Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: Monocyte subpopulations` content in healthy individuals and CRC patients.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques:

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: The distribution of CD163+ and CCR2+ peripheral blood monocytes in patients with colon and rectal cancers. Individual profiles of CCR2+ and CD163+ monocyte subsets for each patient with rectal and colon cancers. (A) , The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after NAC and after surgical resection in rectal cancer patients. (B) , The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after surgical resection in colon cancer patients. (C) , Associations of CCR2-expressing monocyte subsets with hematogenous and lymphatic metastasis in rectal cancer patients. (D) , Associations of CD163-expressing monocyte subsets with hematogenous and lymphatic metastasis in colon cancer patients. M 0 , metastasis-negative status, M 1 , metastasis-positive status. N 0 , lymph node-negative status, N 1-3 , lymph node-positive status.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: An activator of glycolysis PFKFB3 is overexpressed in colon cancer and is indicative for higher risk of tumor relapse in colon cancer but not rectal cancer. (A) , Сolon cancer tissue is massively infiltrated by PFKFB3-positive monocytes. IF/confocal microscopy analysis was performed for 10 colon tumor tissues. The infiltration of CD14+CD68+PFKFB3+ cells was found in all samples. Representative image is given from one patient. Scale bar corresponds to 50 µm in main image and 20 µm in zoom image. (B) , Spearman correlation coefficients between PFKFB3 expression, M2 macrophage gene expressions and predicted cell abundance scores, FDR<0.05. (C) , Predicted cell composition of CD45+ AOIs and hierarchical clustering of AOIs. (D) , Difference in monocyte and macrophage cell abundance scores between the CD45+ AOIs in colon and rectal cancers (the Mann-Whitney U test was applied). (E) , PFKFB3 gene expression is elevated in patients with recurrence and larger tumor size in colon cancer. Variance in PFKFB3 expression was stabilized via the variance stabilizing transformation (VST). (F) , PFKFB3 had prognostic significance for the DFS and OS. High-risk group had worse survival rates compared to low-risk group. ROC analysis and Kaplan–Meier method were applied.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Confocal Microscopy, Expressing, MANN-WHITNEY, Gene Expression, Transformation Assay

Journal: Frontiers in Immunology

Article Title: Immunization With Bovine Herpesvirus-4-Based Vector Delivering PPRV-H Protein Protects Sheep From PPRV Challenge

doi: 10.3389/fimmu.2021.705539

Figure Lengend Snippet: Effect of BoHV-4-A-PPRV-H-ΔTK vaccination in ovine PBMC populations. PBMCs obtained from the three treatment groups [PBS (black), BoHV-4-A-ΔTK (blue), and BoHV-4-A-PPRV-H-ΔTK (red)] were stained with different monoclonal antibodies and analyzed by flow cytometry at different time points [pre-immunization (D0), pre-challenge (D42), and days 2, 4, 7, 9, 11, and 14 post-challenge (PC)]. Average percentage of (A) CD4 + cell population, (B) CD8 + cell population, (C) WC1 + (γδ T cell) population, (D) B-cell + population, (E) CD14 + cell population, (F) CD14 + CD16 + cell population, and (G) CD16 + CD14 − cell population for each sheep group were plotted. * p < 0.05 one-way ANOVA test (BoHV-4-A-PPRV-H-ΔTK vs . BoHV-4-A-ΔTK and PBS control groups). The black arrow (D42) denotes the time of virulent PPRV ICV’89 challenge in all animals.

Article Snippet: The following antibodies were used to label the different PBMC subpopulations: anti-ovine CD4 (clone 44.38), CD8 (clone 38.65), and WC1 (clone 19.19), anti-human CD14 (clone TÜK4) and CD16 (clone KD1) (all from Bio-Rad), and anti-bovine B cell marker (clone BAQ44A) (Kingfisher Biotech).

