Journal: Molecular Therapy. Nucleic Acids

Article Title: Retroviral Replicating Vector Delivery of miR-PDL1 Inhibits Immune Checkpoint PDL1 and Enhances Immune Responses In Vitro

doi: 10.1016/j.omtn.2016.11.007

Figure Lengend Snippet: RRV-U6-miRGFP Has Higher GFP Downregulation Activity than RRV-U6-shGFP and Does Not Trigger IFN Response (A) RRVs produced from transient transfection in 293T were used to infect U87-MG/GFP cells at a MOI of 0.1 (see Figure S1 for MOI of 1 and 10). The GFP downregulation activity was evaluated and quantified by flow cytometric analysis for MFI. Both uninfected cells and cells infected with RRV-yC2 were included as a positive control. The percentage of GFP downregulation is relative to the uninfected cells. The data shown represent mean ± SD of triplicates performed from one of multiple independent experiments. (B) Vector stability of RRV-U6-shGFP and RRV-U6-miRGFP using genomic DNA from infected U87-MG/GFP cells at 30 days post infection was analyzed by endpoint PCR. The DNA molecular marker (1 Kb Plus marker, Invitrogen) is included in the first lane. The right and left arrows indicate the expected size of the PCR products for the RRV-shGFP (514 bp) and for the RRV-miRGFP (844 bp), respectively. (C) Induction of OAS1 gene expression induced by IFN response in U87-MG cells infected with RRV-U6-shGFP or RRV-U6-miRGFP at a MOI of 0.1, 1, and 10. U87-MG cells treated with exogenous recombinant human IFNα were included as positive control. The values are presented as the means ± SD of triplicates.

Article Snippet: PDL1 upregulation by IFNγ was performed in maximally RRV-shPDL1 and RRV-miR-PDL1 infected cells by adding 500 IU/mL recombinant human IFNγ (R&D System, cat. no. 285-IF-100/CF) for 36 hr before harvesting the cells for flow cytometric analysis.

Techniques: Activity Assay, Produced, Transfection, Infection, Positive Control, Plasmid Preparation, Marker, Gene Expression, Recombinant

Journal: Molecular Therapy. Nucleic Acids

Article Title: Retroviral Replicating Vector Delivery of miR-PDL1 Inhibits Immune Checkpoint PDL1 and Enhances Immune Responses In Vitro

doi: 10.1016/j.omtn.2016.11.007

Figure Lengend Snippet: Downregulation of PDL1 by RRV-miRPDL1 Restores PHA-Stimulated T Cell Activation In Vitro (A) PHA-induced PBMCs were cultured alone or co-cultured with Mel 103 cells or Mel 103 cells overexpression PDL1 (Mel 103/LV-PDL1). IFNγ production in the supernatant was measured by ELISA. (B) PHA-induced PBMCs were cultured alone or co-cultured with LN-18 parent cells or LN18 cells infected with RRV-miRPDL1 (LN18/KD). IFNγ production in the supernatant was measured by ELISA. (C) PHA-stimulated PBMCs were cultured alone or co-cultured with RRV-miRGFP infected MDA-MB-231BR cells in the presence or absence of anti-PDL1 and anti-PD1 antibody and compared to that co-cultured with RRV-miRPDL1 infected MDA-MB-231BR cells. The intracellular IFNγ expression in the CD3 + /CD8 + gated population was analyzed by flow cytometry. The values present percentage of IFNγ + cells from one of multiple experiments.

Article Snippet: PDL1 upregulation by IFNγ was performed in maximally RRV-shPDL1 and RRV-miR-PDL1 infected cells by adding 500 IU/mL recombinant human IFNγ (R&D System, cat. no. 285-IF-100/CF) for 36 hr before harvesting the cells for flow cytometric analysis.

Techniques: Activation Assay, In Vitro, Cell Culture, Over Expression, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: BCL-XL Overexpression Protects Pancreatic β-Cells against Cytokine- and Palmitate-Induced Apoptosis

doi: 10.3390/ijms24065657

Figure Lengend Snippet: BCL-XL overexpression protects rat β-cells against cytokine- and palmitate-induced apoptosis. INS-1E cells were infected with adenoviral (ad) vectors encoding luciferase (adLUC, blue dots) or rat BCL-XL (adBCL-XL, red dots). After 48 h of recovery, cells were left untreated (NT) or treated with either IFNγ + IL-1β (10 and 100 U/mL, respectively) ( a , c , e ) or 0.5 mM palmitate ( b , d , f ) for 24 h. ( a – d ) Protein expression was measured by Western blot. Representative images of three independent experiments are shown in ( a , b ) and densitometry results are presented for BCL-XL ( c , d ). ( e , f ) Apoptosis was assessed using HO/PI staining. Results are the means ± SEM of the 3–5 independent experiments, where each dot represents an independent experiment. ** p ≤ 0.01 and *** p ≤ 0.001 vs. untreated (NT) and infected with the same adenoviral vector; # p ≤ 0.05, ## p ≤ 0.01, and ### p ≤ 0.001, as indicated by bars. Two-way ANOVA.

