Journal: Communications Chemistry

Article Title: Synthesis of Acinetobacter baumannii Lipid A(s) and derivatives and their structure-immunostimulatory activity relationships

doi: 10.1038/s42004-025-01628-6

Figure Lengend Snippet: A Inverted binding mode of lipid A moiety of E. coli LPS in TLR4/MD-2 complex. B . Inverted binding mode of A. baumannii acyl 6 lipid A ( 2 ) in TLR4/MD-2 complex. C . Retained binding mode of IV A ( 6 ) in MD-2 receptor.

Article Snippet: To confirm the agonistic lipid A(s) to be TLR4 targeting, THP1-DualTM cells were treated with 5 μg mL −1 of anti- h TLR4 antibody (InvivoGen, San Diego) and mouse IgG1 isotype control antibody (the negatiuve control; InvivoGen, San Diego) separately for 2 h. The antibody-pretreated THP-1 cells were then subjected to activation with each of the E. coli lipid A 5, A. baumannii acyl 7 1 , acyl 6 2 at different concentrations for 20 h. Comparison with the treatment with the control antibody, the treatment with the anti- h TLR4 antibody completely abolished the NF-κB activation (See Supplementary Fig. ).

Techniques: Binding Assay

Journal: Communications Chemistry

Article Title: Synthesis of Acinetobacter baumannii Lipid A(s) and derivatives and their structure-immunostimulatory activity relationships

doi: 10.1038/s42004-025-01628-6

Figure Lengend Snippet: A Binding modes of lipid A moiety of E. coli LPS. B Binding mode of A. baumannii acyl 6 lipid A ( 2 ). C Binding mode of A. baumannii acyl 7 lipid A ( 1 ). D Binding mode of E. coli acyl 6 lipid A ( 5 ). E Binding mode of A. baumannii symmetric acyl 6 lipid A derivative ( 4 ). F Binding mode of S . Typhi acyl 7 lipid A ( 7 ). Cationic residues of TLR4, MD-2, and TLR4* are shown in blue, wheat, and green, respectively. The distances between the PO 4 groups and the residues are depicted by black dotted lines.

Article Snippet: To confirm the agonistic lipid A(s) to be TLR4 targeting, THP1-DualTM cells were treated with 5 μg mL −1 of anti- h TLR4 antibody (InvivoGen, San Diego) and mouse IgG1 isotype control antibody (the negatiuve control; InvivoGen, San Diego) separately for 2 h. The antibody-pretreated THP-1 cells were then subjected to activation with each of the E. coli lipid A 5, A. baumannii acyl 7 1 , acyl 6 2 at different concentrations for 20 h. Comparison with the treatment with the control antibody, the treatment with the anti- h TLR4 antibody completely abolished the NF-κB activation (See Supplementary Fig. ).

Techniques: Binding Assay

Journal: RSC Chemical Biology

Article Title: Signals from the sea: the structural peculiarity of lipid A and weak immunostimulatory lipopolysaccharide from Rheinheimera japonica †

doi: 10.1039/d5cb00134j

Figure Lengend Snippet: Stimulation of HEK-Blue™ hTLR4 (A) and HEK-Blue™ hTLR2 (B) cells. SEAP levels (OD 620 nm) following stimulation with 1, 10, and 100 ng mL −1 of E. coli O111:B4 LPS ( E. coli LPS) or R. japonica KMM 9513 T ( R. japonica ) LPS were evaluated using the Quanti-Blue assay. Data are shown as mean ± standard deviation. Significant differences between R. japonica LPS and E. coli LPS (**** p -value <0.0001) and between PAM3CSK4 and NS ( ### p -value <0.001) were evaluated via Student's t test. (C) and (D) Competition assays between R. japonica LPS and E. coli LPS for human TLR4. Fold of NF-kB activation in HEK Blue hTLR4 cells primed with the potent TLR4 antagonist Rhodobacter sphaeroides (RS) or R. japonica LPS (0.1–1–10–100 ng mL −1 ) for 4 hours and then stimulated with 100 ng mL −1 (C) or 10 ng mL −1 (D) of E. coli LPS for 16 hours. Data are expressed as a percentage versus 10 ng mL −1 or 100 ng mL −1 E. coli LPS values considered as 100% and shown as mean ± standard deviation. Significant differences between R. japonica or RS LPS and E. coli LPS (** p -value <0.01; *** p -value <0.001; **** p -value <0.0001 vs E. coli LPS) were evaluated via Student's t test.

