Background: A co-inhibitory molecule, B7-H4, is believed to negatively regulate T cell immunity by suppressing T cell proliferation and inhibiting cytokine production. However, the mechanism behind B7-H4-mediated tolerance remains unclear. Methods: Balb/c $(H-2^d)$ mice were fed with dendritic cell line, DC2.4 $(H-2^d)$ every day for 10 days. Meantime, mice were hydrodynamically injected with recombinant plasmid expressing B7-H4 fusion protein (B7-H4.hFc) or hFc via tail vein. One day after last feeding, mice were immunized with allogeneic B6 spleen cells. 14 days following immunization, mice were challenged with B6 spleen cells to ear back and the ear swelling was determined the next day. Subsequently, a mixed lymphocyte reaction (MLR) was also performed and cytokines profiles from the reaction were examined by sandwich ELISA. Frequency of immunosuppressive cell population was assayed with flow cytometry and mRNA for FoxP3 was determined by RT-PCR. Results: Tolerant mice given plasmid expressing B7-H4.hFc showed a significant reduction in ear swelling compared to control mice. In addition, T cells from mice given B7-H4.hFc plasmid revealed a significant hyporesponsiveness of T cells against allogeneic spleen cells and showed a significant decrease in Th1 and Th2 cytokines such as IFN-${\gamma}$, IL-5, and TNF-${\alpha}$. Interestingly, flow cytometric analysis showed that the frequency of CD4+CD25+FoxP3+ Tregs in spleen was increased in tolerant mice given recombinant B7-H4.hFc plasmid compared to control group. Conclusion: Our results demonstrate that B7-H4 synergistically potentiates oral tolerance induced by allogeneic cells by increasing the frequency of FoxP3+ CD4+CD25+ Treg and reducing Th1 and Th2 cytokine production.
Naegleria fowleri is the cause of primary amoebic meningoencephalitis in man, IL-2 levels after stimulation of T lymphocytes by PHA or N.fowleri lysates. the amounts of T lymphocyte subsets and the blastogenic responses of T lymphocytes in mice after Infected with pathogenic N. fowleri were studied comparing between two study groups, one $1{\;}{\times}{\;}10^4$ trophozoites inoculated mice and the other $1{\;}{\times}{\;}10^5$ trophozoites inoculated mice. All experimental samples were obtained on the day 7, 14 and 24 after inoculation. The mice inoculated with $1{\;}{\times}{\;}10^4$ trophozoites showed a 14.3% mortality rate, and 72.2% in the mice inoculated with $1{\;}{\times}{\;}10^5$ trophozoites. The IL-2 levels on day 14 of two experimental groups were significantly decreased as compared with the control group. Thy 1.2+T cells in the total spleen Iymphocytes of $1{\;}{\times}{\;}10^5$ trophozoites inoculated group on day 7 were significantly increased compared with the control group. There was no significant difference between $1{\;}{\times}{\;}10^4$ trophozoites inoculated group and the control group. $L3T4^{+}{\;}T$ cells and $Ly2^{+}$ T cells in the total spleen Iymphocytes of $1{\;}{\times}{\;}10^5$ trophozoites inoculated group on day 7 were sigrlificantly increased compared with the control group. The DNA S fraction of T cells in the spleen of $1{\;}{\times}{\;}10^5$ trophozoites inoculated group was significantly increased on day 7. The amount of S fractions of DNA were sequentially decreased on day 14 and 24 but they were also signiacantly increased compared with the control group. The results obtained in the experiments indicats that cell mediated immunity after N.fowleri infection acts on very important host's protection immunity around the 7th day after infection. IL-2 level was much suppressed on day 14 which resulted from the exhaustion of host immune response. It was observed that the level of IL-2 production ability and the amounts of T lymphocytes subsets and the blastogenic responses of T lymphocytes were not well correlated during the observation period.
