Effects of <i>Lactobacillus paracasei</i> MCC1849 on the population of T cells and gene expression in PP cells in OVA-immunized mice.

<p>(A) The proportion of Tfh cells (CD4⁺ CXCR5⁺ PD-1^high cells) and Th1 cells (CD4⁺ T-bet⁺ cells) in PPs were analyzed via FCM. (B) Gene expression related to T cell differentiation was measured via real-time RT-PCR analysis. The level of gene expression was normalized to that of GAPDH mRNA expression in the control group. Data are shown as the mean ± SD. n = 14. Data are representative two independent experiments. *p<0.05, **p<0.01 compared to the control.</p

Effects of <i>Lactobacillus paracasei</i> MCC1849 on the population of IgA-related cells in the intestinal tissues of OVA-immunized mice.

<p>(A, B and C) The proportion of IgA⁺ B220⁺ cells and IgA⁺ plasmablasts (IgA⁺ B220^- cells) in PP, MLN and LP cell samples were analyzed via FCM. Data are shown as the mean ± SD. N = 14. Data are representative two independent experiments. *p<0.05, **p<0.01 compared to the control.</p

Effects of <i>Lactobacillus paracasei</i> MCC1849 on total IgA production in mice.

<p>(A) Mice were treated with or without MCC1849 for 5 weeks. Total IgA concentrations in homogenized small intestine and serum samples were determined via ELISA. (B) The proportions of IgA⁺ B220⁺ cells and IgA⁺ plasmablasts (IgA⁺ B220^- cells) in PPs were analyzed via FCM. (C) Gene expression related to the differentiation of IgA⁺ cells was measured via real-time RT-PCR analysis. The level of gene expression was normalized to that of GAPDH mRNA expression in the control group. Data are shown as the mean ± SD. N = 16. *p<0.05, **p<0.01 compared to the control.</p

Orally administered heat-killed <i>Lactobacillus paracasei</i> MCC1849 enhances antigen-specific IgA secretion and induces follicular helper T cells in mice

<div><p>Antigen-specific immunoglobulin (Ig) A plays a major role in host defense against infections in gut mucosal tissue. Follicular helper T (Tfh) cells are located in germinal centers and promote IgA production via interactions with germinal center B cells. Several studies have demonstrated that some lactic acid bacteria (LAB) strains activate the host’s acquired immune system, inducing IgA secretion in the intestine. However, the precise molecular mechanisms underlying the effects of LAB on IgA production and Tfh cells are not fully resolved. <i>Lactobacillus paracasei</i> MCC1849 is a probiotic strain isolated from the intestine of a healthy adult. In this study, we investigated the effects of orally administered heat-killed MCC1849 on IgA production in the intestine and on Tfh cell induction <i>in vivo</i>. We found that orally administered MCC1849 induced antigen-specific IgA production in the small intestine, serum and lungs. We also observed that MCC1849 increased the proportion of IgA⁺ B cells and Tfh cells in Peyer’s patches (PPs). In addition, MCC1849 increased the gene expression of IL-12p40, IL-10, IL-21, STAT4 and Bcl-6 associated with Tfh cell differentiation. These results suggest that orally administered MCC1849 enhances antigen-specific IgA production and likely affects Tfh cell differentiation in PPs.</p></div

Effects of <i>Lactobacillus paracasei</i> MCC1849 on IL-12 production.

<p>Murine splenocytes were cultured with various heat-killed <i>Lactobacillus</i> strains (10 μg/ml) for 2 days. Data shown are the mean ± SD of the levels of IL-12p70 which are the representative of three independent experiments.</p

Effects of <i>Lactobacillus paracasei</i> MCC1849 on total IgA and OVA-specific IgA production in OVA-immunized mice.

<p>(A, B) Mice were treated with or without MCC1849 for 5 weeks. All mice were orally immunized on days 14, 21, and 28 with OVA and cholera toxin. On day 35, mice were euthanized and dissected. Data show the total IgA and OVA-specific IgA concentrations in homogenized small intestine, small-intestine lavage fluid, homogenized colon, colon contents, serum and lung samples. AU: arbitrary unit. Data are shown as the mean ± SD. N = 14. Data are representative three independent experiments. *p<0.05, **p<0.01 compared to the control.</p

CD4⁺ T cells differentiated with lamina propria APCs from mice fed LG2809 had stronger suppressive activity.

<p>(A and B) BALB/c mice were fed live LG2809 (1 mg/day) or saline for 7 days. CD4⁺ CD25^- T cells from DO11.10 mice were cultured with 1 μM OVA323–339 and CD3^- LP cells from BALB/c mice fed LG2809 or saline as APCs. After 72 hrs, the CD4⁺ T cells were purified by MACS. The activated CD4⁺ T cells were incubated with responder CD4⁺ CD25^- T cells from DO11.10 SPL cells (at ratios of 0.4:1 and 1:1), plus APCs and 1 μM OVA323–339. After 48 hrs, IL-2 in the supernatant was measured by ELISA (A). The activated CD4⁺ T cells were incubated with APCs and 1 μM OVA323–339. After 48 hrs, IL-10 in the supernatant was measured by ELISA (B). Data are shown as means ± SD (n = 4). Data are representative of two independent experiments. Statistical differences were analyzed by Student’s <i>t</i>-test. *<i>p</i><0.05, significantly different vs. saline group; n.s., not significant.; ^###<i>p</i><0.001, significantly different vs. responder DO11.10 CD4⁺ CD25^- T cells alone.</p

