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

Article Title: Il-12 antagonism enhances apoptotic death of T cells within hepatic allografts from Flt3 ligand-treated donors and promotes graft acceptance.

doi: 10.4049/jimmunol.166.9.5619

Figure Lengend Snippet: FIGURE 1. A, Enhanced expression of immunoreactive IL-12 (red) in both the graft and the spleen of recipients of murine liver allografts from FL-treated donors was completely reversed by host treatment with anti-IL-12 mAb. Tissues were removed from C3H mice 4 days after transplantation of B10 livers from normal control, or FL-treated donors, with or without anti-IL-12 treatment (500 mg i.v., day 0 and day 2). Controls received hamster IgG instead of anti-IL-12 mAb. Cryostat sections were stained with specific goat anti-mouse IL-12 p40 Ab, using the ABC method. Goat IgG was used as isotype control. Magnification, 3200. B, Two-color immunohistochemical staining for CD11c (DC; brown) and IL-12 p40 (blue) in liver from an FL-treated donor was performed as described in Materials and Methods. Left, numerous CD11c1 cells in portal areas (right; detail) also stain for IL-12 (and consequently appear purple); other interstitial leukocytes (CD11c2) also appear IL-121. Left, magnification, 3200; right, (detail) magnification, 31000. The results are representative of three experiments, with three animals in each experimental group.

Article Snippet: Endogenous peroxidase activity was quenched in 2% H2O2 before addition of goat anti-mouse IL-12 (p40) Ab (1:200; 1 h at room temperature (RT); Santa Cruz Biotechnology, Santa Cruz, CA) followed by donkey anti-goat IgG (1:400; 30 min, RT).

Techniques: Expressing, Transplantation Assay, Control, Staining, Immunohistochemical staining

Journal:

Article Title: Interleukin-18 (IL-18) Enhances Innate IL-12-Mediated Resistance to Toxoplasma gondii

doi:

Figure Lengend Snippet: Expression of IL-18 during toxoplasmosis. (A) Total RNA from the spleens of uninfected SCID mice or SCID mice infected for 7 days was extracted, and RPA analysis was performed as described in Materials and Methods. Similar results were observed in two experiments. (B) Densitometric analysis of RNA levels and expression relative to the housekeeping L32 gene. The results shown are the means ± the SD of the data presented. (C) Levels of IL-18 and IL-12 were measured by using ELISA in the serum from uninfected mice or mice infected for 7 days. The data shown are the means ± the SD from four independent experiments with three to six mice per group.

Article Snippet: Cultures were stimulated with cytokines and/or RH strain tachyzoite lysate antigen (TLA) or cytokine(s) with or without antibody for 24 h. For the depletion of cytokines in the cultures, 30 μg of anti-IL-12 (C17.8), 20 μg of rat anti-IL-18 MAb (R&D Systems, Inc.), or 30 μg of rabbit anti-IL-18 polyclonal antibody per ml was added to the culture.

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Interleukin-18 (IL-18) Enhances Innate IL-12-Mediated Resistance to Toxoplasma gondii

doi:

Figure Lengend Snippet: IL-18 induced production of IFN-γ by splenocytes from infected SCID mice is dependent on IL-12. Splenocytes from SCID mice infected for 7 days were stimulated with 30 μg of TLA, 10 ng of IL-12, or 10 ng of IL-18 per ml in combination with 30 μg of rat IgG or anti-IL-12 (C17.8) per ml for 24 h, and the production of IFN-γ in the supernatants was measured by ELISA. The data shown are the means ± the SD from a single experiment done in triplicate. Similar results were observed in two additional experiments.

Article Snippet: Cultures were stimulated with cytokines and/or RH strain tachyzoite lysate antigen (TLA) or cytokine(s) with or without antibody for 24 h. For the depletion of cytokines in the cultures, 30 μg of anti-IL-12 (C17.8), 20 μg of rat anti-IL-18 MAb (R&D Systems, Inc.), or 30 μg of rabbit anti-IL-18 polyclonal antibody per ml was added to the culture.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Interleukin-18 (IL-18) Enhances Innate IL-12-Mediated Resistance to Toxoplasma gondii

doi:

Figure Lengend Snippet: IL-18-mediated resistance is dependent on endogenous IL-12. (A) SCID mice were infected with T. gondii and treated with IL-18 or PBS alone or in combination with anti-IL-12 or rat IgG. The treatment with anti-IL-12 resulted in complete abrogation of serum levels of IL-12p40 on days 3, 5, and 7 postinfection. The serum levels of IFN-γ on day 5 postinfection were measured by ELISA (A), and the percentage of infected PECs on day 7 postinfection calculated (B) as described in Materials and Methods. The data shown are the means ± the SD of the pooled data from three experiments with three to five mice per group. (C) Effect of depletion of endogenous IL-12 on the survival of SCID mice treated with IL-18. Similar results were observed in a repeat experiment with four mice per group.

