Journal: The Journal of Experimental Medicine

Article Title: A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation required for cytokine expression

doi: 10.1084/jem.20060136

Figure Lengend Snippet: CyP specifically inhibits LPS stimulation of DCs. (A) DCs were stimulated for 16 h with different TLR agonists (1 μg/ml LPS, 10 μg/ml PGN, 20 μg/ml poly(I:C), and 2.5 μg/ml R848), 20 ng/ml IL-1β, or 1 μg/ml soluble CD40L in the absence or presence of 20 μg/ml CyP. Data are expressed as the percentage of the response (TNF production, black bars; IL-6 production, white bars) obtained with the specific agonists in the absence of CyP and represent the mean ± SD of four independent experiments. Inhibition of LPS was found to be statistically significant (P < 0.0001), whereas inhibition of all other stimuli was found to be nonsignificant (P > 0.05). (B) DCs were challenged with graded numbers of DH5α bacteria in the absence (white bars) or presence (black bars) of 20 μg/ml CyP. TNF was measured in the 20-h culture supernatants by ELISA. CyP did not affect bacterial growth. One representative experiment of three is shown. (C) DCs were stimulated with LPS, 10 ng/ml IFN-γ, soluble CD40L, or R848 alone or in the indicated combinations in the absence (white bars) or presence (black bars) of CyP. IL-12p70 was measured in the 24-h culture supernatants. One representative experiment of four is shown.

Article Snippet: Cytokine production was measured in the supernatants of DCs (0.5 × 10 6 cells/ml) stimulated for 16–24 h using matched paired antibodies specific for human TNF, IL-6, and IL-12p70 (DuoSet ELISA development kits; R&D Systems).

Techniques: Inhibition, Bacteria, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: The novel and potent CD40 agonist KHK2840 augments the antitumor efficacy of anti‐ PD ‐1 antibody and paclitaxel

doi: 10.1111/cas.16366

Figure Lengend Snippet: KHK2840 is a novel human CD40 agonist. (A) Representative sensorgrams of hCD40 (left), cynomolgus monkey CD40 (middle left), rat CD40 (middle; right), and mouse CD40 (right) to KHK2840. KHK2840 was captured on the sensor chip immobilized with anti‐human IgG, and then dilutions of recombinant CD40 proteins (0.0625, 0.125, 0.250, 0.500, 1.00 μg/mL) were injected. (B) KHK2840 (red), isotype control (human anti‐DNP IgG2, gray), and positive control (human anti‐DNP IgG1, blue) bind to FcγRIIIa (left, 158F; middle, 158 V) and C1q. Data represent the mean ± SD of three experiments. (C) The in vitro expression of CD80, CD86, and IL‐12p70 in hmoDCs induced by KHK2840 (red) and isotype control (human anti‐DNP IgG2, gray). In IL‐12p70, values below the lower limit of quantification (LLOQ) were replaced with the LLOQ. Data represent the mean ± SD of three wells. The same experiments were repeated in a total of six donors. (D) The in vitro expression of CD86 and CD95 induced in Ramos cells by 0.01 (filled circle), 0.1 (blank circle), 1 (filled square) and 10 μg/mL (blank square) KHK2840 after 0.5, 4, 24, and 48 h of treatment. KHK2840 was washed out at the indicated time points. Cells were analyzed after a total of 48 h of incubation. Data represent the mean ± SD of three wells.

Article Snippet: Human IL‐12p70 in the supernatant was measured using a Human IL‐12p70 Quantikine ELISA kit (R&D Systems).