Article Snippet: In addition, Normocin, Blasticidin, HEK selection, E. coli O111:B4 LPS ( E. coli LPS), Rhodobacter sphaeroides (RS) LPS (#tlrl-prslps), Quanti-Blue assay, PAM3CSK4 and anti-hTLR4 neutralization antibody (# mabg-htlr4-2) were obtained from InvivoGen (Toulouse, France).

Techniques: Standard Deviation, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Recombinant SARS-CoV-2 Spike Protein Stimulates Secretion of Chymase, Tryptase, and IL-1β from Human Mast Cells, Augmented by IL-33

doi: 10.3390/ijms24119487

Figure Lengend Snippet: Full-length SARS-CoV-2 spike stimulates IL-1β secretion from human mast cells via TLR4 signaling. LADR mast cells (2.5 × 10 5 cells) were pretreated with anti-TLR2 Ab (2 μg/mL), anti-TLR4 Ab (2 μg/mL), or anti-ACE2 Ab (2 μg/mL) for 1 h and then stimulated with recombinant full-length SARS-CoV-2 S (10 ng/mL) for 1 h and 24 h, respectively. Secretion of chymase ( A ), tryptase ( B ) , and IL-1β ( C ) was determined by specific ELISAs. All conditions were performed in triplicate for each dataset and repeated 3 times ( n = 3). The horizontal brackets indicate the corresponding levels of significance when present ( p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****)).

Article Snippet: LADR Mast Cell Treatments : LADR mast cells were stimulated with recombinant full-length SARS-CoV-2 S (1–10 ng/mL; ab281471, Abcam, Cambridge, UK) or RBD (1–10 ng/mL; ab273065, Abcam, Cambridge, UK), and/or preincubated with the following: (1) anti-TLR2 antibody (2 μg/mL, 1 h; maba2-htlr2, InvivoGen, San Diego, CA, USA), (2) anti-TLR4 antibody (2 μg/mL, 1 h; mabg-htlr4, InvivoGen, San Diego, CA, USA) and (3) anti-ACE2 antibody (2 μg/mL, 1 h; ab108209, InvivoGen, San Diego, CA, USA).

Techniques: Recombinant

Journal: Journal of Biological Methods

Article Title: Utilizing a human TLR selective ligand in a humanized immune system mouse model to investigate human TLR4 signaling

doi: 10.14440/jbm.2023.408

Figure Lengend Snippet: AZ617 is a human-specific, TLR4-dependent agonist that activates NFκB. A. Stimulation of human PBMCs, but not mouse splenocytes, with AZ617 for 24 hours effected a concentration-dependent release of NFκB-related cytokines IFNγ, IL-1β, TNFα, and IL-6. Representative graphs generated from 4 independent experiments. B. AZ617 activity was reduced when human PBMCs were pre-treated with an anti-human TLR4 blocking reagent for one hour prior to stimulation. Representative graphs derived from 2 independent experiments. Unpaired t-test **p#x003C;0.01, ***p#x003C;0.001. Error bars represent standard error of the mean (SEM).

Article Snippet: For TLR4-dependency studies, human PBMCs were pre-incubated with 12.5 μg/mL anti-human TLR4 blocking antibody (Invivogen, mabg-htlr4) for 1 hour at 37ºC prior to stimulation with 50 ng/mL AZ617 for an additional 24 hours.

Techniques: Concentration Assay, Generated, Activity Assay, Blocking Assay, Derivative Assay

Journal: Cancers

Article Title: ATP128 Clinical Therapeutic Cancer Vaccine Activates NF-κB and IRF3 Pathways through TLR4 and TLR2 in Human Monocytes and Dendritic Cells

