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:

Article Title: CD40-CD40 Ligand Interactions Augment Survival of Normal Mice, but Not CD40 Ligand Knockout Mice, Challenged Orally with Salmonella dublin

doi:

Figure Lengend Snippet: Effect of CD40 ligand on secretion of IL-12p70 from Salmonella-infected macrophages from BALB/c mice. Uninfected cells were cultured in medium alone (0) or with recombinant soluble trimeric CD40 ligand (1 μg/ml) (CD40L). Salmonella-infected cells (3:1 ratio of Salmonella bacteria to macrophages) were cultured in medium alone (SAL) or with recombinant soluble trimeric CD40 ligand (1 μg/ml) (SAL + CD40L) as indicated. At 48 h after exposure, the supernatant was removed from each culture and the amount of IL-12p70 present was quantified by a capture enzyme-linked immunosorbent assay. This experiment was performed twice with similar results. Results are shown as triplicate determinations (short bars) with the means indicated (long bars).

Article Snippet: Briefly, a capture antibody against murine IL-12p70 (clone G297-289; PharMingen, San Diego, Calif.) was applied as a coating to microtiter plates (Nunc Maxisorp, Naperville, Ill.) at 10 μg/ml for 18 h. After washing and blocking with phosphate-buffered saline containing 2% BSA, culture supernatants were added to each well for 2 h. Unbound material was washed off, and biotinylated anti-mouse IL-12p70 antibody (clone C17.8; PharMingen) was added at 5 μg/ml for 2 h. Bound antibody was detected by addition of streptavidin-alkaline phosphatase (Southern Biotechnology Associates, Birmingham, Ala.) for 30 min, followed by addition of nitrophenyl phosphate (Sigma Chemical Co.).

Techniques: Infection, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Oncology Reports

Article Title: Spirulina lipopolysaccharides inhibit tumor growth in a Toll-like receptor 4-dependent manner by altering the cytokine milieu from interleukin-17/interleukin-23 to interferon-γ

doi: 10.3892/or.2017.5346

Figure Lengend Snippet: Spirulina LPS induces limited or does not induce the production of IL-6 and IL-23 in vivo , but augments T cell-dependent IL-12 induction. (A) C3H/HeN and C3H/HeJ mice were injected i.p. with E. coli LPS or Spirulina LPS (200 µg). Serum IL-6, TGF-β, or IL-23 levels were assessed 4 h later. Data represent mean ± SE of 4–5 mice per group. *P<0.05 compared with prior to injection (pre). (B) Whole spleen cells from DO11.10 mice were cultured in the presence of E. coli or Spirulina LPS without OVA for 5 days and B-cell proliferation was estimated. *P<0.05 compared with no LPS. (C) Serum IL-12 levels were measured in tumor-bearing C3H/HeN or C3H/HeJ mice treated as in . Data represent mean ± SE of 5 mice per group. *P<0.05 compared with saline. (D) Tumor-bearing C3H/HeN mice were treated with Spirulina LPS and with anti-CD4 and/or anti-CD8, or rat IgG antibodies as in . Serum levels of IL-12 were measured on day 14. *P<0.05 compared with saline.

Article Snippet: For IL-23(p19/40), rat anti-mouse IL-23p19 (G23-8, no. 14-7232-85, eBioscience, San Diego, CA, USA) and biotinylated anti-IL-12p40 (C17.8, BD Biosciences) were used.

Techniques: In Vivo, Injection, Cell Culture