Techniques: Recombinant, Injection, Control, Positive Control, In Vitro, Expressing, Incubation

Journal: Cancer Science

Article Title: The novel and potent CD40 agonist KHK2840 augments the antitumor efficacy of anti‐ PD ‐1 antibody and paclitaxel

doi: 10.1111/cas.16366

Figure Lengend Snippet: The in vivo CD40 agonistic activity of KHK2840 in hCD40 bacterial artificial chromosome (BAC) Tg mice. (A) Pharmacodynamics of CD80‐ (top left), CD86‐ (top right) and CD95‐ (bottom left) positive B cells in peripheral blood of hCD40 BAC Tg mice after the intravenous administration of vehicle and KHK2840. Dots represent individual data. Bars indicate averages of groups ( n = 4–5 mice per group). (B) Pharmacodynamics of IL‐12p70, IFN‐γ, IL‐6, IL‐10, TNF‐α, IL‐1β, and CXCL1 in peripheral blood of hCD40 BAC Tg mice after the intravenous administration of vehicle and KHK2840. Values below the lower limit of quantification (LLOQ) were replaced with the LLOQ. Dots represent individual data. Bars indicate averages of groups ( n = 4–5 mice per group).

Article Snippet: Human IL‐12p70 in the supernatant was measured using a Human IL‐12p70 Quantikine ELISA kit (R&D Systems).

Techniques: In Vivo, Activity Assay

Journal: Cancer Science

Article Title: The novel and potent CD40 agonist KHK2840 augments the antitumor efficacy of anti‐ PD ‐1 antibody and paclitaxel

doi: 10.1111/cas.16366

Figure Lengend Snippet: KHK2840 is a potent CD40 agonist. (A) The ex vivo expression of CD69, CD86, and CD95 induced by KHK2840, APX‐005 M, and CP‐870,893 in human peripheral blood B cells. Fresh human blood was treated with KHK2840, CP‐870,893, and APX‐005 M overnight in vitro. Data represent the mean ± SD of six donors. (B) IL‐12p70 production in peripheral blood of B16.F10 tumor‐bearing hCD40 bacterial artificial chromosome Tg mice 1 day after a single intravenous administration of vehicle or 0.57 or 1.2 mg/kg KHK2840 and 4.3 and 8.92 mg/kg CP‐870,893. Values below the lower limit of quantification (LLOQ) were replaced with the LLOQ. Dots represent individual data. Bars indicate averages of groups.

Article Snippet: Human IL‐12p70 in the supernatant was measured using a Human IL‐12p70 Quantikine ELISA kit (R&D Systems).

Techniques: Ex Vivo, Expressing, In Vitro

Journal: NPJ Vaccines

Article Title: Toxoplasma WH3 Δ rop18 acts as a live attenuated vaccine against acute and chronic toxoplasmosis

doi: 10.1038/s41541-024-00996-9

Figure Lengend Snippet: Detection of cytokines and T. gondii -specific IgG level in the sera of mice at 30/75/125 dpv with WH3 Δ rop18 by ELISA test. a Strategy diagram of the experimental process. b IFN-γ, c IL-12p70, d TNF-α, e IL-10 ( n = 5) and f Toxoplasma -specific total IgG and IgG1 and IgG2a ( n = 8). Serum was derived from unvaccinated mice as a negative control. *** P < 0.001, ** P < 0.01, * P < 0.05, ns not significant.

Article Snippet: In the meantime, the expression levels of cytokines interferon-gamma (IFN-γ), interleukin 12p70 (IL-12p70), tumor necrosis factor TNF (TNF-α) and interleukin 10 (IL-10) were detected by using ELISA kits (ProteinTech Group, Inc., USA) according to the manufacturer’s recommendations.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Negative Control

Journal: NPJ Vaccines

Article Title: Toxoplasma WH3 Δ rop18 acts as a live attenuated vaccine against acute and chronic toxoplasmosis

doi: 10.1038/s41541-024-00996-9

Figure Lengend Snippet: a Schematic illustration of the experimental procedure. b , d Percentage of CD3 + CD8 + T cells and CD3 + CD4 + T cells detected with FCM ( n = 4). c , e Increased percentage of Th1 (CD4 + IFN-γ + ) cell population in the mouse spleens following WH3 Δ rop18 immunization compared to the control ( n = 4). f Spleen cells cultured in vitro were stimulated with 10 µg/mL WH3 WT soluble antigens. Subsequently, the levels of IFN-γ, TNF-α, IL-12p70 and IL-10 in the splenic cell culture supernatant were detected by ELISA ( n = 5). Unpaired t -tests were used for statistical analysis. Bars = mean ± SEM. * P < 0.05, *** P < 0.001.

