anti human ccl2 neutralizing antibody  (R&D Systems)

Journal: Journal of Neuroinflammation

Article Title: Sigma-1 receptor regulates HIV-1 and methamphetamine-induced endothelial/pericyte barrier impairment via strain-specific inflammatory responses and mitochondrial dysregulation

doi: 10.1186/s12974-026-03750-1

Figure Lengend Snippet: METH enhances HIV-1 NL4-3 replication in HBVPs independently of Sigma-1R. A - B Impact of METH (25 µM) on HIV-1 replication as measured by p24 antigen release levels in HBVPs infected with HIV-1 NL4-3 ( A ) or JR-CSF ( B ). Data are means ± SEM ( n = 3). C - D Impact of METH (25 µM) on HIV-1 replication as measured by HIV-1 Gag mRNA expression levels in HBVPs infected with HIV-1 NL4-3 ( C ) or JR-CSF ( D ) ( n = 4–5). E Impact of pretreatment with S1RA (10 µM) for 6 h on NL4-3 HIV-1 replication in the presence and absence of METH ( n = 3–4). F Impact of HBVP pretreatment with the CXCR4 chemokine receptor antagonist AMD070 (5 µM) for 1 h on HIV-1 NL4-3 replication in the presence and absence of METH ( n = 4). G The heat map demonstrating the impact of HIV-1 NL4-3 infection and/or METH treatment for 72 h on gene expression profile of 42 ISGs in HBVPs ( n = 6). Genes with high expression levels are represented in shades of red, while those with low expression levels are shown in shades of green. Gene names are shown on the x axis. Red arrows indicate genes that were significantly differentially regulated in the HIV-1 NL4-3 + METH group compared to the control group, as determined by the RT² Profiler PCR Array ( p < 0.05). H - K RT-qPCR analysis of mRNA expression of CCL2 ( H ), MX2 ( I ), IFI30 ( J ), and PRKD2 ( K ) in HBVPs infected with HIV-1 NL4-3 and/or treated with METH ( n = 12). L Impact of blocking endogenous CCL2 with anti-human CCL2 neutralizing antibody on p24 release in HIV-1 NL4-3-infected HBVPs, with or without METH, at 72 h post-infection ( n = 6). M Impact of pretreatment with the CXCR4 chemokine receptor antagonist AMD070 (5 µM) for 1 h on CCL2 release in the presence and absence of METH at 72 h post-infection ( n = 6). Data are means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Abbreviations as in Fig. ; CCL2 - C-C motif chemokine ligand 2

Article Snippet: To neutralize CCL2, cultures were incubated with an anti-human CCL2 neutralizing antibody (8 μg/mL; R&D Systems, Cat# AF-279-NA) or with normal goat IgG as a control (R&D Systems, Cat# AB-108-C).

Techniques: Infection, Expressing, Gene Expression, Control, Quantitative RT-PCR, Blocking Assay

Journal: Journal of Neuroinflammation

Article Title: Sigma-1 receptor regulates HIV-1 and methamphetamine-induced endothelial/pericyte barrier impairment via strain-specific inflammatory responses and mitochondrial dysregulation

doi: 10.1186/s12974-026-03750-1

Figure Lengend Snippet: Synergistic impact of METH and CXCR4-Tropic HIV-1 on pericyte-dependent endothelial barrier breakdown via CXCR4/CCL2-driven viral replication and Sigma-1R-mediated mitochondrial and inflammatory dysregulation. This proposed model depicts intersecting pathways through which CXCR4-tropic HIV-1 and METH synergistically compromise endothelial barrier integrity via viral replication, Sigma-1R-mediated mitochondrial dysfunction, and modulation of IL6-associated inflammatory response. Notably, METH-enhanced replication of CXCR4-tropic HIV-1 in pericytes appears to occur independently of the Sigma-1R signaling ( www.BioRender.com )

Article Snippet: To neutralize CCL2, cultures were incubated with an anti-human CCL2 neutralizing antibody (8 μg/mL; R&D Systems, Cat# AF-279-NA) or with normal goat IgG as a control (R&D Systems, Cat# AB-108-C).

