Fig 1: In vivo studies with mLiTCo-AlbuPharmacokinetic study after a single intravenous (i.v.) dose (2 mg/kg) of mLiTCo (n = 3) and mLiTCo-Albu (n = 4) in BALB/c mice. Data are shown as mean ± SD (A). Average tumor volume growth of BALB/c mice bearing CT26hEGFR tumors treated with PBS, anti-4-1BB 3H3 mAb, or mLiTCo-Albu. Data are presented as the mean ± SD Significance was determined by one-way ANOVA adjusted by the Bonferroni correction for multiple comparison test (B and C). Liver weights from mice (mean ± SD, n = 5/group) treated with PBS, anti-41BB 3H3 IgG mAb, or mLiTCo-Albu. Significance was determined by unpaired Student t test. (D). Sera from treated mice were collected from peripheral blood on days 0 and 21 of treatment, and levels of INF-? (E) and IL-6 (F) were measured by ELISA (mean ± SD, n = 3 per time point). Significance was determined by unpaired Student t test. Average tumor volume growth of BALB/c mice bearing CT26hEGFR tumors treated with PBS, mLiTCo-Albu, or anti-PD1 RMP 1.14 mAb alone or in combination (G and H). Data are presented as the mean ± SD. Significance was determined by one-way ANOVA adjusted by the Bonferroni correction for multiple comparison test.
Fig 2: Immunohistochemical (IHC) analysis of the expression of three inflammatory cytokines in M5 canine melanoma tumors implanted in BALB/c nude mice. (a,b) Representative images showing IL-1ß (a) and TNF-a (b) expression. (c–e) Quantitative analyses of the IHC expression of IL-1ß (c), TNF-a (d), and serum IL-6 (e). The mice were administered the following treatments three times per week: saline (control), 2 mg/kg cisplatin (cisplatin), 100 mg/kg PTS (PTS), or 100 mg/kg PTS and 2 mg/kg cisplatin (cisplatin + PTS). Data are presented as mean ± SD, n = 7 per group. ** p < 0.01 and *** p < 0.001 vs. control; ## p < 0.01 vs. cisplatin; ++ p < 0.01 vs. PTS. Scale bars: 50 µm.
Fig 3: C. albicans induces pro-inflammatory cytokines in whole-brain and BV-2 cells. C57BL/c mice were challenged i.v. with 25,000 CFU of C. albicans after which whole brains were harvested at the indicated days for analysis by real-time quantitative PCR (RT-qPCR), western blot, or ELISA. a, b Nuclear factor kappa B (NF-?B) expression as assayed by RT-qPCR (a) or western blot (b; p65 subunit) over 14 days. c Densitometric analysis of the data from b. d–f IL-1ß, IL-6, and TNF cytokine levels from whole-brain homogenates as assessed by ELISA. g BV-2 cells were seeded for 6 h in 24-well plates (1 × 105 cells per well) and then incubated with C. albicans (200 viable cells per well) for 16 h after which secreted IL-1ß, IL-6, and TNF were quantitated by ELISA. h BV-2 cells were seeded same as above and incubated with lysates of C. albicans (equivalent 200 viable cells per well), irradiated C. albicans (200 cells per well), secreted aspartic proteases (SAP, 1 µM), or inhibited secreted aspartic proteases (1 µM) for 16 h and the same cytokines were quantitated by ELISA (n = 4. mean ± S.E.M, ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using two-tailed Student’s t-test (g) or one-way ANOVA (a, c–f, h) followed by Dunnett’s test for multiple comparison). Data are shown as representative of two independent experiments
Fig 4: Immunohistochemical expression of (a) IL-1ß and (b) TNF-a in A549 tumors from BALB/cByJNarl mice and the comparative immunohistochemical expressions of (c) IL-1ß and (d) TNF-a; as well as (e) IL-6 and (f) IL-10 levels in serum. All experiments were conducted on the A549 tumor masses from BALB/cByJNarl mice. Findings are reported for the control group and treatments: 5-FU (100 mg/kg/week), zotarolimus (2 mg/kg/day), and zotarolimus (2 mg/kg/day) combined with 5-FU (100 mg/kg/week). All data are presented as mean ± standard deviation, n = 7 per group. ** p < 0.01 and *** p < 0.001 compared with the control group. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the 5-FU group. +++ p < 0.001 compared with the zotarolimus group. Scale bars = 60 µm.
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