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: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: rFlaA: Betv1 induces IL-1β and pro-inflammatory cytokine secretion from both human and mouse macrophages. C57BL/6J peritoneal macrophages (A) , human buffy coat-differentiated macrophages (B) , or PMA-differentiated THP-1 macrophages (C) were stimulated with either LPS as a positive control or the indicated equimolar amounts of either rBet v 1, rFlaA, rFlaA + rBet v 1, or rFlaA:Betv1 for 24 h. Supernatants were collected and checked for the secretion of IL-1β, IL-6, IL-12, and TNF-α by ELISA. Data are the mean results ± SD from either three independent experiments (A, C) or samples collected from four donors (B) . Statistical significances are indicated as **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: Cytokines in the supernatants of mouse cell cultures were analyzed using the following antibody combinations: IL-1β (capture antibody: anti-IL-1β monoclonal mouse antibody (#14-7061-85, eBioscience, Frankfurt, Germany, 1:500) plus detection antibody: anti-IL-1β monoclonal mouse biotin-conjugated antibody (#13-7112-81, eBioscience, 1:500)), IL-6 (capture antibody: anti-IL-6 monoclonal mouse antibody (#14-7061-85, eBioscience, Frankfurt, Germany, 1:500) plus detection antibody: anti-IL-6 monoclonal mouse biotin-conjugated antibody (#13-7062-85, eBioscience, 1:500), TNF-α (capture antibody: anti-TNF-α monoclonal mouse antibody (#14-7325-85, eBioscience, 1:500) plus detection antibody: anti-TNF-α monoclonal mouse biotin-conjugated antibody (#13-7326-85, eBioscience, 1:500), IL-12 p70 (capture antibody: anti-IL-12 monoclonal mouse antibody (#14-7122-85, eBioscience, 1:500) plus detection antibody: anti-IL-12 monoclonal mouse biotin-conjugated antibody (#MM121B, Invitrogen, 1:500).

Techniques: Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: Both NLRP3- and NLRC4-inflammasome activation contributes to rFlaA:Betv1 induced IL-1β and pro-inflammatory cytokine secretion from THP-1 macrophages. PMA-differentiated wild-type (WT), NLRP3-, NLRC4-, or ASC-deficient THP-1 macrophages were stimulated with either LPS as a positive control, or equimolar amounts of rFlaA + rBet v 1, rFlaA:Betv1, rFlaA *D1 :Betv1, or rFlaA ΔDC0 :Betv1 for 24 h (A) . Supernatants were collected and checked for the secretion of IL-1β, IL-6, IL-12, and TNF-α by ELISA (B) . Data are the mean results of three independent experiments ± SD, and statistical significances are indicated as ns: p-value > 0.05, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: Cytokines in the supernatants of mouse cell cultures were analyzed using the following antibody combinations: IL-1β (capture antibody: anti-IL-1β monoclonal mouse antibody (#14-7061-85, eBioscience, Frankfurt, Germany, 1:500) plus detection antibody: anti-IL-1β monoclonal mouse biotin-conjugated antibody (#13-7112-81, eBioscience, 1:500)), IL-6 (capture antibody: anti-IL-6 monoclonal mouse antibody (#14-7061-85, eBioscience, Frankfurt, Germany, 1:500) plus detection antibody: anti-IL-6 monoclonal mouse biotin-conjugated antibody (#13-7062-85, eBioscience, 1:500), TNF-α (capture antibody: anti-TNF-α monoclonal mouse antibody (#14-7325-85, eBioscience, 1:500) plus detection antibody: anti-TNF-α monoclonal mouse biotin-conjugated antibody (#13-7326-85, eBioscience, 1:500), IL-12 p70 (capture antibody: anti-IL-12 monoclonal mouse antibody (#14-7122-85, eBioscience, 1:500) plus detection antibody: anti-IL-12 monoclonal mouse biotin-conjugated antibody (#MM121B, Invitrogen, 1:500).

