Journal:

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells

doi:

Figure Lengend Snippet: TNF and, to a lesser extent, IL-1 are involved in the permanently induced NF-κB activity present in thymocytes during the coculture with TEC. Thymocytes were freshly isolated (lanes 1 and 2) or cultured for 45 h (lanes 3 to 11) either alone (lane 3 [Control thymocytes]) or in the presence of TEC under conditions avoiding contact (lanes 4 to 9) or allowing contact (lanes 10 and 11). During the coculture, the cells were either left untreated (lane 4 [Control coculture, 45 hours] and lane 10 [Control coculture]) or were treated at the start of the coculture with antibodies respectively raised against TNF (lane 5), IL-6 (lane 6), and IL-1β (lane 8), with the antagonist of the IL-1 receptor (lane 7), or with αTNF plus IL-1 ra plus αIL-6 (lanes 9 and 11). Whole-cell extracts from these various samples were incubated with a 32P-labeled oligonucleotide representing the HIV LTR-derived κB motif. Competition (comp [lane 1]) with a 40-fold molar excess of unlabeled oligonucleotide was used to confirm the specificity of the DNA-binding activity detected. NF-κB activity is indicated. The positions of two nonspecific (n.s 1 and n.s 2) binding activities are indicated. The positions of two nonspecific (n.s 1 and n.s 2) binding activities are indicated. This experiment is representative of three independent experiments, each carried out on a different thymus.

Article Snippet: MAb against IL-1β (B-A15) was purchased from Diaclone Research (Besançon, France).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Labeling, Derivative Assay, Binding Assay

Journal:

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells

doi:

Figure Lengend Snippet: TNF and, to a lesser extent, IL-1 induce NF-κB activity in thymocytes, and IL-7 is required for this effect. Whole-cell extracts were prepared from freshly isolated thymocytes (panel A, lane 1) or from thymocytes cultivated for 30, 60, or 45 h. (A) During the indicated culture times, thymocytes were either left untreated (lane 1, control for freshly isolated; lane 2, control 30 h; and lane 5, control 60 h) or were stimulated with either TNF (lanes 3 and 6), IL-1β (lanes 4 and 7), or TNF in the presence of TEC CM (TEC CM + TNF) (lane 9). TEC conditioned medium was also added alone (lane 8 [TEC CM]). The data shown here are representative of three independent experiments carried out on three thymuses. (B) Thymocytes were cultured for 45 h either untreated (lane 1, control 45 h), with TNF (lane 2), with TEC CM (lane 3), or with TNF in the presence of TEC CM (TEC CM + TNF) preincubated (lane 5) or not (lane 4) with antibody against IL-7. In lanes 6 to 8, the thymocytes were left untreated for 6 h (lane 6) or incubated with an antibody against IL-7Rα (lane 7) or with an IgG1 control serum (lane 8) before being cocultured with TEC for 45 h. The data shown here are representative of two independent experiments carried out on two thymuses. (C) Cells were left untreated for the 45 h of the culture (lane 1, control 45 h) or treated with IL-1β (lane 2), TNF (lane 3), IL-1β plus TNF plus IL-6 plus GM-CSF (lane 4), IL-7 (lane 5), IL-7 plus IL-1β (lane 6), or IL-7 plus TNF (lane 7). This experiment is representative of three independent experiments carried out on three thymuses. (D) Thymocytes were left untreated (lane 1, control) or stimulated with IL-7 (lane 2) or IL-7 in presence of anti-TNF and Il-1ra (lane 3). Whole-cell extracts were incubated with a 32P-labeled oligonucleotide representing κB motif. NF-κB activity is indicated. This experiment is representative of two independent experiments, each carried out on a different thymus.

Article Snippet: MAb against IL-1β (B-A15) was purchased from Diaclone Research (Besançon, France).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Labeling

Journal: PLoS ONE

Article Title: Isolation of Highly Suppressive CD25+FoxP3+ T Regulatory Cells from G-CSF-Mobilized Donors with Retention of Cytotoxic Anti-Viral CTLs: Application for Multi-Functional Immunotherapy Post Stem Cell Transplantation

doi: 10.1371/journal.pone.0085911

Figure Lengend Snippet: Expanded CD25+ cells isolated from both G-CSF-mobilized and non-mobilized donors were stimulated with CMVpp65 peptide loaded autologous PBMCs in the presence of Brefeldin A and purified anti CD28-antibody. Histograms illustrate CMVpp65 rechallenge experiments in representative CD25+ expansions from a G-CSF-mobilized and non-mobilized donor, analysed for IL-2, TNF-α, IFN-γ, Granzyme B and IL-10. Open peaks represent isotype matched controls (A). The combined assessment in G-CSF-mobilized ( n = 5) and non-mobilized ( n = 5) donors are summarized (B). ** p <0.01, in an unpaired t test. Gra, granzyme; Mob, mobilized.

Article Snippet: Cells were fixed and permeabilized using Intrastain (DakoCytomation, Ely, UK) according to the manufacturer’s instructions and stained with CD25-APC, CD4-PerCP either PE-conjugated anti-IL-2, anti-TNF-α, anti-Granzyme B, anti-IL-10 or anti IFN-γ and CD69-APC Cy7 (all BD Biosciences) monoclonal antibodies.

