Journal: Kidney Diseases

Article Title: Treatment of Membranous Nephropathy by Disulfiram through Inhibition of Podocyte Pyroptosis

doi: 10.1159/000524164

Figure Lengend Snippet: DSF alleviated C3a/C5a-induced podocyte injury by inhibiting pyroptosis. Podocyte injury was induced with C3a (50 nM) and C5a (50 nM). The inhibitory effects of DSF (250 nM) on C3a/C5a-induced pyroptosis were examined. a Representative images of GSDMD(N)/ZO-1/Nucleus (DAPI) triple immunofluorescent staining of treated podocytes. Scale bars = 20 μm. b Representative Western Blot images of GSDMD, NF-κB p65, p-NF-κB p65 (Ser536), NLRP3, ASC, Caspase-1, IL-18 and the internal control (GAPDH) in treated podocytes. c IL-18 release in treated podocytes was detected. d, e Representative images of PI/Nucleus (DAPI) double fluorescent staining of treated podocytes ( d ) and percentage of PI-positive cells ( e ); arrows, PI-positive cells; scale bars = 40 μm. f LDH release in treated podocytes was detected. The data above represent three independent experiments in duplicate and are shown as the mean ± SD, and ANOVA with LSD-t test (equal variances assumed) or Welch's test with Dunnett's T3 test (equal variances not assumed) was used for multiple comparisons among groups. *, p < 0.05; **, p < 0.01.

Article Snippet: IL-18 release in the culture supernatant of the podocytes and the serum IL-1β/IL-18 levels of rats were tested using human IL-18 ELISA and rat IL-1β/IL-18 ELISA kits (Boster Biological Technology Co. Ltd., Wuhan, China), respectively.

Techniques: Staining, Western Blot

Journal: Kidney Diseases

Article Title: Treatment of Membranous Nephropathy by Disulfiram through Inhibition of Podocyte Pyroptosis

doi: 10.1159/000524164

Figure Lengend Snippet: DSF inhibited the renal pyroptosis signaling pathway in PHN rats. a Representative renal GSDMD(N)/Synaptopodin and GSDMD(N)/ZO-1 double immunofluorescent staining of the rats ( n = 6) in each group; Scale bars = 20 μm. b, c Representative renal immunohistochemical staining ( b ) and semiquantification based on the glomerular IOD/area of GSDMD(N), NF-κB p65, p-NF-κB p65 (Ser536), NLRP3, ASC, Caspase-1, IL-1β, and IL-18 ( c ) of the rats ( n = 6) in each group; Scale bars = 20 μm. d Relative mRNA levels of glomerular GSDMD, NLRP3, ASC, Caspase-1, IL-1β and IL-18 of the rats ( n = 6) in each group. e Representative Western Blot images of renal GSDMD, NF-κB p65, p-NF-κB p65 (Ser536), nuclear NF-κB p65, nuclear p-NF-κB p65 (Ser536), NLRP3, ASC, Caspase-1, Caspase-1 p20, IL-1β, IL-1β (mature form), IL-18 and the internal control (GAPDH, Histone H3) of the rats ( n = 6) in each group. Serum IL-1β ( f ) and IL-18 ( g ) of the rats ( n = 6) in each group on days 1, 5, 8, 15 after model establishment. The data above are shown as the mean ± SD ( c, d, f, g ) and were compared to the PHN group ( f, g ). ANOVA with LSD-t test (equal variances assumed) or Welch's test with Dunnett's T3 test (equal variances not assumed) was used for multiple comparisons among groups. *, p < 0.05; **, p < 0.01.

Article Snippet: IL-18 release in the culture supernatant of the podocytes and the serum IL-1β/IL-18 levels of rats were tested using human IL-18 ELISA and rat IL-1β/IL-18 ELISA kits (Boster Biological Technology Co. Ltd., Wuhan, China), respectively.

Techniques: Staining, Immunohistochemical staining, Western Blot

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 1. Antitumor effect of intratumoral IFN-α gene transfer. (a) Growth of tumors injected with Ad-mIFN. Tumor volumes were measured at indicated days following the intratumoral injection of Ad-mIFN (n = 6) or Ad-AP (n = 8). Relative tumor volumes compared with those at day10 were presented. Data are shown as means ± standard deviation (s.d.). (b) ELISpot assay of IFN-γ-producing cells in response to stimulation of CT26 cells. Twenty-two days after tumor inoculation, splenocytes were isolated from mice injected with Ad-mIFN (n = 4) or Ad-AP (n = 3), and were cultured with CT26 or syngeneic lymphocytes. Data are presented as means ± s.d. (c) Intracellular cytokine staining of IFN-γ-producing cells in response to CT26 cells. The splenocytes from mice injected with Ad-mIFN (n = 4) or Ad-AP (n = 3) at day 22 were incubated with CT26 cells and stained by anti-mouse IFN-γ antibody. The activated cell fractions were analyzed by staining with anti-mouse CD8 antibody. Representative FACS plots (right panel) are shown.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Standard Deviation, Enzyme-linked Immunospot, Isolation, Cell Culture, Staining, Incubation