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: Primers for qPCR

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: LILRB4 is associated with microglial inflammatory phenotypes and morphology after tMCAO. ( A ) Gene expression of M1-associated phenotype markers (MCP-1, TNF-α, IL-1β, and CD32) and M2-associated phenotype markers (Arg-1, TGF-β, and CD206). ( n = 6; ** p = 0.0061, *** p = 0.0007, **** p < 0.0001, * p = 0.0222; ns p >0.05). ( B , C ) Fluorescence imaging of microglia in the infarct border region in Control, LILRB4-KO, and LILRB4-TG mice. The lower shows Sholl analysis, where the cell body is the center, and the number of points intersecting several concentric circles is calculated. Shows the number, length of microglia processes (branches), scale bar 10 μm. ( n = 10, * p = 0.0397/0.0306/0.0285)

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques: Gene Expression, Fluorescence, Imaging, Control

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: Microglia LILRB4 deficiency increases the CCL2 production. ( A ) qPCR analysis of CCL2, CCL5, CXCL1, CXCL5, CXCL10 in Control and LILRB4-KO mice 1 day after tMCAO. ( n = 6; * p = 0.0109). ( B ) UMAP plots of 19 cell populations identified by single-cell spatial transcriptomics analysis and the expression level of CCL2 among each cell type in mice after stroke. ( C ) Spatially transcriptome heatmaps of expression patterns of CCL2 across tissue sections from sham or stroke mouse. ( D ) qPCR analysis of CCL2 in microglia of Control and LILRB4-KO mice 1 day after tMCAO. ( n = 6; * p = 0.0229)

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques: Control, Expressing

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: Blockade of CCL2 or addition of Arg1 suppress CD8 + T cell activation and migration in co-culture with LILRB4-KD microglia. ( A , B ) Differential expression of LILRB4 in BV2 microglia transfected by knockdown and negative control lentiviral vectors. The expression of LILRB4 in BV2 was detected by PCR ( A ) and Flow cytometry assay ( B ) ( n = 3; ** p = 0.0025/0.0065/0.0057). ( C ) Experimental procedure. Transwell-placed, Control, and LILRB4-KD microglia (without or with CCL2 inhibitor or IgG) were cultured for 4 h under OGD conditions. During reoxygenation, the t-cell-containing Transwell device was placed on a 24-well plate and exposed to medium on its lower surface for 24 h, and the levels of t-cell migration to the lower layer were measured by Flow cytometry. In another experiment, microglia cells were co-cultured with CD8 + T cells. Control and LILRB4-KD microglia were collected and cultured under OGD conditions for 4 h, and CD8 + T cells were added directly to the medium during reoxygenation. One group was added recombinant Arg-1 and another group was not. After 24 h, CD69 and IFN-γ expression in CD8 + T cells were detected by flow cytometry. ( D ) T cell migration after exposure to OGD/R, measured by the number and type of T cells microglia into 24-well plates, with or without CCL2 inhibition. Flow cytometry tests for T cell migration and ratio of CD8 + T cell. ( n = 4; * p = 0.0187/0.0383/0.0104, ** p = 0.0029). ( E ) Control or LILRB4-KD microglia were exposed to OGD/R and co-cultured with CD8 + T cells with or without the addition of recombinant Arg-1. CD8 + T cells were collected for flow cytometry detection of CD69 and IFN-γ expression. ( F ) Quantitation and statistical evaluation of data in ( E ). ( n = 6; * p = 0.0106/0.0427, *** p = 0.0006, ** p = 0.0024/0.0024/0.0029). ( G ) Control or LILRB4-KD microglia were exposed to OGD/R and co-cultured with CD8 + T cells with or without the addition of recombinant Arg-1. Flow cytometry tests for T cell proliferation. ( n = 5; * p = 0.0348/0.0487/0.0153)

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques: Activation Assay, Migration, Co-Culture Assay, Quantitative Proteomics, Transfection, Knockdown, Negative Control, Expressing, Flow Cytometry, Control, Cell Culture, Recombinant, Inhibition, Quantitation Assay