Techniques: Activation Assay, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: NFκB- and SAP/JNK MAP kinase-signaling pathways contribute to rFlaA:Betv1-induced cytokine secretion from THP-1 macrophages. PMA-differentiated THP-1 macrophages were pre-treated with the indicated inhibitor concentrations for 90 min and subsequently stimulated with 27.4 µg/mL rFlaA:Betv1 for additional 24 h (A) . Supernatants were collected and examined for the secretion of IL-1β, IL-6, IL-12, and TNF-α by ELISA (B) . Data are the mean results of three independent experiments ± SD. Statistical comparisons were performed between the indicated samples and rFlaA:Betv1-stimulated samples, with statistical significance shown as *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: Cytokines in the supernatants of mouse cell cultures were analyzed using the following antibody combinations: IL-1β (capture antibody: anti-IL-1β monoclonal mouse antibody (#14-7061-85, eBioscience, Frankfurt, Germany, 1:500) plus detection antibody: anti-IL-1β monoclonal mouse biotin-conjugated antibody (#13-7112-81, eBioscience, 1:500)), IL-6 (capture antibody: anti-IL-6 monoclonal mouse antibody (#14-7061-85, eBioscience, Frankfurt, Germany, 1:500) plus detection antibody: anti-IL-6 monoclonal mouse biotin-conjugated antibody (#13-7062-85, eBioscience, 1:500), TNF-α (capture antibody: anti-TNF-α monoclonal mouse antibody (#14-7325-85, eBioscience, 1:500) plus detection antibody: anti-TNF-α monoclonal mouse biotin-conjugated antibody (#13-7326-85, eBioscience, 1:500), IL-12 p70 (capture antibody: anti-IL-12 monoclonal mouse antibody (#14-7122-85, eBioscience, 1:500) plus detection antibody: anti-IL-12 monoclonal mouse biotin-conjugated antibody (#MM121B, Invitrogen, 1:500).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Human Non-Germinal Center B Cell Interleukin (IL)-12 Production Is Primarily Regulated by T Cell Signals CD40 Ligand, Interferon γ, and IL-10: Role of B Cells in the Maintenance of  T Cell Responses

doi:

Figure Lengend Snippet: B cell–derived IL-12 induces T cells to produce IFN-γ. CD3 + CD4 + CD45RA + enriched T cells (98% CD3 + CD4 + > 75% CD45RA) were stimulated with PMA (1 ng/ml) plus CD28 mAbs (2 μg/ml) with or without rhIL-12 p70 at concentrations ranging from 1 to 1,000 pg or with supernatants (a 50% vol of 200 μl final vol) from tonsillar B cells cultured for 24 h on t-CD40L cells. Where indicated, neutralizing anti–IL-12 mAb (α–IL-12) was added. After 3 d, supernatants were harvested and analyzed for IFN-γ production by ELISA.

Article Snippet: Plates were again thoroughly washed followed by a 2-h incubation using biotinylated anti–IL-12 mAbs (350 ng/ml; R&D Systems).

Techniques: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Characterization of the Myeloid Cell Populations’ Resident in the Porcine Palatine Tonsil

doi: 10.3389/fimmu.2018.01800

Figure Lengend Snippet: Evaluation of antigen processing, T cell stimulatory capacity and cytokine secretion profile following TLR stimulation (A) . Differential endocytosis between cell populations was evaluated with OVA-DQ-FITC by culture of lineage depleted myeloid cells for 1.5 h at 37°C (and 4°C), and FITC fluorescence was assessed by flow cytometry. Histogram shows the mean percentage of cells taking up DQ-OVA from eight pigs (each tested in triplicate), following subtraction of non-specific fluorescence (uptake at 4°C) for each cell population from three independent experiments. CD4 T cells were also assessed as a negative control. (B) Myeloid cells (APC) were sorted and peripheral blood mononuclear cells from allogeneic animals were stained with Violet CellTrace and mixed at a APC:T cell ratio of 1:10 before being culture for 5 days at 37°C. Proliferation of CD4 + CD8α − (CD4 T cells), CD4 − CD8α + (CD8 T cells), and CD4 + CD8α + (memory T cells) was evaluated by flow cytometry. A value of 100 was assigned to the population with the highest proliferation value and all other populations were compared to this value (and repeated for each pig). Data are from three separate experiments and a minimum of four different animals for each cell type. A one-way ANOVA was performed and statistical significance is described by **** p < 0.0001, *** p = 0.0002, ** p = 0.0016, and * p = 0.0108. (C) Isolated tonsil cells were depleted for lineage markers (CD3, CD8α, CD21, and IgM) and stimulated for 12 h in the presence of toll-like receptors agonists CpG, Poly I:C or LPS. After incubation, the myeloid populations were defined using the same antibody panel as described above. IL-12 (top panel) and TNF-α (bottom panel) secretion was assessed by intracellular staining and flow cytometry. For each cell population, each point represents a single pig and the horizontal line represents the mean of at least seven pigs tested in three independent experiments. The mean percentage of secreting cells (non-stimulated) was subtracted from each of the relevant data points. (D) Representative flow cytometry dot plots, showing IL-12 and TNFα secretion associated with cDC1 and CD14 + cells, respectively following CpG stimulation.

Article Snippet: For intracellular staining, cells were treated with BD Cytofix/Cytoperm™ (BD Biosciences, Oxford, UK) for 20 min at 4°C washed with BD Perm/Wash™ before staining with either biotinylated anti-IL-12 (R&D Systems, Abingdon, UK) or directly conjugated anti-TNF-α Brilliant Violet 605 (eBioscience, Hatfield, UK) in Perm/Wash™ buffer.

Techniques: Fluorescence, Flow Cytometry, Negative Control, Staining, Isolation, Incubation