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: PLoS ONE

Article Title: Immune response to recombinant Burkholderia pseudomallei FliC

doi: 10.1371/journal.pone.0198906

Figure Lengend Snippet: (A) HEK-Blue TM -hTLR5 cells (1.4 x 10 5 cells/ml) in HEK-Blue™ Detection medium were incubated with 1, 10, and 100 ng/ml rFliC in three independent experiments. The cells stimulated with flagellin purified from S . Typhimurium (FLA-ST) were used for comparison. After incubation for 24 h, NF-κB activation was determined by monitoring SEAP production. (B) THP1-Dual TM cells (5.6 x10 5 cells/ml) were added into a 96-well plate containing 20 μl of rFliC at indicated concentrations. The supernatant was collected at 24 h after incubation and SEAP activity was measured using the Quanti-Blue assay. The result of THP-1 cell assay was obtained from three independent experiments. Data represent the mean, and error bars represent the standard deviation of the results of three independent experiments conducted in triplicate.

Article Snippet: HEK-Blue TM -hTLR5 cells (Invivogen, USA) were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 μg/ml of Normocin (Invivogen, USA), 30 μg/ml of blasticidin (Invivogen, USA) and 100 μg/ml of Zeocin (Invivogen, USA).

Techniques: Incubation, Purification, Comparison, Activation Assay, Activity Assay, Standard Deviation, Reporter Assay

Journal: iScience

Article Title: Flagellin in blood and Bifidobacterium pseudocatenulatum in gut are associated with live birth upon IVF-frozen embryo transfer

doi: 10.1016/j.isci.2025.111933

Figure Lengend Snippet:

Article Snippet: Serum flagellin level was measured using the HEK-DualTM-hTLR5 reporter cell line (InvivoGen, San Diego, USA) according to the manufacturer’s instructions as described previously.

Techniques: Recombinant, Purification, LAL Assay, Sequencing, Software

Journal: Cell reports

Article Title: Toll-like Receptor 5 Activation by the CagY Repeat Domains of Helicobacter pylori.

doi: 10.1016/j.celrep.2020.108159

Figure Lengend Snippet: Figure 1. Stimulation of TLR5 by Worldwide Hp Strains (A) Hp strains from different continents display a functional T4SS permitting CagA delivery and phosphorylation. Gastric AGS cells were infected in parallel as control. (B) Activation of TLR5 in HEK reporter cells was quantified using NF-kB luciferase reporter gene assays. Data are represented as mean ± SEM. (C) CagA translocation and phosphorylation in AGS cells with T4SS-positive Hp strain G27 involve functional cagA, cagY, and cagL but not flaA, slt, and gmhA genes. (D) Activation of TLR5 in HEK cells using NF-kB luciferase reporter assay depends on functional cagY and cagL. Data are represented as mean ± SEM. (E) SEAP reporter assay of TLR5+ and parental cells infected with strain P1 and isogenic mutants of various known virulence genes. The results confirm that DcagL and DcagY play a role in TLR5 activation but not other T4SS-independent virulence genes. Data are represented as mean ± SEM. (F) IP of cell lysates using a-TLR5 antibodies revealed that CagY and CagL interact with TLR5 on TLR5+ cells but not other cagPAI proteins as control. The data are representative of three independent experiments.Significant differences were defined by p % 0.05 (*), p % 0.01 (**), p % 0.001 (***) and p % 0.0001 (****). n.s. not significant See also Figure S1.

Article Snippet: SEAP reporter gene assay TLR5 activation was also examined by using human TLR5/NF-kB/SEAP reporter HEK293 cells (HEK-Blue-hTLR5) and HEK-Blue Null1 cells (Invivogen) as control.

