Journal: PLoS Pathogens

Article Title: Activation of TLR2 and TLR6 by Dengue NS1 Protein and Its Implications in the Immunopathogenesis of Dengue Virus Infection

doi: 10.1371/journal.ppat.1005053

Figure Lengend Snippet: ELISA was performed to quantify the amount of IL-6 (A) and TNF-α (B) secreted into the supernatant by mock-infected and DV2-infected PBMC. Data represent mean ± SEM of three independent experiments obtained using separate PBMC from three donors. Student’s t- tests were used to compare mean amount of IL-6/TNF-α detected in culture supernatants of DV2-infected PBMC to that of the mock-infected PBMC. ELISA was also performed to quantify the amount of IL-6 (C) and TNF-α (D) secreted into the supernatant by mock-infected, DV2-infected, LPS-treated and MALP-2 treated PBMC on day 2 post-treatment. Data represent mean ± SEM of three independent experiments obtained using separate PBMC from three donors. Student’s t- tests were used to compare mean amount of IL-6/TNF-α detected in culture supernatants of TLR2 and TLR6 blocked PBMC to that of the isotype control PBMC.

Article Snippet: Ultrapure LPS-treated PBMC were added ultrapure lipopolysaccharide (InvivoGen, LPS-EB Ultrapure).

Techniques: Enzyme-linked Immunosorbent Assay, Infection

Journal: PLoS Pathogens

Article Title: Activation of TLR2 and TLR6 by Dengue NS1 Protein and Its Implications in the Immunopathogenesis of Dengue Virus Infection

doi: 10.1371/journal.ppat.1005053

Figure Lengend Snippet: The amount of IL-6 (A) and TNF-α (B) secreted into the supernatant by PBMC treated with dengue viral proteins (capsid, envelope, premembrane, NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5, each 50 μg/ml), His-tag-treated PBMC, untreated PBMC, UV-inactivated DV2-treated PBMC (10 virus particles per cell), LPS-treated PBMC (5 μg/ml, 10 μg/ml, 50 μg/ml) on day 2 post-treatment were quantified using ELISA. Data represent mean ± SEM of three independent experiments obtained using separate PBMC from three donors. Student’s t- tests were used to compare mean amount of IL-6/TNF-α detected in culture supernatants of viral protein-treated PBMC to that of the His-tag-treated PBMC, the mean amount of IL-6/TNF-α detected in culture supernatants of LPS-treated PBMC to that of the untreated PBMC, the mean amount of IL-6/TNF-α detected in the culture supernatants of His-tag-treated PBMC to that of the untreated PBMC and the mean amount of IL-6/TNF-α detected in the culture supernatants of UV-inactivated DV2-treated PBMC to that of the untreated PBMC. The amount of IL-6 (C) and TNF-α (D) secreted into the supernatant by DV NS1 protein-treated PBMC (1 μg/ml) on day 1 to day 3 post-treatment were quantified using ELISA. Data represent mean ± SEM of three independent experiments. Student’s t- tests were used to compare mean amount of IL-6/TNF-α detected in culture supernatants of DV NS1 protein-treated PBMC to that of the His-tag-treated PBMC. The amount of IL-6 (E) and TNF-α (F) secreted into the supernatant by DV NS1 protein-treated PBMC (1 μg/ml) on day 1 post-treatment were quantified using ELISA. TLR2 and TLR6 of the PBMC were blocked prior to the treatment. Data represent mean ± SEM of three independent experiments obtained using separate PBMC from three donors. Student’s t- tests were used to compare mean amount of IL-6 detected in culture supernatants of DV NS1 protein-treated PBMC isotype control to that of DV NS1 protein-treated TLR2/6 blocked PBMC. HEK 293 cells transfected with SEAP reporter plasmid (G), HEK 293 cells transfected with both SEAP reporter plasmid and pDUO-hTLR6/TLR2 plasmid (H) were treated with His-tag (1 μg/ml), DVNS1 recombinant protein (1 μg/ml), LPS (5 μg/ml), MALP-2 (50 ng/ml), mock-infected or DV2-infected (M.O.I of 10). The supernatants of the HEK 293 cell cultures were harvested on day 1 to day 3 post-treatment. The SEAP in the supernatants were quantified using SEAP reporter assay kit. Data represent mean ± SEM of three independent experiments. Student’s t- tests were used to compare mean amount of SEAP detected in culture supernatants of DV NS1 protein-treated HEK 293 cells to that of His-tag-treated HEK 293 cells, mock-infected HEK 293 cells to that of DV2-infected HEK 293 cells, LPS-treated HEK 293 cells to that of untreated and MALP-2 HEK 293 cells to that of untreated HEK293 cells.

Article Snippet: Ultrapure LPS-treated PBMC were added ultrapure lipopolysaccharide (InvivoGen, LPS-EB Ultrapure).