Techniques: Staining, Bioprocessing, Flow Cytometry, Control

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 4. POPG inhibits LPS-induced MAPK and IB phosphorylation and MKP-1 expression. A, POPG liposomes (200 g/ml) were added to monolayer cultures of differentiated U937 cells that received either no treat- ment or 10 ng/ml LPS. After incubating for the indicated times, cells were lysed using buffer containing detergent, protease inhibitors, and phospha- tase inhibitors. Aliquots with 15 g of protein from lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The amount of phosphorylation was detected using phospho-specific antibodies to p38 MAPK, p42/p44 ERK, p46-p54 JNK, and phosphorylated IB. To determine equal loading of proteins between samples, the membranes were probed with rabbit polyclonal p46 JNK, p42/p44 ERK, p38 MAPK, and IB antibodies. The expression of MKP-1 was detected with a polyclonal MKP-1 antibody. B, POPC and DPPG liposomes (200 g/ml) were compared with POPG lipo- somes (200 g/ml) as antagonists of LPS activation of cells using the same conditions as described for panel A. The time of analysis following LPS expo- sure for p38, ERK, and JNK was 30 min, and that for IB and MKP-1 was 60 min.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Expressing, Liposomes, SDS Page, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 9. CD14 binds to solid phase lipids. A, affinity-purified preparations (2 g each) of the extracellular domains of CD14 (sCD14) and TLR4 (sTLR4) and the full-length MD-2 were analyzed by gel electrophoresis under reduc- ing and denaturing conditions. The electrophoretic gels were stained with Coomassie Blue. mol. mass st., molecular mass standards. B, phospholipids (1.25 nmol) in 20 l of ethanol were placed onto microtiter wells, and the solvent was evaporated. Nonspecific binding was blocked with 20 mM Tris buffer(pH7.4)containing0.15 MNaCl,5mMCaCl2(intheupperpanel),or2mM EGTA (in the lower panel) and 5% (w/v) bovine serum albumin (buffer A). Varying concentrations of human CD14 in buffer A were added and incu- bated at 37 °C for 1 h. The binding of CD14 to phospholipids was detected using anti-CD14 monoclonal antibody as described under “Experimental Pro- cedures.” The data shown are the means S.E. from three separate experi- ments with duplicate samples in each experiment.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Affinity Purification, Nucleic Acid Electrophoresis, Staining, Solvent, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 10. PG Inhibits CD14 binding to solid phase LPS. A, various types of PG were coated onto microtiter plates and incubated with CD14 (1 g/ml) at 37 °C for 1 h. The binding of CD14 to PG was detected using anti-CD14 mono- clonal antibody, and the ELISA-based absorbance of CD14 bound to POPG was defined as 100%. Types of PG shown on the graph are: dilauroylphos- phatidylglycerol (DLPG), DMPG, DPPG, and 16:0/18:1 POPG. B, LPS (2 g) in 20 l of ethanol was placed onto microtiter wells, and the solvent was evaporated. After blocking the nonspecific binding with buffer A, the mix- ture of CD14 (1 g/ml) and phospholipid liposomes (20 g/ml) in buffer A, which was preincubated at 37 °C for 1 h, was added and incubated at 37 °C for 1 h. The binding of CD14 to LPS was detected using anti-CD14 mono- clonal antibody. The ELISA-based absorbance of CD14 bound to LPS was defined as 100%. The data shown are the means S.E. from three separate experiments with duplicate samples in each experiment. *, p 0.05, **, p 0.01, when compared with LPS-CD14 binding in the absence of phospholipids.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Solvent, Blocking Assay, Liposomes

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE11.MonoclonalantibodiesspecificfortheLPSbindingsiteinhibit CD14 interaction with POPG and PI. POPG (A) or PI (B) were coated onto microtiter plates. After blocking the nonspecific binding with buffer A, the mixture of CD14 (1 g/ml) and monoclonal antibodies or isotype control IgG (50g/ml)inbufferA,whichwaspreincubatedat37 °Cfor1h,wasaddedand incubated at 37 °C for 1 h. The binding of CD14 to phospholipids was detected using sheep anti-CD14 polyclonal antibody, and the ELISA-based absorbance of CD14 bound to phospholipid was defined as 100%. The data shown are the means S.E. from three separate experiments with duplicate samples in each experiment. *, p 0.05, when compared with CD14 binding in the absence of monoclonal antibody. mIG, mouse Ig. C, CD14 (2 g) was coated onto microtiter plates, and nonspecific binding was blocked with buffer A. Monoclonal antibodies or isotype control IgG (50 g/ml) in buffer A were added and incubated at 37 °C for 1 h. The CD14 was detected using sheep anti-CD14 polyclonal antibody, and the ELISA-based absorbance of solid phase CD14 alone was defined as 100%.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Blocking Assay, Binding Assay, Bioprocessing, Control, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Circulation research

Article Title: CD45 Expression in Mitral Valve Endothelial Cells After Myocardial Infarction

doi: 10.1161/CIRCRESAHA.116.309598

Figure Lengend Snippet: (A-F) Immunohistochemical staining of MV leaflets: anti-CD45 staining (A, B, D, F), control IgG staining (C). anti-CD14 staining (E). Ovine MV leaflets from animals 6 months after IMI (A, C, D, E), control animals (B) and mitral regurgitation (MR) only animals (F). (G-J) Immunofluorescence staining of IMI 6-months MV leaflets. Panel G shows double-staining with anti-CD45 and anti-von Willebrand factor (vWF), an endothelial marker. Arrows mark endothelial cells co-expressing CD45 and vWF. Panel H shows single staining with anti-vWF. Panel I shows single staining with anti-CD45 and Panel J shows DAPI staining only.

Article Snippet: A portion of each excised MV was frozen in OCT compound (Sakura Finetek, Tokyo, Japan), sectioned and stained with primary mouse anti sheep CD45 antibody (AbD Serotec, NC, USA, cat# MCA2220GA) or anti-sheep CD14 (AbD Serotec, cat#MCA920GA) by the avidin-biotin-peroxidase method or by fluorescence double-labeling for von Willebrand Factor (vWF; DAKO, CA, USA, cat#, A0082) as described 20 .

Techniques: Immunohistochemical staining, Staining, Control, Immunofluorescence, Double Staining, Marker, Expressing

Journal: Cell reports

Article Title: The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis

doi: 10.1016/j.celrep.2019.06.007

Figure Lengend Snippet:

Article Snippet: After blocking with 1% BSA and 0.1% tween-20 in 1 × PBS, tissues were incubated with anti-CD14-biotin sheep antibodies (R & D systems) and anti-CD68 mouse antibodies (Abcam).

Techniques: Control, Northern Blot, Recombinant, Purification, Adjuvant, Lysis, Staining, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Microarray, Software