Journal: European journal of immunology

Article Title: Comparison of stable human Treg and Th clones by transcriptional profiling.

doi: 10.1002/eji.200838807

Figure Lengend Snippet: Figure 4. Human Treg clones, but not Th clones, produce the mature and active form of TGF-b. (A) Schematic representation of TGF-b processing. Double lines represent cell membrane. Sites of proteolytic cleavages are indicated by arrow heads. Small bars indicate disulfide bonds. LAP: latency associated peptide. LTBP: latent TGF-b binding protein; TGFBR: TGF-b receptors. (B) Three Treg clones (Treg D1, Treg B2, and Treg B3) and four Th clones (Th C2, Th D1, Th B1, and Th A2) were activated in X-VIVO-10 serum-free medium for 24 h with anti-CD3 and anti-CD28 Ab. Active and total TGF-b concentrations in the supernatants were measured by ELISA, before or after treatment with acid, respectively. Values represent means of duplicate1SD. (C) Four Treg clones (Treg C2, Treg D1, Treg B2, and Treg B3) and four Th clones (Th C2, Th D1, Th B1, and Th A2) were activated for 24 h with anti-CD3 and anti-CD28 Ab in the presence of IL-2, prior to staining with biotinylated polyclonal anti-LAP antibody (filled gray histograms) or isotype control (empty histograms) and Streptavidin-PE. (D) Western blot analysis of cell lysates from Treg and Th clones collected at rest (T 5 0), or 6 or 24 h after activation with anti-CD3 and anti-CD28 Ab. Samples in lanes 4 and 8 were treated with 5 ng/mL of recombinant human TGF-b1 (rhTGF-b1) during the last 15 min of the activation period and serve as positive controls. Samples in lanes 17 and 18 were prepared as detailed in Fig. 3D. Briefly, suppressed (‘‘S’’) or control (‘‘Ctrl’’) clone Th A2 was sorted by FACS out of a co-culture with clone Treg A1 or clone Th A2, respectively. Blots were hybridized with an anti-phosphorylated SMAD2 (P-SMAD2) or anti-b-actin antibody.

Article Snippet: For surface LAP expression, cells activated for 24 h with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 (160 IU/mL) were labeled with a biotinylated polyclonal anti-LAP antibody or an isotype control (R&D Systems, BAF246 and BAF108, respectively), followed by incubation with Streptavidin coupled to PE (BD Pharmingen).

Techniques: Clone Assay, Membrane, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Control, Western Blot, Activation Assay, Recombinant, Co-Culture Assay

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Cellular association and assembly of a multistage delivery system.

doi: 10.1002/smll.201000126

Figure Lengend Snippet: Figure 7. Endothelial targeting with the assembled MDSs. A) HUVECs were incubated with either control IgG fluorescein-labeled MDSs (A) or anti- VEGFR-2 fluorescein antibody-labeled MDSs (B,C) for 2 h at 37 8C (10:1 ratio of particles to cells). C) Antibody-targeted cells are shown at two magnifications,with3Dimagesatthecrosshairsofthehighermagnificationimage(middle).Totheright,lamellopodiaprojectingfromtwocellsmake contactwiththeMDS(63oilimmersionlens;A,B)bar25 mm,C) bars50and7.5 mm;actin:rhodaminephalloidin;nuclei:DRAQ5).D)Flowcytometry dot plot (left) showing microparticle light scatter and a histogram (right) showing microparticle fluorescence before and after conjugation with fluorescentantibody. E) Bar graph (top) showingthe relative association of the MDSswith HUVEC andHMVEC endothelial cells conjugated with either control IgG or anti-PECAM-specific antibody. Below are representative flow cytometry dot plots showing the increase in side scatter in HUVECs after incubation with targeted (anti-PECAM) MDSs.

Article Snippet: Antibody conjugation: Anti-PECAM antibody was obtained from Sigma, while anti-VEGF R2/KDR (vascular endothelial growth factor receptor) and isotype control (IgG1) fluorescein-conjugated antibodies were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Control, Labeling, Conjugation Assay, Cytometry

Journal: Frontiers in Immunology

Article Title: Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions

doi: 10.3389/fimmu.2019.00579

Figure Lengend Snippet: PRRSV-induced IL-1Ra was not involved in suppression of IFN-γ-producing T lymphocytes in both (A) polyclonal and (B) recalled CSFV responses. The supernatants obtained from type 2 PRRSV or mock (MARC-145 cell lysate) were pretreated with anti-IL-1Ra Ab for 2 h prior to addition into the culture. PBL or PBMC were cultured with PHA, CSFV or controls for 48 h, in the presence of the pretreated supernatants. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p < 0.05.

Article Snippet: In some experimental conditions, cells were pre-treated with final concentration of 10 ng/mL polyclonal goat anti-porcine IL-1Ra antibody (R&D system, clone AF780) or polyclonal goat IgG isotype control antibody (R&D system) at 2 h post-inoculation to neutralize PRRSV-induced IL-1Ra which was then cultured for another 22 h. To determine the effect of PRRSV-induced IL-1Ra on MoDC phagocytic activity, the antibody pre-treated MoDC (2 × 10 6 cell) were further incubated with inactivated E. coli -FITC (ThermoFisher Scientific) in complete RPMI at a MoDC: E. coli ratio of 1:50 for 10 min at 37°C.

Techniques: Cell Culture