Article Snippet: Cultures were stimulated with cytokines and/or RH strain tachyzoite lysate antigen (TLA) or cytokine(s) with or without antibody for 24 h. For the depletion of cytokines in the cultures, 30 μg of anti-IL-12 (C17.8), 20 μg of rat anti-IL-18 MAb (R&D Systems, Inc.), or 30 μg of rabbit anti-IL-18 polyclonal antibody per ml was added to the culture.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Journal of gastroenterology and hepatology

Article Title: Evidence for the critical role of interleukin-12 but not interferon-gamma in the pathogenesis of experimental colitis in mice.

doi: 10.1046/j.1440-1746.2003.03024.x

Figure Lengend Snippet: Figure 3 Wasting disease in C57BL/6 wild-type and IL-12 p40–/– mice given a 2,4,6-trinitrobenzene sulfonic acid (TNBS) enema. (a) C57BL/6 wild-type mice were treated with TNBS after intraperitoneal administration of rat anti- mouse IL-12 mAb (), or rat IgG as a control (❍); (b) IL-12 p40–/– mice were treated with TNBS in 50% ethanol () or 50% ethanol alone (). Change in body weight over a 7–8 day period of observation was expressed as a percent of the orig- inal weight at the start of the experiment. Six to nine mice were used for each group. The data shown represent average values. The body weights of mice treated with anti-IL-12 mAb were significantly higher than those of mice treated with rat IgG on day 3, 5, and 7 (indicated by *; P < 0.01). In IL-12 p40–/– mice, no significant difference in weight was observed on day 3, 5, and 7.

Article Snippet: Sections were then incubated with biotin-conjugated rat anti-mouse CD4 mAb, GK1.5 (PharMingen, SanDiego, CA, USA) or goat anti-mouse IL-12 p40 polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 ∞ C overnight.

Techniques: Control

Journal: Journal of gastroenterology and hepatology

Article Title: Evidence for the critical role of interleukin-12 but not interferon-gamma in the pathogenesis of experimental colitis in mice.

doi: 10.1046/j.1440-1746.2003.03024.x

Figure Lengend Snippet: Figure 4 Histological evaluation of the colons in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-administered C57BL/6 wild-type, IL-12 p40–/–, and wild-type mice pretreated with anti-IL-12 p40 mAb. Hematoxylin/eosin-stained colon cross-sections from TNBS-administered anti-IL-12 p40 mAb-pretreated C57BL/6 wild-type (a) wild-type; (b) IL-12 p40–/–; (c) mice. The magnitude of inflammatory changes in the colons was analyzed 7 or 8 days after TNBS administration. Original magnification was 50 ¥. Dif- ferences in inflammatory activity were confirmed by histological grading of the colon sections. Colon specimens in C57BL/6 wild- type or IL-12 p40–/– mice treated with TNBS or 50% ethanol alone were taken on day 8. Each filled circle indicates an individual mouse. Data were pooled from two independent experiments.

Article Snippet: Sections were then incubated with biotin-conjugated rat anti-mouse CD4 mAb, GK1.5 (PharMingen, SanDiego, CA, USA) or goat anti-mouse IL-12 p40 polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 ∞ C overnight.

Techniques: Staining, Activity Assay

Journal: Journal of gastroenterology and hepatology

Article Title: Evidence for the critical role of interleukin-12 but not interferon-gamma in the pathogenesis of experimental colitis in mice.

doi: 10.1046/j.1440-1746.2003.03024.x

Figure Lengend Snippet: Figure 6 Detection of IL-12 in the colonic tissues of 2,4,6- trinitrobenzene sulfonic acid (TNBS)-administered C57BL/6 wild-type or IL-12 p40–/– mice. Expression of IL-12 was exam- ined in the colonic tissues of TNBS-administered C57BL/6 wild-type (a) IL-12 p40–/–; (b) mice by immunohistochemistry with anti-IL-12 mAb. Original magnification ¥ 400.

