Journal: PLoS ONE

Article Title: Novel Process of Intrathymic Tumor-Immune Tolerance through CCR2-Mediated Recruitment of Sirpα + Dendritic Cells: A Murine Model

doi: 10.1371/journal.pone.0041154

Figure Lengend Snippet: ( A ) Thymus was collected at 14 days after Col26 tumor inoculation. Percentage of DC-SIGN + Sirpα + and total Sirpα + cells in thymic CD11c high DCs in WT mice bearing tumor or mice without tumor are shown. ( B ) A double-color fluorescent immunostaining for Sirpα (red) and Col IV (green). Single color image for Sirpα and merged image are shown on the upper and lower, respectively. Scale bar, 50 µm. ( C ) A double-color fluorescent immunostaining for Col IV (red) and CCL2 (green). The right panel shows the thymus which was collected at 1 hr after intravenous injection of rCCL2 (5 μg). Single color image for CCL2 and merged image are shown on the upper and lower, respectively. Scale bar, 50 µm. ( D ) Parental Col26 and Col26-7ND were injected into WT mice. Subsequently, OVA 647 was intravenously injected at 14 days after tumor inoculation. Uptake of OVA 647 in Sirpα − (left) and Sirpα + cDCs (right) were analyzed at 4 hrs after OVA injection. Parental Col26-, Col26-7ND-bearing mouse, and mouse without tumor were represented by a solid-lined, dash-lined, and gray-filled histogram, respectively. Representative results from 3 independent experiments are shown here. ( E ) Enhancement of the capability of OVA uptake by Sirpα + cDCs in tumor-bearing mice. Enhancement of OVA uptake = MFI of OVA 647 captured by Sirpα + DCs in WT or CCR2 −/− mice bearing parental Col26 or Col26-7ND tumor/that in WT or CCR2 −/− mice without tumor. The right unfilled column shows the fold-enhancement of OVA 647 , when OVA 647 was injected at 1 day after three times of daily injections of rCCL2 (2.5 μg/injection). ( F ) Two mg OVA protein was intravenously injected into DO11.10 mice at 14 days after tumor inoculation and 2 days later, expression of CD4 and CD8 in DO11.10 + thymocytes was analyzed. Percentage of DP thymocytes is shown in each panel. Data represent mean ± SD from three independent experiments. ( G ) The numbers of total thymocytes (upper graph) and DP thymocytes (lower graph) are shown. Representative results from three ( B and C ) or four ( D and F ) independent experiments are shown here. Data represent mean ± SD from four independent experiments ( A , E , and G ). *, p <0.01.

Article Snippet: Mouse anti-mouse I-A d (AMS-32.1; BD Biosciences), and hamster anti-mouse CD11c (HL-3; BD Biosciences), and anti-mouse CCL2 (2H5; eBioscience) mAbs were used.

Techniques: Immunostaining, Injection, Expressing

Journal: Scientific Reports

Article Title: Proinflammatory profile of neonatal monocytes induced by microbial ligands is downmodulated by histamine

doi: 10.1038/s41598-019-50227-8

Figure Lengend Snippet: Impaired production of IFN-γ-inducible chemokines by MNCs of NBs in response to LPS. The presence of CXCL10 ( A ), CXCL9 ( B ), IFN-γ ( C ) CXCL8 ( D ), CCL5 ( E ) and CCL2 ( F ) was assessed in the supernatants of MNC cultures from healthy adult subjects (n = 13) and newborns (NBs; n = 9) stimulated with a TLR4 agonist (LPS - 1 µg/mL) or non-stimulated (Uns) for 24 h. Chemokines and cytokines were measured using a cytometric bead array and flow cytometry. Data are shown as the median. *p ≤ 0.05, **p ≤ 0.01,***p ≤ 0.001 compared with adults; # p ≤ 0.05 and ## p ≤ 0.01 compared with baseline.

Article Snippet: To analyse intracellular staining in purified monocytes, staining was performed using the following antibodies: CD3-BV605 (SK7), CD19-Horizon V500 (HIB19, CD16-FITC (3G8), CD14-Horizon V450 (MØP9), IL-10-APC (JES3-19F1), CXCL8 –PE (G265-8), CCL2-PE (2H5, e-Bioscience, Thermofisher, Hwalthan, Massuchsetts, EUA).

Techniques: Flow Cytometry

Journal: Scientific Reports

Article Title: Proinflammatory profile of neonatal monocytes induced by microbial ligands is downmodulated by histamine

doi: 10.1038/s41598-019-50227-8

Figure Lengend Snippet: Histamine exerts an inhibitory effect on LPS-induced CCL2 production in NBs and adults. CCL2 production induced in MNCs from adults (n = 12) and NBs (n = 8) stimulated with LPS (1 µg/mL) in the presence of histamine (10 µM) for 24 h. CCL2 levels in the supernatants of the MNC cultures were measured using a cytometric bead array and flow cytometry. Individual data for histamine treatment of adult cells (open circle) showing no histamine effect (n = 5) or adult (n = 7) or NB (closed circle, n = 8) cells showing an inhibitory effect ( A ) and the frequency of the histamine inhibitory effect on CCL2 or no effect in newborns and adult cells ( B ). Data are shown as bars or before-after. **p ≤ 0.01 compared with LPS stimulation without histamine.

