Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Innate direct anticancer effector function of human immature dendritic cells. II. Role of TNF, lymphotoxin-alpha(1)beta(2), Fas ligand, and TNF-related apoptosis-inducing ligand.

doi: 10.4049/jimmunol.168.4.1831

Figure Lengend Snippet: FIGURE 8. Monocyte-derived immature DCs express cell surface TNFR2, Fas, and LT-R, but not TNFR1, TRAILR1, and TRAILR2. Im- mature DCs were stained intact or permeabilized, and then stained with mAbs specific for TNF family receptors and analyzed using flow cytometry as described in Fig. 7. Similar data were obtained with normal T cell blasts, fibroblasts, melanocytes, and keratinocytes.

Article Snippet: Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 (IgG1) mouse mAb (Caltag Laboratories, Burlingame, CA), M3 anti-human Fas (IgG1) mouse mAb (Immunex, Seattle, WA), anti-human TRAIL (anti-Apo-2L) rabbit PCA (Calbiochem, San Diego, CA), 32A1380 antihuman TRAILR1 (IgG1) mouse mAb (Imgenex, San Diego, CA), 54B1005 anti-human TRAILR2 mouse mAb (Imgenex), isotype control mouse mAbs (Caltag Laboratories), normal rabbit serum (Endogen), biotin-conjugated anti-mouse IgG (H L) goat PCA (Jackson ImmunoResearch, West Grove, PA), biotin-conjugated anti-rat IgG (H L) goat PCA (Jackson ImmunoResearch), biotin-conjugated anti-goat IgG (H L) rabbit PCA, (Vector Laboratories, Burlingame, CA), CyChrome-conjugated anti-human HLA-DR (IgG1) mouse mAb (BD PharMingen, San Diego, CA), and all previously described conjugated and unconjugated Abs used for determination of DC phenotype and purification of monocytes and DCs (47).

Techniques: Derivative Assay, Staining, Cytometry

Journal: Cancer Research

Article Title: Expression of RASSF1A, an Epigenetically Silenced Tumor Suppressor, Overcomes Resistance to Apoptosis Induction by Interferons

doi: 10.1158/0008-5472.can-05-2303

Figure Lengend Snippet: Figure 5. Effect of lentivirus expression of RASSF1A on IFN-induced and Apo2L/TRAIL–induced apoptosis in ACHN cells. A, map of lentiviral RASSF1A construct. B, ACHN cells were transduced with RASSF1A carrying lentivirus or empty virus. Seventy-two hours later, selective antibiotic was added, and the population of clones was grown in selection media for 14 days before assessing apoptotic response to IFN-h (50 units/mL over 5 days) by TUNEL assay. After confirmation of stable expression of RASSF1A by immunoblotting, clonal selection was done to identify clones that expressed similar amounts of RASSF1A protein as achieved by DNMT1 AS (40 nmol/L) over 8 days. Treatment with DNMT1 mismatch control oligonucleotide (MM) and empty lentivirus–transduced cells served as negative controls. In clones expressing RASSF1A at levels comparable with DNA-demethylating treatment, IFN-h (50 units/mL over 5 days) resulted in 20% to 40% apoptotic cells, whereas stable transduction with empty lentivirus (kept in selective antibiotic for equal amounts of time) did not. C, concurrent treatment of ACHN RASSF1A clone 1.3 with 2 Ag/mL TRAIL neutralizing antibody (TRAIL-N AB) and IFN-h (50 U/ml) over 5 days inhibited IFN-induced apoptosis compared with cotreatment with 2 Ag/mL control rabbit immunoglobulin (CTRL AB). D, RASSF1A expression markedly increased sensitivity to apoptosis induction by Apo2L/TRAIL in ACHN cells. NKE cells, even after pretreatment with 5-AZA-dC (200 nmol/L) over 4 days, which alone resulted in moderate apoptosis induction, remained resistant to the apoptosis-inducing effects of Apo2L/ TRAIL. Columns, means of independent experiments; bars, SD.

Article Snippet: Apo2L/tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) was obtained from PeproTech (RockyHill, NJ), rabbit polyclonal TRAIL neutralizing antibody and control rabbit immunoglobulin were from ProSci (Poway, CA).

Techniques: Expressing, Construct, Transduction, Virus, Clone Assay, Selection, TUNEL Assay, Western Blot, Control

Journal: Cancers

Article Title: Chemotherapeutic Agents Sensitize Resistant Cancer Cells to the DR5-Specific Variant DR5-B More Efficiently Than to TRAIL by Modulating the Surface Expression of Death and Decoy Receptors

doi: 10.3390/cancers12051129

Figure Lengend Snippet: Neutralization of decoy receptors improved TRAIL cytotoxic activity. A549 and HT-29 cells were pre-incubated with 1500 nM and 2500 nM doxorubicin (Dox), 50 nM and 25 nM bortezomib (Btz), 100 nM and 50 nM panobinostat (Pst) respectively for 24 h, following incubation with anti-DcR1 and anti-DcR2 antibodies (15 µg/mL each) for 1 h and 1000 ng/mL ligands for another 24 h. Mean ± Standard Deviation ( n = 3). The asterisks indicate significance (* p < 0.05) and (** p < 0.001) relative to cells treated with ligands without anti-DcRs antibodies.

Article Snippet: Antibodies to decoy receptors DcR1 (AF630) and to DcR2 (AF633) were form R&D systems.