Techniques: Virus, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Recombinant, Infection, Reporter Assay

Journal: PLoS Pathogens

Article Title: Activation of TLR2 and TLR6 by Dengue NS1 Protein and Its Implications in the Immunopathogenesis of Dengue Virus Infection

doi: 10.1371/journal.ppat.1005053

Figure Lengend Snippet: Mock-infected, His-tag-treated, DV2-infected, DV NS1 protein-treated, LPS-treated PBMC were lysed and equal total protein was loaded into SDS-PAGE gel. PBMC of two donors were used. The presence of TLR6 protein in the cell lysate was detected using rabbit anti-TLR6 antibody and goat anti-rabbit HRP conjugated antibody on the Western blot (A). Actin was used as loading control. The band intensity was measured using GelQuantNET software program. The intensities of the TLR6 bands were normalized against the intensity of the corresponding actin bands and were plotted in the graph (B). The presence of TLR2 protein in the cell lysate was detected using rabbit anti-TLR2 antibody and goat anti-rabbit HRP conjugated antibody on the Western blot (C). Actin was used as loading control. The intensities of the TLR2 bands were normalized against the intensity of the corresponding actin bands and were plotted in the graph (D). The relative intensities of the bands were indicated on the charts. PBMC were untreated (E & J), mock-infected (F & K), DV2-infected (G & L), His-tag-treated (H & M) and DV NS1 protein-treated (I & N). The PBMC were harvested at day 3 post-treatment. TLR6 was stained red using primary rabbit anti-TLR6 antibody and secondary goat anti-rabbit DyLight 633 antibody (E to I). TLR2 is stained red using primary mouse anti-TLR2 antibody and secondary goat anti-mouse DyLight 633 (J to N). CD14 was stained green using anti-CD14 antibody conjugated with FITC (E to N). Cell nuclei are stained blue with DAPI. Representative images from triplicate experiments obtained using separate PBMC from three donors are shown.

Article Snippet: Ultrapure LPS-treated PBMC were added ultrapure lipopolysaccharide (InvivoGen, LPS-EB Ultrapure).

Techniques: Infection, SDS Page, Western Blot, Software, Staining

Journal: PLoS Pathogens

Article Title: Activation of TLR2 and TLR6 by Dengue NS1 Protein and Its Implications in the Immunopathogenesis of Dengue Virus Infection

doi: 10.1371/journal.ppat.1005053

Figure Lengend Snippet: ELISA was performed to quantify the amount of IL-6 (A) and TNF-α (B) secreted into the supernatant by His-tag (1 μg/ml), DV NS1 recombinant protein (1 μg/ml), MALP-2 (50 ng/ml), Ultrapure LPS (2.5 x 10 4 EU/ml), mock-infected or DV2-infected (M.O.I of 10) wild-type or TLR6 -/- murine peritoneal macrophages on day 2 post-treatment. (C) Wild-type (23 mice) and TLR6 -/- (9) mice were injected with 20 μg of DV NS1 protein at 1–2 day-old via intraperitoneal injection. Control wild-type (18 mice) and TLR6 -/- (9) mice were injected with 20 μg of His-tag. The survival of the mice was monitored from day 1 to day 7 on a daily basis. Kaplan-Meier survival curves were shown. Log-rank test was performed to determine if there was significant difference in survivability between DV NS1-treated wild-type and TLR6 -/- mice. ELISA was performed to quantify the amount of IL-6 (D) and TNF-α (E) in the livers of wild-type mice injected with 20 μg of DV NS1 protein or His-tag at 1–2 day-old via intraperitoneal injection. Each set of samples was in triplicates.

Article Snippet: Ultrapure LPS-treated PBMC were added ultrapure lipopolysaccharide (InvivoGen, LPS-EB Ultrapure).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Infection, Injection

Journal: Nature biomedical engineering

Article Title: Microneedle patch for the ultrasensitive quantification of protein biomarkers in interstitial fluid

doi: 10.1038/s41551-020-00672-y

Figure Lengend Snippet: a, Schematic illustration showing the working principle of immune response induced by LPS administration, cytokine generation and administration of the microneedle patch for longitudinal detection. b, Timeline of endotoxic shock (LPS injection), longitudinal microneedle administration on mice, and blood collection (for validation of the microneedle test). c, Fluorescence map representing mouse IL-6 captured on the microneedles before and at 1, 2.5 and 4 hours post LPS injection. Scale bar, 200 μm. d, Plot depicting concentrations of mouse IL-6 in ISF before and at 1, 2.5 and 4 hours post LPS/saline injection measured using microneedle. (n=3 in each group) Data statistically significant, **** P< 0.0001 by two-way analysis of variance (ANOVA) with Sidak’s multiple-comparison test. e, Plots showing the comparison of concentrations of IL-6 measured in serum and ISF in saline group (left) and LPS group (right). (n=3 in each group) At 4 hours post LPS injection, IL-6 in dermal ISF determined by plasmonic-fluor enhanced microneedle (blue) exhibited good qualitative correlation with that in serum tested by conventional ELISA (grey). Error bars represent standard deviation. Error bars of serum results are technical repeats.

