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Figure Lengend Snippet: Alternate protein profiles in the spinal cord induced by OEC transplantation. (A) The study design for protein array analysis. (B) Principal component analysis among the OEC group, vehicle group, and control group. (C) Heat map presenting differentially expressed proteins in the OEC group, vehicle group, and control group. (D) Antibody array profiles among the three groups. IL-1α, IL-1β, IL-1Ra, Notch-1, and Notch-2 were detected with Raybio cytokine antibody array-3 and cytokine antibody array-4. Their location in the array is labeled by white underscores. (E) ELISA analysis of IL-1α, IL-1β, IL-1Ra, Notch-1, and Notch-2. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-GAPDH (1:1000, ab9485, Abcam) and rat anti-IL-1Ra (1:100, ab124962, Abcam).

Techniques: Transplantation Assay, Protein Array, Ab Array, Labeling, Enzyme-linked Immunosorbent Assay

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Figure Lengend Snippet: Expression of IL-1Ra in the spinal cord after OEC transplantation. (A) IL-1Ra immunostaining in the OEC group, vehicle group, and control group 7 days after transplantation. (B) Quantification of IL-1Ra expression (integrated optical density, IOD). * p < 0.05, **** p < 0.0001; n = 5.

Article Snippet: The membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-GAPDH (1:1000, ab9485, Abcam) and rat anti-IL-1Ra (1:100, ab124962, Abcam).

Techniques: Expressing, Transplantation Assay, Immunostaining

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Figure Lengend Snippet: Secretion of IL-1Ra by OECs under inflammatory stimulation. The expression of IL-1Ra secreted by OECs under different concentrations and exposure time to lipopolysaccharide (LPS). **** p < 0.0001, n = 5.

Article Snippet: The membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-GAPDH (1:1000, ab9485, Abcam) and rat anti-IL-1Ra (1:100, ab124962, Abcam).

Techniques: Expressing

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Figure Lengend Snippet: Co-culture with activated microglia increases the secretion of IL-1Ra from OECs. (A) Western blot analysis of IL-1Ra expression in the supernatant after co-culture with HAPI microglia. (B) IL-1Ra expression in the co-culture supernatant. **** p < 0.0001, n = 5.

Article Snippet: The membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-GAPDH (1:1000, ab9485, Abcam) and rat anti-IL-1Ra (1:100, ab124962, Abcam).

Techniques: Co-Culture Assay, Western Blot, Expressing

Journal: Gene therapy

Article Title: Anti-inflammatory effect by lentiviral-mediated overexpression of IL-10 or IL-1 receptor antagonist in rat glial cells and macrophages.

doi: 10.1038/gt.2010.8

Figure Lengend Snippet: Figure 1 Time-dependent cytokine production by LV-transduced rat cells. Rat IL-10 and rat IL-1ra were amplified using complementary DNA prepared from rat liver and cloned into the pRRLsin transfer plasmids (pRRLsin-PPThCMV-wpre). LVs were produced by transfection of the transfer plasmids (pRRLsin-PPThCMV-transgene-wpre) in combination with the viral core packaging construct pCMVdeltaR8.74 (pPack) and the vesicular stomatitis virus G (VSV-G) envelope protein vector pMD2.G (pEnv). The production of LV-GFP was described before.72 Determination of transducing units (TU) was performed on human embryonic kidney (HEK) 293T cells transducing them with 10-fold dilution series of viral vectors ranging from 1 103 to 1 106. At 48 h after transduction, the cells were immunocytochemically stained with antibodies corresponding to the gene that was overexpressed, that is, GFP (rabbit ant-GFP, Chemicon, Hofheim, Germany, 1:500), rIL-10 (goat anti-rat IL10, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:100) and rIL-1ra (sheep anti-rat IL-1ra, NIBSC, Potters Bar, UK, 1:1000). The number of transduced cells was counted in several fields throughout with a 10 objective (visible field is 2 mm2) and TU was determined using the following formula: TU ml–1 ¼ mean cells/field dilution factor 100 volume (ml). Titers were usually around 109 TU ml–1. Subsequently, NR8383 and C6 cells (1 104) were seeded in 24-well plates with 0.5 ml of the appropriate culture media 1 day before transduction. Infectious particles were added to the cells using 0.1 ml virus, corresponding to 1 105 TU as determined by virus titration. The media were harvested at day 4, 7 and 10 after transduction for cytokine measurement. Cytokine production at each time-point was measured in triplicate. All data were corrected for the time-dependent total cellular protein content. Rat IL-10 (a), and IL-1ra (b) concentrations were measured in cell culture supernatants by rat-specific enzyme-linked immunosorbent assays (NIBSC).73 Data are presented from two separate experiments as mean±s.e.m. (n ¼ 6) and are expressed as pg ml–1. *Po0.05, **Po0.001 vs non-transduced cells. Abbreviation: Vd, virally derived.