Techniques: Neutralization, Activity Assay, Incubation, Standard Deviation

Journal: The Journal of Clinical Investigation

Article Title: Macrophage-epithelial paracrine crosstalk inhibits lung edema clearance during influenza infection

doi: 10.1172/JCI83931

Figure Lengend Snippet: (A and B) IFNα (A) and TRAIL (B) concentrations of AEC mono- and coculture supernatant 24h pi quantified by ELISA. mAEC were monocultured and inoculated in vitro with PBS (ctrl) or PR8 (AEC) or cocultured with BMM without infection (ctrl), with infection of only macrophages (BMM), or with infection of both cell types (AEC/BMM). (C) TRAIL mRNA expression quantified by qPCR in BMM isolated from WT or Ifnar–/– mice noninfected (ctrl) or PR8-infected (IAV) at 16h pi. (D–G) NKAα1 relative MFI on/of mAEC treated with 25U/ml mouse rIFNα or/and 100 pg/ml mouse rTRAIL for 16 hours (D); mAEC derived from Ifnar–/– or Trail–/– mice inoculated with PBS (ctrl) or PR8 (IAV) 24h pi (E); mAEC derived from Ifnar–/– mice inoculated for 2 hours with conditioned media from PBS (ctrl) or PR8 (IAV) WT AEC infected for 16 hours (F); and mAEC derived from WT, Ifnar–/–, or Dr5–/– mice cocultured with BMM from WT or Trail–/– mice and infection of none (ctrl) or both cell types (AEC/BMM) in vitro (G). Value of control conditions were normalized to 1. (H and I) Vectorial water transport of hAEC monoculture 6 hours after inoculation with PBS (ctrl), PR8 (IAV), rIFNα (150 pg/ml), and/or rTRAIL (100 pg/ml) or in coculture with human AM without additional treatment (–), in presence of neutralizing antibodies against human IFNα (0.5 μg/ml) and human TRAIL (0.1 μg/ml) (anti-IFN + anti-TRAIL) or the respective IgG controls (IgG). hAEC maintained barrier integrity in all assay conditions (Supplemental Figure 9). Bar graphs represent means ±SEM of 4–6 independent experiments (A, B, and E–I), 3 independent experiments (C), and 6–8 independent experiments (D). Statistical significance was analyzed by Student’s t test (F) or 1-way ANOVA and post-hoc Tukey (A–E and G–I). *P < 0.05; **P < 0.01; ***P < 0.005.

Article Snippet: For in vitro experiments, recombinant murine TRAIL (R&D Systems, 100 pg/ml), recombinant murine GM-CSF (R&D Systems, 25 ng/ml) recombinant mouse IFNα and -β (PBL Interferon Source, 25 U/ml; R&D Systems, 10–100 ng/ml), recombinant human IFNα (R&D Systems, 150 pg/ml), recombinant human TRAIL (R&D Systems, 100 pg/ml), anti–human IFNα neutralizing antibody (R&D Systems, 0.5 μg/ml), anti–human TRAIL neutralizing antibody (R&D Systems, 0.1 μg/ml), and the following chemical inhibitors were used: AICAR, AMPK inhibitor Compound C, (5Z)-7-Oxozeanol (Curvularia sp), STO-609 (all Calbiochem) and BAPTA-AM (Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, In Vitro, Infection, Expressing, Isolation, Derivative Assay, Control

Journal: British Journal of Cancer

Article Title: Induction of apoptosis in experimental human B cell lymphomas by conditional TRAIL-expressing T cells

doi: 10.1038/sj.bjc.6601407

Figure Lengend Snippet: Tet induces TRAIL overexpression in Jurkat-TR (J-TR) cells. ( A ) Five selected single clones of J-TR or one clone of Jurkat-CO cells (J-CO) were cultured for 48 h in the presence (+, switched on) or absence (−, switched off) of tet (2 μ g ml −1 ). Proteins were extracted and TRAIL expression was assayed by Western blot analysis using a mouse mAb, which specifically detects the 32 kDa TRAIL protein. Expression of the 43 kDa α -ACTIN protein was examined as a control for equal conditions. ( B ) Jurkat-TRAIL (J-TR) clone 5 and Jurkat-CO (J-CO) cells were incubated in the presence (+) or absence (−) of increasing doses of tet (0, 0.1, 0.5, 1, 1.5, 2 μ g ml −1 ). After 48 h, total RNA was harvested and RT–PCR was performed using TRAIL-specific primers. Assaying the RNA expression of GAPDH confirmed equal conditions. ( C ) Jurkat-TRAIL (J-TR) or Jurkat-CO (J-CO) cells were cultured in the presence (open histograms with dotted lines) or absence (filled histograms with solid line) of tet (2 μ g ml −1 ) and analysed by flow cytometry. The number indicates the percentage of TRAIL-positive cells. ( D ) Jurkat-TRAIL (J-TR) clone 5 and Jurkat-CO cells were switched on (2 μ g ml −1 tet) or left switched off. After 48 h, cytospins were prepared and the cell surface expression of TRAIL was analysed by immunofluorescence staining. The representative results shown are from one of three different experiments with similar outcomes.

Article Snippet: Expression of TRAIL was detected by a mouse mAb anti-human TRAIL (BD/Pharmingen), diluted 1 : 250 in PBS-Tween containing 1.5% Roti®-Immunoblock.

Techniques: Over Expression, Clone Assay, Cell Culture, Expressing, Western Blot, Incubation, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Flow Cytometry, Immunofluorescence, Staining