Journal:

Article Title: Pathological Role of Large Intestinal IL-12p40 for the Induction of Th2-Type Allergic Diarrhea

doi:

Figure Lengend Snippet: Selective production of IL-12p40 by the large intestine of diarrhea-induced mice. In A, large intestinal tissues from diarrhea-induced mice were immunostained with anti-IL-12p40 mAb, anti-IL-12p35 mAb, or control IgG. Control non-disease mice section gave no signal above background (data not shown). In B-1, IL-12p40-specific mRNA was expressed selectively in the large intestine of mice with allergic diarrhea. In B-2, quantitive real-time PCR analysis of IL-12p40- and p35-specific mRNA expression was performed. The ratio was obtained as the level of IL-12p40 or p35 expression in non-treated mice as a scale of one. The detailed information for the expression of this ratio is described in the Materials and Methods section. In C-E, IL-12p40 was detected in MØ and DC and epithelial cells in the large intestine. The serial sections of the large intestine from diarrhea-induced mice were stained with anti-IL-12p40 mAb and anti-CD11b mAb (C), with anti-IL-12p40 mAb and anti-CD11c mAb (D). The arrows point to double-positive cells. Large intestinal epithelial cells were stained with anti-IL-12p40 mAb (E).

Article Snippet: After electrophoresis, proteins were transferred to a polyvinylidene difluoride microporous membrane (PVDF Immobilon; Millipore, Bedford, MA) and the membrane was reacted with biotinylated anti-IL-12 (C17.8) followed by incubation with biotin-streptavidin complex (ABC-AP Kit; Vector Laboratories, Inc.).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining

Journal:

Article Title: Pathological Role of Large Intestinal IL-12p40 for the Induction of Th2-Type Allergic Diarrhea

doi:

Figure Lengend Snippet: Induction of IL-12p40 homodimer in the large but not small intestine of diarrhea-induced mice. Large and small intestinal tissue extracts were subjected to immunopreciptation and Western blotting analysis using anti-IL-12p40 (C17.8) mAb under non-reducing conditions (A). The captions above the figure indicate the experimental mouse group receiving different in vivo treatments. Thus, the samples were obtained from SC/PO mice treated with C17.8 or control antibodies. Further, the samples were isolated from mice treated with PO only, SC only, or non-treated mice. The arrow points to IL-12p40 homodimer expression in the large intestine of diarrhea-induced mice. The data represent four independent experiments. In B, at the indicated times after oral administration of OVA, large intestinal tissue extracts isolated from diarrhea-induced mice were assayed for IL-12p40 by the same method as in A. In C, the large intestinal tissue extracts of diarrhea-induced mice were subjected to Western blotting with anti-IL-12p35 Ab as well as anti-IL-12p40. IL-12p70 protein was used as a positive control for the IL-12p35 detection system. As negative control, immunoprecipitation was performed without the tissue specimens (Ab only). The data represent three different experiments.

Article Snippet: After electrophoresis, proteins were transferred to a polyvinylidene difluoride microporous membrane (PVDF Immobilon; Millipore, Bedford, MA) and the membrane was reacted with biotinylated anti-IL-12 (C17.8) followed by incubation with biotin-streptavidin complex (ABC-AP Kit; Vector Laboratories, Inc.).

Techniques: Western Blot, In Vivo, Isolation, Expressing, Positive Control, Negative Control, Immunoprecipitation

Journal:

Article Title: Pathological Role of Large Intestinal IL-12p40 for the Induction of Th2-Type Allergic Diarrhea

doi:

Figure Lengend Snippet: Inhibition of allergic diarrhea disease by the treatment with anti-IL-12p40 mAb. In A, anti-IL-12p40 mAb (C17.8) treatment (thin dashed line) delayed the development of allergic diarrhea when compared with the rat IgG-treated group (solid line). Statistical differences were determined by Wilcoxon rank-sum test and are indicated by **, P < 0.01. Mice with SC only were used as controls (thick dashed line). In B, left, body weight was recovered in allergic diarrhea mice treated with anti-IL-12p40 mAb (C17.8). In B, right, OVA-specific IgE Abs were reduced in the serum of allergic diarrhea mice treated with anti-IL-12p40 mAb (C17.8). The data are expressed as the mean of ± SE and are representative of five independent experiments. Statistical differences between anti-IL-12p40 mAb and control rat IgG-treated mice are indicated as **, P < 0.01.

Article Snippet: After electrophoresis, proteins were transferred to a polyvinylidene difluoride microporous membrane (PVDF Immobilon; Millipore, Bedford, MA) and the membrane was reacted with biotinylated anti-IL-12 (C17.8) followed by incubation with biotin-streptavidin complex (ABC-AP Kit; Vector Laboratories, Inc.).

Techniques: Inhibition

Journal:

Article Title: Pathological Role of Large Intestinal IL-12p40 for the Induction of Th2-Type Allergic Diarrhea

doi:

Figure Lengend Snippet: In vivo treatment with anti-IL-12p40 (C17.8) reduced the predominant antigen-specific Th2 type responses by large intestinal mononuclear cells isolated from diarrhea-induced mice. The mononuclear cells isolated from the large intestine (1.5 × 105 cells/well) were cultured with OVA (1 mg/ml) for 3 days. Culture supernatants were harvested and then assayed for IL-4, IL-13, IL-5, and eotaxin by ELISA assay. These data are expressed as the mean ± SE and are representative of three independent experiments. The statistical differences between anti-IL-12p40 mAb and control antibody treated mice are indicated as **, P < 0.01.

