Journal: Pharmaceuticals

Article Title: Antibodies Enhance the Suppressive Activity of Extracellular Vesicles in Mouse Delayed-Type Hypersensitivity

doi: 10.3390/ph14080734

Figure Lengend Snippet: Macrophages treated with Ts-EVs release inhibitory Mac-EVs that differ in marker’s expression pattern depending on mouse immunizing antigen. ( A ) Macrophages, after 30 min treatment at 37 °C with TNP-Ts-EVs, were cultured in protein-free MDM medium. Yielded supernatant was collected 90 min, 24 h or 48 h later, filtered and ultracentrifuged, and resulting fractions (i.e., pellet–filled bars; and supernatant above–open bars) were used to treat CHS effector cells prior to their transfer into naive recipients ( n = 5 per group) that were immediately challenged with hapten to elicit CHS reaction, measured as ear swelling 24 h later. ( B ) Untreated macrophages or macrophages treated for 30 min at 37 °C with DNA/RNA extracted from either TNP-Ts-EVs or control, non-suppressive EVs, were cultured in protein-free MDM medium for 48 h. Yielded supernatant was filtered and ultracentrifuged, and resulting fractions (i.e., pellet–filled bars; and supernatant above–open bars) were used to treat CHS effector cells prior to their transfer into naive recipients ( n = 5 per group) that were immediately challenged with hapten to elicit CHS reaction, measured as ear swelling 24 h later. ( C ) PCL-Mac-EVs (produced by TNP-Ts-EV-treated macrophages from PCL-sensitized mice) and OVA-Mac-EVs (produced by OVA-Ts-EV-treated macrophages from OVA-immunized mice) were absorbed onto cupper grid, negatively stained with 3% uranyl acetate, and visualized with TEM microscope. ( D ) PCL-Mac-EVs and OVA-Mac-EVs were coated onto latex beads, stained with fluoresceinated antibodies against selected EVs’ markers, including CD9, CD63, CD81 tetraspanins and I-A molecules, and analyzed with flow cytometry. Data are expressed as delta ± SEM. One-way ANOVA with post hoc RIR Tukey test; * p < 0.05, ** p < 0.01.

Article Snippet: Following antigens, haptens and antibodies were used: ovalbumin (OVA), OVA 323-339 peptide (OVA-323, Sigma, St Louis, MO, USA); purified and biotinylated rabbit polyclonal anti-OVA-323-339 IgG antibodies, fluorescein isothiocyanate (FITC)-conjugated rabbit polyclonal IgG isotype antibodies (Innovagen, Lund, Sweden); purified mouse anti-trinitrophenol (TNP) IgG1 monoclonal antibody of A111-3 clone, purified and phycoerythrin (PE)-conjugated rat anti-mouse CD9 monoclonal antibody (clone KMC8), PE-conjugated rat anti-mouse CD63 monoclonal antibody (clone NVG-2), PE-conjugated hamster anti-mouse CD81 monoclonal antibody (clone Eat2), PerCP-Cy- or PE-conjugated rat anti-mouse I-A/I-E monoclonal antibody of M5/114.15.2 clone, FITC- or horseradish peroxidase (HRP)-conjugated streptavidin (all from BD Biosciences, San Diego, CA, USA); PCL (TNP-Cl, Chemtronix, Swannanoa, NC, USA); trinitrobenzene sulphonic acid (TNBSA, Eastman Chemicals, Rochester, NY, USA).

Techniques: Expressing, Cell Culture, Produced, Staining, Microscopy, Flow Cytometry

Journal: Pharmaceuticals

Article Title: Antibodies Enhance the Suppressive Activity of Extracellular Vesicles in Mouse Delayed-Type Hypersensitivity

doi: 10.3390/ph14080734

Figure Lengend Snippet: OVA-Ts-EVs modulate vesicle-mediated interaction of Raji B cells and Jurkat T cells at the immune synapse. ( A ) CD81-GFP-transfected Raji B cells were pulsed with SEE superantigen for 30 min at 37 °C and then cultured with Jurkat T cells in the presence of OVA-Ts-EVs. Twenty four hours later, cells were stained with viability dye and fluoresceinated antibodies against CD19, and analyzed with flow cytometry ( n = 3). Relative changes in the percentage of CD19 neg GFP pos Jurkat T cells caused by SEE-stimulation of Raji B cells were calculated as follows: (percentage of CD19 neg GFP pos events in SEE-stimulated sample)/(percentage of CD19 neg GFP pos events in unstimulated sample); and shown in the graph. ( B ) CD81-GFP-transfected Raji B cells were left untreated (upper panel) or were treated with OVA-Ts-EVs for 4 h at 37 °C (lower panel), both populations were then pulsed with SEE for 30 min at 37 °C, and mixed with CMAC-stained Jurkat T cells (1 × 10 5 cells) in a ratio 1:1. Then, cell mixtures were plated onto Poly-L-Lys-coated slides for 1 h incubation at 37 °C, fixed, blocked, stained with selected primary and then secondary antibodies, mounted on Prolong Gold and analyzed with confocal microscope; scale bar: 10 µm. ( C ) CD81-GFP-transfected Raji B cells were treated with OVA-Ts-EVs for 4 h at 37 °C, pulsed with SEE for 30 min at 37 °C, and cultured with CMAC-stained Jurkat T cells (1 × 10 5 cells) in a ratio 1:1 for 24 h on standard culture plate. Then, cell mixtures were plated onto Poly-L-Lys-coated slides for 1 h incubation at 37 °C, fixed, blocked, stained with selected primary and then secondary antibodies, mounted on Prolong Gold and analyzed with confocal microscope; scale bar: 10 µm. Data are expressed as mean ± SD. Two-tailed Student t -test; * p < 0.05.