Bakground : Complete resection by the surgery has been selected as the treatment of choice in lung cancer patients, but in cases of recurrence after excision or inoperable cases, the importance of anticancer chemotherapy has been emphasized. If one can select a set of the sensitive chemotherapeutic agents before anticancer chemotherapy, it will give more favourable results. Subrenal capsular assay has been recognized as a useful in-vivo chemosensitivity test of thoracic and abdominal tumors and it can be done in a short time for a rapid interpretation of tumor responsiveness to anticancer chemotherapeutic drugs. It has been reported that various kinds of cancer cells can be implantable to the kidney, but so far there is no comparative study of xenogeneic cell implantation on liver, spleen and kidney. The author implanted the human lung cancer cells under the capsule of S.D rat's liver, spleen and kidney respectively and compared the pattern of growth and histology. Material and Method: After incubation of human lung cancer cell line (SW-900 G IV) in RPMI 1640 (Leibovitz L-15 medium) culture media, 3${\times}$3${\times}$3 mm size fibrin clots which contain 108 cancer cells were made. Thereafter the fibrin clots were implanted at subcapsule area of liver, spleen and kidney of S.D. female rat. For immune suppression, cyclosporin-A (80 mg/Kg) was injected subcutaneously daily from post-implantation first day to sixth day. The body weight was measured at pre and post implantation periods. The growth pattern and the size of tumor mass were observed and the pathologic examination and serum tumor marker tests were performed. Result: Body weight increased in both of control and experimental groups. Serum Cyfra 21-1 was not detected. Serum levels of CEA and NSE revealed no significant change. The SCC-Ag increased significantly in implanted group. The growth rate of human lung cancer cells which was implanted on spleen was higher than on liver or kidney. The surface area, thickness, and volume of tumor mass were predominant at spleen. The success rates of implantation were 80% on kidney, 76.7% on spleen and 43.3% on liver. Pathologic examination of implanted tumors showed characteristic findings according to different organs. Tumors that were implanted on kidney grew in a round shape, small and regular pattern. In the spleen, tumors grew well and microscopic neovascularization and tumor thrombi were also found, but the growth pattern was irregular representing frequent daughter mass. Human lung cancer cells that were implanted in the liver, invaded to the liver parenchyme, and had low success rate of implantation. Microscopically, coagulation necrosis and myxoid fibrous lesion were observed. Conclusion: The success rate of implantation was highest in the kidney. And the mass revealed regular growth that could be measured easily. The SCC-Ag was presented earlier than CEA or Cyfra21-1. The Cyfra21-1 was not detected at early time after implantation. The best model for tumor implantation experiment for chemosensitivity test was subrenal capsular analysis than liver and spleen and the useful serum tumor marker in early period of implantation was the SCC-Ag.
Park, Jae-Yong;Kim, Jeong-Ran;Chang, Hee-Jin;Kim, Chang-Ho;Park, Jae-Ho;Jung, Tae-Hoon
Tuberculosis and Respiratory Diseases
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v.45
no.3
/
pp.587-595
/
1998
Background: Recombinant adenovirus hold promise as vectors to carry therapeutic genes for several reasons: 1) they can infect both dividing and non-dividing cells; 2) they have the ability to directly transduce tissues in vivo; 3) they can easily be produced in high titer; and 4) they have an established record of safety as vaccination material. However, one of the major limitation in the use of adenoviruses is that transgene expression is quite short because adenovirusees insert their DNA genome episomally rather than by chromosomal integration, and an immune response against the virus destroys cells expressing the therapeutic gene. Since sodium butyrate has been reported to induce adenovirus-mediated gene expression, we hypothesized that treatment of tumor cells, transduced with herpes simples virus thymidine kinase(HSVtk) gene using adenoviral vector, with butyrate could augment the effect of gene therapy. Methods: We transduced HSVtk gene, driven by the cytomegalovirus promoter, into REN cell line(human mesothelioma cell line). Before proceeding with the comparison of HSVtk/ganciclovir mediated bystander killing, we evaluated the effect of butyrate on the growth of tumor cells in order to rule out a potential antitumor effect of butyrate alone, and also on expression of HSVtk gene by Western blot analysis. Then we determined the effects of butyrate on bystander-mediated cell killing in vitro. Results: There was no inhibition of growth of cells exposed to butyrate for 24 hours at a concentration of 1.5mM/L. Toxic effects were seen when the concentration of butyrate was greater than 2.0mM/L. Gene expression was more stable and bystander effect was augmented by butyrate treatment of a concentration of 1.5mM/L. Conclusion: These results provide evidence that butyrate can augment the efficiency of cell killing with HSVtk/GCV system by inducing transgene expression and may thus by a promising new approach to improve responses in gene therapy using adenoviral vectors.