Article Snippet: The following concentrations were used: recombinant human IL-1β (R&D Systems, Cat. No. 201-LB/CF, Abingdon, UK) at 10 or 50 U/mL for INS-1E and EndoC-βH1 cells, respectively; recombinant human IFNγ (PeproTech, Cat. No. 300-02-250UG, Cranbury, NJ, USA) at 1000 U/mL for EndoC-βH1 cells; and recombinant rat IFNγ (R&D Systems, Cat. No. 585-IF) at 100 U/mL for INS-1E cells.

Techniques: Over Expression, Infection, Luciferase, Expressing, Western Blot, Staining, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: BCL-XL Overexpression Protects Pancreatic β-Cells against Cytokine- and Palmitate-Induced Apoptosis

doi: 10.3390/ijms24065657

Figure Lengend Snippet: BCL-XL overexpression protects human β-cells from cytokine- and palmitate-induced apoptosis. EndoC-βH1 cells were infected with adenoviral (ad) vectors encoding luciferase (adLUC, blue dots) or rat BCL-XL (adBCL-XL, red dots). After 48 h of recovery, cells were left untreated (NT) or treated with either IFNγ + IL-1β (50 and 1000 U/mL, respectively) ( a , c , e ) or 0.5 mM palmitate ( b , d , f ) for 48 h. ( a – d ) Protein expression was measured by Western blot. Representative images of four independent experiments are shown in ( a , b ) and densitometry results are presented for BCL-XL ( c , d ). ( e , f ) Apoptosis was assessed using HO/PI staining. Results are the means ± SEM of the 4–6 independent experiments, where each dot represents an independent experiment. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 vs. untreated (NT) and infected with the same adenoviral vector; # p ≤ 0.05 and ### p ≤ 0.001, as indicated by bars. Two-way ANOVA.

Article Snippet: The following concentrations were used: recombinant human IL-1β (R&D Systems, Cat. No. 201-LB/CF, Abingdon, UK) at 10 or 50 U/mL for INS-1E and EndoC-βH1 cells, respectively; recombinant human IFNγ (PeproTech, Cat. No. 300-02-250UG, Cranbury, NJ, USA) at 1000 U/mL for EndoC-βH1 cells; and recombinant rat IFNγ (R&D Systems, Cat. No. 585-IF) at 100 U/mL for INS-1E cells.

Techniques: Over Expression, Infection, Luciferase, Expressing, Western Blot, Staining, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: BCL-XL Overexpression Protects Pancreatic β-Cells against Cytokine- and Palmitate-Induced Apoptosis

doi: 10.3390/ijms24065657

Figure Lengend Snippet: BCL-XL overexpression alleviates ER stress upon inflammatory or metabolic insults in rat β-cells. INS-1E cells were infected with adenoviral (ad) vectors encoding luciferase (adLUC, blue dots) or rat BCL-XL (adBCL-XL, red dots). After 48 h of recovery, cells were left untreated (NT) or treated with either IFNγ + IL-1β (10 and 100 U/mL, respectively) ( a , c , e ) or 0.5 mM palmitate ( b , d , f ) for 24 h. The mRNA expressions of Chop ( a , b ), Bip ( c , d ), and Xbp1s ( e , f ) were analyzed by RT-qPCR and normalized by Gapdh . Results are the means ± SEM of the 6 to 8 independent experiments. ** p ≤ 0.01 and *** p ≤ 0.001 vs. untreated (NT) and infected with the same adenoviral vector.

Article Snippet: The following concentrations were used: recombinant human IL-1β (R&D Systems, Cat. No. 201-LB/CF, Abingdon, UK) at 10 or 50 U/mL for INS-1E and EndoC-βH1 cells, respectively; recombinant human IFNγ (PeproTech, Cat. No. 300-02-250UG, Cranbury, NJ, USA) at 1000 U/mL for EndoC-βH1 cells; and recombinant rat IFNγ (R&D Systems, Cat. No. 585-IF) at 100 U/mL for INS-1E cells.