Techniques: Functional Assay, Phospho-proteomics, Infection, Control, Activation Assay, Luciferase, Translocation Assay, Reporter Assay

Journal: Cell reports

Article Title: Toll-like Receptor 5 Activation by the CagY Repeat Domains of Helicobacter pylori.

doi: 10.1016/j.celrep.2020.108159

Figure Lengend Snippet: Figure 2. T4SS-Pilus Associated Protein CagY of Hp Can Directly Bind to TLR5 (A) Co-localization of Hp (GFP, green) with TLR5 (red) as judged by confocal immunofluorescence microscopy of parental and TLR5+ HEK293 cells counter- stained with DAPI (blue). Arrows indicate TLR5 signals in proximity to Hp. Dotted lines indicate the cell periphery. Scale bars represent 5 mm. (B) Schematic domain structure of the CagY protein. The shown C-terminal (C-term) B10 domain of the CagY protein shares homology with Agrobacterium tumefaciens VirB10. CagY contains two large repeat regions, NRR (red) and MRR (green). Amino-acid positions and purified fragments from two Hp strains (Q86A and 13A) are indicated. (legend continued on next page)

Article Snippet: SEAP reporter gene assay TLR5 activation was also examined by using human TLR5/NF-kB/SEAP reporter HEK293 cells (HEK-Blue-hTLR5) and HEK-Blue Null1 cells (Invivogen) as control.

Techniques: Microscopy, Staining

Journal: bioRxiv

Article Title: Divergence of the Individual repeats in the leucine-rich repeat domains of human Toll-like receptors explain their diversity and functional adaptations

doi: 10.1101/2024.09.30.615863

Figure Lengend Snippet: Examples of the regular and irregular LRR domains. Left Panels: An example of a regular LRR domain, the porcine ribonuclease inhibitor (PDB:2BNH), the first LRR protein with an experimentally solved structure. The LRR domain of the RNI structure is superimposed over a 3D model of a regular torus. The repeats recognized by an Interpro/Pfam HMM models are highlighted in blue and magenta which are used to characterize different subtypes of LRR repeats – however the classification does not correlate with structural similarity between repeats. 8 repeats out of 17 repeats are recognized by InterPro/Pfam HMMs. The alignment of three consecutive repeats (86-141) with the central LxL pattern are highlighted on the bottom panel. Right panels: Analogous figures for an example of highly irregular LRR domain of hTLR5 (PDB:3J0A). The LRR domain of hTLR5 LRR is superimposed with an ellipsoid based torus which shows a much greater radius and local curvature divergence than the torus used in RI. The repeats recognized by an Interpro/Pfam HMM models are highlighted in blue and magenta with different colors used to identify different subtypes of LRR repeats – however the classification does not correlate with structural similarity between repeats. Only 7 out of 21 repeats are recognized by InterPro/Pfam HMMs in hTLR5. The repeats identified often overlap in position across LRR subtypes and are inconsistent in length ranging from 19-59 with a mean of 30.5. For reference, manual length analysis showed a range of 20-36 with a mean of 25.9. The alignment of three consecutive repeats (498-560) with the central LxL pattern are highlighted on the bottom panel.

Article Snippet: As shown in , for the hTLR5 InterPro/Pfam HMMs ( ) needed 2, out of 16 defined in the database, different HMM models to recognize individual repeats, and even then, more than 1⁄2 of the repeats in most LRR proteins are still not recognized.

Techniques:

Journal: bioRxiv

Article Title: Divergence of the Individual repeats in the leucine-rich repeat domains of human Toll-like receptors explain their diversity and functional adaptations

doi: 10.1101/2024.09.30.615863

Figure Lengend Snippet: Thumbnail figures of the three-dimensional structures of human TLR 1-10, illustrating the distortions from the ideal solenoid structure (RNI). Local curvature moving averages (interval of three, see the Methods section) and repeat lengths are shown the boxes below. Full data available in the supplemental materials. AlphaFold models of the structures of hTLR5, hTLR6, hTLR9 and hTLR10 are shown, as no experimental structures were available as of this writing.

Article Snippet: As shown in , for the hTLR5 InterPro/Pfam HMMs ( ) needed 2, out of 16 defined in the database, different HMM models to recognize individual repeats, and even then, more than 1⁄2 of the repeats in most LRR proteins are still not recognized.

