Journal: Journal of Inflammation Research

Article Title: Therapeutic Effect of C-C Chemokine Receptor Type 1 (CCR1) Antagonist BX471 on Allergic Rhinitis

doi: 10.2147/JIR.S254717

Figure Lengend Snippet: Relative quantification of NF-kB p65, TLR4, and TLR2 protein expression levels. ( A ) Western blot analysis for NF-kB p65, TLR4, and TLR2 relative expression from groups indicated. ( B ) summary graph for relative expression of NF-kB (n=8 per group). ( C ) summary graph for relative expression of TLR4 (n=8 per group). ( D ) summary graph for relative expression of TLR2 (n=8 per group). All values are represented as mean ± SEM. * p<0.05; ** p<0.01; and *** p<0.001 versus the vehicle control group.

Article Snippet: Samples were subsequently treated with anti β-actin antibody (BM3873, Boster Biological Technology, China), NF-kB p65 monoclonal antibody (A10609, Abclonal, USA.), TLR4 antibody (BA1717, Boster Biological Technology, China), or TLR2 antibody (BM4001, Boster Biological Technology, China), and were incubated overnight at 4°C.

Techniques: Quantitative Proteomics, Expressing, Western Blot, Control

Journal: Journal of Neuroinflammation

Article Title: Protection of ischemic post conditioning against transient focal ischemia-induced brain damage is associated with inhibition of neuroinflammation via modulation of TLR2 and TLR4 pathways

doi: 10.1186/1742-2094-11-15

Figure Lengend Snippet: The effect of ischemic postconditioning on TLR2 between 2 hour and 4.5 hour ischemia groups. (A) Representative images of cells with positive immunochemical staining to TLR2, scale bar = 50 μm. (B) Statistics of the number of cells stained with TLR2. (C) Statistics of the transcriptional levels of TLR2. (D) Representative Western blotting images of TLR2. (E) Statistics of the expressional level of TLR2. (F) Representative images under confocal microscope in the sham group. (G) Representative images under confocal microscope in the ischemia group; (n = 4 in each group).

Article Snippet: After the standard histological procedures, sections were incubated overnight at 4°C in the mixture of 1:100 rabbit anti-TLR2 (Boster Biotechnology Company, Wuhan, China)/1:100 mouse anti-NeuN (Millipore Corporation, Billerica, USA), 1:100 rabbit anti-TLR2/1:1,000 mouse anti-GFAP (Millipore Corporation, Billerica, USA), 1:100 rabbit anti-TLR2 (Boster Biotechnology Company, Wuhan, China), 1:100 rabbit anti-TLR4 (Boster Biotechnology Company, Wuhan, China)/1:100 mouse anti-NeuN (Millipore Corporation, Billerica, USA), 1:100 mouse anti-TLR4 (Abcam, Cambridge, USA)/1:1,000 rabbit anti-GFAP (Millipore Corporation, Billerica, USA), 1:100 mouse anti-TLR4 (Abcam, Cambridge, USA).

Techniques: Staining, Western Blot, Microscopy

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Improving the ex vivo expansion of human tumor-reactive CD8 + T cells by targeting toll-like receptors

