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  • br Fig Immune cell population T cell polarization and the

    2022-05-20


    Fig. 6. Immune-cell population, T-cell polarization, and the expression levels of Treg-related and anti-inflammatory cytokine genes in the spleen. The profile of immune cells such as dendritic cells, macrophages, NK cells, CD4+ cells, CD8+ cells, and Tubercidin was determined (A–F). In addition, T-cell polarization was measured by flow cytometry via confirmation of the expression of cell adhesion proteins (G–L). Lastly, the genes associated with Treg and their recruitment and anti-inflammatory cytokines were analyzed by PCR (M–Q). Data are presented as the means ± SD from three independent experiments. Statistical significance was evaluated by Student’s t test (#p < 0.05 Non-induction vs Cancer, *p < 0.05 Cancer vs TL or NL) (Non-induction: group without inoculation with cancer; Cancer: group inoculated with cancer; NL: NL treated group inoculated with cancer; TL: TL treated group inoculated with cancer).
    Fig. 7. Protein levels of cytokines and expression of genes in splenocytes reactivated by anti-CD3 antibody stimulation. The anti-CD3 antibody was used to boost T-cell functions. The expression of genes associated with Tregs and their recruitment and genes of anti-inflammatory cytokines like FOXP3, IL-10, TGF-β, CCR5, and CCL5 was evaluated by PCR (A–E), and the cytokines such as IFN-γ, TNF-α, IL-4, IL-13, and IL-10 were analyzed by an ELISA (F–J). Data are presented as the means ± SD from three independent experiments. Statistical significance was evaluated by Student’s t test (#p < 0.05 Non-induction vs Cancer, *p < 0.05 Cancer vs TL or NL) (Non-induction: group without inoculation with cancer; Cancer: group inoculated with cancer; NL: NL treated group inoculated with cancer; TL: TL treated group inoculated with cancer).
    blocking of Treg cell recruitment according to the gene expression profile (Fig. 6). Additionally, the same trend was observed after re-boosting of splenocytes by the anti-CD3 antibody for 2 days. FOXP3 and CCL5 are known as genes involved in the weakening of immunity by accumulating Tregs. These genes were downregulated, and cytokines such as TNFα and IL-4 that enhance the immune system were upre-gulated by treatment with the TL extract (Fig. 7).
    FOXP3 is an important transcription factor controlling differentia-tion and function of Tregs and is found in tumors like PC and melanoma (Karanikas et al., 2008). The role of FOXP3 in cancer has been reported especially in PC: this tumor can accumulate Tregs in/near the tumor and may ruin the immune system (Wang et al., 2017). Additionally, the 
    same research group discovered that FOXP3 in cancer interacts with CCL5 to turn on the CCL5–CCR5 signaling, which is strongly involved in Treg recruitment, and this effect is attenuated by the CCL5 antibody (Wang et al., 2017).
    Treg recruitment to tumors from peripheral regions is regulated by chemokine receptors, e.g., CCR, and their ligands, such as CCL (Adams
    & Eksteen, 2006). CCL22 in human ovarian cancer is transactivated with CCR4 on Tregs to drive them into the tumor, and CCL17 or CCL22 in gastric cancer have been reported to participate in Treg cell re-cruitment to the tumor (Curiel et al., 2004; Mizukami et al., 2008). In another murine model of PC, CCR5+ Tregs infiltrated the tumor de-pending on tumor CCL5 levels to create a favorable tumor
    Fig. 8. HPLC analysis of β-carotene (A), NL (B), and TL (C). Chromatographic separation was carried out on a C30 column (250 × 4.6 mm, 5 μm particle size) using an isocratic solvent system composed of water and acetone at a 1:9 ratio. The flow rate was adjusted to 1.0 mL/min, the detection wavelength was 450 nm, and 10 μL of each sample was injected. The analytical process was performed at 30 C. β-carotene was detected at RT 40.5 min, and it was observed in TL extract. According to reference previously reported, 1st peak (RT 7.4 min), 2nd peak (RT 7.7 min), 3rd peak (RT 9.2 min) and 4th (RT 10.5 min) were assumed as lutein, zeaxanthin, β-apo-8′-carotenal and β-cryptoxanthin though the resolution between 1st and 2nd peaks were poor (Howe & Tanumihardjo, 2006).