Techniques:

Journal: Oncogene

Article Title: Mobilan: a recombinant adenovirus carrying Toll-like receptor 5 self-activating cassette for cancer immunotherapy

doi: 10.1038/onc.2017.346

Figure Lengend Snippet: Adenoviral constructs and their characterization in vitro . ( A ) Schematic representation of expression cassettes in (a) Mobilan M-0, (b) Mobilan M-VM3, and (c) Ad-mCherry. P, promoter, T, transcription terminator. ( B ) Western blot analysis of Mobilan-directed protein expression in MOSEC cells. (a) Detection with anti-TLR5 antibody; lysates from uninfected (lane 1) and M-0-infected MOSEC cells (lane 2). (b) Detection with rabbit anti-CBLB502 pAb; lysates from M-0-infected MOSEC cells left untreated (lane 1) or treated with a mixture of deglycosylation enzymes (lane 2). ( C ) Activity comparison of E. coli -produced CBLB502 and CBLB502NQs in HEK293-NF-κB-lacZ reporter cells. Cells were incubated with purified proteins for 24 h; β-galactosidase activity (OD414) was measured in cell lysates using ONPG substrate. ( D ) Activity comparison of CBLB502s and CBLB502NQs produced in MOSEC cells. MOSEC cells were infected with M-0 or M-VM3, medium was collected after 48 h and the concentrations of CBLB502s and CBLB502NQs were measured by ELISA after heat-inactivation of residual adenovirus. The indicated amounts of MOSEC-produced proteins or E. coli -produced CBLB502 standard were applied to HEK293-NF-κB-lacZ cells and β-galactosidase was measured as above. ( E ) Western blot analysis of MOSEC cells infected with Ad-mCherry (lane 1), M-0 (lane 2) or M-VM3 (lane 3) using rabbit anti-CBLB502 pAb. The protein with slower mobility in lane 3 is the expected size of unglycosylated CBLB502NQs (31.5 kDa); the faster mobility protein is presumably a partially degraded form of CBLB502NQs. ( F ) Infection with M-VM3 (MOI=3 × 10 4 ) induces NF-κB p65 nuclear translocation in TLR5-negative MOSEC cells 24 h after infection compared with control uninfected MOSEC cells. Antibodies against NF-κB p65 were used for immunostaining. ( G ) Induction of NF-κB-dependent luciferase expression in TRAMP-C2 cells infected by M-VM3. TRAMP-C2 cells carrying an NF-κB-dependent luciferase reporter construct were infected with M-VM3 or Ad-mCherry at the indicated MOIs. Luciferase activity was measured in lysates prepared 48 h post-infection and is shown as a percentage of that in uninfected cells (set at 100%). ( H ) Effect of neutralizing anti-CBLB502 antibodies on TLR5 signaling in M-VM3-infected MOSEC-NF-κB-luciferase reporter cells. Cells were infected with M-VM3 in the presence or absence of an excess of neutralizing rabbit anti-CBLB502 pAb. Luciferase activity was measured after 80-h incubation. Graphs show mean±s.d. of triplicate measurements.

Article Snippet: Rabbit anti-CBLB502 polyclonal antibody (pAb), biotin-labeled goat anti-CBLB502 pAb and 1B04 anti-human TLR5 monoclonal antibody (mAb) were produced by Cleveland BioLabs, Inc. (CBLI; Buffalo, NY, USA).

Techniques: Construct, In Vitro, Expressing, Western Blot, Infection, Activity Assay, Comparison, Produced, Incubation, Purification, Enzyme-linked Immunosorbent Assay, Translocation Assay, Control, Immunostaining, Luciferase

Journal: PLoS ONE

Article Title: Impact of cagPAI and T4SS on the Inflammatory Response of Human Neutrophils to Helicobacter pylori Infection

doi: 10.1371/journal.pone.0064623

Figure Lengend Snippet: Mean fluorescence intensity (MFI) of TLR5 on neutrophils 3, 6, and 24 h after infection (A, B, and C, respectively) with cagPAI + strain 26695, T4SS mutants ( virB4 – and virD4 – ), or cagPAI – strain 8822. The data shown are from three independent experiments and line represent median. The results were obtained as medians ± intervals of percentiles and analyzed using the Kruskal–Wallis one-way test. Differences were considered statistically significant when *P<0.05. MC = mock control.

Article Snippet: Another set of neutrophils was incubated with PE-conjugated anti-human TLR2 and TLR4 (Santa Cruz Biotechnology, Santa Cruz, CA) and fluorescein-isothiocyanate-conjugated anti-human TLR5 (Santa Cruz Biotechnology) and anti-human TLR9 (Imgenex Corp., San Diego, CA).

Techniques: Fluorescence, Infection, Control