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: 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 proNGF immunoreactivity in naïve glabrous skin and following application of CCI . A) In sham operated controls, proNGF (brown precipitate) was found in the upper dermis, particularly in wall of blood vessels, and in small patches of keratinocytes (arrowhead). B) 1 week post-injury, proNGF immunolabeling was detected in mast cells (arrows; identified based on toluidine blue metachromatic counterstaining), in blood vessels in the upper dermis as well as in keratinocytes. C) At 2 weeks post-injury, note the intense immunostaining of keratinocytes, the labeling of some unidentified structures in dermis and a lower number of immunostained mast cells. D) At 4 weeks post-injury, note mast cell immunolabeling. E) Preincubation of antibody with control peptide abolished all specific staining. F) proNGF quantification was done by Western Blot analysis (n = 6) from sham, and animals 1 - 4 week post-injury. OD was normalized against β-actin loading controls. ***p < 0.001, **p < 0.01, *p < 0.05; +SEM.

Article Snippet: Primary antibodies such as mouse anti-TrkA (1:500, MAB1056, R&D Systems, Minneapolis, MN), rabbit anti-proNGF (1:500, ANT-005, Alomone Labs, Isreal), or mouse anti-p75 (1:200, MC192, Novus Biologicals) were incubated overnight in blocking buffer at 4°C.

Techniques: Immunolabeling, Immunostaining, Labeling, Staining, Western Blot

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: Changes in p75 expression on S100-IR Schwann cells . A) In sham-operated rats Schwann cells (green) were detected by means of S100 immunoreactivity in small nerves, with immunoreactivity of varying intensity from bright (arrow), lower in the dermis, to dim, along the dermo-epidermal junction. P75 immunoreactivity (red) was evident surrounding all S100-IR Schwann cells. B) One week following nerve injury, p75 immunostaining was dramatically upregulated in Schwann cells; the intense red color masked the mixture of red and green stainings which could be detected by analysing the separately the S100 and p75 stainings (not shown). C-D) 2 & 4 weeks post-injury a decrease in p75 intensity was observed in that the yellow indicative of S100 co-labelling was able to be visualized. E) proNGF (red) S100 (green) and p75 (blue) triple labelling to demonstrate the relative distribution of Schwann cells with respect to proNGF; note a limited distribution of Schwann cells with proNGF and faint immunoreactivity for p75 in sham-operated controls. F) 2 weeks post-injury, the clear upregulation of p75 immunoreactivity associated with S100 (arrow) was observed, which wrapped around proNGF-IR blood vessels.

Article Snippet: Primary antibodies such as mouse anti-TrkA (1:500, MAB1056, R&D Systems, Minneapolis, MN), rabbit anti-proNGF (1:500, ANT-005, Alomone Labs, Isreal), or mouse anti-p75 (1:200, MC192, Novus Biologicals) were incubated overnight in blocking buffer at 4°C.

Techniques: Expressing, Immunostaining

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: Primary antibodies such as mouse anti-TrkA (1:500, MAB1056, R&D Systems, Minneapolis, MN), rabbit anti-proNGF (1:500, ANT-005, Alomone Labs, Isreal), or mouse anti-p75 (1:200, MC192, Novus Biologicals) were incubated overnight in blocking buffer at 4°C.

Techniques: Staining, Immunostaining

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: 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: RMD Open

Article Title: ProNGF-p75NTR axis plays a proinflammatory role in inflamed joints: a novel pathogenic mechanism in chronic arthritis

doi: 10.1136/rmdopen-2017-000441

Figure Lengend Snippet: p75NTR, TrkA and sortilin expression levels in patients with JIA. (A) mRNA expression levels of TrkA, p75NTR and sortilin in freshly isolated mononuclear cells from peripheral blood of healthy children (CTRL PBMC) and in mononuclear cells from peripheral blood or from synovial fluids of patients with JIA (respectively, JIA PBMC and JIA SFMC) were quantified by real-time PCR analysis. JIA PBMC (n=34) and JIA SFMC (n=66) express low TrkA and high p75NTR mRNA levels compared with CTRL PBMC (n=10). Sortilin, a coreceptor for p75NTR essential for proNGF binding, is highly expressed in JIA SFMC (n=19) and expressed in JIA PBMC (n=13) and in CTRL PBMC (n=7). The results are expressed as arbitrary units (AU) after normalisation with the housekeeping gene GAPDH. (B) p75NTR mRNA levels are higher in SFMC than in PBMC of matched patients with JIA (n=18). (C) Sortilin mRNA levels are higher in SFMC than in PBMC of matched patients with JIA (n=11). (D) Western blot and densitometric analysis of three independent experiments confirmed that p75NTR was the prevalent NGF receptor in JIA mononuclear cells, while TrkA was the most expressed NGF receptor in CTRL PBMC. Sortilin is also highly expressed in JIA mononuclear cells. *p<0.05, **p<0.01, ***p<0.001. JIA, juvenile idiopathic arthritis; NGF, nerve growth factor; PBMC, mononuclear cells isolated from peripheral blood; SFMC, mononuclear cells isolated from synovial fluids.