Journal: Journal of Neuro-Oncology

Article Title: Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines

doi: 10.1007/s11060-024-04672-9

Figure Lengend Snippet: Cytokine expression and increased AKT activity are independent mechanisms without a tumor cell intrinsic effect on rebound growth. ( a ) Luminex-based multiplex assay results showing cytokine secretion during withdrawal after treatment for five days with either DMSO (solvent control) or 5 nM dabrafenib. Data is shown as mean ± SD ( n = 3 independent biological replicates). Two-tailed unpaired t-test, *p-value ≤ 0.05 **p-value ≤ 0.01; no indication: not significant. ( b ) Kinase phosphorylation array of samples treated for five days with 5 nM dabrafenib or DMSO (solvent control) followed by 24 h withdrawal (wd). Arrays consist of two membranes, each target is detected in technical duplicates. Images shown are representatives of two biological replicates. ( c - d ) Western blot analysis of AKT phosphorylation after five days treatment with 5 nM dabrafenib (dabra) followed by up to 72 h withdrawal. Blots shown are representative of three independent biological replicates ( c ). Quantification ( d ) is relative to solvent control (DMSO; dashed line) and shown as mean ± SD ( n = 3 independent biological replicates). One-sample t-test, * p-value ≤ 0.05; no indication: not significant. ( e - f ) Western blot analysis of AKT activity markers after treatment with recombinant cytokines. Treatment for five days with DMSO or 5 nM dabrafenib (dabra) served as negative and positive control for western blots ( e ). Blots shown are representative of three biological replicates ( e ). Quantification ( f ) was done relative to untreated samples (dashed line) and is shown as mean ± SD ( n = 3 independent biological replicates). One-sample t-test; no indication: not significant. ( g ) RT-qPCR analysis of chemokine gene expression after five days treatment with 5 nM dabrafenib (dabra) alone or in combination with varying concentrations of alpelisib (alp). Quantification was done relative to dabrafenib only treatment (0; dashed line) and is shown as mean ± SD ( n = 3 independent biological replicates). One-sample t-test, *p-value ≤ 0.05 **p-value ≤ 0.01; no indication: not significant. ( h ) Viable cell counts during treatment with 5nM dabrafenib (dabra) alone or in combination with a combination of antibodies neutralizing CCL2 (0.5 µg/mL), CX3CL1 (0.25 µg/mL), CXCL10 (0.25 µg/mL) and CCL7 (0.1 ng/mL) (neuABs), IgG control (1 µg/mL), 5 µM alpelisib (alp) or 1 µM ipatasertib (ipa) followed by ten days of withdrawal. Dashed line indicates withdrawal timepoint. Viable cell counts are normalized to treatment start (-5d). Data is shown on a logarithmic scale (base 10) as mean ± SD of at least three biological replicates. ( i ) Viable cell counts during treatment with 5 nM dabrafenib (dabra) followed by withdrawal. During withdrawal cells were either untreated (= solvent) or treated for five days with a combination of antibodies neutralizing CCL2 (0.5 µg/mL), CX3CL1 (0.25 µg/mL), CXCL10 (0.25 µg/mL) and CCL7 (0.1 ng/mL) (neuABs), IgG control (1 µg/mL), 5 µM alpelisib (alp) or 1 µM ipatasertib (ipa), followed by five days of withdrawal. Dashed lines indicate withdrawal timepoints. Viable cell counts are normalized to treatment start (-5d). Data is shown on a logarithmic scale (base 10) as mean ± SD of at least three biological replicates

Article Snippet: For cell count experiments and western blot analysis, cells were treated with antibodies neutralizing CCL2 (0.5 μg/ml; cat. no. MAB279, R&D systems), CX3CL1 (0.25 μg/ml; cat. no. MAB3652, R&D systems), CXCL10 (0.25 μg/mL; cat. no. MAB266, R&D systems) and CCL7 (0.1ng/ml; cat. no. MAB282, R&D systems) or mouse IgG (cat. no. MAB002, R&D systems).

