ccl2 neutralising antibody  (R&D Systems)

Journal: Nature Communications

Article Title: The CCL2-CCR2 axis drives neuromuscular denervation in amyotrophic lateral sclerosis

doi: 10.1038/s41467-025-62351-3

Figure Lengend Snippet: A Proteome profiler analysis of 111 inflammatory proteins in gastrocnemius (GC), tibialis anterior (TA) and extensor digitorum longus (EDL) muscles and spinal cord (SC) samples from late symptomatic (postnatal day 19) hTDP-43 Tg/Tg mice. Fold change in protein levels is presented relative to wild-type (WT) littermate controls. In the GC muscle, CCL2, CCL3, CCL4, CCL5 and CCL6 chemokines showed the greatest increase in protein levels (magnified and highlighted in the top right panel). Exact fold changes are available in the Source Data file. B –E Quantitative analysis of CCL2 ( B ), CCL3 ( C ), CCL4 ( D ) and CCL5 ( E ) proteins in the GC and TA muscles and SC using Legendplex immunoassay. Data is presented as mean ± sem. The Legendplex multiplex immunoassay and statistical analysis was performed with n = 4 mice in the GC, TA and SC of the WT group and TA and SC of the Tg/Tg group. The GC of the Tg/Tg group was quantified in n = 3 mice. Data analysis: In each case ( B – E ), unpaired, one-tailed t-test was performed for direct comparison of WT vs Tg/Tg groups; ( B) gastrocnemius : p = 0.0350; ( C) g astrocnemius : p = 0.0174; ( C) tibialis anterior: p = 0.0318; ( C) spinal cord: p = 0.0083; ( D) gastrocnemius: p = 0.0209; ( E) gastrocnemius: p = 0.0031; ( E) tibialis anterior: p = 0.0456. * = p < 0.05; ** = p < 0.01.

Article Snippet: To assess the effects of local immunomodulatory treatment on NMJ denervation and immune cell infiltration, hTDP-43 Tg/Tg mice received two intramuscular injections (at P7 and P14) of either CCL2 neutralising antibody [Cat# AF-479-NA, RRID: AB_354500, R&D Systems, Minneapolis, MN, USA; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in phosphate buffer saline (PBS)] or isotypic normal goat IgG (Cat# AB-108-C, RRID:AB_354267, R&D Systems; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in PBS) in the severely affected GC muscle.

Techniques: Muscles, Multiplex Assay, One-tailed Test, Comparison

Journal: Nature Communications

Article Title: The CCL2-CCR2 axis drives neuromuscular denervation in amyotrophic lateral sclerosis

doi: 10.1038/s41467-025-62351-3

Figure Lengend Snippet: A–C CCL2 staining (green) in the GC muscle of wild-type (WT; A ) and late symptomatic postnatal day 19 (P19) hTDP-43 Tg/Tg (Tg/Tg; B, C ) mice. Arrows indicate CCL2 + cells in the close vicinity of neuromuscular junctions (NMJs), visualised with α-bungarotoxin (BTX; magenta). C High magnification image of CCL2 + cells around a single NMJ in the GC muscle of late stage hTDP-43 Tg/Tg mice. Innervation is shown through NEFH/SV2a (gray) staining. Asterisks label individual nuclei of CCL2 + cells. 3D-reconstructed version of this image is available as Supplementary movie . D–F Quantitative analysis of the ratio of NMJs with perisynaptic CCL2-expressing cells in presymptomatic ( D ), early symptomatic ( E ) and late symptomatic ( F ) mice. Data is presented as mean ± sem. Scale bar represents 50 µm on ( A, B ) and 10 µm on ( C ). Data analysis: ( D ) Presymptomatic stage (postnatal day 7): n = 4 (WT), n = 3 (Tg/Tg), unpaired, two-tailed t-test, p = 0.0466; ( E ) Early symptomatic stage (postnatal day 15): n = 3 (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0088; ( F ) Late symptomatic stage: n = 4 (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0011. * = p < 0.05; ** = p < 0.01.

Article Snippet: To assess the effects of local immunomodulatory treatment on NMJ denervation and immune cell infiltration, hTDP-43 Tg/Tg mice received two intramuscular injections (at P7 and P14) of either CCL2 neutralising antibody [Cat# AF-479-NA, RRID: AB_354500, R&D Systems, Minneapolis, MN, USA; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in phosphate buffer saline (PBS)] or isotypic normal goat IgG (Cat# AB-108-C, RRID:AB_354267, R&D Systems; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in PBS) in the severely affected GC muscle.