Article Snippet: Sections were then incubated with biotin-conjugated rat anti-mouse CD4 mAb, GK1.5 (PharMingen, SanDiego, CA, USA) or goat anti-mouse IL-12 p40 polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 ∞ C overnight.

Techniques: Expressing, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Diacylglycerol kinase ζ regulates microbial recognition and host resistance to Toxoplasma gondii

doi: 10.1084/jem.20061856

Figure Lengend Snippet: Impairment of proinflammatory cytokine production in DGKζ −/− DCs and BMMφ. (A–D) Measurement of cytokine production in DCs. Splenic DCs from WT and DGKζ −/− mice were left unstimulated or stimulated with 30 or 100 ng/ml LPS or 30 or 100 ng/ml STAg at 37°C for 24 h. IL-12 p40 (A and B) and TNFα (C and D) levels in culture supernatants were determined by ELISA. (E–J) Measurement of cytokine production by BMMφ. BMMφ from WT and DGKζ −/− mice were left unstimulated or stimulated with 100 ng/ml LPS (E and F), 50 ng/ml Pam3CSK4 (G and H), or 50 μg/ml Poly(I:C) (I and J) for 24 h. IL-12 p40 (E, G, and I) and TNFα (F, H, and J) levels in supernatants were detected by ELISA. (K) Cell surface expression of CD11b, TLR4, and other molecules on WT and DGKζ −/− BMMφ. WT and DGKζ −/− BMMφ were stained with a control antibody (dotted green line) or a biotin-labeled anti-TLR4 antibody, followed by PE-conjugated streptavidin staining or fluorescently labeled anti-CD11b, F4/80, CD80, CD86, or FcγR antibodies (WT, blue shaded region; DGKζ −/− , red line). Data shown in A–J are the mean ± SD of each group and represent three experiments.

Article Snippet: Spleens were removed from mice before or 6 h after STAg or LPS stimulation, and frozen sections were processed and stained with a goat anti–mouse IL-12 p40 (BD Biosciences), followed by a double incubation with Alexa Fluor 488–conjugated anti–mouse CD11c and Alexa Fluor 594–conjugated anti–goat antibodies (Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Staining, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Diacylglycerol kinase ζ regulates microbial recognition and host resistance to Toxoplasma gondii

doi: 10.1084/jem.20061856

Figure Lengend Snippet: Impaired TNFα and IL-12 production in DGKζ-deficient mice after TLR-stimulation. (A and B) Measurement of serum cytokine concentrations by ELISA. WT and DGKζ −/− mice were intraperitoneally injected with 10 μg LPS or 5 μg STAg. 6 h after injection, sera were collected for measurement of IL-12 p40 and TNFα production by ELISA. Data shown are the mean ± SD for each group and are representative of three experiments. (C and D) Measurement of cytokine messengers by Northern blot. WT and DGKζ −/− mice were intraperitoneally injected with 10 μg LPS or 5 μg STAg. 2, 3, and 5 h after injection, total splenic RNA was isolated, and IL-12 p40 and TNFα transcripts were measured by Northern blot analysis. Data shown are representative of three experiments. (E) Increased resistance to LPS-induced shock in DGKζ −/− mice. Age- and sex-matched WT mice ( n = 10) and DGKζ −/− mice ( n = 6) were intraperitoneally injected with 20 mg d -galactosamine and 2 μg LPS per mouse, and their survival was observed at the indicated time points. Data shown are representative of two independent experiments. (F) Detection of IL-12 production by immunofluorescence. Spleens were removed from mice 6 h after injection of PBS, LPS, or STAg. Frozen sections of spleens were processed and stained with a goat anti–mouse IL-12 p40 antibody, followed by a double incubation with Alexa Fluor 488–conjugated anti–mouse CD11c and Alexa Fluor 594–conjugated anti–goat antibodies. The cells were counterstained for nuclei using DAPI. Images were captured using an Axiovert microscope with AxioVision software (see Materials and methods). Green, red, and blue represent CD11c + DCs, IL-12, and nuclei, respectively; yellow represents CD11c + IL-12 + after overlay. Bars, 50 μm.