doi: 10.3390/cancers14205134

Figure Lengend Snippet: Functionality of KISIMA vaccine. ( A ) Scheme of ATP128 constructs. ATP128 full length (FL) is comprised of a Cell Penetrating Peptide (CPP) at the N-terminus, a Multiantigenic domain (Mad), and a TLR agonist at the C-terminus (Anaxa). ( B ) THP-1 Dual cells were incubated with increasing concentrations of ATP128 vaccine constructs. After 18 h, supernatant was recovered and either SEAP activity was measured by QUANTI-Blue assay (left panel), or Luciferase activity was measured by QUANTI-Luc assay (right panel). The EC50s of the different constructs were calculated from the obtained dose-response curve using the GraphPad Prism software. Experiments shown here represent the average of % of activation of 3 biological replicates. ( C ) THP-1 Dual cells were incubated with 300 nM ATP128 in presence or not of blocking antibodies anti-TLR. After 18 h, cell supernatants were recovered, assessed, and represented as described above. The values reported here are the mean of 4 biological replicates. Values were compared using a one-way ANOVA test to the untreated sample (positive control—no blocking antibody). ( D ) THP-1 Dual cells wild-type (WT) or knocked-out (KO) for specific TLR were stimulated with 300 nM ATP128. After 18 h, the cell supernatants were recovered, assessed, and represented as described above. The values reported here are a mean of 3 biological replicates. Values were compared via one-way ANOVA test to the WT sample. ( E ) THP-1 Dual cells were incubated with 300 nM ATP128 in presence or not of the TLR4 inhibitor CLI-095. After 18 h, the cell supernatants were recovered, assessed, and represented as described above. The values reported here are a mean of 4 biological replicates. Values were compared via one-way ANOVA test to the untreated sample (positive control). ( F ) THP-1 Dual WT, and TLR2 KO and TLR4 KO cells were incubated with 300 nM ATP128 in presence or not of antibodies anti-TLR and CLI-095. After 18 h, the cell supernatants were recovered, assessed, and represented as described above. The values reported here are a mean of 3 biological replicates. Values were compared via a one-way ANOVA test to the untreated sample in WT cells. **** p -value < 0.0001, *** p -value < 0.001, ** p -value < 0.01, * p -value < 0.1. ns: no significance.

Article Snippet: For THP-1 activation assays: anti-mouse TLR2 (mab2-mtlr2) and anti-human TLR4 (mabg-htlr4) antibodies were purchased from InvivoGen.

Techniques: Construct, Incubation, Activity Assay, Luciferase, Software, Activation Assay, Blocking Assay, Positive Control

Journal: Cancers

Article Title: ATP128 Clinical Therapeutic Cancer Vaccine Activates NF-κB and IRF3 Pathways through TLR4 and TLR2 in Human Monocytes and Dendritic Cells

doi: 10.3390/cancers14205134

Figure Lengend Snippet: ATP128 engages mostly TLR4 for human moDC activation in an ex vivo system. ( A , B ) Human moDC were pre-treated with anti-TLR2, anti-TLR4 or CLI-095 for 1 h, then stimulated for 24 h with 300 nM ATP128. Cells were collected after stimulation, and surface costimulatory molecules ( A ) and HLA class I and II molecules ( B ) were analyzed through flow cytometry. Each shape is representative of a different buffy coat. Histograms of one representative buffy coat are shown below. Values were compared via one-way ANOVA test to the sample stimulated with ATP128 FL. **** p -value < 0.0001, * p -value < 0.05.

Article Snippet: For THP-1 activation assays: anti-mouse TLR2 (mab2-mtlr2) and anti-human TLR4 (mabg-htlr4) antibodies were purchased from InvivoGen.

Techniques: Activation Assay, Ex Vivo, Flow Cytometry

Journal: Cancers

Article Title: ATP128 Clinical Therapeutic Cancer Vaccine Activates NF-κB and IRF3 Pathways through TLR4 and TLR2 in Human Monocytes and Dendritic Cells

doi: 10.3390/cancers14205134

Figure Lengend Snippet: ATP128 activates human moDCs in a profile similar to the TLR4 agonist, MPLA, in an ex vivo system. ( A , B ) Human moDC were incubated for 24 h with 300 nM ATP128 or 600 nM MPLA or 1 ng/mL FSL-1. Cells were collected after stimulation and surface costimulatory molecules ( A ) and HLA class I and class II molecules ( B ) were analyzed through flow cytometry. Each shape is representative of a different buffy coat. Histograms of one representative buffy coat are shown below. Values were compared via one-way ANOVA test to the sample stimulated with ATP128 FL., *** p -value < 0.001, ** p -value < 0.01, * p -value < 0.05.

Article Snippet: For THP-1 activation assays: anti-mouse TLR2 (mab2-mtlr2) and anti-human TLR4 (mabg-htlr4) antibodies were purchased from InvivoGen.