Techniques: Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: The CCL2-CCR2 axis drives neuromuscular denervation in amyotrophic lateral sclerosis

doi: 10.1038/s41467-025-62351-3

Figure Lengend Snippet: A Schematic of experimental design. B–D Representative images of NMJ denervation in the gastrocnemius (GC) muscle of untreated ( B ), IgG-treated ( C ) and CCL2-neutralising antibody-treated ( D ) late symptomatic postnatal day 19 (P19) hTDP-43 Tg/Tg mice. Red arrows indicate fully innervated NMJs, as revealed by the presynaptic NEFH/SV2a staining. Scale bar represents 40 µm. E–G Representative images of the CD45 + leukocyte infiltration in the innervation zone of the GC muscle in untreated ( E ), IgG-treated ( F ) and CCL2-neutralising antibody-treated ( G ) late symptomatic hTDP-43 Tg/Tg mice. Scale bar represents 40 µm. H Qualitative analysis of NMJ innervation, representing the ratio of fully innervated NMJs in the GC muscle. Analysis was performed with n = 7 (untreated) and n = 4 (IgG, CCL2) mice/group and one-way ANOVA with Fisher’s LSD post-hoc, no treatment vs. IgG: p < 0.0001, no treatment vs. CCL2: p < 0.0001. On average 30-40 NMJs were analysed per muscle. I–K Quantitative morphometric analysis of the NMJs revealed improvement in axonal diameter ( I ), nerve terminal area ( J ) and overlap ( K ) variables. The exact p values and statistical analyses are detailed in Table . L, M Quantitative analysis of CD45 + ( L ) and CCR2 + ( M ) cell count in the late symptomatic GC muscle of hTDP-43 Tg/Tg mice. Analysis was performed on n = 8 (untreated) and n = 4 (IgG, CCL2) mice/group. Data analysis: ( L) CD45 analysis: Kruskal-Wallis test and uncorrected Dunn’s post-hoc [Shapiro-Wilk normality test, ALS untreated group: p = 0.0091, ALS IgG group: p = 0.7802, ALS CCL2 group p = 0.3820], no treatment vs. IgG: p = 0.0114, no treatment vs. CCL2: p = 0.0053; ( M) CCR2 analysis: one-way ANOVA with Fisher’s LSD post-hoc, no treatment vs. IgG: p < 0.0001, no treatment vs. CCL2: p = 0.0002. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Data is presented as mean ± sem.

Article Snippet: To assess the effects of local immunomodulatory treatment on NMJ denervation and immune cell infiltration, hTDP-43 Tg/Tg mice received two intramuscular injections (at P7 and P14) of either CCL2 neutralising antibody [Cat# AF-479-NA, RRID: AB_354500, R&D Systems, Minneapolis, MN, USA; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in phosphate buffer saline (PBS)] or isotypic normal goat IgG (Cat# AB-108-C, RRID:AB_354267, R&D Systems; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in PBS) in the severely affected GC muscle.

Techniques: Staining, Cell Counting

Journal: Thorax

Article Title: Evidence for chemokine synergy during neutrophil migration in ARDS

doi: 10.1136/thoraxjnl-2016-208597

Figure Lengend Snippet: Plots showing th elevated bronchoalveolar lavage (BAL) fluid levels of chemokine (C-C motif) ligand (CCL)2 and CCL7 in a human model of acute lung inflammation and in patients with acute respiratory distress syndrome (ARDS). Healthy volunteers were challenged with 0.6% nebulised saline (n=5) or with 50 μg of nebulised lipopolysaccharide(LPS) (n=25) and BAL was performed 6 hours later. The levels of CCL2 (A) and CCL7 (B) were measured in BAL fluid recovered from saline and LPS-challenged volunteers by ELISA (right axis). BAL was also performed on patients with a clinical diagnosis of ARDS and the levels of CCL2 (A) and CCL7 (B) were measured (n=18) in recovered ARDS BAL fluid by ELISA (left axis). BAL fluid was also recovered from a second cohort of patients who underwent surgical oesophagectomy who were either at risk of developing ARDS (n=20) or who went on to develop ARDS (n=20). The levels of CCL2 (C) and CCL7 (D) were measured in recovered BAL fluid by ELISA. Power calculations were based on previous studies with α=0.05; power=0.8; difference between means=1.5 (50% reduction); SD=1.6. Statistical analysis was performed using one-way analysis of variance with Newman-Keuls post-hoc test.

