rats in each group (200X). TLR2 was expressed in each group, and was more obvious in crypts. There was weakly positive staining on the top of intestinal epithelial cytoplasm in groups C (c), D (d), and E (e), while there was strong positive expression on villi and crypts in groups B (b) and A (a).

Article Snippet: The TLR2 immunohistochemistry primary antibody (rabbit anti-rat) was purchased from GenWay Biotech, Inc. (San Diego, CA, USA).

Techniques: Immunohistochemistry, Expressing, Staining

Journal: Genetics and Molecular Research

Article Title: Protective effects of bifidobacteria on intestines in newborn rats with necrotizing enterocolitis and its regulation on TLR2 and TLR4

doi: 10.4238/2015.september.28.2

Figure Lengend Snippet: Figure 3. Immunohistochemistry can detect the expression level of TLR4 in neonatal intestinal tissues of rats in each group (200X). TLR4 in groups C (c), D (d) and E (e) was only weak expressed in rat intestinal epithelial cells, and not expressed in the submucosa. There was an enhanced and increased expression in group A (a), while in group B (b) the expression was significantly increased, showing diffuse or granular distribution.

Article Snippet: The TLR2 immunohistochemistry primary antibody (rabbit anti-rat) was purchased from GenWay Biotech, Inc. (San Diego, CA, USA).

Techniques: Immunohistochemistry, Expressing

Journal: Genetics and Molecular Research

Article Title: Protective effects of bifidobacteria on intestines in newborn rats with necrotizing enterocolitis and its regulation on TLR2 and TLR4

doi: 10.4238/2015.september.28.2

Figure Lengend Snippet: Figure 4. Immunohistochemistry can detect the expression level of NF-кB p65 in neonatal intestinal tissues of rats in each group (200X). There was no nuclear staining of NF-кB p65 in intestinal villi of Group E (e) rats. In groups C (c) and D (d), some nuclei stained can be seen. Nuclear staining increased significantly in group A (a), while Group B (b) showed a large nuclear staining.

Article Snippet: The TLR2 immunohistochemistry primary antibody (rabbit anti-rat) was purchased from GenWay Biotech, Inc. (San Diego, CA, USA).

Techniques: Immunohistochemistry, Expressing, Staining

Journal: International Journal of Biological Sciences

Article Title: Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

doi: 10.7150/ijbs.49332

Figure Lengend Snippet: Pam3CSK4 induces inflammatory responses in AVIC and monocyte co-culture. A. AVICs and monocytes were cultured separately and treated with a low dose of Pam3CSK4 (0.03 µg/ml) for 24 h. Representative immunoblots and ELISA data of 4 separate experiments show that Pam3CSK4 elevates ICAM-1 level moderately and causes a slight increase in MCP-1 production in monocytes while Pam3CSK4 in this low dose has no effect on the levels of ICAM-1, VCAM-1 and MCP-1 in AVICs. B. AVIC and monocyte co-cultures were treated with Pam3CSK4 (0.03 µg/ml) for 24 hours or left untreated. Representative immunoblots and ELISA data show that low levels of ICAM-1, VCAM-1 and MCP-1 are present in untreated control. However, stimulation of TLR2 with a low dose of Pam3CSK4 markedly up-regulates the production of ICAM-1, VCAM-1 and MCP-1 in co-cultures. Data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves, *P <0.05 vs. control. C. Representative images of ICAM-1 immunofluorescence staining from 3 experiments show that both AVICs and monocytes express ICAM-1 (red) following the exposure to a low dose of Pam3CSK4 (0.03 µg/ml). Immunofluorescence staining of vimentin (green) and DAPI counterstaining (blue) were applied to identify AVICs and to outline all nuclei in the co-culture. Original magnification 40× objective.

Article Snippet: Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO).

