Journal: British Journal of Cancer

Article Title: Treatment with interleukin-2 in malignant pleural mesothelioma: immunological and angiogenetic assessment and prognostic impact

doi: 10.1038/sj.bjc.6605438

Figure Lengend Snippet: Correlations between immunological and angiogenetic parameters in the IL-2-treated patients’ group and in the untreated patients’ group

Article Snippet: For TIL-immunohistochemical staining, sections were incubated with the following antibodies: mouse anti-human Foxp3 monoclonal antibody (clone 236A/E7 diluted 1 : 300; Abcam, Cambridge, UK) , mouse anti-human CD8 monoclonal antibody (clone C8/144B, ready to use for the Ventana automated slide stainer; Ventana) , and mouse anti-human CD4 monoclonal antibody (clone 1F6 diluted 1 : 20; Diagnostic BioSystem, Pleasanton, CA, USA).

Techniques:

Journal: British Journal of Cancer

Article Title: Treatment with interleukin-2 in malignant pleural mesothelioma: immunological and angiogenetic assessment and prognostic impact

doi: 10.1038/sj.bjc.6605438

Figure Lengend Snippet: Association of immunological and angiogenetic variables with clinicopathologic parameters

Article Snippet: For TIL-immunohistochemical staining, sections were incubated with the following antibodies: mouse anti-human Foxp3 monoclonal antibody (clone 236A/E7 diluted 1 : 300; Abcam, Cambridge, UK) , mouse anti-human CD8 monoclonal antibody (clone C8/144B, ready to use for the Ventana automated slide stainer; Ventana) , and mouse anti-human CD4 monoclonal antibody (clone 1F6 diluted 1 : 20; Diagnostic BioSystem, Pleasanton, CA, USA).

Techniques:

Journal: British Journal of Cancer

Article Title: Treatment with interleukin-2 in malignant pleural mesothelioma: immunological and angiogenetic assessment and prognostic impact

doi: 10.1038/sj.bjc.6605438

Figure Lengend Snippet: Univariate analysis of overall survival and time to progression in MPM patients treated with IL-2

Article Snippet: For TIL-immunohistochemical staining, sections were incubated with the following antibodies: mouse anti-human Foxp3 monoclonal antibody (clone 236A/E7 diluted 1 : 300; Abcam, Cambridge, UK) , mouse anti-human CD8 monoclonal antibody (clone C8/144B, ready to use for the Ventana automated slide stainer; Ventana) , and mouse anti-human CD4 monoclonal antibody (clone 1F6 diluted 1 : 20; Diagnostic BioSystem, Pleasanton, CA, USA).

Techniques:

Journal: Journal of Virology

Article Title: Potential for Virus Endogenization in Humans through Testicular Germ Cell Infection: the Case of HIV

doi: 10.1128/JVI.01145-20

Figure Lengend Snippet: HIV-1 alternative receptor expression by primary TGCs and HIV-1 binding inhibition. (A) Representative profiles of membrane expression of HIV receptors on the whole population of TGCs (red) and the n (green), 2n (pink), and 4n DNA (blue) TGC populations. Immunolabeling was performed using specific antibodies against CD4, CCR5, CXCR4, CCR3, GalCer, or HSPGs or, in the case of CD206, after detection of biotinylated BSA-mannose (BMA) ligand in the presence (dot-lined histograms) or absence (filled histograms) of mannan competitor. BSA was used as a control (open histograms). Cells were further stained with DRAQ5 for ploidy profile. Specific antibody detection was ensured using isotypes as controls, except for HSPGs, where pronase treatment was used. The right panel shows positive controls for each receptor. (B) The table shows for each receptor the percentage of positive patients and the median and range of percentages of positive cells. (C) HIV binding was measured after incubation of TGCs with HIV R5 SF162 (25 ng p24) in the presence or absence of CD206 BSA-mannose competitor (BMA), galactosylceramide-specific monoclonal antibody, or HSPG competitor (heparin) at the 3 indicated doses. Results represent the mean ± standard error of the mean (SEM) from 3 to 6 independent experiments performed in duplicate and are expressed relative to their respective controls (virus-cell incubation without receptor inhibitors or in the presence of BSA for BSA-mannose). (D) TGCs obtained from 4 donors were incubated for 2 h at 37°C with the env gene deleted or HIV-1 env-expressing pseudoviruses (25 ng p24), and p24 antigen was measured by ELISA. (E) Viral env neutralization was performed in the presence of gp120-specific antibody ( n = 3). In all experiments, background p24 levels measured in wells without cells were deduced from the values obtained with cells. Statistical analysis was performed with the nonparametric Wilcoxon test. *, P < 0.05.

