Journal: Mediators of Inflammation

Article Title: Cytomegalovirus Infection Impairs Immunosuppressive and Antimicrobial Effector Functions of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1155/2014/898630

Figure Lengend Snippet: CMV blocks IDO activity and IDO-mediated antimicrobial effects in human MSC. (a) PBL, stimulated with OKT3, were cocultured with MSC in the presence or absence of CMV. As controls UV-inactivated CMV (uvCMV), the IDO-specific inhibitor 1-L-methyl-tryptophan (1-MT; 1.5 mM), or a neutralising anti-IFN- γ antibody ( α IFN- γ ; 10 ng/mL) was used. After three days IDO activity was determined and is presented as mean kynurenine content ± SEM of three independent experiments, each done in triplicate. (b) PBL (1 × 10 5 /well), stimulated with CD3-directed mAB OKT3, were cocultured with MSC (3 × 10 4 /well) in the absence or presence of CMV (MOI 5). After three days cultures were infected with S. aureus (10–100 cfu/well) and bacterial growth was determined photometrically. As a control, cultures were supplemented with L-tryptophan (Trp; 0.6 mM) at the time point of bacterial infection. Data are given as mean OD (620 nm) ± SEM of three experiments, each done in triplicate. Significant differences ( P < 0.05) as compared to the positive control are marked by asterisks.

Article Snippet: Recombinant human IFN- γ was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Activity Assay, Infection, Control, Positive Control

Journal: Mediators of Inflammation

Article Title: Cytomegalovirus Infection Impairs Immunosuppressive and Antimicrobial Effector Functions of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1155/2014/898630

Figure Lengend Snippet: CMV inhibits IDO induction by recombinant IFN- γ . (a) MSC (2 × 10 4 /well) which were infected with various amounts of CMV (MOI 0.1–10) were stimulated with IFN- γ (300 U/mL). After three days IDO activity was determined and is presented as mean kynurenine content ± SEM of three independent experiments, each done in triplicate. (b) MSC (1.5 × 10 6 /flask) were stimulated with IFN- γ (600 U/mL) in the absence or presence of CMV (MOI 5). Cells were harvested after 24 h and IDO protein was detected in Western blot analysis. β -Actin was utilized as a protein loading control, while the viral pp72 protein served as an infection control.

Article Snippet: Recombinant human IFN- γ was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Recombinant, Infection, Activity Assay, Western Blot, Control

Journal: Immunology

Article Title: Co-immunization with an optimized plasmid-encoded immune stimulatory interleukin, high-mobility group box 1 protein, results in enhanced interferon-? secretion by antigen-specific CD8 T cells

doi: 10.1111/j.1365-2567.2009.03044.x

Figure Lengend Snippet: High-mobility group box 1 (HMGB1) induces strong human immunodeficiency virus (HIV)-specific T-cell responses in mice. Interferon-γ enzyme-linked immunosorbent spot-forming cell assay (ELISPOT). BALB/c mice were immunized three times, each 2 weeks apart, with 25 μg of pVax vector or with pHMGB1 and were killed 1 week later. Splenocytes were harvested and cultured overnight in the presence of R10 (negative control), 10 μg/ml of HIV-1 Gag peptide pools or 10 μg/ml of HIV-1 Env peptide pools against a library of peptides spanning HIV-1 subtype B. (a) Responses to HIV-1 Gag are shown as a stacked group. (b) The total additive response to HIV-1 Env. Spot-forming units (SFU) were quantified using an automated ELISPOT reader, and the raw values were normalized to the number of SFU per million splenocytes. Error bars represent the standard deviation (SD) of ELISPOT results in triplicate wells and the data are representative of three independent experiments.

Article Snippet: 23 , 28 , 29 Briefly, 96-well ELISPOT plates (Millipore, Billerica, MA) were coated with anti-mouse IFN-γ capture antibody and incubated for 24 hr at 4° (R&D Systems).