Article Snippet: In the meantime, the expression levels of cytokines interferon-gamma (IFN-γ), interleukin 12p70 (IL-12p70), tumor necrosis factor TNF (TNF-α) and interleukin 10 (IL-10) were detected by using ELISA kits (ProteinTech Group, Inc., USA) according to the manufacturer’s recommendations.

Techniques: Control, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: NPJ Vaccines

Article Title: Toxoplasma WH3 Δ rop18 acts as a live attenuated vaccine against acute and chronic toxoplasmosis

doi: 10.1038/s41541-024-00996-9

Figure Lengend Snippet: 10 3 WH3 Δ rop18 parasites were i.p. into BALB/c mice for 75 days as a vaccine dose. Then, the mice were challenged with 10 3 WH3 WT or 10 5 WH6 tachyzoites. Unvaccinated mice were treated similarly. a Schematic illustration of the study design. b The vaccinated mice were challenged with 10 3 WH3 WT tachyzoites or c 10 5 WH6 tachyzoites. The survival rate was recorded for 35 days ( n = 10). Gehan-Breslow-Wilcoxon tests. d WH3 WT or WH6 tachyzoites were used for challenges of vaccinated mice, and 7 days later, the parasite burden in ascites were tested by qPCR ( n = 5). *** P < 0.001. e IFN-γ, IL-12p70, TNF-α and IL-10 in sera were tested by ELISA ( n = 5), *** P < 0.001. f , g Toxoplasma -specific total IgG and subclasses of IgG1 and IgG2a in sera were tested by ELISA. Unpaired t -tests were used for s t atistical analysis. Bars = mean ± SEM. *** P < 0.001.

Article Snippet: In the meantime, the expression levels of cytokines interferon-gamma (IFN-γ), interleukin 12p70 (IL-12p70), tumor necrosis factor TNF (TNF-α) and interleukin 10 (IL-10) were detected by using ELISA kits (ProteinTech Group, Inc., USA) according to the manufacturer’s recommendations.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: NPJ Vaccines

Article Title: Toxoplasma WH3 Δ rop18 acts as a live attenuated vaccine against acute and chronic toxoplasmosis

doi: 10.1038/s41541-024-00996-9

Figure Lengend Snippet: 10 3 WH3 Δ rop18 parasites were i.p. into BALB/c mice as a vaccine dose and observed for 125 days. Vaccinated or un-vaccinated mice were then infected with 10 3 RH or 10 5 ME49 parasites. a Schematic illustration of the study design. b 10 3 RH or c 10 5 ME49 tachyzoites were used for challenges ( n = 10). The survival rate was recorded for 35 days. Gehan-Breslow-Wilcoxon tests. d RH or ME49 tachyzoites challenges in vaccinated mice, followed by detection of parasite burden by qPCR in the ascites of animals 7 days later ( n = 5). *** P < 0.001. e IFN-γ, IL-12p70, TNF-α and IL-10 in sera were tested by ELISA ( n = 5), *** P < 0.001, ** P < 0.01. f , g Toxoplasma -specific total IgG and subclasses of IgG1 and IgG2a in sera were tested by ELISA ( n = 10). Unpaired t -tests were used for statistical analysis. Bars = mean ± SEM. *** P < 0.001.

Article Snippet: In the meantime, the expression levels of cytokines interferon-gamma (IFN-γ), interleukin 12p70 (IL-12p70), tumor necrosis factor TNF (TNF-α) and interleukin 10 (IL-10) were detected by using ELISA kits (ProteinTech Group, Inc., USA) according to the manufacturer’s recommendations.

Techniques: Infection, Enzyme-linked Immunosorbent Assay