Journal: Biomaterials

Article Title: An all-in-one adjuvanted therapeutic cancer vaccine targeting dendritic cell cytosol induces long-lived tumor suppression through NLRC4 inflammasome activation.

doi: 10.1016/j.biomaterials.2022.121542

Figure Lengend Snippet: Fig. 2. Generation and characterization of a protein-based all-in-one cancer vaccine (DCpep6-4xE7ΔNLS-FlaB; DEF). (A) The vector maps and amino acid sequences of recombinant proteins are shown; in vivo DC-targeting peptide (DCpep6), tumor antigen (E7ΔNLS), and built-in flagellin adjuvant (FlaB). The E7ΔNLS tumor antigen was prepared by deleting the N-terminal nuclear localization sequence (NLS) of the HPV16 E7 protein. An all-in-one type TCV containing DCpep6, HPV E7ΔNLS and built-in FlaB adjuvant was constructed by recombinant DNA technology in the pET30a+ plasmid. 4xE7ΔNLS-FlaB (EF) was also generated as a non- CD11c+ cell targeting TCV. (B) Characterization of the recombinant proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and sub sequent Western blot analysis using mouse anti-FlaB or anti-E7 serum. Anti-FlaB or anti-E7 serum was induced by intraperitoneal immunization of FlaB or E7FL with complete Freund’s adjuvant. (C) Selection of optimal tumor antigen (E7ΔNLS) for the all-in-one cancer vaccine. C57BL/6 mice were implanted with TC-1 cells in the right midflank. When the tumor size reached approximately 3–5 mm in diameter, the tumor-bearing mice were subcutaneously vaccinated with 200 μl of PBS only (PBS; n = 5), 4 μg of FlaB (F; n = 5), 10 μg of E7 full length (E7FL; n = 5), 10 μg of E7FL plus 4 μg of FlaB (E7FL+F; n = 5), 8 μg of E7ΔNLS (E; n = 5), and 8 μg of E7ΔNLS plus 4 μg of FlaB (E+F; n = 5) in the peritumoral region three times at five-day intervals. The statistical significance of tumor suppression was calculated by two-way ANOVA. (D) Determination of TLR5-dependent NF-κB stimulating activity by FlaB, EF or DEF. The relative NF-κB activities were analyzed by using HEK- Blue™ hTLR5 cells and HEK-Blue™ detection assay systems. (E) Determination of dose-dependent cellular uptake of DEF in CD11c+ mouse BMDCs by FACS. BMDCs were incubated with various concentrations of EF or DEF for 2 h, and then intracellular localization of the recombinant proteins was determined by FACS analysis using anti-FlaB serum gating upon CD11c+ cell population. (F) Determination of the intracellular localization of DEF in BMDCs or RAW264.7 cells by confocal microscopic observation. BMDCs or RAW264.7 cells were incubated with 20 μg/ml of EF or DEF for 2 h, and intracellular localization of the recombinant proteins was determined by confocal microscopic observation. (G) Determination of energy-dependent cellular uptake of DEF in CD11c + cells. BMDCs were incubated with 20 μg/ml of DEF at 37 ◦C, 4 ◦C, or 37 ◦C in the presence of 0.1% NaN3, or 37 ◦C in the presence of 2 μg/ml cytochalasin B (CB) for 2 h, and then intracellular localization of the recombinant proteins was determined by FACS analysis gating upon CD11c+ cell population. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ns, nonsignificant.

Article Snippet: To test whether the EF or DEF proteins maintain TLR5-stimulating activities, we measured TLR5-dependent NF-κB-stimulating activity of the recombinant proteins by using HEK-BlueTM hTLR5 cells (InvivoGen, hκb-htlr-5) and HEK-BlueTM Detection (InvivoGen, hb-det2) assay systems following the manufacturer’s instructions.

Techniques: Plasmid Preparation, Recombinant, In Vivo, Adjuvant, Sequencing, Construct, Generated, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Selection, Activity Assay, Detection Assay, Incubation

Journal: Inflammation Research

Article Title: BacSp222 bacteriocin as a novel ligand for TLR2/TLR6 heterodimer

doi: 10.1007/s00011-023-01721-3

Figure Lengend Snippet: All tested forms of bacteriocin, BacSp222, suc-K20-BacSp222 and -fM-BacSp222, activate TLR2 receptor. HEK-Blue hTLR2, hTLR4 and hTLR5 cells were incubated in medium alone (negative control) or stimulated with BacSp222, suc-K20-BacSp222, -fM-BacSp222, or with positive control agonist (HKLM in case of HEK-Blue hTLR2, LPS for HEK-Blue hTLR4, or flagellin for HEK-Blue hTLR5). After this the SEAP activity was measured in culture media after colorimetric reaction with the Cell Culture Medium for SEAP reagent. The absorbance measured for negative controls (cells incubated with medium without additives) was subtracted from the absorbance measured for subsequent samples. The bars represent the mean ± SD ( n = 3), # p < 0.001 vs positive control

Article Snippet: HEK-Blue hTLR2 and HEK-Blue hTLR6 cells were seeded on a 96-well plate at density 2.5 × 10 4 cells per well in 100 μL DMEM containing 10% (v/v) FBS, 50 units/mL penicillin, 50 μg/mL streptomycin (control) or in 100 μL DMEM containing 10% (v/v) FBS, 50 units/mL penicillin, 50 μg/mL streptomycin and (1) 20 μM sparstolonin B, (2) 200 μM TL2-C29 (InvivoGen, San Diego, CA, USA).

Techniques: Incubation, Negative Control, Positive Control, Activity Assay, Cell Culture

Journal: Inflammation Research

Article Title: BacSp222 bacteriocin as a novel ligand for TLR2/TLR6 heterodimer

doi: 10.1007/s00011-023-01721-3

Figure Lengend Snippet: All forms of BacSp222 activate hTLR2/TLR6 heterodimers. a HEK-Blue hTLR2 and hTLR1or b HEK-Blue hTLR2 and hTLR6 were incubated with medium alone or stimulated with bacteriocins, FSL-1 (positive control for HEK-Blue hTLR2 and hTLR6) or CU-T12-9 (positive control for HEK-Blue hTLR2 and hTLR1). SEAP activity was measured colorimetrically in culture media after reaction with the cell culture medium for SEAP reagent. The absorbance measured for negative controls (the cells incubated with medium without additives) was subtracted from the absorbance measured for subsequent samples. c , d Viability of the cells was analyzed with the MTT method. The bars represent the mean ± SD ( n = 3), # p < 0.001 vs positive control

Article Snippet: HEK-Blue hTLR2 and HEK-Blue hTLR6 cells were seeded on a 96-well plate at density 2.5 × 10 4 cells per well in 100 μL DMEM containing 10% (v/v) FBS, 50 units/mL penicillin, 50 μg/mL streptomycin (control) or in 100 μL DMEM containing 10% (v/v) FBS, 50 units/mL penicillin, 50 μg/mL streptomycin and (1) 20 μM sparstolonin B, (2) 200 μM TL2-C29 (InvivoGen, San Diego, CA, USA).

Techniques: Incubation, Positive Control, Activity Assay, Cell Culture

Journal: Inflammation Research

Article Title: BacSp222 bacteriocin as a novel ligand for TLR2/TLR6 heterodimer

doi: 10.1007/s00011-023-01721-3

Figure Lengend Snippet: The effect of TLR2 inhibitors on BacSp222-induced activation of hTLR2/TLR6 heterodimer. a HEK-Blue hTLR2 and hTLR6 were activated with BacSp222 or FSL-1 (positive control) in media without antagonist or in media containing sparstolonin B or TL2-C29. SEAP activity was measured in culture media and indicates receptor stimulation. b The possible toxicity of inhibitors was excluded using the MTT method. The bars represent the mean ± SD ( n = 3), # p < 0.001 and * p < 0.05 vs cells stimulated without antagonist

Article Snippet: HEK-Blue hTLR2 and HEK-Blue hTLR6 cells were seeded on a 96-well plate at density 2.5 × 10 4 cells per well in 100 μL DMEM containing 10% (v/v) FBS, 50 units/mL penicillin, 50 μg/mL streptomycin (control) or in 100 μL DMEM containing 10% (v/v) FBS, 50 units/mL penicillin, 50 μg/mL streptomycin and (1) 20 μM sparstolonin B, (2) 200 μM TL2-C29 (InvivoGen, San Diego, CA, USA).

Techniques: Activation Assay, Positive Control, Activity Assay