Techniques: Ex Vivo, Incubation, Flow Cytometry

Journal: Cancers

Article Title: ATP128 Clinical Therapeutic Cancer Vaccine Activates NF-κB and IRF3 Pathways through TLR4 and TLR2 in Human Monocytes and Dendritic Cells

doi: 10.3390/cancers14205134

Figure Lengend Snippet: ATP128 induces cytokines release upon activation of both the NF-κB and IRF3 pathways. ( A ) Human moDC were pre-treated with anti-TLR2, anti-TLR4, or CLI-095 for 1 h, then stimulated for 6 h or 24 h (for IL-12) with 300 nM ATP128. Cell supernatants were collected to assess the signature of cytokines secretion upon ATP128 stimulation through a multiplex cytokine assay. The values in the graphs represent the fold increase of the stimulated samples to the medium. Each shape is representative of a different buffy coat. Represented are the cytokines that are mainly induced upon stimulation. Values were compared via one-way ANOVA test to the sample stimulated with ATP128 FL. ( B ) Human moDC were incubated for 6 h or 24 h (for IL-12) with 300 nM ATP128 or 600 nM MPLA or 1 ng/mL FSL-1. Cell supernatants were collected to assess the signature of cytokines secretion through a multiplex cytokine assay (same as above). The values in the graphs represent the fold increase of the stimulated samples to the medium. Each shape is representative of a different buffy coat. Represented are the cytokines that are mainly induced upon stimulation. Values were compared via one-way Anova test to the sample treated with the buffer (negative control *** p -value < 0.001, ** p -value < 0.01, * p -value < 0.05.

Article Snippet: For THP-1 activation assays: anti-mouse TLR2 (mab2-mtlr2) and anti-human TLR4 (mabg-htlr4) antibodies were purchased from InvivoGen.

Techniques: Activation Assay, Multiplex Assay, Cytokine Assay, Incubation, Negative Control

Journal: Cancers

Article Title: ATP128 Clinical Therapeutic Cancer Vaccine Activates NF-κB and IRF3 Pathways through TLR4 and TLR2 in Human Monocytes and Dendritic Cells

doi: 10.3390/cancers14205134

Figure Lengend Snippet: Both CPP and Anaxa are essential for human moDC activation. ( A ) Human moDC were pre-treated with a combination of anti-TLR2 and anti-TLR4 for 1 h, then stimulated for 24 h with 300 nM ATP128 FL, ATP128 w / o CPP, or ATP128 w / o Anaxa. Cells were collected after stimulation and surface costimulatory molecules ( A ) and HLA class I and class II molecules ( B ) were analyzed through flow cytometry. Each shape is representative of a different buffy coat. Histograms of one representative buffy coat are shown below. Values were compared via one-way ANOVA test to the sample stimulated with ATP128 FL. **** p -value < 0.0001, *** p -value < 0.001, ** p -value < 0.01, * p -value < 0.05.

Article Snippet: For THP-1 activation assays: anti-mouse TLR2 (mab2-mtlr2) and anti-human TLR4 (mabg-htlr4) antibodies were purchased from InvivoGen.

Techniques: Activation Assay, Flow Cytometry

Journal: Cancers

Article Title: ATP128 Clinical Therapeutic Cancer Vaccine Activates NF-κB and IRF3 Pathways through TLR4 and TLR2 in Human Monocytes and Dendritic Cells

doi: 10.3390/cancers14205134

Figure Lengend Snippet: The absence of the CPP and Anaxa domain greatly affects the secretion of pro-inflammatory cytokines and type I IFN. Human moDC were treated with a combination of anti-TLR2 and anti-TLR4 and incubated for 6 h with 300 nM ATP128 FL, w / o CPP, or w / o Anaxa. Cell supernatants were collected to assess the signature of cytokines secretion upon ATP128 stimulation through a multiplex cytokine assay. The values in the graphs represent the fold increase of the stimulated samples to the medium. Each shape is representative of a different buffy coat. Represented are the cytokines that are mainly induced upon stimulation. Values were compared via one-way ANOVA test to the sample stimulated with ATP128 FL*** p -value < 0.001, ** p -value < 0.01, * p -value < 0.05.

Article Snippet: For THP-1 activation assays: anti-mouse TLR2 (mab2-mtlr2) and anti-human TLR4 (mabg-htlr4) antibodies were purchased from InvivoGen.

Techniques: Incubation, Multiplex Assay, Cytokine Assay