Image_1_Identification of the Effects of Chondroitin Sulfate on Inhibiting CDKs in Colorectal Cancer Based on Bioinformatic Analysis and Experimental Validation.pdf

With a high occurrence rate and high mortality, the treatment of colorectal cancer (CRC) is increasingly attracting the attention of scholars. Hub genes that determine the phenotypes of CRC become essential for targeted therapy. In the present study, the importance of cyclin-dependent kinases (CDKs) on the occurrence of CRC was identified by data mining of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The results showed that the gene expression levels of CDK1, CDK4, and CDK6 were obviously changed in different stages of CRC. Among the CDKs, CDK4 was suggested as an independent risk factor for CRC based on Cox analysis. Furthermore, chondroitin sulfate (CS), a kind of dietary supplement to treat osteoarthritis, was predicted to treat CRC based on its chemical structure and GEO datasets. Cell assay experiments with the human CRC cell line HCT-116 also verified this prediction. CS inhibited the gene and protein expression levels of CDKs and increased the ratios of apoptotic or dead HCT-116 cells by regulating mitogen-activated protein (MAP) kinase pathways. Our data highlight the essential roles of CDKs in CRC carcinogenesis and the effects of CS on treating CRC, both of which will contribute to the future CRC treatment.</p

LG2809 increased the ratio of CD62L^low CD44^high CD4⁺ T cells with IL-10-producing and suppressive activities.

<p>DO11.10 mice were treated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158643#pone.0158643.g001" target="_blank">Fig 1</a>. (A and B) Whole SPLs from DO11.10 mice of each group were stained with anti-CD4-PerCP, KJ-1.26-FITC, anti-CD44-PE, and anti-CD62L-APC. The expression of CD44 and CD62L on CD4⁺ KJ-1.26⁺ T cells was analyzed by flow cytometric analysis. The mean ratio of CD62L^low CD44^high cells in CD4⁺ KJ-1.26⁺ T cells is shown (B). Data are shown as means ± SD (n = 6). Data are representative of two independent experiments. Statistical differences were analyzed by Student’s <i>t</i>-test. *<i>p</i><0.05. (C) The correlation between the concentration of IL-10 in the culture supernatant of CD4⁺ T cells shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158643#pone.0158643.g001" target="_blank">Fig 1C</a> and the ratio of KJ-1.26⁺ CD62L^low CD44^high cells among CD4⁺ T cells. Closed circles, LG2809/OVA group; open circles, LG2809/water group; closed triangles, saline/OVA group; open triangles, saline/water group. The continuous line is a linear regression for LG2809/OVA group and the dashed line is a linear regression for saline/OVA group. R, Pearson correlation coefficient. Data are representative of two independent experiments. (D) CD62L^high CD44^high and CD62L^low CD44^high cells sorted from CD4⁺ T cells isolated from DO11.10 mice of the LG2809/OVA group were cultured with APCs and 0.3 μM OVA323–339. After 48 hrs, IL-10 in the supernatant was measured by ELISA. (E) The sorted CD62L^high CD44^high and CD62L^low CD44^high CD4⁺ T cells from DO11.10 mice of the LG2809/OVA group were incubated with responder CD4⁺ T cells from DO11.10 SPL (at a ratio of 1:1), plus APCs and 0.3 μM OVA323–339. IL-2 in the supernatant was measured by ELISA. (F) The sorted CD62L^high CD44^high and CD62L^low CD44^high CD4⁺ T cells from DO11.10 mice of the saline/OVA, LG2809/OVA group, and CD4⁺ CD25^- T cells were incubated with APCs and 0.3 μM OVA323-339. The cultures were pulsed with [³H]-thymidine for the last 24 hrs of the 96 hrs culture periods and [³H]-thymidine incorporation was measured. Data are representative of two independent experiments.</p

Table_1_Identification of the Effects of Chondroitin Sulfate on Inhibiting CDKs in Colorectal Cancer Based on Bioinformatic Analysis and Experimental Validation.pdf

With a high occurrence rate and high mortality, the treatment of colorectal cancer (CRC) is increasingly attracting the attention of scholars. Hub genes that determine the phenotypes of CRC become essential for targeted therapy. In the present study, the importance of cyclin-dependent kinases (CDKs) on the occurrence of CRC was identified by data mining of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The results showed that the gene expression levels of CDK1, CDK4, and CDK6 were obviously changed in different stages of CRC. Among the CDKs, CDK4 was suggested as an independent risk factor for CRC based on Cox analysis. Furthermore, chondroitin sulfate (CS), a kind of dietary supplement to treat osteoarthritis, was predicted to treat CRC based on its chemical structure and GEO datasets. Cell assay experiments with the human CRC cell line HCT-116 also verified this prediction. CS inhibited the gene and protein expression levels of CDKs and increased the ratios of apoptotic or dead HCT-116 cells by regulating mitogen-activated protein (MAP) kinase pathways. Our data highlight the essential roles of CDKs in CRC carcinogenesis and the effects of CS on treating CRC, both of which will contribute to the future CRC treatment.</p