Article Snippet: The sections were incubated with a rabbit anti-GFP antibody (final dilution 1/1000, Chemicon), a sheep anti-rat IL-1ra antibody (S373/B1, 1.5 mg ml–1, final dilution 1/1000, NIBSC) or a goat anti-rat IL-10 antibody (final dilution 1/100, Santa Cruz Biotechnology Inc.) using the biotin–avidin–peroxidase method, as previously described.15 All the sections were analyzed with light microscopy (Vanox, Olympus, Tokyo, Japan) and the sections shown are representative of those obtained in the proximity of each injected site or the contralateral side in all animals in the experimental group.

Techniques: Clone Assay, Produced, Transfection, Construct, Virus, Plasmid Preparation, Transduction, Staining, Titration, Cell Culture, Derivative Assay

Journal: Gene therapy

Article Title: Anti-inflammatory effect by lentiviral-mediated overexpression of IL-10 or IL-1 receptor antagonist in rat glial cells and macrophages.

doi: 10.1038/gt.2010.8

Figure Lengend Snippet: Figure 2 Biological activity of LV-rIL-10 and LV-rIL-1ra in vitro. The biological activity of the anti-inflammatory cytokines produced from the different viral vectors was tested in vitro using LPS-stimulated NR8383 (for rIL-10) and rIL-1b/forskolin-stimulated C6 (for rIL-1ra) cells. NR8383 or C6 cells (1 105 cells per well seeded in triplo in a 12-wells plate) were transduced with LV-rIL-10 or LV-GFP (NR8383) 3, 6 and 9 days before treatment with LPS (100 ng ml–1, Escherichia coli serotype 0128-B12; Sigma, St Louis, MO, USA) or with LV-rIL-1ra or LV-GFP (C6) 3, 6 and 9 days before treatment with rIL-1b (Glaxo Imb, Geneva, Switzerland, 0.1 ng ml–1)/forskolin (Sigma-Aldrich, St Louis, MO, USA, 5 mM). At 24 h after stimulation, the media were collected and the protein levels of (a) TNFa (NR8383) or (b) IL-6 (C6) were measured by specific enzyme-linked immunosorbent assays.73–75 As control, non-transduced cells were cultured and treated with the same stimuli. Data are presented as mean±s.e.m. (n ¼ 6) and are expressed as % compared with non-transduced cells. *Po0.05, **Po0.001 vs non-transduced cells.

Article Snippet: The sections were incubated with a rabbit anti-GFP antibody (final dilution 1/1000, Chemicon), a sheep anti-rat IL-1ra antibody (S373/B1, 1.5 mg ml–1, final dilution 1/1000, NIBSC) or a goat anti-rat IL-10 antibody (final dilution 1/100, Santa Cruz Biotechnology Inc.) using the biotin–avidin–peroxidase method, as previously described.15 All the sections were analyzed with light microscopy (Vanox, Olympus, Tokyo, Japan) and the sections shown are representative of those obtained in the proximity of each injected site or the contralateral side in all animals in the experimental group.

Techniques: Activity Assay, In Vitro, Produced, Transduction, Control, Cell Culture

Journal: Gene therapy

Article Title: Anti-inflammatory effect by lentiviral-mediated overexpression of IL-10 or IL-1 receptor antagonist in rat glial cells and macrophages.

doi: 10.1038/gt.2010.8

Figure Lengend Snippet: Figure 5 Effect of central administration of LV-rIL-10 and LV-rIL-1ra on protein expression levels of TNFa or IL-6 respectively. To determine cytokine protein levels, the dissected brain regions were homogenized in lysis buffer containing 10 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 100 mM phenylmethylsulphonyl fluoride, 10 mg ml–1 leupeptin, 10 mg ml–1 pepstatin and 10 mg ml–1 aprotinin. Homogenates were cleared by centrifugation (14 000 revolutions per minute for 30 min at 4 1C). (a) TNFa protein expression levels were measured by enzyme-linked immunosorbent assay (ELISA) in cortex and hippocampus of animals infected with LV-GFP or LV-rIL-10. Animals were injected with LPS at 5 days after infection with LV-rIL-10 and killed 4 h later. (b) IL-6 protein expression levels were measured by ELISA in cortex and hippocampus of animals infected with LV-GFP and LV-rIL-1ra. Animals were injected 5 days after infection with IL-1b/forskolin and killed 4 h later. Data are presented as mean±s.e.m. (n ¼ 6). *Po0.05 vs LV-GFP.