Article Snippet: After electrophoresis, proteins were transferred to a polyvinylidene difluoride microporous membrane (PVDF Immobilon; Millipore, Bedford, MA) and the membrane was reacted with biotinylated anti-IL-12 (C17.8) followed by incubation with biotin-streptavidin complex (ABC-AP Kit; Vector Laboratories, Inc.).

Techniques: In Vivo, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Pathological Role of Large Intestinal IL-12p40 for the Induction of Th2-Type Allergic Diarrhea

doi:

Figure Lengend Snippet: Suppression of allergic diarrhea development in IL-12p40 KO mice. In A, the incidence of allergic diarrhea was reduced in the IL-12p40 KO mice when compared with wild-type mice immunized subcutaneously and then given OVA repeatedly by the oral route (SC/PO). In B, the large intestinal LP mononuclear cells from IL-12p40 KO mice did not produce IL-4. Mononuclear cells isolated from the large intestine were restimulated with OVA for the assessment of IL-4 synthesis as described in Figure 4A. The data are expressed as the mean ± SE and represent three different experiments.

Article Snippet: After electrophoresis, proteins were transferred to a polyvinylidene difluoride microporous membrane (PVDF Immobilon; Millipore, Bedford, MA) and the membrane was reacted with biotinylated anti-IL-12 (C17.8) followed by incubation with biotin-streptavidin complex (ABC-AP Kit; Vector Laboratories, Inc.).

Techniques: Isolation

Journal: The Journal of Experimental Medicine

Article Title: Interferon-α and Interleukin-12 Are Induced Differentially by Toll-like Receptor 7 Ligands in Human Blood Dendritic Cell Subsets

doi: 10.1084/jem.20020207

Figure Lengend Snippet: TLR-7 ligands induce the production of IL-12 and IFN-α from MDCs and PDCs, respectively. (A and B) After 24-h culture with various stimuli, concentration of IL-12 p40+p70 (A) and IFN-α (B) in the culture supernatants of MDCs, PDCs, and PBMCs were measured by ELISA. The data are shown by means ± SEM of five independent experiments. (C) After 5-h culture with R-848 (10 −6 M), intracellular staining of IL-12 and IFN-α in MDCs and PDCs, together with the staining of surface expression of CD40, was performed. Percentages of the respective cytokine producing DCs are indicated. This figure represents the results from one of three experiments.

Article Snippet: 10 μg/ml brefeldin A (Sigma-Aldrich) was added during the last 1 h. After the stimulation, MDCs and PDCs were stained with Cy-Chrome-labeled CD40 (5C3; BD PharMingen), and then fixed, permeabilized (FIX and PERM kit; Caltag Laboratories), and stained with FITC-labeled anti–IL-12 p40+p70 mAb (B-P24; DIACLONE Research) or unconjugated mouse anti–human IFN-α mAb (MC-16; Genzyme).

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

Journal: Journal of Immunology Research

Article Title: GK-1 Improves the Immune Response Induced by Bone Marrow Dendritic Cells Loaded with MAGE-AX in Mice with Melanoma

doi: 10.1155/2014/158980

Figure Lengend Snippet: BMDCs phenotype. Levels of molecules of the major histocompatibility complex II (MHCII), CD40, CD80, CD86, and IL-12 in BMDCs were measured after different treatments: control (without treatment: WT), LPS, GK-1, TNF α , TNF α /GK-1. Treatment with LPS, TNF α , and TNF α /GK-1 induces increased expression of MHCII, CD40, CD80, and CD86. When BMDCs were treated only with GK-1 an increase in the production of IL-12 was found. (a) Mean fluorescence intensity (MIF) of MHCII. * P < 0.05, *** P < 0.001. (b) BMDCs phenotype after 10 days of differentiation. 91.9% differentiation was induced (91.9% of CD11c+ cells). Red: isotype control. Blue: BMDCs. (c) Percentage of CD40+ BMDCs after treatment. * P < 0.05. (d). MFI of CD40 in BMDCs. ** P < 0.001, *** P < 0.0001. (e) Percentage of CD86+ BMDCs. * P < 0.05, ** P < 0.001. (f) MFI of CD86 in BMDCs. * P < 0.05, ** P < 0.001, c P < 0.05 TNF α versus GK-1. (g) Percentage of CD80+ BMDCs. * P < 0.05. (h) MFI of CD80 in BMDCs. ANOVA, Tukey. *** P < 0.0001, c P < 0.0001 TNF/GK-1 versus GK-1, + P < 0.0001 TNF versus GK-1. (i) Percentage of IL-12+ BMDCs. (j) MFI of IL-12 in BMDCs. * P < 0.05, ** P < 0.001, *** P < 0.0001. Mean ± SEM n ≥ 3.

Article Snippet: Monoclonal antibodies for staining of cells analyzed by flow cytometry, CD3-biotin, CD8-CyCrome, CD11c-allophycocyanin, CD40-biotin, CD86-biotin, Ia/Ie-phycoerythrin, IL-12-biotin, IFN γ -biotin, and IL-10-biotin, were purchased from Pharmingen, BD Bioscience, USA.

Techniques: Expressing, Fluorescence