Techniques: Isolation, Purification

Journal: Obesity (Silver Spring, Md.)

Article Title: Tight Association between Macrophages and Adipocytes in Obesity: Implications for Adipocyte Preparation

doi: 10.1002/oby.20634

Figure Lengend Snippet: Most CLS macrophages express TNFα while a few express both TNFα and IL-10. Images presented are a LSCM 2D projection of a 3D image stack. Adipose tissue from obese mice that was fixed and stained with BODIPY 558/568 for lipid (gray), Hoechst for nuclei (blue), anti-F4/80 for macrophages (red), anti-TNFα (green), and anti-IL-10 (magenta). The panel shows a four micron 2D projection captured with a 63× objective. The majority of CLS macrophages produce TNFα and some appear to produce both TNFα and IL-10 (arrows) while others have no cytokine expression (F4/80 only).

Article Snippet: For complex 5-color staining, fixed tissue and floating layer adipocytes were stained utilizing directly conjugated fluorescent anti-mouse monoclonal antibodies: anti-TNFα conjugated FITC (eBioscience), anti-IL-10 conjugated PE (BD Bioscience) and anti-F4/80 conjugated AlexaFluor647 (Invitrogen) in PBS staining buffer with 1% BSA.

Techniques: Staining, Expressing

Journal: Obesity (Silver Spring, Md.)

Article Title: Tight Association between Macrophages and Adipocytes in Obesity: Implications for Adipocyte Preparation

doi: 10.1002/oby.20634

Figure Lengend Snippet: The adipocyte floating layer has high expression of TNFα and IL-10 in obese mice compared to non-obese mice. (a) qRT-PCR of floating layer cells showing relative amounts of TNFα compared to IL-10 mRNA normalized to GAPDH. Obese mice had a 40-fold increase in TNFα (open boxes) while only inducing a 10-fold increase in IL-10 production (black boxes). * p<0.05 in all figures. (b) Floating layer adipocyte with attached macrophages. Floating layer cells stained with BODIPY (gray) for lipid to denote adipocytes, F4/80 (red) for macrophages, TNFα (green), IL-10 (magenta), and nuclei (blue). There was no IL-10 expressed in either cell type in this example (field not shown). TNFα can clearly be seen to be associated with only the macrophages, but not the adipocyte (F4/80 vs. TNFα panels and overlay). The adipocyte nucleus is indicated by the white arrow in the overlay and right side view, while macrophage nuclei are indicated by the blue arrows. The figure is an LSCM 2D projection of a 3D image stack using a 63× objective. The side views are 1um thick sections imaged from the corresponding dotted white lines parallel to the section.(c) Macrophages in the floating adipocyte layer ingest lipid and produce TNFα. At high magnification using LSCM and 63× objective a 2D projection shows a macrophage (red) with a single nucleus (blue arrow) remained attached to and ingested lipid (gray) in the floating layer. TNFα (green) expression was associated with the macrophage (white arrows) and not the attached or ingested lipid. No IL-10 is produced. (d) Floating layer macrophage(s) with engulfed lipid produced IL-10 to a lesser extent. Individual panels (left) show a one micron section of a LSCM image using a 63× objective. The floating layer cells were stained for F4/80 (red), TNFα (green) and IL-10 (magenta) and the overlay depicts all of these fields plus a brightfield image. The macrophage, shown with engulfed lipid, is expressing mostly IL-10, but there is some expression of TNFα. A 2D projection of the LSCM image shows the extent of IL-10 production in the macrophage surrounding the lipid.

Article Snippet: For complex 5-color staining, fixed tissue and floating layer adipocytes were stained utilizing directly conjugated fluorescent anti-mouse monoclonal antibodies: anti-TNFα conjugated FITC (eBioscience), anti-IL-10 conjugated PE (BD Bioscience) and anti-F4/80 conjugated AlexaFluor647 (Invitrogen) in PBS staining buffer with 1% BSA.

Techniques: Expressing, Quantitative RT-PCR, Staining, Produced

Journal: bioRxiv

Article Title: Recombinant Fc-fusion vaccine of RBD induced protection against SARS-CoV-2 in non-human primate and mice

doi: 10.1101/2020.11.29.402339

Figure Lengend Snippet: a) ELISPOT assay for IFN-γ in PBMCs from macaca fascicularis. Data are shown as mean ± SEM. Significance was calculated using unpaired t-test (n.s., not significant). b). SARS-CoV-2 RBD-specific IL-4 + T cells and TNF-a + T cells in CD4 + T and CD8 + T cells from macaca fascicularis PBMCs were detected by flow cytometry.

Article Snippet: Then cells were first stained with Percp conjugated anti-monkey CD4 (RM4-5, BD, 561115), Percp conjugated anti-monkey CD8a (RPA-T8, BD, 560662), at 4°C for 25 min. PE conjugated anti-monkey IL-4 (8D4-8, BD, 551774), and PE conjugated anti-monkey TNF-a (MAb11, BD, 557068) using the Cytofix/Cytoperm fixation/permeabilization solution kit (BD) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunospot, Flow Cytometry