Journal of the Korean Society of Food Science and Nutrition
/
v.40
no.7
/
pp.949-955
/
2011
[ ${\beta}$ ]Glucan is a polysaccharide expressed on the cell walls of fungi. It is known that ${\beta}$-glucan is recognized by a family of C-type lectin receptors, dectin-1, which is expressed mainly on myeloid immune cells, including macrophages, neutrophils and dendritic cells. Raw 264.7 cells were treated with ${\beta}$-glucan from Schizophyllum commune. ${\beta}$-Glucan was not cytotoxic up to 400 ${\mu}g$/mL as measured by MTT assay. To measure the activity of macrophages, NO and TNF-${\alpha}$ assays were performed in Raw 264.7 cells. Treatment with ${\beta}$-glucan for 24 hr significantly increased production of NO and TNF-${\alpha}$ compared with control groups (p<0.05), indicating activation of macrophages. To measure inhibition of breast cancer cell proliferation, MTT assay was performed in MDA-MB-231 cells. Cell viability was significantly decreased in the group treated with 400 ${\mu}g$/mL of ${\beta}$-glucan for 48 hr (p<0.05) compared to the control group. However, tumor volume was decreased in the groups administered 200 ${\mu}g$ of ${\beta}$-glucan/mouse compared to the control group. These results indicate that ${\beta}$-glucan inhibits breast cancer cell growth through the induction of apoptosis.
No, Hoon-Jeong;Moon, Gu;Moon, Seok-Jae;Won, Jin-Hee;Moon, Young-Ho;Park, Rae-Gil
THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
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v.6
no.1
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pp.81-97
/
2000
Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.
Kim, Do-Soon;Park, Jueng-Eun;Seo, Kwon-Il;Ko, Sung-Ryong;Lee, Jong-Won;Do, Jae-Ho;Yee, Sung-Tae
Journal of Ginseng Research
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v.30
no.3
/
pp.117-127
/
2006
Ginseng is a medicinal herb widely used in Asian countries. Dendritic cells(DCs) play a pivotal role in the initiation of T cell-mediated immune responses, making them an attractive cellular adjuvant for use in cancer vaccines. In this study, we examined the effects of Red-ginseng(water extract, edible and fermented ethyl alcohol extract, crude saponin) on the DCs phenotypic and functional maturation. Immature DCs were cultured in the presence of GM-CSF and IL-4, and the generated immature DCs were stimulated by water extract, edible and fermented ethyl alcohol extract, crude saponin and LPS, respectively, for 24hours. The expression of surface co-stimulatory molecules, including MHC(major histocompatibility complex) class II, CD40, CD80 and CD86, was increased on DCs that were stimulated with crude saponin, but antigen-uptake capacity was decreased. The antigen-presenting capacity of Red-ginseng extracts-treated DCs as analyzed by allogeneic T cells proliferation and IL-2, $IFN-{\gamma}$ production was increased. Furthermore, $CD4^+$ and $CD8^+$ syngeneic T cell(OVA-specific) proliferation and $IFN-{\gamma}$ production was significantly increased. However, $CD4^+$ syngeneic T cell secreted higher levels of IL-2 in responding but not $CD8^+$ syngeneic T cell. These results indicate the immunomodulatory properties of Red-ginseng extracts, which might be therapeutically useful in the control of cancers and immunodeficient diseases through the up-regulation of DCs maturation.
