Journal:

Article Title: Bacterial Probiotic Modulation of Dendritic Cells

doi: 10.1128/IAI.72.6.3299-3309.2004

Figure Lengend Snippet: Probiotic stimulates IL-10 release in DC cultures. BM cells were grown in GM-CSF. Various concentrations of probiotic or bacteria were added on day 2 for a further 3 days. DC culture supernatants were harvested on day 5, and cytokine levels were measured by ELISA. Results are shown for IL-10 (A) and IL-12 (p70) (B). Error bars represent standard deviations from duplicate wells. P values indicate levels of significance above control PBS. Results are representative of three separate experiments.

Article Snippet: Cytokine detection was done by enzyme-linked immunosorbent assay (ELISA) for interleukin-10 (IL-10) and gamma interferon (IFN-γ) with paired antibodies (PharMingen) and for bioactive IL-12 (p70) with the Duoset DY 419 kit (R&D Systems).

Techniques: Bacteria, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Bacterial Probiotic Modulation of Dendritic Cells

doi: 10.1128/IAI.72.6.3299-3309.2004

Figure Lengend Snippet: Probiotic stimulation of IL-12 (p70). Cells at day 4 of BM DC culture were stimulated for an overnight period by the addition of 107 organisms of probiotic or bacteria/ml until the next day. Supernatants were collected, and IL-12 levels (A) or IL-10 release (B) in supernatants were determined by ELISA. Standard deviation is shown for duplicate wells. The level of significance is indicated by the P value.

Article Snippet: Cytokine detection was done by enzyme-linked immunosorbent assay (ELISA) for interleukin-10 (IL-10) and gamma interferon (IFN-γ) with paired antibodies (PharMingen) and for bioactive IL-12 (p70) with the Duoset DY 419 kit (R&D Systems).

Techniques: Bacteria, Enzyme-linked Immunosorbent Assay, Standard Deviation

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: Communications medicine

Article Title: Fusion peptide is superior to co-expressing subunits for arming oncolytic herpes virus with interleukin 12.

doi: 10.1038/s43856-023-00270-4

Figure Lengend Snippet: Fig. 1 The structures of T-mfIL12 and T-mIL12-IRES. The boxes (top line) represent inverted repeat sequences flanking the long (UL) and short (US) unique sequences of HSV-1 DNA. Oncolytic HSV-1 uses G47Δ as the backbone and contains 1.0 kb deletions in both copies of the γ34.5 gene, a 0.3 kb deletion in the α47 gene, and a 0.9 kb deletion in the ICP6 gene. The lacZ gene and the cytomegalovirus (CMV) promoter-driven interleukin-12 (IL-12) gene are oriented in opposite directions, and inserted into the deleted ICP6 locus. T-mfIL12 contains a cassette carrying the p35 and p40 genes of murine IL-12 linked by two bovine elastin motifs. Murine IL-12 is expressed as a single fusion peptide and folds to become active. T-mIL12- IRES contains a cassette in which the p40 and p35 genes of murine IL-12 are separated by an internal ribosome entry site (IRES) sequence from the encephalomyocarditis virus (EMCV). The p40 and p35 subunits are efficiently co-expressed to form active murine IL-12. T-01 contains a cassette with no transgene.

Article Snippet: Supernatants were collected, and the concentration of IL-12 was measured using mouse IL12 p70 immunoassay (M1270, R&D Systems Inc., IL) with a detection limit of 7.8 pg/ ml.

Techniques: Sequencing, Virus

Journal: Communications medicine

Article Title: Fusion peptide is superior to co-expressing subunits for arming oncolytic herpes virus with interleukin 12.

doi: 10.1038/s43856-023-00270-4

Figure Lengend Snippet: Fig. 2 In vitro replication capabilities and murine IL-12 expressions of T-mfIL12 and T-mIL12-IRES. a In vitro replication assay. Vero cells were infected with G47Δ, T-01, T-mfIL12 candidate (4 clones) or T-mIL12-IRES candidate (4 clones) at a multiplicity of infection (MOI) of 0.01, the progeny virus was recovered 48 h after infection, and the number determined by plaque assay. T-mfIL12 and T-mfIL12-IRES showed no significant difference in replication capability (p = 0.736, t-test). b In vitro murine IL-12 expression. Vero cells were infected with G47Δ, T-01, T-mfIL12 candidate (4 clones) or T-mIL12-IRES candidate (4 clones) at an MOI of 1, and the amount of murine interleukin-12 (IL-12) (p70) secreted was determined by enzyme-linked immune-sorbent assay (ELISA). T-mfIL12 expressed a significantly higher amount of p70 IL-12 than T-mIL12-IRES (p < 0.001, t-test). c The time course of viral yields. Vero cells were infected with G47Δ, T-01, T-mfIL12 or T-mIL12-IRES in duplicate at an MOI of 0.01, the progeny virus was recovered 0 h, 6 h, 24 h and 48 h after infection, and titrated by plaque assay. The time course for the viral yields of T-mfIL12 was comparable to that of T-mIL12-IRES. d In vitro murine IL-12 expression in murine tumor cell lines. Neuro2a, Pr14-2 or TRAMP-C2 cells were infected with T-mfIL12 or T-mIL12-IRES at an MOI of 1, and the amount of murine IL-12 (p70) secreted was determined by ELISA. T-mfIL12 expressed a higher amount of p70 IL-12 than T-mIL12-IRES in all three cell lines. All assays were performed in duplicate. ***, p < 0.001; NS, not significant.