Article Snippet: Following antigens, haptens and antibodies were used: ovalbumin (OVA), OVA 323-339 peptide (OVA-323, Sigma, St Louis, MO, USA); purified and biotinylated rabbit polyclonal anti-OVA-323-339 IgG antibodies, fluorescein isothiocyanate (FITC)-conjugated rabbit polyclonal IgG isotype antibodies (Innovagen, Lund, Sweden); purified mouse anti-trinitrophenol (TNP) IgG1 monoclonal antibody of A111-3 clone, purified and phycoerythrin (PE)-conjugated rat anti-mouse CD9 monoclonal antibody (clone KMC8), PE-conjugated rat anti-mouse CD63 monoclonal antibody (clone NVG-2), PE-conjugated hamster anti-mouse CD81 monoclonal antibody (clone Eat2), PerCP-Cy- or PE-conjugated rat anti-mouse I-A/I-E monoclonal antibody of M5/114.15.2 clone, FITC- or horseradish peroxidase (HRP)-conjugated streptavidin (all from BD Biosciences, San Diego, CA, USA); PCL (TNP-Cl, Chemtronix, Swannanoa, NC, USA); trinitrobenzene sulphonic acid (TNBSA, Eastman Chemicals, Rochester, NY, USA).

Techniques: Transfection, Cell Culture, Staining, Flow Cytometry, Incubation, Microscopy, Two Tailed Test

Journal: PLoS ONE

Article Title: CCL2-Expressing Astrocytes Mediate the Extravasation of T Lymphocytes in the Brain. Evidence from Patients with Glioma and Experimental Models In Vivo

doi: 10.1371/journal.pone.0030762

Figure Lengend Snippet: (A) Detail of CD4 + and CD8 + T-cell immunostaining in the striatum of LPS injected mice. Scale bar: 20 µm. (B) The number of infiltrated T-cells (CD4 + , CD8 + and CD3 + T-cells) correlates with the number of CCL2 + cells in the areas of LPS injection. (C) Intraparenchymal injection of anti-CCL2 antibodies attenuates the LPS-induced infiltration of lymphocytes in the mouse brain parenchyma. The diagram on the top shows the arrangement of the experiment. Graphs show the density of CD8 and CD4 T-cells in the brain parenchyma surrounding the injection site. (D) Astrocytes express CCL2 after LPS injection in mouse brain. Confocal images of the injected areas show the co-localization of GFAP + astrocytes (green) and CCL2 (red) in mouse brain. DAPI (blue) was used as a nuclear counterstaing. * p<0.05 ANOVA-test.

Article Snippet: The following antibodies were used: anti-mouse CCL2 (1∶200, Rabbit, Serotec), anti-mouse CD4 (1∶750, Rat; Serotec), anti-mouse CD8 (1∶750, Rat; Serotec), anti-mouse CD3 (1∶750, Hamster, Serotec) and anti-GFAP (1∶500; mouse, Chemicon, Millipore).

Techniques: Immunostaining, Injection

Journal: Journal of Virology

Article Title: Recombinant Hepatitis C Virus Envelope Glycoprotein Vaccine Elicits Antibodies Targeting Multiple Epitopes on the Envelope Glycoproteins Associated with Broad Cross-Neutralization

doi: 10.1128/jvi.01911-14

Figure Lengend Snippet: Figure 2. Model of E1E2 showing the location of epitopes targeted by the mAbs used in this 715

Article Snippet: Monoclonal antibodies (mAbs) mouse anti-CD81 clone JS-133 81 (BD Biosciences); mouse isotype control IgG1 (R&D Systems); mouse anti-NS5A (9E10); 134 anti-HCV mAbs (IGH526, A4, AP33, HC33.4, 1:7, AR3B, AR4A, AR5A); and human anti-HIV 135 antibody B6 have been described previously (15, 16, 23-31).

Techniques: