Journal: Infection and Immunity

Article Title: Group A Streptococcus Infection of the Nasopharynx Requires Proinflammatory Signaling through the Interleukin-1 Receptor

doi: 10.1128/IAI.00356-20

Figure Lengend Snippet: Anakinra antagonizes GAS colonization of the nasopharynx. Induction of inflammatory cytokines and their contribution to GAS infection. C57BL/6 mice were treated with anakinra (50 μg/kg) or PBS control and inoculated intranasally with 108 CFU of GAS M1T1 5448. Mice were euthanized after 24 or 72 h, and the nasopharynxes were subjected to lavage to quantify GAS CFU by dilution plating (A), quantify cytokines with a Meso Scale Diagnostics multiplex ELISA (means are displayed; comparisons indicated reach significance only for IL-1β and IL-6) (B), and measure levels of IL-1 signaling using a transgenic IL-1 receptor reporter specific for active IL-1α and IL-1β (C). Data are means ± standard deviations (SD) (n = 10 each) and are representative of at least 3 experiments. ND, none detected; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ns, not significant.

Article Snippet: Mice were euthanized after 24 or 72 h, and the nasopharynxes were subjected to lavage to quantify GAS CFU by dilution plating (A), quantify cytokines with a Meso Scale Diagnostics multiplex ELISA (means are displayed; comparisons indicated reach significance only for IL-1β and IL-6) (B), and measure levels of IL-1 signaling using a transgenic IL-1 receptor reporter specific for active IL-1α and IL-1β (C).

Techniques: Infection, Control, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Transgenic Assay

Journal: Infection and Immunity

Article Title: Group A Streptococcus Infection of the Nasopharynx Requires Proinflammatory Signaling through the Interleukin-1 Receptor

doi: 10.1128/IAI.00356-20

Figure Lengend Snippet: SpeB and caspase-1 contribute to IL-1β generation in the nasopharynx. Effects of IL-1 and inflammasome signaling on GAS survival. (A) C57BL/6, casp-1/11−/−, or IL1R1−/− mice were inoculated intranasally with 108 CFU of wild-type GAS M1T1 5448. Mice were euthanized after 24 h, nasopharyngeal lavage fluids were plated to enumerate CFU, and cytokine levels were quantified by ELISA and IL-1R reporter assay. (B and C) Role of SpeB and CovRS in GAS survival. C57BL/6 mice were given anakinra (50 μg/kg) or PBS and infected as described above with wild-type, ΔspeB, or AP covS-frameshift GAS, and CFU and cytokines were examined as described above. (D) Role of host pathways in selection for covS-frameshift SpeB− clones. Isolated colonies from the experiments for panels A, B, and C were screened for SpeB hydrolysis of azocasein, and the fraction where SpeB activity is lost is indicated. ND, none detected (for experiments where no GAS was recoverable from the mouse). Data are means ± SD (n = 5 each) and are representative of at least 3 experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ns, not significant.

Article Snippet: Mice were euthanized after 24 or 72 h, and the nasopharynxes were subjected to lavage to quantify GAS CFU by dilution plating (A), quantify cytokines with a Meso Scale Diagnostics multiplex ELISA (means are displayed; comparisons indicated reach significance only for IL-1β and IL-6) (B), and measure levels of IL-1 signaling using a transgenic IL-1 receptor reporter specific for active IL-1α and IL-1β (C).

Techniques: Enzyme-linked Immunosorbent Assay, Reporter Assay, Infection, Selection, Clone Assay, Isolation, Activity Assay

Journal: Infection and Immunity

Article Title: Group A Streptococcus Infection of the Nasopharynx Requires Proinflammatory Signaling through the Interleukin-1 Receptor

doi: 10.1128/IAI.00356-20

Figure Lengend Snippet: IL-1 mediates microbial interference in the nasopharynx. (A) SpeB-dependent effects on IL-1β and growth. C57BL/6 mice were inoculated intranasally with 108 CFU of GAS, L. lactis, or SpeB-expressing L. lactis. IL-1β was quantified by ELISA and CFU by dilution plating from nasopharyngeal lavage samples collected after 24 h infection. (B) IL-1β-dependent effects on L. lactis growth. C57BL/6 mice treated with anakinra (50 μg/kg) or PBS control were infected and CFU enumerated after 24 h as described above. (C) In vivo competition experiment with GAS and L. lactis. C57BL/6 mice were inoculated with 108 CFU GAS, L. lactis, or a mix of both (5 × 107 each; 1:1), and CFU were enumerated after 24 h as described above. (D) In vitro competition experiment with GAS and L. lactis. GAS (104 CFU), L. lactis (104 CFU), or a mix of both (5 × 103 CFU each; 1:1) were grown 18 h in 3 ml Todd-Hewitt broth at 37°C in 5% CO2; then, the CFU of each were enumerated by dilution and differential plating. Data are means ± SD (n = 5 each) and are representative of at least 3 experiments. ND, none detected; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ns, not significant.

Article Snippet: Mice were euthanized after 24 or 72 h, and the nasopharynxes were subjected to lavage to quantify GAS CFU by dilution plating (A), quantify cytokines with a Meso Scale Diagnostics multiplex ELISA (means are displayed; comparisons indicated reach significance only for IL-1β and IL-6) (B), and measure levels of IL-1 signaling using a transgenic IL-1 receptor reporter specific for active IL-1α and IL-1β (C).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Infection, Control, In Vivo, In Vitro

Journal: BMB Reports

Article Title: Effects of α-lipoic acid on LPS-induced neuroinflammation and NLRP3 inflammasome activation through the regulation of BV-2 microglial cells activation

doi: 10.5483/BMBRep.2019.52.10.026

Figure Lengend Snippet: α-LA inhibits the expression of pro-inflammatory cytokines in LPS-treated BV-2 microglial cells. (A) Effects of α-LA on cell viability. BV-2 microglial cells were incubated with LPS (1 μg/ml) for 30 min followed by treatment with the indicated concentrations of α-LA for 24 h. Thereafter, cell viability was assessed through the MTT assay. (B and C) BV-2 microglial cells were treated with LPS (1 μg/ml) for 30 min followed by treatment with the indicated concentrations of α-LA at the suggested times. The cell-free conditioned culture medium was collected and analyzed with ELISA for TNF-α, IL-6. Data from three independent experiments are presented as means ± S.D. *≤ 0.05, **< 0.01, ***< 0.001 and are related to both LPS-induced cells and α-LA treated cells.

Article Snippet: Both TNF-α and IL-6 were quantitatively measured through an enzyme-linked immunosorbent assay (ELISA) using the mouse TNF-α and IL-6 DuoSet ELISA kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, MTT Assay, Enzyme-linked Immunosorbent Assay