Article Snippet: Supernatants were collected, and the concentration of IL-12 was measured using mouse IL12 p70 immunoassay (M1270, R&D Systems Inc., IL) with a detection limit of 7.8 pg/ ml.

Techniques: In Vitro, Infection, Clone Assay, Virus, Plaque Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Communications medicine

Article Title: Fusion peptide is superior to co-expressing subunits for arming oncolytic herpes virus with interleukin 12.

doi: 10.1038/s43856-023-00270-4

Figure Lengend Snippet: Fig. 5 Immune responses by T-mfIL12 and T-mIL12-IRES. a Immunohistochemistry. Bilateral subcutaneous Neuro2a tumors were generated in A/J mice, left tumors only were inoculated with T-mfIL12, T-mIL12-IRES, T-01 (2 × 105 pfu) or mock on days 0 and 3, and the tumors were harvested on day 6 (n = 3 per group). An increased infiltration of CD4+ and CD8+ lymphocytes were observed in the tumor for all three viruses, both in the treated and the untreated side, most prominently with T-mfIL12 (Fig. 5a). HSV-1 positive cells were observed in the tumor with all viruses in the treated side, but not in the untreated side. HE, hematoxylin and eosin. Scale bars, 100 μm. b In vivo levels of interleukin-12 (IL-12) and Interferon γ (IFNγ). In the unilateral subcutaneous Neuro2a model, established tumors were inoculated with T-01, T-mfIL12, T-mIL12-IRES (2 × 106 pfu) or mock, sera and tumor samples were collected on days 1, 3 and 6 (n = 3 per group), and the levels of mouse IL-12 and IFNγ were measured by ELISA. The intratumoral IL-12 levels for T-mfIL12 were significantly higher than those for T-mIL12-IRES at all time points (p = 0.018, p = 0.016 and p = 0.046 for days 1, 3 and 6, respectively). The levels of IL-12 detected from the serum were remarkably lower than those in the tumor. Correlating with the intratumoral IL-12, the serum IL-12 level for T-mfIL12 was higher than that for T-mIL12-IRES on day 1 (p = 0.047). The IFNγ levels of T-mfIL12 were significantly higher than those of T-mIL12-IRES both in the tumor and serum on day 1 (p = 0.014 and p = 0.027, tumor and serum, respectively). c Immune responses specific to Neuro2a cells. In the unilateral subcutaneous Neuro2a model, established tumors were inoculated with T-01, T-mfIL12, T-mIL12-IRES (5 × 104 pfu) or mock on days 0 and 3, and the spleen was harvested on day 6. By ELISpot assay, splenocytes from T-mfIL12-treated mice showed a significantly higher number of IFNγ release stimulated by Neuro2a cells than those from T-01- and T-mIL12-IRES- treated ones (p = 0.005 and p = 0.004 vs T-01 and T-mIL12-IRES, respectively). No significant difference in number of IL-4 releasing splenocytes was observed among the three virus-treated groups. The IFNγ or IL-4 releases specific to Neuro2a cells were calculated by subtracting the numbers of SaI/N responding spots from those of Neuro2a responding spots. For b and c, Graphs show the means. Dots represent individual data. Bars, SD. *, p < 0.05; **, p < 0.01; NS, not significant; one-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: Supernatants were collected, and the concentration of IL-12 was measured using mouse IL12 p70 immunoassay (M1270, R&D Systems Inc., IL) with a detection limit of 7.8 pg/ ml.

Techniques: Immunohistochemistry, Generated, In Vivo, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Virus

Journal: Communications medicine

Article Title: Fusion peptide is superior to co-expressing subunits for arming oncolytic herpes virus with interleukin 12.

doi: 10.1038/s43856-023-00270-4

Figure Lengend Snippet: Fig. 6 Comparison of in vivo efficacy of T-mfIL12 and direct intratumoral injection with recombinant interleukin-12 (rIL-12). In the bilateral subcutaneous Neuro2a model, rIL-12, T-01 (5 × 104 pfu) without or with rIL-12, T-mfIL12 (5 × 104 pfu) or mock was inoculated into the left tumors only on days 0 and 4 (n = 10 per group). Three different doses were used for rIL-12; 500 ng (a), 50 ng (b) and 1 ng (c). The dose 50 ng represents the intratumoal IL-12 level treated with T-mfIL12 at 2 × 106 pfu and 1 ng represents that at 5 × 104 pfu, the T-mfIL12 dose used in these experiments. a When the dose of 500 ng was used for rIL-12, rIL-12, T-01, T-01+rIL-12 and T-mfIL12 were all significantly more efficacious than mock in the treated side (p < 0.001 vs mock for all). In the untreated side, rIL-12 alone showed no significant antitumor effect compared with mock, whereas T-01+rIL-12 and T-mfIL12 showed a significantly higher efficacy than mock (p = 0.039 and p < 0.001 vs mock, respectively). Further, in the untreated side, T-mfIL12, but not T-01+rIL-12, was significantly more efficacious than rIL-12 alone (p = 0.003). b Results similar to a were obtained when the dose of 50 ng was used for rIL-12. c When the dose of 1 ng was used for rIL-12, rIL-12 alone showed no significant efficacy in both treated and untreated sides, and T-mfIL12, but not T-01+rIL-12, was significantly more efficacious than rIL-12 alone in the treated side. Tumor volume = length × width × height × 0.52. Results represent the mean. Bars, standard error of the mean (SEM). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant; Two-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: Supernatants were collected, and the concentration of IL-12 was measured using mouse IL12 p70 immunoassay (M1270, R&D Systems Inc., IL) with a detection limit of 7.8 pg/ ml.

Techniques: Comparison, In Vivo, Injection, Recombinant

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