Article Snippet: Spleens were removed from mice before or 6 h after STAg or LPS stimulation, and frozen sections were processed and stained with a goat anti–mouse IL-12 p40 (BD Biosciences), followed by a double incubation with Alexa Fluor 488–conjugated anti–mouse CD11c and Alexa Fluor 594–conjugated anti–goat antibodies (Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Northern Blot, Isolation, Immunofluorescence, Staining, Incubation, Microscopy, Software

Journal: The Journal of Experimental Medicine

Article Title: Diacylglycerol kinase ζ regulates microbial recognition and host resistance to Toxoplasma gondii

doi: 10.1084/jem.20061856

Figure Lengend Snippet: Enhanced activation of the PI3K–Akt pathway in DGKζ-deficient Mφ after TLR4 stimulation. (A) Elevated LPS-induced PIP 3 production in DGKζ-deficient BMMφ. WT and DGKζ-deficient BMMφ were rested in phosphate-free RPMI 1640 with 0.5% FBS at 37°C for 3 h, with the last 90 min supplemented with [ 32 P]orthophosphate. Cells were left unstimulated or stimulated with 100 ng/ml LPS for 10 and 60 min. Total lipids were extracted and separated by TLC. PIP 3 (molecular mass ≈ 1.2 kD) was visualized using a Phosphorimager (see Materials and methods). Data shown are representative of three experiments. (B) Enhanced Akt activation in DGKζ −/− Mφ. WT (+/+) and DGKζ-deficient (−/−) BMMφ were stimulated as in , but Western blots were probed using an anti–phospho-Akt (Ser473) antibody. The blot was stripped and reprobed with an anti-Akt antibody to determine equal protein loading. Data shown are representative of three experiments. (C) Restoration of LPS-induced IL-12 production in DGKζ-deficient Mφ by inhibition of PI3K or by exogenous PA. WT and DGKζ-deficient Mφ were left unstimulated or stimulated with 100ng/ml LPS in the absence or presence of indicated concentrations of Ly294002 or PA at 37°C for 24 h. IL-12 p40 levels in supernatants were measured with ELISA. Data shown are representative of three experiments. Data shown are the mean ± SD.

Article Snippet: Spleens were removed from mice before or 6 h after STAg or LPS stimulation, and frozen sections were processed and stained with a goat anti–mouse IL-12 p40 (BD Biosciences), followed by a double incubation with Alexa Fluor 488–conjugated anti–mouse CD11c and Alexa Fluor 594–conjugated anti–goat antibodies (Invitrogen).

Techniques: Activation Assay, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Diacylglycerol kinase ζ regulates microbial recognition and host resistance to Toxoplasma gondii

doi: 10.1084/jem.20061856

Figure Lengend Snippet: Impaired innate and T cell responses to T. gondii infection in DGKζ-deficient mice. C57BL/6 WT and DGKζ −/− mice were intraperitoneally injected with 20 cysts of ME49 strain T. gondii . (A and B) Decreased serum IL-12 and IFN-γ concentrations in DGKζ-deficient mice. Serum IL-12 (A) was measured 5 d after infection, and IFN-γ (B) was measured 7 d after infection by ELISA. Data shown are representative of three experiments. Horizontal lines in A and B indicate the means. (C and D) Recall responses. At day 15 (C) and day 30 (D) after T. gondii infection, splenocytes from WT and DGKζ-deficient mice were left unstimulated or stimulated with STAg at 37°C for 24 h. IFN-γ in the culture supernatant was measured by ELISA. (E) Splenic CD4 and CD8 T cell numbers in WT and DGKζ-deficient mice after T. gondii infection. Splenic T cell subsets from uninfected and T. gondii –infected mice were determined after enumeration and FACS analysis. (F) CD44 and CD62L staining on splenic CD4 and CD8 T cell subsets of CD4 and CD8 T cells as in E. Data shown are representative of three experiments. Data shown in C–F are the mean ± SD.

Article Snippet: Spleens were removed from mice before or 6 h after STAg or LPS stimulation, and frozen sections were processed and stained with a goat anti–mouse IL-12 p40 (BD Biosciences), followed by a double incubation with Alexa Fluor 488–conjugated anti–mouse CD11c and Alexa Fluor 594–conjugated anti–goat antibodies (Invitrogen).

Techniques: Infection, Injection, Enzyme-linked Immunosorbent Assay, Staining

Journal: Immunity

Article Title: Structural activation of pro-inflammatory human cytokine IL-23 by cognate IL-23 receptor enables recruitment of the shared receptor IL-12Rβ1

doi: 10.1016/j.immuni.2017.12.008

Figure Lengend Snippet: A) Schematic representation of protein components participating in IL-23 and IL-12 signaling complexes. EC: Extracellular, PM: Plasma Membrane, CP: Cytoplasm, p19: IL-23p19, p40: IL-12p40. B) Cartoon representation of the IL-23:IL-23R crystal structure. Nanobody 22E11 used as a crystallization adjuvant is shown in gray surface representation. C) Close-up view of the IL-23R:IL-23p19 interface in the vicinity of W156 in IL-23p19. D) Restructuring of IL-23 upon binding to IL-23R. E) Close-up view of the interactions around the AB-loop of IL-23p19. F) Top-down view of the IL-23:IL-23R complex with IL-23R (surface). See also Figure S1, Figure S2, Figure S3, Table S1, and Table S2.

