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Image Search Results
Journal: Frontiers in Immunology
Article Title: Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis
doi: 10.3389/fimmu.2022.818630
Figure Lengend Snippet: proNGF increases the expression of inflammatory cytokines in synovial fibroblasts from RA patients. (A) The addition of exogenous proNGF (200ng/ml) together with IL-1β at suboptimal doses (10pg/ml and 50pg/ml) induces a synergic increase in IL-6 production not observed at higher concentration of IL-1β. The experiments (n = 4) were performed in serum free medium. Data were analyzed by paired t -test (*p < 0.05). (B–D) Inhibition of proNGF binding to p75NTR with LM11A-31 (10nM) decreases inflammatory mediators: IL-6 (B) , IL-8 (C) and MCP1 (D) production was significantly reduced in RA-FLS cells activated using 100 ng/ml TNF-α, 100 ng/ml LPS or 1 ng/ml IL-1β. RA-FLS were cultured for 18 hours in 10% FBS DMEM. The inhibitory effect of LM11A-31 on cytokine release is expressed as percentage decrease (% decrease) from activated cells. (E) To obtain a confirm of the effects of p75NTR blocking, p75NTR was neutralized using a specific anti-p75NTR antibody (2,5 µg/ml) and the release of IL-6 was measured in RA-FLS activated with 100 ng/ml TNF-α, 100 ng/ml LPS or 1ng/ml IL-1β. In this set of experiments (n = 6) cells were cultured in 10% FBS DMEM for 18 hours. The results are expressed as percentage decrease (% decrease) from activated cells.
Article Snippet: RA-FLS and OA-FLS were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 PBS, incubated for 1 hour at RT with 1% BSA, 5% goat serum (Abcam, Cambridge, UK) PBS and then with mouse anti-p75NTR antibody (Merck, Darmstadt, Germany, clone 8211) or
Techniques: Expressing, Concentration Assay, Inhibition, Binding Assay, Cell Culture, Blocking Assay
Journal: Frontiers in Immunology
Article Title: Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis
doi: 10.3389/fimmu.2022.818630
Figure Lengend Snippet: proNGF present in synovial fluids increases the expression of inflammatory mediators in synovial fibroblasts from RA patients. (A) proNGF is by far the most abundant NGF form (100 to 200-fold that of mature NGF) detected in synovial fluids (SF) obtained from six active patients (1-6). (B) To recreate ex vivo inflamed synovia condition, 30% v/v synovial fluid was added to RA-FLS (n = 19) cultured in 10% FBS DMEM. Data were analyzed by paired t -test (***p < 0.001). (C) RA-FLS in 30% v/v synovial fluid were treated with 10 nM of LM11A-31 with LM11A-31 and IL-6 production was measured after 18 hours. The data represent the percentage of inhibition obtained in 19 independent experiments performed using seven different RA-FLS and synovial fluids obtained from different patients (n = 18). Data were analyzed by one sample t -test (***p < 0.001). (D) 30% v/v synovial fluid was added to RA-FLS cultured in 10% FBS DMEM with or without the addition of anti-IL1β (5µg/ml) (n = 9). IL-6 release was measured after 18 hours of incubation. Data were analyzed by one sample t-test (*p < 0.05). (E) The apoptosis rate of RA-FLS treated with 30% v/v of synovial fluids with or without the addition of LM11A-31 at two different doses (10nM used for all our experiment and a ten-fold higher dose 100 nM) was analyzed by Annexin V/PI staining. No modification in the percentage of apoptotic cells was observed following the addition of synovial fluid or after p75NTR inhibition with LM11A-3 with both the doses used. Representative scatterplots of three independent experiments are shown.
Article Snippet: RA-FLS and OA-FLS were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 PBS, incubated for 1 hour at RT with 1% BSA, 5% goat serum (Abcam, Cambridge, UK) PBS and then with mouse anti-p75NTR antibody (Merck, Darmstadt, Germany, clone 8211) or
Techniques: Expressing, Ex Vivo, Cell Culture, Inhibition, Incubation, Staining, Modification
Journal: Frontiers in Immunology
Article Title: Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis
doi: 10.3389/fimmu.2022.818630
Figure Lengend Snippet: The pro-inflammatory p75NTR/proNGF loop in RA-FLS. (A) Inflammatory stimuli activate an autocrine loop involving proNGF and p75NTR. Inflammatory stimuli strongly induce the contemporary expression of p75NTR, the specific proNGF receptor, and of its ligand, proNGF, in RA-FLS. The increased p75NTR expression results in a higher binding capacity of RA-FLS for proNGF, whose endogenous production is strongly induced by inflammatory stimuli. (B) The active p75NTR/proNGF axis enhances inflammatory cytokines production. The high amounts of endogenously-produced proNGF, released by activated RA-FLS, interact with the p75NTR receptors highly expressed on RA-FLS membrane. The activation of p75NTR intracellular pathways (i.e. p38 and JNK) results in an amplified production of inflammatory cytokines (i.e. IL-6). (C) Inhibition of the p75NTR/proNGF loop decreases inflammatory cytokines production. The blocking of p75NTR using LM11A-31, a small molecule that specifically blocks the binding site of p75NTR for proNGF, results in a net reduction of inflammatory cytokine production (i.e. IL-6). The endogenous proNGF released by active RA-FLS cannot bind to p75NTR and, consequently, does not activate p75NTR intracellular pathways that induce inflammatory cytokine production.