Article Snippet: Chemotaxis of isolated human neutrophils was measured in response to human BAL fluid (described above), with or without 10 μg/mL anti-human CCL2 or CCL7 neutralising antibody (anti-human CCL2 279-MC, R&D Systems, anti-human CCL7 AF-282-NA, R&D Systems), 10 μg/mL anti-human CXCL8 neutralising antibody (anti-human IL-8 AB-208-NA, R&D Systems) or 20 μg/mL polyclonal Ig control (R&D Systems) diluted 1:1 in RPMI-1640.

Techniques: Saline, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Thorax

Article Title: Evidence for chemokine synergy during neutrophil migration in ARDS

doi: 10.1136/thoraxjnl-2016-208597

Figure Lengend Snippet: Chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-C motif) ligand (CCL)2 and CCL7 contribute to the neutrophil chemotactic activity of acute respiratory distress syndrome (ARDS) bronchoalveolar lavage fluid (BALF). Human neutrophils were isolated from the blood of healthy volunteers and chemotaxis was measured across 3 µm membranes (ChemoTX, NeuroProbe) in response to media alone, healthy volunteer BALF (n=11) or BALF from patients with ARDS (n=18) (A). Neutrophil chemotaxis was measured as the chemotactic index (number of migrated neutrophils following treatment vs medium control) after 1 hour (healthy BALF, left axis; ARDS BALF, right axis). Neutrophil chemotaxis towards BALF obtained from patients with ARDS was measured in the presence of polyclonal Ig control (20 μg/mL), neutralising anti- CXCL8) (10 μg/mL), anti-CCL2 (10 μg/mL) and a combination (10 μg/mL of each) of the two antibodies (B) or anti-CXCL8 (10 μg/mL), anti-CCL7 (10 μg/mL) and a combination (10 μg/mL of each) of the two antibodies (C). Antibodies were incubated in the presence of ARDS BALF for 20 min prior to the addition of isolated neutrophils. Differences in neutrophil chemotaxis between treatment groups were measured as the percentage of maximal chemotaxis to ARDS BALF alone (n=18 ARDS BALF samples using neutrophils isolated from multiple healthy human volunteers). Statistical analysis was performed using one-way analysis of variance with Newman-Keuls post-hoc test.

Article Snippet: Chemotaxis of isolated human neutrophils was measured in response to human BAL fluid (described above), with or without 10 μg/mL anti-human CCL2 or CCL7 neutralising antibody (anti-human CCL2 279-MC, R&D Systems, anti-human CCL7 AF-282-NA, R&D Systems), 10 μg/mL anti-human CXCL8 neutralising antibody (anti-human IL-8 AB-208-NA, R&D Systems) or 20 μg/mL polyclonal Ig control (R&D Systems) diluted 1:1 in RPMI-1640.

Techniques: Activity Assay, Isolation, Chemotaxis Assay, Control, Incubation

Journal: Thorax

Article Title: Evidence for chemokine synergy during neutrophil migration in ARDS

doi: 10.1136/thoraxjnl-2016-208597

Figure Lengend Snippet: Human neutrophils migrate toward recombinant chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-C motif) ligand (CCL)2 and CCL7. Human neutrophils were isolated from the blood of healthy volunteers and chemotaxis was measured across 3 µm membranes (ChemoTX, NeuroProbe) in response to recombinant human CXCL8, CCL2 and CCL7 (A), over a range of concentrations as stated. Neutrophil chemotaxis was also measured in response to a suboptimal concentration of recombinant human CXCL8 (5 ng/mL) in combination with recombinant human CCL2 (B) or recombinant human CCL7 (C) across a range of concentrations as stated (n=3 independent experiments using different healthy volunteer donors). Neutrophil chemotaxis was measured as the chemotactic index (number of migrated neutrophils following treatment vs medium control) after 1 hour. Statistical analysis was performed using one-way analysis of variance with Newman-Keuls post-hoc test.

Article Snippet: Chemotaxis of isolated human neutrophils was measured in response to human BAL fluid (described above), with or without 10 μg/mL anti-human CCL2 or CCL7 neutralising antibody (anti-human CCL2 279-MC, R&D Systems, anti-human CCL7 AF-282-NA, R&D Systems), 10 μg/mL anti-human CXCL8 neutralising antibody (anti-human IL-8 AB-208-NA, R&D Systems) or 20 μg/mL polyclonal Ig control (R&D Systems) diluted 1:1 in RPMI-1640.

Techniques: Recombinant, Isolation, Chemotaxis Assay, Concentration Assay, Control