Article Snippet: For each mouse in vaccination group, cocaine vaccination solution including 100 μg cocaine-BSA conjugate (Fitzgerald Industries, PART# 80–1037) and 2 μg lipopolysaccharide (InvivoGen, catalog code: vac-3pelps) in 100 μl saline mixed with 100 μl 2% Alhydrogel (InvivoGen, catalog code: vac-alu-250) was freshly prepared prior to vaccination.

Techniques: Injection, Fluorescence, Saline, Comparison, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: The Journal of Immunology Author Choice

Article Title: Salmonella Flagellin Activates NAIP/NLRC4 and Canonical NLRP3 Inflammasomes in Human Macrophages

doi: 10.4049/jimmunol.2000382

Figure Lengend Snippet: Recombinant PrgI triggers the NAIP/NLRC4 inflammasome, whereas flagellin activates the NAIP/NLRC4 and the NLRP3 inflammasome in THP-1 cells. Parental wild-type THP-1 ( A , B , and D ), NLRC4 KO (A and B), NAIP KO ( C ), or NLRP3 KO (D) cells were transfected with PrgI or flagellin (A–D) or stimulated with LPS+ nigericin (B–D) in the presence of NLRP3 inhibitors MCC950 (B and C) or glybenclamide (B). ASC speck formation (B–D) and IL-1β (A) and IL-18 (A–D) release were determined at 4 h poststimulation. Mean with SD of at least three individual experiments (two replicates/experiment) is displayed. Statistical analysis was performed using two-way ANOVA followed by a Bonferroni posttest. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: E. coli 0111:B4 LPS and nigericin were purchased from InvivoGen (LPS: tlrl-3pelps; nigericin: tlrl-nig) or Sigma (LPS: L5293-2ML; nigericin: SML1779-1ML).

Techniques: Recombinant, Transfection

Journal: The Journal of Immunology Author Choice

Article Title: Salmonella Flagellin Activates NAIP/NLRC4 and Canonical NLRP3 Inflammasomes in Human Macrophages

doi: 10.4049/jimmunol.2000382

Figure Lengend Snippet: Flagellin activates the canonical NLRP3 inflammasome in THP-1 cells. ( A ) Schematic figure of canonical NLRP3 activation is shown in black; noncanoncial caspase-4/-5–mediated pathway is shown in gray. ( B ) THP-1 NLRC4 KO cells were transfected with flagellin or LPS or stimulated with extracellular LPS and EtOH or nigericin in the presence of the pan-caspase inhibitor z-VAD-fmk. ASC speck formation and IL-18 release were determined at 4 h poststimulation. Mean with SD of at least three individual experiments (two replicates/experiment) is displayed. Statistical analysis was performed using two-way ANOVA followed by a Bonferroni posttest. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: E. coli 0111:B4 LPS and nigericin were purchased from InvivoGen (LPS: tlrl-3pelps; nigericin: tlrl-nig) or Sigma (LPS: L5293-2ML; nigericin: SML1779-1ML).

Techniques: Activation Assay, Transfection

Journal: The Journal of Immunology Author Choice

Article Title: Salmonella Flagellin Activates NAIP/NLRC4 and Canonical NLRP3 Inflammasomes in Human Macrophages

doi: 10.4049/jimmunol.2000382

Figure Lengend Snippet: Flagellin-mediated NLRP3 inflammasome is dependent on ROS production and cathepsin activity. ( A ) THP-1 cells were transfected with equimolar amounts of PrgI or S. Typhimurium flagellin (ST) or twice the molar amount of B. subtilis flagellin (BS) or stimulated with LPS and EtOH or nigericin in the presence of the NLRP3 inhibitor MCC950. IL-1β and IL-18 release as well as ASC speck formation were determined at 4 h poststimulation. ( B ) THP-1 NLRC4 KO cells were transfected with PrgI, Salmonella -, or B. subtilis –derived flagellin or stimulated with extracellular LPS and EtOH or nigericin in the presence of the ROS inhibitor DPI or the cathepsin inhibitor Ca-047-Me. ASC speck formation and cytokine release were determined at 4 h poststimulation. Mean with SD of three individual experiments (two replicates/experiment) is displayed. Statistical analysis was performed using two-way ANOVA followed by a Bonferroni posttest. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: E. coli 0111:B4 LPS and nigericin were purchased from InvivoGen (LPS: tlrl-3pelps; nigericin: tlrl-nig) or Sigma (LPS: L5293-2ML; nigericin: SML1779-1ML).

Techniques: Activity Assay, Transfection, Derivative Assay