Article Snippet: To analyse intracellular staining in purified monocytes, staining was performed using the following antibodies: CD3-BV605 (SK7), CD19-Horizon V500 (HIB19, CD16-FITC (3G8), CD14-Horizon V450 (MØP9), IL-10-APC (JES3-19F1), CXCL8 –PE (G265-8), CCL2-PE (2H5, e-Bioscience, Thermofisher, Hwalthan, Massuchsetts, EUA).

Techniques: Flow Cytometry

Journal: Scientific Reports

Article Title: Proinflammatory profile of neonatal monocytes induced by microbial ligands is downmodulated by histamine

doi: 10.1038/s41598-019-50227-8

Figure Lengend Snippet: Participation of histamine receptors in the inhibitory effect on CCL2 production by LPS-stimulated MNCs from adults and newborns. Healthy adult (n = 7) and NB (n = 9) MNCs were analyzed for mRNA expression of HRs (H1R, H2R and H4R) and histidine decarboxylase (HDC) via qPCR normalized with GAPDH ( A ). Cultures of MNCs from healthy adults (n = 7) and NBs (n = 9) were incubated with histamine receptor antagonists of H1R (Pyrilamine −100 µM), H2R (Cimetidine - 1000 µM) and H4R (JNJ7777120 - 1 µM) for 1 h at 37 °C and thereafter with histamine (10 µM) and a TLR4 agonist (LPS - 1 µg/mL) for 24 h. The presence of CCL2 in the supernatant was measured using a cytometric bead array and flow cytometry ( B ). Data are shown as bars and before-after. *p ≤ 0.05 compared with adults; **p ≤ 0.01 compared with LPS stimulation.

Article Snippet: To analyse intracellular staining in purified monocytes, staining was performed using the following antibodies: CD3-BV605 (SK7), CD19-Horizon V500 (HIB19, CD16-FITC (3G8), CD14-Horizon V450 (MØP9), IL-10-APC (JES3-19F1), CXCL8 –PE (G265-8), CCL2-PE (2H5, e-Bioscience, Thermofisher, Hwalthan, Massuchsetts, EUA).

Techniques: Expressing, Incubation, Flow Cytometry

Journal: Scientific Reports

Article Title: Proinflammatory profile of neonatal monocytes induced by microbial ligands is downmodulated by histamine

doi: 10.1038/s41598-019-50227-8

Figure Lengend Snippet: Histamine down-regulates CCL2 and CXCL8 genes and protein levels in NB monocytes stimulated by LPS. Purified monocytes from healthy adults (n = 7) and NBs (n = 9) were stimulated with LPS (1 µg/mL – red line) or LPS plus histamine (10 µM – blue line) or non-stimulated (Uns – gray line) for 4 h. mRNA expression of CCL2, CXCL8 and IL10 ( A ) was assessed by qPCR and normalized against LPS stimulation of adult cells. Data are shown as the median ** p ≤ 0.01 compared with adults; # p ≤ 0.05 and ## p ≤ 0.01 compared with LPS stimulation. ( B ) Purified monocytes from healthy adults (n = 4) and NBs (n = 5) were stimulated with LPS or LPS plus histamine for 18 h, and intracellular staining for CCL2, CXCL8 and IL-10 was assessed by flow cytometry. Data show the median fluorescence intensity (MFI). *p ≤ 0.05, **p ≤ 0.01 compared with baseline.

Article Snippet: To analyse intracellular staining in purified monocytes, staining was performed using the following antibodies: CD3-BV605 (SK7), CD19-Horizon V500 (HIB19, CD16-FITC (3G8), CD14-Horizon V450 (MØP9), IL-10-APC (JES3-19F1), CXCL8 –PE (G265-8), CCL2-PE (2H5, e-Bioscience, Thermofisher, Hwalthan, Massuchsetts, EUA).

Techniques: Purification, Expressing, Staining, Flow Cytometry, Fluorescence

Journal: Cell reports

Article Title: RIPK3 Activation Leads to Cytokine Synthesis that Continues after Loss of Cell Membrane Integrity

doi: 10.1016/j.celrep.2019.07.077

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-CCL2 (clone 2H5) PE , BD Biosciences , Cat # 554443; RRID: AB_395394.

Techniques: Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Viability Assay, LDH Cytotoxicity Assay, RNA Sequencing Assay, CRISPR, Plasmid Preparation, Software, Imaging, Flow Cytometry, Real-time Polymerase Chain Reaction