Techniques: Expressing, Activity Assay, Luminex, Multiplex Assay, Solvent, Control, Two Tailed Test, Phospho-proteomics, Western Blot, Recombinant, Positive Control, Quantitative RT-PCR, Gene Expression

Journal: Journal of Neuro-Oncology

Article Title: Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines

doi: 10.1007/s11060-024-04672-9

Figure Lengend Snippet: Increased cytokine secretion upon MAPKi treatment and withdrawal induces increased microglia attraction. ( a ) Schematic of transwell migration assay setup to investigate migration of HMC3 cells towards conditioned media (CM) collected from BT-40. ( b ) Transwell migration assay of HMC3 cells towards conditioned media (CM) collected from BT-40 cells after 24 h of withdrawal after five days treatment with DMSO (solvent control), 5nM dabrafenib (d) or 2.7 nM dabrafenib and 0.3 nM trametinib (d + t). 2% FCS serves as baseline control as CM contains 2% FCS, 0% FCS as negative control and 10% FCS as positive control. Quantification is shown as mean ± SD ( n = 3 independent biological replicates; 2 technical duplicates per condition; 10–12 randomly distributed images were quantified per transwell) relative to 2% FCS. One-sample t-test, * p-value ≤ 0.05; no indication: not significant. ( c ) Transwell migration assay of HMC3 cells towards CM collected 24 h after dabrafenib withdrawal (dabra wd) containing either antibodies neutralizing CCL2 (1 µg/mL), CX3CL1 (0.5 µg/mL), CXCL10 (0.5 µg/mL) and CCL7 (0.2 ng/mL) (neuABs) or IgG (2 µg/mL). 2% FCS serves as baseline control as CM contains 2% FCS, 0% FCS as negative control and 10% FCS as positive control. Quantification is shown as mean ± SD ( n = 3 independent biological replicates; 2 technical duplicates per condition; 10–12 randomly distributed images were quantified per transwell) relative to 2% FCS. One-sample t-test and two-tailed unpaired t-test (comparing IgG to neuABs), *p-value ≤ 0.05 ***p-value ≤ 0.001; no indication: not significant. ( d ) Representative fluorescence images showing HMC3 migrated through the transwell, nuclei were stained with DAPI. Scale bar = 50 µM. ( e - h ) RT-qPCR analysis of CX3CL1 ( e ), CXCL10 ( f ), CCL2 ( g ) and CCL7 ( h ) expression in BT-40 xenograft tumors after six days treatment with dabrafenib (dabra; 100 mg/kg, six doses, once daily) followed by three days of withdrawal (withdrawal). Samples from mice showing tumor progression (#4, #6; Fig. d) during dabrafenib treatment were excluded from the analysis. Quantification was done relative to the median of untreated samples (control). CX3CL1 was undetected in one sample (#2), CCL2 was undetected in two samples (#2, #7) and CCL7 was undetected in six samples (#2, #3, #5, #7, #8, #11), for these samples Ct values were set to 40 (max. number of cycles). Boxplots depict the median, first and third quartiles. Whiskers extend from the hinge to the largest/smallest value no further than 1.5 * IQR from the hinge (where IQR is the interquartile range). Two-tailed unpaired t-test; no indication: not significant (control: n = 3 mice, dabra: n = 4 mice, withdrawal: n = 6 mice)

Article Snippet: For cell count experiments and western blot analysis, cells were treated with antibodies neutralizing CCL2 (0.5 μg/ml; cat. no. MAB279, R&D systems), CX3CL1 (0.25 μg/ml; cat. no. MAB3652, R&D systems), CXCL10 (0.25 μg/mL; cat. no. MAB266, R&D systems) and CCL7 (0.1ng/ml; cat. no. MAB282, R&D systems) or mouse IgG (cat. no. MAB002, R&D systems).

Techniques: Transwell Migration Assay, Migration, Solvent, Control, Negative Control, Positive Control, Two Tailed Test, Fluorescence, Staining, Quantitative RT-PCR, Expressing