Article Snippet: The sections were incubated with a rabbit anti-GFP antibody (final dilution 1/1000, Chemicon), a sheep anti-rat IL-1ra antibody (S373/B1, 1.5 mg ml–1, final dilution 1/1000, NIBSC) or a goat anti-rat IL-10 antibody (final dilution 1/100, Santa Cruz Biotechnology Inc.) using the biotin–avidin–peroxidase method, as previously described.15 All the sections were analyzed with light microscopy (Vanox, Olympus, Tokyo, Japan) and the sections shown are representative of those obtained in the proximity of each injected site or the contralateral side in all animals in the experimental group.

Techniques: Expressing, Lysis, Centrifugation, Enzyme-linked Immunosorbent Assay, Infection, Injection

Journal: Gene therapy

Article Title: Anti-inflammatory effect by lentiviral-mediated overexpression of IL-10 or IL-1 receptor antagonist in rat glial cells and macrophages.

doi: 10.1038/gt.2010.8

Figure Lengend Snippet: Figure 6 Effect of central administration of LV-rIL-10 or LV-rIL-1ra on clinical scores of chronic-relapsing experimental autoimmune encephalomyelitis (cr-EAE). Chronic-relapsing experimental auto- immune encephalomyelitis (cr-EAE) was induced in adult male Dark Agouti (DA) rats (Harlan CBP), weighing 230–250 g. The rats were anaesthetized with ketamin (Kombivet, 50 mg kg–1, intramus- cular (i.m.)) and xyzaline (Rompun, Bayer, 5 mg kg–1, intraperito- neal (i.p.)) and immunized intradermally in the dorsal tail base with 75 mg of recombinant rat myelin oligodendrocyte glycoprotein (rrMOG1–125) emulsified in incomplete Freund’s adjuvant (IFA; Difco, Detroid, MI, USA) together with 10 mM NaAc (pH 3.0). At the same time, a permanent 22-gauge guide cannula with an indwelling dummy cannula (Plastics One) was implanted into the right lateral ventricle of the brain at stereotaxic coordinates: 0.6 mm posterior to the bregma, 1.5 mm lateral and 3.7 mm under the brain surface. Rats were weighed and examined daily for neurological symptoms of EAE that were scored on the following scale: 0, no clinical disease; 0.5, partial loss of tail tone; 1, complete tail atony; 2, paresis, partial hind limb paralysis; 3, complete paralysis of the hind limbs and/or lower part of the body; 4, moribund or dead because of EAE. All experimental procedures were approved by the animal experiment committee of the VU University Medical Center. Rats were given LV-GFP (n ¼ 3), LV-rIL-1ra (n ¼ 3) or LV-IL-10 (n ¼ 3) (5 ml corresponding to approximately 5 106 TU) during remission of cr-EAE (arrow, day 16) via continuous i.c.v. infusion into freely moving rats at a flow rate of 5 ml min–1 using a microsyringe infusion pump (Harvard Apparatus Inc.). Clinical scores of cr-EAE were measured of rats treated with (a) LV-GFP, (b) LV-rIL-10 or (c) LV-rIL-1ra. Data are expressed as mean+s.e.m.

Article Snippet: The sections were incubated with a rabbit anti-GFP antibody (final dilution 1/1000, Chemicon), a sheep anti-rat IL-1ra antibody (S373/B1, 1.5 mg ml–1, final dilution 1/1000, NIBSC) or a goat anti-rat IL-10 antibody (final dilution 1/100, Santa Cruz Biotechnology Inc.) using the biotin–avidin–peroxidase method, as previously described.15 All the sections were analyzed with light microscopy (Vanox, Olympus, Tokyo, Japan) and the sections shown are representative of those obtained in the proximity of each injected site or the contralateral side in all animals in the experimental group.

Techniques: Recombinant, Adjuvant