doi: 10.3389/fbioe.2022.1027619

Figure Lengend Snippet: Co-stimulation with TLR1/2, TLR2/6, and TLR5 agonists enhances the proliferative response of human blood-derived CD8 + T cells to TCR triggering. (A) Expression profiles of surface TLRs in resting CD8 + T cells within human blood mononuclear cells (PBMCs). The T cell subpopulations of human PBMCs were identified by staining the cells with antibodies against CD3, CD4, and CD8; the TLR expression was measured by flow cytometry and expressed as the percentage of positively stained cells. Shown is aggregated data of 12 samples. (B and C) Co-stimulatory effects of different TLR agonists on the proliferation of human PBMC-derived CD8 + T cells. CD8+T cells were purified from human PBMCs, stained with eFluor 670, and then stimulated with ant-CD3 beads, anti-CD3/CD28 beads, or anti-CD3 beads coupled with the indicated TLRs agonists. Cells were harvested 7 days later and dilution of eFluor 670 was determined by flow cytometry to measure cellular proliferation, with representative flow cytometry plot and data summary (n = 3) shown in (B) and (C) , respectively. (D,E) Determination of optimal dosages of TLR agonists required for co-stimulation of ex vivo proliferation of human CD8 + T cells. CD8+T cells purified from human PBMCs were stimulated with anti-CD3 beads in presence of various concentration of TLR1/2 agonist, TLR2/6 agonist, or TLR5 agonist after labelling with eFluor 670. Flow cytometry analyses were performed on day 4 and 6 after stimulation to determine the cellular proliferation through assessment of eFluor 670 dilution. Shown is aggregated data of 3 samples.

Article Snippet: For surface staining, cells were stained in FACS buffer (PBS supplemented with 2% bovine serum albumin) sequentially with a fixable viability dye and a combination of the following antibodies: CD3-ECD (IM2705U, Beckman coulter), CD8-BV711(563677, BD Pharmingen), CD4-percp-cy5.5 (300530, Biolegend), CD14-BV786(563699, BD Pharmingen), TLR1-FITC(sc-47709FITC, Santa Cruz), TLR2-PE-Vio770 (130-099-021, Miltenyi), TLR4-BV421 (312811, Biolegend), TLR5-Alexa Fluor 700 (NBP2-24787AF700, NOVUS biology), TLR6-DyLight 350 (NB100-56536UV, NOVUS biology), CD3-BV421(562426, BD Pharmingen), CD8-APC-H7(580179, BD Pharmingen), PD-1-PE-CF594(565024, BD Pharmingen), CTLA-4-APC(555855, BD Pharmingen), ICOS-FITC(313505, Biolegend), 4-1BB-PE-Cy7 (309818, Biolegend).

Techniques: Derivative Assay, Expressing, Staining, Flow Cytometry, Purification, Ex Vivo, Concentration Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Improving the ex vivo expansion of human tumor-reactive CD8 + T cells by targeting toll-like receptors

doi: 10.3389/fbioe.2022.1027619

Figure Lengend Snippet: Co-stimulation with a combination of TLR1/2, TLR2/6, and TLR5 agonists enhances the transcriptional induction of proliferative-related genes in human blood-derived CD8 + T cells responding to TCR triggering. CD8+T cells purified from human PBMCs were stimulated with anti-CD3 beads in the presence or absence of a combination of TLR1/2 agonist, TLR2/6 agonist, and TLR5 agonist. Cells were harvested 24 h later for extracting total RNA, which was subjected to RNA sequencing. The differentially expressed genes showing >2-fold change between minus and plus TLR agonists groups were clustered based on Gene Ontology (GO) enrichment analysis (A) . Shown in (B) is the heatmap of representative proliferation-related genes that were identified to be co-stimulated by TLR agonists. H1 to H3 represent samples from three healthy individuals.

Article Snippet: For surface staining, cells were stained in FACS buffer (PBS supplemented with 2% bovine serum albumin) sequentially with a fixable viability dye and a combination of the following antibodies: CD3-ECD (IM2705U, Beckman coulter), CD8-BV711(563677, BD Pharmingen), CD4-percp-cy5.5 (300530, Biolegend), CD14-BV786(563699, BD Pharmingen), TLR1-FITC(sc-47709FITC, Santa Cruz), TLR2-PE-Vio770 (130-099-021, Miltenyi), TLR4-BV421 (312811, Biolegend), TLR5-Alexa Fluor 700 (NBP2-24787AF700, NOVUS biology), TLR6-DyLight 350 (NB100-56536UV, NOVUS biology), CD3-BV421(562426, BD Pharmingen), CD8-APC-H7(580179, BD Pharmingen), PD-1-PE-CF594(565024, BD Pharmingen), CTLA-4-APC(555855, BD Pharmingen), ICOS-FITC(313505, Biolegend), 4-1BB-PE-Cy7 (309818, Biolegend).