Techniques: Over Expression, Infection, Luciferase, Quantitative RT-PCR, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: BCL-XL Overexpression Protects Pancreatic β-Cells against Cytokine- and Palmitate-Induced Apoptosis

doi: 10.3390/ijms24065657

Figure Lengend Snippet: BCL-XL overexpression alleviates ER stress upon inflammatory or metabolic insults in human β-cells. EndoC-βH1 cells were infected with adenoviral (ad) vectors encoding luciferase (adLUC, blue dots) or rat BCL-XL (adBCL-XL, red dots). After 48 h of recovery, cells were left untreated (NT) or treated with either IFNγ + IL-1β (50 and 1000 U/mL, respectively) ( a , c , e ) or 0.5 mM palmitate ( b , d , f ) for 48 h. The mRNA expressions of CHOP ( a , b ), BIP ( c , d ), and XBP1s ( e , f ) were analyzed by RT-qPCR and normalized by β-actin. Results are the means ± SEM of the 7 to 8 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 vs. untreated (NT) and infected with the same adenoviral vector; # p ≤ 0.05 and ### p ≤ 0.001, as indicated by bars. Two-way ANOVA.

Article Snippet: The following concentrations were used: recombinant human IL-1β (R&D Systems, Cat. No. 201-LB/CF, Abingdon, UK) at 10 or 50 U/mL for INS-1E and EndoC-βH1 cells, respectively; recombinant human IFNγ (PeproTech, Cat. No. 300-02-250UG, Cranbury, NJ, USA) at 1000 U/mL for EndoC-βH1 cells; and recombinant rat IFNγ (R&D Systems, Cat. No. 585-IF) at 100 U/mL for INS-1E cells.

Techniques: Over Expression, Infection, Luciferase, Quantitative RT-PCR, Plasmid Preparation

Journal: International journal of molecular sciences

Article Title: The Comparison of Immunomodulatory Properties of Canine and Human Wharton Jelly-Derived Mesenchymal Stromal Cells.

doi: 10.3390/ijms25168926

Figure Lengend Snippet: Figure 6. The effect of pro-inflammatory stimulation on the synthesis of IDO1 in human and canine MSC(WJ)s. Western blot. (A,B) Representative blots presenting IDO1 protein expression in human (A) and canine (B) MSC(WJ)s treated for 24 h with different combinations of IFNγ, TNF, and poly(I:C). Yellow arrows indicate treatments chosen for extended analysis. (C–G) Representative blots presenting the effect of chosen treatments: IFNγ and IFNγ + poly(I:C) in human (C) and canine (D) MSC(WJ)s. (E,F) Mean (±SEM) optical density of IDO-1 normalized to β-actin (n = 10) in hMSC(WJ)s (E) and cMSC(WJ)s (F); 3 independent experiments, cells from 6 different donors in each species, n = 10, analyzed using Wilcoxon test, **, p < 0.01. (G) the comparison of the effect of both treatments on hMSC(WJ)s and cMSC(WJ)s. Mean (±SEM) fold change after treatment in comparison to untreated cells. Mann–Whitney U test, ns—statistically non-significant, *, p < 0.05.

Article Snippet: IFNγ and TNF were used in a species-specific manner—human recombinant IFNγ (R&D System, Minneapolis, MN, USA, Cat#: 285-IF) and human recombinant TNF (Peprotech, Cheshire UK, Cat#: 300-01A) were added to hMSC(WJ)s, whereas canine recombinant IFNγ (R&D System, Cat#: 781-CG) and canine recombinant TNF (R&D System, Cat#: 1507-CT) were added to cMSC(WJ)s; all at a concentration of 20 ng/mL.

Techniques: Western Blot, Expressing, Comparison, MANN-WHITNEY

Journal: International journal of molecular sciences

Article Title: The Comparison of Immunomodulatory Properties of Canine and Human Wharton Jelly-Derived Mesenchymal Stromal Cells.

doi: 10.3390/ijms25168926

Figure Lengend Snippet: Figure 7. Transforming growth factor beta 1 (TGFβ1) secretion by MSC(WJ)s. ELISA. Data blanked to medium alone. (A) basal secretion of TGFβ1 by unstimulated human and canine MSC(WJ)s. (B,C) the effect of pro-inflammatory stimulation on TGFβ1 secretion by human (B) and canine (C) cells. (D) comparison of response to recombinant (species-matched) IFNγ (20 ng/mL) on TGFβ1 secretion between human and canine MSC(WJ)s. The graph presents mean delta values (TGFβ1IFNγ— TGFβ1CTRL). ns—statistically not significant, *, p < 0.05; n = 6 for each cell type, assay performed in duplicates.

Article Snippet: IFNγ and TNF were used in a species-specific manner—human recombinant IFNγ (R&D System, Minneapolis, MN, USA, Cat#: 285-IF) and human recombinant TNF (Peprotech, Cheshire UK, Cat#: 300-01A) were added to hMSC(WJ)s, whereas canine recombinant IFNγ (R&D System, Cat#: 781-CG) and canine recombinant TNF (R&D System, Cat#: 1507-CT) were added to cMSC(WJ)s; all at a concentration of 20 ng/mL.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Recombinant