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 2. Intratumoral IFN-α gene transfer reduced the frequency of Tregs in tumors. (a) Frequency of CD4+Foxp3+ Tregs per CD4+ T cells in tumors. Tumors injected with viruses were harvested at days 16, 22 and 28, and processed into single-cell suspension. The percentage of CD4+

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Suspension

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 3. Intratumoral IL-6 concentration was significantly increased by IFN-α gene transfer. (a) IL-6 concentration in tumors. Tumors injected with Ad-mIFN (n = 5) or Ad-AP (n = 5) were harvested at days 16, 22 and 28, and IL-6 concentration was measured by ELISA. (b) Relationship between IL-6 and IFN-α concentration in tumors. Tumors injected with Ad-mIFN (n = 5) or Ad-AP (n = 5) were harvested at day 16, and the concentrations of IL-6 and IFN-α were compared by ELISA. (c) IL-6 production from tumor CD11c+ cells. The CD11c+ and CD11c −cells were isolated from tumors injected with Ad-mIFN (n = 2) or Ad-AP (n = 2) at day 16, and 5 × 104 cells were plated in 96-well plates. After 48 h, supernatants were assayed for the measurement of IL-6 concentration by ELISA. (d) IL-6 production from splenic CD11c+ cells in response to a recombinant IFN-α protein. The CD11c+ and CD11c−cells isolated from naïve splenocytes, and 5 × 104 of CT26 cells were cultured in 96-well plates with depicted concentration of recombinant mouse IFN-α (Miltenyi Biotech). After 48 h, supernatants were assayed for the measurement of IL-6 concentration by ELISA (n = 2 for each group).

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Concentration Assay, Injection, Enzyme-linked Immunosorbent Assay, Isolation, Recombinant, Cell Culture

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 4. IL-6 receptor blockade suppressed IFN-α-mediated Treg reduction in tumors. (a) Schema of experiment. The 1000 μg of the monoclonal anti-IL-6 receptor antibody was intraperitoneally injected into the mice at days 7, 14 and 21 after tumor inoculation. Ad-mIFN or Ad-AP was injected once at day 10 after inoculation. (b) Frequency of CD4+Foxp3+ cells per CD4+ T cells in tumors treated with IL-6R ab. Tumors were harvested at day 22, and CD4+ T cells and CD4+Foxp3+ Tregs were analyzed by flow cytometry (n = 5 for the group of Ad-AP i.t. +IL-6R ab i.p., n = 4 for the other groups). (c) Ratio of CD8+ T cells to CD4+Foxp3+ Tregs in tumors. Frequency of CD8+ T cells within whole tumor cells (left panel). Frequency of CD4+Foxp3+ Tregs within whole tumor cells (middle panel). The number of CD8+ T cells was compared with that of CD4+Foxp3+ Tregs in tumors at day 16 (right panel) (n = 4 for the group of Ad-mIFN i.t.+IL-6R ab i.p., n = 6 for the other groups). IL-6R ab, anti-IL-6 receptor antibody; i.t., intratumoral injection; i.p., intraperitoneal administration; TDLNs, tumor-draining lymph nodes.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Cytometry

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 5. IL-6 receptor blockade partially attenuated IFN-α-mediated tumor growth suppression. (a) Growth of tumors treated with IL-6R ab. Tumor volumes in mice treated with the viruses and/or IL-6R ab were measured at the indicated days (n = 5 for the group of Ad-AP i.t.+IL-6R ab i.p., n = 6 for the other groups). Relative tumor volumes compared with those at day 10 were presented. (b) ELISpot assay of IFN-γ- producing cells in mice treated with IL-6R ab. The splenocytes were isolated from mice as shown in Figure 4a at day 28, and the cells were cultured with CT26 or syngeneic splenocytes (n = 5 for the group of Ad-AP i.t.+IL-6R ab i.p., n = 6 for the other groups).

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Enzyme-linked Immunospot, Isolation, Cell Culture

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 6. Intratumoral IFN-α expression increased the number of Th17 cells in tumors. (a) Expression of RORγt and Foxp3 genes in tumors. The tumors injected with viruses were harvested at day 16 and were subjected to real-time PCR analysis (Ad-mIFN: n = 4, Ad-AP: n = 3). (b) Expression of IL-17 in tumors. The tumors were harvested at day 28, and subjected to RT-PCR for IL-17 expression (n = 3 for the group of Ad-AP i.t.+PBS i.p., n = 2 for the group of Ad-mIFN i.t.+PBS i.p., n = 3 for the group of Ad-mIFN i.t.+IL-6R ab i.p.). (c) Intracellular cytokine staining of IL-17A in CD4+ T cells. The tumors and tumor-draining lymph nodes were harvested at day 22, and IL-17A expressions were analyzed by flow cytometry (n = 3 for the group of Ad-AP i.t.+PBS i.p., n = 4 for the group of Ad-mIFN i.t.+PBS i.p., n = 4 for the group of Ad-AmIFN i.t.+IL-6R ab i.p.) (upper panel). Representative FACS plots of tumors (lower left panel) and tumor-draining lymph nodes (lower right panel) were shown.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Staining, Cytometry

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