Techniques: Derivative Assay, Purification, RNA Sequencing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Improving the ex vivo expansion of human tumor-reactive CD8 + T cells by targeting toll-like receptors

doi: 10.3389/fbioe.2022.1027619

Figure Lengend Snippet: TLR agonists is an advantageous supplement benefiting the ex vivo expansion of human blood-derived PD1+ CD8 + T cells. (A) Exploration of optimal combination of TLR agonists in facilitating the ex vivo expansion of human blood-derived PD1+ CD8 + T cells. PD1+ CD8 + T cells isolated from blood samples of health individuals were continuously cultured for 21 days in medium containing anti-CD3/anti-CD28 beads and IL-7/IL-15, either alone or in the presence of different combination of TLR1/2, TLR2/6, and TLR5 agonists. Cell counting was performed at various time points throughout the culture period to generate the cell expansion curve. (B) Expansion curves of PD1+ CD8 + T cells derived from blood samples of cancer patients with different tumor types. Among the total of fifteen samples, seven were with lung cancer, four with pancreatic cancer, and the rest four with other types of cancers. Five samples from healthy individuals were also included as controls.

Article Snippet: For surface staining, cells were stained in FACS buffer (PBS supplemented with 2% bovine serum albumin) sequentially with a fixable viability dye and a combination of the following antibodies: CD3-ECD (IM2705U, Beckman coulter), CD8-BV711(563677, BD Pharmingen), CD4-percp-cy5.5 (300530, Biolegend), CD14-BV786(563699, BD Pharmingen), TLR1-FITC(sc-47709FITC, Santa Cruz), TLR2-PE-Vio770 (130-099-021, Miltenyi), TLR4-BV421 (312811, Biolegend), TLR5-Alexa Fluor 700 (NBP2-24787AF700, NOVUS biology), TLR6-DyLight 350 (NB100-56536UV, NOVUS biology), CD3-BV421(562426, BD Pharmingen), CD8-APC-H7(580179, BD Pharmingen), PD-1-PE-CF594(565024, BD Pharmingen), CTLA-4-APC(555855, BD Pharmingen), ICOS-FITC(313505, Biolegend), 4-1BB-PE-Cy7 (309818, Biolegend).

Techniques: Ex Vivo, Derivative Assay, Isolation, Cell Culture, Cell Counting

Journal: Arthritis Research & Therapy

Article Title: Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

doi: 10.1186/ar4274

Figure Lengend Snippet: The effect of gene silencing on fHA-mediated IL-6 production in IVD cells . A ) siRNA-mediated knockdown of genes Toll like receptor ( TLR ) 2 , TLR4 , CD44 and RHAMM was confirmed after 30 hours in intervertebral disc (IVD) cells by qRT-PCR ( n = 4). In each case, gene expression was calculated as fold change as compared to untreated cells. The use of a non-specific scrambled siRNA (siRNA (S)), confirmed specificity of gene knockdown. B, C ) Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) ( n = 4) ( B ) and lipopolysaccharide (LPS)- (25 ng/ml) ( n = 3). ( C ) stimulated IVD cells following gene knockdown of TLR2 or TLR4 respectively, as determined by IL-6 ELISA. ( D ) hyaluronic acid fragment (fHA)-treated (20 μg/ml) IVD cells following gene knockdown as determined by IL-6 ELISA ( n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t -test, * P <0.01.

Article Snippet: Cells were then pre-incubated for one hour with either an affinity purified polyclonal rat anti-human TLR2 neutralizing antibody (final concentration 5 μg/ml) (LabForce, Switzerland) or an isotype matched IgG control (Lucerna-Chem, Luzern, Switzerland).

Techniques: Knockdown, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

doi: 10.1186/ar4274

Figure Lengend Snippet: The effect of TLR2 inhibition on fHA-mediated IL-6 production in IVD cells . Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) ( A ) and hyaluronic acid fragment (fHA)-treated (20 μg/ml) ( B ) intervertebral disc (IVD) cells following antibody-mediated neutralization of Toll like receptor (TLR)2 activity ( n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t -test, * P <0.05 as compared to cells treated with non-specific IgG control antibody.

