Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis inhibits autocrine type I interferon signaling to increase intracellular survival.

doi: 10.4049/jimmunol.1801303

Figure Lengend Snippet: (A) Schematic representation of experiment. (B) IRF-3 deficient RAW264.7 macrophages transfected with an IFN-β responsive luciferase gene (Invivogen) were treated with 20μg/ml of the indicated neutralizing antibodies. 1ng/ml IFN-β was added to the cells every 24 hours, and the amount of secreted luciferase was quantified at the indicated timepoints. (C-D) Ifnβ−/− BMDMs were infected Mtb H37Rv at MOI of 3 for 4h and treated with IFN-β as in Figure 1. (C) CFUs were enumerated at the indicated timepoints. (D) Nitrite levels from culture supernatants were determined using the Griess reagent. (E-F) C57BL6/6 or Ifnar1−/− BMDMs were infected with Mtb H37Rv at MOI of 3 for 4h and treated with IFN-β as in Figure 1. (E) CFUs were enumerated at the indicated timepoints. (F) Nitrite levels from culture supernatants were determined using the Griess reagent. All data shown are presented as mean ± S.E.M. of at least three independent experiments.

Article Snippet: Experiments with neutralizing antibodies were performed using InVivo Plus anti-mouse IFNAR1 and InVivo mAB mouse IgG1 isotype control (Bio Xcell).

Techniques: Transfection, Luciferase, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis inhibits autocrine type I interferon signaling to increase intracellular survival.

doi: 10.4049/jimmunol.1801303

Figure Lengend Snippet: (A) Ifn-β−/− BMDMs were infected with Mtb H37Rv at MOI of 3 for 4h and flow cytometry was conducted at 4hpi to measure surface receptor expression levels of IFNAR1 and IFNAR2. (B and C) Ifn-β−/− BMDMs were infected as described in the presence of 300pg/ml IFN-β. Cell lysates were collected at 20 min post infection and immunoblotted for pJAK1 (Y1022/1023), total JAK1, pTYK2 (Y1054/1055), and total TYK2. (D and E) Ifn-β−/− BMDMs were infected as described in the presence of 300pg/ml IFN-γ. Cell lysates were collected at 20 min post infection and immunoblotted for pJAK1 (Y1022/1023), total JAK1, pTYK2 (Y1054/1055), and total TYK2. (F) Ifn-β BMDMs were treated with DMSO or increasing amounts of a JAK inhibitor for 1 hour, then stimulated with 200pg/ml IFN-β for 4 hours. Cell lysates were then collected and immunoblotted for pSTAT1 (Y701), total STAT1, and β-actin. Densitometry was performed using ImageJ software and phosphorylated protein bands were normalized to loading control for each condition. Densitometric ratios are relative to the UI +IFNβ or the UI +IFNγ conditions. Data and densities shown represent one representative experiment out of three.

Article Snippet: Experiments with neutralizing antibodies were performed using InVivo Plus anti-mouse IFNAR1 and InVivo mAB mouse IgG1 isotype control (Bio Xcell).

Techniques: Infection, Flow Cytometry, Expressing, Software

Journal: Nature Communications

Article Title: Modular-designed engineered bacteria for precision tumor immunotherapy via spatiotemporal manipulation by magnetic field

doi: 10.1038/s41467-023-37225-1

Figure Lengend Snippet: A Serum levels of TNF-α, IL-6, IFN-γ, and IFN-α1, as analyzed by ELISA on day 9 in the indicated groups introduced in Fig. ( n = 5). Cytokine concentration of each sample was normalized to Z-score (i.e., standard score), which was calculated as ( X – E[ X ])/σ[ X ]. X , the cytokine concentration of the individual sample; E[ X ], the average concentration of all samples; σ[ X ], the standard deviation of cytokine concentrations of all samples. B Ifna mRNA expression levels in tumor-infiltrating DCs, as determined by RT-qPCR on day 9 ( n = 5 mice). C Scheme and grouping of in vivo therapy to evaluate the role of the type I IFN signaling pathway and CD8 + T cells. The colons of BALB/c mice were inoculated with CT-26-luc cells on day -7, and the mice were treated with engineered bacteria (1 × 10 8 CFU) by colon-specific administration on day 0 and day 3, followed by AMF treatment (310 kHz and 23.8 kA/m) for 80 min at 24 h after colon administration. Anti-CD8 (15 mg/kg, clone TIB210) or anti-IFNAR1 (15 mg/kg, clone R46A2) neutralization antibodies were i.p . injected on days −9, −6, −3, 0, and 3. D Bioluminescence imaging was performed to evaluate tumor growth on day 0, 7, and 14 ( n = 5). E Survival curves of mice from the indicated groups for 80 days ( n = 5 mice). F Semi-quantitative results of bioluminescence intensity in the tumor region on day 14, which were normalized to day 0 ( n = 5 mice). G Schematic illustration of the type I IFN pathway and adaptive immunity activated by AMF-Bac. TLRs, Toll-like receptors. The data ( B , E , F ) are shown as the mean ± SD. Statistical analysis was performed by a two-tailed unpaired t test. Survival significance was analyzed by the log-rank test. * P < 0.05; ** P < 0.01 ; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: The anti-mouse CD47 antibody (catalog No. BE0270, clone name: MIAP301, 1: 1000 for in vitro use, 20 mg/kg for in vivo use), anti-mouse CD8 antibody (catalog No. BE0061, clone name: 2.43, 15 mg/kg for in vivo use), anti-mouse IFNAR1 antibody (catalog No. BE0241, clone name: MAR1-5A3, 15 mg/kg for in vivo use), rat anti-mouse CSF1 neutralizing antibody (catalog No. BE0204, Clone: 5A1, 100 μg/mouse for in vivo use), rat IgG1 isotype control (catalog No. BE0088, Clone: HRPN, 100 μg/mouse for in vivo use), and anti-FcR antibody (catalog No. BE0307, clone name: 2.4G2, 1.0 μg per 1000000 cells in 100 μL volume) were purchased from BioXcell (USA).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation, Expressing, Quantitative RT-PCR, In Vivo, Bacteria, Neutralization, Injection, Imaging, Two Tailed Test