Son, Ji Yoon;Park, Young W.;Renchinkhand, Gereltuya;Han, Jung Pil;Bum, Jin Woo;Paik, Seung-Hee;Lee, Jo Yoon;Nam, Myoung Soo
Journal of Life Science
/
v.26
no.6
/
pp.689-697
/
2016
Kefir is an acidic-alcoholic fermented milk product originating from the Caucasian mountains. Kefir has long been known for its probiotic health benefits, including its immunomodulatory effects. The objectives of this study were to investigate the properties of a fermented whey product and to examine the effects of kefir grains on the in vitro immune-modulation of human mast cell-1 (HMC-1). The results showed that the whey fermented by kefir grains contained the maximum lactic acid bacteria and yeast for 16 hr by 1.83×108 and 6.5×105 CFU/ml, respectively, and lactose and whey proteins were partially hydrolyzed. The experimental whey fermented by kefir grains exhibited an in vitro anti-inflammatory effect on the HMC-1 line for 8, 16, and 24 hr, and this effect induced the expression of interleukin (IL)-4 as a pro-inflammatory cytokine, but not for 48 hr by RT-PCR in HMC-1 cells. In addition, the same phenomenon was observed for the expression of IL-8 as a pro-inflammatory cytokine by the kefir-fermented whey during the same periods of 8-48 hr under the same conditions. These cytokines resulted in the production of IL-4 at 20-25 ng in HMC-1 cells for 8, 16, and 24 hr, whereas 5 ng was produced for 48 hr by the fermented whey. In contrast, IL-8 was produced at 15-20 ng in HMC-1 cells during 4, 8, 16, and 24 hr, while 7 ng was produced at 48 hr. It was concluded that the whey fermented by kefir grains possesses a potential anti-inflammatory function, which could be used for an industrial application as an ingredient of functional foods and pharmaceutical products.
Journal of the Korean Society of Food Science and Nutrition
/
v.45
no.11
/
pp.1564-1570
/
2016
The present study investigated the immunomodulatory effects of high-purity ${\beta}$-1.3/1.6-glucan on macrophages, natural killer (NK) cells, and T cell-mediated factors. Effect of high-purity ${\beta}$-1.3/1.6-glucan on cytotoxicity in macrophages was investigated. Using macrophages, cytotoxicity of high-purity ${\beta}$-1.3/1.6-glucan was evaluated by MTT assay. We treated high-purity ${\beta}$-1.3/1.6-glucan at concentrations of 10, 50, 100, 150, 200, and $250{\mu}g/mL$ in macrophages. High-purity ${\beta}$-1.3/1.6-glucan did not affect macrophage viability. Phagocytic activity was assessed using zymosan. Activity of high-purity ${\beta}$-1.3/1.6-glucan on macrophages significantly increased as compared with zymosan. We treated high-purity ${\beta}$-1.3/1.6-glucan to murine NK cells co-incubated with YAC-1 cells. High-purity ${\beta}$-1.3/1.6-glucan resulted in significantly increased activity of NK cells as compared with the control. In addition, treatment of macrophages with high-purity ${\beta}$-1.3/1.6-glucan resulted in significantly increased activity of T cell-mediated cytokine (IL-2, IL-12, $IFN-{\gamma}$, and $TNF-{\alpha}$) levels and CD4+/CD8+ T cells as compared with the control. In conclusion, high-purity ${\beta}$-1.3/1.6-glucan could enhance the immune response through activation of macrophages, NK cells, and T cell-mediated factors.
Two kinds of Korean rice-wine (Yakju) with different process and ingredients, and Japanese rice-wine (Sake) were chosen for this study, and throughly dried and solubilized in water or cell culture medium. In vitro cytotoxicity assays of the solubilized wine solids exhibited that maximum dilution factors for inhibition of B 16BL6 mouse melanoma cell growth were 16X for herbal medicine-added rice-wine (Korean rice-wine I) and typical Korean rice-wine (Korean rice-wine II), and 8X for Japanese rice-wine. Their cytotoxic effects on HRT18 human colon adenocarcinoma cells were even lower than those on B16BL6 cells. The morphology of the tumor cells were changed by addition of the solubilized wine solids. Inhibitory effect of the rice-wine on in vivo tumor growth and metastasis were monitored after implantation of B16BL6 cells into C57BL/6 mice with daily feeding the solubilized wine solids. Compared to non-fed control groups, B16BL6 tumor growth and metastasis to lung were clearly inhibited by feeding the wine solids, in order of Korean rice-wine I > Korean rice-wine II > Japanese rice-wine. The data of in vitro cytotoxicity and the cell shape changes indicate that the inhibitory effect of tumor progression may be attributed to tumor cell differentiation or immune stimulation induced by certain components in the rice-wine, rather than direct cytotoxicity of the components.
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