Journal: Frontiers in Immunology

Article Title: Properdin Is a Key Player in Lysis of Red Blood Cells and Complement Activation on Endothelial Cells in Hemolytic Anemias Caused by Complement Dysregulation

doi: 10.3389/fimmu.2020.01460

Figure Lengend Snippet: Inhibition of properdin protects endothelial cells from heme-induced complement activation. (A,B) Complement activation on heme-treated endothelial cells is alternative-pathway dependent. HUVECs were stimulated with M199 media or 100 μM hemin, washed and incubated with NHS or Factor B-depleted serum (FB-dpl) (15% final), in the presence or absence of Cp20 (50 μM) or EDTA (20 mM). 54 nM Factor B (FB) was used to restore Factor B-depleted serum activity. (C) Anti-properdin MoAb protects heme-treated HUVECs from C3 fragment deposition. HUVECs were stimulated with 100 μM hemin (or M199), NHS (33% final) and one of the following reagents at the indicated final concentrations: Cp20 (50 μM), Compstatin control peptide (“Cp20 Control,” 50 μM), anti-properdin MoAb 6E11A4 (80 nM), IgG1 isotype control (80 nM), eculizumab (53 nM or 80 nM) or SALO (5 μM). (A–C) C3 deposition (GMFI) was determined as described in “Materials and Methods” and “Fold difference” was calculated by dividing GMFI of each group by GMFI of NHS alone group. (A) is a combination of four independent experiments (each with duplicates), (B,C) are representative of four and two independent experiments, respectively, with duplicates. The data was analyzed by one-way ANOVA with Tukey's multiple comparison test. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, p > 0.05 ns. Statistical analysis between NHS+heme group and each inhibitor group is shown in (C) .

Article Snippet: Compstatin control peptide (Tocris) was used as the control for Cp20.

Techniques: Inhibition, Activation Assay, Incubation, Activity Assay, Control, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

doi: 10.1073/pnas.1417883112

Figure Lengend Snippet: Fig. 3. α-Syn aggregates directly bind to CD11b, which activates Nox2 to induce a migratory conformation of microglia. (A) In vitro binding assays. Myc-DDK– fused human CD11b was incubated with rH α-syn aggregates, and binding was detected via immunoblot for DDK and α-syn. Incubation of CD11b or α-syn alone served as controls. n = 3. (B) In vivo binding assays. Purified rH α-syn aggregates were mixed with WT or CD11b−/−microglial lysates to allow α-syn to react with CD11b, and the mixtures were further incubated in the IgG- or Ab-conjugated magnetic beads. Binding was detected via immunoblot for CD11b and α-syn. n = 3. (C) O2

Article Snippet: After multiple washes, the beads were boiled in Laemmli sample buffer, and the samples were examined by Western blot analysis for α-syn or DDK peptide (OriGene Technologies).

Techniques: In Vitro, Binding Assay, Incubation, Western Blot, In Vivo, Purification, Magnetic Beads

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 1: Expression of p33ING1b and p29ING4 in renal cell carcinoma. (a) mRNA expression of p33ING1b and p29ING4 was increased at early and late stages of disease with higher expression of both genes at late stages of disease compared to early stages (Robson stages III/IV vs. I/II) (p33ING1b and p29ING4: stages I/II and III/IV vs. normal tissue p<0.001 and p<0.0001, respectively; p33ING1b and p29ING4: stages III/IV vs. I/II p<0.01 and p<0.001, respectively). (b) Results of the real time RT-PCR were normalized to kidney normal tissue and expressed as x-fold difference (2- ∆∆Ct). Expression of p33ING1b and p29ING4 by renal carcinoma cells was confirmed (Cy3 red; DAPI blue (nuclear counterstaining)). (c) Sections are representative of n=30 patients/group. Representative western blots of p33ING1b and p29ING4 (loading control ß-actin).

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 4: p33ING1b- and p29ING4-specific T cell reactivity. Four peptides resulted in significantly increased expression of IFN-γ compared to controls, independent of tumor stage (p33ING1b (aa109-118) and aa259-268, and p29ING4 (aa149-158) and aa239-248; p<0.001) Stages I/ II (a) and Stages III/IV (b). Elispot analysis was measured as spots/5 x 105 cells/patient.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Enzyme-linked Immunospot

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 5: p33ING1b- and p29ING4-specific T cell reactivity. Two peptides resulted in increased expression of IL-10 compared to controls; peptide p33ING1b (aa109-118) at all stages and p29ING4 (aa239-248) at late stages (p<0.001). Stages I/ II (a) and Stages III/IV (b). Luminex analysis was measured as pg/ml.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Luminex

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 6: p33ING1b- and p29ING4-specific T cell reactivity. Evaluation of IL-2 expression revealed that only a single epitope (p29ING4 (aa149-158)) resulted in high responsiveness of T cells from RCC patients after stimulation with the peptide (stages I/II vs. III/IV p<0.001). PBMCs from healthy volunteers (n=30) had no significant IL-2 expression after stimulation with all peptide epitopes. Luminex analysis for IL-2 expression was measured as pg/ml and presented as percent stimulation index.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Luminex

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 7: p29ING4 presentation by MHC class I (a) and II (b) molecules. p29ING4 presentation by major histocompatibility complex (MHC) class I and II antigens was shown to be increased in RCC but not in healthy kidneys.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Immunopeptidomics