Techniques: Virus, ELISpot Assay, Enzyme-linked Immunospot, Plasmid Preparation, Cell Culture, Negative Control, Standard Deviation

Journal: American Journal of Physiology - Cell Physiology

Article Title: IFN? regulates retinal pigment epithelial fluid transport

doi: 10.1152/ajpcell.00255.2009

Figure Lengend Snippet: Expression and localization of IFNγ receptors. A: immunoblots of IFNγ receptor subunits 1 and 2 (IFNGR1 and IFNGR2) in human fetal and adult retinal pigment epithelium (RPE) and fetal choroidal (hfCHC) cells. M, molecular weight marker lanes; lane 1, primary hfRPE cell culture; lane 2, native human adult RPE; lane 3, primary hfCHC cells. B: immunofluorescence localization of IFNγ receptor in hfRPE cells. For IFNGR1 and IFNGR2, cross section through the z plane is shown at top. In each case, x-y plane is shown as an en face view of the apical membrane (maximum-intensity projection through the z-axis). ZO-1 (green) stains tight junctions; 4,6-diamidino-2-phenylindole (DAPI, blue) labels nuclei. Inset at higher gain shows a z-section above each panel; note that IFNGR1 and IFNGR2 are mainly located on the basolateral membrane (Ba). Ap, apical membrane.

Article Snippet: The blotted membrane was incubated with antibodies against human IFNGR1 (catalog no. MAB6731, R & D Systems, Minneapolis, MN), IFNGR2 (catalog no. sc-970, Santa Cruz Biotechnology, Santa Cruz, CA), IFNα receptor subunits 1 and 2 (IFNAR1 and IFNAR2, Abcam, Cambridge, MA), IRF-1, IRF-2, and ICSBP (Santa Cruz Biotechnology), and CFTR (CFTR antibody 217, Cystic Fibrosis Foundation Therapeutics, Bethesda, MD).

Techniques: Expressing, Western Blot, Molecular Weight, Marker, Cell Culture, Immunofluorescence, Membrane

Journal: American Journal of Physiology - Cell Physiology

Article Title: IFN? regulates retinal pigment epithelial fluid transport

doi: 10.1152/ajpcell.00255.2009

Figure Lengend Snippet: IFNγ activates JAK-STAT signaling pathway in hfRPE cells. A: IFNγ stimulated tyrosine phosphorylation of STAT1 in hfRPE cells (15 min) and can be blocked by anti-IFNGR1 blocking antibody. GAPDH was used as loading control. B: regulation of interferon regulatory factor (IRF) gene expression in hfRPE cells. Cells were treated with serum-free medium [SFM (Ctrl)], IFNγ, or cycloheximide (CHX) + IFNγ for 4 h. ICSBP, IFN consensus sequence-binding protein.

Article Snippet: The blotted membrane was incubated with antibodies against human IFNGR1 (catalog no. MAB6731, R & D Systems, Minneapolis, MN), IFNGR2 (catalog no. sc-970, Santa Cruz Biotechnology, Santa Cruz, CA), IFNα receptor subunits 1 and 2 (IFNAR1 and IFNAR2, Abcam, Cambridge, MA), IRF-1, IRF-2, and ICSBP (Santa Cruz Biotechnology), and CFTR (CFTR antibody 217, Cystic Fibrosis Foundation Therapeutics, Bethesda, MD).

Techniques: Phospho-proteomics, Blocking Assay, Control, Gene Expression, Sequencing, Binding Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: IFN? regulates retinal pigment epithelial fluid transport

doi: 10.1152/ajpcell.00255.2009

Figure Lengend Snippet: IFNγ-induced alterations in gene expression by quantitative RT-PCR

Article Snippet: The blotted membrane was incubated with antibodies against human IFNGR1 (catalog no. MAB6731, R & D Systems, Minneapolis, MN), IFNGR2 (catalog no. sc-970, Santa Cruz Biotechnology, Santa Cruz, CA), IFNα receptor subunits 1 and 2 (IFNAR1 and IFNAR2, Abcam, Cambridge, MA), IRF-1, IRF-2, and ICSBP (Santa Cruz Biotechnology), and CFTR (CFTR antibody 217, Cystic Fibrosis Foundation Therapeutics, Bethesda, MD).

Techniques: Gene Expression, Quantitative RT-PCR