Article Snippet: Mononuclear cells were treated with or without mNGF or mutated cleavage-resistant proNGF (Alomone Labs, Jerusalem, Israel) at a concentration, respectively, of 100 ng/mL and 200 ng/mL, calculated to yield the same molar concentration as described in neurons.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot

Journal: RMD Open

Article Title: ProNGF-p75NTR axis plays a proinflammatory role in inflamed joints: a novel pathogenic mechanism in chronic arthritis

doi: 10.1136/rmdopen-2017-000441

Figure Lengend Snippet: Mature and immature NGF forms in patients with JIA and RA. (A,B) Only proNGF forms of different molecular weights but not mature NGF were detected by western blot in synovial fluids of 12 patients with JIA (40 μg total protein). (B) Blocking the anti-NGF antibody by adding mature NGF results in the disappearance of the specific proNGF bands observed in synovial fluids of three patients with JIA, as well as of the band of the 25 kDa commercial proNGF that was added as positive control. (C) In JIA and RA synovial fluids, proNGF and mature NGF (mNGF) concentrations were assessed using a newly developed ELISA showing that proNGF is 4.8-fold higher in JIA (n=27) and 16.8-fold higher in patients with RA (n=5) than mNGF. (D) Real-time PCR shows a very low expression of NGF mRNA in mononuclear cells obtained from peripheral blood of healthy donors (CTRL PBMC) or in mononuclear cells from peripheral blood (JIA PBMC) and from synovial fluids of patients with JIA (JIA SFMC). High NGF mRNA expression levels characterised synovial tissues (RA synovia; n=5). Fibroblast-like synoviocytes (FLS; n=3) of patients with RA express more NGF mRNA than FLS from osteoarthritis patients (OA FLS) (n=4) and control fibroblasts (CTRL FB; n=4). Results were expressed as arbitrary units (AU) and obtained after normalisation with the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (E) Both unstimulated (US) and LPS-stimulated JIA SFMC do not release proNGF or mNGF in the supernatants. Commercial proNGF and mNGF were added as positive controls. (F) Western blot shows the stability of exogenous added mNGF and proNGF. After its addition to culture media, proNGF is still detected after 3 hours and undergoes a minor maturation into mNGF in the first 2 hours of incubation (left side of F). mNGF is not degraded in the first 3 hours after its addition (right side of F). Commercial proNGF and mNGF were included as positive controls. (G) Neither proNGF nor mNGF were released in culture media of US or LPS-stimulated JIA SFMC after 18 hours of incubation. Exogenous proNGF or mNGF were detected only at time of supplementation (in agreement with F) but were no longer detectable after 18 hours of incubation. Commercial proNGF and mNGF were included as positive controls. (H) ELISA instead shows that conditioned media of RA FLS (n=5) have higher concentrations of proNGF than control fibroblasts (CTRL FB; n=5) and that the concentration of proNGF is higher than mNGF after 18 hours of incubation. *p<0.05, **p<0.01, ***p<0.001. JIA, juvenile idiopathic arthritis; LPS, Lipopolysaccharide ; NGF, nerve growth factor; PBMC, mononuclear cells isolated from peripheral blood; RA rheumatoid arthritis; SFMC, mononuclear cells isolated from synovial fluids.

Article Snippet: Mononuclear cells were treated with or without mNGF or mutated cleavage-resistant proNGF (Alomone Labs, Jerusalem, Israel) at a concentration, respectively, of 100 ng/mL and 200 ng/mL, calculated to yield the same molar concentration as described in neurons.

