rabbit anti-prongf  (Merck & Co)

Journal: Molecular Pain

Article Title: Neurotrophic factor changes in the rat thick skin following chronic constriction injury of the sciatic nerve

doi: 10.1186/1744-8069-8-1

Figure Lengend Snippet: Distribution of p75 immunoreactivity following nerve injury and relationship to nerve fibers and proNGF . A) In sham-operated animals, p75 was distributed (red) around and with PGP 9.5-IR nerve fibers (green). P75 staining was found more clearly around large cutaneous PGP 9.5-IR nerve fiber bundles and smaller fibers along the dermo-epidermal junction. Where nerve fibers crossed the dermo-epidermal junction into the epidermis, the yellow color representing p75 associating with nerve fibers was lost (arrow). B) At 1 week post-injury, virtually all PGP-9.5-IR nerve fibers disappeared from the upper dermis and epidermis; immunostaining in p75-IR Schwann was very intense. C) At 2 weeks post-injury, a low number of PGP-9.5-IR fibers were detected and were associated with p75 Schwann cells (yellow), however most of the PGP-9.5-IR was restricted to Langerhans cells in epidermis (arrow) D) At 4 weeks post-injury, p75 immunoreactivity decreased co-incidentally with the increase in PGP-9.5 immunoreactivityin the upper dermis (yellow). E-H) ProNGF and p75 association in sham-operated controls and in lesioned animals was loose in that most proNGF immunoreactivity was segregated from that for p75 and there was no obvious co-localization (arrows).

Article Snippet: Immunohistochemical staining with this antibody in either rat or human skin was absent following preadsorption with purified human PGP 9.5 protein [ , ]. proNGF: The rabbit anti-proNGF antibody (Alomone #ANT-005 Lot#AN-03) was generated by injection of a synthetic peptide corresponding to a.a. 84-104 of the precursor form of rat NGF.

Techniques: Staining, Immunostaining

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 rabbit anti-proNGF (Merck AB9040) and Alexa Fluor secondary antibodies (Invitrogen).

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 rabbit anti-proNGF (Merck AB9040) and Alexa Fluor secondary antibodies (Invitrogen).

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 rabbit anti-proNGF (Merck AB9040) and Alexa Fluor secondary antibodies (Invitrogen).

Techniques: Expressing, Binding Assay, Produced, Activation Assay, Amplification, Inhibition, Blocking Assay