Article Snippet: To immunophenotype the cells containing vDNA, DNAscope detection by tyramide signal amplification (TSA) plus Cy3.5 was combined with immunofluorescence targeting cell markers by using rabbit polyclonal anti-DDX4 (1:500; Abcam) for TGCs and mouse monoclonal anti-CD4 (1:100, clone 1F6; Vector) for T-lineage cells.

Techniques: Expressing, Binding Assay, Inhibition, Immunolabeling, Staining, Incubation, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Journal of Virology

Article Title: Potential for Virus Endogenization in Humans through Testicular Germ Cell Infection: the Case of HIV

doi: 10.1128/JVI.01145-20

Figure Lengend Snippet: HIV entry in primary TGCs following cell-associated infection. (A) TGCs prelabeled with CellTracker (blue) were exposed for18 h to cell-free wild-type R5 HIV-1 or mock exposed. Cells were costained for p24 (red) and DDX4 (green), and p24 localization was assessed by confocal microscopy “Mock” shows absence of labeling with DDX4 antibody isotype (IgG). (B) TGCs prelabeled with CellTracker (blue) were exposed for 18 h to R5 HIV-1 GFP-Vpr viral particles (green) or mock exposed. Rare viral entry events (arrows) in cells labeled with DDX4-specific antibody (red) were visualized by confocal microscopy. “Mock” shows absence of labeling with DDX4 antibody isotype (IgG). (C) Potential HIV receptors were assessed on the surface of Tcam-2 cells labeled for DDX4 using specific antibodies against CD4, CCR5, CXCR4, CCR3, galactosylceramide (GalCer), and HSPGs or following detection of BSA-mannose, a ligand for mannose receptor. Immunolabeling was analyzed by flow cytometry. (D) HIV-1 entry (arrows) was assessed in CFSE-labeled Tcam-2 cells (in green), exposed for 4 h to either a primary R5-tropic HIV-1 strain (ES X-2556-3) or HIV R5 JRCSF or mock exposed, before immunolabeling with p24 antibody (red). Nuclei were stained with DAPI (blue). (E) HIV-1 reverse transcription was assessed by quantitative PCR (qPCR) on Tcam-2 cells cultured for 24 h with or without the reverse transcriptase inhibitor nevirapine, following exposure to HIV R5 JR-CSF ( n = 6). (F and G) TGCs prelabeled with CellTracker (blue) were incubated for 18 h with either PBMCs infected with a primary R5-tropic HIV-1 strain or mock infected (F) or with Jurkat cells infected with HIV-1 R5 or X4 strains (G). Cells were costained for p24 (red) and DDX4 (green) or with control IgG (shown here as “Mock”). (A, B, and G) z-stack maximum projections are presented along with cross-sectional yz orthogonal viewing from the indicated positions (a and b) to discriminate entering viral particles (white arrows) from membrane-bound viruses (arrowheads). Nuclei are stained with DRAQ5 (cyan). Scale bars = 10 μm.

Article Snippet: To immunophenotype the cells containing vDNA, DNAscope detection by tyramide signal amplification (TSA) plus Cy3.5 was combined with immunofluorescence targeting cell markers by using rabbit polyclonal anti-DDX4 (1:500; Abcam) for TGCs and mouse monoclonal anti-CD4 (1:100, clone 1F6; Vector) for T-lineage cells.

Techniques: Infection, Confocal Microscopy, Labeling, Immunolabeling, Flow Cytometry, Staining, Real-time Polymerase Chain Reaction, Cell Culture, Incubation

Journal: PLoS ONE

Article Title: Comparative analysis of the oral mucosae from rodents and non-rodents: Application to the nonclinical evaluation of sublingual immunotherapy products

doi: 10.1371/journal.pone.0183398

Figure Lengend Snippet: Mucosal tissues from animal species were processed for immunohistology analysis, as described in Methods. Slides were stained with the following specific antibodies: anti-MHC-II, anti-CD163, anti-CD172a, anti-CD3, and anti-CD4 to detect and quantify positive cells in tissue sections or with toluidine blue to quantify mast cells (magnification x200). Representative photomicrographs of mucosal tissue sections are shown.