Techniques: Co-Culture Assay, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

doi: 10.7150/ijbs.49332

Figure Lengend Snippet: Conditioned medium from Pam3CSK4-treated monocytes induces inflammatory responses in human AVICs. Conditioned medium was collected from monocyte cultures treated with Pam3CSK4 (0.10 µg/ml, Pam3 CM) or untreated controls (control CM). Pam3 CM and control CM, 0.15 ml each, were added to AVIC culture in 0.35 ml growth medium [achieving a 30% (vol/vol) for CM in AVIC culture], and AVICs were treated with control CM or Pam3 CM for 24 hours. A. Representative immunoblots and densitometric data show that Pam3 CM markedly up-regulates ICAM-1 and VCAM-1 expression in AVICs while neither control CM nor 0.03 µg/ml of Pam3CSK4 (equivalent to the concentration in Pam3 CM-treated AVIC culture) demonstrated this effect. *P <0.05 vs. untreated control . B. ELISA data shows that Pam3 CM stimulates AVICs to produce and release MCP-1. *P <0.05 vs. untreated control. C. AVICs were treated with DMSO or CU CPT 22 for 1 hour before Pam3 CM treatment for 24 hours. Inhibition of TLR2 in AVICs markedly attenuated ICAM-1 and VCAM-1 expression induced by Pam3 CM. *P <0.05 vs. untreated control, #P <0.05 vs. Pam3 CM+DMSO. All data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves.

Article Snippet: Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO).

Techniques: Control, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: International Journal of Biological Sciences

Article Title: Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

doi: 10.7150/ijbs.49332

Figure Lengend Snippet: Pam3 CM up-regulates TLR2 expression in AVICs to elicit cellular inflammatory responses. A. AVICs were exposed to Pam3CSK4 (0.03 μg/ml), control CM (30%) or Pam3 CM (30%) for 24 hours. Representative immunoblots and densitometric data show that Pam3 CM elevates TLR2 levels, but not MyD88 levels in AVICs. *P <0.05 vs. untreated control, Pam3CKS4-treated and control CM-treated. B. AVICs were treated with control CM or Pam3 CM for 4 to 24 hours . A representative immunoblot and densitometric data show that cellular TLR2 levels increase at 8, 12 and 24 hours following the exposure to Pam3 CM. *P <0.05 vs. control CM treatment for the same time period. Representative images of immunofluorescence staining confirmed the up-regulation of TLR2 (red) at 8-24 hours of treatment with Pam3 CM. The nuclei were visualized with DAPI (blue) counterstaining. Original magnification 40× objective. C and D. AVICs were transduced with lentivirus expressing control (mock) shRNA or TLR2 shRNA. After expression for 5 days, cells were treated with Pam3 CM for 24 hours. Knockdown of TLR2 abolished TLR2 up-regulation induced by Pam3 CM, and reduced the production of adhesion molecules and MCP-1. *P <0.05 vs. mock transduction, #P <0.05 vs. Pam3 CM + mock transduction. All quantitative data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves in each group.

Article Snippet: Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO).

Techniques: Expressing, Control, Western Blot, Immunofluorescence, Staining, Transduction, shRNA, Knockdown

Journal: International Journal of Biological Sciences

Article Title: Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

doi: 10.7150/ijbs.49332

Figure Lengend Snippet: TNF-α up-regulates TLR2 and contributes to the mechanism underlying the inflammatory responses in AVICs, but recombinant TNF-α is inadequate to induce the inflammatory responses. A. ELISA assay confirmed that Pam3 CM has greater levels of TNF-α in comparison to control CM. Data are presented as mean ± SEM. n=5, *P <0.05 vs. control CM. B. AVICs were treated for 24 hours with Pam3 CM pre-incubated with TNF-α neutralizing antibody (10 µg/ml) or non-immune mouse IgG (10 µg/ml). Neutralization of TNF-α abolished the effect of Pam3 CM on TLR2 up-regulation and attenuated the capacity of Pam3 CM to induce ICAM-1 and VCAM-1 expression in AVICs. *P <0.05 vs. untreated control, #P <0.05 vs. Pam3 CM and Pam3 CM + IgG. C. AVICs were treated with recombinant TNF-α (1.0 or 2.0 ng/ml) or Pam3 CM for 24 hours. TNF-α in the tested doses increased TLR2 levels in AVICs, but had no effect on the expression of ICAM-1 and VCAM-1. Data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves in each group. *P <0.05 vs. untreated control.

Article Snippet: Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Comparison, Control, Incubation, Neutralization, Expressing

Journal: International Journal of Biological Sciences

Article Title: Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

doi: 10.7150/ijbs.49332

Figure Lengend Snippet: Schematic diagram depicting the mechanism underlying the interaction between monocytes and AVICs. Monocytes activated by TLR2 agonist release pro-inflammatory cytokines. Among these cytokines, TNF-α is responsible for up-regulating AVIC expression of TLR2. AVICs with greater levels of TLR2 become hypersensitive to TLR2 stimulation by a low level of activator.

Article Snippet: Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO).

Techniques: Expressing