Journal: PLoS ONE

Article Title: Role of Lactobacillus pentosus Strain b240 and the Toll-Like Receptor 2 Axis in Peyer's Patch Dendritic Cell-Mediated Immunoglobulin A Enhancement

doi: 10.1371/journal.pone.0091857

Figure Lengend Snippet: (A) PP cells (1.5×10 6 cells) were cultured with saline (open circles), 1.2×10 6 counts of heat-killed b240 (closed squares), or 1.2 × 10 7 counts of heat-killed b240 (closed circles) for 1, 3, 5, and 7 days. (B, C) In the presence or absence of heat-killed b240 (4.7×10 6 counts), PP cells (5.8×10 5 cells) were cultured with (B) anti-IL-6 mAb (10 μg/ml), anti-IFN-γ mAb (10 μg/ml), anti-TNF mAb (10 μg/ml), rat IgG1 k isotype control (10 μg/ml), (C) LE540 (1 μM), BCMA-Ig+ TACI-Ig (5 μg/ml each), dimethyl sulfoxide, or human IgG1 Fc antibody (10 μg/ml) for 4 days. The stimulation index of each sample was calculated (for example, (b240-treatment and anti-IL-6 Ab treatment)/(saline-treatment and anti-IL-6 Ab treatment) is the stimulation index for anti-IL-6 Ab treatment). (D) PP cells (5.8×10 5 cells) were cultured with a low dose (light gray), medium dose (dark gray), and high dose (black) of rIL-6 (0.4, 2, or 10 ng/ml), rIFN-γ (0.6, 3, or 15 ng/ml), rTNF (0.08, 0.4, or 2 ng/ml), or heat-killed b240 (4.7×10 6 counts) for 4 days. IgA or cytokine in the culture supernatants was determined by ELISA or CBA. Data are expressed as mean ± SEM (n = 3). (A, B) * P <0.05 versus control group by Dunnett's test. (C) Student's t -test was conducted. (D) Statistical analysis was not conducted. Data are representative of 2 independent experiments producing similar results.

Article Snippet: IFN-α and IFN-β were measured using Mouse Interferon Alpha and Mouse Interferon Beta ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ, USA), respectively.

Techniques: Cell Culture, Saline, Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Role of Lactobacillus pentosus Strain b240 and the Toll-Like Receptor 2 Axis in Peyer's Patch Dendritic Cell-Mediated Immunoglobulin A Enhancement

doi: 10.1371/journal.pone.0091857

Figure Lengend Snippet: (A, C) In the presence (black) or absence (white) of heat-killed b240 (1.6×10 6 counts), (A) purified WT PP IgD + cells (2×10 5 cells/well) were cultured with or without purified WT PP CD11c + B220 − DCs (1×10 5 cells/well), (B) purified WT PP CD11c + B220 − DCs and purified WT PP IgD + B cells were co-cultured with (black) or without (white) anti-IL-6 mAb (10 μg/ml) in the presence or absence of heat-killed b240 (1.6×10 6 counts), and (C) purified WT or TLR2 −/− PP CD11c + B220 − DCs and purified WT or TLR2 −/− PP IgD + B cells were co-cultured for 7 days. IgA and IL-6 in the culture supernatants were determined by ELISA and CBA, respectively. Data are expressed as mean ± SEM (n = 1–3). ND, not detected. * P <0.05 versus (A) saline group by Welch's t -test, (B) isotype Ig group by Student's t- t est, (C) WT DCs + WT B cells + b240 co-culture by Dunnett's test. Data are representative of 2 independent experiments producing similar results.

Article Snippet: IFN-α and IFN-β were measured using Mouse Interferon Alpha and Mouse Interferon Beta ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ, USA), respectively.

Techniques: Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Saline, Co-Culture Assay

Journal: Biochemical Journal

Article Title: Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members

doi: 10.1042/BJ20141523

Figure Lengend Snippet: RAW cells were incubated for 1 h without (−) or with (+) 1.0 μM JQ1 or 1.0 μM BI2536 and then stimulated for 8 h without (−) or with (+) poly(I:C) (10 μg/ml) ( A and C ) or for 4 h without (−) or with (+) LPS (100 ng/ml) ( B and D ). At each time point, the total RNA was extracted from the cells and Ifnb mRNA was quantified by qPCR ( A and B ) and the concentration of IFNβ in the cell culture medium was determined by ELISA ( C and D ). Results are means+S.E.M for triplicate determinations. ( A ) and ( B ) show the fold increase in mRNA levels relative to the values measured in cells that had not been stimulated with LPS or poly(I:C).

Article Snippet: The level of secreted IFNβ in the cell culture medium was determined using the Verikine mouse and human IFNβ ELISA kits (PBL Interferon Source) or the LEGEND MAXTM Mouse IFN-β ELISA Kit (BioLegend) following the manufacturer's protocol.

Techniques: Incubation, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Biochemical Journal

Article Title: Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members

doi: 10.1042/BJ20141523

Figure Lengend Snippet: ( A and B ) Gen2.2 cells were incubated for 1 h with or without BI-2536 (1.0 μM), JQ1 (1.0 μM) or I-BET151 (1.0 μM) and then stimulated for 8 h with CL097 (1.0 μg/ml) ( A ) or for 12 h with ODN1826 (1.0 μM) ( B ). The concentration of IFNβ in the culture medium was measured by ELISA. Results are means+S.D. from two independent experiments each performed in duplicate. ( C ) Gen2.2 cells were incubated for 1 h with or without BI-2536 (1.0 μM), JQ1 (1.0 μM), I-BET151 (1.0 μM) or BI-605906 (5.0 μM) and then stimulated for 1 h with CL097. Staining with anti-IRF5, or DAPI to reveal nuclei, followed by deconvolution microscopy was performed as described in the Materials and methods section. ( D ) Gen2.2 cells were incubated for 1 h with BI-2536 (1 μM) or JQ1 (1 μM), then stimulated for 1 h with the TLR7 agonist CL097 (1 μg/ml), cross-linked and lysed. Chromatin was sheared by sonication and ChIP was performed using anti-IRF5. The enrichment of the Ifnb promoter was measured by qPCR, normalizing to input. Results are means+S.D. similar results were obtained in three independent experiments each performed in duplicate. IP, immunoprecipitation.

Article Snippet: The level of secreted IFNβ in the cell culture medium was determined using the Verikine mouse and human IFNβ ELISA kits (PBL Interferon Source) or the LEGEND MAXTM Mouse IFN-β ELISA Kit (BioLegend) following the manufacturer's protocol.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy, Sonication, Immunoprecipitation