Article Snippet: Cells were then pre-incubated for one hour with either an affinity purified polyclonal rat anti-human TLR2 neutralizing antibody (final concentration 5 μg/ml) (LabForce, Switzerland) or an isotype matched IgG control (Lucerna-Chem, Luzern, Switzerland).

Techniques: Inhibition, Neutralization, Activity Assay, Control

Journal: Molecules

Article Title: Baicalein and Αlpha-Tocopherol Inhibit Toll-like Receptor Pathways in Cisplatin-Induced Nephrotoxicity

doi: 10.3390/molecules27072179

Figure Lengend Snippet: Effect of α-tocopherol and baicalein administration on TLR2 and TLR4 in control and treated groups. All data are expressed as mean ± SEM. Significant vs. control group refers to A, while significant vs. (cisplatin, -tocopherol, and baicalein) treatment groups refers to (B, C, D), respectively.

Article Snippet: Briefly, paraffin-embedded sections were subjected to antigen retrieval citrate buffer for 30 min. Then, the expression levels of Kelch-like ECH associatedprotein1 (Keap-1) (cat:sc-514914, Santa Cruz), toll-like receptor 4 (TLR4) (cat: A5258, AB clonal), and toll-like receptor 2 (TLR2) (cat: A11225, AB clonal) were detected at 1:100 dilution; biotinylated secondary antibody was added after washes, and the slides washed again with PBS.

Techniques:

Journal: Molecules

Article Title: Baicalein and Αlpha-Tocopherol Inhibit Toll-like Receptor Pathways in Cisplatin-Induced Nephrotoxicity

doi: 10.3390/molecules27072179

Figure Lengend Snippet: Microscopic pictures of adrenal sections immunostained against TLR2 from rat groups sacrificed after 10 days showing ( A ) positive cell score, significant vs. control group refers to A, while significant vs. (cisplatin, -tocopherol, and baicalein) treatment groups refers to (B, C, D), respectively, ( B ) control group, and ( C ) marked expression in positive brown tubular expression in Cisplatin group. ( D ) The positive brown tubular expression slightly decreased in renal sections from α-tocopherol group, ( E ) moderately decreased in renal sections from baicalein group, and ( F ) significantly decreased in renal sections from the combined treatment group. Black arrows indicate positive reaction. IHC counterstained with Mayer’s hematoxylin. High magnification ×: 400 bar 50.

Article Snippet: Briefly, paraffin-embedded sections were subjected to antigen retrieval citrate buffer for 30 min. Then, the expression levels of Kelch-like ECH associatedprotein1 (Keap-1) (cat:sc-514914, Santa Cruz), toll-like receptor 4 (TLR4) (cat: A5258, AB clonal), and toll-like receptor 2 (TLR2) (cat: A11225, AB clonal) were detected at 1:100 dilution; biotinylated secondary antibody was added after washes, and the slides washed again with PBS.

Techniques: Expressing

Journal: Molecules

Article Title: Baicalein and Αlpha-Tocopherol Inhibit Toll-like Receptor Pathways in Cisplatin-Induced Nephrotoxicity

doi: 10.3390/molecules27072179

Figure Lengend Snippet: Primers used for quantitative RT-PCR.

Article Snippet: Briefly, paraffin-embedded sections were subjected to antigen retrieval citrate buffer for 30 min. Then, the expression levels of Kelch-like ECH associatedprotein1 (Keap-1) (cat:sc-514914, Santa Cruz), toll-like receptor 4 (TLR4) (cat: A5258, AB clonal), and toll-like receptor 2 (TLR2) (cat: A11225, AB clonal) were detected at 1:100 dilution; biotinylated secondary antibody was added after washes, and the slides washed again with PBS.

Techniques: Sequencing