Article Snippet: The following polyclonal or monoclonal antibodies (mAbs) were used for immunohistology: anti-CD3 (for rats: clone 1F4, Bio-Rad, Oxford, UK; for dogs: Ab828, Abcam, Cambridge, UK; for minipigs: clone 8E6, WSU Monoclonal antibody center, Pullman, WA; for monkeys: clone CD3-12, Abcam), anti-CD4 (for rats: clone OX-35, Bio-Rad; for dogs: clone DH-29A, WSU Monoclonal antibody center; for minipigs: clone 74-12-4, WSU Monoclonal antibody center; for monkeys: clone BC/1F6, Abcam), anti-CD163 (for all species: clone AM-3K, Antibodies online, Paris, France), anti-CD172a (for rats: clone ED9, Bio-Rad; for dogs: clone DG-DH59B, WSU Monoclonal antibody center; for minipigs: clone BL1H7, Bio-Rad; for monkeys: Ab139698, Abcam), anti-MHC-II (for rats: clone OX-6, Bio-Rad; for dogs: clone DG-H42A, WSU Monoclonal antibody center; for minipigs: clone TH21A, WSU Monoclonal antibody center; for monkeys: clone L243, Abcam).

Techniques: Staining

Journal: PLoS ONE

Article Title: Comparative analysis of the oral mucosae from rodents and non-rodents: Application to the nonclinical evaluation of sublingual immunotherapy products

doi: 10.1371/journal.pone.0183398

Figure Lengend Snippet: Mucosal tissues from animal species were processed for immunohistology analysis, as described in Methods. Cell counting was performed on slides labeled with Abs specific for APC (anti-MHC-II, anti-CD163, anti-CD172a) and T cell (anti-CD3 and anti-CD4) markers or stained with toluidine blue for mast cells to evaluate the mean number of positive cells per field using a light microscope (magnification x400). All areas (epithelium (Epith.), Lamina propria (LP) and muscle) were scored. Histograms represent the mean + SEM with n = 3.

Article Snippet: The following polyclonal or monoclonal antibodies (mAbs) were used for immunohistology: anti-CD3 (for rats: clone 1F4, Bio-Rad, Oxford, UK; for dogs: Ab828, Abcam, Cambridge, UK; for minipigs: clone 8E6, WSU Monoclonal antibody center, Pullman, WA; for monkeys: clone CD3-12, Abcam), anti-CD4 (for rats: clone OX-35, Bio-Rad; for dogs: clone DH-29A, WSU Monoclonal antibody center; for minipigs: clone 74-12-4, WSU Monoclonal antibody center; for monkeys: clone BC/1F6, Abcam), anti-CD163 (for all species: clone AM-3K, Antibodies online, Paris, France), anti-CD172a (for rats: clone ED9, Bio-Rad; for dogs: clone DG-DH59B, WSU Monoclonal antibody center; for minipigs: clone BL1H7, Bio-Rad; for monkeys: Ab139698, Abcam), anti-MHC-II (for rats: clone OX-6, Bio-Rad; for dogs: clone DG-H42A, WSU Monoclonal antibody center; for minipigs: clone TH21A, WSU Monoclonal antibody center; for monkeys: clone L243, Abcam).

Techniques: Cell Counting, Labeling, Staining, Light Microscopy

Journal: Journal of immunology research

Article Title: Circulating CD3 + HLA-DR + Extracellular Vesicles as a Marker for Th1/Tc1-Type Immune Responses.

doi: 10.1155/2019/6720819

Figure Lengend Snippet: Figure 1: Identification of Th1-derived EV-dominant proteins. (a) Chemiluminescence images. Th1- and Th2-derived EVs were subjected to western blot analysis with anti-CD3 and anti-CD4 mAbs. (b) Fluorescent nanoparticle tracking image. Th1-derived EVs were stained with Qdot655-conjugated anti-CD3 antibody and analyzed on the NanoSight. Results in light scatter (black line) and fluorescence (grey line) modes. (c, d) Two parts of 2D-DIGE images of Th1-derived EV proteins. (e, f) 2D-DIGE images of Th2-derived EV proteins in the same areas as c and d, respectively. Protein spots that showed differences in the fluorescence intensities were picked up for MS analysis. The protein spots indicated by circles (panels c and e, mw = 37067 and pI = 4 8) and dotted circles (panels d and f, mw = 31322 and pI = 7 8) were identified as alpha- and beta-chains of HLA-DR, respectively. Data from one out of six pairs of EVs are shown. The results were similar for the five out of six pairs.

Article Snippet: CD3 and CD4 proteins were visualized using 5 μg/ml of mouse monoclonal antibodies against CD3 (clone: 4C1, Abnova, Taipei, Taiwan) and CD4 (clone:1F6, Bio-Rad, Hercules, CA) and horseradish peroxidase-labeled goat antimouse IgG (Zymed Laboratories, San Francisco, CA) followed by chemiluminescent detection using a Western blot chemiluminescence reagent (New England Nuclear, Boston, MA).

Techniques: Derivative Assay, Western Blot, Staining