blocking antibody against mouse il-1β  (R&D Systems)

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: In mice, treatment with angiotensin II (Ang II) and β-aminopropionitrile (BAPN) causes acute inflammation before aortic dissection occurs. ( A ) Gross morphology of aortic dissection (AD) in male C57BL/6J mice; aortas in control mice without Ang II or BAPN treatment (Control), aortas without AD after exposure to ANGII (1 µg/kg/µl) and BAPN (5 g/drinking water) (No-AD), and aortas with AD after exposure to ANGII and BAPN (AD). ( B , C ) Flow cytometric analysis showing percentages with plots after gating of CD45 + immune cells excluding doublets ( B ) and cell numbers of total CD45 + cells, neutrophils, Ly6C high monocytes, and macrophages ( C ). (n = 5 controls, n = 4 Non-AD, n = 5 AD). One-way analysis of variance with Tukey’s multiple comparison test; * P < 0.05. ( D ) Uniform manifold approximation and projection (UMAP) dimensionality reduction analysis identifying unique immune populations in male mice exposed to Ang II (1 µg/kg/µl) and BAPN (5 g/l in drinking water) for 1 week. ( E ) Dot plots displaying the signature cell gene expression markers. ( F , G ) CellChat showing the activated interaction between each cell population in the interleukin-1 (IL-1) signaling pathway. ( H ) Feature plots of IL-1β and Il1r1 expression on UMAP. CellChat; a tool that can quantitatively infer and analyze intercellular communication networks from scRNA-seq data. Macs macrophages, Monos monocytes, DCs dendritic cells, NK cells natural killer cells, ILCs Innate lymphoid cells, VSMCs vascular smooth muscle cells, ECs endothelial cells.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 μg every 3 days for 2 weeks (Fig. ).

Techniques: Dissection, Control, Comparison, Gene Expression, Expressing

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: IL-1β + inflammatory macrophages are accumulated before the onset of macroscopic aortic dissection (AD) in mice. ( A ) Re-clustering of monocytes and macrophages into three groups: mouse aortas without Ang II or BAPN (control), mouse aortas without AD after exposure to Ang II and BAPN (non-AD), and mouse aortas with AD after exposure to Ang II and BAPN (AD). ( B ) Violin plots depicting single-cell gene expression of each canonical monocyte or macrophage marker for clustering. ( C ) Proportion of each monocyte-macrophage cluster. ( D ) Heatmap of the top-5 differentially expressed genes in each monocyte and macrophage cluster. ( E ) Trajectory pseudo-time analysis in Monocle3 with Seurat cluster annotations (left) and change in the expression of IL-1β across pseudo-time for the monocyte and monocyte-derived macrophage partitions (clusters 0, 1, 2, 3, 4) in the non-AD and AD groups (right). Macs macrophages, Monos monocytes, Ang II Angiotensin II, BAPN β-aminopropionitrile.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 μg every 3 days for 2 weeks (Fig. ).

Techniques: Dissection, Control, Gene Expression, Marker, Expressing, Derivative Assay

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: Anti-IL-1β neutralizing antibody improves survival rate in mice. ( A ) Kaplan–Meier survival curve tracking death due to ruptured aortic dissection (AD) in male C57BL/6J mice exposed to Ang II (1 µg/kg/µl) and BAPN (1 g/l in drinking water) for 2 weeks after treatment with isotype control or anti-IL-1β neutralizing antibody (isotype IgG or anti-IL-1β neutralizing antibody; 200 β g i.p./mouse/every 3 days, n = 14 sham; n = 14 anti-IL-1β neutralizing antibody). Log-rank (Mantel–Cox) test, *P < 0.05. ( B ) Incidence of AD in AD model mice treated with the isotype control or anti-IL-1β antibody (n = 11/14 sham; n = 7/14 anti-IL-1β neutralizing antibody). ( C ) Blood pressure of AD model mice treated with the isotype control or anti-IL-1β neutralizing antibody (n = 8, sham; n = 9, anti-IL-1β neutralizing antibody). ( D ) Representative images of Elastica van Gieson (EVG) staining in the ascending (upper) and thoracoabdominal aorta (bottom). ( E ) Percentage of EVG-stained area per total tunica media without AD in the ascending aorta (upper) and thoracoabdominal aorta (bottom). *P < 0.05. Ang II Angiotensin II, BAPN β-aminopropionitrile.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 μg every 3 days for 2 weeks (Fig. ).

Techniques: Dissection, Control, Staining

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: Single-cell RNA sequencing reveals a characteristic immune cell landscape in the ascending aorta in patients with Stanford type A acute aortic dissection (AAD). ( A ) Representative contrast-enhanced computed tomography (CT) image of Stanford type A AAD. ( B ) Histological images stained with hematoxylin and eosin (HE) and Elastica van Gieson (EVG). ( C , D ) Uniform manifold approximation and projection (UMAP) dimensionality reduction analysis identifying a unique single-cell immune landscape in the aortas of combined ( C ) and each individual Control or AAD group ( D ) (n = 3 Controls from Li et al. ; n = 2 AAD). ( E ) Dot plots displaying signature cell gene expression markers for each immune cell cluster. ( F ) Comparison of the proportions of each cluster between controls and patients with AADs.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 μg every 3 days for 2 weeks (Fig. ).

Techniques: RNA Sequencing, Dissection, Computed Tomography, Staining, Control, Gene Expression, Comparison

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: Monocytes and inflammatory macrophages expressing IL-1β are accumulated in ascending aorta in patients with Stanford type A aortic dissection (AAD). ( A ) Sub-clustering of myeloid cells by uniform manifold approximation and projection (UMAP) in Controls and AADs (n = 3 Controls from LiY et al ; n = 2 AAD). ( B ) Dot plots displaying signature cell gene expression markers for each subcluster of myeloid cells. ( C ) Featured plots displaying characteristic gene expression of IL1B, NLRP3, and CCL2 in myeloid cells. ( D ) Pie charts showing the proportion of each myeloid cell cluster in the Control and AAD groups. Percentage of partitioned monocytes and monocyte-derived macrophages (clusters 0, 1, 2, and 3). ( E ) Gene ontology (GO) terms showing enriched biological processes (BP) (left) and molecular functions (MF) of clusters 0, 1, 2, 3, and 4. ( F ) Trajectory pseudo-time analysis in Monocle3 with Seurat cluster annotations (left) and change in the expression of IL-1β across pseudo-time for monocytes and monocyte-derived macrophage partitions (clusters 0, 1, 2, and 3) in AAD samples (right). ( G ) Histological staining with EVG, CD68 and, IL-1β. The scale bar represents 100 μm. Macs macrophages, Monos monocytes, cDC conventional dendritic cells, pDCs plasmacytoid dendritic cells.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 μg every 3 days for 2 weeks (Fig. ).

Techniques: Expressing, Dissection, Gene Expression, Control, Derivative Assay, Staining

Journal: Oncotarget

Article Title: Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL

doi: 10.18632/oncotarget.25849

Figure Lengend Snippet: (A) HeLa cells were transiently transfected with SCAT1 and Flag-tagged p210BCR-ABL (p210-Flag). At 43 h after transfection, cells were washed with PBS and then treated with serum-free DMEM in the presence or absence of TNF-α (50 ng/ml) and cycloheximide (CHX) (10 μg/ml) for 5 h. WCL were prepared and subjected to immunoblotting. Bands were visualized by probing with antibodies against Myc tag or Flag tag or actin. (B) HeLa cells were transiently transfected with SCAT1 and p210-Flag or pFlag empty vector. At 24 h after transfection, z-YVAD-fmk (20 μM) was added and further cultured for 19 h; cells were washed with PBS and then treated with serum-free DMEM with or without z-YVAD-fmk (20 μM) and/or TNF-α (50 ng/ml) and cycloheximide (10 μg/ml) for 5 h. WCL were prepared and subjected to immunoblotting. Bands were visualized by probing with antibodies against Myc tag, Flag tag or actin. (C) HeLa cells were transiently transfected with SCAT1 and p210-Flag or a Flag-tagged kinase-dead mutant of p210BCR-ABL (p210KD-Flag) or pFlag empty vector. At 24 h after transfection, imatinib (10 μM) was added and further cultured for 19 h, cells were washed with PBS and then treated with serum-free DMEM with or without imatinib (10 μM) for 5 h. WCL were prepared and subjected to immunoblotting. Bands were visualized by probing with antibodies against Myc tag, Flag tag, phospho Tyr (p-Tyr) or actin. (D) K562 cells were transiently transfected with SCAT1 cDNA. At 43 h after transfection, cells were washed with PBS and then subjected to medium change to serum-free RPMI1640 for 5 h. WCL and conditioned medium were prepared and subjected to immunoblotting. Bands were visualized by probing with antibodies against Myc tag or actin.

Article Snippet: Blocking antibody against mouse IL-1β was purchased from R&D systems. (Minneapolis, MN, USA).

Techniques: Transfection, Western Blot, FLAG-tag, Plasmid Preparation, Cell Culture, Mutagenesis

Journal: Oncotarget

Article Title: Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL

doi: 10.18632/oncotarget.25849

Figure Lengend Snippet: (A) 32D/TetOff-p210 cells were Tet-supplied or depleted and then cultured for 48 h in the presence or absence of imatinib (1 μM). WCL were subjected to immunoblotting. The values of relative band intensity versus the Tet (+) control were shown below each panel. Data are representative of three independent experiments. (B) 32D/TetOff-p210 cells or parental 32D cells were Tet-supplied or depleted and then cultured with IL-3 supplement for 48 h in the presence or absence of imatinib (1 μM). Cell viability was determined by WST-8 assay. * P < 0.01, ** P < 0.001. Data are shown as mean ± SEM ( n = 6) and are representative of three independent experiments. NS indicates no significant difference. (C) 32D/TetOff-p210 cells were Tet-supplied or depleted and then cultured for 96 h in the presence or absence of imatinib (1 μM). Cells were double-stained with annexin V and PI and analyzed by flow cytometry. The proportion of cell population with the representative data is shown in each panel. Four independent double-staining experiments were performed and statistical analysis was executed. * P < 0.01, ** P < 0.001. Data are shown as mean ± SEM ( n = 4).

Article Snippet: Blocking antibody against mouse IL-1β was purchased from R&D systems. (Minneapolis, MN, USA).

Techniques: Cell Culture, Western Blot, Staining, Flow Cytometry, Double Staining

Journal: Oncotarget

Article Title: Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL

doi: 10.18632/oncotarget.25849

Figure Lengend Snippet: (A) 32D/TetOff-p210 cells were Tet-depleted and then cultured for 48 h. WCL were subjected to immunoblotting. Bands were visualized by probing with antibodies against caspase-1 p10, cleaved caspase-3 or cleaved PARP. (B) 32D/TetOff-p210 cells were Tet-depleted or supplied and then cultured in the presence or absence of imatinib (1 μM) for 96 h and then incubated with FLICA 660 active caspase-1 or caspase-3 detection probe and analyzed by flow cytometry. The merged histogram (solid line) with non-stain control (dashed line) is shown in each panel. The proportion of FLICA-positive cell population max is shown in each panel. Three independent FLICA caspase-1/3 assays were performed, and statistical analysis was executed, as shown in lower graphs. * P < 0.05, ** P < 0.01. Data are shown as mean ± SEM ( n = 3).

Article Snippet: Blocking antibody against mouse IL-1β was purchased from R&D systems. (Minneapolis, MN, USA).

Techniques: Cell Culture, Western Blot, Incubation, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL

doi: 10.18632/oncotarget.25849

Figure Lengend Snippet: 32D/TetOff-p210 cells or parental 32D cells were Tet-supplied or depleted and then cultured with IL-3 supplement for 96 h to collect the culture supernatant and WCL. (A) ELISA was performed to determine concentration of IL-1β and TNF-α in the culture supernatant. * P < 0.001, # P < 0.05. Data are shown as mean ± SEM ( n = 3) and are representative of three independent experiments. ND indicates not detected (under the detection limit). NS indicates no significant difference. (B) ELISA was performed to determine concentration of S100A8/A9 in the culture supernatant. * P < 0.05. Data are shown as mean ± SEM ( n = 3). ND indicates not detected (under the detection limit). WCL were subjected to immunoblotting. Bands were visualized by probing with antibodies against S100A8, S100A9 or actin. The values of relative band intensity versus the Tet (+) control are shown under each panel. (C) ELISA was performed to determine concentration of S100A8/A9 in the plasma of 8-month-old WT ( n = 7) and BCR-ABL TG ( n = 9) mice. Data are shown as mean ± SEM. * P < 0.05. (D) Expressions of both S100a8 and S100a9 in the spleen of 8-month-old WT ( n = 5) and BCR-ABL TG ( n = 6) mice were determined by quantitative RT-PCR. Data are shown as mean ± SEM. * P < 0.005. Statistical significance was evaluated by Mann–Whitney U test.

Article Snippet: Blocking antibody against mouse IL-1β was purchased from R&D systems. (Minneapolis, MN, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Quantitative RT-PCR, MANN-WHITNEY

Journal: Oncotarget

Article Title: Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL

doi: 10.18632/oncotarget.25849

Figure Lengend Snippet: 32D/TetOff-p210 cells were Tet-supplied or depleted and then cultured in the presence or absence of imatinib (1 μM) for 96 h. (A) Giemsa staining was performed. Cells with segmented nuclei (indicated by arrows) are shown in each panel. Scale bar, 10 μm. (B) Cells were triple-stained with anti-CD11b-BV421, anti-Ly6C-APC, and anti-Ly6G-PE, or isotype anti-rat IgG2b-BV421, anti-rat IgM-APC, and anti-rat IgG2a-PE. Stained cells were analyzed by flow cytometry. The obtained data were processed by selection of PI - and CD11b + cell population, and then the cell surface expression of Ly6C and Ly6G within the CD11b + cell population was analyzed. Numbers in the plots indicate the percentages of gated cells. (C) Three independent triple-staining experiments, as exemplified in panel B, were performed, and statistical analysis was executed. * P < 0.01. Data are shown as mean ± SEM ( n = 3).

Article Snippet: Blocking antibody against mouse IL-1β was purchased from R&D systems. (Minneapolis, MN, USA).

Techniques: Cell Culture, Staining, Flow Cytometry, Selection, Expressing