Article Snippet: RA-FLS and OA-FLS were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 PBS, incubated for 1 hour at RT with 1% BSA, 5% goat serum (Abcam, Cambridge, UK) PBS and then with mouse anti-p75NTR antibody (Merck, Darmstadt, Germany, clone 8211) or
Techniques: Expressing, Binding Assay, Produced, Activation Assay, Amplification, Inhibition, Blocking Assay
Journal: Oncotarget
Article Title: ProNGF is a potential diagnostic biomarker for thyroid cancer
doi: 10.18632/oncotarget.8652
Figure Lengend Snippet: A–D. Immunohistochemical detection of proNGF was performed with a polyclonal antibody on a series of thyroid cancers (n=40), adenoma (n=40) and normal thyroid tissues (n=80). ProNGF was found in epithelial cells with a marked increased in cancer tissues. Representative pictures are shown for normal thyroid tissue (A), adenoma (B), papillary carcinoma (C), follicular carcinoma (D). Scale = 50μm. E. Quantification of proNGF staining intensities was performed using the Halo™ image analysis platform, h-scores were calculated and used to establish the ROC curves. ProNGF staining intensities were significantly higher for cancers (median h-score = 69.3) than adenomas (median h-score = 35.5) and normal tissues (median h-score = 19.7) (p<0.0001). The box limits indicate the 25th and 75th percentiles with the whiskers extending 1.5 times the interquartile range from the 25th and 75th percentiles (outliers are represented by dots) (****p<0.0001). F–H. ROC curves for proNGF staining intensity levels in thyroid cancers versus adenomas and normal thyroid tissues were established and analyzed using GraphPad™. The area under the curve was 0.99 (95% CI 0.98-1.00, p<0.0001) for cancers versus normal samples (F), 0.84 (95% CI 0.75-0.93, p<0.0001) for cancers versus adenomas (G), and 0.95 (95% CI 0.85-0.96, p<0.0001) for cancers versus adenomas and normal samples (H).
Article Snippet: A
Techniques: Immunohistochemical staining, Staining
Journal: Oncotarget
Article Title: ProNGF is a potential diagnostic biomarker for thyroid cancer
doi: 10.18632/oncotarget.8652
Figure Lengend Snippet: ProNGF expression in thyroid cancers versus adenomas and normal tissues (cohort 1) and associations with clinicopathological parameters
Article Snippet: A
Techniques: Expressing
Journal: Oncotarget
Article Title: ProNGF is a potential diagnostic biomarker for thyroid cancer
doi: 10.18632/oncotarget.8652
Figure Lengend Snippet: A-F. Immunohistochemical detection of proNGF in thyroid cancers of various histological types (n=127 cases), adenomas (n=6) and normal thyroid tissues (n=55) was performed witha monoclonal antibody. ProNGF was found in epithelial cells with a marked increased in cancer tissues. Representative pictures are shown for normal thyroid tissue (A), adenoma (B), papillary carcinoma (C), follicular carcinoma (D), medullary carcinoma (E), and anaplastic carcinoma (F). Scale = 50μm. G. Quantification of proNGF staining intensities was performed using the Halo™ image analysis platform, h-scores were calculated and used to establish the ROC curves. ProNGF staining intensities were significantly higher for cancers (median h-score = 54.6) than adenomas (median h-score = 21.7) and normal tissues (median h-score = 7.2). The box limits indicate the 25th and 75th percentiles with the whiskers extending 1.5 times the interquartile range from the 25th and 75th percentiles (outliers are represented by dots) (****p<0.0001). H. The ROC curve for proNGF staining intensity levels in thyroid cancers versus normal thyroid tissues was established and analyzed using GraphPad™. The area under the ROC curve was 0.98 (95% CI 0.97-0.99, p<0.0001).
Article Snippet: A
Techniques: Immunohistochemical staining, Staining
Journal: Oncotarget
Article Title: ProNGF is a potential diagnostic biomarker for thyroid cancer
doi: 10.18632/oncotarget.8652
Figure Lengend Snippet: ProNGF expression in thyroid cancers of different histological types (cohort 2) and associations with clinicopathological parameters
Article Snippet: A
Techniques: Expressing