Article Snippet: The presence of both IL-12p40 and IL-12Rβ1 in the crosslinked species was ascertained by western blot using polyclonal anti human IL-12p40 and anti-human IL-12 Rβ1 primary antibodies (R&D).

Techniques: Crystallization Assay, Binding Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: Dex up‐regulates the miR‐381 to repress the expression of IRF4‐IL‐9 in neuron cells. A‐C, The expression of IL‐9 in neuron cells after silencing IRF4 detected by RT‐qPCR, ELISA and Western blot analysis, respectively. D, E, The mRNA expression and protein level of IRF4 in neuron cells measured by RT‐qPCR and Western blot analysis, respectively. F, G, The expression of IL‐9 in cells treated with Dex and miR‐381 inhibitor studied using ELISA and Western blot analysis. H, The mRNA expression of IRF4 in neuron cells after treatment of si‐IRF4 and Dex measured by RT‐qPCR. I, J, The expression of IL‐9 in neuron cells after treatment of si‐IRF4 and Dex determined using ELISA and Western blot analysis. * P < .05. The above‐mentioned measurement data were expressed as the mean ± SD and analysed using one‐way ANOVA and Tukey's post hoc test. Each cellular experiment was repeated three times

Article Snippet: The membrane was immersed in IRF4 polyclonal antibody (sc‐6059, 1:500; Santa Cruz Biotechnology), IL‐9 rabbit polyclonal antibody (ab205718, 1:500; Abcam Inc) and GAPDH monoclonal antibody (ab9485, 1:2000; Abcam Inc).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal:

Article Title:

doi:

Figure Lengend Snippet: Dex reduces the inflammatory response by down‐regulating the IRF4‐IL‐9 to alleviate cerebral ischaemic injury. A‐C, The mRNA expression of IRF4 and IL‐9 in OGD‐treated neuron cells treated with Dex and oe‐IRF4 detected by RT‐qPCR, ELISA and Western blot analysis, respectively. D, Apoptosis of OGD‐treated neuron cells treated with Dex and oe‐IRF4 measured by flow cytometry. E‐G, The mRNA expression and protein expression of IRF4 in brain tissues of MCAO rats treated with Dex and oe‐IRF4 assessed by RT‐qPCR, ELISA and Western blot analysis, respectively. H‐J, The expression of IRF4 in brain tissues of MCAO rats observed by RT‐qPCR, ELISA and Western blot analysis. K, mNSS evaluation on the neurological function of MCAO rats. L, M, Cell apoptosis in brain tissues of MCAO rats detected by TUNEL staining (scale bar = 100 μm). N, Cell apoptosis in brain tissues of MCAO rats using flow cytometry. O, BrdU‐positive cell rate was detected by BrdU. P, Ki‐67 staining to detect the number of Ki‐67‐positive expression. * P < .05. The above‐mentioned measurement data were expressed as the mean ± SD, and compared using one‐way ANOVA and Tukey's post hoc test. Non‐parametric test (Kruskal‐Wallis test) was used to compare the neurological scores. n = 12. Each cellular experiment repeated three times

Article Snippet: The membrane was immersed in IRF4 polyclonal antibody (sc‐6059, 1:500; Santa Cruz Biotechnology), IL‐9 rabbit polyclonal antibody (ab205718, 1:500; Abcam Inc) and GAPDH monoclonal antibody (ab9485, 1:2000; Abcam Inc).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, TUNEL Assay, Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: The mechanism of Dex in cerebral ischaemic injury with the involvement of miR‐381. The use of Dex induced highly expressed miR‐381, which led to an inhibition of inflammation response in rats with cerebral ischaemic injury by repressing IRF4‐IL‐9

Article Snippet: The membrane was immersed in IRF4 polyclonal antibody (sc‐6059, 1:500; Santa Cruz Biotechnology), IL‐9 rabbit polyclonal antibody (ab205718, 1:500; Abcam Inc) and GAPDH monoclonal antibody (ab9485, 1:2000; Abcam Inc).

Techniques: Inhibition