Techniques: Western Blot, Blocking Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Incubation, Concentration Assay, Isolation

Journal: RMD Open

Article Title: ProNGF-p75NTR axis plays a proinflammatory role in inflamed joints: a novel pathogenic mechanism in chronic arthritis

doi: 10.1136/rmdopen-2017-000441

Figure Lengend Snippet: Ex vivo effects of proNGF and NGF on JIA mononuclear cells. (A) No changes in cell viability were observed when mononuclear cells from synovial fluid (SFMC) of patients with JIA were treated for 24 hours with proNGF or mNGF, with or without LPS stimulation. Apoptosis was assessed by Annexin V flow cytometry analysis. The results represent one of three independent experiments performed in duplicate. (B) While proNGF addition alone did not induce the production of interleukin (IL)-6 in unstimulated SFMC, in LPS-activated cells proNGF induced IL-6 release in a dose-dependent manner with a maximal effect at 200 ng/mL (n=4). (C) proNGF stimulation significantly increased IL-6, IL-1β, IL-8 and tumor necrosis factor-α (TNF-α) mRNA expression levels in LPS-stimulated SFMC (n=18) of patients with JIA after 3 hours of incubation, but did not modify IL-10 mRNA expression in either CTRL or JIA mononuclear cells. Mature NGF treatment did not significantly modify proinflammatory cytokine levels or IL-10 mRNA expression in JIA mononuclear cells, while it increased IL-10 mRNA levels in CTRL PBMC. Results are expressed as arbitrary units (AU) and obtained after normalisation with the housekeeping gene GAPDH. (D) proNGF treatment significantly increased IL-6 release after 18 hours (measured by ELISA) in LPS-stimulated SFMC from patients with JIA (n=16) compared with PBMC of healthy CTRL (n=6). Mature NGF treatment did not affect IL-6 protein levels. *p<0.05. JIA, juvenile idiopathic arthritis; LPS, Lipopolysaccharide ; NGF, nerve growth factor; PBMC, mononuclear cells isolated from peripheral blood.

Article Snippet: Mononuclear cells were treated with or without mNGF or mutated cleavage-resistant proNGF (Alomone Labs, Jerusalem, Israel) at a concentration, respectively, of 100 ng/mL and 200 ng/mL, calculated to yield the same molar concentration as described in neurons.

Techniques: Ex Vivo, Flow Cytometry, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Isolation

Journal: RMD Open

Article Title: ProNGF-p75NTR axis plays a proinflammatory role in inflamed joints: a novel pathogenic mechanism in chronic arthritis

doi: 10.1136/rmdopen-2017-000441

Figure Lengend Snippet: proNGF and NGF activated different intracellular pathways. (A) The neutralisation of p75NTR using either a specific anti-p75NTR antibody (a-p75; 2.5 µg/mL) or using the specific pharmacological inhibitor LM11A.31 (10 nM), decreased interleukin (IL)-6 release in LPS-activated SFMC of patients with JIA (n=16) treated with proNGF. On the contrary, the blocking of TrkA with a neutralising antibody (a-TrkA; 3 µg/mL) did not affect IL-6 release. (B) Western blot shows an increased phosphorylation of p38 and JNK after 5 minutes of proNGF stimulation in LPS-treated SFMC. mNGF treatment did not affect the phosphorylation of these downstream molecules. The result is representative of one out of three independent experiments. Results of the densitometric analysis of all experiments are expressed as arbitrary units (AU). (C) In LPS-activated SFMC of three different patients with JIA, preincubation with the inhibitor LM11A-31 (10 nM), which blocks the binding of proNGF to p75NTR, abolished the phosphorylation of p38 and JNK induced after 5 minutes of proNGF addition. Results of the densitometric analysis of all experiments are expressed as arbitrary units (AU). *p<0.05, **p<0.01, ***p<0.001. JIA, juvenile idiopathic arthritis; LPS, Lipopolysaccharide ; NGF, nerve growth factor; SFMC, mononuclear cells isolated from synovial fluids.

Article Snippet: Mononuclear cells were treated with or without mNGF or mutated cleavage-resistant proNGF (Alomone Labs, Jerusalem, Israel) at a concentration, respectively, of 100 ng/mL and 200 ng/mL, calculated to yield the same molar concentration as described in neurons.

Techniques: Blocking Assay, Western Blot, Binding Assay, Isolation